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Title: The Nature of Animal Light

Author: E. Newton Harvey

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PREFACE
CONTENTS
BIBLIOGRAPHY
INDEX

Monographs On Experimental Biology
EDITED BY

JACQUES LOEB, Rockefeller Institute
T. H. MORGAN, Columbia University
W. J. V. OSTERHOUT, Harvard University

THE NATURE OF ANIMAL
LIGHT
BY

E. NEWTON HARVEY, Ph.D.

MONOGRAPHS ON EXPERIMENTAL BIOLOGY
PUBLISHED
FORCED MOVEMENTS, TROPISMS, AND ANIMAL CONDUCT
By JACQUES LOEB, Rockefeller Institute
THE ELEMENTARY NERVOUS SYSTEM
By G. H. PARKER, Harvard University
THE PHYSICAL BASIS OF HEREDITY
By T. H. MORGAN, Columbia University
INBREEDING AND OUTBREEDING: THEIR GENETIC AND
SOCIOLOGICAL SIGNIFICANCE
By E. M. EAST and D. F. JONES, Bussey Institution, Harvard University
THE NATURE OF ANIMAL LIGHT
By E. N. HARVEY, Princeton University

IN PREPARATION
PURE LINE INHERITANCE
By H. S. JENNINGS, Johns Hopkins University
THE EXPERIMENTAL MODIFICATION OF THE PROCESS OF
INHERITANCE
By R. PEARL, Johns Hopkins University
LOCALIZATION OF MORPHOGENETIC SUBSTANCES IN THE EGG
By E. G. CONKLIN, Princeton University
TISSUE CULTURE
By R. G. HARRISON, Yale University
PERMEABILITY AND ELECTRICAL CONDUCTIVITY OF LIVING
TISSUE
By W. J. V. OSTERHOUT, Harvard University
THE EQUILIBRIUM BETWEEN ACIDS AND BASES IN ORGANISM
AND ENVIRONMENT
By L. J. HENDERSON, Harvard University
CHEMICAL BASIS OF GROWTH
By T. B. ROBERTSON, University of Toronto
COÖRDINATION IN LOCOMOTION
By A. R. MOORE, Rutgers College
OTHERS WILL FOLLOW

Monographs on Experimental Biology

THE NATURE OF ANIMAL LIGHT
BY

E. NEWTON HARVEY, Ph.D.
PROFESSOR OF PHYSIOLOGY, PRINCETON UNIVERSITY

PHILADELPHIA AND LONDON J. B. LIPPINCOTT COMPANY
COPYRIGHT, 1920. BY J. B. LIPPINCOTT COMPANY
Electrotyped and Printed by J. B. Lippincott Company.
The Washington Square Press, Philadelphia, U. S. A.

EDITORS' ANNOUNCEMENT

The rapid increase of specialization makes it impossible for one author to
cover satisfactorily the whole field of modern Biology. This situation,
which exists in all the sciences, has induced English authors to issue series
of monographs in Biochemistry, Physiology, and Physics. A number of
American biologists have decided to provide the same opportunity for the
study of Experimental Biology.
Biology, which not long ago was purely descriptive and speculative, has
begun to adopt the methods of the exact sciences, recognizing that for

permanent progress not only experiments are required but quantitative
experiments. It will be the purpose of this series of monographs to
emphasize and further as much as possible this development of Biology.
Experimental Biology and General Physiology are one and the same
science, in method as well as content, since both aim at explaining life from
the physico-chemical constitution of living matter. The series of
monographs on Experimental Biology will therefore include the field of
traditional General Physiology.
Jacques Loeb,
T. H. Morgan,
W. J. V.
Osterhout.

PREFACE

Bioluminescence, the production of light by animals and plants, has always
excited the admiration of the layman and the wonder of the scientist. It is
not surprising that an enormous literature dealing with the subject has
grown up. A large part of this literature, however, is made up merely of
reports that a certain animal is luminous, or records of especially brilliant
phosphorescence of the sea. Among those who have inquired somewhat
more carefully into the nature and causes of light production may be
mentioned the names of Beijerinck, R. Boyle, Dahlgren, Dubois,
Ehrenberg, Krukenberg, Mangold, McDermott, Molisch, Panceri, Pflüger,
Phipson, Quatrefages, Spallanzani, and Trojan. Several of these men have
written comprehensive monographs on the subject.
It is not the purpose of this book to deal with every phase of
bioluminescence. Volumes could be written on the evolutionary side of the
problem and the structure and uses of luminous organs. These questions can
only be touched upon. Neither is it my purpose to discuss the ultimate cause
of the light, whether due to vibration of electrons or to other causes. That
problem must be left to the physicist, although it is highly probable that a

study of animal light will give important information regarding the nature
of light in general, and no theory of light can be adequate which fails to
take into account the extraordinary powers of luminous animals.
We shall be concerned largely with the physical characteristics of animal
light and the chemical processes underlying its production. Great advances
have been made since the first early guesses that the light was due to
phosphorus and was a kind of oxidation. Although the problem cannot be
considered as solved, it has been placed on a sound physico-chemical basis.
Some material is oxidized. Exactly what this material is and why light
accompanies its oxidation are the two more fundamental problems in the
field of Bioluminescence. How far and with what success we have
progressed toward a solution of these problems may be seen from a perusal
of the following pages.
It gives me pleasure to acknowledge the kindness of Dr. W. E. Forsythe of
the Nela Institute, Cleveland, Ohio, in reading and criticizing the
manuscript of Chapter III, and of Professor Lyman of Harvard University
for a similar review of Chapter II. I am also deeply indebted to my wife for
reading the proof and to Dr. Jacques Loeb and Prof. W. J. V. Osterhout for
many suggestions throughout the book. My thanks are also due to Prof. C.
Ishikawa of the Agricultural College, Imperial University of Tokio, Japan,
for his generous assistance in providing Cypridina material. Finally I wish
to acknowledge the support of the Carnegie Institution of Washington,
through its director of Marine Biology, Dr. Alfred G. Mayor. Without this
support much of the work described in this book could not have been
accomplished.
E. N. H.
Princeton, N. J.,
October, 1919.

CONTENTS
CHAPTER PAGE
I. Light-Producing Organisms 1
Early records and theories. "Shining fish and flesh."
"Burning of the sea." Distribution of luminous organisms
in plant and animal kingdoms. Secondary luminosity.
False luminosity. St. Elmo's fire. Ignis fatuus. Flashing of
flowers. Luminosity in man. Use to man of photogenic
organisms.
II. Luminescence and Incandescence 20
The complete spectrum. Radiation and temperature. "Cold
light." Thermoluminescence. Phosphorescence and
fluorescence. Triboluminescence and piezoluminescence.
Crystalloluminescence. Chemiluminescence.
III. Physical Nature of Animal Light 40
Purkinje phenomenon. Color and spectra of animal light.
Polarization. Efficiency of animal light. Infra-red
radiation. Ultra-violet radiation. Luminous efficiency and
visual sensibility. Production of radiation penetrating
opaque objects. Intensity of animal light. Summary.
IV. Structure of Luminous Organs 67
Photochemical and chemiphotic changes. The eye and the
luminous organ. Intracellular and extracellular
luminescence. Continuous and intermittent luminescence.
Periodicity of luminescence. Luminous bacteria. Noctiluca
and photogenic granules. Chætopterus and luminous gland
cells. Cypridina. Luminous glands. The firefly. Luminous
organs (photophores) with lenses, reflectors, opaque and
color screens. Uses and purpose of animal light.
V. The Chemistry of Light Production, Part I 85

Boyle's and Spallanzani's experiments. Shining wood and
burning coal. Oxygen and luminescence. Carbon dioxide
and luminescence. Heat production during luminescence.
Luminescence and respiration. Water and luminescence.
Phipson's noctilucin. Luciferin and luciferase. Photogenin
and photophelein. Proluciferin. Oxyluciferin. Pyrophorin
or luciferescein. Chemiluminescent reactions.
"Biozymoöxyluminescence."
VI. The Chemistry of Light Production, Part II 114
Pyrophorus luciferin and luciferase. Pholas luciferin and
luciferase. Cypridina luciferin; stability, hydrolysis by
acid and enzymes, adsorption, precipitation, salting out,
solubility, distribution. Cypridina luciferin a proteose?
Cypridina luciferase and properties. Cypridina luciferase
an albumin. Specificity of luciferase. Action of fat solvent
anæsthetics. Action of cyanides. Oxyluciferin. Nature of
oxidative reaction.
VII. Dynamics of Luminescence 143
Minute amounts of material for luminescence. Reaction
velocity and chemiluminescence. Temperature and
chemiluminescence. Oxidation in steps. Concentration and
bioluminescence. Temperature and bioluminescence.
Oxidation with and without luciferase. Reaction velocity
and color of bioluminescence.

THE NATURE OF ANIMAL LIGHT

CHAPTER I
LIGHT-PRODUCING ORGANISMS
The fact that animals can produce light must have been recognized from the
earliest times in countries where fireflies and glowworms abound, but it is
only since the perfection of the microscope that the phosphorescence of the
sea, the light of damp wood and of dead fish and flesh has been proved to
be due to living organisms. Aristotle mentions the light of dead fish and
flesh and both Aristotle and Pliny that of damp wood. Robert Boyle in 1667
made many experiments to show that the light from all three sources, as
well as that of the glowworm, is dependent upon a plentiful supply of air
and drew an interesting comparison between the light of shining wood and
that of a glowing coal. Boyle had no means of finding out the true cause of
the light and early views of its nature were indeed fantastic. Even as late as
1800 Hulme concludes from his experiments on phosphorescent fish that
the light is a "constituent principle of marine fishes" and the "first that
escapes after the death of the fish." It was only in 1830 that Michaelis
suspected the light of dead fish to be the result of some living thing and in
1854 Heller gave the name Sarcina noctiluca to the suspected organism. In
1875 Pflüger showed that nutrient media could be inoculated with small
amounts of luminous fish and that these would increase in size, like
bacterial colonies, and we now know that the light of all dead fish and flesh
is due to luminous bacteria.
In the early part of the nineteenth century it was surmised that the light of
damp wood was connected with fungus growth because of a similarity in
smell. In 1854 Heller recognized minute strands, which he called
Rhizomorpha noctiluca, as the actual source of the light. We now know that
all phosphorescent wood is due to the mycelium of various kinds of fungi
and that sometimes the fruiting body of the fungus also produces light.
The phosphorescence or "burning of the sea," which is described by so
many of the older explorers, is also due entirely to living organisms, both
microscopic and macroscopic. The latter are mostly jelly-fish (medusæ) or
comb jellies (Ctenophores) and give rise to the larger, more brilliant flashes

of light often seen in the wake or about the sides of a steamer at night. The
former are various species of dinoflagellates or cystoflagellates such as
Noctiluca (just visible to the naked eye) which collect at the surface of the
sea and often increase in such numbers that the water is colored by day
(usually pink or red) and shines like a sheet of fire when disturbed at night.
Although Noctiluca was recognized as a luminous animal in 1753 by Baker,
the light of the sea was a mysterious phenomenon to the older observers.
MacCartney, speaking before the Royal Society in 1810, outlines the
various older theories as follows: "Many writers have ascribed the light of
the sea to other causes than luminous animals. Martin supposed it to be
occasioned by putrefaction; Silberschlag believed it to be phosphoric; Prof.
J. Mayer conjectured that the surface of the sea imbibed light, which it
afterwards discharged. Bajon and Gentil thought the light of the sea was
electric, because it was excited by friction.... I shall not trespass on the time
of the Society to refute the above speculations; their authors have left them
unsupported by either arguments or experiments, and they are inconsistent
with all ascertained facts upon the subject. The remarkable property of
emitting light during life is only met amongst animals of the four last
classes of modern naturalists, viz., mollusca, insects, worms, and
zoöphytes." MacCartney recognized the true cause of the light, although he
had little idea of the vast number of marine forms which are luminous and
omits entirely any reference to the fishes, many of which produce a light of
their own when living, apart from any bacterial infection.
A survey of the animal kingdom discloses at least 36 orders containing one
or more forms known to produce light and several more orders containing
species whose luminosity is doubtful. In the plant kingdom there are two
groups containing luminous forms. The distribution of luminous organisms
is brought out in the accompanying classification of plants and animals.
Those orders are printed in italics which contain species whose self-
luminosity is fairly well established. It will be noted that further
subdivisions into orders is not given in classes of animals which lack
luminous forms.

TABLE 1
DISTRIBUTION OF LUMINOUS ORGANISMS IN PLANT AND ANIMAL
KINGDOMS

Plant Kingdom
I. Thallophyta
Algæ
Cyanophyceæ (Blue-green Algæ)
Chlorophyceæ (Green Algæ)
Phæophyceæ (Brown Algæ)
Rhodophyceæ (Red Algæ)
Lichenes (Lichens, symbiotic growth of algæ and fungi)
Fungi
Myxomycetes (Slime moulds)
Schizomycetes (Bacteria)
Bacterium, Photobacterium, Bacillus, Pseudomonas,
Micrococcus, Microspira, Vibrio.
Phycomycetes (moulds)
Ascomycetes (Sac fungi, yeasts, some moulds)
Basidiomycetes (Smuts, rusts, mushrooms)
Ustilaginæ (Smuts)
Uridineæ
Auriculariæ (Judas ears)
Tremellineæ (Jelly fungi)
Hymenomycetes (Mushrooms)
Agaricus, Armillaria, Pleurotus, Panus, Mycena,
Omphalia, Locellina, Marasinium, Clitocybe,
Corticium.
Gasteromycetes (Stinkhorns and puff-balls)

II. Bryophyta
Hepaticæ (Liverworts)
Musci (Mosses)

III. Pteridophyta
Equisetineæ (Horsetails)
Salviniæ (Salvinia, Marsilia, etc.)

Lycopodineæ (Club Mosses)
Filicineæ (Ferns)

IV. Spermatophyta
Gymnospermæ (Cycads, Ginkgo, Conifers)
Angiospermæ (Mono- and Dicotyledonous flowering plants).

Animal Kingdom
I. Protozoa. (One-celled animals)
Sarcodina
Rhizopoda
Heliozoa
Radiolaria
Thallassicola, Myxosphæra, Collosphæra, Collozoum,
Sphærozoum.
Mastigophora
Flagellata
Choanoflagellata
Dinoflagellata
Ceratium, Peridinium, Prorocentrum, Pyrodinium,
Gonyaulax, Blepharocysta, Amphidinium, Diplopsalis,
Cochlodinium, Sphærodinium, Gymnodinium.
Cystoflagellata
Noctiluca, Pyrocystis, Leptodiscus, Craspedotella.
Sporozoa
Infusoria

II. Porifera (Sponges)
Calcarea
Hexactinellida
Desmospongiæ

III. Cœlenterata
Hydrozoa (Hydroids and Jelly-fish)
Leptomedusæ or Campanulariæ
Medusa form—Eutima, Phyalidium (Oceania).

Hydroid form—Aglaophenia, Campanularia,
Sertularia, Plumularia, Cellularia, Valkeria, Obelia,
Clytia.
Trachomedusæ
Geryonia, Lyriope, Aglaura
Narcomedusæ
Cunina
Anthomedusæ or Tubulariæ
Medusa form—Thaumantias, Tiara, Turris, Sarsia.
Hydroid form—?
Hydrocorallinæ
Siphonophora
Abyla, Praya, Diphyes, Eudoxia, Hippopodius.
Scyphozoa (Jelly-fish)
Stauromedusæ
Peromedusæ
Cubomedusæ
Carybdia
Discomedusæ
Pelagia, Aurelia, Chrysaora, Rhizostoma, Cyanæa,
Dianea, Mesonema.
Actinozoa (Corals, Sea-fans, Sea-pens, Sea-anemones)
Actinaria
Madreporareia
Antipatharia
Alcyonaria
Alcyonium, Gorgonia, Isis, Mopsea
Pennatulacea
Pennatula, Pteroides, Veretillum, Cavernularia.
Funicularia, Renilla, Pavonaria, Stylobelemon,
Umbellularia, Virgularia?
Ctenophora (Comb-jellies)
Cydippida
Pleurobranchia.
Lobata
Mnemiopsis, Bolinopsis, Leucothea (Eucharis).
Cestida

Cestus.
Beroida
Beroë.

IV. Platyhelminthes
Turbellaria (Flat-worms)
Trematodes (Parasitic flat-worms)
Cestodes (Tape-worms)
Nemertinea (Nemertines)

V. Nemathelminthes
Nematoda (Round worms)
Gordiacea (Hair worms)
Acanthocephala (Acanthocephalids)
Chætognatha (Sagitta)

VI. Trochelminthes
Rotifera (Wheel animalcules)
Gastrotricha (Chætonotus)
Kinorhyncha (Echinoderes)

VII. Molluscoidea
Bryozoa (Corallines)
Entoprocta
Ectoprocta
Membranipora, Scrupocellaria, Retepora? Flustra?
Brachiopoda (Lamp shells)
Phoronidea (Phoronis)

VIII. Annulata
Archiannelida (Primitive worms, including Dinophilus)
Chætopoda (True worms)
Polychæta
Chætopterus, Phyllochaetopterus, Telepsaris, Polynoë,
Acholoë, Tomopteris, Odontosyllis, Lepidonotus,
Pionosyllis, Phyllodoce, Heterocirrus,
Polyopthalamus?
Oligochæta

Lumbricus, Photodrilus, Allolobophora (Eisemia),
Microscolex, Nonlea, Enchytræus, Octochætus.
Gephyrea (Sipunculus)
Hirudinea (Leeches)
Myzostomida (Myzostomus)

IX. Echinodermata
Asteroidea (Star-fish)
Ophiuroidea (Brittle-stars)
Ophiurida
Ophiopsila, Amphiura, Ophiacantha, Ophiothrix,
Ophionereis.
Euryalida
Echinoidea (Sea urchins)
Holothuroidea (Sea Cucumbers)
Crinoidea (Feather-stars)

X. Arthropoda
Crustacea (Crabs, lobsters, shrimps, etc.)
Phyllapoda
Ostracoda
Halocypris, Cypridina, Pyrocypris, Conchœcia,
Cyclopina.
Copepoda
Metridia, Leuckartia, Pleuromma, Oncæa,
Heterochæta.
Cirripedia
Phyllocardia
Schizopoda
Nyctiphanes, Nematoscelis, Gnathophausia, Euphausia,
Stylochiron,Boreophausia, Mysis?
Decapoda
Sergestes, Aristeus, Heterocarpus, Hoplophorus,
Acanthephyra, Pentacheles, Colossendeis
Stomatopoda
Cumacea
Amphipoda

Isopoda
Onychophora (Peripatus)
Myriapoda (Centipedes and Millepedes)
Symphyla
Chilopoda
Geophilus, Scolioplanes, Orya.
Diplopoda
Pauropoda
Insecta (Insects)
Aptera (Spring-tails)
Lipura, Amphorura, Neanura
Orthoptera
Neuroptera
Teleganoides and Cænis of the Mayflies? Termites?
Hemiptera
Diptera (Flies)
Bolitophila and Ceroplatus larvæ, Thyreophora?
Coleoptera (Beetles)
Pyrophorus, Photophorus, Luciola, Lampyris,
Phengodes, Photuris, Photinus, etc.
Lepidoptera
Hymenoptera
Arachnida (Spiders)

XI. Mollusca
Amphineura (Chiton)
Pelecypoda (Bivalves)
Protobranchia
Filibranchia
Pseudo-Lamellibranchia
Eu-lamellibranchia
Pholas
Septibranchiata
Gasteropoda (Snails, periwinkles, slugs, etc.)
Prosobranchiata
Ophisthobranchiata
Phyllirrhoë, Plocamopherus.

Pulmonata
Scaphopoda (Dentalium)
Cephalopoda (Squids and Octopus)
Tetrabranchiata
Dibranchiata decapoda
Onychoteuthis, Chaunoteuthis, Lycoteuthis,
Nematolampas, Lampadioteuthis, Enoploteuthis,
Abralia, Abraliopsis, Watasenia, Ancistrocheirus,
Thelidioteuthis, Pterygioteuthis, Pyroteuthis,
Octopodoteuthis?, Calliteuthis, Histioteuthis,
Benthoteuthis, Hyaloteuthis, Eucleoteuthis,
Chiroteuthis, Mastigoteuthis, Cranchia, Liocranchia,
Pyrgopsis, Leachia, Liguriella, Phasmatopsis,
Toxeuma, Megalocranchia, Leucocranchia,
Crystalloteuthis, Phasmatoteuthis, Galiteuthis,
Corynomma, Hensenioteuthis, Bathothauma, Rossia?,
Heteroteuthis, Iridoteuthis, Sepiola, Rondeletia,
Inioteuthis, Euprymna, Melanoteuthis?.

XII. Chordata
Adelochorda (Balanoglossus)
Balanoglossus, Ptychodera, Glossobalanus
Urochorda (Ascidians)
Larvacea
Appendicularia?
Thaliacea
Salpa, Doliolum?
Ascidiacea
Pyrosoma, Phallusia
Acrania (Amphioxus)
Cyclostomata (Cylostomes)
Pisces (Fishes)
Elasmobranchii
Centroscyllium, Spinax, Paracentroscyllium, Isistius,
Læmargus, Euproctomicrus, Benthobatis?
Holocephalii
Dipnoi

Teleostomi
Stomias, Chauliodus, Melanostomius, Pachystomias,
Bathophilus, Dactylostomius, Malacosteus,
Astronesthes, Ophozstomias, Idiacanthus,
Bathylychnus, Macrostomius, Gonostoma, Cyclothone,
Photichthys, Vinciguerria, Ichthyococcus, Lychnopoles,
Diplophos, Triplophos, Valenciennellus, Maurolicus,
Argyropelecus, Sternoptyx, Polyipnus, Ipnops?
Neoscopelus, Myctophum, Halosausus,
Xenodermichthys? Macrurus? Photoblepharon,
Anomalops, Porichthys, Leuciocornus, Mixonus?
Bassozetus? Oneirodes, Ceratias, Gigantactis,
Chaunax, Malthopsis, Halicmetus, Monocentris,
Lamprogrammus.
Amphibia (Frogs, Toads, Salamanders)
Reptilia (Snakes, Lizards, Turtles)
Aves (Birds)
Mammalia (Mammals)

The only groups of the plant kingdom which are known to produce light are
some of the bacteria and some of the fungi and the dinoflagellates
(Peridineæ) if one is to include them among the plants. Many different
species of phosphorescent bacteria have been described, differing in cultural
characteristics and structural peculiarities and grouped in the genera,
Bacterium, Photobacterium, Bacillus, Microspira, Pseudomonas,
Micrococcus, and Vibrio. Specific names indicating their light-producing
power such as phosphorescens, phosphoreum, luminosum, lucifera, etc.,
have been applied.
All the fungi which are definitely known to produce light belong to the
Basidiomycetes, the largest and most highly developed of the true fungi.
Either the mycelium alone or the fruiting body alone, or both, may be
luminescent.

Among animals the best known forms are the dinoflagellates; Noctiluca;
hydroids; jelly-fish; ctenophores; sea pens; Chætopterus and other marine
worms; earthworms; brittle stars; various crustaceans; myriapods; fireflies
and glowworms, the larvæ of fireflies; Pholas dactylus and Phyllirrhoë
bucephala, both molluscs; squid; Pyrosoma, a colonial ascidian; and fishes.
Luminous animals are all either marine or terrestrial forms. No examples of
fresh water luminous organisms are known. Of marine forms, the great
majority are deep sea animals, and it is among these that the development
of true luminous organs of a complicated nature is most pronounced. Many
of the luminous marine animals are to be found in the plankton, while the
littoral luminous forms are in the minority. Some members of all the above
groups are found at one or another of our marine laboratories with the
possible exception of Pholas, Phyllirrhoë and squid. Although earthworms
and myriapods which produce light are found in the United States, they are
rather rare and seldom observed forms.
Not only adult forms but the embryos and even the eggs of some animals
are luminous. The egg of Lampyris emits light within the ovary and freshly
laid eggs are quite luminous. The light does not come from luminous
material of the luminous organ adhering to the egg when it is laid but from
within the egg itself. Pyrophorus eggs are also luminous. The segmentation
stages of Ctenophores are luminous on stimulation, as noted by Allman
(1862), Agassiz (1874) and Peters (1905), but the eggs themselves do not
luminesce. Schizopod larvæ (Trojan, 1907), Copepod nauplii (Giesbrecht,
1895), Chætopterus larvæ (Enders, 1909), and brittle star plutei (Mangold,
1907) also produce light.
Apparently there is no rhyme or reason in the distribution of luminescence
throughout the plant or animal kingdom. It is as if the various groups had
been written on a blackboard and a handful of sand cast over the names.
Where each grain of sand strikes, a luminous species appears. The
Cœlenterates have received most sand. Luminescence is more widespread
in this phylum and more characteristic of the group as a whole than any
other. Among the arthropods luminous forms crop up here and there in
widely unrelated groups. In the mollusks, excluding the cephalopods, only
two luminous species are known. Several phyla contain no luminous forms
whatever. It is an extraordinary fact that one species in a genus may be

luminous and another closely allied species contain no trace of luminosity.
There seems to have been no development of luminosity along direct
evolutionary lines, although a more or less definite series of gradations with
increasing structural complexity may be traced out among the forms with
highly developed luminous organs.
While the accompanying list of luminous genera aims to be fairly complete,
there are no doubt omissions and some inaccuracies in it. Anyone who has
ever tried to determine what animal is responsible for the occasional flashes
of light observed on agitating almost any sample of sea water will realize
how difficult it is to discover the luminous form among a host of non-
luminous ones, especially if the animal is microscopic in size. It is not
surprising, then, to find many false reports of luminous animals in the
literature of the subject and we cannot be too careful in accepting as
luminous a reported case. The difficulty lies chiefly in the fact that all
luminous organisms with the exception of bacteria, fungi, and a few fish,
flash only on stimulation, and, while it is easy enough to see the flash, the
animal is lost between the flashes. The only safe way to detect luminous
organisms is to add a little ammonia to the sea water. This slowly kills the
organisms and causes any luminous forms to glow with a steady, continuous
light for some time, a condition accompanying the death of the animal. Not
all observers, however, have followed this method. One must always be on
guard against confusing the light from a supposed luminous form with the
light from truly luminous organisms living upon it. The reported cases of
luminosity among marine algæ are now known to be due to hydroids or
unicellular organisms living on the alga.
We know also that many non-luminous forms may become infected with
luminous bacteria, not only after death, but also while living, so that their
luminescence is purely secondary. Giard and Billet (1889-90) succeeded in
inoculating many different kinds of amphipod crustacea (Talitrus,
Orchestia, Ligia) and isopod crustacea (Porcellio, Philoscia) with luminous
bacteria, in some cases passing the infection from one to the next through
nine individuals. Curiously enough the bacterium did not produce light on
artificial culture media but did when growing in the body of the crustacea,
which were killed in about seven days by the infection. The species of
Talitrus and Orchestia might easily have been taken for truly luminous
animals if not carefully investigated.

