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BROWN UNIVERSITY

PROVIDENCE, RHODE ISLAND

CONTRIBUTIONS

FROM THE

BIOLOGICAL LABORATORY

(formerly Anatomical Laboratory )

ISSUED wear RIL, 1916 ‘

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PROVIDENCE, R. I. \

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PREFACE

The papers which are collected in this eighth volume of Contribu-
tions have been written by officers or students in the Department of
Biology of Brown University, and have recently appeared in various
scientific journals. In the table of contents and on the title page of each
paper will be found the place and time of publication. At the end of
the volume is a complete list of the papers published in the preceding

volumes of this series.

106.

107.

108.

109.

IIo.

Iit.

113.

TAREE OF “CONTENTS
Volume VIII

Tue Puystorocicat Errecrs oF ALKALOIDS oF ZYGADENUS INTERMEDIUS.
BY PHILIP H. MITCHELL AND GEORGE SMITH.

American Journal of Physiology, Vol. XXVIII, No 6, September 1, rgrr.

SEASONAL VARIATION IN THE BacTertaL ConTENT oF OvysTERrs.
BY FREDERIC P. GORHAM.
American Journal of Public Health, Vol. II, Old Series Vol. VIII, 1912,
PP 24-27.

Tue Sanitary REGULATION OF THE OysTER INDUusTRY.
BY FREDERIC P. GORHAM.
American Journal of Public Health, Vol. II, Old Series Vol. VIII, 1912,
Pp 77-84.

Superior SANITARY QuaLity oF Ruope Istanp OysTeRs.
BY FREDERIC P. GORHAM.

The Providence Medical Journal, Vol. XIII, 1912, pp 46-53.

Some BrocHEMICAL PRoBLEMS IN BACTERIOLOGY.
BY FREDERIC P. GORHAM.

Science, N. S., Vol. XXXV, No. 897, March 8, 1912, pp 357-362.

A ConTRiIBuTION To Our KNowLeDGE or THE Gas METABOLISM OF BACTERIA.

First Paper. The Gaseous Products of Fermentations of Dextrose, by B. Coli,
by B. Typhosus and by Bact. Welchii.

Second Paper. The Absorption of Oxygen by Growing Cultures of B. Coli and
of Bact. Welchii.

BY FREDERICK G. KEYES AND LOUIS J. GILLESPIE.

The Journal of Biological Chemistry, Vol. XIII, No. 3, 1912, pp 291-310.

How Do Isoronic Soprum CHLoRIDE SoLuTION AND OTHER PARTHENOGENIC
Acents IncrEasE OXIDATION IN THE SEA Urcuin’s EaoG?

BY J. F. MCCLENDON AND P. H. MITCHELL.

Journal of Biological Chemistry, Vol. X, No. 6, 1912, pp 459-472.

Tue SIGNIFICANCE OF THE TIME AT wHiIcH Gas Is Propucep IN Lactose
Perrone BILE.

BY WILLTAM W. BROWNE.

American Journal of Public Health, Vol. III, No. 9, 1913, pp 944-956.

114.

Tig:

116.

10 Ye

118.

119g.

120.

I2I.

122.

123.

A Comparative Strupy or THE SMITH FERMENTATION TuBE AND THE IN-
VERTED VIAL IN THE DETERMINATION OF SUGAR FERMENTATION.

BY WILLIAM W. BROWNE,

American Journal of Public Health, Vol. III, No. 7, 1913, pp 701-704.

Rerort or Mosgurro Conrror For 1913.
BY FREDERIC P. GORHAM.

Report of Superintendent of Health for the year 1913.

Hints oN THE HanpiinG oF CLEAN OysTERs.
BY LESTER A. ROUND.
Prepared for the Commissioners of Shell Fisheries of the State of Rhode Island,
1913.

Tre Rats or ProvipENCE AND THEIR ParaAsITEs.
BY GEORGE H. ROBINSON.

American Journal of Public Health, Vol. III, No. 8, 1913, pp 773-776.

Size oF THE SAMPLE NECESSARY FOR THE ACCURATE DETERMINATION OF THE

SANITARY QUALITY oF SHELL OysTeERs.
BY GEORGE H. SMITH.

American Journal of Public Health, Vol. III, No. 7, 1913, pp 705-708.

Scumucker’s ‘¢ THE MEaninG or EvotLurTion.”’
BY HERBERT E. WALTER.

Science, N. S., Vol. XXXVIII, No. 987, November 28, 1913, p 779.

Genetics, AN InTROopUCTION To THE STupY or HeEreEpitTy.
BY HERBERT E, WALTER

Published by the Macmillan Company.

Tre NecGri Bopires In Rapiks.
BY ERNEST M. WATSON.

Journal of Experimental Medicine, Vol. XVII, No. 1, 1913, pp 29-42.

Tue Propucrion or Acip By THE Bactitus Cort Group.
BY WILLIAM W. BROWNE.

Journal of Infectious Diseases, Vol. XV, No. 3, November, 1914, pp 580-604.

On THE CLASSIFICATION OF STREPTOCOCCI.

OxpsERVATIONS ON HEMOLysIN PRopUCTION BY THE STREPTOCOCCI.

Reprints not available.
BY HAROLD W. LYALL.

Journal of Medical Research, Vol. XXX, pp 487-532.

124.

125.

126.

ra7)

128.

129.

130.

132

133-

Tue SrrucTurE or THE CoTTON FIBER.
BY BENJAMIN S. LEVINE.

Science, N. S., Vol. XL, No. 1042, December 18, 1914, p 906.

Tue Errect oF WatTer-Gas Tar oN OystTErs.
BY PHILIP H, MITCHELL.

From Bulletin of the United States Bureau of Fisheries, Vol. XXXII, 1912,
Document 786, issued February 18, 1914.

Tue OxyGen REQUIREMENTS OF SHELLFISH.
BY PHILIP H. MITCHELL.

From the Bulletin of the United States Bureau of Fisheries, Vol. XX XIII, 1912,
Document 787, issued February 18, 1914.

CoNTRIBUTIONS TO THE BACTERIOLOGY OF THE OYSTER.
BY LESTER A ROUND

The Results of Experiments and Observations Made while Conducting an Investi-
gation Directed and Authorized by the Commissioners of Shell Fisheries of the
State of Rhode Island, 1914.

REPORT ON THE INSPECTION oF Camps, 1913.
BY LESTER A. ROUND.

Health Bulletin of the Rhode Island State Board of Health for February, 1914.

Tue ImporTANCE oF A CONSIDERATION OF THE FIBER ProTEeINs IN THE
Process or BLEACHING CoTTON.

BY BENJAMIN S&S. LEVINE.

Science, N. S., Vol. XLI, No. 1058, April 9, 1915, pp 543-545.

An Epipemic, StmuLatTinG Typxorp, Causep By A PARAGAERTNER ORGANISM.
BY GEORGE H. ROBINSON,

Journal of Infectious Diseases, Vol. XVI, No. 3, May, 1915, pp 448-455.

Isolation, IDENTIFICATION, AND Serum Reactions oF TyPHoIp AND Para-
TYPHOID BAcILti.

BY GEORGE H. ROBINSON.

The Journal of Medical Research, Vol. XXXII, No. 3, July, rg15.

Tue Orp Mepicat Scuoot IN Brown UNIVERSITY.
BY FREDERIC P. GORHAM.

The Providence Medical Journal, Vol. XVI, July, 1915.

Tue Atm AND ConTEeNT oF Hicu Scuoor Brotocy.
BY HERBERT E. WALTER.

School and Society, Vol. II, No. 48, November 27, 1915, pp 757-762.

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106

THE PHYSIOLOGICAL EFFECTS. OF ALKALOIDS OF
ZYGADENUS INTERMEDIUS.
BY PHILIP H. MITCHELL anp GEORGE SMITH.

American Journal of Physiology, Vol. XXVIII, No. 6, September 1,
LOL:

Reprinted from the American Journal of Physiology.
Vol. XXVIII. — September 1, 1911. — No. VI.

THE PHYSIOLOGICAL EFFECTS OF ALKALOIDS
OF ZYGADENUS INTERMEDIUS.

By PHILIP H. MITCHELL anp GEORGE SMITH.
[From the Biological Laboratory of Brown University.]

FA YGADENUS intermedius is one of the several varieties of the

plants formerly called Zygadenus venosus and popularly known
as death camas. They have also been designated by a number of
other names, among which are wild onion, mystery grass, lobelia, and
poison sego lily. Their economic significance, as pointed out by
Chestnut and Wilcox,! is considerable because of their prevalence on
the sheep and cattle ranges of many western states and their highly
toxic effect on the animals, especially sheep, which eat them. Start-
ing up in the spring earlier than the grass but resembling it in form
and texture, these plants are so attractive to sheep that they are an
enemy which the rancher must reckon with. During the season of
1900 in Montana alone 3,030 sheep were reported to be poisoned by
various varieties of Zygadenus.

Chestnut and Wilcox! have carried out some experiments on the
injection of watery and alcoholic extracts of Zygadenus venosus as
well as feeding experiments. ‘They considered that some, at least,
of the plants which they used may have been Zygadenus intermedius.
The sheep and rabbits used in their experiments showed symptoms
of poisoning, such as dizzy movements, slow and labored or convul-
sive respiration, and finally a deep coma which, if sufficient poison
had been administered, terminated fatally. The effects were ap-
parently the same after injection of extracts or feeding of plants, and
were identical with those observed in animals poisoned on the open
ranges. These authors showed that all parts of the plant contain
toxic substances, so that the belief formerly held that animals must
eat the bulb or root to be poisoned is erroneous. They found that of

1 CHEestTNuT and Witcox: U. S. Department of Agriculture Bulletin No. 26,

Igor.
318

Effects of Alkaloids of Zygadenus Intermedius 319

the substances which they tried as antidotes potassium permanganate
was the most effective. For sheep and cattle they advised giving it
in the form of a drench consisting of aluminium sulphate as well as
potassium permanganate dissolved in water.

No further work on the effects of this poison has, so far as the authors
know, been reported. Little is known of the chemical nature of the
active principle. Slade? concluded from certain color tests that the
plants contained the alkaloids sabadine, sabadinine, and veratral-
bine. Heyl and Raiford* have made alkaloidal preparations from
leaves, flowers, bulbs, and roots of the intermedius variety and have
shown that alkaloids are especially abundant in the flowers.

Some alkaloidal material prepared by them was sent to the authors
of the present paper for the purpose of determining its toxicity. The
results of injections into guinea-pigs were sufficiently complex and
interesting to warrant a more detailed analysis of the pharmacological
action of the preparation. This report is concerned with experiments
carried out with that preparation only. It consisted of alkaloids,
presumably a mixture, prepared from various parts of Zygadenus
intermedius according to the methods reported by Heyl and Raiford ®
and held in 17.5 per cent aqueous solution in the form of sulphates.

EXPERIMENTS.

J. The fatal dose for guinea-pigs. — To obtain some idea of the
relative toxicity of the preparation, intraperitoneal administration
to guinea-pigs was employed. Injections of the alkaloidal solution
diluted with sterilized physiological saline were made aseptically.

In Table I the results are shown arranged in the order of the dose
per 100 gm. of guinea-pig. The fatal dose is seen to be between 4.6
and 5.1 mgm. per 100 gm. of animal. It is probably nearer the lower
than the upper of these limits, because the animal which received 4.6
mgm. per 100 gm. recovered only with great difficulty after a long
period, eighteen hours, and seemed at least twice during that time to
be in its death struggle. This injection, therefore, was probably very

2 StapDE: American journal of pharmacology, 1905, Ixxvii, p. 262.
3 Hey and RatrorpD: Journal of the American Chemical Society, 1or1,
2.86 SUT) OF 206.]

320 Philip H. Mitchell and George Smith.

near the fatal dose. In the case of the injection of 5.1 mgm. per 100
gm., on the other hand, death ensued in so short a time, twenty-five
minutes, that this dose was perhaps a little more than the fatal one.
These results indicate therefore that we have a substance of marked
toxicity.

TABLE I.

Weight of
guinea-pig.

gm.

485

INTRAPERITONEAL INJECTIONS INTO GUINEA-PIGS.

Volume of
solution.

Amount of
sulphate.

gm.

0.05

Dose per |
100 gm. of |

animal.

0.0111

|

Result.

Time be-

| tween in-

Time be-
tween in-

jection and | jection and

death.

| recovery.
|

Death

min.

19

hours

0.04 0.0104 Death 23

0.028 0.0090

0.0076

Death 21
31
26
25

0.0245 Death

0.02 0.0060 Death

0.015 0.0051 Death

| Recovery

0.014 0.0046

0.0035 0.0011 Recovery

It seemed important also to test the effect of administration per os.
The undiluted alkaloidal solution as furnished us was put in small
gelatin capsules and fed to guinea-pigs by forcing the capsules into
the back of the mouth through a glass tube. The results are arranged
in Table II, and apparently indicate that a comparatively large
quantity (0.2 gm.) is required for the fatal effect.

The actual fatal dose absorbed from the digestive organs is not,
however, as large as these figures would seem to show. A considerable
quantity of the material fed was vomited up before it was absorbed,
because the substance, as will be explained below, is a very powerful
emetic. Another factor tending to diminish the toxicity when fed
is the power of the organism to effect an oxidative destruction of the
alkaloid before a fatal amount has been absorbed. This effect is
illustrated by the rather quick recovery of guinea-pigs from the
effects of injection of small doses. For example, in the last experi-

Effects of Alkaloids of Zygadenus Intermedius. 29

ment recorded in Table I, 3.5 mgm. produced very profound symptoms
of poisoning, but these had entirely passed away within three hours.
In another experiment, not recorded above, the subcutaneous injec-
tion of 3.7 mgm. produced undoubted effects which had all disap-
peared in an hour and a half. It does not seem necessary then to

TABLE II.

Per Os ADMINISTRATION TO GUINEA-PIGS.

: Time
Dose per eet between
ee ae of | Result. feeding feedine
: and death recoveey

No. of
capsules
used.

Weight of
guinea-pig.

Amount of
sulphate.

gm. min.
0.0175 0.0053 Recovery ste 30

0.140 0.040 Recovery? ApoE 12
ours

ele About 12
0.21 0.0755 Death 2 niet ie

1 Last two capsules given one hour after the first two.
2 Occurred during the night and was not exactly known.

conclude from these experiments that gastro-intestinal digestion
destroys the alkaloid or diminishes its toxicity, but it seems reasonable
to conclude that the tendency to vomiting, the slowness of absorp-
tion, and the rate of destruction of the alkaloid account for the rela-
tively large quantity required to kill when fed.

II. The effects on guinea-pigs. — After subcutaneous or intraperi-
toneal injection or after feeding, the alkaloid produces the same
symptoms of poisoning. There are marked insalivation and frequently
repeated vomiting, which begin very soon after the substance is ad-
ministered and persist nearly as long as any symptoms can be ob-
served. During the first few minutes of the onset of the effects the
animal jumps and runs about the cage excitedly in the intervals be-
tween vomiting. It soon begins, however, to lose control of its muscles.
The hind legs are usually affected first, but finally all the limbs become
useless and the guinea-pig lies on its side completely prostrated.
The respiration, at first rapid, has by this time become slower than
normal and very labored. It is frequently interrupted by short

322 Philip H. Mitchell and George Smith.

periods of very convulsive breathing. Although quite unable to
make any co-ordinate movements, the animal is exceedingly
irritable and responsive to reflex stimuli. The lightest touch will
start a struggle, and as the symptom complex advances there comes
a stage when the struggles become spasmodic and later typical tetanic
spasms occur. The guinea-pig now behaves quite like an animal
poisoned with strychnine, and at the slightest irritation, such as a
jar of the cage or a breath of wind, exhibits profound tetanus. All
of its muscles are rigid and the body stiffened out in an extended
position sometimes for ten seconds at a time. The heart rate as
detected by allowing the guinea-pig to lie in the observer’s hand so
that the cardiac impulse may be felt is found to be much slower than
normal, and this condition persists until recovery is quite far ad-
vanced or death occurs. Defecation takes place at frequent intervals
throughout the development of effects. Micturition is also generally
observed. If the fatal dose has been given by injection, death occurs
after an interval of about twenty to thirty minutes. No relation-
ship between the dosage administered intraperitoneally and the time
required to kill is to be seen in our results. Two typical protocols
follow:

Experiment No. 1. Jan. 30, 1911. — Female guinea-pig; weight, 845 gm.

Dose: 0.05 gm.; volume, 2 c.c.

3.25 P.M. Intraperitoneal injection.

3.27 P.M. Vomiting; claws mouth.

3-29 P.M. Spasmodic twitching; retching; jumping; gagging;
squealing.

3.30 P.M. Defecation; vomiting; lies on side. Cannot right itself.

3.32 P.M. Respiration very irregular.

3-33 P.M. Short, recurring tetanic spasms.

3-35 P.M. Respiration shallow and infrequent; spasms _ stop.
Trembling.

3.36 P.M. Perfectly limp.

3.38 P.M. Infrequent, spasmodic respiration.
3.40 P.M. Muscular spasms.

3.41 P.M. Respiration, 17 per minute.

3.44 P.M. Lid reflex absent.

3.45 P.M. Tetanus.

3-46 P.M. Dead. MHeart in complete diastole. Peristalsis of the
intestines very marked.

Effects of Alkaloids of Zygadenus Intermedius. 323

Experiment No. 2. Jan. 30, 1911.— Male guinea-pig; weight, 352 gm.
Dose: 0.0037 gm.; volume, I.5 C.c.

Subcutaneous injection.

Insalivated.

Vomits a little.

Vomits repeatedly.

Labored respiration interrupted by rapid respiration.

Control of limbs is lost. Labored and slow breathing.
Respiration, 49-50 per minute.

Slight spasm.

Respiration very irregular; heart fast. Spasm.
Respiration, 24 per minute; sometimes nine seconds

elapse between breaths. Tetanic spasms; heart rate, 81 per minute.

Respiration, 23 per minute; still tries to right itself

although unable to control limbs.

Spasms.
Tetanus in back muscles when touched; very bad

Heart rate, 42 per minute.
Respiration, 29 per minute; lid reflex present.
Very acute spasms; tetanus continues for three minutes

Acute spasms; lid reflex present.
Spasms; great respiratory distress.
Hyperpneea alternating with apneea.
Spasms; tetanus; defecation.

Heart rate, 48 per minute.

Spasms; defecation; much urine passed.
Heart very slow.

Spasms; eyes closed. Reflex present.
Respiration, 32 per minute.

Spasms; respiration, 46 per minute.
Respiration, 23 per minute.

Acute spasms; defecation.

Spasms; tries to gain feet.

Copious flow of urine.

Acute spasms; spasms now follow slightest stimulus.

4.06 P.M.
4.09 P. M.
4.11 P.M.
4.13 P.M.
4.39 P.M.
Spasm.
4.40 P.M.
4.41 P.M.
4.42 P.M.
4.44 P.M.
4.46 P.M.
4.49 P.M.
4.52 P.M.
4.53 P.M.
spasms.
4.58 P.M.
5.00 P.M.
5.02 P.M.
without stopping.
5. ee, M.
BLAS Ps iM.
pe] (Pe Me
5.21 P.M.
5-29 P.M.
5-32 P.M.
e325. NA
5-35 P.M.
5-39 P.M.
5.42 P.M.
5.54 P.M.
5-55 P.M.
5-55 P.M
6.01 P. M.
6.12 P.M.
6.30 P. M.

Regained control of fore limbs. Spasms no longer

follow stimuli.
6.57 p.M. Labored and spasmodic respiration.

324 Philip H. Mitchell and George Smith.

6.59 P.M. Respiration easier.

7.01 P.M. Heart beat, roo per minute.

7.10 P.M. Vomits again. Appears nearly recovered.
Jan. 31, 1911. — 9.00 A.M. Entirely recovered.

III. The effects on frogs. — The effects of administration to frogs
were not very striking. It was thought that because of the tetanic
spasms in guinea-pigs this poison might affect the spinal cord some-
what as strychnine does. Experiments with frogs did not fulfil that
expectation. Two injections into the dorsal lymph sac of frogs with
the brain pithed in which 0.175 gm. of material was administered in
one case and twice that amount in the other caused some slight irrita-
bility, but the animal soon became entirely limp and unresponsive to
stimuli. To another frog about 0.1 gm. was injected without pith-
ing any part of the nervous system. Some twitchings and spasmodic
movements occurred, but in a few minutes the animalseemed paralyzed,
gave no further response to stimuli, and soon died. In all three frogs
there was a noticeable reddening of the skin, and the blood vessels in
the web of the foot seen under the microscope appeared to be dilated.

IV. The effects on dogs. — To more accurately trace the action of
the alkaloidal preparation, intravenous injection into dogs was em-
ployed. The animals, seven in all, were anesthetized in each case
with ether followed by A-C-E- mixture. The alkaloidal material
diluted with physiological salt solution was injected into the right
femoral vein, while a tracing of blood pressure in the left carotid
artery was recorded by the aid of a mercury manometer. A record of
respiratory movements made by using a simple cord and pulley de-
vice to connect a recording lever with a skin suture in the thoracic
or abdominal wall of the dog was simultaneously obtained.

The first injection invariably caused a fall in the blood pressure
within a few seconds. Its amount was roughly proportional to the
amount of material injected. 0.0035 gm. of the alkaloidal sulphate
given in r c.c. of a fifty-fold dilution of the original preparation to a
dog of 13 kilos caused a fall of blood pressure equal to 41.6 mm. of
mercury, while the injection of 0.0105 gm. in 5 c.c. of the same dilu-
tion in a dog of 20 kilos produced a fall of 82 mm. The proportion-
ality, however, was not always maintained. The quickness of the
recovering rise also depended on the amount injected, and if more
than ro mgm. of the alkaloidal sulphate was given the recovery was

Effects of Alkaloids of Zygadenus Intermedius. 325

very slow. In no case did the blood pressure ever return to quite as
high a level as that recorded before the first injection. The cause of
this depressor effect is not a simple one. The most potent factor in
the initial drop is the marked slowing of the heart rate. That this
is due for the most part to an effect on the cardio-inhibitory centre
appears from the fact that after section of both vagi an injection of
the quantity usually employed did not cause the usual striking de-
pression of the heart rate but only a very slight decrease. A second
factor involved in the fall of blood pressure is vaso-dilation. Even
when the pulse had quite recovered to its initial rate as seen in several
tracings, the blood pressure was found to be still low, and only re-
covering so slowly that after some half-hour’s observation it did not
reach its original level. Also, the arterial pressure fell too much
when injections were given after double vagotomy to be accounted
for apparently by the slight slowing of the heart.

The effect on respiration is, in general, to slow it by a prolongation
of the expiratory phase. This effect is slightly modified after the
vagi have been severed.

A tracing to show the effect of injection without previous vagotomy
is given (Fig. 1). The tracing seems to show that the alkaloid acts
on the cardio-inhibitory and respiratory centres, or at least produces
effects which involve them.

The results so far discussed are those obtained by an initial injec-
tion of small quantities of the alkaloid. Subsequent injections or
the administration of larger quantities produced more complex effects.
Second, third, and fourth injections of quantities comparable to that
ordinarily used for our first, produced a successively smaller effect in
decreasing the already lowered blood pressure. After a considerable
quantity, for example, 0.0665 gm. had been given, a compensatory
hastening of the heart rate produced a slight rise in blood pressure,
and this effect was obtained after section of the vagi with compara-
tively small quantities of the alkaloid. Fig. 2 shows the effect of in-
jection of 0.0105 gm. into a dog of 8.2 kilos after a previous injection,
about twenty minutes earlier, of the same quantity. The rise in blood
pressure amounts to 36 mm. of mercury, and the change in the heart
rate is from 117 per minute before the injection to 157 per minute
shortly after. In this case the vagus nerves were severed at the be-
ginning of the experiment and the first injection gave a slight fall in
pressure.

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uf a ag gt

da

326

Effects of Alkaloids of Zygadenus Intermedius. 227

ty

i)

WATT

UME

faannnnnel

R
EP

Te

Tracing of carotid pressure partly superimposed on respiration curve

Figure 2.— Effect of a second injection, after section of the vagi.

At A 0.0175 gm. of alkaloid in 5 c.c. of saline

Time = 0.5 sec.

because the pressure was lowered from the effects of the first injection.

was injected.

When sufficiently large quan-
tities of the alkaloid had been
administered to a dog, its heart
showed an effect distinctly dif-
ferent from that produced by
the previous injection of small
quantities. Instead of the de-
crease of the heart rate with
increase in the force of the beat
seen in the initial injections or
the increase in rate with slight
decrease in force observed after
further injections, there ap-
peared in the more advanced
stages of poisoning a marked
fluttering of the heart. A trac-
ing of such an effect is shown
in Fig. 3. Sometimes the heart
action as shown by the blood
pressure curve would produce
a series of very irregular beats
interrupted at quite regular in-
tervals by groups of three or
four regular but very quick,
shallow beats.

The effects of larger quan-
tities of the poison on respira-
tion were also noteworthy. In-
stead of the mere slowing of
respiration, there appeared a
considerable irregularity char-
acterized by movements more
or less convulsive. This effect
in a mild degree can be seen
im fie. 2:

One of the most interest-
ing effects of this substance
was that on intestinal peristal-
sis. Even the smallest dose
administered, 0.0035 gm. given

328 Philip H. Mitchell and George Smith.

intravenously to a dog of 20 kilos, caused in a few minutes un-
mistakable intestinal rumblings. In all the dogs used defecation
occurred several times, even though stools had previously been passed
at the time of etherizing. In the course of an hour, after three or
more injections of small quantities, fluid or semi-fluid feces were

BP
isnt Ng yen i

breve EMIT

wn

bs nx a Baba)

Ficure 3.— The effect on the heart of cumulative action of the alkaloid. Upper trac-
ing shows carotid pressure, lower one respiration. Time = 0.6 sec. At A 0.0175
gm. of the alkaloid in 5 c.c. of saline was injected. The vagi were intact. By
means of three previous injections, 0.0315 gm. of the substance had been given.

observed in each case. Micturition also took place in many of the
dogs during the experiment. This involuntary evacuation of in-
testines and bladder in anesthetized dogs was plainly the result of
the action of the alkaloid, because effects followed quickly after in-
jection and in the case of defecation were proportional to the amount
given. Such results confirm our observations with guinea-pigs. The
influence of the substance on the vomiting mechanism of dogs was
not tested, as in guinea-pigs, because no injections were made with-
out previous anesthesia.

Several of the dogs while still anesthetized were killed by intra-
venous injection of a fatal dose. In each case the heart beat became
very irregular and soon stopped. Invariably the heart failed before
respiration. At the moment respiration ceased, however, the dog
passed into a death struggle exhibiting tetanic spasms of the entire

Effects of Alkaloids of Zygadenus Intermedius. 329

body. The convulsion was always brief, and though in two cases fol-
lowed by a few spasmodic respiratory movements was never, so far
as we observed, succeeded by any revival of the heart beat. The
heart was found in the opened thorax in complete diastole, as was
seen also in post mortem examination of the guinea-pigs.

No attempt to determine the fatal dose for dogs was made. The
smallest quantity which gave a fatal result was 0.105 gm. in 3 C.c.
of saline injected intravenously into a dog of 10 kilos without previous
injections.

Our experiments have led to the following conclusions:

t. The alkaloidal preparation from Zygademus intermedius slows
the heart rate by action apparently on the cardio-inhibitory centre.

2. It slows respiration by an effect involving the respiratory centre.

3. It causes vaso-dilation.

4. In quantities approaching the fatal dose it hastens the heart?
rate and produces both irregularity of the heart beat and convulsive
respiration.

5. The fatal dose given intravenously to dogs stops the heart be-
fore respiration ceases.

6. The fatal dose for guinea-pigs is between 4.6 and 5.1 mgm. per
100 gm. of animal.

7. It has a very powerful action, whether injected or fed, both as
a purgative and an emetic.

The authors wish to acknowledge with thanks the assistance of
Mr. H. A. Swaffield, who aided in some of the experiments.

107

SEASONAL VARIATION IN THE BACTERIAL CONTENT
OF OYSTERS.

BY FREDERIC P. GORHAM.

American Journal of Public Health, Vol. II, Old Series, Vol. VIII,
1912, pp. 24-27.

‘ va We a iat J

i

Reprinted from American Journal of Public Health, January, 1912.

SEASONAL VARIATION IN THE BACTERIAL
CONTENT OF OYSTERS
FREDERIC P. GORHAM,
Brown University.

In the course of the investigation of the sanitary condition of the oyster
layings in Narragansett Bay and its tributaries by the Rhode Island Shellfish
Commission,* it became apparent that analyses of oysters from a certain bed
made in the summer did not agree with analyses of oysters from the same bed
made in the winter. With the advent of cold weather there seemed to be so
great an improvement in the sanitary quality of the oysters that oysters taken
from beds in close proximity to the outfalls of large sewers showed in the colder
months entire absence of any evidence of contamination, if judged solely by the
results of analyses.

The question of the cause of this improvement during the winter at once
arose. Did it mean that the bacterial content of the salt water varied with the
season to such an extent as to cause this difference, or did it mean that the bac-
terial content of the oysters themselves varied at different seasons?

An investigation was immediately begun to answer these questions, if
possible. The work of the summer of 1910 showed that all of the oysters on
beds in the Providence and Warren Rivers and in the upper parts of Narra-
gansett Bay were so badly polluted by sewage that they would be condemned as
unfit for food even when judged by the most lenient standards. The analyses of
oysters from these beds showed the presence of the colon bacillus in the shell
water of every oyster in amounts as small as one one-hundredth of a cubic centi-
meter and even less. Analyses of the water, both chemical and bacteriological,
showed it to be heavily charged with sewage.

In December, 1910, it appeared that oysters from these polluted areas gave
on anatysis very different results. Indeed it was impossible to find a single laying
in any of these localities which would not pass inspection if judged by the anal-
ysis alone. The condition of the water however seemed to be unchanged.

In December, 1910, two of these Providence River beds, numbers 8 and 44
on the 1919 map of the Rhode Island Shellfish Commission were selected for
the purpose of studying the results of analyses made during the succeeding five
months, or until the waters warmed up and assumed their normal summer condi-
tion. Similar studies were made of two other beds in the Warren River, num-
bers 204 and 205, for ihe purpose of camparison with conditions in the Provt-
_ dence River because the City of Providence began during this period the routine
disinfectioa of its sewage by means of bleaching powder. But as the sewage from
the City of Providence was not the only source of pollution in the Providence
_ River, later examinations showed that this disinfection of Providence sewage had
_very little effect on the total colon content of the waters of the river. Bac-

¥ + Read before the Laboratory Section of the American Public Health Association, Havana, Cuba,
December, 1911.

; * See Preliminary Report upen the Sanitarv Condition of Rhode Tsland Ovster Beds, by FP.
Gorham, in the Annual Report of the Rhode Island Commissioners of Shell Fisheries made at the

January Session of the Legislature, 1911.

2 THE AMERICAN JOURNAL OF PUBLIC HEALTH

terial analyses of the water over these beds were made at the same time
that the oysters were examined, and records were made of the temperature and
density of the water, conditions of the tides, winds, etc.

Bed number 8 is about one-third of a mile from the main sewer outlet of
the City of Providence which discharges an average of 21,000,000 gallons of
disinfected sewage per day into a river already heavily polluted. Bed 204, in the
Warren River, is nearly opposite the town of Warren which discharges its
untreated sewage into the river by several small sewers. Bed number 44, in the
Providence River, and bed number 205, in the Warren River, are some distance
below any direct sewage contamination, but the results of the analyses made
in the summer showed these to be so badly polluted that they would be con-
demned under the standards adopted by the State of Rhode Island and the
United States Pure Food Board.

All analyses were made according to the Standard Methods adopted by the
Laboratory Section of the American Public Health Association. The average
total count gives the total number of bacteria present in the shell water of the
five oysters examined in each lot. The presence of B. coli in the five oysters
examined was tested by the bile tube and subsequent isolation and identification
of the organism.

SEASONAL VARIATION IN THE BACTERIAL CONTENT OF OYSTERS.

Average Total Proportion of Five B. coli Temperature
Bacterial Count Oysters showing Score. present of water,
Date. of Shell Water B. coli in in water
of Hive Oysters cc. | O FLCC, O!OLcc: in
Bed No. 8. Providence River.
DEC 20; TOTO on oe 1000 3 I o 4 O.OICC. —I°
(janenla etOGleer cee 750 5 3 I 41
fanyeo Ge one ey ens 80 4 3 fe) 23 0.OIcc. To
ame 27 Stet h. Sets tiiete 23 5 3 te) 32
Hie be elOget es tiers cise tices 130 2 2 fe) 4 LCC Ont
Hebe 2O meee ey 140 oO fe) oO fe) 0.0001 cc. Ta
Marsares: aay te 200 5 4 ) 4I 0.0Ice. 75s
Atpralma nice rrck de ne 275 5 2 fo) 23 O.0Icc. 8.5°
Aprila8. si2css ee 700 5 5 4 410 0.000Icc. 12oe
Mia yal obe coe sree erent 1700 5 5 5 500 0.000T cc. T5
Bed No. 44. Providence River.
amt 7 A WOM to cs. vers 425 5 5 I 140 0.25°
HebrelOe.:oteaacoocar 250 4 oO Oo 4 On
Hebe 2Ou se eieter- cis site 240 5 I (a) 14 0.5°
IMIG aE. go dlosn.a alec 100 5 2 (0) 23 2s
Apmiligia’) aac cutie ee ca 210 2 0) to) 2 8.5°
Aprilia oct. 1000 5 5 4 410 Dr 75e
MEN PA sacs das amooe II00 5 5 4 410 14.75°
Bed No. 204. Warren River.
Jains DR, Wow coon adore 600 5 4 I 50 Ow
HED oRIO We oatanre eee 140 oO fe) oO fo) Ice. Oy
eb 2p ey ado arclcieice 400 ) ) oO ) 0.0ICcC., 0.75°
Manchiateccisa: ace 750 aii Siti OT ii 0.75°
iMiarchimiiraess eee 60 I 0) fo) I 0.OICe. .
NMiarchigiAwenem crete 3400 e) fe) ) (0) 0.0Icc. Sse
INDUS. aaa ada io 1050 5 iS 4 410 0.0TCC. ing
Bed No. 205. Warren River.
DEG N22 IO LO- Aer 250 3 (0) oO 3 —I°
IES, TO MCN a sch youn 32 fo} fo) fo) (e) mcs On
Bebe 2si ser eee 450 4 B oO 14 O.OTcc. if
Migimeln 2h on Ss cee esc oe 600 2 2 I 5 0.75°
Nia cheeiinn a manne ace 85 2 I fe) 3 O.0Ice. 2e
April A eer. ee et 325 I I Co) 2 0.01cCc, 8.25°
Ap mili Senora eee 4000 5 5 5 500 0.OICe. Diese

+ Only three oysters used.

|

SEASONAL VARIATION IN THE BACTERIAL CONTENT OF OYSTERS 3

Five oysters were examined in each case and the accompanying table shows
the number in which B. coli was found in one, one-tenth and one one-hundredth
cubic centimeter amounts of the shell water. The score of the several lots of
oysters is the numerical method of indicating the results of the colon test as
adopted by the Committee on Standard Methods. A perfect score, that is no
colon bacilli found, would be zero, while if every oyster of the five showed colon
bacilli present in one one-hundredth of a cubic centimeter of the shell water the
score would be 500. The standard adopted by the United States Pure Food Board
is that all oysters showing a score of 32 or above be condemned. The tempera-
ture of the water ranged from —1 degree C. to 15 degrees C., from the low tem-
peratures of the winter to the higher temperatures of the spring.

The conclusions to be drawn from these observations seem to be somewhat
as follows: The number of B. coli in the water of these rivers during the win-
ter apparently does not decrease and this organism is present practically all the
time with such variations as we might expect with tidal conditions. More ex-
tended series of observations might show that the temperature had some affect
on the colon content but this is not apparent from these investigations.

The most interesting and important results are seen in the B. coli content of
the shell water of the oyster. During the cold weather the number present varies
somewhat as might be expected in the examination of different lots of the same
oysters, but is never very high, and in most cases these oysters would pass the
standard required by the government and state authorities. But just as soon as
the water begins to warm up the number gradually increases, until with the
warming of the water to 12 or 15 degrees the number reaches the maximum,
and practically every oyster shows the presence of B. coli in one one-hundredth of
a cubic centimeter. This is the condition which holds throughout the summer.

We must conclude then that the reduction of the number of B. coli in the
oyster in cold weather is not due to any decrease in the number present in the
seawater but is due to some peculiar condition in the oyster itself.

The only reasonable explanation of such a condition to my mind is that
with the advance of the cold weather, the oyster assumes a condition of rest or
hibernation, during which time the ciliary movement ceases, the current of water
over the gills stops, and the process of feeding is suspended. No organisms are
taken in from the water outside, and those inside are gradually eliminated so
that the total number of organisms is reduced very considerably and the oyster
becomes practically free from B. coli.

These observations enable us to give a reasonable scientific explanation for
the common belief that oysters are not fit to eat in the summer time, but improve
greatly in the winter. Perhaps it would be well, as the result of these observa-
tions, to limit the oyster season even more than at present, particularly in the case
of oysters from polluted areas, by excluding the months of September and Oc-
tober, March and April, from the open season.

It is a fact well known to all oyster growers that there are two periods in
the year when oysters are fat and two when they are lean. Oysters all fatten up
in the late spring preparatory to the discharge of their sexual products in July
and August. After these products are discharged there is a period of leanness

| THE AMERICAN JOURNAL OF PUBLIC HEALTH

which continues until the advent of the colder weather, about November and
December, when the oysters fatten up again and are supposed to be in the very
best condition for marketing. It is at this time that they apparently store up a
reserve of material to carry them through the period of hibernation during the
rest of the winter.

Of course, these conditions would apply only to oysters in our cold northern
waters. There is some evidence to show however that a similar period of hiberna-
tion occurs in oysters growing farther south, but further observations are neces-—
sary to confirm this.

From these facts we must conclude that in warm weather the results of bac-
terial analysis tally very well with the actual conditions as determined by the
sanitary survey and therefore analyses may be used to determine whether or not
certain oysters may be sold for human consumption. But during the cold weather
oysters judged by analyses alone would be pronounced good although they came
from within a short distance of a larger sewer outfall.

We probably do not want to eat oysters which come from the immediate
neighborhood of sewer outlets, even though they appear on analysis to be free
from colon bacilli. Therefore, in order to exclude these oysters from the market
in the winter time, the only reasonable method would be to set definite limits
from sewer outlets within which it shall be unlawful to take oysters or other —
shellfish for use as food.

108

THE SANITARY REGULATION OF THE OYSTER
INDUSTRY.
BY FREDERIC P. GORHAM.

American Journal of Public Health, Vol. II, Old Series Vol. VIII, 1912,
pp. 77-84.

Reprinted from American Journal of Public Health, February, 1912.

THE SANITARY REGULATION OF THE
OYSTER INDUSTRY.

FREDERIC P. GORHAM,
Brown University.

There are two distinct questions involved in any attempt to regulate the
sanitary quality of shellfish. First, the question of protection of the public
against shellfish which are actually dangerous, that is, those which may contain
disease germs and therefore may be the cause of the spread of disease; and
second, the question of the protection of the public against shellfish which are
merely dirty, which, in the language of the United States Pure Food ruling,
consist “in whole or in part of a filthy, decomposed or putrid animal or vege-
table substance,’ which is interpreted as meaning shellfish taken from water
which contains sewage material even though this material has been so diluted
or disinfected that it carries no disease-producing organisms.

I believe that we should separate these two ideas in our minds very clearly.
For I am sure that we are all of the same mind in regard to the first question,
but in regard to the second, we may differ in our interpretation of what is meant
by “dirty.” And when it comes to the enactment of laws which shall properly
regulate these matters we should certainly consider the distinction between
dangerous and dirty. For what is dangerous should certainly be prohibited, but
where between the different degrees of dirtiness shall we draw the line showing
which should be used as food and which should not?

In regard to the first of these questions, the protection of the public health,
we have some accurate scientific data on which to base our opinions. Let me
briefly review them.

Many shellfish, oysters in particular, are often eaten raw. These shellfish
are sometimes taken from sewage-polluted waters and contain therefore the
organisms which are present in sewage. These organisms may in certain cases
be pathogenic. As long ago as 1880 it was suspected that shellfish might occa-
sionally be the cause of epidemics of typhoid fever. Observations have been
continued since then and now we have on record several well-authenticated cases
where typhoid fever epidemics have been traced to the eating of raw polluted
shellfish.

But we must consider all the facts in these cases and be perfectly fair in
our conclusions. We must not condemn altogether the eating of raw shellfish,
thus denying ourselves the enjoyment of a most delectable delicacy, and at the
same time doing damage to a very large industry, until all the evidence is in.
For as far as our knowledge goes at present no epidemics thus far studied have
been due to eating shellfish grown in polluted waters, but in every case to eating
shellfish removed from their growing beds and “floated,” “fattened” or “fresh-
ened” in polluted waters. The reason for this is apparent at once. Beds on which
oysters are grown are rarely situated directly at the mouths of sewers. While

Read before the Massachusetts Association of Boards of Health, January 25, 1912.

2 THE AMERICAN JOURNAL OF PUBLIC HEALTH

the presence of a little sewage in the water may hasten the growth of oysters,
any considerable amount of it exhausts the oxygen, deposits a smothering mud
upon the bottom, and makes it impossible for oysters to live. The typhoid or-
ganism is not an hardy one. It probably lives but a short time under the unfav-
orable conditions found in sea-water. Those that do survive are so diluted in
the enormous amount of water ebbing and flowing with the tides, that few if
any of them reach the oyster beds. And if a few should reach the oysters I
have an idea that they would be digested and destroyed by the feeding appara-
tus of the oyster himself long before such oysters reach the market.

We have evidence which bears on this point in the City of Providence. The
“floating” of oysters is not practiced in Rhode Island, and all oysters come di-
rectly from the growing beds. Until last winter there was nothing to prevent
the use of oysters from beds in the upper part of Narragansett Bay and from
the Providence River, which were badly polluted with sewage. Yet in 1910
Dr. Chapin wrote in regard to Providence: “raw oysters are very popular; they
are consumed in restaurants in large numbers, and form a course in a large
proportion of banquets and dinners. Many oysters are grown in the upper part
of the bay in water grossly polluted with sewage and in the water and in the
oysters colon bacilli are found. Yet Providence has a typhoid death-rate less
than half that of the average American city. Oysters are not eaten to any extent
in August when typhoid fever begins to increase and they are largely consumed
in winter and spring when there is little of the disease. Very few oysters are
eaten by laboring people, but at present laboring people furnish fully their share
of typhoid fever.”

Taking these facts altogether, then, it seems to me that there is no evidence
whatever that oysters taken directly from their growing beds, even though these
beds may be polluted by sewage to a certain extent, have ever caused disease.

But we cannot say the same of “floated” oysters. It is to them that the
finger of suspicion points. For here we have very different conditions. Here
we find instances where oysters have been placed directly at the mouths of
tvphoid-laden sewers or in typhoid-laden creeks for hours just previous to sale.
No better scheme ‘for the direct infection of such shellfish could be found unless
the germs were inserted directly between the shells.

As to whether or not the practice of floating in itself is a proper one 1s a
subject which has been hotly discussed for some time. But in regard to floating
in polluted water there can certainly be no difference of opinion.

The arguments usually advanced in favor of floating are that it removes
the grit and mud and slime from the interior of the oysters and improves their
flavor and keeping qualities. Those advocating the practice usually fail to add
also that it bloats and swells the meats by the absorption of a large amount of
water which can then be sold at the price of oysters. It has been said that the
profit from this increase in bulk is often sufficient to pay all expenses of opening
the oysters. From this standpoint then floating constitutes fraud and adulter-
ation under the Pure Food and Drugs Act. Those who are unbiased in their
opinion all agree that the only real advantage gained by floating is the cleansing
from mud and slime, and this can be accomplished just as well by holding the
oysters in clean salt water for a while. The fact that it is practically impossible

SANITARY REGULATION OF THE OYSTER INDUSTRY oO

to find any stream of fresh water which is free from all pollution should deter
any conscientious oyster grower from carrying on this practice. That floating
is indeed dangerous and accomplishes nothing in the improvement of the stock
is recognized by the Pure ood Board because they are at present considering
the withdrawal of their sanction of the practice in any form and prohibiting it
altogether.

I think you will all agree with me then in answering the first question, when
I say that as far as the public health is concerned, for the present at least, all
that is necessary is to prohibit this practice of floating oysters. Until further
evidence is produced oysters taken directly from the beds on which they are
cultivated may be considered safe in practically all cases.

Investigations have not been carried far enough as yet to say whether the
same applies to clams, quahogs, and mussels or to the natural set of oysters
which are occasionally found in small quantities in heavily and directly polluted
waters.

This brings us to a consideration of the second question. How much sew-
age pollution, not in itself dangerous, but merely dirty, shall we permit in our
oysters or other shellfish before we prohibit their sale? This opens up the
whole matter of standards of purity. Think of the years of discussion on this
subject in our studies of water supplies. Shall colon bacilli in 1 cubic centimeter,
10 cubic centimeters, or 100 cubic centiemters condemn water? It was only when
the sanitary survey came to our rescue that we were able to answer this question
satisfactorily. We are at present trying to work out standards in our milk
analyses. Shall it be 500,000 bacteria per cubic centimeter, 100,000 per cubic
centimeter or 10,000 per cubic centimeter? The conditions which enter into
the shellfish question are just as complicated as in water and in milk. Before
satisfactory laws can be passed regulating the industry much more light must
be thrown on the subject than we have at present.

At first the Pure Food and Drug Board adopted the practice of condemning
a lot of oysters if three out of five examined showed colon bacilli in one-tenth
of a cubic centimeter of the shell liquor. We in Rhode Island followed their
lead, and under the law passed at the January session, 1910, we passed or con-
demned oyster beds in Narragansett Bay by this standard. This gave us re-
sults which coincided with the conditions shown by the sanitary survey.

The examination of beds was made in the summer so that the certificates
could be issued at the opening of the oyster season in the fall. But on examin-
ing some of these beds again later in the winter it was found that if this same
standard was used every bed in Narragansett Bay and Providence River would
easily pass inspection. Indeed beds in immediate proximity to sewer outlets
showed the absence of colon bacilli in all five oysters examined even in cubic
centimeter amounts of the shell liquor, a far better result than that given by
oysters from far down the Bay, beyond all sources of immediate pollution during
the summer.

Obviously the same standard could not be used in winter as in summer, and
the question immediately arose ought we to prohibit the use of oysters when
they come from near a sewer outlet when analyses show them to be as free from
the colon bacillus as many of our best drinking waters? Simply because they

| THE AMERICAN JOURNAL OF PUBLIC HEALTH

are in polluted water should they be condemned though analysis shows them to
be better than what we consider the best oysters in the summer? Under such
conditions our sanitary survey fails us. We have no standard by which to judge
the purity of such shellfish. We are at a loss as how to proceed. Must we have
a separate standard for summer and winter?

Of course in a measure we have this now, for no very considerable quantity
of oysters is marketed at present in the warmer weather. The old idea as to
the oysters being fit to eat only in the R months seems in a way to be borne out
by this observation of seasonal variation in the bacterial content.

Almost the same question has arisen in regard to the use of oysters grown
in waters into which disinfected sewage is discharged. Disinfected sewage is
not dangerous but is it dirty? Ought such oysters to be used for human food?

I believe that the only satisfactory way to handle this matter is to adopt a
certain distance from discharging sewers within which it shall be unlawful to
take shellfish for food.

These are some of the problems which are troubling us at present. But
while we are working at them a very considerable amount of good has already
been accomplished in the improvement of the industry along sanitary lines.

In the first place we have seen placed on the statute books of the State of
khode Island laws which regulate the industry and protect it. Pollution of the
waters of the State in which shellfish are grown is prohibited under severe pen-
alty. The State Shellfish Commission must make examinations of and issue
sanitary certificates for any bed on which oysters are grown for consumption as
food before such oysters can be taken from the beds and sold.

The Commissioners are required to inspect all opening and packing houses
and the methods in use therein and must issue certificates as to their sanitary
condition before shellfish can be sold therefrom. They are also given the power
to make any regulations concerning the construction and operation of such oyster
houses as they may see fit in order to place them in proper sanitary condition.

As a result of such legislation we have been able to make a series of analy-
ses of oysters from all the beds in Narragansett Bay and its tributaries at differ-
ent seasons; also chemical and bacteriological analyses of the water at the sur-
face, at the bottom, and of the mud at the bottom of a considerable portion of the
Bay; a study of the tides, currents, temperatures, and densities of these waters;
and a complete sanitary survey showing every sewer outlet, every source of
pollution entering the waters of the Bay.

In addition to this, the oystermen have been awakened to the seriousness of
the matter. Because of the activities of the United States authorities and those
of the State, they have been forced to examine into their methods of conducting
business and to make every effort to improve their methods along sanitary lines.
They have co-operated nobly in the matter and have helped to bring about a
complete revolution in their business, which has in many cases involved changes
from methods which were as old as the industry itself.

But best of all has been the effect this campaign for clean shellfish has had
upon the entire removal of some of the sources of pollution from the tidewaters
of the State. Ultimately, of course, this is the goal which we seek, the return
of our bays and streams to their earlier condition of purity. It may be a long

SANITARY REGULATION OF THE OYSTER INDUSTRY D

time indeed before this can be accomplished, but we are glad to record that a
beginning has been made.

The Commissioners of Shellfish summoned before them the parties inter-
ested in sewage disposal in the several cities and towns which were using Narra-
gansett Bay as a common sewer. At this hearing they were required to tell what
efforts they were making to tomply with the law which prohibited the pollution
of the tidewaters. They were informed that they must take steps immediately
to remedy the conditions. As a result already some of them have moved in the
matter. The City of Providence is already conducting an investigation of its
methods of sewage disposal and has begun the disinfection of its effluent. Other
towns are doing something. Much remains to be done. Rhode Island even has
a bone to pick with Massachusetts, for there seems to be some interstate traffic
in sewage which we hope in time to see remedied.

In conclusion, then, were I asked along what lines at present can we best
effect improvement of the shellfish industry I would say:

1. Prohibition of the practice of floating oysters.

2. Prohibition of the use for food of shellfish taken from waters directly
polluted with sewage, that is, within a certain distance of discharging sewers.

3. Careful sanitary regulation and supervision of the methods of handling
and packing shellfish.

4. Efforts to secure the removal of sewage pollution from the tidewaters
where shellfish are grown, or at least the disinfection of all sewage effluents

which enter tidewater. é
Discussicn.

Prof. Wurpepte. The subject of oyster sanitation is a most interesting one
and it may interest you to know that a committee has been studying the subject,
in the American Public Health Association, for the past year or two. A pre-
liminary report was made at Havana a few weeks ago and this will be published
in due time. (This Journal, January, 1912.—Ed.)

What Prof. Gorham has said in regard to the winter conditions of oysters
seems to be true not only of oysters in the Providence River but also of oysters
found in this country farther south. During the winter season the bacteriolog-
ical analyses of all oysters seem to be better than during the summer season.
It looks as if the oyster was a more intelligent being than we have given him
credit for, because, knowing that we appreciate his society only in the winter,
he apparently takes particular care to protect himself against sewage and other
filth during this season in order that he may not do us damage or injure his
own reputation. I have sometimes wondered why, if that is the case, it is
necessary to go to the expense of disinfecting sewage in the way that Prof.
Phelps has outlined. If the oyster protects himself why is it necessary to
spend money to further protect him? In reply, there is this to be said,—that
the oyster is no Pharisee. Neither during the winter, nor at any time, does
he attempt to keep clean the outside of his shell and there is a real danger at
all times that polluted mud that has not been thoroughly washed off may get
into the half shell in which the oyster is served. Many oysters have a small
cavity near the hinge which is difficult to clean and which is often filled with
mud when served. If this mud gets into the bowl, as it may easily do, there is
a danger that it may also get into one’s mouth.

6 ’ THE AMERICAN JOURNAL OF PUBLIC HEALTH

Then of course there is the further question of general cleanliness. No
one desires to eat food that is unclean. Our committee is trying hard to devise
some practical standard which can be applied to bring this about. Prof. Gorham
has marked out some of the ways in which this can be done. One other
method has been suggested and the committee is working along this idea at the
present time and will doubtless report upon it later, viz., a method of “scoring”
the oyster beds and the oyster houses, very much as milk farms are now
scored. By this method some appropriate authority would give a good score
to the oysterman who raises oysters on clean grounds and who harvests and
markets his oysters in a clean way, and a low score to the man who is careless
in handling his goods and grows his oysters very near a sewer.

I think the whole question of the sanitation of the oyster is one that is
rapidly clearing itself up. To some it may seem to be growing more and more
complicated, but now that the oystermen are co-operating with the sanitarians
we are learning many facts hitherto unknown, and I think that in a short time
we shall be able to establish certain standards that will satisfy the sanitarians
and that can be lived up to by the oyster growers.

Mr. Wittrams. When IJ accepted the invitation of my local board of health
to attend this meeting I knew that I would be greatly rewarded by listening
to the papers that were to be read here, and | was especially interested, of
course, in the paper that Professor Phelps was to read. I have had a number
of very interesting meetings with Professor Phelps and Professor Sedgwick
with regard to the disinfection of sewage.

New Bedford, as you may know, has been doing its best to become a large
city and in doing so it has had to meet a few of the ills that come with
municipal growth and development, one of which is the very serious pollution of
the river upon which it is located. This body of water which we call the Acushnet
river, is really only a tidal arm of Buzzard’s Bay. But we have succeeded in
polluting it to such an extent that a few years ago the State Board of Health
prohibited the further taking of shell fish from its waters. These shell fish, of
course, did not include oysters. They did include the famous little neck clams,
which, I understand, we have distributed to quite a considerable extent over
various parts of the country.

In order to relieve ourselves from a very serious situation and to save a
business which furnishes employment to a great many of our citizens the city
undertook, two years ago, to provide for a further disposal of its sewage. We
have now started upon the construction of an intercepting sewer which will
collect all the sewage of the city and deposit it at a point in Buzzard’s Bay
something over half a mile from the most extreme point of land in New Bed-
ford. Furthermore the sewage will be delivered in water about 30 feet deep
at low tide where it will also come in contact with the great tidal movement
of the water of Buzzard’s Bay.

When we announced our intention some of our neighboring shore towns
became seriously alarmed as to what the effect might be upon their shores, and
so, to relieve them of any cause for alarm, we consulted with Professor Sedg-
vick and Professor Phelps and thereby learned of their great interest in the
disintection of sewage. Disinfection of sewage, of course, has been generally

SANITARY REGULATION OF THE OYSTER INDUSTRY 7

known about in one way or another for a good mnay years, but up to this time
none of us knew of any practicable way of disinfecting raw sewage.

You probably understand that our situation was such that we could not
very well introduce sand filters, because we didn’t have the sand areas suitable
for the purpose, and contact filters were also a very expensive method of treat-
ment for a city situated as we are. And so we have decided that the proposition
of Professors Sedgwick and Phelps was worthy of our consideration. We
have gone into the subject to a considerable extent; and we have decided that
we will provide at a point near the outlet of our intercepting sewer for disin-
fecting the crude sewage by the addition of chloride of lime, or ordinary bleach-
ing powder if later it is definitely decided to adopt this method of treatment.
While our plant is not yet built we are preparing plans with that object in view.

Gentlemen, that, in a few words, places New Bedford’s position before
you. I thank you very much.

Prof. PHetps. There have been, Mr. Chairman, two things said here about
the oyster that I want to say do not apply to the clam. First, Professor Gorham
says the oyster loves clean waters and will not grow near sewers. That cer-
tainly does not apply to the soft shell clam. Then Professor Whipple tells us
the oyster shuts up and fasts in winter. That, according to my observation,
the soft shell clam does not do. And so, however we may remove suspicion
from the oyster, I think this clam situation is going to still be with us. It is
certainly true that the clam is inherently a filthy beast. He seems to prefer
polluted waters and grows fatter and faster in them. The oyster may be wise
and we may possibly trust to his discretion, but the clam, proverbially happy,
needs the guardianship of the State to keep him from falling into evil ways.

Mr. STEPHEN DeM. Gace. I have been much interested in Professor Gor-
ham’s paper and am particularly pleased that he has drawn the distinction so
sharply between polluted or dangerous oysters and oysters that are merely dirty.
The shellfish situation in Massachusetts is quite different from that in Rhode
Island and in the States further south. In these States the oyster is the only
mollusk of any economic importance. The hard and soft clams apparently are
unable to grow south of New Jersey and the few beds of these clams which
existed at one time along the shores of Long Island Sound and in Narragansett
Bay have been practically depleted. Even the clams for the famous Rhode
Island clambakes are now brought in from Massachusetts sources. On the
other hand, north of Massachusetts, the oyster industry is so small as to be
negligible, but the gathering of hard and soft clams and of scallops is an im-
portant industry. Massachusetts lies intermediate between these zones and along
different portions of her coast all of these various types of shellfish are gathered
in greater or less quantities. In 1909, the Massachusetts Fish and Game Com-
mission published a special report on the Mollusk Fisheries in the State and
some statistics from that report showing the relative amount and value of these
different shellfish gathered annually may be of interest. The number of men
employed in the oyster industry was 159, in the scallop industry 645, in quahaug
or hard clam industry 745, and in soft clam industry was 1,360. The catch of
oysters is about 115,000 bushels per year valued at $148,000, of scallops 86,000
bushels valued at $164,000 of hard clams 144,000 bushels valued at $195,000,

8 rHE AMERICAN JOURNAL OF PUBLIC HEALTH

and of soft clams about 154,000 bushels valued at about $150,000. It will be
observed that the value of both the hard and soft clam industries is greater
than that of the oyster industry and that the number of bushels of hard and
soft clams produced is each much greater than the number of bushels of oysters
produced. Furthermore, there is a still greater difference in the food value of
these various shellfish owing to differences in thickness of shell and differences
in actual food value of their bodies. Computed on this basis the oyster industry
produces only about 8 per cent., while the hard clam industry produces about
17 per cent. and the soft clam industry produces over 37 per cent. of the food
value of the entire shellfish industry.

For more than ten years the State Board of Health has been studying the
question of the pollution of these various kinds of shellfish, and in certain
instances, areas which are dangerously polluted have been closed. The question
of the pollution of clam flats is the most serious problem, however, as not only
is the soft clam industry more important from an economic viewpoint, but the
flats from which soft clams are found are widely distributéd along the shores
of the State and as has already been stated, the soft clam will grow and thrive
in shallow water in close proximity to public sewers, while the other mollusks
mentioned are natural inhabitants of deeper waters where pollution is reduced
to a greater or less extent by dilution.

109

SUPERIOR SANITARY QUALITY OF RHODE ISLAND
OYSTERS.
BY FREDERIC P. GORHAM.
The Providence Medical Journal, Vol. XIII, 1912, pp. 46-53.

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SUPERIOR SANITARY QUALITY OF RHODE
ISLAND OYSTERS

By FREDERIC P. GORHAM, BROWN UNIVERSITY
Sanitary Expert, Rhode Island Shellfish Commission

It is a very serious matter when any suspicion is cast on the fair
name of the Rhode Island oyster. It is no wonder then that all lovers
of this most delicious shellfish were up in arms at once when Dr. Wiley
and his associates on the Board of Food and Drug Inspection descended
upon these Plantations and informed us that certain of our Rhode
Island oysters must not be shipped out of the State because they were
polluted with sewage.

We have known for some time that a constantly increasing amount
of sewage was being poured into Narragansett Bay, but we had failed
to realize that this sewage might injuriously affect the shellfish. In-
deed, as long ago as 1904 in a paper in this Journal by Dr. C. A. Ful-
ler* the conditions existing at that time in reference to the pollution
of the oyster beds was pointed out, and the suggestion made that
something should be done to prohibit the sale of polluted shellfish and
to prevent the further increase of the pollution. We did not realize
that the time had come for action until the Washington authorities
brought the matter most forcibly to our attention by prohibiting the
sale of certain Rhode Island oysters in interstate trade and haling
some of the oyster growers into court.

By a ruling of the United States Board of Food and Drug Inspec-
tion oysters which are grown in sewage-polluted waters, or which have
been fattened by the process of “floating” or “drinking” in sewage-
polluted waters, are prohibited from interstate trade because they con-
tain an added “poisonous or other added deleterious ingredient which
may render such article injurious to health” or because they consist “in
whole or in part of a filthy, decomposed or putrid animal or vegetable
substance.”

Whether such oysters would be the cause of disease depends
entirely on whether typhoid germs are present in the sewage. In

*Providence Medical Journal, 5, 1904, 39
Reprinted from THE PROVIDENCE MEDICAL JOURNAL, March, 1912

2)

every community of any size there is a possibility that typhoid germs
may be present in the sewage. It is true these germs die out rather
rapidly in the sewage, and particularly when it is discharged into salt
water. There is no evidence whatever to show that oysters taken
directly from beds on which they are grown have ever been so pol-
luted as to be the cause of disease; but there is very good evidence to
show that oysters which have been removed from these beds for pur-
poses of “floating,” and placed at the mouths of sewers or in waters
where the pollution is immediate and direct, have caused serious epi-
demics of typhoid. As the practice of “floating” is not carried on in
Rhode Island, there is no danger from this source. And there is no
evidence to show that there has ever been any disease caused by eating
oysters from Rhode Island waters. Indeed, Dr. Chapin in his recent
book on the “Sources and Modes of Infection” says, “raw oysters are
very popular; they are consumed in restaurants in large numbers, and
form a course in a large proportion of banquets and dinners. Many
oysters are grown in the upper part of the bay in water grossly pol-
luted with sewage, and in the water and in the oyster colon bacilli are
found. Yet Providence has a typhoid death rate less than half that
of the average American city. Oysters are not eaten to any extent in
August, when typhoid fever begins to increase, and they are largely
consumed in winter and spring, when there is little of the disease.
Very few oysters are eaten by laboring people, but at present labor-
ing people furnish fully their share of typhoid fever.”

But whether dangerous or not, oysters grown in sewage-polluted
waters are dirty and we do not want them on the market, so any rea-
sonable steps taken by the federal authorities to limit the sale of such
oysters will be welcomed by all advocates of pure and clean food.

When the United States authorities made the first move toward
condemning certain shipments of oysters and ordered certain dealers
to appear in court, the oyster growers of the state were immediately
aroused. They at once appealed to the Rhode Island Shellfish Com-
mission for assistance. This body is charged by the state with the
care of its shellfish industries. Last year the Shellfish Commission
paid into the treasury of the state $133,341 as rentals from the 20,846
acres under cultivation. The capital invested in the business was
stated by the last census to be $1,031,738, with a total value of all
products, shell and shucked oysters and seed, of $2,930,750.. In the
neighborhood of 25,000 gallons of oysters were opened in the state on
every day of the oyster season. This places the oyster industry as the
third or fourth on the list of large industries of the state.

3

It is no wonder then that the appeal of the oyster growers to the
Shellfish Commission met with ready response. The importance of
the industry warranted thorough investigation of any criticism of the
product, and also warranted every effort to correct whatever injurious
conditions might be found.

The Shellfish Commission set to work immediately. They secured
an appropriation from the legislature for the work, and, acting under
the existing laws, which were sufficient to cover the matter, they began
a thorough investigation of the whole situation.

In order to secure accurate information as to the conditions in
Narragansett Bay with reference to sewage pollution at the present
time, a comprehensive plan for a complete sanitary survey was out-
lined and work begun. The engineer of the commission undertook to
locate and map every discharge pipe of whatever sort emptying into
tidewater. Twelve plans were prepared, on which the sewer outlets
were indicated. These covered practically the whole shore line of the
state, so that the sources of pollution are known at least. In addition
to this the sewer commissioners of the largest towns concerned in pol-
luting the bay were summoned before the Shellfish Commission at a
public hearing, and under oath testified to the conditions in their sev-
eral towns, the number of sewers, the ownership thereof, the number
of houses connected with the several sewers, the presence or absence
of sewage treatment plants and the efficiency of those plants. These
hearings called the attention of the authorities and the public to the
conditions, and because of this publicity it was hoped to secure the
active co-operation of the towns concerned in remedying the unsatis-
factory conditions.

Under the direction of the writer a careful study of the distribu-
tion of this pollution in the waters of the bay, with special reference
to the oyster beds, was undertaken. A series of “stations” covering
the entire upper part of the bay were located by the engineer, and at
these stations observations were made as to the amount of pollution
and as to other conditions which might affect shellfish. Chemical
analyses of the water at the surface and at the bottom were made at
these stations under different conditions of tide; bacteriological
analyses of the surface and deep water were made also under different
tidal conditions. Samples of the mud and shellfish at the bottom were
also analyzed bacteriologically, with special attention to the presence
or absence of sewage organisms. Besides the above analyses, data in
regard to wind, weather, tide, salinity and temperature were secured.

4

Very early in the work it became apparent that very many factors
enter into the question as to whether the water and shellfish at a given
locality are polluted or not. Besides wind and tide, the temperature of
the water appears to have a very considerable effect, so that it became
necessary to extend observations throughout the year, so as to include
all seasons. Obviously it would be unfair to the oyster grower to con-
demn a given locality from observations in the summer time, if that
same locality in the winter time, when the oysters are marketed, is
entirely free from contamination. Such conditions seem to hold in
certain parts of the bay.

A new law was passed which required that sanitary certificates be
issued by the Shellfish Commission for every oyster bed in the state,
and it prohibited the sale of oysters from beds which did not hold a
certificate showing that the given bed has passed inspection. For the
purpose of issuing these certificates samples of oysters were taken
from each of the 296 leased oyster beds of the state and subjected to
bacteriological analysis. The regular methods used by the Federal
authorities and recommended by the American Public Health Associa-
tion Committee on Standard Methods of Shellfish Analysis were used.
The standard adopted was that used by the Federal Laboratory also,
and required that the colon bacillus be absent from amounts as large
as one-tenth of a cubic centimeter in three out of five oysters.

In our study of these oysters from the different beds it soon be-
came apparent that we could not draw a hard and fast line between
the beds which passed and those which did not pass. We found an
intermediate zone of beds which could not be definitely put into either
of the above classes. In these intermediate beds we found conditions
variable, different samples, taken on different days, might vary, the
~ oysters might be bad and yet the water analysis might be good. Obvi-
ously such beds were on the border line between the polluted area
and the unpolluted area, and different conditions of wind and tide
determined the different conditions at any one time. For such beds
as these we determined to issue conditional certificates, which stated
that “the bed or beds covered by this certificate are in a questionable
area and are still under investigation. This certificate is therefore
temporary and a final certificate will be issued later.” This meant
that the use of oysters from such a bed must be determined by the
conditions at the time, and if the oysterman wished to make sure that
a lot of oysters from such a bed was in proper condition to be mar-
keted he must have an examination of the oysters made at the time

5

’ they were taken. With an improvement of the conditions of sewage
pollution in the bay, such beds as these ought to be the first to show
changes for the better.

As a result of our examination of the oyster beds of the state, we
made three classes. First, there were those which were so badly con-
taminated that they were refused all certificates; second, those which
were questionable, and third, those which were free from pollution.
Certificates were refused for 3,986 acres; conditional certificates were
given for 3,188.2, leaving 12,864.8 acres which were given full cer-
tificates as to their sanitary quality.

While the Shellfish Commission did not attempt to supervise the
taking of oysters from these beds, indeed exercised no police duty at
all in the matter, yet we believe that to a very considerable extent the
oystermen co-operated in the matter and refrained from marketing
oysters from prohibited areas. Indeed we know that many of the
larger dealers went to a very considerable expense to remove their
oysters from polluted beds to clean water before marketing them, and
also we know that in some cases oysters were marketed from non-
certified beds; but on the whole the oystermen co-operated, and we
believe that the oyster shipments from Rhode Island were, on the
average, of better sanitary quality during the past year than ever
before. At any rate the oyster growers had the information at hand
for knowing the exact condition of their oysters, and it was at their
own risk, and it was endangering their business reputation, if they
shipped oysters from polluted areas.