Tarchanoff (1901) has injected luminous bacteria into the dorsal lymph sac
of frogs with the result that the animals continued to glow for three to four
days, especially about the tongue. I remember once while collecting
luminous beetles in Cuba, I was astounded to find a frog which was
luminous. Expecting this animal to be of great interest, I examined it further
only to find that the frog had just finished a hearty meal of fireflies, whose
light was shining through the belly with considerable intensity.
Infection with luminous bacteria is especially liable to occur in any dead
marine animal. The flesh is an excellent culture medium. I have seen non-
luminous species of squid, recently killed, covered with minute growing
colonies, quite evenly spaced, so as to closely resemble luminous species
whose light is restricted to scattered light organs over the surface of the
body.
Indeed Pierantoni (1918) has carried this idea to extremes. He believes that
in the luminous organs of fireflies, cephalopods and Pyrosoma, luminous
symbiotic bacteria occur which are responsible for the light of these
animals, and he claims in the case of cephalopods and Pyrosoma to have
been able to isolate these in pure culture on artificial culture media. In the
firefly they can be seen but not grown and in luminous animals where no
visible bacteria-like structures are apparent he believes we are dealing with
ultra-microscopic luminous bacteria similar to the pathogenic forms
suspected in filterable viruses. While the assumption of ultra-microscopic
organisms makes the refutation of Pierantoni's views a somewhat hazardous
task, no one can deny that even an ultra-microscopic organism will be killed
by boiling with 20 per cent. (by wt.) HCl for 6 hours. As we shall see, the
luminous material of Cypridina, an ostracod crustacean, can withstand such
prolonged boiling with strong acid. The light of one animal at least, and I
believe many others also, cannot be due to any sort of symbiotic organism.
Apart from these cases where light is actually produced but is not primary,
not produced by the animal itself, there are many forms whose surface is so
constituted as to produce interference colors. This is true in many cases
among the birds and butterflies whose feathers and scales are iridescent.
Some of these have been erroneously described as luminous. Perhaps the
best known case among aquatic animals is Sapphirina, a marine copepod
living at the surface of the sea, and especially likely to be collected with

other luminous forms. Its cuticle is so ruled with fine lines as to diffract the
light and flash on moving much as a fire opal. Needless to say no trace of
light is given off from this animal in a totally dark room.
It has often been supposed that the eye of a cat or of other animals is
luminous. The eyes of a moth, also, can be seen to glow like beads of fire
when it is flying about a flame. Both of these cases are, however, purely
reflection phenomena and due to reflection out of the eye again of light
which has entered from some external source. The correct explanation was
given by Prevost in 1810. The eye of any animal is quite invisible in
absolute darkness. The same explanation applies to the moss, Schistostega,
which lives in dimly illuminated places and whose cells are almost
spherical, constructed like a lens, so as to refract the light and condense it
on the chloroplasts at the bottom of the cells. Some of this light is reflected
out of the cells again and gives the appearance of self-luminosity. The alga,
Chromophyton rosanoffii, is another example of apparent luminosity, due to
reflection from almost spherical cells.
There are several light phenomena known which have nothing to do with
living organisms. Commonest of these is St. Elmo's fire ("corposants" of
English sailors), a glow accompanying a slow brush discharge of electricity,
which appears as a tip of light on masts of ships, spires of churches or even
the fingers of the hand. It is best seen in winter during and after snowstorms
and is a purely electrical phenomenon.
Less well known is the Ignis fatuus (Will-o'-the-Wisp, Jack-o'-Lantern,
spunkie), a fire seen over marshes and stagnant pools, appearing as a pale
bluish flame which may be fixed or move, steady or intermittent. So
uncommon is this phenomenon that its nature is not well understood, but it
is believed to be the result of burning phosphine (PH3 + P2H4), a self-
inflammable gas, generated in some way from the decomposition of organic
matter in the swamp. The difficulty with this explanation is that phosphine
is not known as a decomposition product of organized matter. Methane
(CH4), a well-known decomposition product of organic matter and
abundantly formed in swamps, will burn with a pale bluish flame and some
have thought the Ignis fatuus to be the result of this gas. As methane is not
self-inflammable there remains the difficulty of explaining how it becomes
lighted. Although still a mystery, it is possible that this light is also of

electrical origin or that in some cases large clusters of luminous fungi have
been observed.
The flashing of flowers, especially those of a red or orange color, like the
poppy, which many observers have noticed during twilight hours, is a
purely subjective phenomenon due to the formation of after images in eyes
partially adapted to the dark. This flashing, first observed by the daughter of
Linnæus, is never observed in total darkness or in the direct field of vision,
but only in the indirect field as during a sidelong glance at the plant.
There are some cases of luminosity on record in connection with man
himself. (See Heller, 1854). Before the days of aseptic and antiseptic
surgery, wounds frequently became infected with luminous bacteria and
glowed at night. The older surgeons even supposed that luminous wounds
were more apt to heal properly than non-luminous ones. We know that
luminous bacteria are non-pathogenic, harmless organisms and the presence
of these forms even on dead fish or flesh never accompanies but always
precedes putrefaction. As recorded by Robert Boyle, no harm has come
from eating luminous meat, unless it may also have become infected with
pathogenic forms.
A few cases of luminous individuals have been noted in which the skin was
the source of light, especially if the person sweated freely. It is possible that
here we are again dealing with luminous bacteria upon the accumulations of
substances passed out in the sweat, which serves as a nutrient medium.
There are also on record, in the older literature, cases of luminous urine,
where the urine when freshly voided was luminous. If these observations
are correct and they may, perhaps, be doubted, we are at present uncertain
of the cause of the light. Bacterial infections of the bladder are not
inconceivable although luminous bacteria are strongly aerobic and would
not thrive under anaerobic conditions. I can state from my own experiments
that luminous bacteria will live in normal human urine, but not well. In
albuminous urines it is very likely that they would live better, and it is
possible that the luminous urines reported are the results of luminous
bacterial infection. On the other hand, the light may be purely chemical, due
to the oxidation of some compound, an abnormal incompletely oxidized
product of metabolism, which oxidizes spontaneously in the air. We know

that sometimes these errors in metabolism occur, as in alkaptonuria, where
homogentistic acid is excreted in the urine and on contact with the air
quickly oxidizes to a dark brown substance. Light, however, has never been
reported to accompany the oxidation of homogentistic acid, although it does
accompany the oxidation of some other organic compounds. (See Chapter
II.)
Finally, we may inquire to what extent luminous animals may be utilized by
man. Leaving out of account the use of tropical fireflies for adornment by
the natives of the West Indies and South America and the use for bait, in
fishing, of the luminous organ of a fish, Photoblepharon, by the Banda
islanders, we find that luminous bacteria are of value for certain purposes in
the laboratory.
These methods are all due to Beijerinck (1889, 1902). He has, for instance,
used luminous bacteria for testing bacterial filters. If there is a crack in the
filter the bacteria will pass through and a luminous filtrate is the result, but
a perfect filter allows no organisms to pass and gives a dark filtrate.
Luminous bacteria are also very sensitive to oxygen and cease to luminesce
in its absence. By mixing luminous bacteria with an emulsion of
chloroplasts (from clover leaves) in the dark, allowing the bacteria to use up
all the oxygen, and then exposing the mixture to light of various colors, the
effect of different wave-lengths in causing photosynthesis could be studied.
Only if the chloroplasts are exposed to a color in the spectrum which
decomposes CO2 with liberation of oxygen do the bacteria luminesce, and
when this oxygen is used up by the bacteria, the tube again becomes dark.
Beijerinck has also worked out a method of testing for maltose and diastase
with luminous bacteria, based on the fact that a certain form,
Photobacterium phosphorescens, will only produce light in presence of
maltose or diastase which will form maltose from starch.
Although Dubois and Molisch have both prepared "bacterial lamps" and
although it has been suggested that this method of illumination might be of
value in powder magazines where any sort of flame is too dangerous, it
seems doubtful, to say the least, whether luminous bacteria can ever be used
for illumination. Other forms, perhaps, might be utilized, but bacteria
produce too weak a light for any practical purposes. The history of Science

teaches that it is well never to say that anything is impossible. It is very
unlikely that any luminous animal can be utilized for practical illumination,
but there is no reason why we cannot learn the method of the firefly. Then
we may, perhaps, go one step further and develop a really efficient light
along similar lines. To what extent our inquiry into the "secret of the firefly"
has been successful may be gleaned from the following pages.

CHAPTER II
LUMINESCENCE AND INCANDESCENCE
Modern physical theory supposes that light is a succession of wave pulses
in the ether caused by vibrating electrons. The light to which we are most
accustomed—sunlight, electric light, gaslight, etc.,—is due to electrical
phenomena connected more or less directly with the high temperature of the
source of the light. Every solid body above the temperature of absolute zero
is giving off waves of different wave-length (λ) and frequency (ν) but of the
same velocity (υ), in vacuo, 180,000 miles, or 300,000 kilometres a second.
In fact, υ (a constant)=λν, so that it is only necessary to designate the wave-
length in order to characterize the waves. This is radiant energy or radiant
flux.
As everyone knows, the long waves given off in largest amount from
objects at comparatively low temperatures give the sensation of warmth. As
we raise the temperature, in addition to these longer heat waves, those of
shorter and shorter wave-length are given off in sufficient quantity to be
detected. At 525° C., rays of about λ=.76µ in length are just visible as a
faint red glow to the eye. As the temperature increases still shorter wave-
lengths become apparent, and the light changes to dark red (700°), cherry
red (900°), dark yellow (1100°), bright yellow (1200°), white-hot (1300°)
and blue-white (1400° and above). Above λ=.4µ the waves again fail to
affect our eye, and, although they are very active in producing chemical
changes, we have no sense organs for perceiving them. Thus, a white-hot
object liberates radiant energy or flux of many different wave-lengths
corresponding to what we know as "heat, light and actinic rays." All can be
dispersed by prisms of one or another appropriate material to form a wide
continuous spectrum, such as that indicated in Fig. 1. Radiant energy of
λ=.76µ to λ=.4µ, evaluated according to its capacity to produce the
sensation of light, is spoken of as visible radiation or luminous flux.
Below the infra-red comes a region of wave-length as yet uninvestigated,
and beyond this may be placed the Hertzian electric waves of long wave-

length used in wireless telegraphy. Above the ultra-violet comes another
region as yet uninvestigated, and then Röntgen rays (X-rays) and radium
rays, of exceedingly short wave-length. These last types need not concern
us except in that we may later inquire if they are given off by luminous
animals. The shortest of the ultra-violet are known as Schumann and Lyman
rays. These relations are brought out in Table 2.
TABLE 2.
Wave-lengths of Various Kinds of Radiation
Wave-lengths of light are usually given in Ångstrom units. One micron
(µ)=.001 mm.=1000 millimicrons (µµ)=10,000 Ångstrom units (Å) or tenth
metres=10-10 metres or 10-8 centimetres. The entire scale of wave-lengths
extends from 106 to 10-9 centimetres.

Hertzian electric waves (upper limit not reached) above 12 km. to .16 cm.
Unexplored region .16 cm. to 310µ
Infra-red 310µ to .76µ
Visible light 7600 Å to 4000 Å
Ultra-violet 4000 Å to 320 Å
Unexplored region 320 Å to 12 Å
X-rays 12 Å to 0.2 Å
Radium γ rays 0.2 Å and shorter

Fig. 1.—Schematic representation of various
types of radiation to form a wide continuous
spectrum.
The total radiant energy which a body emits is a function of its temperature
and for a perfect radiator, or what is known as a black body, the total
radiation varies as the fourth power of the absolute temperature, T. (Stefan-
Boltzmann Law). The radiant energy emitted at different wave-lengths is

not the same but more energy is emitted at one particular wave-length
(λmax.) than at longer or shorter ones, depending also on the temperature. If
the various waves are intercepted in some way, their relative energy can be
measured by an appropriate instrument and spectral energy curves can be
drawn, showing the distribution of energy throughout the spectrum. Fig. 2
gives a few of the curves, and it will be noted that the maximum shifts
toward the shorter waves the higher the temperature. In fact, for a black
body λmax.×T=2890, and at 5000° C. (about the temperature of the sun)
λmax. lies within the visible spectrum. In gas or electric lights it lies in the
infra-red region. The area enclosed by these spectral energy curves
represents the total energy emitted, and, knowing this and the area enclosed
by the curve of visible radiation, it is easy to determine how efficient a
source of light is as a light-producing body. We shall inquire more fully into
this question in Chapter III, in considering the efficiency of the firefly as a
source of light.

Fig. 2.—Distribution of energy throughout the
spectrum of the sun, electric arc, and gas light
(after Nichols and Franklin). Ordinates show
the relative intensities of different wave-
lengths emitted. The notches in the curve
represent absorption bands and the dotted line
represents what the radiation from the sun
would be if no selective absorption occurred.
V=violet and R=red end of visible spectrum.
(Courtesy Macmillan Co.)
A body which emits light because of its (high) temperature is said to be
incandescent and we speak of temperature radiation. We know, however, of
many cases where substances give off light at temperatures much below

525° C. They do not follow the Stefan-Boltzmann law. The light emission is
stimulated by some other means than heat. Such bodies we speak of as
luminescent, and in this category belong all luminous animals. The
distinction between light and luminescence was first pointed out by
Wiedemann (1888). It is usual to classify luminescences, according to the
means of exciting the light, into the following groups:

Thermoluminescence
Phosphorescence and Fluorescence
Photoluminescence
Cathodoluminescence
Anodoluminescence
Radioluminescence
Triboluminescence and Piezoluminescence
Crystalloluminescence
Chemiluminescence

The luminescence which appears in a vacuum tube when an electric current
is passed through it is sometimes spoken of as electroluminescence. As
electroluminescence and also thermoluminescence are really special cases
of phosphorescence or fluorescence and tribo-and crystalloluminescence are
closely allied, the classification has only the merit of emphasizing the
means of producing light. Let us examine each kind in turn in order that we
may place the light of animals, organoluminescence or bioluminescence (or
biophotogenesis), in one of these classes. All are examples of "cold light,"
light produced at temperature far below those observed in incandescent
solids. In this category should be placed also the light from salts in the
bunsen flame, for flame spectra and line spectra in general, while only
obtained at relatively high temperatures, are not to be confused with the
purely temperature radiation from the incandescent particles of carbon in a
gas or candle light. The sodium or lithium flame, etc., is not a simple
function of temperature and has been spoken of as a luminescence,
pyroluminescence. As the luminescence of organisms could in no manner be
regarded as a pyroluminescence, occurring at temperatures far above those
compatible with life, a consideration of this form of luminescence will be
omitted. Some other low temperature flames are known, such as that of CS2
in air, rich in ultra-violet rays, despite its relatively low temperature. While

these are of interest to the physicist and chemist, they can have no direct
bearing on the luminescence of animals and their consideration will also be
omitted. (See Bancroft and Weiser, 1914-1915.)
Thermoluminescence.—Some substances begin to emit light of shorter
wave-length than red, well below 525°. This is thermoluminescence.
Diamond, marble, and fluorite are examples. Only certain varieties of
fluorite show the phenomenon well. A crystal of one of these varieties
heated in the bunsen flame on an iron spoon will give off a white light long
before any trace of redness appears in the iron. Other crystals may
luminesce in hot water. In all, this luminescence is dependent on a previous
illumination or radiation of the crystal. If kept in the dark for a long time no
trace of light appears when fluorite is placed at a temperature of 100°, but
after a short exposure to the light of an incandescent bulb, although no light
can be observed in the fluorite at room temperature, quite a bright glow
appears at 100°. Calcium, barium, strontium, magnesium and other
sulphates containing traces of manganese sulphate, show a similar
phenomenon after exposure to cathode rays (Wiedemann and Schmidt,
1895 b). They emit light during bombardment, but this soon ceases when
the rays are cut off. If the sulphates are now heated they give off light, red
in the case of MgSO4 + MnSO4, green in the case of CaSO4 + MnSO4. The
power to emit light on heating may be retained for months after the
exposure to cathode rays. The emission of light by bodies after previous
illumination or radiation is called phosphorescence and will be considered
below. It would seem that the cases of thermoluminescence with which we
are acquainted are really cases of phosphorescence intensified by rise of
temperature. The spectrum of thermoluminescent bodies, also, is similar to
that of phosphorescent ones. (See Fig. 3.) However, not all phosphorescent
materials are also thermoluminescent. The production of light by animals is
quite another phenomenon from thermoluminescence.
Phosphorescence and Fluorescence.—Although the word phosphorescence
has been used in a very loose way to indicate all kinds of luminescence, and
particularly that of phosphorus or of luminous animals, to the physicist it
has a very definite meaning, namely, the absorption of radiant energy by
substances which afterwards give this off as light. Phosphorescence does
not strictly apply to the light of white phosphorus. If the radiant energy is
light (visible or ultra-violet) we speak of photoluminescence, if cathode rays

we have cathodoluminescence, if anode rays, anodoluminescence, and if X-
rays (Röntgen rays) we have radioluminescence. Inasmuch as the α, β, and
γ rays of radium correspond to the anode, cathode, and X-rays, respectively,
radium radiation also produces luminescence in many kinds of material. If
the material gives off the light only during the time it is radiated we speak
of fluorescence; if the light persists we speak of phosphorescence. The
distinction is perhaps a purely arbitrary one, as there are a great many
substances which give off light for only a fraction of a second (1/5000 sec.
in some cases) after being illuminated (photoluminescence). Some
substances also, which fluoresce at ordinary temperatures, will
phosphoresce at low temperatures. Phosphorescence is exhibited chiefly by
solids, fluorescence also by liquids and vapors.
Special means must be used to observe a phosphorescence of short
duration. E. Becquerel has devised an apparatus for doing this, a
phosphoroscope. It consists of revolving disks with holes in them between
which the object to be examined is placed. The holes are so arranged that
the object is first illuminated and then completely cut off from light. The
observer looking at it through another hole sees it at the moment it is not
illuminated and can thus tell if it is phosphorescing. By determining the rate
of revolution of the disks it is easy to calculate how long the
phosphorescence persists.
While relatively few solids phosphoresce after exposure to light at ordinary
temperature a large number of these acquire the property at the temperature
of liquid air. Included in the list are such biological products as urea,
salicylic acid, starch, glue and egg shells. The temperature also affects the
wave-length and hence the color of the light given off. Usually the higher
the temperature the shorter the wave-length, but in the case of some bodies
(SrS) the wave-lengths become longer at the higher temperature.
The best known cases of phosphorescence which occur at room temperature
and the group to which the word phosphorescence is commonly applied, are
those of the alkaline earth sulphides (BaS, CaS, SrS) and ZnS. An Italian,
Vicenzo Cascariolo, is said to have discovered the Bologna stone (BaSO4)
which, by calcination with charcoal, gave an impure phosphorescent BaS or
lapis solaris. Canton's phosphorus (CaS) was later prepared "by heating a
mixture of three parts of sifted calcined oyster shells with one part of

sulphur to an intense heat for one hour." Hulme spoke of it as the "light
magnet of Canton," because of its power of attracting and absorbing light.
The pure sulphides do not show this property. Only if small amounts of
some other metal such as Cu, Pb, Ag, Zn, Sb, Ni, Bi, or Mn are present, will
the sulphide phosphoresce. One part of impurity in a million is often
sufficient. Such mixtures, together with a flux of Na2SO4, Li3(PO4)2 or
some other fusible salt constitute a "phosphor." A "phosphor" is in reality an
example of a solid solution and is the basis of some kinds of luminous
paints.
The intensity and duration of a phosphorescent light depend chiefly on the
nature of the exciting rays, the color chiefly on the impurity present but the
alkaline earth metal also exerts an influence. Rise in temperature increases
the intensity but diminishes the duration, so that the total amount of light
emitted is about constant at different temperatures.
The spectrum of most phosphorescent substances is made up of one or more
continuous bands having maxima at different wave-lengths. In the light
incident on a phosphorescent substance are also bands of light rays which
are absorbed and whose wave-lengths are more efficient than others in
stimulating phosphorescence. These bands in the phosphorescent light are
usually of longer wave-length than those in the light which excites the
phosphorescence. This fact is known as Stokes' Law, but it has been found
not to be universally true. Curiously enough, red and infra-red rays have the
power of annulling phosphorescence after a momentary increase in
brightness and phosphorescing materials have been used to determine if
infra-red rays are given off in the light of the firefly. Ives (1910) showed
that infra-red radiation had no power of quenching the light of the firefly as
it does the phosphorescent light of Sidot blende (ZnS), one fact tending to
show that the firefly's light is not due to phosphorescence. Fig. 3 is a
reproduction of a photograph of the phosphorescence spectrum of ZnS.

Fig. 3. Spectrum of zinc sulphide
phosphorescence (after Ives and Luckiesh).
Photographs were taken by a special device
one minute (middle) and fifteen minutes
(bottom) after exposure to the light of the
mercury arc and compared with a helium
spectrum (top). In the middle photograph, the
mercury exciting lines are visible. It will be
noted that the narrow band of phosphorescent
light does not shift its position during decay of
phosphorescence.
Other facts show that the light of luminous animals is in no sense a
phosphorescence and is quite independent of previous illumination of the
animal. Luminous bacteria will continue to luminesce although they are
grown in the dark for many weeks. Indeed strong light has a bactericidal
action on these forms similar to that with ordinary bacteria. With some
marine forms light has an inhibiting effect. They lose their power of
luminescence during the day and only regain it at dusk or when kept in the
dark for some time. Indeed, ordinary light never has the effect of causing
luminescence in the same sense as it causes phosphorescence of CaS.
Fluorescence is most efficiently excited by the cathode rays of a vacuum
tube. They not only cause the residual gas in the tube to glow
(electroluminescence) by which their path may be followed with the eye,
but also a vivid fluorescence of the glass walls of the tube, yellow green

with sodium glass, blue green with lead and lithium glass. LiCl2 in the path
of cathode rays gives off a blue light; in the path of anode rays a red light;
NaCl a blue cathodoluminescence and a yellow anodoluminescence. The
spectrum of the latter is a line spectrum of Li or Na, showing the
characteristic red or yellow lines similar to those observed where Li or Na
is held in the bunsen flame. The spectrum of the salts under excitation of
cathode rays is a short continuous one in the blue region. Fluorescent
spectra in general are of this nature, made up of short bands of light in one
or more regions.
Diamonds, rubies and many minerals fluoresce brilliantly in the path of
cathode rays. Some specimens of fluorite (CaF2) show the phenomenon
especially well, whence the name fluorescence. Fluorescent screens of
barium platinocyanide, willemite (Zn2SiO4), Sidot blend (ZnS) or Scheelite
(Ca tungstate) are frequently employed to render visible X-rays. The
luminous paint most used at the present time is ZnS containing a trace of
radium salt. The rays of the radium continually emitted cause a steady
fluorescence of the ZnS. Indeed, if one examines the paint on the hands of a
watch with a lens the flash of light from the impact of alpha particles on the
ZnS can be distinctly seen, as in the spinthariscope.
Some animal tissues and fluids, especially the lens of the eye, will
luminesce in the path of radium rays, as shown by the experiments of Exner
(1903), but there is no evidence that luminous animals are especially active
in this respect. Ultra-violet rays have the same action.
The luminous material of practically all luminous forms, if dessicated
sufficiently rapidly, can be obtained in the form of a dry powder which will
give off light when moistened with water. Coblentz (1912) has exposed this
dry material to light, to the ultra-violet spark, and to X-rays and in no case
has a phosphorescence or fluorescence ever been observed. I have
examined the action of radium upon Cypridina light. There was no
intensifying or diminishing effect of twenty milligrams of radium (probably
the bromide) on a luminous solution of Cypridina material, nor was
phosphorescence or fluorescence excited in a non-luminous extract of the
animal. We must conclude that animal light is not a fluorescence of any
substance due to radiation produced by the animals themselves.

Many solutions show fluorescence in strong lights. This is especially
marked in quinine sulphate, mineral oils, eosin, fluorescein, esculin,
rhodamin, chlorophyll, etc. The fluorescence of eosin in 10-8 grams per
cubic centimetre is visible in daylight and 10-15 grams per cubic centimetre
in the beam from an arc lamp. It is difficult to realize that the bluish
fluorescence of quinine sulphate is really an emission rather than a
reflection of light. But a test tube of quinine sulphate solution held in the
ultra-violet region of a spectrum will glow with a pale blue light, although it
is not illuminated with any rays that are visible to our eyes. Concerning this,
Stokes, to whom the word fluorescence and much of our knowledge of the
subject is due, says, "It was certainly a curious sight to see the tube"
(containing quinine sulphate solution) "instantaneously lighted up when
plunged into the invisible rays; it was literally 'darkness visible.'" Quinine
sulphate absorbs the ultra-violet converting these rays into visible blue
ones. Its spectrum is a short continuous one. Most fluorescent substances
convert short into longer wave-lengths (Stokes' Law), but some may cause
the reverse change.
A substance, fluorescent in solution, has been found in a few luminous
animals, notably in several species of fireflies and also in a non-luminous
beetle. It is called pyrophorine or luciferesceine. Dubois (1886) has ascribed
to pyrophorine the power of absorbing invisible rays and transforming them
into visible ones, thus increasing the animal's light. That this is not the case
has been shown by the work of Coblentz (1909). He photographed the
spectrum of the firefly's light and the fluorescent spectrum of luciferesceine.
The latter is almost complementary to the former (see Fig. 4) and no trace
of the fluorescent spectrum appears in the spectrum of the light of the
firefly. McDermott (1911 a) has studied the properties of luciferesceine and
regards it merely as an incidental material found in many animals of the
Lampyridæ (in some non-luminous forms) and having no connection with
the light production. A trace of alkali usually increases and acid inhibits the
fluorescence of solutions.

Fig. 4.—Spectrum of fluorescent substance
found in fireflies below (2) and of firefly
luminescence above (2) compared with helium
vacuum tube (1) (after Coblentz).
Triboluminescence and Piezoluminescence.—Under this head are grouped a
number of light phenomena which at first sight may appear to be electrical
in nature but in reality are not. The light is produced by shaking, rubbing, or
crushing crystals, and only crystalline bodies appear to show
triboluminescence or piezoluminescence. A striking case is that of uranium
nitrate. Gentle agitation of the crystals is sufficient to give off sparks of
light which much resemble the scintillations of dinoflagellates when sea-
water containing these animals is agitated. If Romberg's phosphorus, which
is fused CaCl2, is rubbed on the sleeve, it glows with a greenish light.
Lumps of cane sugar rubbed together will glow. Saccharin crystals will also
light if shaken and Pope (1899) found that the bluish light of saccharin was
bright enough to be visible in a room in daytime. It only appeared from
impure crystals and freshly crystallized specimens. Other crystals, also,
have been found to lose their power of lighting after a time.

Among biological substances, cane sugar, milk sugar, mannite, hippuric
acid, asparagin, r-tartaric acid, l-malic acid, vanillin, cocaine, atropin,
benzoic acid, and many others show triboluminescence. A long list is given
by Tschugaeff (1901), by Trautz (1905), and by Gernez (1905). The
spectrum is a short continuous one, the waves emitted depending on the
kind of crystal. Thus the color of the light varies among different santonin
derivatives from yellow to green. In saccharin it is blue.
Although the light produced by some living organisms resembles
triboluminescence in that it may be evoked by rubbing or shaking the
animals, it is in reality fundamentally different since it is dependent on the
presence of oxygen whereas triboluminescence is not.
Crystalloluminescence.—Crystalloluminescence is observed when
solutions crystallize. It was described by Bandrowski (1894, 1895) in
arsenious oxide, in NaF, or if HCl or alcohol is added to hot saturated NaCl
solution. A bluish light with sparkling points appeared. All well
authenticated cases are exhibited by simple inorganic salts and these are
also all triboluminescent. The reverse is not true, however; many
triboluminescent substances are not crystalloluminescent.
Crystalloluminescence is much less widespread than triboluminescence.
Trautz (1905) has studied the matter in a number of compounds and comes
to the conclusion that the light is really a special case of triboluminescence
in which the growth of individual crystals causes them to rub together. The
light becomes much brighter on stirring a mass of crystals which exhibit
crystalloluminescence. While in some cases crystalloluminescence is
unquestionably due to the triboluminescence of crystals rubbing against
each other it is not in every case, as has been clearly shown by the work of
Weiser (1918 b). He studied luminescence of saturated aqueous alkali
halide solutions (NaCl, KCl, etc.,) upon addition of alcohol or of HCl. The
salt crystallizes out under these conditions and Weiser found that the light is
brightest when the conditions of concentration of alcohol or of HCl are such
as to cause heaping up of Na and Cl ions. He believes that the bluish light
which appears is due to the combination of ions in the reaction, Na+ + Cl- =
NaCl. Only if this proceeds rapidly enough does luminescence occur.
Weiser studied also the crystalloluminescence and triboluminescence of
AsCl3 and of K2SO4. By photographing the luminescence through color

screens of different absorptive power (Weiser, 1918, a) a spectrum of the
light could be obtained, and it was found to be identical in both the tribo-
and crystalloluminescent light; in the case of AsCl3, a band in the green-
blue, blue and violet. Weiser believes the light in this case also to come
from recombination of the ions, As+++ + 3Cl- = AsCl3, and that
crystalloluminescence in general is due to rapid reformation of molecules
from ions broken up by electrolytic dissociation while triboluminescence is
due to rapid reformation of molecules from ions broken up by violent
disruption of the crystal. Of course in triboluminescent organic crystals
which do not dissociate into ions, some other reaction must be responsible
for the light. One thing seems certain, that the two types of luminescence
are similar. As Bigelow[1] remarks, "It is altogether probable that the cause
of this" (crystalloluminescence) "whatever it may be, is the same as the
cause of triboluminescence, whatever that may be."
[1] Theoretical and Physical Chemistry, 1912, p. 516.

Crystals are not found in the luminous organs of animals with the exception
of the fireflies. In these a layer of cells occurs (see Chapter IV) filled with
minute crystals of one of the purine bodies (xanthin or uric acid). One
might surmise that the light of the animal was a crystalloluminescence
accompanying the formation of these crystals. It is easy to show, however,
that the light comes not from the crystal layer but from another layer of
cells containing large granules. It is also dependent on the presence of
oxygen while crystalloluminescence takes place in the absence of oxygen.
The crystal layer possibly serves as a reflector. Its significance will be
discussed in a later chapter.

Fig. 5.—Dubois's figures
showing transformation of
photogenic granules to crystals
(after Dubois).
The light of luminous organisms is quite generally associated with granules.
In one of the centipedes (Orya barbarica), which produces a luminous
secretion, Dubois (1893) has described the transformation of these granules
into crystals and at one time he supposed the light to be a
crystalloluminescence. He later reversed this opinion and, certainly,
examination of his drawings which are reproduced in Fig. 5 does not
convince one of the actuality of crystal formation.
The phenomenon of lyoluminescence, described by Wiedemann and
Schmidt (1895) as a light accompanying the solution of colored (from
exposure to cathode rays) crystals of Li, Na, or K chlorides, is probably due
to a triboluminescence from stirring of the crystals during solution.
Chemiluminescence.—As the name implies, chemiluminescence is the
production of light during a chemical reaction at low temperatures. This

does not mean that the other types of luminescence are not connected with
chemical reactions—using the word reaction in a broad sense—for we have
reason to believe that in some cases spectra are not characteristic of the
element as such but are rather characteristic of a particular reaction in
which the element takes part (dissociation into ions, changes from
monovalent to bivalent condition, etc.) and that this is the reason one
element may show various spectra under different conditions (Bancroft,
1913). The chemiluminescences are rather oxidation reactions involving the
absorption of gaseous or dissolved oxygen and may be very easily
distinguished from all the previously mentioned luminescences by this
criterion. They should, perhaps, more properly be called oxyluminescences.
The glow of phosphorus is the best known case, recognized since
phosphorus was first prepared by Brandt in 1669. It is interesting to note
that when first prepared phosphorus was regarded as a peculiarly persistent
type of phosphor, i.e., a material akin to the impure alkaline earth sulphides.
Fresh cut surfaces of Na and K metal will glow in the dark for some time,
especially if warmed to 60°-70° (Linnemann, 1858). A film of oxide is
formed over the surface, showing definitely that oxidation has occurred.
Ozone oxidizes organic matter with an accompanying glow (Fahrig, 1890;
Otto, 1896). The light from ozone acting on pyrogallol solution is especially
bright under certain conditions.
Radziszewski (1877, 1880) gives a long list of substances, chiefly essential
oils, which luminesce if slowly oxidized in alcoholic solutions of alkalis.
Formaldehyde, dioxymethylen, paraldehyde, metaldehyde, acroleïn,
disacryl, aldehydeammonia, acrylammonia, hydrobenzamid, lophin,
hydroanisamid, anisidin, hydrocuminamid, hydrocinamid, besides waxes,
and such biological substances as glucose, lecithin, cholesterin, cholic,
taurocholic, and glycocholic acids, and cerebrin, all luminesce on oxidation.
Radziszewski himself and many other authors have compared the light of
organisms to this type of luminescence. Indeed the incorrect identification
of granules found in the cells of practically all luminous tissues as oil
droplets, is largely due to the influence of Radziszewski's work. Dubois
(1901 b) has added esculin, and Trautz (1904-5) many aldehydes and
phenol derivatives, including vanillin, papaverin, tannic and gallic acids,
besides glycerol and mannite to the list of biological substances oxidizing

with light production. Guinchant (1905) has described oxyluminescence of
uric acid and asparagine, Weitlaner (1911) of substances in humus and
McDermott (1913) of substances in urine and the anaerobic alkaline
hydrolysis products of glue and Witte's peptone. Pyrogallol is especially
prone to luminesce, as was first noticed by Lenard and Wolf (1888) in
developing a photographic plate with pyrogallol developer. Later the
luminescence was studied in some detail by Trautz and Schloringin (1904-
5) who developed the well-known luminescent mixture of pyrogallol,
formaldehyde, K2CO3 and H2O2. As I have shown, pyrogallol can be
oxidized in a great many different ways, and some of these are of great
interest, for they very closely imitate the mechanism for the production of
light in organisms. These are recorded in Table 3, which also includes
various other types of oxyluminescence of general or biological interest.
TABLE 3
Types of Oxyluminescent Reactions

1. Oxidation in air spontaneously.
(a) At ordinary temperatures. [Phosphorus. Fresh-cut surfaces
of Na or K. Thiophosgene and Thio-ethers (RCS.OR).]
(b) At melting or vaporizing points. (Fats, terpenes, sugars,
resins, gums, ether, silk and others.)
2. Oxidation in aqueous or alcoholic alkalies. (Many organic
substances.)
3. Oxidation in hypoiodites, hypobromites, or hypochlorites. (Many
organic substances.)
4. Oxidation in peroxides (H2O2 or Na2O2). (Many organic
substances.)
5. Oxidation in ozone. (Many organic substances.)
6. Oxidation in acid permanganate. (Pyrogallol.)
7. Oxidation in persulfates and perborates. (Formaldehyde,
paraformaldehyde.)
8. Oxidation in perchlorates, periodates, and perbromates. (Palmitic
acid.)
9. Combination of 2 and 4. (Many organic substances.)
10. Combination of 3 and 4. (Many organic substances.)