In fact, we believe that these certificates had a very considerable
educational as well as commercial value. The public became immedi-
ately interested in knowing whether the oysters which they were buy-
ing were from certified beds, and the oystermen were educated to the
fact that unless their oysters were from certified beds the public
didn’t want them and the Federal Pure Food Inspectors did. So on
the whole the certificate matter was a good thing all around, even
though strict police supervision was not had to enforce the exact pro-
visions of the law. We believe that in the future certified oysters
will be the only oysters that can secure a market either in our own
state or in interstate trade.

The new law required also that every opening house be inspected
by the commissioners and that oysters must not be opened for market
in a house which had not received a certificate showing that it was in
proper sanitary condition. This regulation of the opening houses

6

was, perhaps, a more difficult matter than the certification of the beds.
It required not only inspection of the houses, but education of
the owners. They must be told what to do to improve the insanitary
conditions. One inspection was not sufficient, the houses must be
visited again and again to see that the requirements had been carried
out. But the commissioners went immediately about the work.

A book of instructions was drawn up by the sanitary expert of the
commission, placards were prepared showing the rules which must be
complied with, and the official inspector began his visits. He visited
every house in the state, talked with the owners, informed them of
the conditions which were wrong, told them how to improve them,
gave them the literature describing the requirements, and posted the
placards on the walls of the opening houses. He promised to return
shortly to see what improvements had been effected.

Of course, everything could not be accomplished in one year. A
very satisfactory beginning was made, however. Many of the own-
ers spent thousands of dollars in putting their houses into better shape.
In the majority of cases an attempt was made to comply with every
request of the authorities, and on their side the authorities tried to
make every request a reasonable one. Of course, there was some
opposition, and several visits were required to some houses to get the
owner started on the improvements. Much remains to be done, but
with the co-operation of federal and state authorities to make only
reasonable requirements, and the oystermen to try to carry out these
requirements at the earliest possible moment, the opening houses of
Rhode Island should soon be above all suspicion.

But the efforts of the state authorities did not stop with the
attempt to regulate the sanitary condition of the industry. They
went still further, and made an attempt to prohibit the further pollution
of the waters of the state, and so far as possible to abate the pollu-
tion already present. After the facts of the case had been secured
by the sanitary survey and by the public hearings, the authorities of
the several towns were warned by letters from the attorney general
of the state that present conditions were contrary to law, and that
they must take steps to remove their sewage from tide water at the
earliest possible moment or prosecution would follow. Indeed an
earnest attempt on the part of the Shellfish Commissioners, the law
department of the state, and the oyster-growers was made to bring
about an improvement in conditions.

Here again work went on slowly, but we can see some gain.

7

The city of Providence, the greatest offender in the matter, was the
first to co-operate. Not only did they begin at once a very general
overhauling of their sewage system, but they installed a disinfecting
plant at the Field’s Point precipitation works. Recent experiments
have shown that ordinary bleaching powder, if used in proper
amounts, will so effectually disinfect a sewage effluent as to render it
free from all objectionable micro-organisms and therefore of such a
quality that it may be discharged immediately into a stream or into
salt water. Experiments carried on in co-operation between the city
of Providence and the State Shellfish Commission at the Field’s Point
plant showed that it was possible without great expense to so treat
the Providence sewage that there could be no objection to it on the
ground that the effluent contained injurious bacteria.

The Providence authorities also began plans for the installation
of additional sewers, which at the earliest possible moment would
remove all the sewage from tide water and carry it to the disposal
plant, where it could be properly treated. We could have asked no
heartier co-operation than that we secured from the city of Provi-
dence. Some of the other towns also began to inquire into the prob-
lem, and appeared to be somewhat interested in the matter. Others
have not yet moved at all. Indeed some of them have even tried to
dlock further activities of the Shellfish Commission. Of course, the
installation of sewage disposal plants in towns is slow work, particu-
larly during the period when a study of the situation is going on, but
we have every hope that the ball which has been set rolling will gather
momentum as it goes, that the examples already set will be followed
by others. There is no reason why Narragansett Bay should be
turned into an open sewer because the several towns are unwilling to
expend the small sum necessary for the proper disposal of their
wastes. If we could but secure united action by all the parties con-
cerned we would soon find the waters of our beautiful bay returning
to their original pure condition, to the advantage of the shellfish, the
summer boating, the summer residents, and the general good health
of the state.

Many new facts regarding the oyster and its relation to sewage
pollution have been brought out by these investigations. Many
problems have arisen in the course of the work. The relation of the
oysters themselves to the colon bacilli, whether these organisims mul-
tiply in the oyster or whether they tend to die out, their distribution
in the oyster and the length of time required for oysters to purify

8

themselves when removed to clean water; all of these problems have
begun to receive attention. And then the matter of standards by
which to judge the purity of oysters, both in the shell and shucked,
has received much attention. At first we were inclined to believe
that the standards used by the government authorities were too high.
When we began work during the summer months, there were many
localities far removed from all sources of pollution where oysters did
not come up to the standard. But when we followed up these locali-
ties during the winter, when oysters were being marketed, we found
that the oysters from these same localities easily measured up to the
standard required. In fact, from our year’s work, we are inclined
to believe that the government standard is a good one if used during
the summer season, but localities must not be condemned for winter
use by examinations made during the warmer weather. I believe that
oysters which cannot pass the government standard in the colder
months are certainly growing too near to sources of pollution to be
used for food. In fact, we found in the winter many oyster beds
situated very near to sewer outlets which easily passed this standard,
and in the end it may be necessary to abandon the methods of judg-
ing oysters by the results of analysis, and adopt a regulation prohibit-
ing the taking of shellfish from within a certain distance of a sewer
outlet.

In conclusion then, we believe that in Rhode Island, in the short
space of one year, a great deal has been accomplished in the cam-
paign for clean oysters, simply by active co-operation on the part of
all concerned. Government and state officials, oyster-growers and
those concerned in sewage disposal must all work together to bring
about, in the shortest possible time, in the tidal waters of our entire
coast, a condition most favorable to the further development of the
oyster industry.

I believe that it can be truthfully said that never before has such
a superior quality of oysters been marketed as those which came
from Rhode Island waters this year. No other state has so success-
fully and energetically attacked the problem of clean oysters, and
nowhere has so much been accomplished as here.

If those efforts are continued, and if, as soon as possible, the
present sources of pollution are removed, we shall see a remarkable
extension of the oyster industry in the state, and the Rhode Island
oyster will come to occupy a position unequalled by any oysters in
the world.

110

SOME BIOCHEMICAL PROBLEMS IN BACTERIOLOGY.
BY FREDERIC P. GORHAM.
Science, N. S., Vol. XX XV, No. 897, pp. 357-362, March 8, 1912.

[ Reprinted from Science, N.S., Vol. XXXV., No.
897, Pages 857-362, March 8, 1912]

SOME BIOCHEMICAL PROBLEMS IN
BACTERIOLOGY?

Tue Society of American Bacteriologists
stands, primarily, for pure as distin-
_ guished from applied bacteriology. In
these days when the applications of the
science are becoming so immensely im-
portant, and therefore so enticing to the
investigator, there is danger that our
thoughts turn not often enough to the
broader aspect of the science, upon which,
as a foundation, all of its applications
must ultimately rest. We as a society
must make it our special duty to see that
these foundations are laid broad and firm,
upon the very bed rock of truth itself.

We have as a society been interested for
some time in the preparation of standard
and uniform methods of describing bac-
terial species. This is of fundamental im-
portance, leading as it does to uniformity
of method and completeness and compara-
bility of results. When we couple with
this the use of the standard methods of the
laboratory section of the American Pub-
lic Health Association we have gone a
long way toward the standardization of
our work, and have begun the foundation
upon which can be built the science of
pure bacteriology.

But we must ever beware that we be-

1President’s address before the Society of

American Bacteriologists at the Washington meet-
ing, December 28, 1911.

>
“

come not slaves to standardization and
uniformity. It is well enough to proceed
by standard methods, but we must not be
tied by them. We must ever be ready to
abandon the old and adopt the new, when
the new marks the way of progress.

At this time I wish to bring to your at-
tention some of the lines along which we
are making little progress, I believe, be-
cause of the false security which we are
taking in our standard methods.

First, suppose we consider our ordinary
culture media. We have drawn up ridicu-
lously exact procedures for mixing and
boiling and filtering and titrating our
broth, agar and gelatin, and when we are
through we believe we have a standard,
uniform product. But indeed it is not so.
Not only are the results obtained in differ-
ent laboratories unlike, but two lots of the
same medium made at different times in
the same laboratory are extremely unlike,
when measured by the delicate physiolog-
ical properties of organisms which respond
to the slightest of chemical differences.
Far better, it is true, are the results se-
cured at the present time by the use of
standard methods, than before their intro-
duction, and I do not for a moment want
to decry our standard methods, but I
simply want to warn against the false se-
curity which their use may give. For
however careful we may be in the process,
the final result can never be uniform as
long as the ingredients used are themselves
variable. No medium can be standardized,
that is, can be exactly duplicated at
another time or place, if it contains such
variable materials as beef extract, either

ra
v

freshly made or commercial, peptone, gela-
tin, agar, blood serum, bile, ete. I am in-
elined to think that in order to get uniform
results, particularly in our study of the
delicate physiological properties upon
which we depend so largely for the differ-
entiation of bacterial species, the time has
come for us to abandon altogether the use
of all complex and variable animal and
vegetable products, and in their places to
substitute materials of definite, known,
chemical composition. From my work of
the past few years I am led to believe that
for every organism it is possible to prepare
a synthetic medium containing chemically
pure salts, upon which these organisms will
gerow and grow well. Such a medium as
this we are able to duplicate exactly any-
where and at any time.

In the past we have been inclined to
think that the physiological properties of
many organisms were too variable to be of
much use in species determination. I am
coming more and more tosthink that it is
not so much the properties of the bacteria
which are variable as the environment
in which we have attempted to study them.
We talk about rejuvenation of organisms
to restore them to normal conditions. Our
attempts at rejuvenation are attempts to
make normal organisms adapt themselves
in short order to an abnormal environ-
ment. Could we but supply the proper en-
vironment we should find the organisms
responding in an entirely normal and uni-
form manner. It is the environment, our
culture media, that need rejuvenating and
not the organisms. This rejuvenation
will come about, I believe, through the

4

adoption of synthetic media of absolutely
known chemical composition. Then the
physiological properties of organisms will
come into their proper place in species
differentiation, for then we can substitute
the exact qualitative and quantitative
tests of the chemist for the inexact de-
termination of the present-day bacteriol-
ogist.

There is scarcely a physiological prop-
erty of the bacteria that is to-day accu-
rately measurable. Variations in the
media are so great that measurements at
present amount to nothing. Chemistry
advanced to its present position as a sci-
ence only when it became quantitative.
Lord Kelvin said: that ‘‘When you can
measure what you are speaking about, and
express it in numbers, you know something
about it; but when you can not measure it,
when you can not express it in numbers,
your knowledge is of a meager and unsatis-
factory kind; it may be the beginning of
knowledge, but ‘you have searcely in your
thoughts advanced to the stage of science.’’
Bacteriology must then become quantita-
tive before the real foundations of the sci-
ence are laid, before it can take its
proper place among the sciences. And it
can not become quantitative until we can
measure its reactions in media of known
chemical composition, by methods as exact
and definite as any known to the chemist.
We are always dealing, it is true, with liv-
ing protoplasm, but if we place it under
definitely determined environment, recent
experiments lead us to believe that living
substance always responds in a perfectly

5

definite way both qualitatively and quanti-
tatively.

The very complexity of the society’s
eard for the description of bacterial spe-
cies is to me its own condemnation. It is
our admission of ignorance. Are not the
real diagnostic characters of a species lost
in a maze of unessential characters?
Aside from the morphological data, which
I believe is all-essential, the cultural and
biochemical data could, I am sure, be
simplified to a very considerable extent as
far as species differentiation is concerned.
The forms of cultures and colonies are but
functions of the morphology and methods
of subdivision of the organisms themselves,
as has been so well shown by the recent
work of Graham-Smith* We might then
eliminate from the card many of the com-
plex descriptions of cultures and colonies
on agar and gelatin, retaining perhaps the
agar streak and the gelatin stab, and sub-
stitute therefore information in regard to
their determining factors. And then
among the biochemical data if we could
eliminate all but a few deep-seated physio-
logical characters which are accurately
measurable, and which can be easily de-
termined by means of accurate chemical
tests on synthetic media of known compo-
sition, we would have a simple, accurate,
all-sufficient description of a _ bacterial
species.

One of the weakest parts of bacteriology
to-day is its taxonomy. Our methods of
classification of bacteria are practically

?Graham-Smith, G. 8., ‘‘The Division and Post-

fission Movements of Bacilli when Grown on Solid
Media,’’ Parasitology, 3, 1910, 17.

6

the same to-day that they were in the
earliest days of the science. Migula, it is
true, systematized the scheme of classifica-
tion to a certain extent, Chester contrib-
uted to its more accurate terminology, and
the Winslows gave it a new impetus by
the introduction of the methods of biom-
etry. But when we think of the thousands
of described species among which but
two or three genera are recognized, it
must be apparent at once that some of our
generic names are seriously overworked.
In other biological sciences the classifica-
tion into species, genera, families, orders,
classes, ete., is not only of great conveni-
ence, but it also expresses for us something
of the relationship of the different groups,
something of their probable ancestry and
line of evolution. I see no reason why the
bacteria should not be classified in the
same way. The bacteria are not excep-
tions to the general biological laws. Varia-
tion, selection, heredity, are the factors of
evolution here as elsewhere. It is true
among the unicellular forms we are free
from many of the complications which
enter into our discussions of the origin of
species among multicellular animals and
plants. Sexual reproduction is absent,
there is no differentiation into germplasm
and somatoplasm to prevent the acquisi-
tion of new characters, the environment
presses very closely upon these unicellular
forms and they respond more directly to it.
Generation follows generation with start-
ling rapidity, elimination of the unfit pro-
ceeds rapidly, the struggle for existence is
more severe because of the enormous num-
bers concerned. In the bacteria we ought

7

almost to be able to see the actual process
of evolution since we can place under ob-
servation untold numbers of generations as
compared with the comparatively few gen-
erations of multicellular forms which we
are able to observe. Eons of multicellular
time are literally compressed into a few
unicellular days.

Because of this lack of the regulating
influence of reproduction and the greater
influence of the environment and the rapid-
ity of reproduction, we might expect to
find among unicellular organisms a series
of intergrading forms without divisions
into groups that resemble the species of
higher forms. But this isnotso. Among the
protozoa differentiation has followed definite
lines, and classes, orders, families, genera
and species are well marked. The species
of protozoa are, for the most part, based
on morphological differences, it is true, but
among the bacteria a morphological basis
of classification fails us. The only morpho-
logical differences are the three main di-
visions as to shape, the round, the rod and
the spiral, with slight modifications as to
size, arrangement of flagella, formation of
spores, chemical composition, ete. But in
spite of this lack of morphological basis for
classification we find as distinct groups as
among the protozoa. As the Winslows
say :°

Typhoid germs descend from typhoid germs,
tubercle bacilli from tubercle bacilli. The same
yellow coccus falls on gelatin plates exposed to

the air all over world. The same spore-forming
aerobes occur in every soil, the same colon bacilli

* Winslow and Winslow, ‘‘Systematice Relation-
ships of the Coccacer,’’ p. 1, John Wiley and
Sons, 1908.

8

crowd the intestines of animals and man in every
clime. These fundamental types can not be trans-
formed into each other.

And yet to a considerable extent these
fundamental types are based not on mor-
phology, but upon physiological differ-
ences.

It is among the bacteria that for the first
time among living forms we find physiolog-
ical differences made a basis for classifica-
tion. Are physiological properties valid
eriteria for the separation of species? We
are accustomed to think of morphological
characters as fairly stable, rather difficult
to modify, but of physiological characters
as easily modified and directly dependent
upon the environment. But have we any
reason to assume that physiological charac-
ters are not deep seated also, are not
stable, fully as much as morphological
characters? Are we not in reality dealing
with characters of the same sort as mor-
phological characters except that we can
not see their ultimate basis in structure?
I am not sure but what physiological char-
acters are even of greater importance than
those of form and external appearance,
especially in such simple and undifferenti-
ated forms as the bacteria, since they tes-
tify to deep modifications in the chemistry
and vital properties of the protoplasm
itself. It is upon such chemical properties
as these that these simple organisms de-
pend for their very existence, and not
upon a modification of external appear-
ance.

But before we can proceed far in the
use of physiological differences for species
determination we must be able accurately

9

to determine these delicate characters.
Hitherto this has been impossible because
of the variable and uncertain culture
media in which we have attempted to
study them. With the adoption of media
of definite chemical composition for ma-
king our determinations and measure-
ments, the physiological characters of the
bacteria will assume the importance of the
morphological characters of higher organ-
isms. Then and then only shall we able to
arrive at a natural classification of bac-
terial species which shall express for us
their true relationship.

With the adoption of such accurate
chemical tests of the physiological char-
acters of the bacteria much of the present
apparent variation will pass away and we
shall find the physiological characters of
the different groups as stable as any of
their morphological characters. And
what little variation remains—and we shall
always find some variation as long as we
deal with living organisms—we can
handle easily by the method of biometry.
For, as you know, the methods of statis-
tical variation can apply only in char-
acters which are measurable.

This matter of better methods of spe-
cies identification and a new taxonomy will
be one of the first outcomes of the adop-
tion of simple chemical culture media.

Another important result will be the
‘increased ease with which certain biochem-
ical problems in bacteriology can be at-
tacked. Think for a moment of our en-
deavors to find a suitable method of iso-
lating the colon bacillus. Litmus-lactose-
agar, endo, esculin, neutral red, mala-

10

ehite green, phenol, bile and bile salts and
their various combinations are but expres-
sions of our total ignorance of the chem-
istry involved. The problem should be at-
tacked in an entirely different way. First,
we should determine the simplest synthetic
medium upon which the colon bacillus will
erow rapidly and well. Then by adding
to it the chemical body which is the in-
hibiting agent in the phenol or in the bile
we ought to have the ideal medium for
eolon isolation. A beginning along this
line has been made by Dolt* in his am-
monium lactate, glycerin and malic acid
media. But further work must be done
until we have a simple synthetic substitute
for the complex, variable and inconven-
ient media upon which we depend so much
at present.

And then take the differentiation of the
eolon and typhoid groups by cultural
characters. There must be certain chem-
ical differences inherent in these organ-
isms which could be easily determined by
the use of synthetic media adapted to
each organism.

Another desideratum is a simple syn-
thetic medium to be used as a substitute
for blood serum when used for the diag-
nosis of diphtheria. We have to depend
at present upon the uncertain and unsatis-
factory supply of blood from the abattoirs.
How much better would it be to find a
chemical substitute which would be just as
certain for diagnosis and much easier to

*Dolt, M. L., ‘‘Simple Synthetic Media for the
Growth of B. coli and for its Isolation from

Water,’’ Journal of Infectious Diseases, V., 1908,
616.

en

prepare? I believe that such a substitute
will soon be forthcoming.

We have been gradually accumulating
a knowledge of a considerable number of
important chemical products which are
produced by the activities of bacteria
either by synthesis or decomposition.
Many of these substances are of great im-
portance. Many are produced only as far
as we know at the present time by bac-
teria. How little at present we know of
the chemistry of these products! How
much can be learned by a study of the
formation of these substances in culture
media of known composition! The chem-
istry of ptomaine and toxin formation, of
pigment formation, of enzyme produc-
tion, may be worked out in this way. The
chemist laughs at our present methods of
testing the production of gas, the reduc-
tion of nitrates, the production of indol,
the fixation of nitrogen. And yet when
these processes are tested in synthetic
media how simple are the chemical tests
involved and how accurate may be the re-
sults!

Slowly and laboriously the physiolog-
ical chemist is now trying to work out the
chemistry of protoplasm, of the proteins,
such as the albumens, the peptones and
proteoses. His principal line of attack is
by a study of their decomposition prod-
uets. The brilliant work of Fischer opened
up an entirely new field of research when
he undertook the study of the synthetic
production of the polypeptides from
amino acids by an amide link. Still more
hght might be thrown upon this important
problem by the study of the growth of

12

bacteria in simple chemical solutions. For
in the synthetic culture medium we would
be able to study step by step the synthesis
of protein under conditions accurately
controlled and completely known. For
when bacteria are growing on simple chem-
ical media and are building up untold
millions of bacterial bodies from the
simple salts present, we can almost see
protoplasm in the making.

And finally, aside from the important
chemical information which may in this
way be obtained, I believe that some most
interesting biological information lies
along this path. Who would dare to deny
that some day it might be possible by
some such method as this to discover the
secret of the very origin of life itself!

These then are some of the lines of work
which appear to me to mark progress in
the science of pure bacteriology. Bril-
liant as may be the results of the study of
the applications of bacteriology, fully as
interesting, and hardly less important,
will be the results that come from the ap-
plication of exact chemical methods to
our at present inexact and rather uncertain
bacteriological procedures.

F. P. GorRHAM
Brown UNIVERSITY

111

A CONTRIBUTION TO OUR KNOWLEDGE OF THEGAS
METABOLISM OF BACTERIA.
First Paper—The Gaseous Products of Fermentations of Dex-
trose, by B. coli, by B. typhosus and by Bact. Welchii.
Second Paper—The Absorption of Oxygen by Growing Cultures
of B. coli, and of Bact. Welchii.
BY FREDERICK G. KEYES anp LOUIS J. GILLESPIE.
The Journal of Biological Chemistry, Vol. XIII, No. 3, 1912, pp. 291-310.

a
Y
2 \
; af

Reprinted from THE JouRNAL Or BroLoagicat Cuemistry, Vol. XIII, No. 3, 1912.

A CONTRIBUTION TO OUR KNOWLEDGE OF THE GAS
METABOLISM OF BACTERIA.

FIRST PAPER.

THE GASEOUS PRODUCTS OF FERMENTATIONS OF DEXTROSE
BY B. COLI, BY B. TYPHOSUS AND BY BACT. WELCHII.

By FREDERICK G. KEYES anp LOUIS J. GILLESPIE.

(From the Biological Laboratory of Brown University.)
(Received for publication, October 9, 1912.)
Earlier investigations.

B. coli. The literature on the gases produced by B. coli on
various culture media has already been reviewed by one of us.!
It was found that very little work had been attempted on this
subject by investigators familiar with the properties of gases.

Harden? found that, when B. coli was grown anaérobically on a
non-albuminous dextrose medium based on asparagine, the as-
paragine was reduced to ammonium succinate with a consequent
lessened evolution of hydrogen. He therefore made use of media
made of beef broth and of Witte’s peptone and concluded, from a
study of the solid, liquid and gaseous products, that on these
media dextrose was decomposed with the formation (if not evo-
lution) of equal volumes of hydrogen and of carbon dioxide.
His gas analyses showed for the most part more hydrogen than
carbon dioxide.

One of us* determined for several strains of B. coli the quantities
and the composition of gas obtained in various incubation periods

1 Keyes: Journ. of Med. Res., xxi (N.S. xvi), p. 69, 1909. In this com-
munication the composition of the culture medium was unfortunately given
incorrectly. The medium always contained, in addition to the constit-
uents noted, 1 per cent of Merck’s ‘‘Highest Purity’’ dextrose.

2? Harden: Trans. Jenner. Inst., 1899, ii, p. 126; Journ. Chem. Soc.
(Transactions), Ixxix, p. 610, 1901.

3 Keyes: Journ. of Med. Res., xxi (N. S. xvi), p. 69, 1909.

201

THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XIII, NO. 3.

292 Gas Metabolism of Bacteria

by an exact method from vacuum fermentations of dextrose. A
non-albuminous asparagine medium was used for the most part,
but a dextrose meat bouillon was also tested; and it was found that
a larger percentage of hydrogen was indeed evolved from the albu-
minous medium but that even on this medium carbon dioxide
was produced in considerably greater volume than hydrogen.

B. typhosus. Hesse® studied the gas metabolism of this organism
by growing it in the presence of air and drawing off samples of gas
from day to day for analysis. He stated that carbon dioxide is
evolved and that oxygen is absorbed but he did not calculate his
analytical results. We calculate from his protocols that in one
instance as much as 18 cc. of carbon dioxide was evolved aérobic-
ally from 33 ce. of “glycerin agar-agar.”’

Harden‘ detected no production of gas from anaérobic fermenta-
tions of dextrose by B. typhosus and Pakes and Jollyman’ detected
no formation of gas from sodium formate—the supposed in-
termediate product between dextrose and carbon dioxide and
hydrogen.

Bact. welchii. Dunham’ analyzed the gas evolved by this organ-
ism from 1 per cent dextrose bouillon in Smith fermentation tubes.
The ratio CO.:H»s was 0.43, but his results are not comparable with
ours because he disregarded the loss of carbon dioxide due to solu-
tion in the medium and to diffusion from the open end of the fer-
mentation tube. .

Technique.

Organisms. Cultures were kept in a vigorous state of growth
by means of frequent transfers and isolations by the plate method.
The medium used for B. typhosus and Bact. welchii was the ordi-
nary meat extract peptone agar; in the case of B. coli it consisted

4 We shall give below an analysis from a fermentation on a Witte’s peptone
medium, which gave practically the same figures-as the meat bouillon. We
have always found more CO, than He, and attribute Harden’s contrary
result, as also his negative result with B. typhosus, to insufficient removal of
dissolved carbon dioxide from the culture fluid.

5 Hesse: Zeitschr. f. Hyg., xv, p. 17, 1893.

6 Harden: Journ. Chem. Soc. (Transactions), Ixxix, p. 610, 1901.

7Pakes and Jollyman: Journ. Chem. Soc. (Transactions), Ixxix, p. 459,
1901. '

8 Dunham: Johns Hopkins Hospital Bull., viii, p. 68, 1897.

F. G. Keyes and L. J. Gillespie 293

of 1 per cent dextrose, 0.2 per cent disodium phosphate and 1.5
per cent agar, with eitber 1 per cent asparagine for experiments
on asparagine media or 1 per cent ammonium lactate for experi-
ments on lactate media, and was made neutral to litmus with
sodium hydrate. Test cultivations of B. coli or of B. typhosus
were started from a loopful of culture taken from the surface of a
twenty-four-hour growth on slant agar. Bact. welchii fermenta-
tions were started from stab cultures. Subcultures from the test
cultivations were never used for further tests. The organisms
answered the usual identification tests.

Culture fluids. The culture fluids were sterilized in the absence
of air in fermentation bulbs (to be described) in streaming steam
by the intermittent method. The reaction after boiling was
slightly acid to litmus, except in certain experiments (noted in the
protocols) in which the reaction was made just neutral to phenol-
phthalein by the addition of normal sodium hydrate. When
alkali was added, the amount of carbon dioxide thus introduced
was calculated from a determination of the actual volume of
carbon dioxide liberated from a sample of the sodium hydrate
solution upon acidification in vacuo with sulphuric acid. Since
the medium was subsequently exposed only momentarily to the
atmosphere, the correction applied (a small one) was exact.

Oxygen. Oxygen was prepared by heating potassium perman-
ganate in vacuo and was purified by passing over phosphorus pen-
toxide and over sodium hydrate (not that purified by alcohol). It
was measured dry, the pressure being read on a barometer column.
Generator, burette and barometer were permanently incorporated
in the pump system (fig. 2) by fused glass joints. The generator
was exhausted by the mercury pump and then rinsed out twice
with small quantities of oxygen before the gas was generated for
use in the work.

Control of gaseous environment and collection and analysis of the
gases. Fermentations were conducted either in vacuo® or in the
presence of gases admitted in known quantities after a vacuum
had first been obtained.

The fermentation bulbs (fig. 1) fitted with stopcocks of a special form
described by one of us,!° were about half or three-quarters filled with cul-

® That is, in an atmosphere of water vapor only.
10 Keyes: Science (N.S.), xxviii, p. 17, 1908.

204 Gas Metabolism of Bacteria

ture medium, and were sterilized. After sterilization, the stopcocks of
the bulbs were wiped dry with sterile rolls of filter paper and re-greased with
sterile lubricant. The lubricant was the non-volatile non-antiseptic mix-
ture described by one of us." The medium was then inoculated.

The bulbs were then freed from gases., It was found that this could be
done to any desired degree of completion by the use of a good Sprengel water
pump, which was capable of maintaining the pressure at from 9 to 15 mm.
(The use of a mercury pump gave no better results and was moreover un-
necessarily troublesome.) This method of exhaustion depends upon rins-
ing out the gas with water vapor. In extracting dissolved gases from liquids
the driving force becomes less as the process becomes more nearly complete
and the process comes practically to an end while detectable amounts of
gases are still dissolved, unless by vigorous agitation the liquid is kept welk
mixed and many new boundary surfaces thus continually formed. We
found that, without agitation, pumping for an hour after most of the air
had been removed was not so efficacious as five or ten minutes’ vigorous
shaking interrupted by a few exposures to the action of the pump. This
experience led us to think that the degree of exhaustion is controlled, in
cases where the space above the liquid is kept as free from gases as in these
experiments, by the thoroughness of agitation. The bulbs were therefore
subjected alternately to the action of the pump, by opening the stopcock
of the bulb for an instant, and to a vigorous shaking. This was repeated
until a metallic sound was emitted upon striking the bulb hard with the
finger ends!” and was then further continued until this effect (which cannot
be produced until the vacuum is rather good) was produced only with great
difficulty. As the liquid was shaken, falling drops of liquid clicked metalli-
cally against the sides of the bulb. We believe that we were able to treat
the bulbs with practical uniformity and that before we finished the process
the gas that remained in any bulb was negligible in quantity so far as our
analyses were concerned.

If gas was to be admitted, the bulb was now connected to the pump sys-
tem by a short piece of heavy walled rubber tubing, which was well painted
with hot adherent grease. Both ends of the tube were bound tightly on with
copper wire. The tubing connecting the bulb with the gas burette was evac-
uated by the use of the mercury pump, and the gas, having been measured,
was pushed over into the bulb. By careful manipulation practically all
the gas could be passed over into the bulb without admitting any mercury.

11 Keyes: Journ. Amer. Chem. Soc., xxxi, p. 1271, 1909.

12 Special experiments have shown us that this sound is occasioned by
the tearing apart of the liquid with the liberation of a bubble or two of gas
and the consequent collapse of the newly formed walls of liquid. Thus the
range of concentrations of dissolved gases at which this effect is possible,
should be, as it in fact is, limited in both directions.

18 Except possibly in the case of B. typhosus, where the total gas evolu-
tion was very small.

F. G. Keyes and L. J. Gillespie 295

Fic. 1. FERMENTATION BULB
FITTED WITH VACUUM
STOPCOCKS.

B, bottle, secured by cotton
plug, serving to keep sterile the
capillary lead. Capacity of
bulb, about 300 cc.

Fic. 2. THE PUMP SYSTEM.

B, oxygen measuring burette; D,
delivery tube—it connects with gas
analysis apparatus at C; M, manometer
column, provided with meter stick; ¢, t,
air traps; V, valve tight to ascending
mercury. Fermentation bulb connects
at F. Mercury reservoirs connect by
heavy rubber tubing at R, R, R.

After incubation at about 37°C., the gases in the bulb were
recovered by the mercury pump and analyzed. In every instance,
the whole obtainable quantity was pumped out and the whole
quantity was used for the analysis or else a measured portion after
the gases were well mixed. The gas absorptions were conducted
according to the method of Hempel. The burettes were filled with
mercury. Carbon dioxide was absorbed by strong caustic soda solu-
tion (after the total volume had been measured dry); oxygen, by
phosphorus or when necessary by alkaline pyrogallol; and hydrogen

2096 Gas Metabolism of Bacteria

was determined by exploding with a quantity of air sufficient to
avoid the burning of nitrogen. Other gases were absent. Some-
times the Hempel pipettes were used for the reagents; but when
small amounts of gas were to be analyzed, they were generally
measured under diminished pressure, and treated with absorbing
reagents in tubes inverted over mercury, as in the method given
by Travers.44 Sometimes the diminution of pressure was read on
an open manometer; and sometimes the actual pressure was
read on a barometer (the space of which was saturated with water
vapor) sealed to the burette. The work was done in a cellar where
the temperature was sufficiently constant. All gas volumes have
been calculated for 0°C., 760 mm. and dryness. The medium was
always strongly acid after incubation so that no carbon dioxide
was retained chemically.

Experiments with B. Coli.

Experiment 1. ‘B. coli was grown in vacuo for 48.2 hours on 250 cc. of a
medium containing 1.00 per cent each of Witte’s peptone and Merck’s
‘‘Highest Purity’’ dextrose in distilled water, prepared hot and filtered.

Obamas teerec tnt ak ay smgciy os puck oa chee eee 137.0 ce.

(CLO be Ri tibet a5 sin @ cits So arc ee Ree che! Scr esarcack auc 56.1 per cent.
Te se Beets SecA ek SearetG SREP A ee Ween eee eae Sd ee tg 43.0 per cent.
COs, per gram of dextrosesy.4.. 72-0 aa sere ee TOU Ce
EVEtLOs . © Oar orp seer et nate leck, co's essere Se eae pene Bul

The ratio 1.28 was found by one of us" for beef infusion broth containing
1 per cent each of dextrose and Witte’s peptone.

It seemed better to work with a medium containing no sub-
stances of unknown composition. One such, described by Dolt,'®
has the advantage that no amino group is present so that retention
of hydrogen by the type of reaction found by Harden for aspara-
gine is excluded. It consisted of 1.00 per cent ammonium lactate,
1.00 per cent dextrose and 0.200 per cent disodium phosphate in
distilled water, prepared without heating. In table I are given
some experiments upon this medium, arranged according to the
length of incubation.

The influence of phosphate, of nitrate and of increased dextrose
percentage is shown in table II. The medium contained 1.00 per

4 Travers: Study of Gases, 1901, p. 28.

15 Keyes: Journ. of Med. Res., xxi (N.S. xvi), p. 69, 1909.
16 Dolt: Journ. Inf. Dis., v, p. 616, 1908.

F. Gi. Keyes. and L. J. Gillespie 297

TABLE I.