11. Oxidation with H2O2 and hæmoglobin or vegetable oxidases.
(Pyrogallol, gallic acid, lophin, esculin.)
12. Oxidation with H2O2 and MnO2, Fe2Fe(CN)6 Mn(OH)2 +
Mn(OH)3 Ag2O, chromium oxide, cobalt oxide. (Pyrogallol.)
13. Oxidation with H2O2 and ferrocyanides, chromates, bichromates,
permanganates, Fe salts, and Cr salts. (Pyrogallol, esculin.)
14. Oxidation with H2O2 and collodial Ag. Pt. Pd. Au. (Pyrogallol.)

The spectrum of chemiluminescent reactions has been described in a few
instances as continuous but no definite measurements of its extent have
been made. Radziszewski (1880) found the light of lophin oxidized in
alcoholic caustic alkali, examined with a two-prism spectroscope, to give a
continuous spectrum, brightest at E, with the red and violet ends lacking.
Trautz (1905, p. 101) states that the pyrogallol-formaldehyde-Na2CO3-
H2O2 reaction gives a continuous spectrum from the red to the blue green
with maximum brightness in the orange. Weiser (1918 a) has studied the
spectra of some chemiluminescent reactions by photographing the light
behind a series of color screens. He finds also that the spectra are short,
with maximum intensity in various regions. Thus, amarin oxidized by
chlorine or bromine, extends from the yellow to greenish blue with a
maximum in the green while phosphorus, dissolved in glacial acetic acid
and oxidized with H2O2, luminesces from yellow green to violet.

The spectra of luminous animals are quite similar to those of
chemiluminescent reactions. Moreover, as we have seen,
chemiluminescence is essentially an oxyluminescence, since oxygen is
necessary for the reaction. All luminous animals also require oxygen for
light production. Therefore, bioluminescence and chemiluminescence are
similar phenomena and they differ from all the other forms of luminescence
which we have considered. The light from luminous animals is due to the
oxidation of some substance produced in their cells, and when we can write
the structural formula of this photogenic substance and tell how the
oxidation proceeds, the problem of light production in animals will be
solved.

CHAPTER III
PHYSICAL NATURE OF ANIMAL LIGHT
Interest in the light of animals from a physical standpoint has centred
around questions of quality, efficiency and intensity, but in only one group
of luminous animals, the beetles, have accurate measurements of these
characteristics been made. This is due in part to the abundance of these
forms and their appeal to human interest and in part because they are among
the brightest of luminous organisms. Weak lights are not only difficult to
measure but, when dispersed to form spectra, give bands so faint that their
limits are very difficult to see and more so to photograph. Very few
organisms produce light visible to the fully light-adapted eye. Although
their light may seem quite bright to the dark-adapted eye, the dark-adapted
eye is a poor judge of the quality, i.e., the color of a light. This is because of
the Purkinje phenomenon, a change in the region of maximum sensibility of
the retina with change in intensity of the light. For an equal energy
spectrum, to the normal, completely light-adapted eye, yellow-green light
of wave-length, λ = .565µ, appears the brightest, but when the light is made
fainter the maximum shifts first to the green and then to the blue. The dark-
adapted eye can see green or blue better than yellow and for this reason
weak lights will appear more green or blue than stronger ones of the same
energy distribution. Also two weak lights of the same spectral composition
may appear different in color if they differ much in intensity. This is
illustrated in Fig. 6.

Fig. 6.—Visibility curves for three
illuminations showing the shift in region of
maximum visibility, or Purkinje phenomenon
(after Nutting).
The shift in sensibility of the eye occurs in illuminations of between 0.5 and
50 metre-candles and represents a change from central cone vision (high
intensities) to peripheral rod vision (low intensities). The fovea centralis
lacks rods and this part of the eye becomes practically color blind at very
low intensities of light. Below 0.5 and above 50 metre-candles visibility
varies but little with change in intensity. It is clearly necessary then to
distinguish between the physical objective phenomenon of light and the
physiological subjective sensation of light.
It is a fact that different luminous animals produce light of quite different
colors as judged by our eye. A range of spectral tints has been described
which extends from red to violet but "yellowish," "greenish" and "bluish"
tints are commonest. Indeed one or two animals possess several luminous
organs emitting lights of different colors. This is true in a South American
firefly, Phengodes, whose lights are red and greenish yellow, and in the
deep sea squid, Thaumatolampas diadema, which produces lights of three
colors, two shades of blue and red. The red light in the case of the squid
appears to be due to a red color screen formed by the chromatophores, but
in Phengodes no screen is present.
TABLE 4
Wave-lengths of Fraunhofer Lines and Prominent Lines in Line Spectra

FRAUNHOFER LINES

Line Color Wave-lengths (µµ = µ/1000) Source
A Red 759.4 (band) Oxygen in atmosphere.
a Red 718.5 (band) Water vapor atmosphere.
B Red 686.7 Oxygen vapor atmosphere.
C Red 656.3 Hydrogen in sun.
D1 D2 Yellow589.6, 589.0 Sodium in sun.
E Green 527.0 Calcium in sun.
b1 b2 b4 Green 518.4, 517.3, 516.8 Magnesium in sun.
F Blue 486.1 Hydrogen in sun.
G Violet 430.8 Calcium in sun.
H K Violet 396.9, 393.4 Calcium in sun.

BUNSEN FLAME LINES

Source Color Wave-lengths (µµ = µ/1000)
Potassium Red 769.9, 766.5 (double)
Lithium Red 670.8
Sodium Yellow589.6, 589.0 (double)
Thallium Green 535.1
Magnesium Green 518.4
Strontium Blue 460.7

PLÜCKER TUBE LINES

Source Color Wave-lengths (µµ = µ/1000)
Mercury Yellow579.0, 576.9
Green 546.1
Blue 491.6, 435.8
Violet 407.8, 404.7
Hydrogen Red 656.3

Blue 486.1, 434.1
Helium Red 728.2, 706.5, 667.8
Yellow587.6
Green 504.8, 501.6, 492.2
Blue 471.3, 447.2
Violet 438.8, 402.6, 388.8

As we have seen, difference in color of the light does not necessarily
indicate difference in spectral composition because of the Purkinje effect.
However, examination of the spectrum of various luminous forms has very
clearly indicated that the different colors are really due to light rays of
different wave-length and are not the result of any subjective phenomena.
To facilitate comparison, spectral lines and colors are given in Table 4. The
first adequate observations on the spectra of luminous animals were made
by Pasteur (1864), who studied Pyrophorus and found a continuous
spectrum unbroken by light or dark bands. Lankester (1868) discovered a
similar continuous spectrum in Chætopterus insignis and placed its limits
from line 5 to 10 on Sorby's Scale (about λ = 0.55µ to λ = 0.44µ). Young
(1870) first recorded the limits of the firefly spectrum as a little above C (λ
= .6563µ) to F (λ = .4861µ). Since then a number of luminous forms have
been examined and all are found to give short continuous spectra (not
crossed by light or dark bands or lines) lying in different color regions.
Thus, Conroy (1882) examined the glowworm (Lampyris noctiluca) light
and observed a band extending from λ = 0.518µ to λ = 0.656µ. Dubois
(1886) states that the spectrum of Pyrophorus noctilucus, the West Indian
"Cucullo," extends from slightly further than the Fraunhofer B line to the F
line, while Langley and Very (1890), working on the same form, placed the
limits at λ = 0.468µ to λ = 0.640µ. It consists, then, of a broad band chiefly
in the green and yellow. But, "would the light not extend farther were it
bright enough to be seen?... if the light of the insect were as bright as that of
the sun would it not extend equally far on either side of the spectrum?" "It
is impossible to increase the intrinsic brilliancy by any optical device, but if
it be impossible to make the light of the insect as bright as that of the sun, it
is on the other hand quite possible to make the light of the sun no brighter
than that of the insect ..." Langley and Very investigated this question,
forming a solar spectrum from sunlight of the same intensity as that of
Pyrophorus and a Pyrophorus spectrum together in the same field of the

spectroscope. The latter was very much shorter than the solar spectrum,
showing that its length was not due to weakness of the red and blue rays but
to their absence. Later Ives and Coblentz (1910) photographed the spectrum
of a firefly (Photinus pyralis), together with that of a carbon glow lamp, on
plates sensitive to all wave-lengths of visible rays under conditions which
would have recorded all visible radiations given off. They found the
spectrum to extend only from λ = 0.51µ to λ = 0.67µ (Fig. 7). Another
species of firefly (Photuris pennsylvanica) was found by Coblentz (1912) to
give a spectrum extending from λ = 0.51µ to λ = 0.59µ (Fig. 8). The
Photinus light extends much further into the red and it is easy to distinguish
between Photinus and Photuris in nature, merely by the reddish tint of the
light of the former. These photographic records show conclusively that the
color of the light of luminous animals is not a subjective phenomenon due
to the Purkinje effect and the low intensity of the light, but is real, an actual
difference in spectral composition of the light emitted. Neither is it due, at
least in the fireflies examined, to the existence of color screens which
absorb certain rays, allowing only those of a definite color to pass. The
spectra of forms thus far investigated are reproduced in Fig. 9 and recorded
in Table 5. It will be noted that they vary considerably in position but are all
of the same type. The spectrum of Cypridina hilgendorfii is the longest thus
far investigated (λ = .610µ to λ = .415µ), extending well into the blue, and
the light of this form is very blue in appearance.

Fig. 7.—Spectra of carbon glow lamp, A,
firefly (Photinus pyralis); B, and helium
vacuum tube, C (after Ives and Coblentz).

Fig. 8.—Spectra of helium vacuum tube (1);
carbon glow lamp (2); the firefly, Photinus
pyralis (3); and the firefly Photuris
pennsylvanica (4) (after Coblentz).

Fig. 9.—Spectra of various luminous animals
(after McDermott). 1. Portion of the visible
solar (grating) spectrum showing Fraunhofer
lines. 2. Pyrophorus noctilucus (Langley and
Very.) 3. Lampyris noctiluca (Conroy). 4.
Photinus pyralis (Ives and Coblentz). 5.
Photinus consanguineus (Coblentz). 6.
Photuris pennsylvanica (Coblentz). 7.
Phengodes laticollis (McDermott). 8.
Bacterium phosphoreum, B. phosphorescens or
Bacillus photogenus (Molish). 9.
Photobacterium indicum (Barnard). 10.
Mycelium X (Molish). 11. Luminous bacteria
(Förster). 12. Agaricus sp.? (Ludwig). 13.
Fluorescent spectrum of luciferesceine of
Photinus pyralis (Coblentz). Only the extreme
ends of the bands are shown and no attempt is

made to indicate the relative density of
different portions of the spectra.

Table 5.—Limits of Spectra of Various Luminous Organisms

Emission Method and
Light Spectrum (µ) Observer
maximum remarks
Eye observation,
Cypridina Harvey,
0.610-0.415 Zeiss comparison
hilgendorfii 1919
spectroscope.
Chætopterus 0.55-0.44 Lancaster,
Eye observation.
insignis (approximately) 1868
Pyrophorus Dubois,
0.72-0.486 Eye observation.
noctilucus 1886
Eye observation
Pyrophorus Langley and comparison
noctilucus .640 - .468 0.57 and Very, with solar
(thoracic light) 1890 spectrum of equal
intensity.
Pyrophorus
noctilucus
.663 - .463
(abdominal
light)
Photographic
Ives and
Photinus comparison with
.67 - .51 .552 Coblentz,
pyralis carbon glow lamp
1909
of equal intensity.
Photographic
Photuris Coblentz, comparison with
.59 - .51
pennsylvanica 1912 carbon glow lamp
of equal intensity.
Photinus .65 - .52 .578 Coblentz, Photographic
consanguineus 1912 comparison with

carbon glow lamp
of equal intensity.
Phengodes McDermott,
.65 - .52 Eye observation.
laticollis 1911 e
Lampyris Conroy,
.656- .518 Eye observation.
(glow worm) 1910
Eye observation
Young,
Photinus .670- .487 direct vision
1870
spectroscope.
G to F
extending Barnard,
Bacteria Photographic.
toward D for 1902
long exposure
Somewhat Fisher,
Bacteria Eye observation.
beyond G to D 1888
Eye observation
Förster, Zeiss. Abbe
Bacteria .58 - .43
1887 microspectral
ocular.
Photographic,
Bright Forsyth,
Bacteria >.500 to .350 quartz
band at .4 1910
spectroscope.
Eye observation,
Agarious 0.56-0.48 Ludwig,
Sorby Brown
melleus (approximately) 1884
microspectroscope.
Eye observation,
Xylaria .54 - .46 Ludwig,
Sorby Brown
hypoxylon (approximately) 1884
microspectroscope.
Eye observation,
Micrococcus Ludwig,
b into the violet Sorby Brown
Pflugeri 1884
microspectroscope.
Eye observation,
Molish,
Mycelium X .570 - .480 Zeiss comparison
1904, book
spectroscope.
Bacterium .570 - .450 Molish, Eye observation,
phosphoreum 1904, book Zeiss comparison

spectroscope.
Eye observation,
Bacterium Molish,
.570 - .450 Zeiss comparison
phosphorescens 1904, book
spectroscope.
Eye observation,
Bacillus Molish,
.570 - .450 Zeiss comparison
photogenes 1904, book
spectroscope.
Eye observation,
Pseudomonas Molish,
.570 - .450 Zeiss comparison
lucifera 1904, book
spectroscope.

As first shown by Dubois (1886) for Pyrophorus, and confirmed by myself
for Cypridina, the light is not polarized in any way. I may add that the
Cypridina light like any other light may be polarized by passing through a
Nicol prism.
Several writers [Dubois (1914 book)], Fischer (1888), Molisch (1904 book)
have noticed that the light of luminous bacteria changes in color if grown
on different culture media. Light which is "silver white" on dead fish
becomes "greenish" on salt-peptone-gelatin media and more yellow on salt-
poor media. Peron (1804) and Panceri (1872) describe the light of
Pyrosoma as yellow to greenish after death of the animal and reddish on
stimulation; then fading out through orange, yellow, greenish and azure
blue. Polimanti (1911) describes the normal light of Pyrosoma as greenish,
and states that as the animals die, or if they are kept at temperatures above
the optimum, the light becomes more red. McDermott (1911, b) noticed that
the light of fireflies placed in liquid air became decidedly reddish just
before going out and on rewarming the first light to appear was reddish
followed by the proper shade at higher temperatures. I have frequently
observed a more reddish color from luminous tissues of the firefly upon the
addition of coagulants such as alcohol, and have noted that the light of
Cypridina becomes weaker and more yellow at both low (0°) and high (50°)
temperatures. The meaning of these color changes will be discussed in
Chapter VII.

The efficiency of any light may be defined in several different ways: (1) By
the percentage of visible wave-lengths in the total amount of radiation
emitted, i.e., visible radiation divided by total (heat, visible, actinic)
radiation; (2) by considering, in addition to visible radiation ÷ total
radiation, the sensibility of the eye to different wave-lengths, visible
radiation × visual sensibility ÷ total radiation. Visible radiation × visual
sensibility is spoken of as luminosity; (3) by the amount of light (expressed
in candles) produced in relation to a given expenditure of energy or in
relation to the cost of the energy expended. Thus, of the radiation emitted
from an incandescent electric lamp only a small per cent. is light, the rest
being heat and actinic rays. It is therefore very far from being 100 per cent.
efficient. If there were no infra-red or ultra-violet in the radiation from an
incandescent lamp its efficiency would be 100 per cent. if we disregarded
visual sensibility. But if we take into account the fact that the eye is most
sensitive to yellow green, a source of light, even though emitting only
visible radiation, would not be 100 per cent. efficient unless its maximum of
emission corresponded also with the maximum of visual sensibility. We
shall return to this question in a later paragraph. Looking at the question
from the standpoint of energy consumption, the carbon incandescent lamp
gives one mean spherical candle for 4.83 watts (watt = 107 ergs per sec.),
while the tungsten lamp gives one mean spherical candle for 1.6 watts,
about one-third the energy, and the latter is consequently more efficient.
As we know practically nothing of the energy transformations occurring
during the process of light production in organisms, all statements regarding
the efficiency of their light are based on relations between the visible
radiation and total radiation. This involves a measurement of rays in the
infra-red region (heat rays) and ultra-violet region (actinic rays) as well as
the light rays proper, and any other radiant energy produced. While all
spectroscopic investigations show that the spectrum of luminous animals
never extends to the limits of the visible spectrum in either the red or violet,
it is possible that bands occur in the infra-red or ultra-violet, and special
methods must be employed to detect these. Radiations of all kinds, if
converted into heat on striking the blackened surface of a thermopile,
bolometer, or radiometer can be measured by changes in temperature and
the relative amounts of energy represented be compared in a common unit,
the calorie. By proper screening, all rays except the visible light rays can be

cut off from the measuring instrument and the amounts of energy
represented in light and in total radiation thus be determined.
Dubois (1886) first studied this problem in Pyrophorus by the use of a
thermopile and galvanometer and found a small amount of radiation from
the luminous region in excess of that from a non-luminous region. It
amounted to a galvanometer deflection of 0.95° and was increased 0.3°
during the flash of the insect on electrical stimulation. This increase of 0.3°
is possibly due to heat produced on muscular contraction. In any case the
amount of heat radiated in comparison with that of the candle is very small
indeed. A more careful study has been made by Langley and Very (1890)
with the bolometer. They point out first of all that the total radiation from
the most powerful luminous organ (the abdominal one) of Pyrophorus
which affected their bolometer slightly, would, in the same time (10
seconds), be sufficient to raise the temperature of an ordinary mercurial
thermometer having a bulb 1 cm. in diameter by rather less than 2.3 × 10-6°
C. We may thus gain some idea of the magnitude of the measurements to be
made. The radiation from Pyrophorus which affected their bolometer was
shown to be due merely to the "body heat"[2] of the insect, and it is largely
cut off by a plate of glass which is opaque to all wave-lengths of 3µ or
more. These waves are given off by bodies at temperatures below 50° C.
and belong "to quite another spectral region to that in which the invisible
heat associated with light mainly appears." Langley and Very then
compared the radiation from a non-luminous bunsen flame and the
Pyrophorus light, interposing a plate of glass in each case to cut off the
waves longer than 3µ, and found several hundred times more radiation in
the case of the bunsen burner but, nevertheless, perceptible radiation from
Pyrophorus. The former consisted of radiant heat shorter than λ = 3µ and
extending up to the visible light rays (λ = 0.7µ since the bunsen flame
emitted no light). The very slight effect of the Pyrophorus radiation must be
due to wave-lengths between λ = 3µ and λ = 0.468µ, the limit of the
Pyrophorus spectrum in the blue. Langley and Very assumed it to be due
entirely to the band of visible light, λ = 0.640µ to λ = 0.468µ, and assumed
that no invisible heat rays were produced. All of the energy of Pyrophorus
light would therefore lie in the visible region and its efficiency (light rays ÷
heat + light + actinic rays) would be 100 per cent. Later, Langley (1902)
reinvestigated the radiation of Pyrophorus and could detect no heating

whatever with the bolometer. "A portion of the flame of a standard sperm
candle, equal in area to the bright part of the insects, gave under the same
circumstances, a bolometric effect of such magnitude that had the heat of
the insect been 1/80,000 as great as that from the candle, it would certainly
have been recognized." Coblentz (1912) also, using a vacuum thermopile of
Pt and Bi, was unable to detect any infra-red radiation from Photinus
pyralis, but found that the temperature of this firefly is slightly lower than
the air. These temperature measurements will be discussed in a later
chapter.
[2] Langley and Very evidently supposed that the body temperature of the firefly,
like the mammal or bird, is higher than its surroundings.

The assumption of Langley and Very that the small amount of Pyrophorus
radiation passing glass is all light has been called into question by Ives
(1910), who points out that Langley and Very failed to use a screen which
would cut off either the visible rays or the invisible rays between 3µ and
0.7µ. They really left the question open as to whether the effect of
Pyrophorus light on their bolometer was due to the visible band of rays or
to this plus another band in the infra-red. "The firefly's actual efficiency as a
light source is dependent to a large degree on the radiation being confined
to the visible region. If there should be found infra-red of quantity
comparable to the visible, the firefly, while still a very efficient source
would not be, as usually supposed, the example of an ideally efficient light
produced by nature."
Ives investigated the question further by the phosphor-photographic
method. "In brief it consists of this: Phosphorescence, which is excited in
various substances by exposure to short waves (blue, violet or ultra-violet),
is destroyed by exposure to longer waves (orange, red, infra-red). Thus, a
surface of Balmain's paint or of Sidot blende, excited to phosphorescence
and then exposed in a spectrograph, will have areas of reduced brightness
wherever long-wave energy has fallen upon it. If this surface is then laid on
a photographic plate for a short period, a permanent record is obtained on
the plate after development." Preliminary tests showed that the method was
applicable in the case of weak light such as the firefly spectrum and also if
the light is intermittent like the firefly. With Sidot blend (ZnS) the
extinguishing action extends from λ = 0.6µ to λ = 1.5µ. A sheet of deep
ruby glass, which cut off all the visible rays of the firefly but allowed infra-

red to pass, was placed between the firefly light and a surface of
phosphorescent Sidot blend which was exposed to the firefly flashes for
three and a half hours. No extinction of phosphorescence occurred, while
without the ruby glass, extinction, due to the orange rays of the visible
firefly light was noticeable in 20 minutes. There is thus no infra-red of an
intensity at all comparable to the visible as far as λ = 1.5µ, the lower limit
of the phosphor-photographic method. Coblentz (1912) had examined the
transparency of the dry chitinous integument of various fireflies (Fig. 10) in
the infra-red and reports it to be fairly transparent down to λ = 2.8µ, opaque
between λ = 2.8µ and λ = 3.8µ, transparent again to λ = 6µ, and opaque
beyond that. The infra-red could, then, if it were emitted, largely pass
through the integument which is similar in absorption properties to complex
carbohydrates. Transparency of the integument to the ultra-violet was not
studied.

Fig. 10.—Transmissivity of the integument of
fireflies to infra-red radiation (after Coblentz.)
Although photographs of the spectrum of firefly (Photinus) light show that
it extends only to the beginning of the blue, Forsyth (1910) reports ultra-
violet radiation in luminous bacteria. He exposed a plate for 48 hours to the
spectrum of bacterial light dispersed by a quartz prism and got a continuous
band from λ = 0.50µ (the lower limit of sensitivity of the plate) to λ =
0.35µ. However, McDermott (1911 d) was unable to observe fluorescence
of p-amino-ortho-sulpho-benzoic acid, which responds to the ultra-violet
light. Molisch (1904, book) photographed bacterial and fungus light
through glass and through a piece of quartz and found no difference in

density on the plate. As the exposure was brief, to avoid saturation, and as
the ultra-violet, which passes quartz but not glass, has a much greater action
on the plate than visible light, we must conclude that ultra-violet is absent.
Ives (1910) investigated the spectrum of Photinus pyralis, using a quartz
spectroscope, and found no evidence of ultra-violet radiation, at least as far
as λ = 0.216µ.
It will thus be seen that the radiation from the firefly has been very
carefully studied and that no waves are given off from λ = 1.5µ to λ =
0.216µ with the exception of the short band (λ = 0.67µ to λ = 0.51µ) in the
visible, and it is highly probable that no radiation is given off with wave-
lengths longer than λ = 1.5µ. The firefly light remains, then, 100 per cent.
efficient, differing from all our artificial sources of light, the best of which
does not approach this value. As Langley and Very express it in the title to
their paper, it is "the cheapest form of light," not cheapest in the sense of
that we can reproduce it commercially at less cost than other lights, but
cheaper in the sense that it is the most economical in the energy radiated.
This energy is all light and no heat. "Cold light" has actually been
developed by the firefly and concerning which "we know of nothing to
prevent our successfully imitating."

Fig. 11.—Spectral energy curves of various
fireflies and the carbon glow lamp (after
Coblentz).
I have already pointed out that we may also consider the efficiency of a
light in relation to the sensibility of our own eye. That is, we take into
account not only the energy distribution in the spectrum of the light but also
the fact that different wave-lengths of an equal energy spectrum affect our
eye very differently. As the normal light-adapted eye is most sensitive to
yellow green of λ = 0.565µ, monochromatic light of this wave-length will
appear much brighter than monochromatic light of any other wave-length
with the same energy. Monochromatic light of λ = 0.565µ will then be the
theoretically most efficient possible, when we consider the energy radiated
in relation to the sensitivity of our eye. This is the usual method of
determining the luminous efficiency of artificial lights and is obtained from
a knowledge of the radiated energy and the visual sensibility. Reduced
luminous efficiency = light (radiated energy × visual sensibility) or
luminosity ÷ total radiated energy.

Fig. 12.—Visibility curves of various
investigators obtained by different methods
(after Hyde, Forsyth and Cady).

Fig. 13.—Luminous efficiency of the 4-watt
carbon glow lamp, shaded area ÷ total area

(after Ives and Coblentz).

Fig. 14.—Luminous efficiency of the firefly,
shaded area ÷ total area (after Ives and
Coblentz).
The spectral energy curve for the firefly has been worked out by Ives and
Coblentz (1910), using a photographic method in which the intensities of
different wave-lengths of the firefly (Photinus pyralis) light is compared
with that of a carbon glow lamp by measuring the amount of photochemical
change produced on panchromatic photographic plates. Fig. 11 gives the
energy curves of various fireflies and the carbon glow lamp in the same
spectral region. The visual sensibility curve used by Ives and Coblentz is
that of Nutting (1908, 1911), based on Konig's data. It is reproduced in Fig.
6. The latest visibility curve is that of Hyde, Forsyth and Cady (1918),
reproduced in Fig. 12. It is based on observations of twenty-nine
individuals. As individuals vary considerably in their sensibility to different
wave-lengths, the visibility curve represents an average, but it is the only
standard we have with which to evaluate the energy we call light. Color-

blind individuals would have a visibility curve very different from normal
individuals. Composite curves showing the luminous efficiency of the 4-
watt carbon glow lamp and the firefly, both in relation to visibility, are
given in Figs. 13 and 14, respectively. In these figures the luminous
efficiency is the shaded area ÷ total area, 0.43 per cent. for the carbon glow
lamp and 99.5 per cent. for the firefly, "these numbers representing the
relative amounts of light (measured on a photometer) for equal amounts of
radiated energy—a striking illustration of the wastefulness of artificial
methods of light production. From the specific consumption of the tungsten
lamp (1.6 watts per spherical candle) and the mercury arc (.55 watts per
spherical candle) we obtained by comparison with the carbon filament that
their luminous efficiencies are 1.3 and 3.8 per cent. The most efficient
artificial illuminant therefore has about 4 per cent. of the luminous
efficiency of the firefly." This is calculated to be .02 watts per candle. More
recent determinations (Coblentz, 1912), using a new sensibility curve of
Nutting's (1911) for a partially light-adapted eye, give the reduced luminous
efficiency as 87 per cent. for Photinus pyralis, 80 per cent. for Photinus
consanguineus and 92 per cent. for Photuris pennsylvanica.

Fig. 15.—Spectral energy, luminosity and
visibility curves (after Gibson and McNicholas)

A. Spectral energy curve of Hefner lamp.
B. Spectral energy curve of acetylene flame.

C. Spectral energy curve of tungsten (gas-
filled) glow lamp.
D. Spectral energy curve of black body at
5000° absolute (sunlight).
E. Spectral energy curve of blue sky.
Hg. Spectral energy curve of Heræus quartz
mercury lamp.
Lv. Visibility curve for human eye.
La. Luminosity of Hefner lamp.
Le. Luminosity of blue sky.
The luminous efficiencies of various forms of artificial illuminants have
been calculated by Ives (1915) and are given together with that of the firefly
in Table 6. Fig. 15 gives spectral energy curves for various illuminants
reduced to 100 at λ = .590µ, luminosity curves for the Hefner lamp and blue
sky, and a visibility curve worked out by Coblentz and Emerson (1917)
from observations on 130 individuals.
Table 6
Luminous Efficiencies of Various Illuminants

Efficiency
(visible radiation
Illuminant and commercial Lumens ×
Commercial rating
description per watt visual sensibility
÷
total radiation)
Carbon incandescent lamp oval anchored
4 watts per mean horiz. c. 2.6 0.0042
(treated) filament
Tungsten incandescent lamp, vacuum type 1.25 watts per mean horiz. c. 8.0 .013
600 C. P. 20 amp., 0.5 w. p. c. Series
Mazda, type c 19.6 .032
type C.
Carbon arc (open) 9.6 amp. clear globe 11.8 .019
Open arc, yellow flame, inclined trim 10 amp. D. C. 44.7 .072
Quartz mercury arc 174-197 volt, 4.2 amp. 42.0 .068
Glass mercury arc 40-70 volt, 3.5 amp. 23.0 .037
Nernst lamp 4.8 .0077
Acetylene 1 L per hr. consumption .67 .0011
Petroleum lamp .26 .0004
Open flame gas burner Bray 6 high pressure .22 .00036
Incandescent gas lamp, low pressure .350 lumens per B. T. U. per hr. 1.2 .0019
Incandescent gas lamp, high pressure .578 lumens per B. T. U. per hr. 2.0 .0032
Firefly 629.0 .96

The firefly light by the above method of calculating efficiency is not 100 per cent. efficient because its maximum
(λ = 0.567µ) does not correspond with the maximum sensibility of the eye (λ = 0.565µ), but taking into
consideration also other effects of color, the firefly light would be a still more inefficient and trying one for
artificial illumination, as all objects would appear a nearly uniform green hue. Indeed the distortion would be even
greater than with the mercury arc, whose objectionable green hue is so well known. "We may say, therefore, that
the firefly has carried the striving for efficiency too far to be acceptable to human use; it has produced the most
efficient light known, as far as amount of light for expenditure of energy is concerned, but has produced it at the
(inevitable) expense of range of color. The most efficient light for human use, taking into account both color and
energy-light relationships, would be a light similar to the firefly light containing no radiation beyond the visible
spectrum, but differing from it by being white." (Ives, 1910.) Although the spectral energy curve for Cypridina
light has not been worked out, it will be noted that the Cypridina spectrum is much longer than that of the firefly,
more nearly approaching the spectrum of an incandescent solid giving white light. It approaches, but does not
attain the ideal.
Although Muraoka (1896) and Singh and Maulik (1911) have described radiations coming from fireflies which
would pass opaque objects and affect a photographic plate, and Dubois reports the same from bacteria, the
existence of such radiation has been denied by Suchsland (1898), Schurig (1901) and Molisch (1904 book). The
experiments of Molisch on luminous bacteria are of greatest interest, for they are very carefully controlled and
show without a doubt that black paper or Zn, Al, or Cu sheet will allow no rays from these organisms to pass that
will affect a photographic plate, even after several days' exposure. The visible light of luminous bacteria will affect
the plate after one second exposure. Moreover, Molisch has pointed out the errors of those who claim to have
found penetrating radiation in luminous forms. It seems that certain kinds of cardboard, especially yellow varieties,
or wood, will give off vapors that affect the photographic plate. The action is especially marked with damp
cardboard at a temperature of 25°-35° C., and Dubois and Muraoka must have used such cardboard to cover their
plates. A piece of old dry section of beech or oak trunk, placed on a photographic plate for 15 hours in a totally
dark place, will register a beautiful picture of the annual rings of growth, medullary rays, junction of bark and
wood, etc. Russell (1897) had previously found that many bodies, both metals and substances of organic origin
(gums, wood, paper, etc.), placed in contact with photographic plates, would affect them, and concluded that
vapors and not rays were the active agents. As a dry piece of wood has a very definite smell, there is something
given off which can affect our nose and there is no reason why it should not change, by purely chemical action, the
photographic plate. This action of wood on the plate is prevented by interposing a sheet of glass. Frankland (1898)
has described similar vapors coming from colonies of Bacillus proteus vulgaris and B. coli communis which affect

a photographic plate laid directly over the colonies in an open petri dish. There is no effect if the glass cover of the
petri dish is between plate and bacteria. There is, then, no specific emission of X-rays or similar penetrating
radiation from luminous tissues which will affect the photographic plate through opaque screens.
A similar conclusion is reached if we attack the problem in another way. X-rays and radium rays (Becquerel rays)
cause fluorescence of ZnS, barium platinocyanide, willemite (Zn2SiO4), and calcium tungstate. Coblentz (1912)
showed that the firefly will cause no fluorescence of a barium platinocyanide screen and I have been unable to
detect fluorescence of zinc sulphide, barium platinocyanide, zinc silicate (willemite) or calcium tungstate shielded
from Cypridina light by black paper, although the light of this organism is quite bright enough to cause
phosphorescence of zinc sulphide without the black paper. The samples of the above four substances all showed
fluorescence in presence of radium rays, but only the ZnS phosphoresces after exposure to light rays, although the
willemite was phosphorescent after exposure to the ultra-violet.
While photometry at low intensities is a difficult procedure at best, if the light varies in intensity or is a flash,
accurate measurements become well-nigh impossible. The figures given for intensity of animal luminescence
must, therefore, be accepted with a realization of the difficulties of measurement. By candle is meant the
international candle, unless otherwise specified, equal to 1.11 Hefner candles (H. K.) 0.1 pentane lamp and 0.104
carcel units. It is a measure of intensity.