B. coli in vacuo.

; |

wxr.xo.|YONME OF] mows | TO | co, | mh | eencnan| 90
ce. hours ce. per cent | percent | ce.
2 100 23.3 27.60 Olt | 14.10
3 25 40.0 7.21 49.5 48.1 14.380 | 1.03
4 100 44.3 28 .40 51.3 48.1 14.55 | 1.06
5 100 58.2 28 .60 ol.1 | 47.0 14.60 | 1.09

cent of ammonium lactate and various quantities of dextrose,
sodium phosphate and ammonium nitrate, as noted. In experi-
ments 7, 8 and 9 Merck’s ‘‘ Highest Purity” dextrose was used;
in experiment 6, the same recrystallized from alcohol; in experi-
ment 10, a nitrate-free dextrose (Merck’s “Pure’’) recrystallized.

The effect of an increase of phosphate is to increase the total gas
formation, probably by delaying acid inhibition, and to increase
the formation of carbon dioxide more than hydrogen.

The effect of nitrate is to decrease slightly the carbon dioxide
formation and to use up most (or all) of the hydrogen that would
otherwise be produced. The latter effect has been noted by Pakes
and Jollyman.!7 Merck’s “Highest Purity” dextrose was found
by the phenolsulphonic acid method to contain 0.04 per cent of
sodium nitrate, which could be reduced to 0.01 per cent by recrystal-
lization. Assuming proportionality, the result of experiment 9
would indicate that the amount of nitrate present in 1 gram of
the ‘Highest Purity’ dextrose could use up 0.012 ce. of hydrogen,
a quantity which is negligible for B. coli fermentations but which
would be of great significance in B. typhosus fermentations. The
ammonium lactate and sodium phosphate were found free from
nitrates by the same (controlled) test.

In another part of our work, to be communicated in a second
paper, we have studied somewhat fully the effect of oxygen upon
B. coli fermentations of dextrose in an asparagine medium. The
following figures show the results for an aérobic fermentation by
B. coli of a medium composed of 1.00 per cent ‘Highest Purity”

17Pakes and Jollyman: Journ. Chem. Soc. (Transactions), lxxix, p. 386,
1901.

Gas Metabolism of Bacteria

298

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PG. Keyes and L. J. Gillespie 299

dextrose, 1.00 per cent ammonium lactate and 0.100 per cent di-
sodium phosphate:

Experiment 11. B. Coli.

Ox Wo CHPACHON GLE ane sto eee nas <a eo ort eeeien BERD ONCCe
NCU AGIONVatio donee ove tale fs ets nce os SE .108. hours.
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CLOTS i Pa dges this SEP Ee Geo te Hee: Ee Pe ment eae Pn OE eo 5.18 cc

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|Rauii@, COCO Ed ah hat terre gee ae ee eR 5.4
ree UREE CANCE p< lacy deft anes aad cb dt ne es 2.01 ce.

Experiments 3, 4, 5 and 10 show that it is possible to find con-
ditions such that nearly equal volumes of CO, and Hz are obtained
but that the CO, is always in slight excess. Evidently the pres-
ence of oxygen, of nitrates or of sodium phosphate, since all these
substances tend to increase the ratio of carbon dioxide to hydrogen,
prevents the realization of the value unity for this ratio. Dolt}8
found that B. coli required either phosphates or nitrates for its
growth. We have found that in the absence of nitrates anaérobic
growth vanishes if the phosphate content is sensibly reduced below
the lowest concentration used in the experiments given above, so
that it appears that, in spite of a systematic error due to the greater
solubility of carbon dioxide in the culture liquid, it is not possible
to collect equal volumes of carbon dioxide and hydrogen. This
means that if, as Harden concluded, the decomposition of dextrose
_ by the action of B. coli results in the formation of an equal number
of molecules of carbon dioxide and hydrogen, according to the
“classical”? fermentation of Duclaux,!® there occurs also a process
which either uses up hydrogen or produces carbon dioxide and
which is therefore oxidational. This process seems to be neces-
sary for growth.

Experiments with B. typhosus.
A few experiments were made with two strains of the typhoid

bacillus. Neither strain could be grown on Uschinski’s solution,
on Fraenkel’s modification or on various other simple media.

18 Dolt: Journ. Inf. Dis., v, p. 616, 1908.
19 Duclaux: Traité de microbiologie, iv, p. 49, 1901.

300 Gas Metabolism of Bacteria

The culture fluid consisted of 1.00 per cent each of Witte’s peptone
and dextrose in distilled water, prepared hot and filtered. In the
first four experiments Merck’s “ Highest Purity” dextrose was used
and in the fifth a preparation made by recrystallizing Merck’s
“Pure” (nitrate-free) dextrose from alcohol.

The following analysis for experiment 5 typifies the procedure
followed in the analysis of small quantities of gases. The explo-

Analysis of experiment 6.

| OBSERVED | MANOMETER | TEMPER- | VOLUME AT| MEAN

VOLUME | READINGS | aes ATURE 760 MM. | VOLUME
| (422007) 1528). 1245) 399 290) )| 42 1504
Total gas.....|{ 6.80 | 357 114| 243 | 2180s) 2anee
| | 5270) Wy 4i3~ Iss) 0295 D249 4 12),220
(1.00 | 264 138) 126 22.5 | 0.166
BLOOM 47 eat 26 9350) || Onlin
After: KOH). 4|8 2e35 al Shenlse 5D 0/170; 1, Omir
TSO) 200 1345 70 22.9 | 0.166
1.40. | 234 135 99 0.182 }
| |
2.60 | 544 130] 414 OS on 1.420
3.75) 425. 126)|) +299 1.470
Air added....|{ 5.15 | 339 120| 219 1.480} | 1.458
5.80 | 309 118) 191 23.0 | 1.460
4.60 | 365 123) 242 1.460
|
Pass spark....| 4.60 | 368 123| 244 1.460
Added elec- |]
trolytic gas
a oe ose 3.90) | 442 129 318 1.310]
arte 5.60 | 296 119| 177 1.300) ae
re ; SG, 80) ||) 2258- 9 een amelie 1.290
a eel aos 40)) |, 410) eto eae aaee 2310 1.286
ploded on | |
passin g|
spark.

The result (volumes at room temperature, but dry without correction
because the barometer space was saturated with moisture) is therefore
COBRA Eee Laces 2.012 Residual... ac scene Os Ooe:
1 [Pee gente: ai et arene emer cx Olean UP Sums eo ro uieclan neee eer 2.173
All gas volumes were further reduced to 0°C.

F. G. Keyes and L. J. Gillespie 301

sive gas was calculated as hydrogen, and the closeness with which
the sums of the analytical percentages thus found approach 100
per cent indicates that the explosive gas was hydrogen. On so
small a quantity of gas as that remaining for the hydrogen deter-
mination the non-production of carbon dioxide upon explosion
could not be certainly proved.

Since the presence of dextrose is of great importance for growth
of bacteria in the absence of oxygen, it is not possible to show by
the omission of dextrose what is probably the truth: that the gases
obtained came from the dextrose. Nevertheless a significant
amount of fermentable sugar could not have been present in the
peptone, since it was found that B. coli produced anaérobically on
a 1 per cent solution of Witte’s peptone in forty-eight hours at
37°C. only 0.095 ec. of COz (88 per cent of the total gas) per gram
of peptone, and B. typhosus produced under the same conditions
less than 0.02 ec. of CO, per gram of peptone.

These results show that carbon dioxide and hydrogen are evolved
by the action of B. typhosus on dextrose, but that the amounts
produced are very much less than those produced by B. coli, and
the ratio of carbon dioxide to hydrogen is many times higher in
the case of B. typhosus. Upon the nitrate-free dextrose (experi-
ment 5) considerably more hydrogen is produced. The increase
per gram of dextrose is about 0.027 ec., whereas the volume of
hydrogen which the amount of nitrate in question can use up in a
B. coli fermentation we have found above to be 0.012 ce.

Just as the amount of nitrate present as impurity in the dex-
trose, while it introduced no significant error in the results for B.
coli, made a large difference in the results for B. typhosus (where
the total amount of gas was much smaller), so may the principles
in the peptone which increase the ratio with B. coli make a greater
difference with B. typhosus. It is possible that this is wholly
responsible for the difference in the values of the ratio found for
the two organisms.

Experiments with Bact. welchiv.

Bact. welchii was grown anaérobically on a medium consisting
of 1 per cent each of Witte’s peptone and Merck’s ‘“ Highest
Purity” dextrose in distilled water. No alkali was added.

302 Gas Metabolism of Bacteria
TABLE IV.
Bact. welchi in vacuo.

CO2

INCUBA- 102
eee VOUUMEVOR| la yeecat TOTAL ‘> 2 RESIDUAL]
EXP. NO.| wmpIuM art a GAS ous Bs GAS Pde lialakced | He
cc. hours (ey per cent | per cent | per cent | ce.
| é ty « . 2Qq « | me
te] 45100 120 | 61.8 | 60,0-|) 39:3 | 05°) 86.99] uNes
2 | 50 336 | 63.5 | 58.5 | 41.2 | 0.3 74.2 | 1.43

The results differ chiefly from those found for B. coli upon this
medium in the larger volumes of gas found per gram of dextrose.

Comparison of the three microérganisms.

Some of the foregoing experiments give a basis for a compari-
son of the three organisms, namely, those anaérobic fermentations
of a medium consisting of 1 per cent each of Witte’s peptone and
dextrose. In table V are given the maximum volumes of carbon
dioxide evolved per gram of dextrose and the ratio COe:Hs. The
lower value of the ratio given under B. typhosus is that obtained
with nitrate-free dextrose.

TABLE V.
B. TYPHOSUS | B. COLI BACT. WELCHII
Sy uc 6 9 ata ie! |
Carbon dioxide in cubic centimeters 2.1 SORT 1a 74.

Ratio, COciko esas nae eee from 19 to 44 Heda, t)| 1.48

* 48.2 hours; possibly not maximum amount obtainable.

SUMMARY.

The gas evolution accompanying the growth of certain bacteria
on culture media containing dextrose has been studied by an exact
method.

I. The principal results for B. coli are:

A. Dextrose-peptone media yield considerably larger volumes
of carbon dioxide than of hydrogen upon anaérobic fermentation.
The volume-ratio CO2:Hs is 1.31.

B. A suitable ‘synthetic’? medium (composed of ammonium
lactate, disodium phosphate and dextrose) yields anaérobically
nearly equal volumes of the two gases. The ratio CO,:Hs is always

F. G. Keyes and L. J. Gillespie 303

greater than unity and has a mean value of 1.06 for a medium of
given composition.

1. The presence of oxygen raises the value of this ratio.

2. Increase of phosphate content also raises the value.

3. The phosphate cannot be reduced sensibly in quantity, or
substituted by a salt less objectionable.

4. The value 1.06 for the ratio CO,:H2 is minimal. This means
that if the principal gas reaction consists of a liberation of an
equal number of molecules of carbon dioxide and hydrogen from
dextrose there also occurs an accompanying gas reaction of the
nature of an oxidation.

If: B. typhosus produces anaérobically from a dextrose-peptone
medium small volumes of carbon dioxide and an explosive gas,
probably hydrogen. The ratio CO2:H:2 is never lower than 19.

Ill. Bact. welchii produces anaérobically from a dextrose-pep-
tone medium large volumes of carbon dioxide and hydrogen.
The ratio CO2:He is 1.48.

Reprinted from Tue JourRNAL or BrotocicaLt Cuemistry, Vou. XIII, No. 3, 1912.

A CONTRIBUTION TO OUR KNOWLEDGE OF THE GAS
METABOLISM OF BACTERIA.

SECOND PAPER.

THE ABSORPTION OF OXYGEN BY GROWING CULTURES OF B.
COLI AND OF BACT. WELCHII.

By FREDERICK G. KEYES anp LOUIS J. GILLESPIE.

(From the Biological Laboratory of Brown University.)
(Received for publication, October 9, 1912.)

We have studied the gas production of B. coli and of Bact.
welchii for various incubation periods in the presence of oxygen
admitted to the fermentation bulbs in known quantities after a
vacuum had first been obtained. The technique employed has
been described by us in the preceding paper.

From the data so obtained we can derive information concern-
ing the rate at which oxygen is absorbed and concerning the rela-
tions existing among the quantities of oxygen absorbed and the
quantities of carbon dioxide and hydrogen evolved.

We have not found in the literature any work on these points
for any microdrganism where the atmosphere over the culture was
accurately controlled and the analyses were made on portions of
gas accurately sampled.

Experiments with B. colt.

The culture medium consisted of 1.00 per cent each of Merck’s
“Highest Purity’ dextrose and asparagine and 0.200 per cent of
disodium phosphate and was made neutral to phenolphthalein with
sodium hydrate. Corrections were made for the carbon dioxide
thus introduced, as explained in the first paper, and the values of
the corrections are given with the analyses.

The results of the gas analyses are given in table I.

395

306 Gas Metabolism of Bacteria

The values of the ratio, COs:H», vary enormously, and are many
times the value previously obtained by one of us! for anaérobie
fermentations. ;

In an experiment described in the preceding paper (Exp. 11, p.
299) the retention of oxygen was found to be 2.0 ce., whereas the
decrease in yield of hydrogen (7.e., that amount which, together
with the amount actually found, would make the ratio CO.:H»
equal to 1.06, the mean value for anaérobic fermentations on the
same medium) is 3.9 ce. or only 0.1 cc. less than that which the
oxygen could have oxidized. The medium for which this result
was obtained was based on ammonium lactate. If this result _
were the rule, it would indicate that the oxygen was almost quanti-
tatively taken up by nascent hydrogen from the dextrose. Similar
calculations from the data here presented show, however, that this
is not the rule (at least in the case of the asparagine medium)
but that the missing volumes of hydrogen are sometimes greater
and sometimes smaller than twice the volumes of absorbed oxygen.

The volume of oxygen absorbed per unit volume of carbon
dioxide evolved (the “respiratory quotient”) is given. The mean
value is 0.135, with a probable error of = 0.02.

The relation between the volume of oxygen admitted to the
culture, the volume of oxygen not absorbed and the duration of
incubation at 37°C. can well be seen from a calculation of the
values of the expression : - log a where ¢ is the time in hours,
V is the volume of oxygen admitted, and »v is the volume of the

unabsorbed oxygen. Since re here equals the ratio of the corre-

sponding pressures, the given expression is that for the constant
of a monomolecular gas reaction. It is in fact nearly constant.
The values are given.in table III. We should note, however,
that the fermentation bulbs were not shaken during incubation,
so that although a certain degree of agitation was imparted to the
medium by the brisk evolution of gas, the rate of oxygen absorp-
tion may, nevertheless (namely, if relatively fast), have been
limited by the rate of distribution. In this case the given expres-
sion should be constant.

1 Keyes: Journ. of Med. Res., xxi (N.S., xvi), p. 69, 1909.

F. G. Keyes,and L. J. Gillespie 307

The data given in table I may properly be compared with those
‘obtained for anaérobic fermentations induced by B. coli on the
same medium, and given by one of us in a previous paper,” with the
following results: (1) Smaller volumes of carbon dioxide are pro-
duced aérobically than anaérobically, for all periods of time. (2)
For the same amount of carbon dioxide, less hydrogen is obtained
aérobically than anaérobically. The presence of oxygen therefore
appears to lessen the production of gases from dextrose and also
either to cause some output of carbon dioxide by a respiratory
process or to cause a disappearance of hydrogen (presumably) by
oxidation.

Experiments with Bact. welchiv.

Similar experiments were made with Bact. welchii. The me-
dium consisted of 1.00 per cent each of Witte’s peptone and Merck’s
“Highest Purity’ dextrose in distilled water, prepared hot and
filtered. No alkali was added. Other conditions were the same
as before, except that all durations of incubation were much longer
and the pressures of oxygen were much smaller.

1 V
The values of the expression : log “p are given in table III. They

are very nearly constant and are about one-third the value found
for B. coli.
All other results are given in table I]. The mean value for

Oz
the respiratory quotient ( ) is 0.014, 7.e., one-tenth the value

CO
found for B. coli, with a probable error of + 0.002.

The ratio COz:Hz is slightly raised by the presence of oxygen,’
and does not vary for this organism in great degree as it does for
B. coli.

As with B. coli, the ‘‘missing”’ volumes of hydrogen are not equal
to twice the volumes of oxygen absorbed but are sometimes greater
and sometimes smaller. It is perhaps of significance in this con-
nection that both the media used for these organisms permit side-
reactions which prevent the evolution of equal volumes of carbon
dioxide and hydrogen by anaérobic fermentation of dextrose, as
discussed in the first paper of this study.

2 Keyes: loc. cit.
Values for anaérobic fermentations are given in the first paper of this
series.

THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XIII, NO. 3.

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Gas Metabolism of Ba

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F. G. Keyes and L. J. Gillespie

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310 Gas Metabolism of Bacteria

TABLE III.

volume Oz» admitted

1
Values of —: log : bate ee iy
l e volume O» recovered

{ = time in hours; common logarithms.

HOURS B. COLI HOURS BACT. WELCHII
24 0.0059 240 0.0013
66 0.0030 240 0.0010
72 0.0040 336 0.0005
74 0.00389 384 0.0011
119 0.0028 | 432 0.0015
167 0.0030 480 0.0013

192 0.0027

Mean value 0.0035 + 0.0003

0.0011 + 0.0001

SUMMARY.

The absorption of oxygen by growing cultures on dextrose media
of B. coli and of (the strict anaérobe) Bact. welchit has been studied
by an exact method. Data afforded by complete gas analyses are
given. The following comparisons are made possible:

1. For both microérganisms, the absorption of oxygen simulates
a monomolecular reaction.

2. The mean values of the respiratory quotients, although the
probable error of each is large, are widely different for the two
microorganisms.

3. With varying pressures of oxygen, the ratio CO2:He varies
enormously in the case of B. coli, but varies only slightly in the
ease of Bact. welchit.

We think that the numerical differences in the results for the
two microérganisms may possibly be referable to differences in
experimental conditions; if so, probably to the differences in
oxygen pressures.

112

HOW DO ISOTONIC SODIUM CHLORIDE SOLUTION
AND OTHER PARTHENOGENIC AGENTS
INCREASE OXIDATION IN THE

| SEA URCHIN’S EGG?
BY J. F. McCLENDON anp PHILIP H. MITCHELL.
Journal of Biological Chemistry, Vol. X, No. 6, 1912, pp. 459-472

Reprinted from Tur JouRNAL or BroLtoaicau CuEemistry, Vol. X, No. 6, 1912

HOW DO ISOTONIC SODIUM CHLORIDE SOLUTION AND
OTHER PARTHENOGENIC AGENTS INCREASE
OXIDATION IN THE SEA URCHIN’S EGG?

By J. F. MCCLENDON anp P. H. MITCHELL.

(From the Embryological Laboratory of Cornell University Medical College,
New York City, the Physiological Laboratory of Brown University, Provi-
dence, R. I., and the U. S. Bureau of Fisheries, Woods Hole, Mass.)

(Received for publication, November 4, 1911.)

Loeb has shown that —OH ions favor development.!. Our own
experiments (Table 6) as well as those of O. Warburg? demonstrate
that increase in the alkalinity of the medium increases oxidation
in fertilized eggs.

According to Warburg, the egg is impermeable to —OH ions or
fixed alkalies, because, although eggs stained with neutral red
are changed to yellow by NH; of a concentration which increases
the oxidation only one-tenth, similar eggs are not changed to
yellow by fixed alkalies of a concentration at which oxidation is
greatly increased.

These facts are, however, capable of another interpretation.
The egg is filled with lipoid particles which take up the neutral
red to such an extent as to render the surrounding protoplasm and
sea water colorless. The ~OH ions cannot freely enter the lipoids
in order to change the color of the neutral red. On the other hand
ammonia is lipoid-soluble and can enter.

It might be objected that the ammonia cannot react with the
dye in the non-aqueous medium owing to the suppression of ioni-
zation, but whether the dye is driven out of the lipoids or changed
to yellow in situ, the fact remains that it does become yellow.

Harvey’ observed that the addition of but a small quantity of
alkali to sea water containing fertilized eggs stained with neutral

1 Loeb: Chemische Entwicklungserregung des tierischen Eies. Berlin, 1909.
2 Warburg: Zeitschr. f. physiol. Chem., |x, p. 305, 1910.
3’ Harvey: Journ. of Exp. Zoology, x, p. 507, 1911.

459

460 Oxidation in the Sea Urchin’s Egg

red, changed the eggs to yellow. However, the eggs were injured
by the alkali and probably their permeability was increased. It
is possible that alkalies or ~OH ions in any concentration enter
the fertilized eggs but must be present in sufficient concentration
to set free ammonia or change the lipoids in order to affect the dye.

We may assume, then, unless more conclusive evidence indicates
the contrary, that the ~OH ions increase oxidation after penetrat-
ing the egg.

One of us has shown that unfertilized sea urchin’s eggs are poorly
permeable to salts and their ions, but become more permeable
after fertilization or the initiation of parthenogenetic develop-
ment.! Not only is the permeability increased but the oxidation
rate is increased. Warburg observed that oxidation increases
from five to seven times on fertilization (compare our Table 5,
experiment III). He found also a large increase after the initia-
tion of parthenogenetic development caused by hypertonic sea
water,” fatty acid, alkalies or traces of the heavy metals,® silver
or copper. Our own experiments, given below, confirm and extend
these findings. There appears then to be some relation between
permeability and oxidation, and the present paper is an attempt
to determine what this relation is.

The living cell may be compared to a furnace, and R. Lillie*
advanced the view that increase in permeability opens the draughts
so to speak, allowing the escape of carbonic acid, and hence oxi-
dation is increased. He supposes that the accumulation of car-
bonic acid and perhaps other end-products checks the oxidation,
and increase in permeability to carbonic acid allows oxidation to
proceed. The difficulty with this hypothesis lies in the fact that
living cells have been shown by Overton and others to be freely
permeable to substances which are easily soluble in fats and oils,
or especially in lecithin and cholesterin. Carbon dioxide is solu-
ble in oils and probably enters cells easily, at least there is evidence
to show that red blood corpuscles are freely permeable to this
gas. Fatty oils are permeable only to the undissociated mole-
cules and not to the ions. Since the proportion of ions of carbonic

1 McClendon: Amer. Journ. of Physiol., xxvii, p. 240, 1910.
2 Warburg: Zettschr. f. physiol. Chem., |xii, p. 1, 1908.

3 Warburg: Jbid., Ixvi, p. 305, 1910.

4 Lillie: Biol. Bull., xvii, p. 188, 1909.

VOLUME (DILUTION)

J. F. McClendon and P. H. Mitchell 461

acid would ordinarily be small, what conditions in the egg might
favor ionization of CO.?

Not all of the alkali metals in the egg are combined with min-
eral acids; some are combined with proteins. This has been shown
by the senior author with electric conductivity measurements of
hens egg yolk given in the accompanying curves. The continu-
ous line represents the conductivity of yolk freed from protein
granules, and the dotted line, yolk containing an excess of protein

-001 -002 -003
ELECTRIC CONDUCTIVITY

granules, separated by the centrifuge. The granules impede the
current as shown by the fact that the granule-containing yolk is a
poorer conductor than granule-free yolk. But on dilution, the
granule-containing yolk becomes the better conductor. There-
fore ions are set free from the granules on dilution.

The carbonic acid formed within the egg would react with
the alkali albuminates with the formation of alkali carbonates
and bicarbonates, which, notwithstanding hydrolysis, would
liberate a considerable quantity of carbonic acid anions.’ The
dissociated carbonic acid, being unable to escape from the unfer-
tilized egg would lower the ~OH ion concentration and thus reduce
oxidation. As the undissociated molecules of carbonic acid escape,
more are formed by the slow oxidation in the egg.

On fertilization, the permeability to the anions of carbonic
acid is greatly increased. They migrate out of the egg, and nega-
tive ions enter the egg to take their place. Since the —OH ions of
the sea water are the fastest negative ions, they enter the egg and

1 At about molecular concentration the equivalent electric conductivity
of NaCl is 76; of NazCOs, 45, at 18°.

462 Oxidation in the Sea Urchin’s Egg

increase oxidation. However, if some ~Cl or ~SO,4 ions entered
the egg they would tend to decrease the ~OH ion concentration
within the egg. But the senior author has shown that the ferti-
lized Fundulus egg is impermeable to ~Cl ions, for when placed in
distilled water, or in solutions of nitrates or sulphates, practically
no chlorine comes out of the eggs.!. We may assume, therefore,
that carbonic acid accumulates in the unfertilized egg until the
reaction is neutral or slightly acid. But on fertilization, the per-
meability to carbonic acid anions is increased and the concentra-
tion of this acid is diminished so that the reaction is neutral or
slightly alkaline. The increased alkalinity is the cause of the
increased oxidation. We will now see to what extent our experi-
ments bear out this assumption.

We observed that oxidation is about doubled when the egg is
made parthenogenetic with carbonated sea water (Table 5, experi-
ment II) or alkaline isotonic sodium chloride (Table 3, experi-
ments I and II). In some cases the eggs, being physiologically
different from those in other experiments, did not show the mor-
phological signs of development in this solution, and oxidation was
not doubled (Table 2: Table 3, experiment III; cf. Table 5, experi-
ment I). The eggs begin development while in the alkaline solu-
tion, but eggs made parthenogenetic by treatment with neutral or
acid solutions (neutral sodium chloride or carbonated sea water)
begin development only when transferred to natural sea water or
other alkaline solution. This bears out our hypothesis, for if the
increased permeability remains after the egg is returned to an alka-
line medium, a chance is given for an increase in alkalinity in the
interior, which was lacking in the non-alkaline solution.

Certain facts may seem to contradict our assumption, but prob-
ably they merely limit its application:

1. A slight increase in OH ions may cause even the unfertilized
egg to absorb more oxygen (Table 2) and a greater increase causes
it to develop. This does not necessarily show permeability of the
unfertilized egg to hydroxyl ions. The increased alkalinity
slowly causes an increased permeability of the egg and thus leads to
parthenogenesis, but the degree of alkalinity of the medium neces-
sary to induce development of the unfertilized egg is far greater

1 These experiments will be published later in the American Journal of
Physiology.

J. F. McClendon and P. H. Mitchell 463

than that necessary for the development of the fertilized egg or
the egg already made parthenogenetic.

2. The fertilized egg of Arbacia punctulata (but not of some other
sea urchins) may develop in a natural medium, as Loeb observed
and which we have confirmed. In other words, a hydroxy] ion
concentration in the medium, greater than that of distilled water,
is not necessary for development of this egg made freely per-
meable to carbonic acid. However this fact does not set a limit
to the alkalinity of the egg interior. The egg probably contains
more Na than Cl ions, and if it be impermeable to Na or Cl, the
escape of carbonic acid might cause the egg interior to become
alkaline or at least neutral. Eggs made parthenogenetic in some
ways (neutral sodium chloride, for instance) do not develop unless
transferred to an alkaline medium, but this may be due to the possi-
bility that these parthenogenetic eggs are not quite as permeable
as are fertilized eggs. The same may be inferred from oxidation
measurements. Neutral sodium chloride causes the unfertilized
egg to absorb more oxygen than it does in sea water (Table 4)
but the increase is slight, and morphological development does not
commence. If the eggs are then transferred to sea water or other
alkaline solution, some of them may develop.

It appears therefore that increase in permeability is a gradual
process. Although some eggs are so permeable as to be able to
develop in an neutral medium others are less permeable and do
not develop, or develop only in an alkaline medium. By treating
eggs with parthenogenetic agents in various concentrations or for
various lengths of time we may induce various degrees of per-
meability. Even fertilized eggs may be made more permeable by
treatment with parthenogenic agents, and a corresponding increase
in oxidation may be observed (Table 6). In these experiments
the oxidation of the eggs in sea water was measured about ninety
minutes after fertilization: they were then placed in isotonic,
alkaline sodium chloride solution, in which the oxidation increased
one-half, when returned to sea water the oxidation fell below its
previous level in the same medium. According to Loeb, this
indicates death of some of the eggs (20 per cent).

The experiments just described explain the discrepancy between
the results of Warburg and those of Loeb. Warburg! found that

1 Warburg: loc. cit.

464 Oxidation in the Sea Urchin’s Egg

the oxidation of the fertilized egg in isotonic sodium chloride solu-
tions containing a trace of sodium cyanide, is much greater than
in sea water containing the same concentration of sodium cyanide.
Loeb! confirmed this determination, but observed further that
if the eyanide is omitted (from both) no increased oxidation in
sodium chloride solution occurs. The cyanogen in both sea water
and sodium chloride solution depresses oxidation. Since sodium
eyanide liberates "OH ions, we may conclude that the increase
in oxidation in the sodium chloride solution used by Warburg was
due to the increased penetration of hydroxyl ions, following in-
crease of permeability.

In our experiments no cyanide was used, and the alkalinity of
the sodium chloride solution was not greater than sea water, yet
oxidation was increased. In Loeb’s experiment the tendency of
increased permeability to increase oxidation was counteracted
by the effect of lower alkalinity, which decreases oxidations.

Alkaline sodium chloride solution also favors oxidation in eggs
that have reached later stages of development, morula or blastula
(Table 7). In this experiment, the rate of oxidation in sea water
was rising gradually (see next section) before the eggs were placed
in the alkaline soduim chloride solution, but in the latter a sudden
increase of more than 50 per cent was observed.

MATERIALS AND METHODS.

The eggs of the sea urchins, Arbacia punctulata, were used. The
animals were washed in a strong stream of fresh water and opened
with precautions against introducing spermatozoa among the
eggs. The ovaries were removed and placed in the first solution
to be used, sea water or neutral van’t Hoff’s solution. The mass
was strained through bolting cloth of such a grade as to allow but
one egg to pass through one mesh at a time. The eggs were
repeatedly precipitated by gravity in fresh portions of the solution
in order to remove coelomic fluid cells (elaeocytes), and trans-
ferred with a small quantity of fluid to the determination flask.

The rate of oxidation in the various solutions was measured by
comparison of the dissolved oxygen in the solution before and
after the eggs had been suspended in it during a definite period.

1Loeb and Wasteneys: Biochem. Zeitschr., xxviii, p. 340, 1910.

J. F. McClendon and P. H. Mitchell 465

Winkler’s thiosulphate method of oxygen determination (iodo-
metric), as described in Treadwell’s Quantitative Analysis, was
used.

From 3 to 7 cc. of eggs were used in each experiment, but the
actual volume of the eggs was not measured until after the oxygen
determinations. j

We tried a number of methods for filling the determination flask
and sample bottles without an uncertain loss or gain of oxygen.
Loeb collected the water in the sample bottle under petroleum.
Although petroleum absorbs five times as much oxygen as water
does, the oil would tend to reduce currents adjacent to the air-
water surface, and thus reduce oxygen exchange. Using paraffin
oil, we found that it was extremely difficult to prevent a little oil
from sticking within the flask, and abandoned the method. Per-
haps kerosene would have worked better, yet the quantity of kero-
sene that would dissolve in the water might vitiate the experiments.

Since the sea water and the solutions were shaken up and satu-
rated with air at the given temperature before beginning the exper-
iment, the control sample might have been taken under air, with-
out change. But after loss of oxygen in the determination flask,
a gain in oxygen would result from such treatment. By intro-
ducing the solution through a tube passing through a doubly
perforated stopper, and extending to the bottom of the sample
bottle, the exposed surface of the water was made as small and
quiet as possible. By maintaining a constant rate of flow the
error could be made to bear an approximately constant ratio to the
oxidation, no matter whether the sample bottle was filled with air
or some other gas. We tried the effect of introducing the sample
under air, and also under hydrogen, and decided that the latter
method was preferable for oxygen-low samples. In order to make
all errors fall in the same direction we also collected the oxygen-
high samples under hydrogen.

The experiments were so regulated that the oxygen content of
the determination flask at the end of the exposure would not fall
very low. However, Warburg failed to observe a decrease in the
rate of oxidation in low oxygen concentration.

The water was forced out of the determination flask rapidly into
the sample bottle by hydrogen under pressure. The determination

466 Oxidation in the Sea Urchin’s Egg

flask held 332 ec., the two sample bottles 152 and 142.6 cc. respec-
tively.

The determination flask was placed in a thermostat which was
kept 2° above the temperature which the air had reached at the
beginning of the experiment. In most cases the time of exposure
was one hour. During the first half-hour the eggs were distributed
throughout the solution once every five minutes by rotating and
rocking the flask, during the last half hour they were allowed to
settle to the bottom.

Although the majority of the eggs settle to the bottom in ten
minutes, at the end of one-half hour there are always a few which,
on account of swelling or fragmentation, have failed to precipitate.
To prevent these going over into the sample bottle and causing
an error due to absorption of iodine, Loeb placed filter paper over
the outlet. We found that a relatively hard filter paper was neces-
sary to retain all fragments of eggs, and that this interfered with
the rapid transfer of the solution. The error due to eggs in sus-
pension is negligible, especially since it was practically constant
in all of our experiments. For instance: a sample bottle filled with
water contained 8.09 parts per million of oxygen, while water from
the same jar run into a sample bottle in which had been placed
about one-hundred times as many eggs as the water from the deter-
mination flask, contained 7.85 parts per million, an error of 0.24
parts per million due to eggs in suspension. As in our experi-
ments, differences of 0.1—3.0 parts per million were obtained, an
error of 0.024 parts per million would not reverse the results.

When the eggs were fertilized or placed in parthenogenic solu-
tions they lost sufficient red pigment (MeMunn’s Echinochrome) to
color the water a straw yellow. In order to ascertain whether this
organic matter would vitiate the results, we took two sample bottles,
into one of which was placed a mass of elaeocytes containing about
fifty times as much echinochrome as is lost from the eggs in one
experiment, and syphoned into each tap water from the same
jar. Tap water causes these cells to liberate their pigment. The
bottle containing water only, was found to hold 6.85 parts per
million of oxygen, while that contaminated by elaeocytes was
titrated as 6.36 parts per million of oxygen. We repeated this
using three sample bottles. ‘Two of these filled with water gave

J. F. McClendon and P. H. Mitchell 467

6.86and 6.91 parts per million respectively and the third contamin-
ated with elaeocytes liberating about one hundred times as much
pigment as is liberated in the experiments with eggs, gave 6.09
parts per million as the titration, showing an error of 0.8 parts per
million. Probably this loss was due chiefly to the broken up
cells, but if due entirely to the pigment, the error in our experi-
ments would be only .008 parts per million.