Amount of light, or light flux, measured in lumens, is that emitted in a unit solid angle (area/r2) by a point source
of one candle-power. One candle-power emits 4π lumens. The latest figure for the mechanical equivalent of light at
λ = .566 is .0015 watt (Hyde, Forsyth and Cady, 1919), i.e., 1 lumen = .0015 watt. One watt is 107 ergs (one joule)
per second.
The illumination (of a surface) is that given by one candle at one metre, the candle metre (C.M.) or lux. The
surface then receives one lumen per square metre. A metre kerze (M.K.) is the illumination given by one Hefner
candle at one metre distance.
The brightness of a surface is measured in lamberts or millilamberts. A lambert is "the brightness of a perfectly
diffusing surface radiating or reflecting one lumen per square cm." A millilambert is 1/1000 lambert. For further
definitions the reader is referred to the reports of the committee on nomenclature of the Illuminating Engineering
Society.
Dubois (1886) states that one of the prothoracic organs of Pyrophorus noctilucus has a light intensity of 1/150
Phœnix candle of eight to the pound (probably about equivalent to 1/150 candle) and that 37 or 38 beetles (each
using all three light organs) would produce light equivalent to one Phœnix candle. Langley (1890) found that to the
eye the prothoracic organ of Pyrophorus noctilucus gave one-eighth as much light as an equal area of a candle and
the actual candle-power of the insect was 1/1600 candle. It may be remarked in passing how widely divergent
these observations are.
For the flash of the firefly (Photinus pyralis) Coblentz (1912) found variation from 1/50 to 1/400 candle, the
predominating values being around 1/400 candle. A continuous steady glow is sometimes obtained from this insect
and it proved to be of the order of 1/50,000 candle.
Steady sources of light can be more easily measured and we have two records of the light intensity from luminous
organisms with continuous light. One of these is a fish, Photoblepharon palpebratus, with a large luminous organ
under the eye, of flattened oval shape, 11 × 5 mm., which glows continuously without change of intensity. The
organ can be darkened by a screen similar to an eyelid which pulls up over it. Steche (1909) reports the intensity to
be .0024 M.K.[3]
[3] The metre-kerze is a unit of illumination, not of intensity, and is incorrectly used by Steche.

Luminous bacteria probably glow with less intensity than any other organism. The light from a single organism
cannot be seen but that from a colony is visible to the dark-adapted eye. Even so we must remember that the eye is
an exceedingly delicate instrument which can detect very small energy changes. The "minimum radiation visually
perceptible" has been calculated by Reeves (1917) to be in the neighborhood of 18 × 10-10 ergs per second and the
light from a small colony of luminous bacteria represents little more radiation than this.

Lode (1904, 1908), by a modified grease spot photometer method, ascertained that the light of his brightest
bacterial colony of Vibrio rumple had an intensity of 7.85 × 10-10 H.K. per sq. mm. or 0.785 H.K. per 1000 sq.
metres (=0.562 German-normal candles per 1000 sq. metres). In round numbers this is about one German-normal
candle per 2000 sq. metres, or two to three times this area for the light from an ordinary stearin candle. Lode
calculated that the dome of St. Peter's at Rome, if covered with bacteria, would give little more light than a
common stearin candle. An ordinary room of 50 sq. metres wall and ceiling area would give out only 0.039
German-normal candle. It does not seem likely that luminous bacteria will ever come into vogue for illuminating
purposes. Friedberger and Doepner (1907) by a photographic method, not entirely free from error, found that one
square millimetre of lighting surface of a bouillon culture of photobacteria gave 6.8 × 10-9 German-normal
candles, about ten times Lode's value. Even at this rate commercial lighting by luminous bacteria does not appear a
promising field for investors.
To sum up, we may say that light from animal sources is in no way different from light of ordinary sources, except
in intensity and spectral extent. It is all visible light, containing no infra-red or ultra-violet radiation or rays which
are capable of penetrating opaque objects. It is not polarized as produced, but may be polarized by passing through
a Nichol prism. Like ordinary light, animal light will also cause fluorescence and phosphorescence of substances,
affect a photographic plate, cause marked heliotropism of plant seedlings (Nadson, 1903) and stimulate the
formation of chlorophyll (Issatschenko, 1903, 1907). Because of the weakness of bacterial light, etiolated
seedlings do not become green to the eye (Molisch, 1912 book), but a small amount of chlorophyll is formed
which can be recognized by the spectroscope because of its absorption bands.

CHAPTER IV
STRUCTURE OF LUMINOUS ORGANS
The production of light is the converse of the detection of light. In the first case chemical energy is converted into
radiant energy; in the second case radiant energy is converted into chemical energy. The lantern of the firefly is an
organ of chemi-photic change; the eye is an organ of photo-chemical change. While it is theoretically probable that
all reactions which proceed in one direction under the influence of light, will proceed in the opposite direction with
the evolution of light, the formation of luciferin from oxyluciferin (described in Chapter VI) is the only one
definitely known. Perhaps we may place in this category also the instances of photoluminescence, but the chemical
reaction involved cannot be pointed out.
We know of no animal whose eyes, the organs, par excellence, of photochemical change, give off light in the dark.
All cases of luminous eyes have been conclusively shown to be purely reflection phenomena. The eyes of a cat
only glow if some stray light is present which may enter and be reflected out again. Photochemical reactions and
chemiluminescent reactions do have this in common, however, that they are largely but not exclusively oxidations.
Whether all photochemical changes in the eyes in animals require oxygen or not, is unknown, but all animal light-
producing reactions, without exception, are oxidations, and light is only produced if oxygen is present. Some
material is oxidized.
In general, we may divide luminous organisms into two great classes according as the oxidizable material is
burned within the cell where it is formed or is secreted to the exterior and is burned outside—intracellular and
extracellular luminescence. Many animals with intracellular luminescence have quite complicated luminous
organs. It is an interesting fact that a great similarity may be observed between the evolution of the complex organs
of vision and of these complicated organs. In the simplest unicellular forms certain structures within the cell serve
as the photochemical detectors of light, while in luminous protozoa, similarly, granules scattered throughout the
cell are oxidized with light production. In the higher forms the eye contains groups of photosensitive cells
connected with afferent nerves, lenses, and accessory structures for properly adjusting the light, while luminous
organs contain groups of photogenic cells in connection with efferent nerves, lenses, and accessory structures for
properly directing the light. It is interesting to note that in the two groups where the eye has attained its highest
development, the cephalopods and vertebrates, here also the luminous organ is found in greatest complexity and
perfection. In intermediate stages of evolution the eye and luminous organ so closely approach each other in
structure that it is still a mooted question whether certain organs found in worms and crustacea are intended for
receiving or producing light.
We may also divide luminous forms into two groups according as the oxidation of luminous material goes on
continuously, independently of any stimulation of the organism; or is intermittent, oxidation and luminescence
occurring only as a result of stimulation, using the word "stimulation" in the same sense in which it is used in
connection with nerve or muscle tissue. Bacteria, fungi, and a few fish produce light continuously and
independently of stimulation. Its intensity varies only over long periods of time and is dependent on the nature of
the nutrient medium or general physiological condition of the organism. All other forms give off no light until they
are stimulated. Stimulation may of course come from the inside (nerves) or outside. Only under unfavorable
conditions, such as will eventually lead to the destruction of the luminous cells, do these forms give off a
continuous light. This has often been spoken of as the "death glow," and is to be compared with rigor in muscle
tissue.
Some of the fish which produce a continuous light possess a movable screen similar to an eyelid which can be
drawn across the organ, thus shutting off the light, so that the animal appears to belong to the group which flashes
on stimulation. This is true of Photoblepharon, while Anomalops can rotate the light organ itself downward, so as
to bring the lighting surface against the body wall and thus cut off the light (Steche, 1909). Other fish
(Monocentris) are unable to "turn off" their light.
Animals which flash spontaneously on stimulation through nerves from within, possess a very varied rhythm. The
different species of fireflies can be distinguished by the character of their flashing (McDermott, 1910-17; Mast,
1912). Fig. 16 shows the method of flashing of some common eastern North America species. The glowworm light

lasts for many seconds and then dies out. This interval of darkness persists for some minutes and is then followed
by another period of glowing. Some fireflies have a light which may be described as partially intermittent. It lasts
for hours, but may become more dim or be intensified on stimulation.

Fig. 16.—Chart showing relative intensities
and durations of flashes of American fireflies
(after McDermott). One cm. vertically =
approximately 0.02 candle power; one cm.
horizontally = approximately one second. The
flash of the males ( ♂ ) is at the left; that of
females (♀) at right of chart.
Some forms only produce light at certain seasons of the year. According to Giesbrecht (1895) this is true of the
copepods, which only light in summer and autumn, and according to Greene (1899) in the toad-fish; Porichthys,
which can only be stimulated to luminesce during the spawning season in spring and early summer.
Some animals possess a periodicity of luminescence. They only luminesce at night and fail to respond to
stimulation or are difficult to stimulate during the day. Bright light has an inhibiting effect. Perhaps correlated with
this is the fact that most luminous forms are strongly negatively heliotropic. Fireflies lie hidden in the day, to
appear about dusk and the ostracod crustacean, Cypridina, is difficult to obtain on moonlight nights.
The Ctenophores were the first forms in which the inhibiting effect of light was noticed. This was described by
Allman (1862) and has been confirmed by a number of observers, especially Peters (1905). Massart found that
Noctiluca was difficult to stimulate during the day and Ceratium, according to both Zacharias (1905) and Moore
(1908), only luminesces at night, or if kept in darkness, for some little time. Crozier[4] finds a persistent day-night
rhythm of light production when Ptychodera, a balanoglossid, is maintained for eight days in continued darkness.
The animal is difficult to stimulate during the period which corresponds to day and luminesces brilliantly and at
the slightest touch during the period which corresponds to night.
On the other hand, a great many forms are able to luminesce quite independently of previous illumination.
According to Crozier[4] Chætopterus luminescence is not affected by an exposure to 3000 metre-candles for six
hours.
[4] Private communication.

In the case of animals with extracellular luminescence we may speak of luminous secretions and true luminous
glands. A large number of forms possess luminous glands or gland cells, including some of the medusæ, the
hydroids (probably), the pennatulids (?), the molluscs (Pholas and Phyllirhoë) (probably), some cephalopods
(Heteroteuthis and Sepietta), most annelids, ostracods, copepods, some schizopods (Gnathophausia) and decapod
(Heterocarpus and Aristeus) crustaceans, all myriapods, and the balanoglossids. The remaining organisms burn

their material within the cell. These include the bacteria, fungi, protozoa, some medusæ (?), ctenophores
(probably), most cephalopods, a few annelids (Tomopterus (?)), ophiuroids (?), some schizopod (Nyctiphanes,
Euphasia, Nematocelis, Stylochiron) and decapod (Sergestes) crustacea, all(?) insects, Pyrosoma, and fishes
(selachians and teleosts). It is among this latter type that the most complicated luminous organs have been
developed. While a description of all the types of luminous organs and luminous structures cannot be attempted
here (excellent descriptions have been given by Dahlgren and Mangold) it is necessary to understand the structural
conditions in a few of the forms whose physiology has attracted most attention.
Luminous bacteria are so small that the light from a single individual cannot be seen. It is almost impossible to
make out structural differences within the cell and we cannot definitely state in just what special region, if any, the
luminescence is produced. We do know that the light is intracellular and that filtration of the bacteria from their
culture medium gives a dark sterile filtrate absolutely free from any luminous secretion.
Among protozoa, in certain forms at least, it is easy to observe that luminescence is connected with globules or
granules which were considered by the earlier observers to be oil droplets. Thus, in Noctiluca (Figs. 17 and 18),
when the animal is violently stimulated or in the presence of reagents which slowly kill it, the whole interior
appears a mass of starry points of light which can be traced to minute granules along the strands of protoplasm
(Quatrefages, 1850).

Fig. 17.—Noctiluca miliaris, showing
photogenic granules in cytoplasm. n, nucleus;
c, cytoplasmic strands containing photogenic
(large) and other (small) granules; p, pharynx;
f, flagellum; o, oral groove; t, tentacle; s,
spines at base of tentacle; v, vacuoles. Drawn
by E. B. Harvey.

Fig. 18.—Noctiluca miliaris as it appears
during luminescence (after Quatrefages).
Upper left and middle, low power; below, high
power; upper right, a crushed fragment still
luminescent.
Turning to the multicellular forms, we find the simplest development of luminosity in those animals which possess
gland cells producing a luminous secretion. These cells may be scattered over the surface of the animal as in
Chætopterus (Fig. 19) or Cavernularia, or restricted to certain areas [Pholas, (Fig. 19),] or more definitely
localized to form an isolated group of gland cells as in Cypridina. True multicellular glands also occur. In every
case, however, we find that the luminosity of these uni- or multicellular glands is connected with the presence of
granules. They are often spoken of as luciferine granules, although it is not certain whether they are made up of
luciferin or luciferase (see Chapter IV) or both. They are most similar to the zymogen granules found so
abundantly in gland cells and thought to be the precursors of various enzymes. According to Dahlgren (1915), the
luciferine granules stain blue-black by iron hæmatoxylon after fixation at the boiling point, and photogenic cells
can be detected by this method of selective staining. Dubois (1914, book), who regards them as examples of
bioprotein, comparable to the chondriosomes and handed on from one generation to another, gives them the name
of vacuolides or macrozymases. In some forms he has described their transformation into crystals and believed at
one time that animal light was a crystalloluminescence. His figures of the crystal transformation are not very
convincing. Pierantoni (1915) has considered the granules to be symbiotic luminous bacteria, but this is certainly
not the case.

Fig. 19.—Diagram of Pholas (right) and
Chætopterus (left) to show distribution of
luminous areas (after Panceri).
The light of Chætopterus comes from a material mixed with a mucous secretion formed over almost the whole
body surfaces of the animal. A section of the epithelium shows large mucous-producing cells and smaller granule-
containing light cells (Fig. 20). These appear to be under nervous control, as a strong stimulation in one part of the
body causes luminescence which spreads over the whole surface of the worm. The animal becomes fatigued rather
readily, however. In the pennatulids, such as Cavernularia, we have also the formation of a luminous secretion
over the whole surface of the body and the individual animals in this colonial form are also connected with nerves.
A stimulation in any local region, as Panceri (1872) first showed (Fig. 21), will cause a wave of luminosity to
spread from this point until it extends over the whole surface of the colony. In Pennatula the rate of this luminous
wave is about 5 cm. per second.

Fig. 20.—Sectional view of the luminous
epithelium of Chætopterus (after Dahlgren). cu,
cuticle; l. c., light cells, some showing
discharge of secretion; d. l. c., discharged and
emptied light cells; m. c., mucous cells.

Fig. 21.—Diagram of Pennatula, showing by
arrows the course of a wave of luminosity
which spreads over the colony from the point
stimulated (s) (after Panceri).
Pholas dactylus possesses similar light cells to those of Chætopterus, but they are restricted to narrow bands on the
siphon and mantle and a pair of triangular spots near the retractor muscles. Nerves pass to the luminous regions.
In many luminous animals the light secretion formed over the surface of the body is small in amount and adheres
to the animal because it is embedded in the mucous skin secretions. In those forms which possess a true localized
light gland the luminous secretion when expelled into the sea water (if the animal be a marine form) may persist as
a luminous streak for some time and exhibit diffusion and convection movements. The most beautiful examples of
luminous secretions are found among the ostracod crustacea.

Fig. 22.—Luminous gland of Cypridina
hilgendorfii (after Yatsu). 2, longitudinal
section. 4, transverse section.

Fig. 23.—Single enlarged gland cell of
Cypridina (after Dahlgren). P, nucleus and
plasmasome; C, cytoplasm; F, secretion fibrils;
D, reservoir duct filled with large yellow
granules; O, valve-like outer opening of cell at
surface of body.
In Cypridina hilgendorfii the luminous gland is situated on the upper lip near the mouth. It is made up of elongate
(some 0.7 mm. in length), spindle-shaped cells, each one of which opens by a separate pore with a kind of valve.
The openings are arranged on five protuberances. Muscle fibres pass between the gland cells in such a way that by
contracting the secretion can be forced out. In the sea water the secretion luminesces brilliantly and the Japanese
call these forms umi hotaru, or marine fireflies. Fig. 22 is a diagram showing the structure. Watanabe (1897), who
first studied this form, and also Yatsu (1917) have described two kinds of granule-containing cells, one with large
yellow globules, 4-10µ in diameter (Fig. 23), the other with small colorless granules 0.5, in diameter. I have
observed in the living form these two types and also large colorless globules of the same size as the yellow
globules. All dissolve when extruded into the sea water. Dahlgren[5] has described from sections four types of cells
containing (1) large globules, (2) small granules, (3) a fat-like material, (4) a mucous material. Just what the
significance and nature of these types of substance is cannot be stated at present. At least one, probably two, are
concerned in light production. The others may possibly form digestive fluids which act on the food of the animal.
[5] Private communication soon to be published.

Turning now to the animals possessing light cells with intracellular luminescence we find in general that such light
cells are localized to form definite light organs and that these may be single, as in the common fireflies, paired, as
the prothoracic light organs of Pyrophorus, or scattered over the surface of the body, as in so many shrimps,

cephalopods and fishes, when they are often called photophores. The light cells proper are often associated with
reflectors, lenses, opaque screens and color screens.

Fig. 24.—Distal portion of malpighian tubule
of Bolitophila, showing modification to form
photogenic organ (after Wheeler and Williams).
MT1, MT4, malpighian tubules forming
photogenic organ; R, reflector; M, muscle; T,
trachea.
The insects possess the simplest types of intracellular light organs, a mass of photogenic cells, which, in the
common firefly (a lampyrid beetle) of Eastern North America, has probably been developed from the fat body,
while in the New Zealand glowworm, the larva of a tipulid fly (Bolitophila luminosa), part of the Malpighian
tubule cells have acquired photogenic power (Wheeler and Williams, 1915). This is illustrated in Fig. 24.
The photogenic organ of the firefly is made up of two kinds of cells, a dorsal mass of small cells several layers
deep, the reflector layer, and a ventral mass of large cells with indistinct boundaries, the photogenic layer (Fig. 25).
The photogenic cells contain a mass of granules, spherical in the male and short rods in the female. The photogenic
cells are divided into groups by large tracheal trunks which pass into the light organ and branch to form tracheoles
connected with tracheal end cells. The exact distribution varies in different species, but in all the arrangement is
such as to give a very abundant oxygen supply. Each group of photogenic cells is surrounded by a clear ectoplasm
containing no granules. The tracheoles pass through this and either end openly within the photogenic cells or
anastomose with tracheoles from neighboring tracheæ. Nerves, but no blood-vessels—which are absent in insects
—enter the organ. It is difficult to determine if the nerves supply the tracheal end cells or the photogenic cells.

Fig. 25.—Sectional view of photogenic organ
of the firefly (after Williams), showing reflector
or crystal layer (U) above and photogenic cells
(P) below. C, cuticula; T, trachea; c, capillaries
of tracheal end cells; H, hypodermis; EC,
tracheal end cells; N, nerve.
The dorsal reflecting layer is made up of cells containing numerous minute crystals of some purin base, either
xanthin or urates, or both. They have a white milky appearance and while they are certainly not good reflectors in
the optical sense, they do act as a white background, scatter incident light, and partially prevent its penetration to
the internal organs of the firefly. Although a few crystals similar to those of the reflector layer are found in the
photogenic cells and in other cells of the body, it is known that the photogenic cells are not transformed into the
reflector cells. The two layers are distinct and permanent from an early stage in development.
Curiously enough, the light organ of the larva of the firefly (glowworm) is quite distinct from that of the adult.
Like so many other structures in insects, the adult organ is developed anew from potential photogenic cells during
the pupal period. Even the egg of the firefly is luminous and glows with a steady light, and during the pupal period
light may sometimes be seen coming from the thoracic region.
In the firefly there is no true lens, the light merely shining through the cuticle which is transparent over the light
organ, whereas over the rest of the body it is dark and pigmented. In the deep sea shrimp, Acanthephyra debelis,
with light organs scattered over the surface of the body, the cuticle covering the light organ forms a concavo-
convex lens, behind which are the photogenic cells (Kemp, 1910). As may be seen from Fig. 26, the lens is made
up of three layers which suggests that it may be corrected for chromatic aberration—a veritable "achromatic
triplet." In an allied form, Sergestes (Fig. 27), the lens is of two layers and double convex. Optical studies of these
lanterns have been made by Trojan (1907). The course of the light rays is shown in Fig. 28. The lens of these
organs is also bluish in color which suggests that they may serve also as color filters. Behind the photogenic cells
is a mass of connective tissues through which enters the nerve, for the light of these organs is under the control of
the animal and may be flashed "at will."

Fig. 26.—Sectional view of photogenic organ
of Acanthephyra debelis (after Kemp). n, nerve;
s. l., sheathing layer of cells; g, cone of
refractive granules at end of nerve strand; c,
cellular layer; i. l., m. l., o. l., inner, middle and
outer layer of lens.

Fig. 27.—Sectional view of photogenic organ
of Sergestes prehensilis (after Terao). bm,
basement membrane; cs, connective strands of
photogenic layer; hy, hypodermis; l1, l2, l3,
layers of lens; le, lens epithelium; n, nerve; ph,
photogenic cells; pi, pigment layer; r, reflector;
th, theca.

Fig. 28.—Diagram of photogenic organ of
Nyctiphanes Conchii, to show pathways of light
rays arising in the light cell layer (after
Trojan). p, pigment; ri, inner reflector; lp, light
cells; rf, refractor; f, focus; l, lens; A-A, axis;
a1-a4, b1-b4, light rays reflected from ri; c1-c4,
light rays passing directly outward; d1-d9 and
e1-e9, light rays which have passed refractor
and lens respectively.
All gradations in complexity of light organs may be found from the condition in the shrimp just described to that
found among the squid and fish. Figs. 29 and 30 are sections of two of the more complicated types found in squid.
The explanation given to the various structures is that of Chun (1903) to whom we are indebted for a careful
histological investigation of these forms. It will be noted that in addition to photogenic and lens tissues there are
various types of reflector cells and a line of pigment about the whole inner surface of the organ to effectively
screen the animal's tissues from the light. In one form (Fig. 30) chromatophores are found about the region where
the light is emitted and these no doubt serve as color filters. There are also an abundant blood supply and nerves
passing to the organ. Figs. 30 and 31 are sections through light organs of fishes.
We thus see that light organs may be very simple and also very complicated. The latter must have evolved from the
former, although it is not always possible to point out the intermediate stages. It is not within the scope of this
book to discuss bioluminescence in its evolutionary aspects. It may be worth while, however, to point out briefly
what is known concerning the use of the light to the animal. There are four possibilities.

Fig. 29.—Sectional view of photogenic organ
of a squid, Abraliopsis (after Chun.) refl1, refl2,
reflectors; lac., lacunar spaces; chr., pigment
screen of chromatophores; chr.1,
chromatophore; phot., photogenic cells; l, lens;
co., cuticle; v, blood vessel; fibr., connective
tissue.
(1) The light may be of no use whatever, purely fortuitous, an accompaniment of some necessary or even
unnecessary chemical reaction.
This appears to be the case in the luminous bacteria and fungi and perhaps the great majority of forms which make
up the marine plankton, Noctiluca, dinoflagellates, jelly-fish, ctenophores and even the sessile sea pens.

Fig. 30.—Sectional view of photogenic organ
of a squid, Calliteuthis (after Chun). phot.,
photogenic cells; l, l1, lens; n, nerve; spec.,
"Spiegel"; pg., pigmented screen; c. fusif.,
spindle-shaped reflector cells; chr.,
chromatophore color screen.

Fig. 31.—Sectional view of photogenic organ
of a fish, Stomias (after Brauer). p, pigment
screen; dr, dr1, photogenic gland cells; l, lens.
We know that luminous bacteria occasionally lose the power of lighting and that on certain culture media they
develop as non-luminous forms. Luminescence is not indispensable to them. The same is true of some of the fungi
but Noctiluca and other animals are not known in a non-luminous condition, although we can see no definite value
to the organism of this power of luminescence.

Fig. 32.—Sectional view of photogenic organ
of a fish, Argyrophelecus affinis (after Brauer).
p, pigmented screen; dr., photogenic cells; r, r1,
reflector?; l, lens?; s, sclera; g, connective
tissue.
In the case of sea pens, however, we might suppose that the light acts as an attraction to small organisms on which
the sea pen feeds, although these creatures only luminesce when stimulated in some way, which rather detracts
from the above suggestion.
(2) The light may act as a warning to scare away predacious animals which would otherwise feed on the luminous
organism. Perhaps this is the case in the sea pens, although these forms possess nematocysts which should serve as
adequate protection. The marine worm, Chætopterus, is brightly luminous and lives its whole life in an opaque
parchment tube. If this tube were torn open by a predacious form we might conceive that the attacking animal
would be alarmed by the light and refrain from destroying the worm. The Chætopterus, however, could not rebuild
another tube and its light would only protect it in the night time. These cases will suffice to indicate the difficulties
and perplexities of the problem. Perhaps we may add one more guess and suppose that the light of certain fishes is
actually for blinding or distracting their enemies or blinding the forms on which they feed. Until this use of
luminous organs has actually been observed, we can give little credence to it.
(3) The light may serve as a means of recognition or a sex signal to bring the sexes together for mating. It would
seem from the work of Mast and of McDermott that this is the case in the common fireflies and it may be the case
in the toad-fish, Poricthys, which is only luminous in the spawning season and in the worm, Odontosyllis, of
Bermuda, which is brilliantly luminous while swarming when the eggs and sperm are shed. It is non-luminous at
other times (Galloway and Welch, 1911.)
(4) Finally, it is possible that animals with complex luminous organs, such as squid, fish and shrimp, actually use
these as lanterns. It is significant that most of them are deep sea forms, living in a region of perpetual darkness,
and it is perfectly logical to suppose that they make use of their light organs for illuminating purposes.

The whole problem of the use and purpose of luminous organs is an exceedingly complex and difficult one. We
have, perhaps, said enough to indicate this and may add that in most cases, so far as opinion is based on actual
evidence and observation, that of the layman is of as great value as that of the scientist.

CHAPTER V
THE CHEMISTRY OF LIGHT PRODUCTION, PART I
Two experiments, both performed very early in the history of Bioluminescence, are of great importance in
understanding the nature of animal light. Boyle (1667), as already mentioned, proved the necessity of air for the
luminescence of wood and fish and Spallanzani (1794) showed that parts of luminous medusæ gave no light when
dried but if moistened again would emit light as before. We see then, that air (oxygen), water, and some photogenic
substance are necessary for the light production. Spallanzani's experiment, which has been confirmed for a great
many luminous forms, shows also that animal luminescence is not a vital process, in the same sense that the
conduction of a nerve impulse is a vital process. A nerve loses its characteristic property of conduction on drying
or maceration while luminous cells still possess the power to luminesce after drying or maceration. Using the
terminology of the older physiology we may say that "living protoplasm" is not necessary for light production.
The experiments of Boyle (1626-91) are of great interest, especially those in which he studied the behavior of
shining wood under the receiver of his air pump. On October 29, 1667, he wrote:
"Exp. I.: Having procured a Piece of shining Wood, about the bigness of a groat or less, that gave a vivid Light,
(for rotten Wood) we put it into a middle sized Receiver, so as it was kept from touching the Cement; and the Pump
being set a-work, we observed not, during the 5 or 6 first Exsuctions of the Air, that the splendor of the included
Wood was manifestly lessened (though it was never at all increased;) but about the 7th Suck, it seemed to glow a
little more dim, and afterwards answered our Expectation, by losing of its Light more and more, as the Air was still
farther pumped out; till at length about the 10th Exsuction, (though by the removal of the Candles out of the
Room, and by black Cloaths and Hats we made the place as dark as we could, yet) we could not perceive any light
at all to proceed from the Wood.
"Exp. II.: Wherefore we let in the outward Air by Degrees and had the pleasure to see the seemingly extinguished
Light revive so fast and perfectly, that it looked to us almost like a little Flash of Lightning, and the Splendor of the
Wood seemed rather greater than at all less, than before it was put into the Receiver."
Boyle proved that light from the wood was able to pass a vacuum and later showed that "shining fish" behaved as
the "shining wood," but that a piece of white hot iron would not regain its light on readmitting air to the exhausted
receiver and that the iron lost its glow under the air-pump merely because it cooled off. A piece of glowing coal,
however, did lose its light in the absence of air and regained it on again admitting air, provided the air had not been
removed for too long. Boyle was apparently impressed with the similarity of the light giving process in glowing
coal and shining wood as he draws a comparison between the two which brings out the fundamental similarity of
combustion processes.
"Resemblances:
VII. The Things wherein I observed a Piece of shining Wood and a burning Coal to agree or resemble each other
are principally these five:
1. Both of them are Luminaries, that is, give Light, as having it (if I may so speak) residing in
them; and not like Looking-glasses, or white Bodies, which are conspicuous only by the incident
Beams of the Sun, or some other luminous Body, which they reflect....
2. Both shining Wood and a burning Coal need the Presence of the Air (and that too of such a
Density to make them continue shining)....
3. Both shining Wood and a burning Coal, having been deprived, for a Time, of their Light, by the
withdrawing of the contiguous Air, may presently recover it by letting in fresh Air upon them....
4. Both a quick Coal and shining Wood will be easily quenched by Water and many other
Liquors....

5. As a quick Coal is not to be extinguished by the Coldness of the Air, when it is greater than
ordinary; so neither is a Piece of shining Wood to be deprived of its Light by the same Quality of
the Air....
Differences:
1. The first Difference I observed betwixt a live Coal and a shining Wood is, that whereas the
Light of the former is readily extinguishable by Compression (as is obvious in the Practice of
suddenly extinguishing a piece of Coal by treading upon it), I could not find that such a
Compression as I could conveniently give without losing sight of its operation, would put out, or
much injure the Light, even of small Fragments of shining Wood....
2. The next Unlikeness to be taken notice of betwixt rotten Wood and a kindled Coal is, that the
latter will, in a very few Minutes, be totally extinguished by the withdrawing of the Air; whereas
a Piece of shining Wood, being eclipsed by the Absence of the Air, and kept so for a Time, will
immediately recover its Light if the Air be let in upon it again within half an hour after it was first
withdrawn....
3. The next Difference to be mentioned is, that a live Coal, being put into a small close Glass, will
not continue to burn for very many Minutes; but a Piece of shining Wood will continue to shine
for some whole Days....
4. A fourth Difference may be this: that whereas a Coal, as it burns, sends forth Store of Smoke or
Exhalations, luminous Wood does not so.
5. A fifth, flowing from the former, is, that whereas a Coal in shining wastes itself at a great Rate,
shining Wood does not....
6. The last Difference I shall take notice of betwixt the bodies hitherto compared is, that a quick
Coal is actually and vehemently hot; whereas I have not observed shining Wood to be so much as
sensibly lukewarm."
It should be clearly borne in mind that if we place luminous organisms, say bacteria or fungi, in an atmosphere
devoid of oxygen and find that no light is produced, this may merely mean that certain functions of the cell are
interfered with, including light production, but does not necessarily indicate that oxygen is actually used up in the
photogenic process. If we find, however, that extracts of luminous cells or luminous secretions devoid of cells
cease to light when the oxygen is removed and again luminesce when it is returned, we may be quite certain that
the photogenic process itself requires free oxygen. As luminous extracts of fireflies, pennatulids, ostracods, Pholas
and others give off no light when the oxygen is removed, we may safely conclude that for these luminescences,
oxygen is necessary. Bacteria, fungi, and Noctiluca, whose light also disappears in absence of oxygen, although
they are whole cells, we may by analogy also assume to require oxygen in the photogenic process.
Some of the earlier workers on fireflies and Noctiluca obtained light even after placing these organisms in absence
of oxygen, but they did not realize how low is the amount of oxygen necessary to produce light. It is difficult to
remove traces of oxygen from the water, traces which are nevertheless sufficient to cause luminescence. If the
organisms are numerous, as in an emulsion of luminous bacteria, they will themselves use up all the oxygen and
the liquid soon ceases to glow except at the surface in contact with air. We may gain an idea of the amount of
oxygen necessary for luminescence from an experiment of Beijerinck (1902). He mixed luminous bacteria with an
emulsion of clover leaves containing chloroplasts and kept the two in the dark until all the oxygen was used up and
the bacteria ceased to glow. If now a match was struck for a fraction of a second, sufficient oxygen was formed by
photosynthesis to cause the bacteria to luminesce for a short time.
Exact figures on the minimal concentration of oxygen for luminescence cannot be given. The luminescent
secretion of Cypridina hilgendorfii will still give off much light if hydrogen containing only 0.4 per cent. of
oxygen is bubbled through it, i.e., a partial oxygen pressure of 1/250 atmosphere (3.04 mm.Hg). However, addition
of a fresh emulsion of yeast cells to a glowing Cypridina secretion is sufficient to rapidly extinguish the light,
because the yeast is capable of utilizing the last trace of oxygen in the mixture. Light only appears when, by
agitation, we cause more air to dissolve. The minimal concentration of oxygen for luminescence of Cypridina lies

somewhere between 3.04 mm. and the amount which living yeast fails to extract from solution, a concentration
approaching zero. It is probably nearer the latter figure.
As the oxygen pressure is increased from 0 to about 7 mm., the intensity of the Cypridina luminescence increases
and at the latter figure the light is just as bright as if the solution were saturated with air (152 mm.O2). Thus, the
luminescence requires only a low pressure of oxygen and the similarity to the saturation of hæmoglobin with
oxygen is obvious. Just as hæmoglobin is nearly saturated with oxygen at low pressures and becomes bright red in
color, so the luminous material becomes saturated with oxygen at low pressures and glows intensely.
Boyle also made many experiments to show that air was necessary for the life of animals and the germination of
seeds and showed that repeatedly respired air was unfit for further breathing. About the same time R. Hooke
discovered the true meaning of respiratory movements and by forcing a blast of air continuously through the lungs
with bellows, was able to keep animals alive. He concludes "that as the bare Motion of the Lungs, without fresh
air, contributes nothing to the life of the Animal, he being found to survive as well as when they were not moved as
when they were; so it was not the Subsiding or Movelessness of the Lungs that was the immediate cause of death,
or the stopping of the circulation of the Blood through the Lungs, but the Want of a sufficient Supply of fresh Air."
The cause of death on collapse of the lungs could not be better stated to-day. Thus combustion, respiration and
luminescence of flesh or wood were early recognized as related phenomena.
Although the "gas sylvestre" (CO2) of burning charcoal and fermentation of wine was known to van Helmont
(1577-1644) and Mayow (1646-1679) in 1674 showed that "spiritus nitroærens" (oxygen) was responsible for the
life of animals and for combustion, a century elapsed before the true significance of these gases became known. In
the meantime the phlogiston theory of combustion had been developed, Black (1728-1799) in 1755 had
rediscovered carbon dioxide ("fixed air") in the expired air and Priestley (1733-1804) and Scheele (1742-1786)
had both rediscovered oxygen ("dephlogisticated air") in 1774. About the same time Lavoisier overthrew the
phlogiston doctrine and showed that in the combustion of organic substances water and CO2 are formed.

Later it was realized that this slow combustion did not take place in the lungs, or in the blood, but in the tissues
cells themselves and respiration in the chemical sense has come to mean this universal slow combustion in the
cells of the body rather than the breathing movements of the lungs themselves. In anaerobic respiration, CO2 is
given off, but no oxygen absorbed. In aerobic respiration, oxygen is absorbed and CO2 given off. In addition we
know of many substances which oxidize by taking up oxygen without giving off CO2. We have seen that oxygen
must be absorbed for luminescence of animals and we may now inquire whether CO2 is given off and the relation
between respiration and light production.
To determine if CO2 is given off during luminescence it is necessary to work with fairly pure luminous materials,
obtained from luminous organisms. It is impossible to use the living organisms themselves as the CO2 continually
respired becomes a very disturbing factor. From Cypridina, a small crustacean, two materials soluble in water may
be prepared (luciferin and luciferase), which will give a brilliant luminescence on mixing. It is possible to
determine the H-ion concentration of the two solutions separately and of the mixture of the two after the
luminescence has occurred.
If CO2 is produced during luminescence the H-ion concentration of the luminous solution should increase.
Measurements made electrometrically with the hydrogen electrode have failed to demonstrate any increase in
acidity. The Ph of both solutions and of a mixture of the two is 9.04. This would indicate that CO2 is not produced.
As both luminous solutions contain proteins and the luminous substances themselves are probably proteins, which
have a high buffer value, a method of this kind is none too sensitive. However, we can definitely state that not
enough CO2 is produced to be detected and that this may be due to the buffer action of the luminous substances
themselves. After all, unless luminescence is connected with respiration, we should hardly expect CO2 to be
produced.
Another method of testing CO2 production is to measure the amount of heat produced during luminescence.
Substances burned during respiration give off considerable heat, one gram of glucose to CO2 and H2O, as much as
4000 calories. We have seen in Chapter III that no infra-red radiation is produced in the light of the firefly. This
does not mean, however, that no heat is produced by the reaction which produces the luminescence. A temperature
change of a few thousandths or hundredths of a degree would evolve no measurable radiation. Coblentz (1912)

first studied the problem of heat production in the firefly, using a thermocouple as the measuring instrument. He
came to the conclusion that the temperature of the insect was slightly lower than the temperature of the air and that
the luminous segments were slightly hotter than the non-luminous segments, whereas a dead firefly is of the same
temperature as its surroundings. No definite increase or decrease in temperature could be established during the
flash of the firefly. However, further work on the firefly is much to be desired.
The use of a living animal for such measurements introduces a possible source of error in that any contraction of
the muscles of the animal will produce heat which may add to an increase or mask a decrease of temperature
during luminescence. Utilization of extracts of luminous animals containing the luciferin and luciferase mentioned
above avoids the complications due to muscular contraction. By bringing the solutions of luciferin and luciferase
to the same temperature and then mixing them one can measure any increase or decrease of temperature which
occurs during the luminescence which results from mixing. We can thus gain some idea of the heat of oxidation of
luciferin.
As a determination of heat production is of considerable interest the method will be given in some detail. Although
the experiment sounds very simple, it is actually somewhat difficult to carry out. The attainment of temperature
equilibrium between two solutions is very slow when one wishes to obtain them to within 0.001° C. of the same
temperature. After many attempts, the following arrangement of apparatus (Fig. 33) was found most satisfactory.
About 10 c.c. luciferin solution was placed in the inner tube (D) of a special non-silvered thermos bottle (A). About
1 c.c. of luciferase solution was placed in a very thin-walled glass tube (E) which was immersed in the luciferin
solution and connected with a small motor so that it could be slowly but constantly rotated, thus stirring the
solutions. Thermocouples (L and M) of advance (.008 in)—copper (No. 30, B and S, enamel insulated) wire were
paraffined and placed in each tube and the copper wires connected through a copper double throw switch (C) with
a Leeds and Northrup d'Arsonval wall galvanometer (No. 34637, silver strip suspension) of 35 ohms resistance and
310 megohms sensitivity. The constant temperature junctions (N) were placed in a large Dewar flask (B) filled with
water at approximately the same temperature as the luciferin solution. One mm. galvanometer scale division
represented 0.003° C. and the division readings could be estimated to tenths. By means of a glass rod (F) placed in
the tube containing luciferase solution, this tube could be broken and the luciferase and luciferin solution mixed.

Fig. 33.—Apparatus for determining heat
production during luminescence of luciferin.
A, special thermos tube. B, Dewar flask for
constant temperature junctions. C, double
throw switch. D, tube containing luciferin
solution. E, tube containing luciferase solution.
F, glass rod for breaking E. G, rubber stopper
with groove, K, for pulley cord. H, cork
closing thermos tube. J, brass sleeve in H

allowing rotation of E. L, thermojunction in
luciferase solution. M, thermojunction in
luciferin solution. N, constant temperature
junctions.

Fig. 34.—Curve showing temperature change
when two tubes containing water at the same
temperature are mixed. 0.1 galvanometer scale
division = 0.003° C. Dots represent readings of
thermocouple in tube D; crosses readings of
thermocouple in tube E.
It was found that even after the luciferase and luciferin solutions came to the same temperature within the thermos
bottle, this was not necessarily the same as that of the room and a slow rise or fall occurred as indicated by a slow
drift of the galvanometer coil. Readings of each thermocouple on the galvanometer scale were therefore taken at
one-minute intervals for some time before and after mixing the luciferin and luciferase solutions and plotted as
curves. Control experiments were also carried out in exactly the same manner as the luciferin-luciferase
experiments, but water was placed in the two tubes instead of luciferin and luciferase. Figs. 34 and 35 give typical
experiments with water and with luminescent solutions, respectively.

Fig. 35.—Curve showing temperature change
when luciferin and luciferase solutions at the
same temperature are mixed. 0.1

galvanometer scale division = 0.003° C. Dots
represent readings of thermocouple in
luciferin solution; crosses, readings of
thermocouple in luciferase solution.
With both control (water) and luciferin experiments there was a slight rise in temperature on mixing the liquids in
the two tubes. The average rise of five control (water) experiments was .0054° C. and the average rise of five
luciferin experiments was .0048° C.
The average rise in temperature is no doubt due to heat from friction in mixing of the liquids and breaking of the
glass tube. The difference in the average rise of control and of luciferin experiments is so small (.0006° C.) as to
have little significance. We may therefore conclude that if any temperature change occurs during the luminescent
reaction it is certainly less than 0.001° C. and probably less than 0.0005° C., too small to be measured by this
method.
To prepare the luciferin solution, two grams of dried Cypridina were dissolved in 20 c.c. hot water and 10 c.c. of
this 10 per cent. solution was used in the thermos bottle in the above experiments. If we assume that 1 per cent. of
the dried Cypridina is luciferin, 0.01 gram of luciferin on oxidation was not able to raise the temperature of the 10
c.c. (in reality 11 c.c., since 1 c.c. luciferase solution was mixed with the 10 c.c. luciferin solution) .001° C. This
means that 1 gram luciferin liberates at least less than 10 calories during the luminescence accompanying
oxidation.
Since 1 gram glucose liberates 4000 calories on complete oxidation to CO2 and H2O, it will be seen that the
oxidation of luciferin is a very different type of reaction from the oxidation of glucose. As we shall see, it is
probably similar to the oxidation of reduced hæmoglobin or the oxidation of leuco methylene-blue to methylene
blue. According to Barcroft and Hill (1910), 1.85 calories are produced per gram of hæmoglobin oxidized. I have
been unable to find figures for the heat exchange during oxidation of leuco-dyes, but it is no doubt also small.
Since luciferin evolves no measurable amount of heat on oxidation, we have very good evidence in support of that
obtained by electrometric measurements of H-ion concentration, that no carbon dioxide is produced during
luminescence of luminous animals.
In most animal cells it is perfectly clear that luminescence does not accompany respiration, since respiration is a
continuous process, whereas light is only produced on stimulation. It is true that on stimulation respiration is
accelerated, and we might suppose that luminescence is an accompaniment of accelerated respiratory oxidations;
but this is not the case, for in luminous animals a rise in temperature of ten degrees centigrade will accelerate the
respiratory oxidations 250 per cent. without necessarily causing the production of light.
In fungi and bacteria, on the other hand, which continually emit light, it is quite natural to suppose that the light is
an accompaniment of respiration, just as we know the heat of these forms to be. This view was accepted by such of
the earlier workers as Fabre in 1855, who found that luminous portions of a mushroom, Agaricus olearius, gave
off more CO2 (4.41 c.c. CO2 per gram in 36 hours at 12° C.) than non-luminous portions (2.88 c.c. CO2 per gram
in 36 hours at 12° C.). This experiment has never been repeated and there are many reasons besides luminescence
why one piece of fungus might have a more rapid respiratory rate than another piece. It is not true that rapidly
respiring plant tissues, such as germinating seeds or the spadix of Araceæ, are luminous, although they produce
considerable heat.
On the other hand, it is very easy to prove that luminescence, even in bacteria, is not connected with respiration.
Thus, Beijerinck (1889 c) found that of several species of luminous bacteria studied by him, one, Bacterium
phosphorescens, was a facultative anaërobe and would grow, i.e., multiply, but not luminesce in the absence of
oxygen. Some forms, ordinarily producing light, will grow, but fail to luminesce at high temperatures. Beijerinck
(1915) has recently found that these individuals may, by continued cultivation at high temperatures, form non-
luminous strains which fail to luminesce when again brought into lower temperatures, favorable for luminescence.
These non-luminous mutants occasionally give rise to atavistic brilliantly luminous forms. Beijerinck also finds
that after exposure of Photobacter splendidum to ultra-violet or strong sunlight, radium or mesothorium rays,
luminescence continues but no growth occurs. There is thus ample evidence that growth and respiration are
properties quite distinct and separable from luminescence. Indeed, respiration increases continuously up to a
relatively high maximum whereas luminescence falls off rapidly above a relatively low optimum. McKenney
(1902) found also that Bacillus phosphorescens could grow rapidly in 0.5 per cent. ether without producing light.

Luminescence has been compared in bacteria to pigment formation, as rather definite cultural conditions are
necessary for realization of both chromogenic and photogenic function. Some pigment-formers, as Bacillus
pyocyaneus, which produces a water-soluble green pigment, remain colorless under anaërobic conditions. A
colorless chromogen is formed, which oxidizes to the green pigment in the air. If this colorless chromogen
produced light during its oxidation as well as green pigment, we would have a case of both chromogenic and
photogenic function combined in one species of bacterium. Luminescence involves something more than
respiration, an oxidation of a very definite and particular kind.
Since Spallanzani's observation that the luminous material of medusæ could be dried, and upon moistening would
again give light, many confirmatory observations have been made on other forms. Pyrosoma, Pholas, Phyllirrhoë,
fireflies, Pyrophorus, copepods, ostracods, pennatulids, fungi, and bacteria can all be dessicated and the
photogenic material preserved for a greater or less time. In a dessicator filled with CaCl2, dried luminous bacteria
lose, after a few months, their power to give light on being moistened. On the other hand, ostracods and copepods
will still luminesce after years of dessication. The luminous material in the latter case appears capable of indefinite
preservation, but it is possible that the quick loss of photogenic power with dried luminous bacteria is merely an
indication that they contain very little photogenic substance and that the dried ostracods would also in time lose
their power to luminesce. It is certainly a fact that the amount of luminous material in a single gland cell of an
ostracod is vastly greater than that in the same mass of bacterial colony.
When the dried powdered luminous material of an ostracod is sprinkled over the surface of water, it goes into
solution and leaves luminous diffusion and convection trails plainly visible in the water. Many luminous marine
forms give off a phosphorescent slime when they are handled, which adheres to the fingers. It is not surprising that
this luminous matter should have early received a name. In 1872, Phipson called it noctilucin and described some
of its properties. He regarded the luminous matter which can be scraped from dead fish (luminous bacteria) and the
mucous secretion of Scolopendra electrica or the luminous matter of the glowworm to be this material, noctilucin,
which, "in moist condition, takes up oxygen and gives off CO2 and when dry appears like mucin." Phipson says
that it forms an oily layer over the seas in summer (he probably refers to masses of dinoflagellates), is liquid at
ordinary temperatures and less dense than water, smells a little like caprylic acid, is insoluble in water but miscible
with it, insoluble in alcohol and ether, dissolves with decomposition in mineral acids and alkalies and contains no
phosphorus. We can see from this description that the word "noctilucin" does not indicate a chemical individual,
but it is the earliest attempt to definitely designate the luminous substance.
The idea of a definite substance oxidizing and causing the light has been upheld by a number of investigators, and
many years later Molisch called this substance the photogen. He contrasts the "photogen theory" with certain other
views of light production, which may be spoken of as "vital theories," notably those of Pflüger (1875), who looked
upon luminescence as a sign of intense respiration, and of Beijerinck (1915), who regarded the light as an
accompaniment of the formation of living matter from peptone.
Fortunately biological science has advanced beyond the stage where a living process can be explained by calling it
a vital process, and we must fall back upon the idea of a photogen oxidizing with light production. Indeed, it is
now possible to go much further than this and describe the properties of the photogen, but we must not lose sight
of the fact that it was recognized very early in the history of Bioluminescence, that water, oxygen, and a
photogenic substance were necessary for light production.
A very great advance in our knowledge of the chemistry of the problem was made by Dubois in 1885. He showed
that if one dips the luminous organ of Pyrophorus in hot water, the light disappears and will not return again. Also
if one grinds up a luminous organ the mass will glow for some time but the light soon disappears. If one brings the
previously heated organ in contact with the unheated triturated organ it will again give off light. Later, Dubois
showed that the same experiment could be performed with the luminous tissues of Pholas dactylus. A hot-water
extract of the luminous tissue, and a cold-water extract of the luminous tissue, allowed to stand until the light
disappears, will again produce light if mixed together. Dubois (1887 b) advanced the theory that in the hot-water
extract there is a substance, luciferin, not destroyed by heating, which oxidizes with light production in the
presence of an enzyme, luciferase, which is destroyed on heating. The luciferase is present together with luciferin
in the cold-water extract, but the luciferin is soon oxidized and luciferase alone remains. Mixing a solution of
luciferin and luciferase always results in light production until the luciferin is again oxidized. Similar substances
have been found by me in the American fireflies, Photinus and Photuris, the Japanese firefly, Luciola, and in the
ostracod crustacean, Cypridina hilgendorfii. Crozier[6] reports that they exist also in Ptychodera, a balanoglossid. I

have been unable to demonstrate their existence in luminous bacteria; in the annelid, Chætopterus; the pennatulids,
Cavernularia and Pennatula; the squid, Watasenia; and the fish, Monocentris japonica. E. B. Harvey (1917) could
not demonstrate them in Noctiluca. There are several reasons why the existence of such bodies might be difficult
to demonstrate, but these reasons cannot be considered here. We thus see that the photogen is in reality of dual
nature, that two substances are necessary for light production and that they may be very readily separated because
of difference in resistance to heating. In this respect Bioluminescence is similar to some other biological processes,
notably to certain immune reactions and to certain enzyme actions.
[6] Private communication.

Thus, for the hæmolysis of foreign red blood corpuscles, a specific immune body (amboceptor or substance
sensibilatrice) not destroyed by moderate heating, and a thermolabile complement (alexin) are necessary.
For the alcoholic fermentation of glucose by the zymase of yeast juice two substances are also necessary. The
zymase is made up of a heat resistant, dialyzing component, the co-enzyme, and a non-dialyzing substance,
destroyed on boiling, the enzyme proper. Both must be present for alcoholic fermentation of glucose to proceed
and the two may be separated by dialysis or by their difference in resistance to heating. Several other
characteristics of living cells are known to depend on the joint action of two substances, one thermolabile, the
other thermostable. The reducing action of tissues, according to Bach, requires a reducing enzyme proper or
perhydridase and some easily oxidizable substance, such as an aldehyde. The aldehyde has been spoken of as the
co-enzyme.
Because of the necessity of thermostable and thermolabile substances for light production in luminous animals and
because I was unable to oxidize the thermostable material of Cypridina with such oxidizing agents as KMnO4,
H2O2, blood and H2O2, BaO2, etc., I called the heat resistant substance of Cypridina, "photophelein" (from phos,
light and opheleo, to assist), comparable to co-zymase, and the heat sensitive substance of Cypridina, "photogenin"
(from phos, light and gennao, to produce), comparable to the zymase proper of yeast. In mode of preparation and
properties, the photophelein of Cypridina was also comparable to the luciferin of Pholas and the photogenin of
Cypridina to the luciferase of Pholas. I also regarded photogenin as the source of the light (hence the name),
because a solution of Cypridina photogenin (=Pholas luciferase) will give light on mixing with crystals of salt and
other substances which could not possibly be oxidized. I later found, however, that this result was due to the fact
that the photogenin solution contained some of the thermostable substance (luciferin) bound (combined or
adsorbed), and that this was freed by the salt crystals and oxidized with light production. I have consequently
abandoned the view that the system of substances concerned in light production is similar to the zymase—co-
zymase system of yeast—and have adopted Dubois' term, luciferase (=photogenin) for the thermolabile material,
and luciferin (=photophelein) for the thermostable material.
The luciferin of Cypridina differs from that of Pholas in that it will not oxidize with light production with any
oxidizing agents that I have tried, and will give no light with luciferase from Pholas. It does, however, oxidize
spontaneously in solution, although no light accompanies this oxidation.
I believe that for accuracy and definiteness we must designate the luciferins and luciferases from different animals
by prefixing the generic name of the animal and speak of Pholas luciferin, Cypridina luciferase, Pyrophorus
luciferase, etc. In extracts of many non-luminous animals Dubois has found oxidizing agents which can oxidize
Pholas luciferin with light production and I have confirmed this for Pholas, but I have not found any such
substances in non-luminous animals which will oxidize Cypridina luciferin with light production. I have found in
extracts of non-luminous animals substances which will liberate the bound luciferin in a concentrated Cypridina
luciferase solution. The luciferin can then be oxidized by the luciferase and light appears. Their effect is similar to
that of salt crystals and I suggest that they be called photopheleins, substances that assist in the luciferin-luciferase
reaction by liberating bound luciferin. One of the best ways of freeing a solution of luciferase from bound luciferin
is to shake with chloroform. We can then do away with the disturbing effects of bound luciferin.
It is obvious that luciferin must be formed from some precursor in the cell and following the usual biochemical
terminology, Dubois has called it proluciferin or preluciferin, and believes that it is converted into luciferin by an
enzyme co-luciferase. The experiments to prove the existence of proluciferin were first made by Dubois on Pholas
in 1907 and have since been amplified (1917 a; 1918 a and b).
In order to understand these experiments it must be borne in mind that Dubois prepares luciferin from Pholas in
three ways: (1) By precipitating the viscid luminous fluid from the siphons with 95° alcohol and dissolving the

precipitate in water (1901a, 1907). (2) By extracting the luminous organs with 90° alcohol in a closed vessel for
twelve hours and filtering (1896). (3) By heating the viscid luminous fluid to 70° C. Apparently Pholas luciferin is
sparingly soluble in alcohol as it can be obtained either in an alcoholic extract (method 2) or by precipitation with
alcohol (method 1). Proluciferin (called preluciferine in a later paper, 1917 a, 1918 a), is prepared by methods 1 or
2 except that fatigued siphons, from which luciferin has been removed by washing, are used (1907, 1917 a, 1918
a). Preluciferin can also be obtained on boiling an extract of the luminous organ of Pholas because luciferin (at
70°), luciferase (at 60°) and a co-luciferase are all destroyed below the boiling point (1917 a).
Co-luciferase is prepared (1) by heating a luciferase solution to 65° (1917 a) or (2) by extracting with water
portions of the siphon of Pholas which have previously been macerated and well extracted with alcohol (1918 a).
Long-continued treatment with alcohol apparently destroys the luciferase without affecting the co-luciferase. On
mixing a solution of preluciferin with one of co-luciferase and allowing them to stand for 8-10 hours, luciferase is
formed and can be recognized by the fact that it will give light with a crystal of KMnO4. Preluciferine does not do
this.
Recently Dubois (1918 a) states that preluciferine is nothing but taurine and that taurine occurs in large quantities
in Pholas and is transformed into luciferine by the action of co-luciferase. Not only taurine, but also Byla's
peptone, egg lecithin, and esculin can be converted into luciferine by co-luciferase, and since esculin, a glucoside,
is so transformed, Dubois believes this proves that co-luciferase belongs to the hydrolases. Indeed, it proves too
much. Luciferin must have an extraordinary chemical structure if it can be formed by hydrolysis of such diverse
compounds as peptone, lecithin, esculin and taurine. A glance at the structural formula of esculin and taurine is
sufficient to emphasize the diverse nature of these two substances.

Taurine Esculin
I believe that in these experiments Dubois has been working with an oxidation product of luciferin, what I have
called oxyluciferin, rather than a pro-substance. The mode of preparation of Pholas preluciferin and Pholas co-
luciferase is such as could be used in the preparation of Cypridina oxyluciferin, and it seems more logical to look
for the presence of Pholas oxyluciferin in one or both of Dubois' extracts rather than believe that luciferin can be
formed from both taurine and esculin. When the co-luciferase solution stands with the preluciferin solution we
would in reality have not the formation of luciferin from preluciferin, but the formation of luciferin from
oxyluciferin, by some reducing agent in the mixture. Indeed, in a very recent paper Dubois (1919c) takes the view
that his co-luciferase is a reducing enzyme which forms luciferin by reduction (presumably from oxidized
luciferin) and no mention is made of preluciferin.
It is, of course, obvious that when luciferin oxidizes, some oxidation products must be formed. Most observers
have assumed the oxidation products of luciferin to be relatively simple and to represent a rather complete
breaking down of the luciferin molecule. Carbon dioxide was mentioned by Phipson (1872) as being formed. We
have just seen that no carbon dioxide is formed during the oxidation of Cypridina luciferin and there is evidence
that no fundamental change at all occurs. It is for this reason that I have called the oxidation product of luciferin
oxyluciferin.[7] As we shall later see, the change luciferin oxyluciferin is to be compared to the oxidation of
colorless dyes (leuco-compounds) to the colored dye. The chemical properties of oxyluciferin are similar to those
of luciferin and the oxyluciferin can be readily reduced to luciferin again.
[7] is unfortunate that Dubois (1918 b) has used the term oxyluciferine in a quite different sense from the present use. He
regards oxyluciferine as a substance still capable of giving light by autoöxidation, and represents the steps in luminescence
as follows:

"Co-luciférase + preluciférine = luciférine.
Luciférase + luciférine = oxyluciférine.
Oxyluciférine + oxygéne = lumiérè."

I should represent them as follows:

Luciferin + oxygen ⇆ oxyluciferin.
The reaction proceeds to right with light production only in presence of luciferase.