In experiment 3, the eggs were divided into two equal portions
and placed simultaneously in two determination flasks of equal
capacity. Therefore the eggs in the two solutions were in the same
stage of ripeness. In each of the other experiments the eggs were
all placed in one flask and treated successively with the various
solutions. In case of fertilized eggs Warburg observed! that the
oxidation rate rose steeply from fertilization to the 2-cell stage then
gradually to the 64-cell stage. In order to determine whether this
would be a great source of error on our experiments, we measured
the oxygen used by a mass of fertilized eggs in sea-water during
successive periods in the first six hours of development, and found
it to vary from 7.93 to 9.76 tenths of a milligram (Table 7). With-
in the duration of the majority of our experiments, however, the
variation was only from 7.93 to 8.96 tenths of a milligram or 11.5
per cent, and would be less between successive exposures. This
possible source of error would not reverse the results of the major-
ity of our experiments.

In making solutions of the same alkalinity as the sea water, a
colorimetric method was used, with phenolphthalein as indicator.
At the end of the season, the sea water was diluted with heavy
rains and failed to color phenolphthalein. The eggs behaved
abnormally, though whether this was due to the decrease in alka-
linity or salinity of the sea water or to some other cause was not
determined.

In making the eggs parthenogenetic with carbon dioxide, they
were placed with a small quantity of sea water in a “sparklet
syphon” and charged under slight pressure for about one minute.
At the end of five minutes they were poured into a large volume of
sea water and this was syphoned off and fresh sea water added.

1 Warburg: Zeitschr. f. physiol. Chem., |x, p. 448, 1909 and loc. cit.

468 Oxidation in the Sea Urchin’s Egg
RESULTS OF EXPERIMENTS.

I. Experiments with Unfertilized Eggs.

TABLE 1.

Oxidation in neutral van’t Hoff’s solution contrasted with that in neutral
NaCl solution.

| a a & a
Be
§ BS ac) hale) a
H HO ZZ ZZ p
EXPERIMENT | SOLUTION Bin aa Oo Re oa
USED a5 at Be BE a REMARKS
Ro a a Zi(= <5 cS)
paige) 28 en
H Aa a °
ce nO min. | min aS
Neutral
First period. .| { van’t 4 24 | 30—/ 30 2.49
Hoff
No ‘‘fertili-
Second period f ee { 4 24 30 30 Myles oe =
\ ¥ NaCl if membrane
formed.
TABLE 2.

Oxidation in neutral van’t Hoff’s solution contrasted with that in sea water
and alkaline NaCl solution.

EXPERIMENT SOLUTION 5 $ Sg 6 g 6 5 : REMARKS
WEED iS g f ate She pees 3
Be | 2B | Be | pe a
> & i=) f=) °
ce 6 min min ae
Neutral
First period. .| { van’t 24 30 30 | 2.29
Hoff.
Second period Sea water 24 30 30 | 2.65
“‘Fertiliza-
M NaCl tion mem-
Third period +—OH 24 30 30) 3.12 branes’”’ in
ions very small
per cent.
Fourth period| Sea water 24 30 30 | 3.48

J. F. McClendon and P. H. Mitchell

Oxidation in sea water contrasted with that in alkaline NaCl solution.

TABLE 3.

469

Pa esc as, he
§ & 2 ze | 2s 6
EXPERIMENT phy g m2 FE Fe E z 2 S z REMARKS
Bo | Go | ga | aa
of Qe pa pa 2)
> a a a °
cc HO; min min aT
I. One-half
of eggs..... Sea water 23 30 30 | 4.80
“e <) pi
Second half z gulete egme
-OH 2
of eggs..... ae 23) i 30 4) 30) [6:90 eee
IL. First formed.
period...... Sea water 24.5| 35 | 25 | 3.38
™ NaCl “Fertiliza-
Second period { +~OH 24.5| 35 25 | 7.80 tion ra
aia brane
f :
Ill. First puned
period..... .| Sea water| 4.5 | 24 30 30 | 6.04
“‘Fertiliza-
¥% NaCl tion mem-
Second period) ; +~OH 4.5 | 24 30 30 | 6.47 branes” in
ions very small
per cent.
TABLE 4.
Oxidation in sea water contrasted with that in neutral NaCl.
ee | 5 | 8 3
pee |e see oa i aee te
EXPERIMENT parte is 3 E 5 i E 4 é 5 a REMARKS
se | ge | ge | 2 | &
> a Qa a °
ce. °¢ min min os
I. First
period...... Sea water | 5 23 30 30 | 2.65
(No “‘fertili-
._ If Neutral zation mem-
Second period \ ™ NaCl 5 2 30 30 | 3.60 Beat
formed.
II. First
period....| Seawater | 2.3 | 23.5 | 30 30 | 1.22
Second period { oom } 2.3 |23.5| 30 | 30 |1.66| Ibid.

470 Oxidation in the Sea Urchin’s Egg

TABLE 5.

Effect of CO.-parthenogenesis on oxidation. Effect of fertilization.

Hae bce! Seu FS gale
scuevcow all Wee | eae al Coal foal ate
PXPPRIMENT ane 3 2 s i & a E = a REMARKS
B es So me a a Da
> Be Lee A é
cc EG: min min. aan
I. First
period.....| Seawater | 4.5 | 23.5 | 30 30 4.58
| “Pertiliza-
After CO2 tion mem-
treatment. .| Sea water AP al2o2On| cou 30 6.00 branes”’ in
small per
cent.
LeShinst
period...... Seawater | 4.5) 24 | 30 | 30 | 4.11
After COz Good mem-
treatment. .| Sea water 4.5| 24 30 30 9.91 brane
III. First formed.
period...... Sea water 25 30 30 2.52
After fertili- 98 per cent of
ZAaGlONs ss. < Sea water 5 25 30 30 | 12.94 eggs seg-
mented.
SUMMARY.

1. The presence of —OH ions in the medium, increases the rate
of oxidation in fertilized eggs of the sea urchin.

2. The oxidation rate of unfertilized eggs is increased by fer-
tilization or any treatment which causes them to develop partheno-
genetically.

In 1 and 2 we merely confirm and extend the observations of
Warburg.

3. Since it was shown by the senior author that fertilization or
parthenogenesis means increased ionic permeability of this egg, and
that the Fundulus egg, even after fertilization, is impermeable to
—Cl ions, the increase in permeability probably applies so far as the
anions are concerned, to OH and ~HCO; or ~CO; ions. The car-
bonic acid anions are more concentrated within, and their outward
diffusion would cause a potential gradient which would pull other

J. F. McClendon and P. H. Mitchell 471

IT. Experiments with Fertilized Eggs.

TABLE 6.
Oxidation in neutral van’t Hoff’s solution contrasted with that in sea water
and alkaline NaCl. —
BH | =)
s [Eb lee | ee! -
EXPERIMENT penile 3 @ F z & FA z 2 is BAAS ES
BS | ge | 32 | 3B] 2
SF ee Wack Aol se
ce °C min min. | mgr./10
I. First Neutral (ne
period...... en 2G) ee 30 30 6.47 some sea
Hoff water.
Second period) Sea water 3.6 | 23 30 30 7.43
Third * NaCl
period...... {# —OH 3.6 | 23 30 30 | 12.28
ions
Fourth Very few
period ....| Seawater} 3.6] 23 30 30 5.97 { eggs dead.
Il. First | (Neutral <a a
period...... van’t 4 23 30 30 3.91 Hoff col.
ne and ferti-
lized in it.
Second period Sea water | 4 23 30 30 Daud
M™ NaCl
Third period fs —OH 4 23 30 30 7.53
ions
Fourth period) Sea water | 4 23 30 30 6.27

anions, hence ~OH ions, into the egg, thereby increasing the inter-
nal concentration of "OH ions. This increase in ~OH ions proba-
bly causes the increased oxidation.

4. The increase of —OH ions in the medium causes even in
unfertilized eggs, an increased oxidation. This is not interpreted
as indicating that the unfertilized egg is normally permeable to
—-OH ions, but that increased alkalinity causes increased permea-
bility.

5. Increase in permeability is a gradual process. Beginning
with the relatively impermeable unfertilized egg, and denoting
degree of permeability by numerals, we have the following series,

472 Oxidation in the Sea Urchin’s Egg

TABLE 7.

Effect of alkaline NaCl on oxidation six hours after fertilization.

Bel SelB ant ae
=) n
ror 6 | #8 | ze | zz p
SXPE . SOLUTION a ay o8 oF - :
EXPERIMENT USED 5 ~ | ee S % Ps E a REMARKS
Ro | g e ae Pas be
5 A ge p@ p@ K
> & A a °
4 2 ae Se , _|—_
ce 5 min min —
Thirteen
i iod 7 99 20 30 3.90 minutes
First period..| Sea water 2 : WPT 3,
lization.

bo
iS)
bo
S
ide)
S
ee
a)
a

2-cell stage.
4-cell stage.
16-cell stage.
32-cell stage.

Second period Sea water vp

Third period | Sea water U

Fourth period Sea water U 22 20
7
7

wNn
woe
oo 0 09
oos
CoS
Ke © ow
wo &

Fifth period..| Sea water

Sixth period..| Sea water 22 | 20 | 30 | 9.76) Manyretarded.
Seventh i eriNia Cl M bl
period... + —OH T 22) \ 020.) 180" | ae eza { eee
“ong tulae.

Eighth period Sea water li 22 20 30 8.98) All alive.

I. slightly increased oxidation, II. greater increase in oxidation
rate, and imperfect ‘‘fertilization membrane”’ formation, III. oxi-
dation still further increased, membrane formation perfect, fol-
lowed by segmentation of the egg, IV, ditto except that oxidation
is still further increased, and the eggs die sooner or later if oxida-
tion is not reduced, V. oxidation enormous, membrane formation
but no segmentation, premature death.

It is supposed that the primary effect of many toxic substances
is an abnormal increase in the permeability of the egg, and ferti-
lized eggs are more susceptible because they are already more per-
meable than unfertilized eggs.

113

THE SIGNIFICANCE OF THE TIME AT WHICH GAS
IS PRODUCED IN LACTOSE PEPTONE BILE.
BY WILLIAM W. BROWNE.
American Journal of Public Health, Vol. III, No. 9, 1913, pp. 944-956.

THE SIGNIFICANCE OF THE TIME AT WHICH
GAS IS PRODUCED IN LACTOSE
PEPTONE BILE

WILLIAM W. BROWNE, Pu. D.

[Reprinted from the AMERICAN JOURNAL oF Pustic Heatrtn, Vol. 3. No. 9.]

THE SIGNIFICANCE OF THE TIME AT
WHICH GAS IS PRODUCED IN
LACTOSE PEPTONE BILE.

Wituiam. W. Browne, Pu.D.,
College of the City of New York.

During the summer of 1912, routine bacteriological examinations of
the oysters of Narragansett Bay were made with the hope of determining
the extent of the pollution of the oyster beds of Rhode Island by the sewage
of cities and towns bordering on the bay. The examinations were con-
ducted under the direction of Prof. F. P. Gorham of Brown University
to whom the writer wishes to extend his thanks for the data here used.
Throughout the entire series of experiments, the examinations of the
oysters were made in general, according to the methods proposed in The
Second Progress Report of the Committee on Standard Methods of Shell
Fish Examination (December, 1911). In all cases, meat extract (3 grams
to the liter) was used in the preparation of the media. Ease of preparation
rendered its use invaluable in a work of this nature.

Lactose peptone bile was used as a presumptive test to indicate the
presence of members of the Bacillus coli group and other lactose fermenters
of intestinal origin. Inverted vials were used for the collection of the gas
and the tubes were always incubated at 37° C. All inoculation into lactose
peptone bile were made in duplicate and the gas was read and recorded
at the end of 24-, 48- and 72-hour periods. In this paper are compiled the
results obtained by comparing the number of tubes showing gas at the
various periods.

During the period covered by this paper, 119 samples of oysters were
examined. The beds from which these oysters were dredged varied greatly
in character and position ranging from the highly polluted areas near
the outlets of the sewers of the cities and towns through questionable areas
which were sometimes coli-positive and again coli-negative to areas which
were comparatively free from pollution near the ocean. During the exam-
ination of these 119 samples of oysters 3,570 tubes of lactose peptone bile
were inoculated from which 1,929 produced gas distributed over the 24-,
48- and 72-hour periods as the following table will show:

TABLE I.
LACTOSE PEPTONE BILE TUBES SHOWING GAS.
Lime ini ours 2.35 ssi sts een ines Q4 48 72
Number of Tubes Showing Gas.......... 148 1361 420
IER Cen tea secre wk eS Se OE Sak 7.6 70.5 ole2

From the above table it can be seen that very few of the intestinal
organisms, generally found in oysters, are able to produce gas in lactose
944

Gas Produced in Lactose Peptone Bile 945

peptone bile by the end of the 24-hour period since out of 1,929 which
showed gas only 148 or 7.6 per cent. produced gas at the end of that period.
By the end of the 48th hour, however, 1,361 tubes or 70.5 per cent. pro-
duced gas showing that 48 hours represents the periods in which more
than a majority of the intestinal organisms usually found in the oysters,
produce their gas in lactose peptone bile. Gas was recorded at the end
of the 72-hour period in 420 or 21.2 per cent. The gas produced by the
end of the 72d hour probably represents the result of growth of attenuated
forms and spore formers since we find more of this type of gas production
in places which are distant from sources of pollution as will be explained
in a later part of this paper.

An examination of the map of the State of Rhode Island reveals that
Narragansett Bay is divided naturally into several well-defined areas by the
presence of islands and peninsulas. The author has taken advantage of
this fact and for the purposes of comparison, has divided that portion of
the bay from which oysters for analysis were taken into eight distinct
areas. Some of these areas include rivers which flow into the bay while
others include large portions of the bay itself. Oysters were taken from
the beds situated in almost every portion of these well-defined areas and
at various intervals during the summer of 1912, so that any comparison
that may be made between the various districts in the bay seem to the
writer to be both reasonable and fair.

Plate I shows the divisions into which the author has divided the bay.
A brief geographical description of each district will follow together with
its likelihood of being polluted as might be judged both from bacteriological
examinations and a superficial sanitary survey.

District I.

District I includes what is known as the Kickemuit River, a small shallow
stream flowing into Mount Hope Bay from the north. Because of its
shallowness and protection from winter storms, this river offers an ideal
place for the cultivation of oysters. Extended bacteriological examina-
tions have shown this river to be a variable locality. The examinations
of one period showing the oysters to be of a high sanitary quality in so
far as the presence of fecal organisms is concerned while only a few days
later, examinations have revealed gross pollution. As there are no towns
on its banks, this pollution must necessarily come from Mount Hope Bay.
At the present time, it is thought that the wind and tide play a very im-
portant part in the varying conditions of this river.

District II.

This district includes Mount Hope Bay and extends from north to south
from the state line to Bristol Ferry. The bay has two outlets one empty-
ing into Narragansett Bay at Bristol Ferry while the other opens into
the Sakonnet River. Bacteriological examinations reveal the oysters
from Mount Hope Bay to be grossly polluted during the greater part of

‘

946 The American Journal of Public Health.

the year. ‘This condition might be expected since this bay receives the
sewage of the City of Fall River and of the cities and towns along the
Taunton River.

Districr III.

District III is known as the Sakonnet River which is nothing more
than an outlet for Mount Hope Bay. Being very wide, however, and
near the ocean, its waters are greatly diluted and as a result, we find the
water of the Sakonnet River to be in a relatively high state of purity
bacteriologically.

District IV.

District IV includes all the beds situated in Bristol Harbor and in the
waters west of the Island of Rhode Island. Repeated bacteriological
examinations have shown this region to be open to great pollution as it
is the meeting place of the waters of Narragansett Bay, Bristol Harbor
and Mount Hope Bay.

District V.

District V includes the beds in the Warren River which for the greater
part of the year are subject to gross pollution.
District VI.

District VI may be designated as that region where the Providence
River merges into Narragansett Bay. Bacteriological examinations show
evidences of pollution.

District VII.

This district known as Upper Narragansett Bay although open to more
or less pollution from the Providence River seems to be almost able to
cere for about all the pollution which reaches it. Examinations have re-
vealed pollution but not in quantity.

District VIII.

District VIII includes that portion of the bay directly south of Dis-
trict VII and will be designated as Lower Narragansett Bay. Bacteriologi-
cal examinations have failed to reveal evidences of much pollution.

In summary of the above description, the eight districts or areas may
be roughly divided into three main classes as to their possibility of be-

—

coming polluted.
TABLE II.
CLASSIFICATION OF THE EIGHT DISTRICTS.

A. Open to Gross Pollution:

Moun tiHope Bay: js cia setscors- Weare errr acters ernie tots ci Sonedeltt oloroleteaye eho nro tare District II.
Bristol arbor 2: ck Sec area eee ea oats SE So eno On cetera he ePaate “calves
WiarrensRuivern fst eee SPT Ree TL atone So oeiae ee ca acount eee\ ia
Providence Riveni:c) sere oe ascites Enters aust Mei eres Seas eae ee se eases SrouValls
B. Variable Locality:
Ka ckeminituRiver Societe: cee oll tee alec hoes heel eae eco ors anatase District I.
C. Relatively Free from Pollution:
Sakonriet Ravers ees. eaten Ceres oe hate Seat Or TOL tueys Sea terete ohare District IT.
Upper Narragansett. Bay 2232. Gece tas » emote eee e ieee See ene ‘Soule

Lower Narragansett Bay.,,....... sees eee renee eee rene eneeeeeees VETTE

ee

alia st teen! hee,

Gas Produced in Lactose Peptone Bile 947

From the various beds which happened to be situated in the designated
sections, oysters were taken to the laboratory and examined bacteriologi-
cally as outlined in the early part of the paper. In the following tables,
are compiled the results obtained by recording the presence of gas in the
lactose peptone bile tubes at the end of the 24-, 48- and 72-hour periods.

TABLE III.
NUMBER OF LACTOSE PEPTONE BILE TUBES SHOWING GAS FROM
DISERICT Ie
KICKEMUIT RIVER.
Bed. 24 hours. 48 hours. 72 hours.
4 6
16 0
15 0
15 1
17 4
18 5
26 1
0 20

Cm oO em OO TO HH

| coo WSCC oOHSCOWOHS

213 71
73.4 24.4

wo DD
i=)

Per cent.

TABLE, IV
NUMBER OF LACTOSE PEPTONE BILE TUBES SHOWING GAS FROM
DISTRICT IL.
MOUNT HOPE BAY.

Bed. ‘ 94 hours. 48 hours. 72 hours.
1 4 10 2
2 1 Q1 1
3 0 10 0
4 0 10 g
5 1 6 1
6 1 6 0
ff 9 6 0
8 1 10 g
9 0 16 1

10 6 6 0

11 0 12 1

12 12 3 1

13 3 18 1

14 5 24 0

15 0 3 3

43 161 15
Per cent. 19.6 73.4 6.8

948 The American Journal of Public Health

TABLE V.
NUMBER OF LACTOSE PEPTONE BILE TUBES SHOWING GAS FROM
DISTRICT III.
SAKONNET RIVER.

Bed. 24 hours. 48 hours. 72 hours.

1 0 0 1
Q 0 2 4
3 0 1 5
4 0 1 5
5 1 3 4
6 0 3 3
Te 0 1 4
8 0 9 4
9 0 13 Q

1 33 32

Per cent. 1s 50.0 48.4
TABLE VI.

NUMBER OF LACTOSE PEPTONE BILE TUBES SHOWING GAS FROM
DISTRICT IV.

BRISTOL HARBOR.

Bed. 24 hours. 48 hours. 72 hours.
1 0 9 0
2 0 19 0
3 13 15 2
4 0 14 1
5 13 8 4
6 0 16 0
Uf 0 20 0
8 0 19 Q
9 0 4 3

10 0 9 0
itt 0 9 6
12 0 10 8
13 0 6 4
14 0 4 4
15 2 ¢f 4
16 Q 3 1
17 Q Q1 0
18 0 4 1
19 3 5 8
20 1 22 3
Q1 0 28 0
Q2 0 30 0
23 0 30 0
24 0 30 0
25 0 0 15
26 0 13 2
Q7 0 4 2

36 349 70

Per cent. 7.9 76.7 15.3

Gas Produced in Lactose Peptone Bile 949

A careful study of Plate II brings out some very interesting points con-
cerning the production of gas in lactose peptone bile. As stated earlier
in this paper, a very small percentage of the tubes show gas by the end
of 24 hours. We would naturally expect those districts which are open

TABLE VII.
NUMBER OF LACTOSE PEPTONE BILE TUBES SHOWING GAS FROM
DISTRICT V.
WARREN RIVER.

Bed. 24 hours. 48 hours. 72 hours.
1 0 26 0
2 1 17 4
3 13 11 3
4 5 10 0
5 0 11 3
6 0 25 1
7 0 18 2
8 0 23 0
9 0 11 3

19 152 14
Per cent. 10.2 82.1 Uot
TABLE VIII.

NUMBER OF LACTOSE PEPTONE BILE TUBES SHOWING GAS FROM
DISTRICT VI.

PROVIDENCE RIVER.

Bed. 24 hours. 48 hours. 72 hours.

1 ve 26 0

Q Q ge 5

3 1 13 4

4 0 19 2

5 2 17 1

6 1 8 +

7 8 eA 1

8 0 13 i;

9 0 7 1
10 0 10 4
11 0 13 g
12 on 5 3
13 0 21 0
14 0 30 0

16 225 34
Per cent. 5.8 81.8 12-3

to gross pollution producing a larger percentage of tubes in 24 hours than
those tubes inoculated with material from regions which are comparatively
free from pollution. And such is the case. The arrangement of the dis-
tricts beginning with the areas which show the largest percentage of tubes

- in 24 hours is as follows:

950 The American Journal of Public Health

L, (Mount Hope Bay wn, en ccrens baie sornaws ate 19.6 per cent.
2. Lower Narragaiseth Gy yi. nore ites eee ote 11.4

TABLE IX.
NUMBER OF LACTOSE PEPTONE BILE TUBES SHOWING GAS FROM
DISTRICT VII.
UPPER NARRAGANSETT BAY.

Bed. 24 hours. 48 hours. 72 hours.
1 0 19 1
Q 0 16 1
3 0 10 6
4 1 "/ i:
5 0 18 8
6 1 Q7 0
a 0 6 8
8 0 10 Uh
9 0 4 6
10 0 8 12
11 0 4 0
12 3 7 Q
13 1 11 Q
14 0 3 17
15 0 Q 11
16 0 11 2
6 163 86

Per cent. 2°23. 63.9 B37

TABLE X.

NUMBER OF LACTOSE PEPTONE BILE TUBES SHOWING GAS FROM
DISTRICT VIII.

LOWER NARRAGANSETT BAY.

Bed. 24 hours. 48 hours. 72 hours.

1 1 15 12

Q 3 12 1

3 0 5 6

4 0 5 5

5 4 5 0

6 0 5 Q

Tf 10 1 0

8 0 9 17

9 0 4 5

10 0 0 10

11 0 4 12

12 0 0 7

13 1 1 6

14 0 0 5

15 Q 0 9

Q1 66 97
Per cent. 11.4 35.8 52.7
37 Warren Rivers. .4 tke eee oe eee 10.2 per cent.

Ay. bristol Harbors.3.0 1 ee ee oe eae 7.9

.

Gas Produced in Lactose Peptone Bile 951

PLATE T

NARRAGANSETT BAY
SHOWING DIVISIONS inte DISTRICTS

LOCATION 2 STATIONS
SAKONNET RIVER

r | c
32 33
fF Ga 43 a=
BRUSH NECK
46 48 0 51
5,
F Cn, = 25
Brgy, §
cxmain yee 64 65 66 61%
2y ES 2
F + +

POTOWOMUT
NEI
80 $1 82,
F }
Gast
e. 09 00 101
- a +
ALLENS
17
F
134
c Tv +
Fee

150 151 152 153
QUONSET PT.

952 The American Journal of Public Health
Os (PrOVideHGGHVeR = sin 4 Poas hes ee ee 5.8 per cent.
6: Upper Narragansett Bay 43.0). lee at 2:3
T. Kiekemurtittverne< io: te mies a ee seals FeO
HH f
f eH
a Sai esaieas teat
HEH
' ities = iH 1
paeaas : iE HEE :
+t BiH
aes: sues
ne rey y io eoeaeas
+. Spret 1 r
saasyuecgannay susrastvessassestess
c s pecs ai i ie
sosuuessessusesas cous EEE EE EEEEEEEEEEEEE geasgese! reeuaa
82) Sakonnetlivier:< supe ceas eee ke erate eee eee 1.5 per cent.’
f

From this list it can be seen that districts like Mount Hope Bay, Bristol
Harbor, Warren River and Providence River which are open to pollution

ig eee

Gas Produced in Lactose Peptone Bile 953

roughly group themselves at the top of the list while districts which are
distant from sources of pollution like the Sakonnet River, Kickemuit
River and Upper Narragansett are found at the bottom of the list. The
district designated as Lower Narragansett Bay has cast its lot with the
polluted areas as a result of a polluted sample from bed seven.

This division is still more apparent at the end of the 48th hour. The
arrangement of the districts beginning with the districts having the largest
percentage showing gas at the end of 48 hours is as follows:

ie ABECN ULV GR scat mnt ott She aliaicamtoiaseee Dea) 82.1 per cent.
PET eMEE MAW ER ie. ois Gi: De tis clio coats ae 2 81.8
SP ERMIGU OL ELAE ODL ote ota ones in bg vic aoe neta wee 16.4
mare A CHTPMERS ENCE ERY. su turet es duis aco tietew 5 a's cee 8» 73.4
Pee EHIME IVEIe cb mdceso Sos os oc wa Nek wate -ateie%e et 73.4
GullipperNartavanselisbay.... 20... 4s. si. Ja. a eos 63.9
Pe Une TL ALINER torn 3or 0s os alesse On a eed s 50.0
Sr Omen NaAbraatIsehE DAY... ohana. site. wine sabe ns 35.8

Here we find those grossly polluted areas showing a larger percentage of
gas tubes than those districts which are comparatively free from pollu-
tion. For instance, the percentage of tubes showing gas from the Warren
River is over twice that percentage shown by the gas tubes from Lower
Narragansett Bay which now has taken its rightful position among the
less polluted areas. The Kickemuit River which has by extended bacterio-
logical examinations been proved to be a very changeable district, oc-
cupies a position in the center of the list as we should expect.

At the end of the 72d hour the arrangement of the districts in the order
of percentages of tubes showing gas is quite reversed as the the following
list will show:

Peck awere arragansehy bay 2 ote Oew iiss. vs ee os 52.7 per cent.
ors aviini a Al 1 Wier or pia ig et 2 ep A8 .4
=. Upper Narragansett Daya. o.. 5.6.2: 42s ese os cat!
ce LSTA tial 2a oi oe ay ee i a Q4. 4
‘Sig ba O28 Eris ay ee hope bet ie Noe oo 15.3
Gee FO VINeNCe uly GR tiie ck ite ese oo Foes Fas 12:3
RAW TETCH PyIVET AP See ea ae fe ee bic one 7.6
et NOME PEO PO Asay ere fe oA as waits dia ccusts 0 oho 6.8

Those regions which are comparatively free from pollution produce
much larger percentage than regions which are open to pollution. At the
end of the 48-hour period, the percentages of tubes showing gas from the
Warren River was over twice as large as that percentage from the Lower
Narragansett Bay for the same period. At the end of the 72d hour, the
positions are reversed and the percentage of showing gas from the Lower
Narragansett Bay is over seven times as large. The same comparison may
be made between the other districts with the same result. The Kickemuit

954 The American Journal of Public Health

holds the same general position in the center of the list. Generally speak-
ing those districts which were at the top of the list for the 24- and 48-hour
periods are found at the bottom at the end of the 72d hour.

The only explanation of these results seems to the author to be that
those districts which lie near the source of pollution must necessarily be
exposed to vast numbers of healthy vigorous intestinal organisms which
only recently have left their native haunts and when these organisms are
placed in a suitable culture medium, like lactose peptone bile, a rapid growth
with the production of gas must result. Hence, we find that the intestinal
organisms isolated from oysters taken from polluted regions produce the
greater part of their gas by the end of the 48th hour.

The beds which are situated in districts more remote from the sources
of pollution present a different condition. The intestinal organisms which
finally reach these beds, must certainly be in an attenuated condition
and greatly reduced in numbers due to their long journey through the salt
waters of the bay. Hence the growth of the organisms and with that, of
course, the production of gas is retarded. The comparison between those
districts near the source of pollution which produce the greater part of their
gas by the 48th hour and those districts distant from the sources of pollu-
tion which produce the greater part of their gas after the 48th hour, may
be seen in the following table:

District. Gas produced at 48 Gas produced at 72 hours
hours in per cent. in per cent.
Mount-Hope Bay a5 i. site ae 93.0 6.8
Warnenthuivier v4, <:..ch kom e ee 92.3 {fas
iProyvadence Raver. icc. ee 0 1o 1923
iBristolttlarbor. = \0¢ fee east 84.6 15.3
Kickemuit River? << o> user 75 4 24.4
Wpper Narra bay ssi. es ae 66.2 Sane :
BakeonnetRiver ss. ssa. sera ae DL AS . 4
Trower: Narra, Baye a2 sen eee 47.2 Ole

From the above table it may be seen that the percentage of tubes show-
ing gas by the end of the 48th hour decreases as we proceed from those
districts open to gross pollution towards the districts which are free from
pollution. At the end of the 72d hour things are just opposite. The per-
centage of tubes which show gas increase as we proceed from the greatly
polluted towards the less polluted areas. With the exception of the Lower
Narragansett Bay District over 50 per cent. of the tubes show their gas
by the end of the 48th hour.

This same conditon was found to be true for the Sakonnet River which
is really an outlet for the waters of Mount Hope Bay from which it re-
ceives the greater portion of its pollution. The town of Tiverton also con-

Re

Gas Produced in Lactose Peptone Bile 955

tributes a small part. During the summer nine samples of oysters were
taken from different portions of the river ranging from the point of greatest
pollution near Mount Hope Bay to those beds situated near the ocean,
Those beds which were near Mount Hope Bay produced the greater part
of their gas by the end of the 48th hour while those nearer the ocean showed

ae Ease
oe
Ge

pegeaeasiae ar ta ete EEE

SHEE
i Ly

| the greatest number at the end of the 72d hour. Plate III, Curve 1,
represents the gas produced from oysters taken from beds nearest the ocean
and so on up to Curve 9 which represents the gas produced by oysters
taken from beds nearest the sources of pollution. The other districts of

956 The American Journal of Public Health

the bay, on account of the complicated sources of their pollution, would
not lend themselves to this treatment.

From the data gathered together in this paper, one cannot help but be
impressed by the strict division obtainable by recording the number of
tubes of lactose peptone bile showing gas at the end of the 24-, 48- and 72-
hour periods. ‘Those tubes inoculated from oysters taken from highly
polluted areas produce almost all their gas by the end of the 48th hour
while those tubes inoculated from oysters taken from regions more dis-
tant from the source of pollution produce a greater amount at the 72d
hour. The writer is of the opinion that this method might be used as a
supplement to the regular oyster analysis in the determination of whether
the contamination is recent or remote by considering the temporal condi-
tions of gas production in lactose peptone bile.

SUMMARY.

1. Lactose peptone bile tubes inoculated with the shell liquor of oysters
taken from 119 different beds of Narragansett Bay produce the greater
part of their gas by the end of the 48th hour.

2. Tubes of lactose peptone bile inoculated with the shell liquor of
oysters taken from polluted districts produce almost all their gas by the
end of the 48th hour.

3. Tubes of lactose peptone bile inoculated with the shell liquor of
oysters taken from regions which are comparatively free from pollution
produce a larger part of their gas by the end of the 72d hour.

4. Consideration of this temporal factor in the production of gas in
lactose peptone bile might aid in the determination of whether the con-
tamination of oysters is recent or remote.

114

A COMPARATIVE STUDY OF THE SMITH FERMEN-
TATION TUBE AND THE INVERTED VIAL IN
THE DETERMINATION OF SUGAR
FERMENTATION.

BY WILLIAM W. BROWNE.

American Journal of Public Health, Vol. III, No. 7, 1913, pp. 701-704.

A COMPARATIVE STUDY OF THE SMITH
FERMENTATION TUBE AND THE
INVERTED VIAL IN THE DE-
TERMINATION OF SUGAR
FERMENTATION.

Wiiu1am W. Browne, Pu.D.,
; College of the City of New York.

During the summer of 1912, a sanitary survey of Narragansett Bay was
made under the direction of Prof. F. P. Gorham of Brown University to
determine the extent of the pollution of the oyster beds of the State of
Rhode Island by sewage of the neighboring cities and towns.

Lactose peptone bile was used as a presumptive test to indicate the pres-

~ ence of the members of the Bacillus coli group and other lactose fermenters

of intestinal origin. In the early part of the investigation, the regular
Smith fermentation tube, as used at the Lawrence Station, was employed
in determining the production of gas in lactose peptone bile, but later shell
vials inverted in regular laboratory test tubes were substituted for the
fermentation tube.

Immediately the question arose—which was the more efficient in a
presumptive work of this kind, the fermentation tube or the inverted vial?
This paper is the result of a brief comparative study of the two varieties.

The technic was very simple. Fifty-two oysters from a bed situated in a
portion of the bay which was known to be polluted were taken to the
laboratory and opened into sterile Petri dishes as proposed in the Second
Progress Report of the Committee on Standard Methods of Shell Fish —
Analysis, Laboratory Section, American Public Health Association, 1911.
From the Petri dishes, the shell liquor was inoculated directly into the
lactose peptone bile tubes. The fermentation tubes were inoculated in the
following dilutions:

ltube’ 1ce.c. Shell liquor.
2tube 0.1 c. c. Shell liquor.
2 tube 0.01 c. c. Shell liquor.

Searcity of fermentation tubes forced us to economize by using only one
tube in the cubic centimeter dilution.

The laboratory tubes containing the inverted. vials were inoculated by
Mr. George H. Smith, whom the writer wishes to thank for his labors, with
the shell liquor from the same Petri dishes in the following dilutions:

2tubes 1c.c. Shell liquor.

Beas 5 0.1 ¢. ec. Shell liquors.