Finally, we have the fluorescent substance of Pyrophorus and fireflies, which Dubois first called pyrophorin, but
later, adopting McDermott's terminology, speaks of as luciferesceine. This Dubois regards as a substance
intensifying the light and modifying its color by changing invisible into visible rays. As we have seen, this theory,
while attractive, will not stand the test of critical examination.
Phipson's noctilucin, while the first name for the photogen of luminous animals, is too vague a substance,
chemically, to warrant a retention of the term. Of the names, luciferin, luciferase, preluciferin or proluciferin, co-
luciferase, photogenin, photophelein, oxyluciferin, luciferesceine, I believe that only proluciferin, luciferin,
oxyluciferin, luciferase and photophelein stand for substances which are really significant for the theory of light
production. Luciferin is the heat resistant, dialyzable substance which takes up oxygen and oxidizes with light
production in the presence of the heat sensitive, non-dialyzing, enzyme-like luciferase. The luciferin must come
from some precursor, proluciferin, but I have been unable to demonstrate the existence of this body in Cypridina
and know nothing definite of its properties. The luciferin oxidizes to oxyluciferin which has the same chemical
properties as the luciferin itself and may be reduced to luciferin again by reducing substances in luminous and
other animals or by inorganic reducing agents. Photophelein is a name for substances in various animal or plant
extracts which are capable of liberating luciferin from some bound condition in solutions containing luciferase.
Under this term are included a number of unknown, probably quite different substances, some of which are
thermostable and others thermolabile.
We have seen that Bioluminescence is an oxyluminescence, that the light is probably due to the oxidation of a
compound, luciferin, in presence of air and water and that the oxidation is accelerated by an enzyme-like
substance, luciferase. We also saw in Chapter 2 that light production is of fairly common occurrence during the
oxidation of many organic compounds, provided the oxidation is carried out in the proper way. Many of these
organic compounds must be oxidized by relatively strong alkali or such strong oxidizing agents as would have a
very deleterious action on living cells. In 1913, Ville and Derrien, in a short note to the French Academy, "Catalyse
Biochemique d'une Oxydation Luminescente," show that lophin could be oxidized by vertebrate blood in the
presence of H2O2. In the same year Dubois (1913) found that esculin, the glucoside from horse chestnut bark,
would also oxidize and luminesce in presence of blood and H2O2. In these cases the hæmoglobin of the blood acts
as a catalyst, transferring oxygen from the H2O2 to esculin or lophin and is to be compared to luciferase, except
that luciferase does not require the presence of H2O2.

As the hæmoglobin does not lose this power on boiling, whereas luciferase does, the analogy is far from perfect.
Many oxygen carriers are known, however, which may be destroyed on boiling their solutions, namely, the
peroxidases of plant juices. Esculin will not luminesce with peroxidase and H2O2, but pyrogallol or gallic acid
will. If one mixes a test tube containing pyrogallol solution + H2O2 with potato or turnip juice or almost any plant
extract, a yellowish luminescence appears. The plant extract loses the power to cause such luminescence on boiling
and the peroxidase will not dialyze. It is, of course, comparable to luciferase and acts on the thermostable,
dialyzable pyrogallol-H2O2 mixture, which is comparable to luciferin. Curiously enough, although many
hydroxyphenol and amino-phenol compounds can be oxidized by peroxidase and H2O2, only pyrogallol and gallic
acid will oxidize with light production. Many other oxidizers can take the place of the peroxidase. A list of these is
given on page 151. No other peroxide can take the place of H2O2 with peroxidases as oxidizers, but a few can
replace H2O2 with other oxidizers. This is brought out in Table 7.

Table 7
Peroxides Giving Light with Pyrogallol and Oxidizers

Oxidizer. H2O2 Benzoyl Ozonized Na2O2 BaO2 MnO2 PbO2 K Na K
(Equal 3 per hydrogen turpentine (powder) (powder) (insoluble (insoluble persulfate perborate perchlorate

parts added cent. peroxide (one drop) powder) powder) M/10 M/20 M/10
to a (insoluble
mixture of powder)
M/100
pyrogallol
and the
peroxide)
Turnip juice + - - - - - - -
1 percent
Faint
blood + - - - - - - - -
flash
extract
M 20
K4Fe(CN)6 + - - - - - - - - -
M 100 Faint Fair
KMnO4 + - - - - - - -
flash flash
M 10 FeCl3 + - -
M 100 CrO3 + - -
Na Faint Faint Fair Fair
+ - - - - -
hypobromite flash flash flash flash
Ca Fair
+ - - - - - - Faint -
hypochlorite flash
MnO2 +
Mn(OH)3
sol in + - -
peptone
Colloidal
+
Ag

Our knowledge of the existence of such analogous, purely organic chemical oxidations, which proceed with light
production, greatly strengthens Dubois' theory that the luciferin-luciferase reaction really represents a catalytic
oxidation of similar nature. As Dubois (1914 a) expresses it, we are dealing in luminous organisms with "1° une
luminescence; 2° une chemiluminescence; 3° une oxyluminescence; 4° une zymoluminescence.
"Ou si l'on bien admettre que les zymases sont encore quelque chose de vivant, une Biozymoöxyluminescence."
Perhaps it is not really necessary to admit that the enzymes are living in order that we may adequately visualize the
nature of the photogenic process.
In the next chapter the properties of the three principal substances, luciferin, oxyluciferin and luciferase, will be
studied more carefully.

CHAPTER VI
THE CHEMISTRY OF LIGHT PRODUCTION, PART II
Since Radziszewski's experiments on the oxidation of oils in alcoholic solutions of alkali, most of the early
workers on Bioluminescence tacitly assumed that the oxidizable material was fat or a fat-like substance. Support
was given to this view by the occurrence in cells of granules or globules from which the light was seen to come.
We now know that these bodies are not fat droplets and that neither luciferin nor luciferase are soluble in such fat
solvents as ether, chloroform, benzol or benzine. Phipson's description of the properties of noctilucin are too crude
and inaccurate to be considered. Dubois did not study the chemical properties of luciferin and luciferase from
Pyrophorus, the first form with which he worked, except to point out that Pyrophorus luciferase was destroyed on
heating and was precipitated by alcohol while the Pyrophorus luciferin was not so affected. Luciferin was found
only in the luminous organ of Pyrophorus, not in the blood; luciferase probably exists throughout the animal.[8]
[8] Private communication from R. Dubois.

Pholas luciferin.—In a series of papers since 1887 Dubois has studied the chemical properties of Pholas luciferin
and Pholas luciferase. He finds the luciferin to be destroyed above 70° C., to dialyze slowly, to oxidize with light
production in the presence of Pholas luciferase, KMnO4, H2O2, hæmatine and H2O2, BaO2, PbO2, hypochlorites,
and the blood of various marine mollusks and crustacea. It is insoluble in fat solvents but forms a colloidal solution
in water from which it is precipitated unchanged by picric acid, alcohol at 82°, and (NH4)2SO4. It is not
precipitated by NaCl, MgSO4 or acetic and carbonic acids, except in presence of neutral salts. It forms an insoluble
alkali albumin with NH4OH. Dubois (1887 a) stated at one time that it could be crystallized and has spoken of it as
belonging to several different classes of substances, proteose, nucleoprotein, albumin. Most recently he describes
luciferin as a natural albumin having acid properties. It occurs only in luminous, not in non-luminous animals, and
is found in all parts of the mantle, especially the siphons. It does not occur in non-luminous parts of the mollusk.
No photographs of luciferin crystals have ever been published.
Pholas luciferase.—Pholas luciferase has all the properties of an enzyme, is destroyed at 60° C., is non-
dialyzable, insoluble in fat solvents, but forms a colloidal solution in water. It is not affected by 1 per cent. NaF but
its activity is suspended in saturated salt solutions, sugar or glycerine, and it may be preserved in this way, its
activity returning on dilution. It is digested by trypsin and slowly destroyed by the fat solvent anæsthetics, such as
chloroform. For this reason Dubois regards it as an oxidizing enzyme similar to the oxydones of Batelli and Stern.
Because he found iron in an extract of Pholas dialyzed for a long time against running water, Dubois considers that
it is associated with iron, and reports that it will oxidize the ordinary oxidase reagents, such as pyrogallol, gum
guaiac, a-naphthol and para-phenylene-diamine, etc. It remains to be proved, however, that luciferase and not the
oxidizing systems such as occur in all cells are responsible for the coloration of these reagents. Dubois has found
luciferases or substances capable of giving light with Pholas luciferin in the blood of many non-luminous
crustacea and mollusks (in Barnea candida, Solen, Cardium edulis, Ostræa and Mytilus).
Cypridina luciferin.—Despite the large amount that has been written on luminous animals, Dubois' work on
Pholas and my own on Cypridina and the firefly are the only truly chemical studies that give us any idea of the
nature of the photogenic substances in any luminous animal. In many ways Cypridina luciferin is similar to Pholas
luciferin, but the two differ in a sufficient number of points to make certain that they are not identical substances.
As I have emphasized above, we should speak not of luciferin and luciferase but of the luciferins and the
luciferases. The luciferins, as the oxidizable substances, must claim first attention. They are more simple
substances than the luciferases. If we are to produce light artificially in the same way that animals do, the luciferins
must be synthesized. The luciferin of Pholas will luminesce with KMnO4 and other oxidizing agents, and,
although I have not yet succeeded in oxidizing Cypridina luciferin with oxidizing agents, I have no doubt but that
some inorganic catalyzer will be found to take the place of luciferase and accelerate oxidation of Cypridina
luciferin with light production.
The most remarkable peculiarity of Cypridina luciferin is its stability. In my first paper on Cypridina I stated that
luciferin was not destroyed by momentary boiling but would be destroyed if boiled four or five minutes; also, that
it was unstable at room temperatures and would disappear from solution in the course of a day or so. The reason

for this is that luciferin oxidizes even in absence of luciferase and will then no longer give light with luciferase.
This spontaneous oxidation, which occurs without light production, can be prevented by keeping the luciferin in a
hydrogen atmosphere or by the addition of acid. Under these conditions the luciferin can be boiled without
destruction or preserved for months without deterioration. The rapid disappearance of luciferin from neutral or
alkaline solution on boiling in the air is entirely due to the more rapid oxidation at the boiling point. As the
oxidation product, oxyluciferin, can be readily reconverted into luciferin again, we can not consider luciferin
unstable in the sense that its molecule is actually destroyed as is the case when luciferase is boiled.
Not only is luciferin stable on boiling but it will actually withstand boiling for 10 hours with 20 per cent. HCl (by
weight, sp. gr. = 1.1) or with 4 per cent. H2SO4. After one day of boiling with 20 per cent. HCl the luciferin was
completely destroyed and with 4 per cent. H2SO4 destruction was almost complete. In these cases there was no
question of a mere oxidation to oxyluciferin, as no oxyluciferin could be demonstrated after boiling with such
strong acids. An actual destruction, probably an hydrolysis of the luciferin molecule, occurred. We shall have
occasion to refer to this again in considering the protein nature of luciferin. It must be borne in mind that many
proteins require four or five days' boiling with 20 per cent. HCl for complete hydrolysis to amino-acids. Luciferin
forms a solution in water, probably colloidal, although the luciferin will dialyze through parchment or collodion
membranes. It is rather readily adsorbed by various finely divided materials such as bone black, Fe(OH)3, kaolin,
talc and CaCo3. It is not destroyed by any of the enzyme solutions which I have tried. These include such as are
widely divergent in action: pepsin HCl, trypsin, erepsin, salivary and malt diastase, yeast invertase, urease, rennin
and the enzymes of dried spleen, kidney and liver substances.
By extracting the dried Cypridinas ground to a powder, the solubility of luciferin in non-aqueous solvents could be
easily studied, and by adding such reagents as dilute acids, alkalies, neutral salts and the alkaloidal reagents to an
aqueous solution of luciferin the general biochemical behavior of luciferin can be quite accurately stated. For
convenience the results of this study are given in Table 8.
Table 8
Properties of Photogenic Substances from Cypridina

Property Luciferase Luciferin
Salting out
By saturation NaCl Not precipitated Not precipitated.
By half saturation MgSO4 Do. Do.
By saturation MgSO4 Nearly completely precipitated Partially precipitated.
By saturation MgSO4 + acetic acid ... Do.
By half saturation (NH4)2SO4 Slightly precipitated Not precipitated.
By saturation (NH4)2SO4 Completely precipitated Nearly completely precipitated.
By saturation (NH4)2SO4 + acetic acid ... Nearly completely precipitated.
Solubility in
Methyl alcohol Insoluble Soluble.
Ethyl alcohol Do. Do.
90 per cent. Do. Do.
70 per cent. Do. Do.
50 per cent. Slightly soluble Do.
Propyl alcohol Insoluble Do.
Isobutyl alcohol Do. Fairly soluble.
Amyl alcohol Do. Slightly soluble.
Benzyl alcohol Do. Soluble.
Acetone Do. Fairly soluble.
90 per cent. Do. Soluble.
70 per cent. Slightly soluble Do.
50 per cent. Fairly soluble Do.
Ethyl acetate Insoluble Do.

Ethyl propionate Do. Fairly soluble.
Ethyl butyrate Do. Do.
Ethyl valerate Do. Slightly soluble.
Ethyl nitrate Do. Very slightly soluble.
Glycerine Do. Soluble.
Glycol Do. Do.
Ether Do. Insoluble.
Chloroform Do. Do.
Carbon disulfide Do. Do.
Carbon tetrachloride Do. Do.
Benzol Do. Do.
Toluol Do. Do.
Xylol Do. Do.
Petroleum ether Do. Do.
Anilin Do. Do.
Glacial acetic acid Do. Fairly soluble.
Alkaloidal Reagents
Phosphotungstic acid Completely precipitated Very nearly completely precipitated.
Phosphotungstic and acetic acids ... Very nearly completely precipitated.
Phosphotungstic acid and HCl ... Completely precipitated.
Tannic acid Nearly completely precipitated Nearly completely precipitated.
Tannic and acetic acids ... Nearly completely precipitated.
Tannic acid and HCl ... Nearly completely precipitated.
Picric acid Nearly completely precipitated Not precipitated.
Picric and acetic acid ... Do.
Picric acid and HCl ... Do.
K4Fe(CN)6 and acetic acid ... Do.
Heavy Metal Salts
Basic lead acetate Completely precipitated Not completely precipitated.
Neutral lead acetate Nearly completely precipitated. Not completely precipitated.
Neutral lead acetate and acetic acid ... Not precipitated.
Mercuric chloride Not precipitated Not completely precipitated.
Mercuric chloride and acetic acid ... Almost completely precipitated.
Uranyl nitrate and acetic acid ... Not completely precipitated.
Acids and Alkalies
NaOH Not precipitated Not precipitated.
NH4OH Do. Do.
Acetic acid Do. Do.
H2CO3 Do. Do.
Trichloracetic acid Do. Do.

Because the luciferin is almost completely precipitated by saturation with (NH4)2SO4, we may conclude that it
occurs in water in the colloidal state. This excludes it from belonging to one of the numerous groups of
biochemical compounds occurring in true solution and places it among the known groups of colloidal substances,
the soaps, proteins, polysaccharides, phospholipins, galactolipins (cerebrosides), tannins or saponins. It is not a
polysaccharide because nearly completely precipitated by phosphotungstic acid, nor a soap because not
precipitated by calcium salts, nor a phospho- or galactolipin because insoluble in benzine, hot or cold. It gives no
tannin or saponin tests. Only the protein group remains, and of the eighteen protein classes recognized by the
American Society of Biochemists, the general properties of luciferin indicate that it should be placed among the
natural proteoses, somewhere on the borderland between the proteoses and peptones. The fact that luciferin will
dialyze, although almost completely salted out by (NH4)2SO4, is strong evidence in favor of placing it in such a
position.

On the other hand, luciferin has two properties which to say the least are unusual for proteins. I refer to its
solubility in alcohols, acetone, esters, etc., and non-digestibility by trypsin or erepsin, which have almost universal
proteolytic power.
The best known class of proteins soluble in alcohol is the prolamines of plants, but the prolamines are insoluble in
water and in absolute alcohol. Zein, the prolamine of corn, is soluble in 90 per cent. ethyl, methyl, and propyl
alcohols, in glycerol heated to 150° C., and in glacial acetic acid. Recently Osborne and Wakeman (1918) have
described a protein from milk having solubilities similar to those of gliadin, the prolamine of wheat. Welker (1912)
has described a substance, obtained from Witte's peptone, giving the biuret, Millon, and Hopkins-Cole tests, which
is soluble in water and absolute alcohol but not in ether, and it is possible that others of the peptones are soluble in
absolute alcohol. On the other hand, some proteins in the absence of salts form colloidal solutions in strong alcohol
from which they may be precipitated by an appropriate salt. As the absolute alcohol extract of Cypridinæ was
made from dry material containing the salts of sea water, some salt was present, but there is always the possibility
of sol formation.
If we extract dried Cypridinæ, which have previously been thoroughly extracted with benzine or ether, with 800
c.c. of boiling absolute alcohol for an hour, filter the alcohol extract through blotting paper and hardened filter
paper, quickly evaporate the filtrate to dryness on the water bath, and dissolve the residue in a small quantity of
water saturated with CO2,[9] we obtain a yellow opalescent solution which gives a bright light with luciferase. This
solution contains some protein or protein derivatives as it gives a very faint Millon reaction, a good positive
ninhydrin test, reddish blue in color, but no biuret reaction. It precipitates with tannic and phosphotungstic acids
but not with picric, acetic, trichloracetic, or chromic acids. The extract gives a faint Molisch reaction for
carbohydrates. As the evidence points to the presence of some protein products in the absolute alcohol extract of
Cypridinæ, it is possible that this protein is luciferin. It should be emphasized, however, that the Millon reaction
was very faint, although the ninhydrin was quite marked and the biuret negative.
[9] To make the solution slightly acid and prevent oxidation of the luciferin.

Although luciferin is not digested by trypsin, even after five days at 38° C., it does hydrolyze with mineral acids
after about 16 hours' boiling. Some proteins, the albuminoids and racemized proteins, resist tryptic digestion but
yield to acid hydrolysis. We know also that some NH-CO linkages of proteins are broken down with great
difficulty by trypsin as it is difficult to obtain a tryptic digest of protein which does not give the biuret reaction, and
the work of Fischer and Abderhalden has shown that certain artificial polypeptides are not digested by pure
activated pancreatic juice.
We have, then, three possibilities: Luciferin is (1) either a natural proteose not attacked by trypsin, or (2) if
attacked by trypsin its decomposition products (presumably amino-acids) still contain the group oxidizable with
light production, or (3) it is not protein at all. I have been unable to oxidize with light production various mixtures
of amino-acids (from tryptic digestion of beef and casein, or the acid hydrolysis products of luciferin itself) by
means of luciferase, and consequently am led to believe that Cypridina luciferin is either a new natural proteose,
soluble in absolute alcohol and not digested by trypsin or that it belongs to some other group than the proteins. The
absence of a biuret reaction would point in that direction and the question must await further study.
Cypridina luciferin is found in the luminous gland of the animal and possibly in parts non-luminous as well as in
the luminous organ. This is true of the luciferin from fireflies which is found throughout the body of Luciola,
Photuris and Photinus.
Cypridina luciferase.—Luciferase, on the other hand, has all the properties of a complex protein. It will not
dialyze through collodion or parchment membranes, is soluble only in aqueous solvents, and hence precipitated by
alcohol and acetone, digested by proteolytic enzymes, readily changed by contact with dilute acid and alkali and
irreversibly coagulated on boiling. It is completely salted out of solution by saturation with (NH4)2SO4 and nearly
completely precipitated by the alkaloidal reagents. Its other properties are given in Table 8. Taken together, they
point to the group of albumins as the class of proteins with which luciferase most closely agrees.
If luciferase is not a protein it is so closely bound up with protein that it cannot be separated. This is characteristic
of many enzymes and luciferase is also an enzyme. We can determine this by finding out whether luciferase will
accelerate the oxidation of a large amount of luciferin, for such is the test of a catalytic substance. If we take 1 c.c.
of a dilute solution of luciferase (1 Cypridina to 50 c.c. water) and add to it successive 1 c.c. portions of

concentrated luciferin (1 Cypridina to 2 c.c. solution) as soon as the light from the preceding addition has
disappeared, after four 1 c.c. additions, no more light is produced. The luciferase is therefore used up and cannot
oxidize more than a certain quantity of luciferin. In this experiment, however, we added a concentration of
luciferin from one Cypridina 100 times that of the luciferase from one Cypridina, i.e., four additions each 25 times
as concentrated. We have, of course, no way of telling what the absolute amount (in milligrams) of luciferin or
luciferase is in a single Cypridina, but we do know that the luciferase from one Cypridina cannot oxidize luciferin
from more than 100 Cypridinas. If the ratio of luciferin to luciferase in a single animal is 100:1, it would mean that
luciferase could oxidize 10,000 times its weight of luciferin. A large excess of luciferin but not an indefinite
quantity can be oxidized by luciferase, and I believe this is sufficient justification for considering luciferase an
enzyme, although it is not an ideal example of an organic catalyzer. Quite a number of enzymes are known to be
diminished during the course of the reaction they accelerate or to be poisoned by their reaction products. Enzyme
reactions inhibited by the formation of reaction products again proceed if these are removed or diluted. However,
light does not again appear in a mixture of weak luciferase with excess of luciferin upon dilution with water, so
that the luciferase cannot have been merely inhibited by some reaction product but must have been actually used
up during the reaction. It should be noted in passing that the peroxidases, ordinarily spoken of as oxidizing
enzymes, are used up in the reaction and can only oxidize limited amounts of oxidizable substances, a quantity
almost in proportion to the concentration of peroxidase present.
Whether luciferase is an oxidizing enzyme made up of an albumin associated with some heavy metal as iron,
copper or manganese is uncertain. From analyses of whole Cypridina, kindly made for me by Prof. A. H. Phillips
of Princeton University, all three of these metals, which we know to be associated with biological oxidations, are
present, and it is quite possible that one of them is concerned with the oxidation of luciferin.
Although I have tested a great many oxidizers, organic and inorganic, and a large number of oxidizing enzymes
from blood and tissue extracts of animals rich in iron, copper and manganese, I have found no material which is
capable of taking the place of Cypridina luciferase. Peroxidases or oxidases of plants, hæmoglobin, hæmocyanin,
extracts of mussels, manganese containing blood of various marine crustacea and mollusks will give no light on
mixing with luciferin. Such active oxidizers as KMnO4, H2O2, BaO2, and many others, will not oxidize Cypridina
luciferin with light production, although they can oxidize Pholas luciferin with light production.
The action of Cypridina luciferase is very highly specific. It is found only in the luminous organ of Cypridina
hilgendorfii, not in non-luminous parts and not in a non-luminous species of Cypridina closely related to
hilgendorfii.
Luciferins and luciferases from closely allied luminous forms will mutually interact to produce light, but no light
appears if these substances come from distantly related forms. Thus firefly (Photuris) luciferin will give light with
Pyrophorus luciferase and vice versa, but Cypridina luciferin will give no light with firefly (Luciola) luciferase or
vice versa, nor with Pholas luciferase or vice versa. The faint luminescences sometimes observed on mixing firefly
or Cypridina luciferase with boiled extracts of non-luminous forms, or of distantly related luminous forms, are
probably caused by photophelein in the boiled extract.
Like the plant peroxidases, Cypridina luciferase is not readily affected by the action of chloroform, toluol, etc.
Unlike the plant peroxidases, it will not oxidize (i.e., produce coloration) in either presence or absence of H2O2,
any of the hydroxyphenol or aminophenol compounds, such as pyrogallol, a-naphthol, para-diamino-benzine, gum
guaiac, etc., commonly used as peroxidase reagents. Neither will luciferase produce light with any substances,
such as oils, lophin, pyrogallol, gallic acid, esculin, etc., which we know to be capable of oxidation with light
production by other means. The luciferases are very highly specific and act only upon the luciferins of the same or
closely related species. They must be placed by themselves in a new class of oxidizing enzymes.
According to Dubois, Pholas luciferase is rather readily destroyed by chloroform and my own observations
indicate that this is true also of firefly luciferase, so that a certain amount of variation exists in the group of
luciferases.
None of the luminescent animals which I have studied are at all affected by cyanides. The luminescence continues
in extracts of Cypridina, firefly, and Cavernularia, or in Noctiluca and luminous bacteria after addition of small or
high (m/40) concentrations of KCN. In this respect the luciferases are very different from many types of oxidizing
enzymes which are inhibited by exceedingly weak concentrations of cyanide. It should be borne in mind, however,

that while KCN inhibits catalase and the catalytic decomposition of H2O2 by Pt or Ag, it does not affect the
catalytic decomposition of H2O2 by thallium.

Oxyluciferin.—When luciferin is oxidized it must be converted into some substance or substances and I believe
this change involves no fundamental destruction of the luciferin molecule as it is a reversible process. I shall speak
of the principal (if not the only) product formed as oxyluciferin.
If we assume that the oxidation of luciferin changes the molecule but slightly, we at once think of comparing the
change luciferin ⇆ oxyluciferin with the change reduced hæmoglobin ⇆ oxyhæmoglobin. The condition is,
however, not so simple as this, for oxyhæmoglobin will again give up its oxygen providing the partial pressure of
oxygen is made sufficiently low, whereas oxyluciferin will not do this, at least in the dark. We can not reduce
oxyluciferin solution by exhausting the oxygen with an air-pump.
There is another oxidation-reduction system which can also be easily reversed, but not by merely removing the
oxygen from the solution—that is, the reduction of a dye such as methylene blue to its leuco-base. I believe the
change which occurs when luciferin is oxidized is similar to that which occurs when the leuco-base of methylene
blue or sodium indigo-sulphonate is oxidized to the blue dye. Oxidation of leuco-dye bases occurs spontaneously
in presence of oxygen and appears to consist in the removal of hydrogen from the leuco-base with formation of
water. Reduction of these dyes may be effected in the same ways that oxyluciferin can be reduced. In the case of
methylene blue, reduction consists in the addition of two hydrogen atoms. Whether a similar change occurs when
oxyluciferin is reduced or whether oxygen is actually added as in formation of hæmoglobin cannot be definitely
stated at present. We may write equations representing these possibilities as follows:
C16H20N3SCl (leuco-methylene blue) + O ⇆ C16H18N3SCl (methylene blue) + H2O

Hæmoglobin + O ⇆ oxyhæmoglobin.
Let us now turn to the methods which may be used in reduction of oxyluciferin. We may then endeavor to write an
equation which will represent the fundamental changes in the luminescence reaction.
My attempts to reduce the oxidation product of luciferin started from the observation that if one places a clear
solution of luciferase in a tall test tube, although it may give off no light at first when shaken, after standing a day
or so a very bright light would appear on shaking. This was especially true when the luciferase had become turbid
and ill-smelling from the growth of bacteria. Thinking that the bacteria produced a substance which could be
oxidized by the luciferase, I tried growing bacteria and also yeast on appropriate culture media, and after some
days of growth mixing the culture media containing the products of bacterial or yeast growth with luciferase,
expecting to obtain light; but no light appeared. However, if a little crude luciferase solution was added to the
bacterial or yeast cultures and then allowed to stand for some hours, light appeared whenever they were shaken.
Indeed such cultures behaved much as a suspension of luminous bacteria which has used up all the oxygen in the
culture fluid and will only luminesce when, by shaking, more oxygen dissolves in the culture medium. Realizing
that in bacterial cultures in test tubes, anaërobic conditions soon appear, and also the strong reducing action of
bacteria upon many substances (for instance, nitrates or methylene blue) under anaërobic conditions, it struck me
that the bacteria might be reducing the oxidation product of luciferin to luciferin again. We must remember that
since crude luciferase solution is a cold-water extract of a luminous animal allowed to stand until all the luciferin
has been oxidized, it must contain oxyluciferin as well as luciferase and will give light if the oxyluciferin is again
reduced and oxygen admitted. This appears to be the correct explanation of the above experiments.
Oxyluciferin may also be readily reduced by the use of the blood of the horse-shoe crab (Limulus) allowed to stand
until bacteria develop. This experiment is of special interest because the blood contains hæmocyanin, which is
colorless in the reduced condition and blue in the oxy-condition. The color change thus serves as an indicator of
the oxygen concentration in the blood. A sample of foul-smelling Limulus blood full of bacteria will become
colorless on standing in a test tube for 10 to 15 minutes, but the blue color quickly returns if shaken with air. Such
a blood has the power of reducing oxyluciferin through the activity of the bacteria which it contains. Fresh blood
has very little if any reducing action.
Not only bacteria but also tissue extracts have a strong reducing action in absence of oxygen. Thus, muscle tissue
stained in methylene blue will very quickly decolorize (reduce) the methylene blue if oxygen (air) is kept away,
but the blue color immediately returns if air is admitted. Oxyluciferin (i.e., a solution of luciferin which has been

completely oxidized by boiling or standing in air until it no longer gives light with luciferase) if mixed with a
suspension of ground frog's muscle and kept in a well-filled and stoppered test tube for some hours, is reduced to
luciferin and gives a bright light if now poured into luciferase solution. Frog muscle suspension alone, or
oxyluciferin alone, give no light with luciferase, nor will a mixture of frog muscle suspension and oxyluciferin, if
shaken with air for several hours. Only if this last mixture be kept under anaërobic conditions is the oxyluciferin
reduced.
The reducing action of tissues is said to be due to a reducing enzyme (reducase or reductase), itself composed of a
perhydridase and some easily oxidized body such as an aldehyde. In the presence of the perhydridase the oxygen
of water oxidizes the aldehyde and the hydrogen set free reduces any easily reducible substance which may be
present. There is a perhydridase in fresh milk, spoken of as Schardinger's enzyme, which is destroyed by boiling. If
some aldehyde is added, fresh milk will reduce methylene blue to its leuco-base or nitrates to nitrites, upon
standing a short time. If shaken with air the blue color returns. There is no reduction unless an aldehyde is added or
unless some boiled extract of a tissue such as liver is added. The boiled-liver extract has no reducing action of its
own, but supplies a substance similar to the aldehyde which has been spoken of as co-enzyme. The aldehyde is
oxidized to its corresponding acid. Milk will reduce methylene blue without aldehyde if bacteria are present in
large numbers. There is no reduction if the milk, methylene blue, and aldehyde are agitated with air. The
temperature optimum is rather high, 60° to 70° C.
I find that milk is a favorable and convenient medium for the reduction of oxyluciferin and that it acts without the
addition of an aldehyde or the presence of bacteria. There is probably a substance acting as the aldehyde in the
luciferase-oxyluciferin solution. No light appears if milk is added to a luciferase-oxyluciferin solution, but if the
mixture is allowed to stand in absence of oxygen light will appear when air is admitted. The air can be
conveniently kept out by filling small test tubes completely with the solution and closing them with rubber
stoppers.
As almost all animal tissues contain reductases it is not surprising to find that a freshly prepared and filtered
extract of Cypridina containing oxyluciferin and luciferase, which gives no light on shaking, will, on standing in a
stoppered tube for 24 hours at room temperature in the dark give light when air is admitted. While this may be due
to the development of bacteria with a reducing action, it does not seem likely, as under the same conditions
methylene blue is not reduced in 24 hours, and there is no turbidity or smell of decomposition in the tube. In 48
hours bacteria appear and methylene blue is also reduced. If we add chloroform, toluol or thymol to the tubes of
Cypridina extract to prevent the growth of bacteria, and allow them to stand 48 hours, upon admitting air the tube
with chloroform gives no light but the tubes with toluol and thymol do give light, although it is not so bright as if
they were absent. I believe that these substances have a destructive action on the reductases, most complete in the
case of chloroform. Dubois (1919c) also has recorded the occurrence of a reducing enzyme in Pholas, a
"hydrogenase," which is able to form hydrogen from cane sugar, and luciferin from a boiled extract of Pholas. He
now regards it as identical with his co-luciferase.
I have not been able to demonstrate that a Cypridina extract will reduce methylene blue, or nitrates to nitrites,
either with or without the addition of acetaldehyde. This may be due to the fact that oxyluciferin, which is also
present, may be reduced more readily than either nitrates or methylene blue, and so is reduced first.
We can also reduce oxyluciferin by means which do not involve the use of animal extracts. Perhaps the best of
these is reduction by palladium black and sodium hypophosphite. The latter is oxidized in presence of palladium
and nascent hydrogen is set free. The nascent hydrogen reduces any easily reducible substance which may be
present, such as methylene blue or oxyluciferin. Oxyluciferin is not reduced by palladium alone or hypophosphite
alone, but methylene blue is reduced by palladium black alone.
If hydrogen sulphide is passed through a solution of methylene blue the dye is very quickly reduced and becomes
colorless. If the H2S is driven off by boiling the colorless methylene-blue solution, the blue color again returns on
cooling. Oxyluciferin can also be reduced by H2S.