2 “ 0.01 e. ec. Shell liquors.
701

ge

702 The American Journal of Public Health

The inoculated tubes were incubated at 37° C. and readings were made
at the end of the twenty-fourth and forty-eighth hour. Pressure of outside
work prevented further readings. No attempt was made to isolate specific
organisms as the writer assumed that the production of gas in lactose
peptone bile was indicative of the presence of some lactose fermenter of
intestinal origin. The presence of gas was recorded by the plus (+) and
(—) sign respectively since percentages of gas produced in fermentation
tubes and inverted vial are of no quantitative value.

The following table will show the comparative gas production of lactose
fermenters isolated from oysters taken from regions known to be polluted
in fermentation tubes and inverted vials:

TABLE I.
PERCENTAGE OF EFFICIENCY.

Cusic CENTIMETER.

24 hours. 48 hours.
No. of Tubes peste No. of Tubes P :
Showing Gas. Seer Showing Gas. ace”
Ferment. Tube.......... 44 84.6 49 94.3
Inverts Vialienes eee eee 49 92.3 50 96.1

OnE Trento Cusic CENTIMETER.

Rerment: Tubes .c.0. soe) 45 86.5 47 90.3
invert. Viale ones 31 59.5 44 84.6

One HunpreptH Cupic CENTIMETER.

Ferment. Tube.......... 17 32.6 29 55.7
Invert) Vials eee oe 12 23.0 22 42.3

An examination of the above table will show that in the cubic centimeter
dilution the inverted vial was slightly more efficient than the fermentation.
At twenty-four hours the inverted vial was 8 per cent. more efficient but
at the end of the forty-eighth hour they were nearly equal. In the tenth
cubic centimeter dilution, we find just the reverse to be true. The fer-
mentation tube is much more efficient at both the twenty-fourth and
forty-eighth hour periods. At the end of the twenty-fourth hour the
difference of their percentage of efficiency is 27 per cent. while at the end
of the forty-eighth hour period, as in the cubic centimeter dilution, the

4
J

4

Sugar Fermentation 703

difference is reduced to about 5 per cent. The same condition holds true
in the hundredth dilution, except there is still a large difference in per-
centage of efficiency even at the end of the forty-eighth hour. From this
little work, it would seem that the inverted vial is more efficacious in the
low dilutions and the fermentation tube is more efficacious in the higher
dilutions, as regards the production of gas in lactose peptone bile by lactose
fermenters isolated from polluted oysters. .

In a comparative work of this kind many irregularities in gas production
have appeared which might be interesting to note. They have also appeared
with great regularity in our main work on the Sanitary Survey. Carefully
planned controls have failed to eliminate them from the work. Conclusions
seem to point to some fault either in the method of making dilutions, since
these irregularities were more apparent in the higher dilutions, or to the
general inefficiency of lactose peptone bile. One of the most glaring
irregularities which have appeared from time to time was the presence of

- gas in one of the duplicates and not in the other. The frequency of this

condition may be noted in the following table:

TABLE II.
GAS PRESENT IN ONE DUPLICATE AND ABSENT IN THE OTHER.

FERMENTATION TUBE.

24 hours. 48 hours.
0.lc¢.e. 0.01 c. c. Ole. c. 0.01 c. c.
MlterOr tUDES:. . ¢.< ss osc 22 13 22 21
MEPTCOMGS hci eicicPats cso. chk. 42.3 25.0 42.3 40.3

INVERTED VIAL.

24 hours. 48 hours.

0.0l ce. lec. | 0.1 ec. c. | 0.01 c. ¢c.

No. of tubes....... 9 15 10 1 9 14
4 LG 28.8 19.2 1.9 L736 26.9

From the above table it can be easily seen just how frequently these
irregularities occur. They are more prevalent in the case of the fermenta-
tion tube than in the inverted vial. They also seem to occur in the higher
dilution more frequently than in the lower dilution. The presence of

:

704 The American Journal of Public Health

these irregularities certainly demonstrates the fact that in presumptive
work with lactose peptone bile we should not depend entirely upon the
gas production in a single tube but should use duplicates at least and even
triplicates, if possible.

SUMMARY.

As regards the production of gas in lactose peptone bile by intestinal
organisms isolated from oysters:

1. Inverted vials seem to be more efficacious in the low dilutions than the
fermentation tubes. ;

2. Fermentation tubes seem to be more efficacious in the higher dilutions
than the inverted vials.

Irregularities show that it is not safe to depend upon a single tube to
demonstrate fermentation but duplicates should always be used and, if
possible, triplicates.

115

REPORT OF MOSQUITO CONTROL FOR 1913.
BY FREDERIC P. GORHAM.
Report of Superintendent of Health for the year 1913.

~~ Ai

‘f va ;
Rie

; 7) rt Re i /

ia
fl

REPORT ON MOSQUITO CONTROL FOR 1913.
BY FREDERIC P. GORHAM.

To the Superintendent of Health:
Dear Sir: The following is my report on mosquito work for

the year 1913:
INTRODUCTION.

Previous IVork. During the years 1900 and 1901, at the
suggestion of Dr. C. V. Chapin, it was my privilege to devote
considerable time to the study of mosquitoes in the city of
Providence. ‘These investigations were undertaken to deter-
mine the species present, their breeding places and their life
histories. Primarily, of course, the object was to study the
relation between the distribution of malaria and the breed-
ing places of the malaria-carrying mosquitoes with the view
of reducing malarial diseases by restricting the propagation
of the mosquitoes. A second object was the determination
of the breeding places of the common mosquitoes in order
to determine whether it might not be possible to diminish
or entirely abate the mosquito nuisance in the city. Some
of the results of these investigations will be found in the
19th annual report of the Superintendent of Health for the
year ending December 31, 1901.

From that time up to the past year little work along these
lines has been done in the city although observations on the
distribution of mosquitoes have been carried on by both Dr.
Chapin and myself and a few experiments in oiling catch
basins in certain sections of the city have been made by Drs.
Chapin and King and by the.Men’s Club of the Free Evan-
gelical Congregational Church.

Beginning of the work of the present year. During the
later month of 1912, however, interest in the subject was
renewed and under the leadership of Alderman John Kelso of
the Second Ward, the Board of Aldermen began a considera-
tion of the problem. At the request of the joint standing

2 REPORT OF SUPERINTENDENT OF HEALTH.

committee on finance, Dr, Chapin, on November 29, 1912, pre-
sented a report on the feasibility and cost of controlling the
mosquito nuisance in Providence as follows:

Health Department, City Hall,
Providence, R. L.,
November 29, 1912.

Henry A. Grimwood, Esq.

Chairman Joint Standing Committee on Finance,

Dear Sir :-—

In accordance with your request I hereby present a report
on the feasability and cost of controlling the mosquito nuisance
in Providence.

Mosquitoes breed only in water.

If the breeding places be destroyed or the mosquitoes be
prevented from breeding, they will be exterminated.

Complete extermination is almost impossible, but by per-
sistent and intelligent effort their numbers can be reduced to
a very small minimum. In Havana, after two years of work
by Dr. Gorgas, the insects were no nuisance at all, and indeed
it was not easy to find a single specimen at a season when
they were formerly very abundant. But this work involves
a very considerable expense. A good deal of effort has been
made to get rid of mosquitoes in various parts of New Jer-
sey, especially in Newark, and also on Staten Island, and in
the Borough of Queens. The town of Brookline, Massachu-
setts has for the past ten years carried on a steady warfare
against the pests. On a recent visit to Newark, New York
and Brookline, I found that the men who have been carry-
ing on this work are very enthusiastic about its success, but
enquiry among other citizens indicates that while mosquitoes
have been greatly reduced in numbers they have by no means
been exterminated. Still, in Brookline, where conditions are
most like those in Providence, people in whom I have every
confidence tell me that they now sleep out-of-doors with-
out any discomfort from mosquitoes, while formerly this was
impossible. It was too late in the season for me to observe
personally the presence of mosquitoes.

In Providence there are three classes of places in which
mosquitoes breed.

FOR THE YEAR 1913. 3

1. Swamps, marshes, pond holes and the reedy banks of
streams. A map has been prepared which shows these breed-
ing places and it will be noticed that most of them are in the
suburbs away from the centres of population. It will also be
noticed that a number of important breeding places are in
other cities and towns immediately adjoining Providence. Sev-
eral breeding places are in the millponds along the Mosshas-
suck and Woonasquatucket Rivers and I am informed by the
mill men, who use the water in finishing their goods, that they
would consider the application of oil injurious. The only al-
ternative would be to clear out the brush and weeds, but ex-
cept in a few instances these areas are not near the centres of
population and their eradication might be postponed for the
present.

2. The catch basins along the streets, of which there are
about 5,500. It is believed that most of the mosquitoes in the
central part of the city are from this source.

3. Various receptacles on private property, such as catch
basins, cisterns and old cellars, barrels, kegs, buckets, cans,
fountains, aquaria, etc., in yards and receptacles on dumps.

The ultimate aim should be to permanently abolish as many
of these breeding places as possible. A thorough inspection
of private premises should result in the elimination of most
of the smaller receptacles for water above referred to. The
catch basins must be retained in substantially their present
form. ‘The swamps and marshes can be drained, filled or
cleared of weeds and in the latter case fish will take care of the
mosquitoes. The cost of draining and filling would be very
considerable and for this and other reasons this part of the
work had best be extended over several years.

The best way of immediately attacking the mosquito prob-
lem is by oiling the breeding places. This if properly done
will in the catch basins and many pondholes and ditches ef-
fectually stop the breeding. It is much more difficult to suc-
cessfully oil large swamps with bushes, grass and cat-tails,
and it is often necessary to do some cutting of grass, and some
ditching. Much encouragement was given to the oiling of
swamps by the work of Prof. Gorham at Meshanticut last
summer, where he succeeded in practically preventing the
breeding of mosquitoes in several acres of thickly wooded
swamp by oiling alone.

If it is proposed to undertake the work of mosquito eradi-
cation in Providence it is recommended:

4 REPORT OF SUPERINTENDENT OF HEALTH.

1. That all breeding places be oiled at intervals of about
two weeks from the first of April to the last of September.

2. ‘That a systematic inspection be kept up during the great-
er part of the same period to discover minor breeding places
on private property.

3. That legislation be secured to provide for the destruc-
tion of breeding places on private property.

4. That as many of ‘the swampy places as possible be
drained or filled thereby materially reducing the cost of oiling
and more effectually preventing the breeding of mosquitoes.

It is estimated that for the oiling four gangs of three men
and a horse and wagon in each gang would be necessary. At
$2.00 per day for each man and for the horse and wagon
this would for 160 working days amount to $1280 for each
gang. It is estimated that 150 barrels of oil would be needed
at about $3.50 per barrel.

Probably four inspectors would be required in April and
May and two for the rest of the season. At $2.00 per day
this would amount to about $1000.

The total cost of oiling and inspection for one season is
therefore estimated at,

Hour jcangs Olre@tlers i: yankees eee ee $5,120 00
150i barrelstor oilatyS3- 500s nave. wee: 525 00
TRS PEGLORS Geiss ocean hee Rete an ca hee 1,000 00
SUPET VISION) Gi. cP Nah epee ees eel Use 1,000 00
Contingeireies | 45 Age ee islet SAO hg 355 00

$8,006 00

The above represents the entire cost of treatment for a
single season, but does not include permanent drainage im-
provements. ‘lhe latter are urged as the best means of com-
bating the mosquito nuisance and as far as permanent drain-
age can be brought about by just so much will the cost of
oiling in subsequent years be diminished. I have consulted
with the City Engineer in regard to an estimate of the cost
of draining or filling the various breeding places and with
him made a tour of the city, pointing out the principal locali-
ties, and he informs me that he is at present at work upon
such an estimate.

Respectfully submitted,
CHARLES WV J GEA PAINE
Superintendent of Health.

FOR THE YEAR 1913. 5

This report was presented to the Board of Aldermen on
January 9, 1913, by Alderman Kelso, together with a reso-
iution appropriating $10,000, to be expended by the Super-
intendent of Health in a campaign of mosquito extermination.
Soon after the resolution was passed in concurrence and ap-
proved by the mayor.

Later in the year, owing to lack of funds in the City treas-
ury, $5,000 was withdrawn from the mosquito appropriation
and still later on June 23, 1913, another appropriation of
$2,500 was made. ‘This gave a total of $7,500 to be expended
for mosquito work.

ORGANIZATION AND EQUIPMENT.

In March, 1913, the writer was placed in charge of the
work for the season. The information acquired in the earlier
years was, of course, immediately available, and consequently
there was but little investigation to be done before the actual
organization of the work could be begun. Dr. Chapin made a
trip to New York, Newark and Brookline, Mass., to study
the methods of work there and the writer also visited Brook-
line for the same purpose.

Subdivision of the city and routing. First of all the map
showing the location of all standing water where mosquitoes
might breed which had been prepared in the earlier investi-
gations was brought up to date and made as complete and ac-
curate as possible. All the localities were then listed on sep-
arate cards, and divided into four sections, covering four parts
of the city, with the idea that each section should be cared for
by a separate gang. The cards in each section were then
arranged in the order in which the locality could be visited
in the least time and with the minimum amount of travel. This
was possible only by actually going over the ground personally
and studying the layout of each locality. Copies of the cards
in this order for each section were then made and later given
to the several gangs. These constituted their “route books”
and were carefully followed throughout the year.

6 REPORT OF SUPERINTENDENT OF HEALTH.

By the courtesy of the Sewer Department we were per-
mitted to make copies of their route books, showing the loca-
tion of all the sewer catch basins in the city. ‘There were nine
of these routes covering the 5,500 catch basins in the city.
When we began the work of oiling the catch basins these
routes were followed by the oiling gangs.

Equipment. Meanwhile bids were being secured for fur-
nishing horses and wagons suitable for the work. ‘These
were finally secured from Mr. A. H. Barrfey at the rate of
$2.00 per day.

ach wagon was equipped with cleats for supporting a
barrel of oil in the rear, a lock box for holding tools, rubber
boots, etc., under the seat in front, and an outfit of two gal-
vanized iron pails, a shovel, an iron rake, two long-handled
brooms, a whisk broom and an oil faucet.

The cost of this outfit was as. follows:

OU aAUC ET es TERN, Raed RHEL RY Ogi Te Oa $231
ARE oy: BL ag WA ae A ING le Sv Bias oer a EN oe
Srovie lege s ease eens Sav ehe in Bea e dere teas le etre) 2 el eae .69
Rake ee Gaia) Sag ee cS ORT A ae ee 35
2 ROOMS) AC. 2 Ore hae ros Ae eC ORES eta 58
Whisks: brOOniains. auc ieispersoicve oo evsis eareiec ce See eee 09

At first ordinary watering pots were included in the out-
fit, but these were later discarded as they used too much oil
and were not convenient. Later a cup was added for applying
oil to the catch basins.

In addition to the outfit on each team a bush scythe was
provided to be used in common by all the gangs as needed,
and a flat-bottom punt which was small and light so that it
could be carried on the wagon yet was strong enough to
support a man and a pail of oil. ‘This was used in common
by the different gangs. The scythe, snath and stone, cost
$1.60 and the boat $7.50.

Labor. Each of the four gangs consisted of two laborers
and a foreman. ‘The foremen were paid at the rate of $2.25

FOR THE YEAR 1913. 7

per day and the laborers $1.85, with the understanding that
they were to furnish their own boots. Early in the work it
was found that rubber boots became practically useless after
a few weeks, so rapidly did the oil attack the rubber. Later
the men adopted high leather boots which served very well.
The men started at 7 A. M., or earlier if they preferred, and
worked for 9 hours each day. During August they worked
but one half day Saturdays.

Oil. Ordinary fuel oil was used, obtained from the Stand-
ard Oil Co. The price paid was 9 cents per gallon during
April and part of May, 81-2 cents during the remainder of
May, June and part of July, and 8 cents for the rest of July,
August and September. This was the price paid per gallon
delivered in barrels. There was a rebate for the barrels re-
turned which made the net price paid per gallon range from
7.6 cents the highest, to 6.6 the lowest.

At first the oil was applied without dilution, but later it
was found that an emulsion made by mixing one half oil and
one half water, mixed thoroughly just before using, spread
much more easily and the same amount of oil appeared to go
farther than when the whole oil was used. This emulsion
was particularly useful where the grass was high, as it seemed
to reach the water much more easily than the clear oil.

As far as possible we tried to apply this oil at the rate of one
gallon to 1200 square feet, although often it was necessary
to use a greater amount where there was a current in the
water or where the grass and reeds were thick.

Daily Report Cards. The following cards were provided or
which the man in charge of each gang reported daily the work
of his gang.

8 REPORT OF SUPERINTENDENT OF HEALTH.

GANG NO. DATE
LABORERS AND

LOCALITY TIME TIME AMOUNT OF

STARTED FINISHED OIL USED

IN CHARGE

METHODS OF WORK.

Applying the Oil. For petrolizing swamps and _ standing
water each of the four gangs consisted of 3 men and a large
wagon equipped as previously mentioned. On reaching the
water to be oiled, the brush would be cut with the scythe
whenever it was necessary in order to reach the water, and
any grass, sticks or debris raked from the water as far as
practicable. In applying the oil the boss would precede with
a bucket of oil or the oil emulsion previously mentioned and
with the small whisk broom scatter the oil as evenly as pos-
sible over the water surface.

The whisk broom was found fully as effective as any
sprinkler on the market and far more economical. Spots
twenty feet or more distant can easily be covered by dipping
the broom in the bucket of oil and flicking the oil from the
broom. ‘The men soon become experts at this work. It may
be that some form of knapsack sprayer would be more con-
venient and we shall make investigations along this line be-
fore another season. ‘The two laborers would follow with
their long handled brooms and thoroughly puddle the oil into
all parts of the water, taking particular care to see that every
little space among the grass, weeds or brush was covered with

FOR THE YEAR 1913. 2

the oil film. The puddling is a most essential part of the
process and should not be omitted. One man could spread
more than enough oil to keep the other two busy and many
times the oiler had to stop and take a hand at the puddling.
Usually about one barrel of oil could be spread in one day, but
in certain localities where large areas were treated, two bar-
rels would be used.

Oiling Catch Basins. In oiling the catch basins one man
would remain in charge of the wagon and the laborers would
walk along, one with a pail of oil and a tin cup for applying
the oil, about 4 ounces to each basin, the other with the hook for
lifting the covers and a pole with which to spread the oil in
the basin. About 400 catch basins could be oiled in one day
although at times as many as 800 have been oiled by one gang
in one day.

Draining. In many localities, particularly in the early spring
before any oil was applied, some intelligent ditching, even such
as could be done by the three men in each gang, greatly reduced
the area of the water to be oiled. Particularly after the first
application of oil and after the men had become somewhat
familiar with the -territory, a very considerable amount of
draining was accomplished thus greatly lessening the amount
of oil and labor required in the subsequent treatment.

Probably the greatest amount of ditching was on the salt
marshes, particularly in the neighborhood of Butler Hospital
and Pitman street on the Seekonk, and Allen’s Avenue at the
City Dump and Old Maid’s Cove. At the Butler Hospital we
had the active cooperation of the Hospital authorities and
much of the ditching was done by them.

Along the west side of the Blackstone Boulevard and _ be-
tween the Boulevard and Hope Street, much ditching was
done by our own gangs. In this neighborhood the installation
of the new boulevard sewer and the building of catch basins by
the sewer department between the Boulevards, and by the
Swan Point Cemetery owners on their land to the west of the
Boulevard, will materially reduce the amount of standing water
another year.

10 REPORT OF SUPERINTENDENT OF HEALTH.

Frequency of Applying Oil. We endeavored as far as
possible to keep a film of oil on all standing water throughout
the season. In those localities where considerable current
existed, a more frequent application of o1l was necessary than
in those cases where the water was quiet. ‘There was always
a tendency for the film of oil to be carried by the wind and
in large open areas of standing water the film would shift
about from shore to shore with changes in the wind. As far
as possible we tried to visit every locality throughout the
season at least once every ten days or two weeks. If a con-
siderable film of oil remained, of course, no more was applied.
In the catch basins regular application of oil was made once
every two weeks irrespective of rain fall or cleaning of the
basins.

Supervision of Gangs. At the beginning of the season and
until the men in charge of the gangs became familiar with
their work and with their several territories, it was necessary
for the writer to be almost continually present to direct and
instruct the men in their work. By the use of an automobile
this was made possible so that each gang could be personally
instructed as to the treatment of each locality. Later it be-
came necessary simply to be present when the gangs started
out each morning to see that they were fully equipped with
men and materials and that they knew where to go and what
to do. Then a single inspection each day sufficed to keep the
work going.

Length of the Season. Ojiling of the swamps and standing
water was begun April 3, 1913 and was continued until Sep-
tember 137 1913 when all mosquito work was suspended on
account of lack of appropriation. Oiling of catch basins was
begun on June 6, 1913 and continued until September 13, 1913.

Inspection of Dumps, Yards, Etc. Other communities
which have attempted mosquito control work have laid particu-
lar stress on the necessity of careful inspection of the dumps
and the destruction of bottles and cans which hold water and
breed mosquitoes. We were prepared to begin such inspection

FOR THE YEAR 1913. 1

and to have our gangs visit the dumps and destroy all mos-
quito breeding receptacles. During the whole mosquito breed-
ing season, however, frequent and thorough inspection failed
to show that mosquitoes were breeding in these places. Con-
sequently the gangs were not withdrawn from the oiling for
work on the dumps.

The necessity of a very careful inspection of yards, on the
other hand, was very early noted. Barrels and tubs and un-
used garbage cans and private catch basins which were breed-
ing mosquitoes, were far more common than we had ever sup-
posed. Owing to our limited funds but one inspector was
employed in this work and only a portion of the city was
covered. ‘The number of mosquito breeding places which he
located and abated, however, warrants us in planning for a
very thorough inspection of this sort another season. About
every greenhouse, stable, lumber yard, various sorts of recep-
tacles were found holding water and breeding mosquitoes
enough to disturb the whole neighborhood. More active co-
operation on the part of householders would greatly help this
part of the work.

In many yards and about stables and garages frequently
private catch basins are located in which mosquitoes were
found breeding. Whenever such basins were located they
were added to our lists of city catch basins and were regularly
oiled by the catch basin gangs.

Later in the season our attention was called to the traps
on the sewer pipes leading from each house. ‘These usually
have an air vent which opens in the yard ‘between the house
and the street and is frequently open or covered with a perfo-
rated top. If the houses are closed for any length of time
the water in these traps might breed mosquitoes, although a
rain fall would flush them completely as the eaves of the houses
are connected with these pipes. No mosquito larvze were
found in these during the past season, but they should be con-
sidered a possible breeding place.

12 REPORT OF SUPERINTENDENT OF HEALTH.

RESULT OF THE WORK.

As was to be expected in the first year of such work com-
plete success was not attained. While some parts of the city
which had suffered severely from the pest in past years were
practically free from mosquitoes throughout the season, other
parts of the city were not. Careful records were made of
the distribution of mosquitoes during the course of the work
with the hope that the sources of the trouble might be dis-
covered and abated. In most cases by the time the mos-
quitoes became abundant it was too late to accomplish much
during the present year, but from the experience gained this
year, more care can be taken in subsequent years.

The district in the neighborhood of the Blackstone Boule-
vard, Cole Avenue, Sessions Street and Morris Avenue has
always been an abundant mosquito territory. Most of the
mosquitoes in this section breed in the swamps between ‘the
boulevards, between the Boulevard and Hope Street, and along
Cole and Rochambeau Avenues. These swamps are always full
of water in the spring and by the middle of summer are
usually dry. It is the earlier varieties of mosquitoes which
give the most trouble in this section. We were prepared to
wage an active campaign in this part of the city from the very
first of the season, but in spite of our efforts, even as early
as the 18th of May, mosquitoes were quite abundant, par-
ticularly in the woods along Cole Avenue. ‘The mosquitoes
were of the striped leg variety of wood mosquito and were
identified as Ochlerotatus subcantans.

The great extent of these swamps and the depth of the
water made it almost impossible to keep a film of oil on
all portions of them and undoubtedly some of the earlier
broods of this species, which is our earliest of spring varieties,
escaped our efforts to destroy them. The only possible way
of dealing with this locality is to drain it and fortunately the
construction of the new sewer along Hope Street and in the
Soulevard will do this to a very considerable extent so that
next season we may hope for better results in this part of
the city.

FOR THE YEAR 1913. 13

As far as could be learned, other parts of the city were
free from mosquitoes during all of May and June. There were
no other localities within the territory which we were oiling
which could not be thoroughly treated.

Such reports as the following were very gratifying to those
in charge of the work.

“THE WAR ON MOSQUITOES.

Results So Far Show Wisdom of Further Appropriations.

To the Editor of the Sunday Journal:

The Health Department deserves the highest commendation
for its effective work in its campaign against mosquitoes. It
is little short of remarkable that certain sections of the city
that were almost unhabitable a year ago are at this time, al-
most free of the pest.

Last summer the plague of mosquitoes in and about Black-
stone Park was so great that it was impossible to make use
of this playground with any comfort; and those living in the
Vicinity were driven indoors. No amount of the various smelly
compounds or of tobacco smoke made it possible to sit out of
doors in the evening; and the neighborhood was one to be
shunned. This year there have been so few mosquitoes. that
they are almost unnoticeable.

Surely there has never been a better demonstration of
efficient work for the public good. It is certainly appre-
ciated by everyone who has been tormented in previous years;
and it is to be hoped that the department will not be handi-
capped through a lack of means to carry on a fight which has
so far been wonderfully successful.

Providence, June 30. Warren B. Lewis.”

With the coming of July the common house mosquito, Culev
pipiens, the mosquito which breeds in sewers, catch basins, rain
water barrels and stagnant pools everywhere, was expected.
In previous years the pest has been abundant quite generally
throughout the city. On July 7, we recorded the capture of the
first one of this species on Ivy Street, and on July 9 and 16

14 REPORT OF SUPERINTENDENT OF HEALTH.

others were caught at Brown University, on July 21 and 22
several were taken in the neighborhood of Butler Avenue, and
at Swan Point Cemetery. But during July these noted were
isolated cases and the reports from nearly all sections of the
city were that mosquitoes were either absent altogether or if
a few were noted they were reported as nowhere nearly as
abundant as in past seasons.

In August conditions were nearly the same as in July. Now
and then reports would come in as to the abundance of mos-
quitoes in certain localities, most frequently from places near
the outskirts, where a favorable wind might carry them in
from outside the city. But on the whole more reports of the
absence or scarcity of mosquitoes were received than of their
abundance.

The same favorable conditions continued inte September
and numerous reports came to us of people returning to the
city after the summer vacation who slept with wide open
windows without screens, something entirely impossible in
other years. Indeed we had reports of people who had slept
without screens all summer without inconvenience.

In those places where mosquitoes were noted we tried to
determine the source from whence they came. At this time
mosquitoes were abundant in East Providence and over the
city lines in Pawtucket and Cranston and along the banks
of the Mosshassuck and Woonasquatuck Rivers, which were
not being oiled on account of the objection of the mill ewners,
and we tried to find whether wind in certain directions brought
these mosquitoes into the city. In certain sections of the city
there was some evidence that this was the case. In parts of
the northern section of the east side when the wind had been
blowing from the north or northwest, mosquitoes were more
abundant and in all probability were blown in from over
the Pawtucket line or from the neighborhood of the Mosshas-
suck River swamps. In the Elmwood section, after a strong
southwest wind, mosquitoes were apparently driven in from
Cranston and the neighborhood of the Mashapaug region.

FOR THE YEAR 1913. 15

In all probability some mosquitoes came from breeding
places which had escaped our attention within the city itself,
such as water barrels, private catch basins, etc., which even
the little inspection of private premises which we were able to
do showed to be extremely common. During another season

more thorough inspection will, we hope, exterminate even these
few.

On the 12th and 23rd of July, when favorable reports were
being received from practically all parts of the city, we visited
the Olneyville district in the neighborhood of the swamp
back of the Weybosset Mills. We had not oiled this swamp
because of the objecton of the mill owners, who were using
the water from the swamp. ‘The waterof the swamp was alive
with mosquito larve. Adult mosquitoes were abundant in
the whole neighborhood. The people interviewed reported
mosquitoes as abundant as in past years. This comparision
showed the effectiveness of the work in other sections of the
city which in former years had been as badly infested as
was the Olneyville section.

The thorough oiling of the catch basins also was certainly
having its effect for no mosquito larvae were found in any
of them, and yet a visit to Pawtucket on the 18th of August
showed that every catch basin in the neighborhood examined,
which was just over the city line from Providence, was
swarming with larve and adults.

Only one salt marsh mosquito was captured in the city by
our inspector during the season. This was O. taeniorhynchus
and was taken on July 20 at Brown University. Even in the
neighborhood of the salt marshes at Butler Hospital and on
Allen’s Avenue and at Field’s Point the salt-marsh varieties
were not troublesome.

'The only observation of the malaria-carrying variety of mos-
quito made during the season was on August 4 when several
larve were observed along the banks of the Mosshassuck
River at Livingstone Street. At this time large numbers of

16 REPORT OF SUPERINTENDENT OF HEALTH.

C. pipiens were found breeding along the banks of the river
and from this time till the end of the season the breeding of
mosquitoes along the banks of this river was prevented by
assigning a man to patrol the banks and sweep the larve from
the sheltered pools into the swift current where they were car-
ried down stream. It was impossible to keep oil on these
places even if the mill owners had permitted its use.

FINANCIAL STATEMENT.
Season of 1913.
Superintendent ssa ciny schon soak eras te cco ee eee $1000 00

Oiling Brigade—

Foreman at $2.25 per day
Laborers at $1.85 per day

dnspectorssatihs-2oyper day. sec senior 3143 69
FHorses-and, Wagons-ats2.WO0sper day lsu. ose een eee 1054 00
iN Fea Phoe yt Ne: stata Lots ae OR GR Ie SSA ee Pe Sa fed sh Cee ae 1982 16
Tools—
BOOMS! Verh sare Rie into at $.29 $19.68
Wiitskabroonpmei ere cae oe > 09 Pl
Galvanized iron pails ........ oe esl 74 2.16
Watering potsucn ieee aN ES)! 2.75
Oil Miaucetsyieee see cee eee 1.86
Rakes) er eee ce raireiom seperti: Reni) 1.40
Shovels weet eet eae GS) 2.76
Scythe, snath and stone ...... 1.60 34 32
5S) 8 UA Raya COEUR Resta D hee LAL De Bi ale eM earn TAN 7 50
Miscellaneous—
F. P. Gorham, trip to Brookline ... $2.30
ORB lankwBooksieceer nee Lobe eee 4.28
Wed er pate itcweee we Gierorcienh dere eretois 80
WockuboxesmloOnmswagOnsSs aacee a. ae 11.04
Rrimtingeatimescards)-erec cierto: 4.42
Book, “The Mosquito in North and
CentraleAmientcay acs cece ee 10.00 32 84
WRotalWwE spenSeSwactaye sess cine: ceiek tiers soon eker tae Pheer eee $7254 51
ND PEO PLIAELO Mey. ede OTTO: SiG elles exe ee Seer care Ieee 7500 00
Balan Ger de eee ance eb yer in aietie ike een Rete L Aa ont: $245 49

Respectfully submitted,
F. P. GORHAM.

116

HINTS ON THE HANDLING OF CLEAN OYSTERS.
BY LESTER A. ROUND.

Prepared for the Commissioners of Shell Fisheries of the State of Rhode
Island, 1913.

State of Rhode Island and Provideure Plantations.

HINTS ON THE HANDLING
OF CLEAN OYSTERS

PREPARED FOR THE
Commissioners of Shell Fisheries
OF THE

STATE OF RHODE ISLAND

BY

LESTER A. ROUND, A. M.,

INSPECTOR OF OYSTER HOUSES

PROVIDENCE, R. I.
E. L, FREEMAN COMPANY, PRINTERS

1913

GENERAL CONSIDERATIONS.

In discussing oysters from a sanitary point of view, we must
keep in mind always that oysters are a food product which to a large
extent is consumed in a raw or uncooked form, and for this reason
every precaution should be taken to keep them in as wholesome con-
dition as possible. No one wants to eat food which is known to be
contaminated with sewage or other filth, or which has been handled
in such a manner as to allow contamination. Probably no other
food resembles milk as much as oysters. Both are raw foods and in
the preparation of the two for market, there are a great many paral-
lels. These are readily understood by every oysterman who gives
the subject thought. We all know of the dangers in the careless
handling of milk; too often these dange1s have proven themselves
real in milk borne epidemics of scarlet fever, diphtheria, typhoid,
etc. From these unfortunate experiences, of which nearly every
city has too many instances, health officers and sanitarians in general
have come to realize the dangers which are likely to follow careless
and unsanitary methods in the milk industry. It is only quite
recently, however, that sanitary experts have considered the danger
which sometimes follows uncleanly methods in the handling of shell-
fish and the necessity of observing the same care in the preparation of
oysters for the market that is observed in the handling of milk.
It was not until these dangers had shown themselves in the form of
typhoid fever epidemics and other intestinal disturbances, that this
phase of the oyster industry began to receive serious consideration.
It has now been proven on several occasions that these dangers do at
times exist and that certain rules and regulations in the sanitation of
oysters is Just as necessary as in the milk industry.

Recently a great deal has been said and written regarding the dan-
ger of eating shellfish, especially oysters. Some of these statements
have been just; but a great deal of this criticism has been unjust.
Articles of a sensational nature have appeared in the newspapers and
magazines, leading one to suppose that all oysters and other shell-
fish are a grave source of danger to the public health and that people
are running a severe risk when they partake of a few oysters on the
half-shell. A great deal of this sensational criticism is no doubt due

—

3

to the fact that the danger has been but recently recognized and for
this reason has been over emphasized. When we consider the
problem carefully, we find that the dangers from oysters and other
shellfish are not to be compared with dangers existing in the public
water supplies of many of our large cities or in the consumption of raw
milk. Probably a great many cases, and whole epidemics of typhoid
really due to shellfish, have been laid to the water or milk supply,
because until recently the question of shellfish and disease had not
been considered. Yet, allowing for all these instances the number of
cases of disease due to eating shellfish is surprisingly small when com-
pared to the number of cases definitely shown to be due to either an
infected milk or water supply. Though this comparison shows the
oyster to be the infecting cause in only a comparatively small number
of cases, yet we must not forget that the oyster has caused some
epidemics, sufficiently large to create national attention, and for
this reason demands that certain regulations of a sanitary nature,
both in the production and handling of our oysters and other shellfish
must be observed in order to restore the public confidence in the con-
sumption of one of our cheapest, most nutritious and most wholesome
of raw food products. The purpose of this folder is to point out to the
oystermen certain precautions which will enable them to produce a
more cleanly and wholesome product and to restore to the oyster a
confidence which has been too severely and too unjustly criticised.

The first consideration in the production of a clean product is of
course to have a clean supply, one which is free from suspicion.
-If the oysters are already polluted as they are taken from the beds,
no amount of care in their handling and preparation can free them
from this condition. This is a matter which the Commissioners of
Shell Fisheries regulate, through the bacteriological examination of
all the oyster beds of the State. The exact sanitary condition of any
bed is known at all times and this information is on file in the office of
the Commission. No oysterman who has any regard for the public
welfare or for his own reputation, will use oysters which are not free
from suspicion. This information can be obtained from the Com-
mission upon application and permission can be obtained to use the
oysters from any bed which has been found to be of a sanitary con-
dition sufficiently high to pass the standard set by the Commissioners
and the Federal Government,

FLOATS.

When he is sure that he has a clean supply of oysters; that his
oyster beds are free from objectionable sewage contamination, the
real sanitary work of the oysterman begins. It used to be common
practice on the part of the oystermen to remove oysters from the
beds and place them in floats for a few days, under the pretense of
allowing the oysters to cleanse themselves. These floats were almost
always located in the mouth of a fresh water stream which was heavily
polluted with sewage from the towns which lay along its banks.
What was the result? To the oysterman the oysters became very
plump and “fat”? from the fresh water which they absorbed. But,
along with this water, they also became laden with sewage and the
germs which sewage contain. If there were typhoid cases in the towns
up-stream and the excreta from these patients was discharged into
the river, there was great danger of the oysters taking up these organ-
isms, for oysters act as filters and filter out all kinds of micro-organisms.
Many cases and whole epidemics of typhoid have been traced to
oysters which had been floated in such places. In New York, in
1910, over one hundred cases of typhoid were traced to oysters from
Jamaica bay which had been stored in thismanner. If space allowed,
many other epidemics might be cited. The writer once had occasion
to examine oysters kept in such a float and found that in spite of the
fact that they were in a high state of purity when taken from the bed,
when they were placed in such a float they very quickly became pol-
luted.

From this it can be seen that the use of floats in general is a very
bad practice and that floats should never be used, unless the oyster-
man is assured by a bacteriological and sanitary examination that his
float is properly placed in water which is clean at all times, for if an
epidemic of typhoid should be traced to his oysters, it would seriously
affect, if not ruin his business.

CLEANLINESS OF OPENING HOUSES.

The point of next importance to the oysterman is the cleanliness of
the oyster house, for if the opening house is unsanitary it is impossible
to keep the oysters in a high state of purity. The walls and ceilings
of many opening houses demand greater attention than is given them.
Every year the walls and ceilings of all houses should be painted or

5

whitewashed. If they are already painted and in good condition they
may be washed and thoroughly cleaned. Painting or whitewashing
has several important features to recommend it. First; before painting
or whitewashing it is necessary to clean the walls and ceiling thoroughly
to remove dirt, cobwebs, ete., which would form a source of contami-
nation. Second; both paint and whitewash are germicides and so kill
the germs that are left on the walls and ceilings and form a smoother
surface which prevents to some extent the lodging of bacteria. Third;
a very important point concerns itself with the appearance of the
house. If the walls are painted some light color it gives much better
light to the operators and a much better appearance—a look of neat-
ness to the house—provided it is kept clean. This latter effect,
namely, the appearance, has a very marked effect upon visitors and
this alone would influence a large percentage of people to give their
trade to the better kept house. It is a pleasure to see that a great
many of our oystermen take scrupulous pains to keep everything
about their oyster houses in a clean and tidy manner.

The appearance and cleanliness of the open house necessarily
has a marked effect upon the employees. If a house is kept in a neat
and cleanly condition, instinctively the employees pay greater atten-
tion to their own personal cleanliness and appearance. The con-
dition of the one necessarily reflects itself in the condition of the
other and is worth the extra effort for this reason alone.

CONSTRUCTION OF HOUSE.

The nature of the material of which a house is constructed plays a
very important part in the ease with which it can be cleaned. The use
of cement instead of wood for benches and floors has a very important
advantage in this respect and every oysterman should consider this
matter seriously when about to reconstruct or build anew. Though
the use of cement would entail a greater initial cost, yet the greater
durability and case of cleaning, together with the higher sanitary
qualities, should be a good argument for cement construction.

CLEANING OF HOUSE AND BENCHES.

Great care should be exercised in keeping the bins and benches as
clean as possible Every time a bin is emptied and before refilling, it
should be cleaned out thoroughly with a hose, if running water is
provided, or with a stiff broom and water in places where running

6

water cannot be obtained. This will remove mud and vegetable
matter, which, if left upon the bench, would decay and produce ill-
smelling results, to say nothing of the unsanitary aspects of such de-
composing material. Kvery day the floor should be washed throughly
in the same manner to remove any vegetable or animal matter which
might otherwsie collect and decay witb the same result as on the
benches.

All the utensils, as opening cans, knives, colanders, ete., should be
cleaned and rinsed as often as they are emptied. The mucus from
the oyster contains a very large number of bacteria and this should be
washed off from all the utensils with which it comes in contact.

TOILET FACILITIES.

The necessity of having a well arranged toilet, has been overlooked
by a great many if not most people. The evils of an ill constructed
toilet are fully appreciated but by a few. Records show that during
the Spanish-American War more soldiers died from typhoid fever than
were killed by Spanish bullets. A similar statement has been made
regarding our Civil War. Surprising as it may seem to most people,
the greatest factor in both these instances in the spread of this
disease was the improper location and regulation of the toilets. In
some cases perhaps the toilets were so located as to infect the drink-
ing water, while in most cases, probably in all, the vaults were open
to access by flies, which a few minutes later winged their way to the
cook house or mess tent and proceeded at once to smear the victuals
with typhoid bacilli from their germ laden feet and bodies. This
last statement is no fairy tale, it is an established fact. When we
realize how easily a fly might carry from the toilet to an oyster pail
1,000 to 100,000 colon. bacilli, we can see how a contribution of this
kind might seriously affect the sale of a shipment of oysters, if they
should be examined by the Government. Add to this that wherever
we find colon bacilli in large numbers we expect sooner or later to find
typhoid bacilli, the matter takes on a serious aspect.

To overcome this danger of fly infection, the toilet should be built
tightly and all openings from the vault, the windows, etc., should be
screcned to prevent flies from either entering or leaving. The door
should be so constructed that it would shut tightly and should have a
spring which would keep it shut at all times to prevent the entrance or
egress of flies. Let it not be that this will prevent proper ventila-

7

tion. The idea is simply to have no cracks large enough to admit
flies and to screen all windows, vents, etc., used for ventilation.

The arrangement of the toilet should be as convenient as possible,
and every encouragement should be given the employees to exercise
care, for we must realize that they return immediately to the handling
of a raw food which someone will eat in a few hours. A supply of
running water should be handy and the necessity of thoroughly and
carefully washing the hands upon leaving the toilet must be em-
phatically impressed upon the employees.

The use of screens in the opening houses would prevent the entrance
of flies and avoid contamination from this source. ‘This is important,
for in a thickly settled community, flies travel quite rapidly and may
come from other peoples’ premises over which the oysterman has no
control.

HANDLING OF THE OPENED OYSTERS.

For washing the oysters as they come from the openers, several
methods are in use. It is a difficult matter to say which is the best
in all respects on account of the number of factors involved. The
old process of washing with a stream of running water on a perforated
bottom or colander, would appear to give satisfactory results and is
the method used in most of our houses. There is no question, how-
ever, but that this method may be improved upon. The purpose of
washing is of course to remove all kinds of foreign matter, as bits of
shells, dirt, ete. But as much care should be used as possible to wash
out the mucus from the gills of the oysters. This mucus contains
more germs than any other part of the oyster. The purpose of this
mucus is go catch and entangle the food of the oyster and together
with the otber constituents of the food, it also catches a large per-
centage of the bacteria contained in the water which is filtered through
the gills. The mucus simply acts as a net and does its work quite
efficiently. Now, the more of this mucus we can remove the fewer
organisms our oysters will contain and the better our product will be
from a sanitary point of view, and the better they will keep. Even
though great precaution is used to keep the bacterial content of opened
oysters at a very low point, yet the numbers will increase and the
smaller number of bacteria we have in our oysters at the outset, the
less danger there will be of large numbers developing some hours
later. This is especially true of long shipments when the oysters are
on the road for several days. The writer has seen oysters which were

reasonably low in bacterial content when shipped, reached their
destination 48-72 hours later in such a condition that they were
unsalable. This fact also brings up the important point of having the
oysters properly iced at all times, both in the opening house before
shipment and afterwards.

The government has fixed a standard for the B. coli content of
opened oysters in interstate commerce. If oysters are found to con-
tain more B. coli than this standard, they are seized and condemned.
The oysters may have been in good condition when shipped, but
either insufficient washing to remove the mucus and with it the
bacteria, or improper refrigeration, or both, caused the bacteria to
increase out of all proportion. This is a.pomt which every oysterman
should look after. Experiments are now being tried with rotary
washers and the results are being watched with interest. The device
which will wash oysters the most thoroughly—remove the most
mucus—in the shortest possible time—without injuring the oysters,
should prove the best method. We should be interested to have
practical oystermen work out methods according to their own ideas.
It is gratifying to see that this is being done in some places.

SOAKING OYSTERS.

On the other hand, oystermen should not go to the extreme and
soak their oysters for long periods. This, as all oystermen are too
well aware, produces the same effect as “floating” or “fattening”’
shell oysters in brackish water and constitutes an infrmgement of the
Federal pure food law on account of the adulterant, 7. e., water which
isadded. The Government experts maintain that soaking more than
half an hour shall not be allowed. In spite of this Government regula-
tion the inspector has quite recently found instances where oysters
were soaked over night and even from Saturday until Monday morn-
ing. So far the State has made no particular efforts to stop the prac-
tice and it is hoped such steps will not be necessary.

CLEANLINESS OF EMPLOYEES.

Cleanliness on the part of the employers is Just as important as in
any other part of the oyster industry. No person about the opening
house should be allowed to touch opened oysters or vessels with
which the oysters come in contact unless his hands are clean and free
from contamination. It is always best to give the hands a thorough

9

washing before handling opened oysters or their containers. The
hands of the openers, as well as their opening dishes, should be rinsed
after each emptying of the opening cans.

Perhaps the most important precaution on the part of the people
who handle the oysters and one which the oystermen should take
the greatest care to see that it is fulfilled, is the thorough washing
of the hands after the use of the toilet. This is a matter which cannot
be too rigidly enforced. It is almost incredible how many oysters
could be contaminated by a speck of fecal material too small to attract
attention. When one stops to consider that a bit of fecal matter no
larger than the head of a pin might contain several million germs, the
fact becomes evident. In this respect the oysterman must think
of the kitchen in his own house and the condition which he would
tolerate there and apply this to his opening house, for both are places
where food is prepared to be eaten raw to a large extent, the only
difference being that the period before consumption on the part of
the oysters is lengthened by a few hours.

THE WATER SUPPLY.

The quality of the water which is used to wash the oysters is a
thing which should be watched carefully. In spite of the most care-
ful vigilance in other parts of the industry, a polluted water supply
used to wash the oysters might endanger the oysters to the same
extent as “floating”? them in polluted water. During the last year
the inspector made examinations of all water supply when not derived
‘from a regular town or city supply and reported the results to the
oysterman whenever there was suspicion and made sanitary surveys
and gave suggestions for its improvement whenever necessary. When-
ever a hew supply is contemplated, the sanitary quality of the new
supply should be ascertained at once in order to save any unnecessary
expense, if the supply proved to be unsafe.

STERILIZATION.

The matter of sterilization is comparatively new to oystermen and
it is interesting to see how it has been treated. Usually the sterilizer
is connected with the heating plant and so one furnace is made to
perform the two-fold function. In the late summer and early fall
the weather is still quite warm and there is no need of steam for warm-
ing the building. In this case, of course, there is no sterilization, for

10

*t seems to the oysterman a needless waste of fuel to run the plant
when the weather is still so warm. So many men have said when the
subject of sterilization was mentioned: “It has been so warm that
we haven’t started our sterilizer yet. In the winter when we have
steam we sterilize every night, but now we only sterilize once a week,”’
or maybe not at all. Or perhaps they say: “ We are not doing very
much business now, it isso warm. We have only a few men working
In the winter when we are doing a good business, we use our sterilizer
every day.”’ But the very time of year when people are inclined to
neglect sterilization is the very time they should insist upon it most.
The very purpose of sterilization is to kill bacteria which might
multiply too rapidly and spoil the product. Under suitable conditions
such as are furnished in warm weather, a single bacterium may
multiply to thousands in a few hours and millions in a day, while in
the winter time when the temperature is around freezing no growth
would take place for weeks or even months. Every oysterman
knows how much more important is frequent and careful icing in
summer than in winter. Just so much more important is sterilization
in warm weather than in cold. The purpose of icing is to keep the
temperature so low that bacteria cannot grow; while the purpose of
sterilization is to kill the bacteria so that there will be none or very
few left to multiply when opportunity is given. From a practical
standpoint, however, we have two questions to consider: (1) What
shall be sterilized? (2) How shall sterilization be accomplished?

In answering the first question, namely, what shall be sterilized,
we can consider as a general rule that every utensil that is used more
than once in handling or holding oysters should be sterilized. This
would include all returnable containers, opening pans and measures,
knives, tubs, whether iron or wood, but especially the latter, washing
basins, colanders, etc. There is no particular reason for sterilizing
non-returnable containers, provided they are clean. Simply rinsing
in clean pure water is all that is necessary. But, in the case of other
utensils, sterilization should be effected every day.

There are two methods of sterilization that oystermen might use,
both of which are feasible. The most reliable is the use of steam
either as “live steam” or under pressure. If “live steam”’ is used it
should be employed for one hour, but with a pressure of 15-20 pounds,
20-30 minutes would be sufficient. Whcnever possible steam should
be preferred.

11

The other method may’ be called chemical sterilization with
calcium hypochlorite or, as it is familiarly known, bleaching powder.
This is a very strong oxidizing agent and a small quantity will act very
quickly and kill all the bacteria. In using bleaching powder one
should use the proportion of one level teaspoonful to 30 gallons of
water. This should not be mixed and kept on hand, but used immedi-
ately upon mixing because the sterilizing effect is soon lost when mixed
with water. The utensils to be sterilized should be filled with water
and the powder added in proper proportion and stirred in and dis-
solved. A large tank might be used into which all the utensils to be
sterilized might be put and then filled with water. The bleaching
powder can then be mixed in concentrated form in a pail of water and
added to the tank. This should be allowed to act for one-half an
hour and then draw off and the utensils rinsed in clean water and
allowed to drain and dry.

(It must be understood that the commissioners do not recommend
the use of bleaching powder instead of steam, but merely as an exped-
ient when steam sterilization is not feasible.)

HEALTH OF THE EMPLOYEES.

The health of the employees and their familics is a matter which
should interest every oysterman. Frequent inquiry among the
employees should be made for any cases of sickness—especially in-
fectious diseases as typhoid, dysentery, scarlet fever, diphtheria,
etc. The employer should make this a very important matter and
should demand that every case of sickness of any kind among the
employees or their families be reported to them in order that no case
might go by undetected. A great many epidemics and isolated cases
of typhoid and other intestinal disturbances have been traced to
infected oysters, but so far as the writer is aware no cases of scarlet
fever, diphtheria, measles or other infectious diseases have been
traced to this source, and yet a great many epidemics of the latter
diseases have been caused by an infected milk supply and there is
reason to believe that oysters might carry such infection if the con-
ditions were suitable. What person, oysterman or otherwise, would
allow food prepared for him by a person in whose family there existed
a case of scarlet fever, typhoid, diphtheria or any other infectious
disease? And yet, the people who handle the oyster in an opening
house are preparing a raw food, which will be consumed in a few hours.

12
When we consider that the opening house is but a kitchen for the
preparation of food, we necessarily get a different point of view and
different ideas concerning cleanliness. We firmly believe that many
changes will be made when the oystermen consider their opening
house from this point of view and compare certain processes and
methods now existing with those of the well appointed home.

Within the last four years great advances have been made in the
sanitation of oysters and oyster beds and in matters of cleanliness,
both in the handling of oysters and the appointments of the house.
The Commissioners appreciate and are deeply grateful for the attitude
which the oystermen have taken in this matter. Almost to a man the
oystermen of Rhode Island have entered into this phase of the subject
zealously and with the firm resolution of making the Narragansett
bay oyster the finest product in the country, both as regards whole-
someness and cleanliness. The efforts of our most progressive oyster-
men in this line are worthy of the truest admiration and highest
respect and the Commissioners take this opportunity of extending to
the oystermen of Rhode Island their sincerest appreciation for the
work which they have done. Rhode Island was the first to take up
this matter seriously and from the first has taken the lead in its cam-
paign for clean oysters. But the work is only at its beginning; greater
things are to be expected in the future than have been accomplished
in the past and the Commissioners stand ready to aid the oystermen in
every way possible in maintaining for the Narragansett Bay Oyster
the superior reputation it at present enjoys.

117

THE RATS OF PROVIDENCE AND THEIR PARASITES.
BY GEORGE H. ROBINSON.
American Journal of Public Health, Vol. III, No. 8, 1913, pp. 773-776.

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[Reprinted from AMERICAN JOURNAL oF Pusiic Hearn, Vol. 3, No. 8]

THE RATS OF PROVIDENCE AND
THEIR PARASITES.

GrorGE H. Ropinson, A. M.
From the Providence Health Department and the Biological Laboratory of
Brown University.

That rats are a factor in the spread of bubonic plague is an established
fact. It has been confirmed beyond the shadow of a doubt, in India, in
California, in England, and in other regions where plague is endemic.
Rats dead of the disease have been found time after time in houses where
outbreaks have occurred and immediate deratisation has effectively stopped
further spread of the epidemic. These facts and a great amount of experi-
mental work show that the rodent disease and that of human beings are
identical.

Bubonic plague is originally a disease of rodents. Its home is thought to
be the region of Thibet,! its natural host being the Tarbagan of that region.
Other rodents are easily susceptible to the disease, as the hares of England,
the ground squirrels of California, and the various species of the Muridze
found in all temperate and tropical regions of the globe. Owing to their
universal distribution and great abundance the rats are to be considered
as the greatest factor in the spread of bubonic plague.

When rats were first connected with the spread of the disease it was
thought that they distributed the germs by contact with human food.
The plague bacilli can be found in the mucous secretions, feces and urine?
of infected rats and so there is some evidence in support of this theory.
In 1898 Simond working in India demonstrated that plague could be
transmitted through the bites of fleas. Later work by the Indian Plague
Commission’ and others has shown the rat flea to be the probable distrib-
utor of the disease.

To the cities along the Altantic coast of this country the spectre of plague
is not very real but with the outbreak of the disease in the West Indies in
1912 it took on a more than ghostly substance to those conversant with
the situation. American cities have an abundance of rats, the rats are
presumably well supplied with fleas and it seemed only necessary for the
introduction of the germs in order to bring about an epidemic. The
Federal authorities required that all ships running between the infected
region and American ports be fumigated and advised the municipal health
officers to conduct an examination of rats.

In accordance with this recommendation an examination of the rats of
Providence was undertaken under the direction of Dr. Charles V. Chapin,
Superintendent of Health. The object of the work was to determine if

1 Preble, Paul, The Tarbagan (Arctomys bobac) and Plague, Public Health Reports X XVII, 1912, 31.
2 Baxter-Tyrie, C. C., Plague in Queensland, Journal of Hygiene, V, 1905, 315.
3 Report of Plague Investigations in India, Journal of Hygiene, VII, 1907, 395.

773

774 The American Tourwal of Public Health ;

possible the presence of any epizobtic among rats, the kinds of rats and the
number and kinds of fleas on the rats. At first the work was confined to
the water front, but later its scope was extended to the entire city.

The work began on July 18, 1912, and continued with several short in-
tervals, up to the last of December. From the beginning of the examina-
tion until the first of September the rats were taken in traps along the
water front. A daily patrol of the shore was also made in order to pick
up any which might have died. Only three were found dead. About the
first of November a bounty of five cents was offered for every live rat
brought to the laboratory and 142 were purchased.

The technic of the examination was as follows: the rats were caught alive
in the French wire traps and carried to the laboratory; there they were
placed in a reservoir of illuminating gas for several minutes killing the
rats and stupefying the fleas; the rats were then immersed in a weak solu-
tion of corrosive sublimate or formalin and left over night; during this time
the putrefactive bacteria entered the circulation but a shorter exposure
to the fluid was not sufficient to penetrate the thick fur and kill the para-
sites; the wet fur was then turned aside with forceps, the parasites were
removed and their position on the body was recorded; the lymph glands
were then exposed, the abdomen was opened and the appearance of the
organs especially the liver was noted.

During the months July, August and September, 118 rats, and from
October to January, 223 were examined, making a total of 341. These
were evenly divided as to sex: 333 were the brown rat (Mus norvegicus),
2 were the roof rat (Mus alexandrinus), 1 was the black rat (Mus rattus), 4
were apparently crosses between the brown and roof rats, and 1 appeared
to be a cross between the black and brown rats. The two specimens of
the roof rat were taken from the New York-Providence boat and so were
doubtless New York rats.

The size ranged from 18 to 46 c. m. (7 to 18 inches). Studying the brown
rats by the biometric method we find them grouped about two distinet
modes, those of 24 and 36 c. m. in length. Thus we have a clear differen-
tiation into the adults and those which are still immature. Of these 59
per cent. were adults and 41 per cent. were young.

The results of the examination for fleas were as follows:

‘Total numberiof-rats examined #7502 12a oe eee ene 341
Total number of rats infected with fleas.................. 195
‘Total: ninmber of teas. en. cs ot eels cu ee eee 2053
Average fleas*per Tabs ie eee hid yc.) Mater ete See ae cna eee 6
Average fleas per flea-infected-rat.s) 0... 2.8. ee 2s ll

Largest number of fleas from one rat. ...... 22.4 ...0.. 00. 300

The Rats of Providence and Their Parasites 175

Per cent. of fleas:

Menoapay iia cheopis:mothseluld | [e023 se. eed ecw we 75
Weratonny bia tasclatiissbOSG. f0%'o. civ. Selec a es wet net 22
Cltenapaylns mast. Dses > kbc oni os OL eek es eee os o:5
Crenneapoanis cnmmis- Curtis. 4 ogo bit ines ace eres 0.5

By way of comparison it is interesting to note that in California the
average fleas per flea-infected rat was 3.5,? and a yearly average of 2.7 was
found. In India the average fleas per rat was 4.0.3 All who have experi-
mented are agreed that the two most numerous species which we found,
Xenopsylla cheopis and Ceratophyllus fasciatus, will bite man. Concern-
ing the others there is some dispute but since they comprise only 3 per cent.
of all they are of little consequence.

During the plague epidemics in India and California much importance
was made of the location of the primary buboes on the rats as indicating
the point of moculation or the flea bite. In India‘ 74.3 per cent. of the
buboes were located in the cervical region. No data on the regional dis-
tribution of fleas on rats in India is available but on guinea pigs artificially
infected by the Commission, 88.9 per cent. of the fleas were found on the
head and neck. Pound?’ in Australia reports fleas as most commonly
occuring on the head and neck of rats. In San Francisco® 72.4 per cent.
of the buboes were found in the groin and none in the cervical region while
73 per cent. of the fleas on the rats were found on the hind quarters.

With these results in view the location of the fleas on the Providence
rats was noted and the following results obtained; 16 per cent. on the head
and neck, 16 per cent. on the fore legs and shoulders, 41 per cent. on the
body, and 27 per cent. on the hind legs and pelvic region. These results
hardly coincide with those found in other places. If we compare the rela-
tive areas of the different regions we find that they stand in about the same
ratio as the percentages of fleas found on them. So it seems quite just to
believe that the fleas are distributed with comparative uniformity over the
body of the rat. However, with the oncome of cold weather there was
noticed a slight change in position from the exposed portions of the body
to the more thickly furred pectoral and pelvic regions. The exact figures
were as follows:

° July-September October-December
Head and neck 15 per cent. 16 per cent.
Front legs and shoulders 15 per cent. 16 per cent.
Body 46 per cent. 37 per cent.
Hind legs and hips 24 per cent. 31 per cent.

1 Acknowledgment is made of the. assistance of Mr. Nathan Banks of the Bureau of Entomology in
confirming the identification of the fleas.

2 McCoy and Mitzmain, The Regional Distribution of Fleas on Rodents, Parasitology, II, 1909, 301.

3 Seventh Report of Plague Investigations in India, Journal of Hygiene Plague Supplement, January,
1913, 217.

4 Report of the Indian Plague Commission, Jour. of Hygiene, VII, 1907, 386.

5 Pound, Report of Plague in Queensland, 1900-1907, 143.

*McCoy, G. W. A Report on Laboratory Work in Relation to the Examination of Rats for Plague at
San Francisco, Cal. Public Health Reports, XXIII, 1908, 1051.

776 The American Journal of Public Health

The average fleas per rat for the months July to September was 10.2 and
that for October to December was 3.7, showing a marked reduction during
the colder months.

‘Twenty-one per cent. of all the rats were found to be infected with mites
and they became more numerous with the onset of cold weather. Lezelaps
echidninus Berlese was the species found. In one case, at the Providence
Coal Co. stable, a specimen of Myonyssus decumani, Tiraboschi, said to
occur on Mus decumanus in Italy, was found. It is interesting to note
that a short while previous to the capture of its host an Italian barkentine
had docked near that point. The common rat louse Polyplax spinulosus
Burmeister was found on 24 per cent. of the specimens. On 12 per cent.
were found open sores.

No numerical study of the occurrence of internal parasites was made
except in cases where the liver was affected. This condition was found in
7 per cent. of the rats and was due to the encysted form of the cat tapeworm
Teenia crassicollis and to the ova of some unknown parasite found also by
McCoy! in California. One noticeable and possibly significant fact is that
from two meat markets from which were obtained 15 and 22 rats, respec-
tively, both these parasites were present in the liver in 25 per cent. of each
lot. A bladder worm was present in all of six cases which were examined.

The question as to whether there is a relation between the filthiness of
the home of the rat and the number of fleas is of interest. The dirty rats
of the stables and docks might be expected to bear more fleas than would
the cleaner rats of other localities. It was found in these investigations,
however, that the average fleas per rat from the stables, and docks was
especially low, in but few cases exceeding one flea. On the other hand the
averages from the cleaner places were generally higher. The average from
a restaurant from which 40 rats were taken was 36 fleas. That from a
creamery was 22, from a dwelling house 10, from another 8, from a drygoods
store 10, from a bar room 1, from two markets 1 each. The reason for this
is doubtless that the fleas breed in the nests of the rats? and are only on the
bodies of the rats to feed. So a damp, cold location of the nests would
strongly inhibit the breeding of the fleas.

The conclusions from our study of the rat situation in Providence are
that rats are present in large numbers and cannot be exterminated until
the city is rebuilt with ratproof construction; these rodents are supplied
with as many fleas as are those in plague infected regions; opportunity
for the fleas to bite human beings is always present as the rats are for the
most part closely associated with man. It would seem, therefore, that
only the germs of plague are lacking to complete the mechanism of an
epidemic in Providence.

1McCoy, G. W. Pathological Conditions found in Rats, Public Health Reports, XXIII, 1908, 1365.
2 Reports on Plague Investigation in India, Journal of Hygiene, VIII, 1908, 241.

118

SIZE OF THE SAMPLE NECESSARY FOR THE ACCU-
RATE DETERMINATION OF THE SANITARY
QUALITY OF SHELL OYSTERS.

BY GEORGE H. SMITH.

American Journal of Public Health, Vol. III, No. 7, 1913, pp 705-708.

[Reprinted from the American JouRNAL or Pusiic Hearn, Vol. 3, No. 7]

SIZE OF THE SAMPLE NECESSARY FOR
THE ACCURATE DETERMINATION
OF THE SANITARY QUALITY
OF SHELL OYSTERS.

GEORGE H. SMITH.

During the past three years while working at Brown University and
for the Rhode Island Shell Fish Commission under the direction of Prof.
F. P. Gorham, I have had occasion to witness and assist in the analysis
of a large number of samples of oysters. Many times throughout this
work the fact has been noted that in oysters taken from the same oyster-
laying there was a very great variation in the colon content. Just how
great this individual variation was or how important a part it played in
oyster analysis has, so far as I know, never been determined. Thus,
during the past winter and spring I have been conducting a series of
experiments with a view to determining these facts.

It has generally been the custom, I believe, for a sample to consist of
five oysters. In fact the Committee on Standard Methods of Shell-fish.
Examination of this Association in their “Second Progress Report”
recommends that this number be used. Furthermore, I think that it is
quite generally the practice to condemn as unfit for food those oysters
which upon analysis and which according to the American Public Health
Method of Rating Oysters for B. coli give a score of thirty-two or more,
and to allow those oysters scoring twenty-three or less to be used as food,
provided the sanitary survey does not show good reason why they should
be condemned. Now in a sample composed of so few individuals as this,
any one individual differing greatly from the others will influence the result
very greatly, and the rating of the sample may be determined largely by
this one oyster, whereas, if the sample were larger, the importance of one
oyster would be greatly decreased. Thus it became apparent that the
factor of chance might play quite an important part in the result of the
analysis. When the analysis being made is for the purpose of determining
whether these oysters are suitable for food or not this factor of chance
assumes great weight. It is of vast importance to the oyster dealer in
such cases just what oysters are chosen for the analysis. Also the health
official should be concerned for it may very readily give him a false idea
of the condition of the oysters which he is considering.

Not only in my own work has this variation been noticed but in exam-
ining the records of the analyses of some 230 oyster samples made by
the Rhode Island Shell Fish Commission it was noted that in but seven
of these did all of the five oysters comprising the sample show the same

5 705

706 The American Journal of Public Health

degree of pollution, and of these seven, five were so badly contaminated
that the colon bacillus was found in every dilution.

The factor of chance of which I have spoken is a variable quantity and
is of far greater importance and value in some cases than in others. Ob-
viously, in badly polluted beds and in layings which are free from con-
tamination, it plays but a very small part. It is in the middle region,
that is, in those layings that are questionable, intermediate between the
good and bad, that it reaches its highest value.

Thus these experiments were with the view of determining how great
this factor might be and if possible to devise some method which would
eliminate it and give a more true index of pollution. To this end I have
made analyses of six different oyster layings in Narragansett Bay whose
locations were such that one would expect to find both extremely bad and
good oysters and also some of an intermediate condition. From each bed
at one time were taken at least 125 oysters. These oysters were trans-
ported to the laboratory and divided arbitrarily into samples of five oysters
each. These samples were then subjected to the usual analysis in so far
as time would permit. By this I mean that lactose-peptone-bile was
inoculated with the shell-liquor in the cubic centimeter, tenth and hun-
dredth cubic centimeter dilutions and gas production in this media was
considered to indicate the presence of some member or members of the
colon group. In but few cases was the colon isolated. The gas production
was noted at 12 hours, 18, 24 and every 12 hours up to 96 hours. I may
here say that by far the greater part (some 97 per cent.) occurred during
the first 48 hours. The results of the analyses were as follows:

Of the first series of 25 samples, which were taken from the upper por-
tion of the Bay where pollution was to be expected, all of the samples
showed contamination to a considerable degree. None of the samples
gave a score of less than 41. Some of them gave scores of 500.

Of the second series comprising 25 samples from a bed which from the
sanitary survey would be classed as in the questionable area the scores
ranged from 5 to 320. Six of the samples gave a score of 23 or less. The
remaining 19 scoring higher than 32.

In the third series taken from a bed located in the same general region
as the former, 23 of the 25 samples gave a score of 32 or higher. The
remaining two samples scoring 23. The scores here ranged from 23 to 410.

The fourth series gave exactly the same results in so far as the scores
were concerned. The individual content of the oysters, however, was
somewhat different.

Series five, which represents oysters taken from a laying much farther
down the Bay and which, from the sanitary survey would be judged favor-
ably located, gave a result which was rather disappointing in that but one
sample of the 25 gave a score of 23 while the remaining 24 samples scored
from 32 to 410.

Quality of Shell Oysters 707

The sixth series of 25 samples was taken from that bed which, from the
sanitary survey, should be the least likely of pollution in that it was the
one farthest out to sea and the most distant from any sewerage outlets.
This bed gave scores ranging from 2 to 320. Fifteen of the samples
scored 23 or less, the remaining 10 being 32 or greater.

Thus it will be seen that in layings which are uniformly bad the part
played by chance in the selection of the five oysters is of comparatively
little importance. Also in layings which are good, although, unfortunately,
I failed to find any, I think it is fair to draw a similar conclusion.

It is in the layings that are intermediate, where there is a wide varia-
tion among the individual oysters, that the importance of what oysters
are chosen is manifest.

This is perhaps brought out the most clearly in series VI. These oysters
were chosen at random and the result was that fifteen samples were com-
paratively good and ten were bad. If, however, by some power I had been
able to choose these oysters with a previous knowledge of their colon
content it would have been easily_possible to have had all 25 samples score
23 or less. In fact, in this series, mathematically the chances were 2 to 1
that any given sample chosen at random would score less than 32.

Again in series II it is possible that 14 of the samples might have scored
less than 32 while in fact only 6 did so. Here the mathematical chance
of obtaining a sample at random which would be considered good is 1 in 8.
For purposes of comparison I will say that while in series I, two samples
scoring 23 could be derived yet the chance of obtaining such a sample
is but 1 in 7,500. From a consideration of this work it would appear that
in many cases at least the analysis of five oysters may fail to give a correct
index of the degree of pollution present in the laying. The only remedy
for this that I can see is to use a larger number of oysters for the analysis
and perhaps place more weight on the number of bacillus coli present per
cubic centimeter in a composite sample of the shell-liquor.

From the examination of my analyses it would appear that the choosing
of any number of oysters less than 15 for a sample will not give consistent
results.