If one adds some Mg powder to oxyluciferin and then dilute acetic acid in successive additions as the acetic acid is
used up in formation of Mg acetate, the oxyluciferin will be reduced relatively quickly. Nascent hydrogen is
produced in the reaction and is no doubt the active reducing agent.

Dilute acid favors the reduction of oxyluciferin. If one saturates an oxyluciferin solution with CO2 or adds a little
dilute acetic acid, HCl, HNO3 or H2SO4, to it, a certain amount of reduction will occur. No reduction occurs if the
solution is saturated with pure hydrogen, even if allowed to stand 24 hours. The action of the acid begins when the
solution of oxyluciferin, ordinarily slightly alkaline (Ph = 9), is made neutral (Ph = 7.1) as indicated in Table 9.
The action of the acid must be on the oxyluciferin, as no luciferin or other enzymes destroyed on boiling are
present.
Table 9
Effect of Acid on Reduction of Oxyluciferin

Ph Luminescence with
Solution Remarks
luciferase
20 c.c. Oxyluciferin alone 9.01 Negative
20 c.c. Oxyluciferin + .05
8.8 Negative
c.c. 5 per cent. acetic acid
20 c.c. Oxyluciferin + .15
7.1 Fair
c.c. 5 per cent. acetic acid
20 c.c. Oxyluciferin + .30 Acid forms precipitate in
5.9 Good
c.c. 5 per cent. acetic acid this oxyluciferin sol.
20 c.c. Oxyluciferin + .50 Acid forms precipitate in
Good
c.c. 5 per cent. acetic acid this oxyluciferin sol.
20 c.c. Oxyluciferin + .75 Acid forms precipitate in
Good[10]
c.c. 5 per cent. acetic acid this oxyluciferin sol.
[10] Light disappears quickly because of the effect of the acidity on the luciferase.

It is possible that the action of bacteria (which produces CO2), muscle tissue (which
contains lactic acid), milk (in which lactic acid may be formed by bacteria), or Mg +
acid, in forming luciferin, is not the result of their reducing power but of their acidity.
Fortunately we can test this matter by the use of reducing fluids which are not acid. If
they also form luciferin from oxyluciferin, a reduction must occur. Nascent H can be
generated by the action of NaOH on Al, or when finely divided Mg or Zn or Al is
placed in water. With Mg the water becomes only slightly alkaline from formation of
almost insoluble Mg(OH)2. If we add some Al powder and dilute NaOH to an
oxyluciferin solution, H is given off and luciferin is formed. As oxyluciferin cannot be
formed by the addition of alkali alone we must have in this experiment a reduction of
oxyluciferin in alkaline medium by the nascent H produced. Luciferin can also be
formed by merely adding Al or Zn or Mg dust to an oxyluciferin solution. Methylene
blue can also be readily reduced to its leuco-base by Zn dust or Al + NaOH.
Indeed, if one adds some Al or Zn or Mg powder to a solution of luciferase, light will
appear whenever the solution is shaken. Luciferase solution must always contain the
oxidation product of luciferin, oxyluciferin. In presence of nascent H this is reduced to
luciferin, and since the reaction of the medium is alkaline and luciferase is present this
is oxidized with light production, when, by shaking, air is dissolved. The light can
never become very bright except at the surface because of the deficiency of oxygen in
the solution. It would seem, then, that the action of bacteria, yeast, muscle cells, etc.,
on oxyluciferin must be due not entirely to their acid reaction but to their reducing
power as well.
The above experiment is a very striking and instructive one. Given a test tube of
luciferase solution containing, as it does, oxyluciferin, add some Zn dust or Mg

powder, and the evolution of hydrogen begins. Conditions are now favorable for the
reduction of oxyluciferin and this occurs. Shake the contents of the tube to dissolve
oxygen and light appears. Allow the tube to stand and the light soon disappears. Shake
again and the light reappears. The luminescence reduction and oxidation process can
be demonstrated many times.
A similar experiment can be performed with luciferase and oxyluciferin solution by
addition of NH4SH. This will serve also as another example of the reduction of
oxyluciferin in an alkaline medium. Whenever we shake a tube of luciferase,
oxyluciferin and NH4SH, light will appear. When the tube is at rest it becomes dark.
Even the merest touch is sufficient to agitate the tube contents, cause solution of
oxygen and appearance of light. It is just as if we stimulate the tube to produce light
and I believe the phenomenon has a deeper significance and a more fundamental
similarity to the phenomena of stimulation than may at first appear. What more simple
means of controlling a process can we think of than by admission or withdrawal of
oxygen? The firefly turns on its light by stimulation through nerves of the luminous
organ. Noctiluca flashes on stimulation of any kind, even the slightest agitation
causing a brilliant emission of light. If the stimulation process means merely the
admission of oxygen to the photogenic cells we have a mechanism in the cell itself for
automatically producing the light. The admission of oxygen results in aërobic
conditions and luciferin in presence of luciferase can then oxidize to oxyluciferin with
luminescence. When the oxygen is used up, the light ceases, anaërobic conditions
prevail, and the oxyluciferin is reduced to luciferin again. Thus, luciferin is reformed
during the rest period of Noctiluca or between the flashes of the firefly. What more
efficient type of light than this is to be desired?
Again, methylene blue offers an interesting parallel to oxyluciferin. A little NH4SH
added to methylene blue solution will reduce (decolorize) it to the leuco-base. If the
tube is now shaken the blue color returns. On standing reduction again occurs. The
process can be repeated a number of times, the reaction going in one or the other
direction, depending on the oxygen content of the mixture.
As methylene blue contains no oxygen, its reduction consists in the addition of two
atoms of hydrogen. When leuco-methylene blue oxidizes, water is formed by the
union of these two atoms of hydrogen with oxygen, thus:
C16H20N3SCl + O ⇆ C16H18N3SCl + H2O
(leuco-methylene blue) (methylene blue)

Briefly—MH2 + O ⇆ M + H2O

To reduce methylene blue we can add the two hydrogen atoms directly from nascent
hydrogen formed in the solution or we can split up water by a catalyzer in the

presence of some substance, which will take up the oxygen of water, thus:
NaH2PO2 + H2O + Pd = NaH2PO3 + H2 + Pd
(Sodium hypophosphite) (Sodium phosphite)
This reaction occurs in presence of finely divided palladium. Methylene blue can be
reduced by the H2 and the hypophosphite oxidized.

Since oxyluciferin can be reduced by palladium and sodium hypophosphite (Harvey,
1918), it is probable that we can write the equation for reduction of oxyluciferin and
oxidation of luciferin in a similar manner to that of methylene blue:
Luciferin + O ⇆ Oxyluciferin + H2O

Briefly—LH2 + O ⇆ L + H2O.

Just as in the case of methylene blue the reaction proceeds in the right hand direction
spontaneously if the pressure of O is sufficiently high. If luciferase is also present we
have luminescence.
LH2 + O + luciferase ⇆ L + H2O + luciferase (luminescence)

The reaction proceeds in the left hand direction under low oxygen pressure, in the
presence of nascent hydrogen or with some catalyzer which is able to split water,
transferring the H2 to oxyluciferin and the O to an acceptor (A). NaH2PO2 plays the
part of the acceptor.
L + H2O + A + Pd = LH2 + AO + Pd.

This appears to be the way in which the reducing enzymes or perhydridases
(comparable to the Pd) of living tissues act (Bach, 1911-13) and the action of yeast
cells, bacteria, muscle suspensions, etc., in reducing oxyluciferin must occur in the
same manner.
If we assume that the LH2 (luciferin) compound is dissociated to even the slightest
extent into L and hydrogen, the hydrogen ion will shift the equilibrium toward the
formation of that substance which involves the taking up of hydrogen. Consequently
we may obtain a partial formation of luciferin by adding an acid to oxyluciferin.
Reduction of the H-ion concentration tends to shift the equilibrium in the opposite
direction. Consequently, addition of alkali favors the oxidation of luciferin, and it is
quite generally true that biological oxidations are favored by an alkaline reaction. In
addition oxygen in alkaline medium has a higher oxidation potential than in neutral or
acid media. I believe this is the explanation of the action of acid in formation of
luciferin from oxyluciferin.

Addition of acid is not the only means of favoring the formation of luciferin from
oxyluciferin. Any reaction which proceeds in one direction with evolution of light
should, theoretically, proceed in the opposite direction under the influence of light. So
far as I know the case of a reaction, photogenic in one direction and photochemical in
the other direction, has never been described, unless we are to accept the cases of
phosphorescence, for instance, the absorption of light by CaS and its emission in the
dark. However, the reaction which occurs during phosphorescence cannot be stated.
It is a fact that light will cause the reduction of oxyluciferin. A tube of oxyluciferin
exposed to sunlight for six hours, or the mercury arc for two hours, will be partially
converted into luciferin. It will luminesce when luciferase is added, while a control
tube kept in darkness shows no trace of luciferin. The action is more marked with the
ultra-violet as a solution of oxyluciferin in a quartz tube showed more reduction than
one in a glass tube when exposed for the same length of time to the quartz mercury
arc. The reduction is not dependent on the formation of acid under the influence of
light since two tubes of oxyluciferin, one kept in darkness and the other exposed to
sunlight for six hours, had the same reaction, Ph = 9.3. Of course some reducing
substance might be formed under the influence of light but this is not very probable.
We may therefore write the reaction for luminescence in the following way:

darkness
alkali
luciferase
luciferin (LH2) + O ⇆ oxyluciferin (L) + H2O (luminescence)
perhydridase
acid
light

Acid and light favor reduction while alkali and darkness favor oxidation in the
luciferin ⇆ oxyluciferin reaction. Whether the luciferin be really oxidized by removal
of H2 or whether by addition of oxygen is, of course, uncertain, but the analogy with
methylene blue is striking and may serve as a working hypothesis until the
composition of luciferin and its oxidation product are known.
While I have not studied the properties of oxyluciferin as fully as those of luciferin, so
far as I can judge, both substances give the same general reactions and possess
identical properties. Both crude luciferin and crude oxyluciferin solution are yellow in
color, but I do not believe that either pure luciferin or oxyluciferin are yellow in color,
because an ether or benzine extract of Cypridina is also yellow, although luciferase,
luciferin, and oxyluciferin are insoluble in ether and benzine. The yellow pigment

which can be observed to make up part of the luminous gland of Cypridina is not
luciferin or luciferase. It may be a pigment related to urochrome.
When tests are applied and precipitating reagents are added to crude luciferin and
crude oxyluciferin solution, they give identical results in each case. A more complete
account of the chemistry of luciferin has been given in this chapter, and there is no
need of duplicating these statements regarding oxyluciferin. Like luciferin, the
oxyluciferin will pass porcelain filters, dialyze through parchment or collodion
membranes, and is undigested by salivary diastase, pepsin HCl, Merck's pancreatin in
neutral solution, and erepsin. The salivary diastase and the pancreatin (containing
amylopsin, trypsin, and lipase) were allowed to digest for four days at 38° C. without
showing any evidence of digestive action.
As luciferin is so easily oxidizable a substance, we should expect to find that it will
reduce just as glucose will reduce. However, a concentrated solution of luciferin has
no reducing action on Fehling's (alkaline Cu), Barfoed's (acid Cu), Nylander's
(alkaline Bi) or Knapp's (alkaline Hg) reagent. Glucose will reduce methylene blue in
alkaline (not in neutral solution), but luciferin will not reduce methylene blue in
alkaline or neutral solution. It would seem, then, that luciferin must contain no
aldehyde group. If so, we should expect to obtain reduction of some of the above
reagents. Just what group is concerned in the oxidation is unknown at the present
time, and in the absence of more experimental data, speculation regarding it can be of
little value.

SUMMARY

In summing up we may say that the luminescence of at least three groups of luminous
animals, the beetles, Pholas, and Cypridina, has been definitely shown to be due to
the interaction of two substances, luciferin and luciferase, in presence of water and
oxygen. Luciferin and luciferase have quite different properties and may be easily
separated from each other by various chemical procedures. As the luciferins and
luciferases from different luminous animals have somewhat different properties, they
may be designated by prefixing the generic name of the animal from which they are
obtained.
Cypridina luciferin differs from Pholas luciferin in that it can not be oxidized with
light production by KMnO4, H2O2, with or without hæmoglobin, or similar oxidizing
agents. Cypridina luciferase differs from Pholas and firefly luciferase in that it is not
readily destroyed by the fat-solvent anæsthetics, such as chloroform, ether, etc.
When Cypridina luciferin is oxidized, no fundamental splitting of the molecule
occurs, because the product, oxyluciferin, can be readily reduced to luciferin again.
This reduction is brought about under conditions similar to those necessary for the

reduction of dyes, such as methylene blue. Oxyluciferin can be reduced to luciferin,
which will again give light with luciferase, by the reductases of muscle tissue, liver,
etc., or by bacteria; by Schardinger's enzyme of milk; by H2S; by the nascent
hydrogen from the action of acetic acid on magnesium or of water or NaOH on
aluminium, zinc or magnesium; and by palladium black and sodium hypophosphite,
all well-known reducing methods. Reduction of oxyluciferin no doubt occurs even in
presence of luciferase if oxygen is absent, and reduction of oxyluciferin no doubt
occurs in animals which burn luciferin within the cell, thus tending for conservation
of material. Dilute alkali favors oxidation and dilute acid favors the reduction. Light
favors the reduction of oxyluciferin.
Apparently luciferin and oxyluciferin have identical chemical properties. Neither is
digested by the enzymes: malt diastase, ptyalin, yeast invertase, pepsin, trypsin,
steapsin, amylopsin, rennin, erepsin, urease or enzymes occurring in the water extracts
of dried spleen, kidney, or liver. Luciferase is destroyed only by pepsin (probably),
trypsin, erepsin, and something in spleen and liver extract.
Luciferase is unquestionably a protein and all its properties agree with those of the
albumins. Although used up in oxidizing large quantities of luciferin, it behaves in
many ways like an enzyme and may be so regarded.
Luciferin, on the other hand, is not digested by proteolytic enzymes, is dialyzable,
almost but not completely precipitated by saturation with (NH4)2SO4, and is soluble
in absolute alcohol, acetone, and some other organic solvents, but not in the strictly
fat-solvents like ether, chloroform, and benzol. There are, however, certain CO-NH
linkages which are not attacked by proteolytic enzymes and some peptones soluble in
absolute alcohol, so that these two characteristics do not bar it from the group of
proteins. Luciferin, in fact, has many properties in common with the proteoses and
peptones but its chemical nature cannot be definitely stated at present.

CHAPTER VII
DYNAMICS OF LUMINESCENCE
One of the most extraordinary things regarding luminescence in general is the small
amount of material necessary to cause a visible emission of light. To take an extreme
case, the flash of light resulting from the impact on ZnS of a single α particle, a
helium atom, is visible to the naked eye. Addition of one part in a million of some
heavy metal to pure CaS will confer phosphorescent properties on the latter. We are
forced to believe that the heavy metal enters into some reaction during illumination
which is reversed with light emission after illumination and a very small amount of
heavy metal is necessary. Pyrogallol in water, 1:5,000,000 (m/512,000), can be
oxidized with light production by K4Fe(CN)6 and H2O2 (Harvey, 1917) and m/100
pyrogallol + H2O2 will give a visible light with colloidal platinum in 1:250,000
concentration (Goss, 1917).
Luciferin and luciferase from Cypridina will also luminesce in exceedingly small
concentration. If one grinds a single Cypridina in a mortar with water and dilutes the
extract to 25,600 c.c., light can be observed if luciferin is added to this dilute
luciferase solution. By determining the volume of the luminous gland of Cypridina
and even assuming that this volume is all luciferase, one can calculate that one part of
luciferase in 1,700,000,000 parts of water will give light when luciferin is added.
Likewise, a similar dilution of luciferin will give visible light when luciferase is
added.
The sensitivity of our eye is largely responsible for the detection of so small an energy
change. As we have seen, recent determinations have proved that the dark adapted eye
can detect 18 × 10-10 ergs per second. From the heat of complete oxidation of
pyrogallol it is possible to calculate the amount of pyrogallol necessary to give 18 ×
10-10 ergs if completely oxidized. This quantity is infinitesimally small. When
pyrogallol is oxidized by K4Fe(CN)6 and H2O2, it is not completely oxidized and
probably only a small amount of the energy is converted into light; otherwise we
should be able to see the luminescence of a very much weaker concentration of
pyrogallol. As the reaction luciferin ⇆ oxyluciferin is so easily reversible, very little
energy must be liberated, and, as experiments indicate, very little heat, if any,
accompanies light production. Even though this be true, it is still possible for a very
small amount of luciferin to produce a very large amount of light.
A very small amount of luciferase only is necessary because it behaves as an enzyme
and follows the general rule that catalysts act in minute concentrations.

On the assumption that luciferase is an enzyme, an organic catalyst oxidizing luciferin
with light production, we may appropriately inquire into the relation between the
concentration of luciferin and luciferase and intensity and duration of luminescence.
Oxygen tension, hydrogen ion concentration and temperature must be maintained
constant as these all affect both intensity and duration of luminescence. Before
considering luciferin and luciferase, however, let us study a few well-known
chemiluminescent oxidations with special reference to concentration of reacting
substances and temperature.
The effect of temperature on luminescence is of special interest because it gives us a
means of analysis for determining if the luminescence depends on reaction velocity.
We know that photochemical reactions are very little affected by temperature because
the reaction is dependent on the absorption of light, a physical process, and this
increases only a small per cent. for a rise of temperature of 10° C. To put it in the
usual way, its temperature coefficient (Q10) for a 10° interval is usually less than 1.1.
On the other hand, we should expect photogenic reactions, in which some of the
chemical energy is converted into radiant energy, to give off much more light the
greater the reaction velocity. As reaction velocity increases so rapidly with
temperature (Q10 = 2 to 3), luminescence intensity should rapidly increase with
increase in temperature.
Trautz (1905), from his extensive study of the chemiluminescence of phenol and
aldehyde compounds came to the conclusion that luminescence intensity was
proportional to reaction velocity. He based his conclusions largely on the effects of
temperature and concentration of reacting substances and went so far as to declare that
any reaction would produce luminescence if the reaction velocity were sufficiently
increased. It is quite true that increasing the temperature does increase the intensity of
chemiluminescence, but this is only within certain limits. As we raise the temperature,
chemiluminescence becomes more intense but we soon reach a temperature for
maximum luminescence and above this the intensity diminishes. This is especially
well seen in the action of various oxidizers on pyrogallol and H2O2 recorded in Table
10. At 100° C. practically no light is produced by many oxidizers which are
themselves unaffected at 100°. If we are to connect reaction velocity with intensity of
luminescence we must conclude that the evolution of light is dependent rather on an
optimum than a maximum reaction velocity.
TABLE 10
Temperature and Light Production. The Oxidizer is Mixed with an Equal Amount of
M/100 Pyrogallol + 3 per cent. H2O2

Temperatures
Oxidizer
0-2° 20° 50° 75° 98-100°

Turnip juice Faint Good Good Bright Negative.
1 per cent. blood extract Faint Fair Good Fair.
m/20 K4Fe(CN)6 Negative Good Bright Good.
m/100 KMnO4 Fair Good Bright Bright Faint flash.
m/50 K2Cr2O7 Negative Fair Faint Fair Negative.
m/100 CrO3 Negative Good Bright Bright Faint.
m/10 KCr alum Negative Faint Faint Faint Negative.
m/10 NH4Fe alum Negative Faint Faint Faint Very faint.
MnO2 Negative Fair Fair Fair Negative.
NaClO Bright flash Bright flash Bright flash Fair flash.

Quite a number of instances are known in which increasing the mass of reacting
substances leads not to an increase but to an actual cessation of luminescence. This
fact does not confirm the theory that reaction velocity is a determining factor in
luminescence. The conditions for the luminescence of white phosphorus are most
interesting and unusual. (See van't Hoff, 1895; Ewan, 1895; Centnerszwer, 1895;
Russell,1903; Scharff, 1908.) Phosphorus will only begin to luminesce at a certain
small pressure of oxygen. This "minimum luminescence pressure" of oxygen is very
low, so low that earlier observers, failing to remove traces of oxygen, thought that
luminescence might occur in absence of oxygen. Curiously enough there is also a
"maximum luminescence pressure" of oxygen above which no luminescence occurs.
Phosphorus will not luminesce in pure oxygen. Between the minimum and maximum
is an "optimum luminescence pressure" where luminescence of the phosphorus is
brightest. The exact values of these pressures vary with degree of water vapor present
and with temperature. According to Abegg's Handbuch der anorganischen Chemie,
the maximum luminescence pressure with water vapor present, is 320 mm. Hg at 0°
and increases 13.19 mm. Hg for each degree rise in temperature. This means that for a
definite temperature, say, 20°, phosphorus will not luminesce with an oxygen pressure
of 583 mm. Hg, but will luminesce with pressures under this. If, however, we raise the
temperature, luminescence will occur with an oxygen pressure of 583 mm. Hg.
A somewhat analogous case is presented by the oxidation of pyrogallol solution in
contact with ozone, except that in this reaction too high a concentration of pyrogallol
will hinder the oxidation. I have not studied the effect of varying concentrations of
ozone. If oxygen, passed through an ozonizer (the silent electric discharge tube), is
bubbled through m/100 pyrogallol, no luminescence occurs at 0°, a fair luminescence
at 20°, a good luminescence at 50°, and a bright luminescence at the boiling point. If
the pyrogallol is of m concentration, no luminescence occurs at 0° or 20°, a fair
luminescence at 50°, and a bright luminescence at the boiling point. For a definite
temperature, say 20°, no light appears if the pyrogallol is of m concentration, but if we

raise the temperature, luminescence can occur. The similarity to phosphorus is
obvious. Thus the "maximum luminescence pressure" of pyrogallol increases with
increase of temperature.
We have already seen that pyrogallol can also be oxidized, if H2O2 is present, by a
great variety of substances, such as peroxidases of potato or turnip juice, hæmoglobin,
KMnO4, K4Fe(CN)6, CrO3, MnO2, hypochlorites and hypobromites, or colloidal Pt
and Ag. For convenience we may collectively speak of these as oxidizers. They are
recorded in Table 13. No light occurs if H2O2is absent. In the case of some of these
oxidizers pyrogallol will luminesce in dilute concentrations but not in strong. Also,
dilute pyrogallol will luminesce with a dilute solution of oxidizer but not with a
concentrated solution of oxidizer. The effect of rise in temperature in these cases also
is to increase the "maximum luminescence concentration" of pyrogallol and the
"maximum luminescence concentration" of oxidizer. Table 11 shows this effect of
temperature with K4Fe(CN)6 and varying concentrations of pyrogallol, and Table 12
shows the effect of temperature with pyrogallol and varying concentrations of
K4Fe(CN)6. Table 10 shows the relation between temperature and intensity of
luminescence with pyrogallol and various oxidizers. The terms faint, fair, good, and
bright are purely relative designations of brightness as estimated by the eye, for
accurate measurements of weak intensities are very difficult to make.
From Table 10 it should be noted that the intensity of luminescence of pyrogallol
oxidized with most oxidizers is actually less at the boiling point, a fact which I have
repeatedly verified. Let us now see how these facts are to be explained. If we assume
that luminescence is dependent on reaction velocity, the intensity of luminescence
should increase with increasing temperature. Up to a certain limit this is what we find,
but at temperatures above this limit the intensity of luminescence actually decreases.
The duration of luminescence also decreases. There is an optimum temperature for
luminescence in many cases and we can only conclude that luminescence depends not
on a very rapid reaction velocity but on a certain definite reaction velocity. Assuming
that this is true, how can we account for the anomalous fact that in high
concentrations of oxygen, phosphorus will not luminesce or that in high
concentrations of pyrogallol, there is no luminescence in presence of ozone or of
oxidizer and H2O2. Of course with high active mass of oxygen (in case of
phosphorous luminescence) or of pyrogallol (in case of pyrogallol luminescence) the
reaction velocity must be greater than the optimum. If that is the case, then lowering
the temperature should reduce the reaction velocity to the optimum and light should
appear. However, as we have seen, not lowering but raising the temperature causes
luminescence with high oxygen concentration or high pyrogallol concentration.
TABLE 11
Temperature, Concentration of Pyrogallol, and Light Production. An Equal Amount of

m/20 K4Fe(CN)6 is Mixed with Pyrogallol + 3 per Cent H2O2

Concentration
Temperatures
of
pyrogallol
(after mixing) 0-2° 10° 20° 30° 50° 75° 98-100°
Very
m/4 Negative NegativeGood Faint Fair Faint
faint
m/40 Negative Faint Faint Faint Good Bright Good
Bright
m/400 Faint Fair Good Good Good Bright
flash
Bright Fair
m/4,000 Bright Bright Bright Bright Negative
flash flash

TABLE 12
Temperature, Concentration of Ferrocyanide and Light Production. An Equal Amount
of K4Fe(CN)6 is Mixed with m/100 Pyrogallol + 3 Per Cent H2O2

Concentration of
Temperatures
K4Fe(CN)6
exposed to light (after
0-2° 10° 20° 30° 50° 75° 98-100°
mixing)
Faint
Half saturated at 20° C Negative Faint Fair Fair Good Good
flash
Very Very Good
One-sixth saturated at 20° C Fair Good Good Bright
faint bright flash

Table 13
Substances Giving Light with Pyrogallol and Hydrogen Peroxide

Equal volume added to mixture Light Light Blueing Blueing Liberation
of 1 part M/100 pyrogallol or 1 with with of gum of gum of oxygen
part 3 per cent H2O2 + 1 part pyrogallol pyrogallol guaiac guaiac from
M/100 pyrogallol; hence, + H2O2 + H2O2 H2O2

concentrations final mixture are
one-half that given
Potassium (K4Fe(CN)6
1 - Bright + +
ferrocyanide M/10-M/20)
Potassium (K3Fe(CN)6 Very faint
2 - - - Very slow
ferricyanide M/10-M/1,250) to -
Potassium (K2CrO4 M/20-
3 - Good + +
chromate M/100)
Potassium (K2Cr2O7 M/50-
4 - Good + +
bichromate M/100)
Potassium (KMnO4 M/50-
5 - Bright + - +
permanganate M/200)
Potassium (KOH M-
6 - - - - Very slow
hydroxide M/6,250)
Potassium (KClO3 M/10)
7 - - - - -
chlorate
Potassium (K2S2O8 M/10-
8 - - - - -
persulfate M/128)
Potassium (Cr2(SO4)3.K2SO4 Very Very
9 chromium - Faint -
M/10) slow slow
alum
Ferric (Fe2(SO4)3.
10 ammonium - Faint + Very slow
(NH4)2SO4 M/10)
alum
Ferric (FeCl3 M/10-
11 - Fair + Slow
chloride M/250)
Ferrous (FeSO4 M/10-
12 - Fair - + Slow
sulfate M/6,250)
Copper (CuSO4 M/5-
13 - - - + Very slow
sulfate M/125)
14 Chromic acid (CrO3 M/100) - Bright + +
Chromic (Cr2(SO4)3 2 per
15 - Faint - + Slow
sulfate cent)
16 Chlorine water - - + +
17 Bromine water - - + +
18 Iodine in KI - - + +
Sodium (Cl water + Faint flash
19 + ++
hypochlorite NaOH) Bright

Sodium (NaOBr, bromine Faint flash
20 + ++
hypobromite water + NaOH) Bright
Sodium
21 (I in KI + NaOH) - Faint + +
hypoiodite
(Ca(OCl)2
Calcium
22 saturated - Good + ++
hypochlorite
solution)
23 Turnip juice - Bright - + ++
24 Turnip juice heated to 70° - Faint - + Very slow
25 Turnip juice boiled - - - - -
26 Albumin solution - - - - -
27 Albumin solution + KMnO4 - Good + - ++
Albumin solution + KMnO4
28 boiled 1 min. and filtered - Good + - ++
(no precipitate forms)
29 Gelatin solution - - - - -
30 Gelatin solution + KMnO4 - Good - - ++
Gelatin solution + KMnO4
31 boiled 1 min. and filtered - Good + - ++
(no precipitate forms)
32 Colloidal Ag - Bright + +
33 Colloidal Pt - Bright + +
34 Colloidal Fe(OH)2 (dilute) - - - + -
35 Sodium nucleoproteinate (liver) - - - + -
Sodium nucleoproteinate
36 - - - - -
(mammary gland)
37 Sodium nucleate (yeast) - - - - -
Squid blood (Sepia esculenta).
38 - Fair ++
Contains hemocyanin
Squid blood (Sepia esculenta)
39 - Good -
boiled
Lobster blood (Palinurus
japonicus).
40 - Faint ++
Contains hemocyanin and
tetronerythrin, a lipochrome
Lobster blood (Palinurus
41 - Fair -
japonicus) boiled
42 Annelid blood (Laonome - Good
japonica).

Contains chlorocruorin
Annelid blood (Laonome
43 - -
japonica) boiled
Luminous pennatulid extract
44 - - - + ++
(Cavernularia haberi)
Luminous ostracod extract
45 - - +
(Cypridina hilgendorfii)
Luminous protozoan extract
46 - - - - -
(Noctiluca miliaris)
Firefly (Luciola viticollis)
47 - - ++
extract, luminous organs
Ferrous ferrocyanide
48 (Fe Fe(CN) ) - Faint + +
2 6
49 Zinc ferrocyanide (Zn2Fe(CN)6) - - + Very slow
50 Chromic oxide (Cr2O3) - - - Slow
51 Chromic hydroxide (Cr(OH)2) - - - Slow +
52 Manganese dioxide (MnO2) - Good Slow Slow ++

I believe the explanation of these phenomena lies rather in another direction and that
the effect of the temperature and concentration of reacting substances affects not only
the reaction velocity but also the reaction products. While intensity of luminescence
undoubtedly increases with increasing reaction velocity, the luminescence itself
probably accompanies only one stage in the formation of a series of oxidation
products. This stage is favored at a definite temperature and mass of reacting
substances. Thus, in the oxidation of phosphorus several intermediate oxides are said
to be formed. The oxidation takes place in steps and probably the luminescence is
connected with only one of the steps in a chain of reactions. It is probable that a
certain oxygen pressure and temperature favors that particular step at the expense of
the others and so this oxygen concentration and temperature correspond to the
optimum for luminescence.
The supposition that certain definite oxidation products of pyrogallol must be formed
in order to produce light is borne out by the fact that pyrogallol must be oxidized in a
particular way to obtain luminescence. The blackening of pyrogallol with absorption
of oxygen in presence of alkali is a very well-known reaction, but luminescence does
not accompany this type of oxidation. I have tried mixing all concentrations of
pyrogallol and all concentrations of alkali in an endeavor to obtain some light, but
always with negative results. Likewise my attempts to obtain light during the
electrolysis of salt solutions containing pyrogallol by means of the nascent oxygen at

various kinds of anodes have met with negative results. A similar case is presented by
luciferin which oxidizes spontaneously (most rapidly in presence of alkali) without
light production and only produces light when oxidized in presence of luciferase.
To sum up the results of the dynamics of chemiluminescence we may say that certain
oxyluminescences occur only if the substance is oxidized in a particular way under
definite conditions of temperature and concentration and that this is probably due to a
favoring of one step (with which the luminescence is associated) in a chain of
oxidations. Providing temperature and concentration are such as to favor the step
responsible for luminescence, then higher temperature and greater concentration result
in increased intensity of luminescence.
Let us now turn to luminous organisms and consider the effect of temperature and of
concentration of reacting substances (oxygen, luciferin and luciferase) on the
luminescence. We have already seen that luminescence of a luciferin-luciferase
mixture begins with an extraordinarily low oxygen tension and increases in intensity
with increasing tension of oxygen, but that very soon an oxygen tension is reached
where a maximum luminescence is obtained and further increase of oxygen tension
gives no brighter light. In this respect the luminescence intensity—oxygen tension
curve is no doubt very similar to the hæmoglobin saturation—oxygen tension curve.
Hæmoglobin is about 50 per cent. saturated at 10 mm. oxygen pressure, 80 per cent.
saturated at 20 mm. oxygen pressure and completely saturated at pressures of oxygen
well below the pressure of oxygen in air (152 mm. Hg). As the optimum oxygen
tension for luminescence of luciferin is also well below that of air, mixtures of
luciferin and luciferase luminesce with equal brilliancy whether air or pure oxygen is
bubbled through them. To obtain an excess of oxygen it is only necessary to keep the
solution saturated with air and statements regarding concentration of luciferin and
luciferase and intensity or duration refer to excess of oxygen. Investigators who have
studied the effect of increase in oxygen pressure on luminous animals have come to
the same conclusions. High pressures of air or oxygen do not increase the intensity of
luminescence (Dubois and Regnard, 1884).
The hydrogen ion concentration of crude solutions of luciferin and luciferase, made
by extracting whole Cypridinas with hot or cold water is fairly constant, about Ph = 9,
determined electrometrically. Such solutions have a high buffer value and the Ph does
not change during oxidation of luciferin so that this variable is automatically
controlled.
Because of difficulties in measuring low intensities of light which are constantly
changing, no figures on light intensities can be given, but it is easy to establish the
following facts: The greater the concentration of luciferin or luciferase the more
intense the luminescence. The greater the concentration of luciferin the longer the
duration of luminescence and the greater the concentration of luciferase, the shorter

the luminescence lasts. Thus, if we mix concentrated luciferin and weak luciferase we
get a bright light which lasts for a half hour or more, gradually growing more dim.
Concentrated luciferase and weak luciferin give a bright flash of light which
disappears almost instantly. Concentrated luciferase and concentrated luciferin give a
brilliant light which lasts for an intermediate length of time and weak luciferin and
weak luciferase give a faint luminescence which lasts for an intermediate length of
time.
These facts can all be explained by regarding luciferase as a catalyzer which
accelerates the oxidation of luciferin and by assuming that intensity of luminescence
is dependent on reaction velocity, i.e., on rate of oxidation. Contrary to the condition
for phosphorus and for pyrogallol there appears to be no optimum concentration of
luciferase or luciferin, but the luminescence intensity increases gradually with
increasing concentration of luminous substances up to the point where pure (?)
luciferin and pure (?) luciferase, as secreted from the gland cells of the animal, come
in contact with each other. This, the maximum brightness, is not to be compared with
the light of an incandescent solid, but is nevertheless visible in a well-lighted room,
out of direct sunlight.
The effect of temperature on Cypridina luminescence also bears out the preceding
conclusions. For a given mixture of luciferin and luciferase the light becomes more
intense with increasing temperature up to a definite optimum and then diminishes in
intensity. The diminution in intensity above the optimum is due to a reversible change
in the luciferase so that its active mass diminishes. This change becomes irreversible
in the neighborhood of 70° (depending on various conditions), where coagulation of
luciferase occurs. Light will appear at 0° but it is far less intense than light at higher
temperatures and it is more yellow in color. The light of optimum temperatures is
quite blue. The weaker light at temperatures above the optimum is also more yellow
in color. I believe this difference in color is a function of the slowed reaction velocity,
for a mixture of luciferin and luciferase which gives a bluish luminescence at room
temperature, will give a weaker and yellowish luminescence if diluted with water.
Dilution with water will slow the reaction velocity. If the difference in color were not
real but due to change in color sensitivity of the eye with different intensities of such
relatively weak light (Purkinje phenomenon), the weaker light should appear more
blue. As the weaker light appears more yellow, I therefore believe the color difference
is actual and not subjective.
A minimum, optimum, and maximum temperature for luminescence is observed in all
luminous organisms. The minimum is usually very low. Luminous bacteria will still
light at -11.5° C. The power to luminesce under ordinary conditions is not destroyed
by exposure to liquid air, for, on raising the temperature, light again appears
(Macfayden, 1900, 1902). Almost all organisms will luminesce at 0° C., and the
luminescence minimum probably represents the point at which complete freezing of

the luminous solution occurs. It is very low with bacteria because they are solutions in
capillary spaces of very small size, a condition tending to lower the freezing point.
The luminescence maximum represents the point at which luciferase is reversibly
changed so as to be no longer active. If the temperature is again lowered the luciferase
again becomes active and light reappears. Some degrees above this, and in all forms
well below the boiling point, luciferase is coagulated and destroyed. As the
coagulation point of proteins depends on many factors, such as time of heating, salt
content, acidity, etc., so the luciferases of different animals coagulate at different
temperatures depending on these conditions. Some of the more reliable observations
on these critical temperatures are collected in Table 14.
Table 14
Temperature Limits of Luminescence for Luminous Organism

Organism Author and date Minimum Optimum Maximum
Pseudomonas javanica Eijkman, 1892 -20° 25-33° 45°
Bacterium phosphorescens Lehmann, 1889 -12° ... 39.5°
Bacterium phosphoreum Molish, 1904, book -5° 16-18° 28°
Light bacteria Tarchanoff, 1902 -7° 15-25° 37°
Light bacteria Harvey, E. N., 1913 -11.5 15-20° 38°
Mycelium X Molish, 1904 ... 15-25° 36°
Lampyrids Macaire, 1821 -10 33° 46-50°
Pyrophorus noctilucus Dubois, 1886 ... 20-25° 47°
Photuris pennsylvanica Lund, 1911 ... ... 50°
Luciola viticollis Harvey, E. B., 1915 <0° ... 42°
Cypridina hilgendorfii Harvey, E. N., 1915 <0° ... 52-54°
Cyclopina gracilis Lund, 1911 ... ... 50°
Phylirrhoë bucephalum Panceri, 1872 44° ... 61°
Pyrosoma Panceri, 1872 <0° ... 60°
Mnemiopsis Leidyi Peters, 1905 9° 21° 37°
Noctiluca miliaris Quatrefages, 1850 1° ... 40°
Noctiluca miliaris Harvey, E. B., 1917 <0° ... 48°
Cavernularia haberi Harvey, E. N., 1915 <0° ... 52°
Watasenia scintillans Shoji, R, 1919 ... 16-31° 49°

We are thus led to the conclusion that intensity of luminescence is dependent on the
velocity of oxidation of luciferin and that with lowered reaction velocity the spectral
composition of the light changes. The maximum emission shifts toward the yellow. I
believe, however, that in Cypridina also, the luminescence intensity depends not only
on reaction velocity but on the particular manner in which luciferin is oxidized.

Cypridina luciferin will luminesce only in presence of Cypridina luciferase and no
light can be obtained from Cypridina luciferin and a host of different oxidizers (with
or without H2O2) such as are able to oxidize pyrogallol. Luciferin will also oxidize in
the air spontaneously but no light is produced. It is easy to show that this spontaneous
oxidation may be much more rapid than an oxidation with luciferase and yet light
appears only in presence of the latter. If a concentrated solution of luciferin is kept
near the boiling point it will be completely oxidized to oxyluciferin in four or five
minutes. No light appears if air or even if pure oxygen is bubbled through it. The
same solution kept at 20° with a small amount of luciferase will luminesce
continuously and not be completely oxidized to oxyluciferin in a half hour. We can,
however, cause the luciferin to oxidize as rapidly at 20° by adding concentrated
luciferase as does the luciferin near the boiling point without luciferase. A bright light
is produced in the former case, none in the latter case. The oxyluciferin formed from
spontaneous oxidation of luciferin appears to be the same as that formed with
luciferase present. Both give luciferin again on reduction. Perhaps the reaction takes
place in two stages, similar to those supposed to occur in other enzyme actions:
luciferin + luciferase = luciferinluciferase
luciferinluciferase + O (or minus H2) = oxyluciferin + luciferase.

We may then assume as a tentative hypothesis that luminescence only occurs during
oxidation (addition of O or removal of H) of the luciferinluciferase compound.
We have just seen that the effect of cooling a Cypridina extract containing luciferin
and luciferase and luminescing with a bluish light, is to reduce the intensity and
change the shade toward the yellow. Velocity of oxidation must be lowered and with
the same concentration of luciferase lowered velocity means more light of the longer
wave-lengths. A very instructive experiment on color of the light can be carried out
with animals having different colored lights and so closely related that their luciferins
and luciferases will interact with each other. Such a case is presented by the American
fireflies, Photinus and Photuris. Photinus emits an orange light, while Photuris emits
a greenish yellow light. The difference in color is especially noticeable when the
luminous organs of the two forms are ground up in separate mortars. As shown by
Coblentz, the difference in color is real, the spectrum of Photinus extending farther
into the red than that of Photuris (see Fig. 8). We can easily prepare luciferin and
luciferase from the two fireflies and make the following mixtures:
Photinus luciferin × Photinus luciferase = reddish light.
Photinus luciferin × Photuris luciferase = yellowish light.
Photuris luciferin × Photuris luciferase = yellowish light.

Photuris luciferin × Photinus luciferase = reddish light.
Thus the color of the light in these "crosses" is that characteristic of the animal
supplying the luciferase. To bring this fact in line with what we have already said
regarding reaction velocity and luminescence, we must believe that the Photinus
luciferase oxidizes at a slower rate than the Photuris luciferase. In this connection it is
of interest to recall that the Photuris light as emitted by the insect becomes reddish at
high temperatures, or if the insect is plunged into alcohol, both conditions which bring
about partial coagulation of the luciferase and reduce its active mass.

BIBLIOGRAPHY

A few of the enormous number of papers on luminescence are included in the list
below. The attempt is made to list only those dealing with the structure, chemistry or
physiology of luminous animals and the physical nature of their light, together with a
small number of general interest. More complete works on light and luminescence
come first and original articles follow. Authors' names are arranged alphabetically,
their papers chronologically. A fairly complete list of literature covering the whole
field of Bioluminescence is given by Mangold, 1910. The 1913 paper of Dubois gives
a bibliography of his own contributions up to this date so that only those papers to
which special reference is made are included below.

BOOKS AND GENERAL WORKS

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INDEX

Abegg, R., 147
Acanthephyra, 78
Agaricus, 99
Agassiz, A., 11
Alkaptonuria, 17
Allman, G. I., 11, 71
Ammonia, 12
Anodoluminescence, 26, 29
Anornalops, 69
Aristeus, 72
Aristotle, 1

Bach, A., 137
Bacteria, luminous, 2, 10, 13, 14, 16, 18, 28, 45, 53, 61, 65, 69, 72, 74, 81, 82,
89, 99, 101, 103
Bacterial lamps, 18
Baker, J., 2
Bancroft, W. D., 36
Bancroft and Weiser, 24
Bandrowski, E., 33
Barcroft and Hill, 98
Barnea, 116
Batelli and Stern, 115
Becquerel, E., 26
Beijerinck, M. W., 18, 89, 99, 100, 102
Bigelow, S. L., 34
Black, 91
Bolitophila, 77
Boyle, R., 1, 16, 85 ff
Brandt, 36
Brittle stars or ophiuroids, 10, 11, 72

Canton's phosphorus, 27
Carbon dioxide and luminescence, 91 ff
Cardium, 116
Cascariolo, V., 27
Cathodoluminescence, 26, 29
Cavernularia, 74, 103
Centnerzwer, M., 147
Cephalopods or Squid, 10, 11, 13, 68, 72, 84, 104
Ceratium, 71
Chætopterus, 10, 17, 42, 71, 73, 74, 83, 103

Chemiluminescence, 36 ff, 45 ff
Chlorophyll formation by animal light, 66
Chromophyton, 15
Chun, C., 79
Coblentz, W. W., 30, 31, 44, 51, 52, 59, 60, 62, 64, 93, 160.
Co-enzyme, 104, 130
Co-luciferase, 107 ff
Color of animal light, 41 ff, 157 ff
Concentration and luminiscence, 145 ff
Conroy, J., 43
Crozier, W. J., 71, 103
Crustacea, 10, 14, 68, 70, 72, 89, 101 ff
Crysalloluminescence, 33 ff, 74
Ctenophores, 2, 10, 11, 71, 72, 82
Cyanides and luminescence, 126
Cypridina, 14, 30, 45, 48, 63, 71, 73, 75 ff, 90, 92, 98, 103, 105 ff, 155 ff

Dahlgren, U., 72, 73, 75
"Death Glow," 69
Dinoflagellates, 2, 10, 32, 82
Dubois, R., 31, 35, 37, 43, 45, 49, 61, 64, 73, 103 ff, 111, 114 ff, 131, 155

Earthworms, 10
Efficiency of animal light, 48 ff
Eggs, luminous, 11
Electroluminescence, 24, 29
Embryos, luminous, 11
Euphasia, 72
Ewan, T., 147
Exner, S., 30
Extracellular luminescence, 68, 71
Eyes, luminous, 15 ff

Fabre J. H., 99
Fahrig, E., 37
Fireflies, 10, 31, 34, 43, 69, 71, 77 ff, 89, 93, 101, 103, 135, 160
Fishes, 1, 3, 10, 18, 64, 69, 72, 84, 85
Flowers, flashing of, 16
Fluorescence, 25 ff, 62
Fluorescent screens, 29
Forsyth, R. W., 53
Frankland, P., 62

Friedberger & Doepner, 65
Frogs, luminous, 13
Fungi or Basidiomycetes, 10, 69, 72, 81, 89, 99, 101

Galloway and Welch, 84
Gernez, D., 32
Giard and Billet, 13
Giesbrecht, W., 11, 70
Glowworms, 1, 10, 43, 77
Gnathophausia, 72
Goss, B. C., 143
Greene, C. W., 70
Guinchant, 37

H-ion concentration and luminescence, 92, 138, 155
Heat production and luminescence, 93 ff
Heliotropism by animal light, 66.
Heller, J. F., 1, 2, 16
Heterocarpus, 72
Heteroteuthis, 72
Hooke, R., 91
Hulme, N., 1
Hyde, Forsyth and Cady, 57, 63
Hydrogenase, 131
Hydroils, 10, 72

"Ignis fatuus," 15
Immune bodies, 104
Infection, with luminous bacteria, 13
Infra red rays in animal light, 48 ff
Intensity of animal light, 63
Intracellular Luminescence, 68, 71
Interference colors, 14
Issatschenko, B., 66
Ives, H. E., 28, 44, 51 ff, 59, 61

Kemp, C., 78

Langley and Very, 43, 50 ff, 64
Lankester, E. R., 42
Lavoisier, 91
Lenard and Wolf, 37
Ligia, 13

Limulus, 129
Linnemann, E., 36
Lode, A., 65
Luciferase, 103 ff. Chap VI (properties);
of Pholas, 114;
of Cypridina, 123 ff
Luciferesceine, 31, 110
Luciferin, 103 ff. Chap. VI (properties);
of Pholas, 114;
of Cypridina, 116 ff
Luciola, 103, 125.
Luminescence, 23 ff
Luminosity, distribution in plant and animal kingdom, 3 to 12
Luminosity, false, 12 ff
Luminous animals, habitat, 10
Luminous animals, uses of to man, 17 ff
Luminous granules, 73, 75
Lyman rays, 21
Lyoluminescence, 35

MacCartney, J., 2, 3
Macfayden, A., 157
Macrozymases, 73
Man, luminosity of, 16
Mangold, E., 11, 72
Massart, J., 71
Mast, S. O., 69
Mayow, 91
McDermott, F. A., 31, 37, 45, 53
McKenney, R. B., 100
Medusæ or jelly fish, 2, 10, 72, 82
Methane, 15.
Michaelis, G. A., 1
"Minimum radiation visually perceptible," 65, 144
Molisch, H., 45, 53, 61, 66, 102
Molluscs, 10, 72
Monocentris, 69, 104
Moore, B., 71
Muraoka, H., 61
Myriapods, 10, 35, 72
Mytilus, 116

Nadson, G., 66
Nematocelis, 72
Noctiluca, 2, 10, 71, 73, 82, 83, 89, 104
Noctilucin, 101
Nutting, P. G., 57, 59
Nyctiphanes, 72, 80

Odontosyllis, 83
Orchestia, 13
Orya, 35
Osborne and Wakeman, 121
Ostræa, 116
Otto, M., 37
Oxygen and luminescence, 1, 67, 85 ff, 147 ff
Oxyluciferine, 108 ff, 127 ff, 158
Oxyluminescence, 36 ff, 111 ff

Paint, luminous, 28, 29
Panceri, P., 45, 74
Pasteur, 42
Penetrating radiation in animal light, 61 ff
Pennatula, 74, 103
Pennatulids or sea pens, 10, 72, 74, 82, 83, 89, 101, 103
Peridineæ, 10
Periodicity of luminescence, 71
Peron, F., 45
Peroxidases, 11, 126, 148
Peters, A. W., 11, 71
Pflüger, E., 1, 102
Phengodes, 41, 42
Phillips, A. H., 125
Philoscia, 13
Phipson, T. L., 101, 109, 110
Pholas, 10, 11, 72, 73, 74, 89, 101, 103, 105 ff, 114 ff, 131
Phosphine, 15
Phosphorescence, 24, 25 ff, 138, 143
Phosphoroscope, 26
Phosphor-photographic method, 52
Phosphorus and luminescence, 38, 39, 147 ff
Photinus, 44, 51, 53, 56, 59, 64, 103, 125, 160 ff
Photoblepharon, 18, 64, 69
Photochemical reactions, 67, 68, 138, 145

Photogen, 102
Photogenin, 105
Photoluminescence, 26, 67
Photophelein, 105, 106, 110
Photosynthesis by animal light, 18
Photuris, 44, 59, 103, 125, 160 ff
Pierantoni, V., 13, 14, 74
Piezoluminescence, 32 ff
Polarization, 45
Polimanti, O., 45
Pope, W. J., 32
Porcellio, 13
Porichthys, 70, 83
Preluciferine or proluciferine, 106 ff
Prevost, B., 15
Priestly, 91
Ptychodera, 71, 103
Purkinje phenomenon, 40, 44, 157
Pyrogallol and luminescence, 37, 111, 148 ff
Pyroluminescence, 24
Pyrophorine, 31, 109
Pyrophorus, 11, 43, 45, 49 ff, 64, 76, 101, 103, 114, 125
Pyrosoma, 10, 13, 45, 72, 101

Quatrefages, A. de., 73

Radiant energy, 20 ff
Radioluminescence, 26
Radium rays or Becquerel rays, 21, 26, 30, 62
Radziszewski, B., 37, 39
Reaction velocity and luminescence, 145 ff
Reductase, 130 ff
Reeves, P., 65, 144
Respiration and luminescence, 91, 92, 99
Rhizomorpha, 2
Romberg's phosphorus, 32
Russel, E. J., 147
Russel, W. J., 62

Sapphirina, 14
Sarcina, 1
Scharff, E., 147

Scheele, 91
Schistostega, 15
Schizopod larvæ, 11
Schumann rays, 21
Schurig, W., 61
Scolopendra, 102
Sea, phosphorescence of, 2
Sepietta, 72
Sergestes, 72, 78
Singh and Maulik, 61
Solen, 116
Spallanzani, L., 85, 101
Spectrum of chemiluminescence, 39
Spectrum of luminous organisms, 42 ff
Spectrum of phosphorescence, 28
Spectrum, range of, 21 ff
Spinthariscope, 30
Steche, O., 65, 69
Stefan-Boltzmann Law, 22, 23
Stimulation and luminescence, 68 ff, 135
Stoke's Law, 28, 31
Stylochiron, 72
Suchsland, E., 61
Sulphides, phosphorescence of, 27
Sweat, luminous, 17

Talitrus, 13
Tarchanoff, J., 13
Temperature and luminescence, 145 ff, 156 ff
Temperature radiation, 23
Thaumatolampas, 42
Thermoluminescence, 24 ff
Tomopterus, 72
Transparency of chitin to infra-red, 52
Trautz, M., 32, 33, 37, 39, 145
Triboluminescence, 32 ff
Trojan, E., 11, 78
Tschugaeff, L., 32

Ultra violet rays in animal light, 53 ff
Urine, luminous, 18
Uses of luminous organs, 81 ff

Vacuolides, 73
van Helmont, 91
van't Hoff, J. H., 147
Vibrio, 65
Ville and Derrien, 111
Visual sensibility, 54 ff

Watanabe, H., 75
Watasenia, 104
Water and luminescence, 85, 101
Weiser, H. B., 33, 34, 39
Welker, W. H., 121
Wheeler and Williams, 77
Wiedemann, E., 23
Wiedemann and Schmidt, 25, 36
"Will-o'-the-wisp," 15
Wood, phosphorescent or shining, 1, 2, 85
Worms or annelids, 3, 72

X-rays or Röntgen rays, 21, 26, 30, 62

Yatsu, N., 75
Young, C. A., 43

Zacharias, O., 71
Zymogen granules, 73

Transcriber's Notes

Uncommon forms for chemical names have
been retained where they occur in the text, e.g.
"atropin" for "atropine"; "asparagin" for
"asparagine", etc.
The spellings of "Sidot blend" and "Sidot
blende" are used interchangeably.
"PH" or PH (subscript H) is used throughout for
the scale of alkali-acidity where the modern
usage is "pH".
On page 173, the citation for Nutting, P. G.:
1908 has page range pp. 261-039. This is as it
appears in the original, but is probably in error.
Minor corrections to formatting and missing
punctuation (mostly in the bibliography) have
been changed without an explicit note.

Changes to the text have been made only in the
case of obvious spelling or type-setting errors.
These are listed as follows:
Page ix: changed "Phoshorescence" to
"Phosphorescence" (II. Luminescence and
Incandescence ... Phosphorescence and
fluorescence.)
Page ix: changed "Biozymoxyluminescence" to
"Biozymoöxyluminescence" (V. The Chemistry

of Light Production, Part I ...
"Biozymoöxyluminescence.")
Page x: changed "chemi-luminescence" to
"chemiluminescence" in two instances
(Reaction velocity and chemiluminescence.
Temperature and chemiluminescence.)
Page 15: changed "th" to "the" (Less well
known is the Ignis fatuus)
Page 26: re-positioned period outside of
parentheses "after being illuminated
(photoluminescence)."
Page 29: changed "platino-cyanide" to
"platinocyanide" (Fluorescent screens of
barium platinocyanide)
Page 29: added missing comma (willemite
(Zn2SiO4), Sidot blend)

Page 34: added missing closing quotation mark
("It is altogether probable that the cause of this"
...)
Page 39: superscript "2" changed to subscript
"2" in Na2CO3 (the pyrogallol-formaldehyde-
Na2CO3-H2O2 reaction).

Page 41: "50-metre candles" changed to "50
metre-candles" (Below 0.5 and above 50 metre-
candles visibility varies ...)
Page 42, Table 4: changed "Fraünhofer" to
"Fraunhofer" in the caption and table heading
(Fraunhofer Lines)
Page 47, Table 5: changed "Forster" to
"Förster" (Bacteria ... Förster, 1887)

Page 56, Fig 12 caption: "Forsythe" changed to
"Forsyth" (after Hyde, Forsyth and Cady).
Page 72: added missing closing parenthesis "the
molluscs (Pholas and Phyllirhoë)".
Page 74: "secretion" changed to "section" (A
section of the epithelium shows large mucous-
producing cells ...)
Page 75: added missing closing punctuation (At
least one, probably two, are concerned in light
production.)
Page 75: changed "intra-cellular" to
"intracellular" (animals possessing light cells
with intracellular luminescence)
Page 81; Fig. 29 caption: added missing comma
(chr.1, chromatophore; ...)
Page 87: added missing closing parenthesis "
(and that too of such a Density to make them
continue shining)."
Page 90: "necesary" changed to "necessary"
(Boyle also made many experiments to show
that air was necessary for the life of animals ...)
Page 93: changed "thermo-couple" to
"thermocouple" (using a thermocouple as the
measuring instrument)
Page 94: "D" changed to "B" (placed in a large
Dewar flask (B) filled with water)
Page 94: "Thermo-couples" changed to
"Thermocouples" (Thermocouples (L and M) of
advance...)

Page 97: "thermo-couple" changed to
"thermocouple" (Readings of each
thermocouple on the galvanometer scale ...)
Page 100: changed "McKenny" to "McKenney"
(McKenney (1902) found also ...)
Page 102: changed "misceable" to "miscible"
(insoluble in water but miscible with it)
Page 103: "demontrate" changed to
"demonstrate" (I have been unable to
demonstrate their existence in luminous
bacteria;)
Page 104: "thermolable" changed to
"thermolabile" ( ...and a thermolabile
complement (alexin) are necessary.)
Page 104: "thermolable" changed to
"thermolabile" (Because of the necessity of
thermostable and thermolabile substances for
light production ...)
Page 105: "thermolable" changed to
"thermolabile" (luciferase (=photogenin) for the
thermolabile material ...)
Page 111: "preslence and H2O3" changed to
"presence of H2O2" (lophin could be oxidized
by vertebrate blood in the presence of H2O2.)

Page 116: "or" changed to "of" ( ... and would
disappear from solution in the course of a day
or so.)
Page 116: changed "oxidizible" to "oxidizable"
(The luciferins, as the oxidizable substances,
must claim first attention.)

Page 123: "contrated" changed to
"concentrated" (1 c.c. portions of concentrated
luciferin)
Page 132: "coluciferase" changed to "co-
luciferase" (He now regards it as identical with
his co-luciferase)
Page 151, Table 13: corrected duplicate
numbering "10" to "11" (11 Ferric chloride)
Page 151, Table 13: corrected duplicate
numbering "14" to "15" (15 Chromic sulfate)
Page 151, Table 13: abbreviated "minute" to
"min." in two entries (boiled 1 min. and
filtered)
Page 158: changed "appear" to "appears" (...
and yet light appears only in presence of the
latter.)
Page 162: added missing closing punctuation
(More complete works on light and
luminescence come first and original articles
follow.)
Page 165: added missing comma (Dubois, R.:
1918a, Sur la Synthèse de la Luciferine.)
Page 165: changed "Biophotogénesis" to
"Biophotogénèse" (Recherches Recentes de M.
Newton Harvey sur la Biophotogénèse)
Page 165: changed "Biophotogénèsis" to
"Biophotogénèse" (Nouvelles Recherches sur la
Biophotogénèse)
Page 166: changed "Oxydations
geschwindigkeit" to

"Oxydationsgeschwindigkeit" (Ueber die
Oxydationsgeschwindigkeit von Phosphor ...)
Page 166: changed "Radiumstrahlem" to
"Radiumstrahlen" (Einige Beobachtungen
ueber die durch Radiumstrahlen in den
tierischen Geweben erzeugte Phosphoreszenz.)
Page 166: changed "neiderer" to "niederer"
(Ueber die Entwicklung von Bakterien bei
niederer Temperatur.)
Page 167: changed "nueue" to "neue" (Ueber
die rosettenförmigen Leuchtorgane der
Tomopteriden und zwei neue Arten von
Tomopteris.)
Page 169: added missing hyphen to "Pflanzen-"
(Ueber das Leuchten im Pflanzen-und
Tierreiche.)
Page 169: changed "Rucksicht" to "Rücksicht"
(mit bes. Rücksicht auf. med. Diagnost. u.
Therapie Wien.)
Page 170: changed "jord." to "jard." (Bull. d.
jard. imp. botan. St. Petersburg)
Page 171: changed "Lichtfaüle" to "Lichtfäule"
(Phosphorezierende Tausendfüssler und die
Lichtfäule des Holzes)
Page 172: changed "Pfluger's Arch" to
"Pflüger's Arch." (Pflüger's Arch., Bd. cxix, pp.
583-601.)
Page 174: changed "Bedentung" to
"Bedeutung" ( ... ihre Bedeutung für die
Principien der Respiration)

Page 175: changed "Lazaro" to "Lazzaro"
(Spallanzani, Lazzaro: 1794, ...)
Page 176: changed "Leuchtvermogen" to
"Leuchtvermögen" (Ueber das Leuchtvermögen
von Amphiura squamata, Sars.)
Page 177: changed "Triboluminescenz" to
"Tribolumineszenz" (Tschugaeff, L.: 1901,
Ueber Tribolumineszenz.)
Page 179: changed "Bandromski" to
"Bandrowski" (Bandrowski, E., 33)
Page 179: changed "Baelli" to "Batelli" (Batelli
and Stern, 115)
Page 179: changed "Centnerswer" to
"Centnerzwer" (Centnerzwer, M., 147)
Page 179: changed "Fire-flies" to "Fireflies"
(Fireflies, 10, 31, 34,...)
Page 180: changed "Forsythe" to "Forsyth"
(Hyde, Forsyth and Cady, 57, 63).
Page 180: changed "Flankland" to "Frankland"
(Frankland, P., 62)
Page 180: changed "Glow-worms" to
"Glowworms" (Glowworms, 1, 10, 43, 77)
Page 181: changed "Piezolumisescence" to
"Piezoluminescence" (Piezoluminescence, 32
ff).
Page 182: changed "Stefan-Boltzman" to
"Stefan-Boltzmann" (Stefan-Boltzmann Law,
22, 23)

Page 182: changed "infrared" to "infra-red"
(Transparency of chitin to infra-red, 52)
Page 182: added missing page references
(Weiser, H. B., 33, 34, 39).
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