As conclusions I may suggest,—

That the present method of analysis and scoring may work injustice
and does not necessarily give a correct index of contamination.

That the sanitary survey, while undoubtedly of great value, is often
misleading.

That a sample composed of five oysters analyzed individually is not
sufficient.

That a standard should be adopted based on the number of bacteria
of the colon group per cubic centimeter of a mixed sample of the shell
liquor of at least fifteen oysters. That is, a standard for oyster-liquor
should be established of a nature similar to that in use for water and milk.

The American Journal of Public Health

708

re

erived from an exam

zed the data d

Is summarl

In the following table
nation of the work here reported:

96 06E 0G OL SI al &P 9F 66 uel [eox tt
$ OLF 0} &3 ima) I oF 69 read I "poo
“Rare
8 OLP 7 &% &@ 6 oP 69 61 gi ayqeuorjsengy
“vale
P OLF 0} &@ &@ 6 14 6¢ Il I ajquuonsongy
"Bale
al 066 97 ¢ 61 9 £6 69 && g ayquuonsonty
*JoyNo aseVMS
é 00¢ 0} OF G3 0 r9 gg 9 0 eB1v] Ivou poywo
-oJT—prq A194,
6S Jopu f)
= i 6G PAOgY | 6§ pun, | -9°0 T9'0 | “9°2 L'O ae) | t 4
2100S S2100G caer panase 10(8) (019) -Aaamg
0} sajdures jo p : ou SULMOYS
= sojdurg | satdureg Arepues
JOON asuRy ® s104sKQ ;
‘i jo "ON jo "ON UI TOD ‘g Sutmoysg s10jsKQ
Seed. - ‘

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6) Al
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96 II
96 I
‘soydures foie
JOLON, :

‘NOILVNINVLINOD JO XAGNI NV SAID OL AUVSSAOAN AIdINVS UALSAO AHL AO AZIS AHL

119

SCHMUCKER’S ‘“‘ THE MEANING OF EVOLUTION.’’
BY HERBERT E. WALTER.
Science, N. S., Vol. XX XVIII, No. 987, p 779, November 28, 1913.

[Reprinted from Sorrnoz, N. 8., Vol. XX XVIII, No. 987, Page 779, November 28, 1913]

SCHMUCKER’S “THE MEANING OF EVOLUTION ”

The Meaning of Evolution. By SamMuen
CurisTIAN ScHMUCKER, Ph.D. New York,
The Macmillan Company. 1913. 12mo.
Pp. 298.

This is a very readable book upon what is
no longer a new theme. Following a literary
“ foreword ” the pre-Darwinian history of evo-
lution is sketched as a background for Dar-
win and Wallace. The historical chapter
about Darwin presents the essentials of his
career in a charmingly vivid and sympa-
thetic manner. Then follows the “ Underly-
ing Idea” of natural selection as the method
of evolution illustrated largely by means of
the English sparrow, of which the author in-
cidentally says (p. 84): “This pestiferous
creature should be exterminated ... but per-
sonally I am taking no share in his destruc-
tion ... I confess that it would be with re-
gret that I should see him disappear from the
landscape.”

Chapters IV. and V. deal with adaptation
for the individual and for the species. The
general attitude toward Lamarck is occasion-
ally rather more conciliatory than the mili-
tant Weismannian would approve of, but this
is not to be wondered at in one who is proud
of having been a student of Professor Cope.
It seems to be very easy to drop into La-
marckian explanations for adaptation. For
instance (p. 89): “ The modern scientist feels
sure not only that the animal is fitted to his
work, but that he has been so fitted by the
work.” It will probably always be a bone of
contention whether the exercise of an organ
determines its structure or the structure of
an organ sets the limits to its exercise.

With respect to protective coloration and
sexual selection the author proposes to retain
the Darwinian interpretation until something
better arises in spite of the recent loss of con-
fidence in the adequacy of these explanations.

The three succeeding chapters upon “ Life
in the Past,” “How the Mammals Devel-
oped,” and “The Story of the Horse” mar-

shal in review some of the classified evidence
in support of animal evolution, while Chapter
IX. takes up “Evolutionary Theories Since
Darwin.”

In this last chapter Weismann, whose
name will doubtless be correctly spelled in
subsequent editions, is justly given promi-
nence because his “work has made us cau-
tious and prevented our lightly accepting a
belief in the influence of the environment.”
Moritz Wagner and Romanes with their iso-
lation theories and the orthogenists receive
attention, and finally Hugo deVries with mu-
tation closes the chapter.

The book could have been written fifteen
years ago so far as any analysis of the signifi-
cant bearing which Mendelism or the pure-
line theory of Johannsen has upon the ques-
tion of evolution.

Chapter X. turns optimistically to the
“Future Evolution of Man” and is sociolog-
ical rather than biological in its treatment,
while the final chapter, “Science and the
Book” gives the impression that the professor
has stepped out of the class room and is
speaking to a church audience and speaking
withal extremely well.

The word “ Evolution” has lost most of its
incendiary character of a generation ago yet
there are no doubt many in whose minds it
still stands contrasted with religion and the
Bible as a faith-destroying invention of god-
less scientists. To all such persons this book
is a welcome message of reassurance and peace
while to others who no longer need to be con-
vinced of the essential truth of the evolution-
ary processes, the pages will be turned with
approving delight.

Dr. Schmucker has stated the facts of the
case in clear non-technical language with
much literary grace and with scientific ac-
curacy, consequently the book is well adapted
to a wide range of readers even outside the
biologically initiated. H. E. Water

Brown UNIVERSITY

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120

GENETICS, AN INTRODUCTION TO THE STUDY OF
HEREDITY.
BY HERBERT E. WALTER.
Published by the Macmillen Company.

GENETICS

AN INTRODUCTION TO THE STUDY
OF HEREDITY

BY

HERBERT EUGENE WALTER

ASSISTANT PROFESSOR OF BIOLOGY
BROWN UNIVERSITY

WITH 72 FIGURES AND DIAGRAMS

Cloth, xiv+272 pp,, bibliogr., index, $1.50 net

PUBLISHED BY

- THE MACMILLAN COMPANY
64-66 Firry AvEeNnuE

New York

< HE subject of heredity,’’ remarks Professor
iit Walter, ‘‘concerns everyone, but many of those
who wish to become better informed regard-

ing it are either too busily engaged or lack the oppor-
tunity to study the matter out for themselves. The recent
literature in this field is already very large, with every

indication that much more is about to follow, which is a

further discouragement to non-technical readers.

“Tt may not be a thankless task, therefore, out of the
jargon of many tongues to raise a single voice to tell the
tale of heredity. There may, too, be a certain advantage
in having as spokesman one who is not at present immersed
in the arduous technical investigations which are making

the tale worth telling.’’

It is evident, therefore, that although the author isa
trained scientist, he fully realizes the difficulties in under-
standing this complicated subject. This realization, com-
bined with Professor Walter’s mastery of the art of clear
and interesting presentation of scientific truths, has enabled
him to write a forceful, accurate and stimulating summary
—for the intelligent, but uninitiated reader—of some of the
more recent phases of the questions of heredity which are

at present agitating the biological world.

The book is, therefore, well adapted for use as a text
in college courses on breeding, heredity or evolution. It is
also a volume from which the busy reader will find it a
pleasure, rather than a task, to gain an acquaintance with
this most vital and absorbing phase of the newer science
which has a most direct bearing upon man and the better-

ment of the race.

CONTENTS

CHAPTER PAGE
I. Intrropvuction.

1. The triangle of life. : : 5 - ps Pe 1
2. A definition of heredity . : : A F A 4
3. The maintenance of life 5
4. Somatoplasm and germplasm. : : : fe eelO

Il. Tur Carriers or THE HERITAGE.

1. Introduction. : d - 5 : hae
2. The cell theory . ‘ ‘ : ; : : Pa
3. Atypical cell. : 2 Sere - - a glib
4. Mitosis. ¢ , ‘ ; 2 : : ls
5. Amitosis . : ; : : : 2 : te 20
6. Sexual reproduction . : : . : : eee 0)
7. Maturation ‘ ‘ : : : 2 eee
8. Fertilization . ; d : : : ‘ mea
9. Parthenogenesis : é : ; : - = 26
10. The hereditary bridge ‘ é / - é Me CAE
11. The determiners of heredity . : : - s “28
12. The chromosome theory . F i : : = eo
13. The enzyme theory of heredity . : ‘ é 5 SE
14. Conclusion A : : : P : ; oo

iI. VaRtaTIon.
1. The most invariable thing in nature . : 5 & 8k
2. The universality of variation . 4 or aes
3. The kinds of variation with respect iB thes —

Nature . - - - : : : eco

a.
b. Duplication . x ‘ : - : Ae
e. Utility’ “».. : ‘ A 2 F Reet!
d. Direction in evolution : : : : . 89
e. Source . ; A - - : : - 40
f. Normality ; : : : , a AO
g. Degree of ae : : c 2 5 am 40
h. Character . ‘ ° a) Ml
i. Relation to an average Siatidaed z : eae Bs
j- Heritability . . : & : aie |

CHAPTBDR

or
e

CONTENTS

Methods of studying variation . 4
Biometry . :
Fluctuating variation F
The interpretation of variation curves
a. Relative variability
b. Bimodal curves
c. Skew polygons .
Graduated and integral TanaticHs
The causes of variation
a. Darwin’s attitude
b. Lamarck’s attitude
c. Weismann’s attitude
d. Bateson’s attitude. . ;

IV. Murarion.

_
°

_
S

a

The mutation theory . i J 5
Mutation and fluctuation .

Freaks :

Kinds of mutation

Species and varieties .

Plant mutations found in nature
Lamarck’s evening primrose

Some mutations among animals

Possible explanations of mutation

A summary of the mutation theory

V. Tue INHERITANCE OF ACQUIRED CHARACTERS.

Shee ca oa ec gO ro

10.

Summary of preceding chapters . :
The bearing of this chapter upon genetics .
The importance of the question .

An historical sketch of opinion .

Confusion i in definitions

Weismann’s conception of neqtcred characters

The distinction between germinal and somatic chars

ters ‘
What variations reappear ?

What may cause germplasm to vary or to acquire

new characters ?

Weismann’s reasons for doubting ee snhentaree of

acquired characters : : :

CHAPTER

CONTENTS

11. No known mechanism for impressing germplasm with

12.

13.

14.
15.

somatic characters
Evidence for the inheritance of Beviitied: chiens
inconclusive .
a. Mutilations
b. Environmental effects
c. The effects of use or disuse
d. Disease transmission
The germplasm theory sufficient to Bit for the
facts of heredity .
The opposition to Weismann
Conclusion

VI. Tue Pore Line.

—_

OPAMP w 10

—
=

~

Ge Sab i)

9:
10.

. The unit character method of attack B

Galton’s law of regression : 2 : A

. The idea of the pure line

Johanssen’s nineteen beans ; : : :
Cases similar to Johanssen’s pure lines

Tower's potato-beetles

Jennings’ work on Paramecium ;
Phenotypical and genotypical distinctions

The distinction between a population and a pure line
Pure lines and natural selection : E :

VII. SEGREGATION AND DOMINANCE.
. Methods of studying heredity . : : -

. The melting-pot of cross-breeding . < é
a. Blending inheritance . : : :
b. Alternative inheritance . : - - <
ce. Particulate inheritance . : A S é
Johann Gregor Mendel . : : A 2 =

Mendel’s experiments on garden peas
Some further instances of Mendel’s law
The principle of segregation
Homozygotes and heterozygotes

The identification of a heterozygote .
The presence and absence hypothesis

Dihybrids pe SR oh on) HS,

PAGE

84:

86
87
88
91
92

94
95
96

102
103
107
108
110
113
115
118

120
120
121
121
121
123
124
128
130
131
132
132
133

CONTENTS

CHAPTER
11. The case of the trihybrid ; - ‘ - -
12. Conclusion
13. Summary

VIII. Reversion to Otp Trrpes AND THE Maxkina or New
ONEs.

1. The distinction between reversion and atavism
2. False reversion
a. Arrested dey ehaent
b. Vestigial structures .
ce. Acquired characters peeriine anc estral ones
d. Convergent variation
e. Regression
3. Explanation of reversion .
4. Some methods of improving old and anienaae new
types .
a. The method of Hallet
b. The method of Rimpau .
c. The method of de Vries .
d. The method of Vilmorin .
e. The method of Johanssen
f. The method of Burbank
g. The method of Mendel
5. The factor hypothesis
a. Bateson’s sweet peas
b. Castle’s agouti guinea-pigs
c. Cuénot’s spotted mice
d. Miss Durham’s intensified mice -
e. Castle’s brown-eyed, yellow guinea-pigs .
6. Rabbit phenotypes .
The kinds of gray rabbits

8. Conclusion 3 x 3 ; i é : ;

=

TX. Buenpine INHERITANCE.

1. The relative value of dominance and segregation.
2. Imperfect dominance

3. Delayed dominance

4. “Reversed” dominance . ; : : :

175
177

i. >» 2 +e 2 ee

CHAPTER

CONTENTS

5. Potency . : - - : . “ 6
a. Total ote
b. Partial potency
c. Failure of potency .
. Blending inheritance
. The case of rabbit ears
. The Nilsson-Ehle discovery
. The application of Nilsson-Ehle’s aalnvation is the
case of rabbit ear-length : :
10. Human skin color . c : 2 : : °

oon a

X. Tue DerTeRMINATION OF SEX.

1. Speculations, ancient and modern
The nutrition theory
The statistical study of sex
Monochorial twins
Selective fertilization
The neo-Mendelian theory of sex
a. Microscopical evidence
1. The “x” chromosome
2. Various forms of x chromosomes
3. Sex chromosomes in parthenogenesis
b. Castration and regeneration experiments .
c. Sex-limited inheritance
1. Color-blindness
2. The English coaeny ath Z
d. Behavior of hermaphrodites in heredity .
7. Conclusion : ‘ - : 2

O om oo 0

XI. Tue Appiication to Man.

1. The application of genetics to man .
2. Modifying factors in the case of man
3. Experiments in human heredity
a. The Jukes
b. The descendants of Tanatian oe
ce. The Kallikak family
4, Moral and mental characters Bakes like phy Seal
ones : :
5. The character of human nits’.
6. Hereditary defects

CHAPTER

CONTENTS

. The control of defects. 4 f A Zs

Inbreeding

. Experiments to test the effects of mibmeeditig
10.
i:

The influence of proximity 2
Inbreeding in the light of Mendelism

XII. Human Conservation.

its
2.
3.
4.

6.

How mankind may be improved Z 4
More facts needed : :
More application of what we know necessary
The restriction of undesirable germplasm through —
a. The control of immigration :
b. More discriminating marriage laws .
c. An educated sentiment
d. The segregation of defectives
e. Drastic measures
The conservation of desirable aevidplasin “5 : ;
a. By subsidizing the fit. : : a
b. By enlarging individual opmonkenee:
c. By preventing germinal waste . :
1. Preventable death . : : < 4
2. Social hindrances ; : <
Who shall sit in judgment? . 5 .

XIII. BrsiiocrRapyy . 5 R ; 4 3 = P :

XIV. Invex F 3 F 5 - i B 5 3 2

PAGE
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2A2

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247

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263

265

121

THE NEGRI BODIES IN RABIKS.
BY ERNEST M. WATSON
Journal of Experimental Medicine, Vol. XVII, No. 1, 1918, pp 29-42.

(Reprinted from the JOURNAL OF EXPERIMENTAL MEDICINE, Vol. XVII, No. 1, 1913.]

THE NEGRI BODIES IN RABIES*

By ERNEST M. WATSON.

(From the Bacteriological Laboratory of Brown University and the Hygienic
Laboratory of the Johns Hopkins Medical School.)

PLATES I AND 2,

Since Negri, in 1903, discovered the bodies contained within the
nerve cells of the central nervous system in cases of rabies, there
has been much speculation as to their identity and importance.
These structures, or Negri bodies, as they are now called, are con-
stantly associated with rabies and we have come to regard them of
prime importance from the standpoint of diagnosis. Their presence
is demonstrated in the cerebral or cerebellar cortex, in the hippo-
campus major, or in the corpus striatum in over 98 per cent. of all
cases with the clinical symptoms of rabies. The occasional failure
to find them is, probably, due to our imperfect methods, to the fact
that the bodies under certain conditions do not take the stain, or that
in certain cases they occur in forms too small to be recognized by the
one-twelfth inch oil immersion lens.

The nature of the Negri bodies is today a mooted question, and
concerning them many and varied views have been advanced.
The clinical history and symptoms of the disease, together with the
nature of the infection and the properties of the virus, point toward
a definite microorganism as the underlying cause. All observers
now agree that the bodies described by Negri are definitely asso-
ciated with rabies. One group of investigators believes the bodies
to be definite parasitic organisms, and the cause of the disease. An-
other group considers them simply cell degenerations caused by the
infecting agent, but incapable of producing the disease. Negri him-
self believed the bodies to be parasites of an animal nature and ipso
facto the cause of rabies. Other Italian investigators including
Volpino, Dominici, Marzocchi, d’ Amato, Bandini, Fasoli, and others,

* Received for publication, September 5, 1912.

29

30 The Negri Bodies in Rabies.

have corroborated Negri’s work and with him agree that the bodies
are of a parasitic nature. In this country Williams and Lowden,
Calkins, and others believe that the Negri bodies are of protozoan
nature. The former authors have worked out a tentative life his-
tory, illustrating undoubtedly a multiplicative cycle in the develop-
ment of the organism and have classed it among the Microsporidia.
Calkins believes it to be more closely allied to the ameba and has
placed it under the classification of the Rhizopods. Other writers
in this country who favor the protozoan identity of the Negri bodies
are Poor, Stimson, Frothingham, Hadley, Harris, and others.

THE NEGRI BODIES.

In the following observations upon the nature of the Negri bodies
the methods and technique employed have been those now generally
used in most of the Board of Health Laboratories in this country.
The examinations of rabic material were made in the regular smear
preparations, and in paraffin sections of the cerebral and cerebellar
cortex, and of the hippocampus major and corpus striatum. In
general it was found that the smear preparation under proper manip-
ulation gave the better results. “The morphology of the bodies by
this method can be brought out definitely and their internal struc-
ture shows clearly, much more so, in fact, than in the paraffin sec-
tions, while the possibility of changing the position of the bodies
within the cell and of mutilating them in any way by pressure, is
reduced practically to nil after a little experience. By the smear
method the general contour and shape of the bodies is the same as
that noted in the paraffin sections, and their arrangement in the cell
shows no appreciable displacement of the cellular protoplasm. <A
further advantage in the smear method is that upon the slide the
layer of brain material is of varying thickness, which fact, after
staining. aids materially in the final conclusions which are based on
a comparative study of very thin, medium, and thick areas.

The stains used have been the Van Gieson stain for rabies, which
at times was modified by the substitution of basic fuchsin instead of
the rose anilin violet, the other proportions remaining the same.
Mann’s method of staining was also used in attempts to bring out
more definitely the inner structure of the bodies.

Ernest M. Watson. 31

Shape.—In general it may be said that the Negri bodies tend to
assume an oval shape. Sometimes, however, they appear round
and occasionally rather irregular, appearing to be in the process of
division. Usually the outline of the bodies is smooth. Less fre-
quently the edges appear irregular.

At times forms resembling a process of budding are noted.
There seems to be a pretty general distribution of the different
shapes in a single animal. If anything the oval shape predomi-
nates, while the division forms are least frequently encountered.

Size.—The size of the bodies seems to vary within rather wide
extremes, from 0.25 to 0.5 to 21 microns. Although in general the
very small ones and the very large ones are met with less frequently
than are those measuring two to nine microns across their longest
diameter. All sizes may be present at the same time in one animal
and frequently in one nerve cell (from Ammon’s horn) bodies meas-
uring one micron and others measuring ten to fourteen microns are
present at the same time.

Number.—TVhe number of bodies present in any individual case
seems to be subject to considerable variation. Sometimes the large
cells from the hippocampus and the cortex of the cerebrum and the
cerebellum are literally filled with bodies, and at other times a search
of several minutes is necessary to reveal a single body. In these
latter cases, however, further search, in the writer’s experience, has
always been rewarded with the finding of an appreciable number of
bodies, although they may be well scattered. When the bodies are
few in number they seem to be uniformly small in size and almost
without exception are quite regular in their morphology. For the
most part the bodies are found with greater regularity in the horn
of Ammon and in the corpus striatum than in either the cerebral
cortex or the Purkinje cells of the cerebellum. A specimen brought
into the laboratory some years ago from a case of human rabies
showed nine well developed bodies in one of the Purkinje cells of the
cerebellum.

Structure —Concerning the structure of the Negri bodies there
has been much speculation and much has been written in attempts
to identify and determine the rdle of the various parts. With the
Van Gieson stain, and also with the basic fuchsin modification of it,

32 The Negri Bodies in Rabies.

the bodies take a characteristic pink stain, lighter and of a more deli-
cate hue than the eosin red color imparted to the red blood cells
which are frequently seen in the same section. With proper stain-
ing solutions, this typical color varies but little from day to day.

The Negri bodies are essentially intracellular parasites and we can
speak of them with certainty only when they are found within the
nerve cells. They never have been demonstrated along the longer
and larger nerve trunks or along the fiber tracts in the cord. There
is, however, good reason to believe that the infection reaches the
central nervous system through the above mentioned channels.
Within the nerve cells the pink color of the bodies is broken by the
regular appearance within the body of the parasite itself of cellular
inclusions termed chromatin granules. These granules are a con-
stant part of the typical Negri body. They take a dark slate blue
_ stain with all the stains which have methylene-blue or hematoxylin
asabase. Their number in the body is variable, being dependent for
the most part upon the size of the parasite. There is usually one
slightly larger and more deeply stained than the rest which occupies
a position more central than the others. This granule has been re-
garded as the nucleus and is present in practically all forms which
are large enough to permit a structural examination of their inner
cytoplasm. In the very small forms only the central chromatin
granule or nucleus is discernible, while in the larger forms the
granules vary from three or four to ten or twelve. In the larger
forms the rather concentric arrangement of the chromatin granules
around the parent nucleus appears like nuclear fragmentation pre-
paratory to a subsequent division of the cytoplasm and these types
have been believed by many to represent a preparatory stage to
direct cell division by fission.

Williams and Lowden have described at length the succeeding
stages in the growth and reproduction of this type of Negri body.
According to them, the bodies are first found as definite tiny forms
0.5 to I micron in diameter, with one chromatin granule or nucleus
taking a deep azure blue stain in the center and enclosed in a delicate
pink staining cytoplasm, while around the whole and blending into
the cell cytoplasm is a definite rim, or ectoplasm, staining a little
deeper pink than the rest of the cell protoplasm. In addition to

Ernest M. Watson. 33

these extremely tiny forms there are larger ones which are similar in
shape and staining reactions, but which contain an extra chromatin
granule or two, probably fragments of the nucleus. Still larger
forms are also noticed which are, in general, similar to the bodies
last described, but which begin to show evidences of division, 1. e.,
constrictions giving rise to hour glass forms. The various steps in
the process of fission have been observed. In the last, only a fine
filmy bit of protoplasm connects the two organisms. In the next
stage, the two bodies have become entirely separate and lie side by
side. In some cases the ectoplasm of the two bodies is in close
proximity, but in others it is separated by a small space through
which the protoplasm of the nerve cell shows as a blue staining line
between the pink staining ectoplasmal rims of the two now separate
individuals.

The above description outlines briefly one undoubted phase in
the life history of the organism. It represents a stage of multiplica-
tive reproduction,—a schizogonous life cycle, or, in speaking of the
Myxosporidia, a stage of plasmotomy carried on within the cells of
the host. This condition we expect on account of the vast num-
bers of parasites that are found in a given host and even in a single
cell of a host, and the great difficulty of conceiving that each para-
site could have arisen from a separate and individual spore infection.
From the above commonly observed conditions, and from the na-
ture and frequency of the findings, we have good reason to believe
that this plasmotomous life cycle is supplemented, either within the
body of the host or elsewhere, by another cycle of a different nature,
this other cycle being concerned with a sexual reproduction.

Bodies corresponding to a sporogonous life cycle of this type
have in two instances in the past few years come under the writer’s
observation, and a general description of these findings is reported
at this time. The two cases and the specimens which showed these
sporogonous types presented rather similar and singular histories.
The dogs came from sections of the state some thirty miles apart
* and during life presented the usual clinical picture of uneasiness,
restlessness, and signs of evident distress, and became very irritable.
During this last stage both animals had bitten people. The dogs
were consequently confined, and in approximately twenty days after

34 The Negri Bodies in Rabies.

the initial signs of malaise both animals died, the reported cause
of death being distemper. Unsuspecting the seriousness of the bites
both dogs were buried, and several days elapsed before they were
dug up in order that their heads might be sent to the laboratory for
examination.

In these two cases the cells of the cerebral cortex, the Purkinje
cells, and the giant cells of the horn of Ammon showed the pres-
ence of the typical Negri bodies with their characteristic nuclei and
chromatin granules as described above under the usual cycle of
an asexual intracellular development. In addition to these were
certain forms quite different from any of the preceding, yet taking
the stain readily and being closely associated with the former, many
of them being in the same cells with the plasmotomous type of body.
These new bodies were seen to vary in size. The smallest ones
measured 0.25 to 0.5 of a micron in diameter (plate 1, figure 4, A).
Some were possibly smaller than this, the difficulties of identification
in these extremely small types being considerable. The smallest
forms present are slightly round or oval, have a definite morphology,
and are regular in outline. They take a deep pink, almost a crimson
stain, and when treated with acid alcohol seem to hold the stain
longer than those of the plasmotomous type. Their protoplasm
stains with no indication of a nucleus or of chromatin granules. At
one end, by careful focusing and with most of the light coming
from the mirror, the condenser being cut out, a small refractive
area is seen which takes the stain less readily than the other portions.
As nearly as could be determined with the immersion lens this light
spot presents an oval outline and gives every indication of being a
capsule, and the whole body with its uniform and homogeneous
consistency seems to have every appearance of a spore.

Bodies of this type are found scattered more or less diffusely
throughout the slides, both inside and outside of the nerve cells.
Forms similar to these in every way but slightly larger are also
found, but are less plentiful than the very small ones (plate 1, figure
1, EH, F; figure 2, E, F, H, J). The next larger body corresponding *
to these forms presents quite a different picture. They measure I
to 11%4 or 2 microns in diameter, stain a brilliant pink, are very
regular in outline, and about them a definite membrane, or rim of

Ernest M. Watson. 35

ectoplasm, can be made out, while their interior contains two, three,
and sometimes four of the definite, tiny, regular oval spores de-
scribed above. All these stain a brilliant pink and occupy com-
pletely the interior of the body, there being no nucleus and no
chromatin granules, nothing in fact which takes the blue stain, and
but little of the residual protoplasm of the parent body which takes
a faint pink stain and shows up in marked contrast to the spores.
Larger forms than this are also observed which include a definite
cell membrane, within which are eight to ten, and sometimes twelve,
of the spore-like bodies (plate 1, figure 2, H, E). They all stain
similarly to the smaller forms and show only a little of the residual
pink staining protoplasm of the original cell and no nucleus or
chromatin granules. Larger forms than these show a broken
ectoplasmal rim with the spores protruding and some are even quite
outside the body itself and in the substance of the nerve cell (plate
I, figure 1, B; figure 2, E, H, I; figure 3, D). They presefit the
same picture as the tiny round or oval spores described above.
Some forms contain as many as twenty to thirty and these were
observed with the ectoplasm intact and the spores completely filling
the inside of the body. These forms measure eight to ten microns
in diameter. In the still larger forms the membrane surrounding
them becomes less distinct, until finally the definite structure of the
rim-like band is lost completely. After losing the membrane the
groups of spores in many cases are seen remaining close together as
before (plate 1, figure 1, B). In general, however, after the dis-
appearance of the cell membrane there is a marked outpushing of
the spores from the center, making the body appear much larger
than in any of the preceding forms and leaving in the center a small
light pink staining area formerly occupied by the spores (plate 1,
figure 2, I; figure 3, D). In these forms nothing which re-
sembled a nucleus is seen nor are there any chromatin granules
present. A later step in the development of the organism is seen
when the spores become further removed from the center and oc-
cupy a much larger area. They form a circle with the pink staining
residual protoplasmic remains of the original Negri body, possibly
too a portion of the ectoplasm, on the inside and the blue staining
cytoplasm of the nerve cell on the outside. The final step in the
dissemination of the spores is seen when the circle of spores has

36 The Negri Bodies in Rabies.

been broken up, and in certain nerve cells they are then seen scat-
tered diffusely over the entire field (plate 1, figure 4, A).

This outlines briefly the successive stages seen in the growth,
reproduction, and dissemination of this new type of Negri body.
The succeeding steps in its growth and development, with the
typical pictures presented in the different stages, and the multi-
plicative or schizogonous life cycle previously described, suggest
immediately a sexual or sporogonous cycle for this new series of
bodies described above.

Other writers have often referred to the possibility of finding a
sexual cycle which in a measure would supplement the knowledge
of the forms already found arid do much toward increasing our
knowledge of this protozoan parasite, which we now believe is the
causative agent of rabies. The above findings, taken in the light of
the multiplicative cycle already observed, are indeed strongly sug-
gestive. Williams and Lowden, as has been stated, have placed the
Negri bodies among the parasitic protozoa in the suborder Micro-
sporidia of the Sporozoa. Organisms of this order in their sporog-
onous life cycle produce numerous oval or pear shaped spores
corresponding closely to those described above, each of which
possesses one polar capsule. This is not seen in the fresh specimen,
but is brought out under suitable treatment with stains and reagents.

From the findings and observations recorded above the writer
believes that we are now justified in placing the Negri bodies a little
more definitely in the suborder of Microsporidia and that we may
regard them as Oligosporogenea, in which forms the trophozoite
produces a single pansporoblast. Its position is perhaps among these
under the family Glugeidz, when we bear in mind that in these the
pansporoblast produces numerous spores.

The smallest forms observed with a single dark staining nucleus
or chromatin granule are undoubtedly on the order of a trophozoite
of a multiplicative cycle characterized ofttimes by an ameboid ap-
pearance and a dense rim of ectoplasm, with a granular endoplasm
containing a nucleus and possibly chromatin granules. These may
increase in size very rapidly until there are numbers of bodies of
different sizes. During this growth they undoubtedly have the
power of endogenous reproduction. This has been observed by
several writers and may take place in different ways, all of which

Ernest M. Watson. 37

are common to the members of this order. In the adult form (1)
simple binary fission may take place, 7. e., an hour glass constriction,
or (2) a simple budding may occur. This plasmotomous division,
or breaking up of the multinucleate cell into numerous parts each
having a nucleus with a bit of surrounding protoplasm has been
termed, in the first case, a simple, and in the latter, a multiple type
of division.

In the youngest stages of the organism there has been described
nuclear fragmentation into several daughter nuclei, supposedly by
a process of multiple amitosis, and this is similar to, if not identical
with, that observed in the young forms of Glugea lophu. This type
early spreads the infection through the tissues of the host and is
probably the most common of all the methods of multiplicative
growth. In the well known forms of Myxobolide and Glugeide it
is undoubtedly the commonest type of growth. In considering the
sporogonous or reproductive cycle, spores undoubtedly begin to
develop at an early period in the life of the trophozoite. The find-
ing of two, three, and four spores in a single small body, or pan-
sporoblast, would seem to prove this. Among the better known and
more closely studied members of the Microsporidia the first step in
spore formation is the gathering of cell protoplasm around some of
the nuclei of the mother endoplasm to form a primitive sphere
(Gurley) or pansporoblast. Whether or not this has been observed
in the case of the Negri bodies it is still difficult to say ; stages which
seemingly show a fragmentation of nuclei have been reported, but
these may indicate a stage in the multiplicative, as well as a prepara-
tion for spore formation in the reproductive life cycle. The pan-
sporoblasts as observed in the development of the Negri bodies have
a definite membrane or envelope around them, and so far at least
the spores they contain have been observed to vary considerably in
number. Furthermore, when first observed they were rather com-
pletely formed so that all steps in their development, particularly the
presence of a sporoblast stage, are not as yet completely understood.
As to the origin of the polar capsules it has not as yet been observed
often enough to state whether or not it arises like many other mem-
bers of this order from a sphere-like vacuole.

38 The Negri Bodies in Rabies.

ANALOGIES BETWEEN THE NEGRI BODIES AND THE BETTER KNOWN
FORMS OF THE SUBORDER OF THE MICROSPORIDIA,

In general, the forms of the suborder of the Microsporidia show
an extremely wide variation in their structure, habitat, and mode of
life. The trophozoite has the following characteristic features which
are well observed in the life cycle of the Negri bodies: (1) It is
characteristically ameboid in shape. (2) Spore formation com-
mences early and continues throughout life. (3) Spores are pro-
duced endogenously, 7. e., within the protoplasm of the trophozoite.
(4) The spores have one or more polar capsules. While it is true
that the Cryptocysts are found more often inhabiting invertebrate
hosts, several forms are known which make their home exclusively
in the tissues of the vertebrates. As parasites they are more de-
structive in their growth to the host cells than any other group of
the Sporozoa. They are in every detail essentially intracellular
organisms differing from the Phzenocysts which are the intercellular
forms, and only exceptionally at the very early stages are they found
within the tissue cells.

The group in the main has two modes of life, one within the body
cavities of the host, and the other within the cells and tissues of the
host. The latter is clearly the manner of life of the Negri bodies.
While muscle and connective tissue are the ones usually selected as
the seat of growth of the organism, some forms attack the nervous
tissue alone, as is seen in the Glugea lophu which has a special affin-
ity for the ganglion cells of the central nervous system of Lophius
piscatorius. The Cryptocysts may restrict their ravages to a single
organ or tract, or they may attack all parts of the body. Naturally
the method of growth may be of two kinds; that in which the
growth is confined to definite foci, and that in which there is a
diffuse infiltration of the tissues. The latter method is peculiar to
the Negri bodies in which the body of the parasite and the proto-
plasm of the cell become closely intermingled, spores are formed,
and the specific tissue becomes actually filled with young forms.

Trophozoite Stage.—This is characterized by its ameboid form
in which an uncertain division can be made out between the coarser
outer region and an inner granular center. The endoplasm has
various inclusions ; in the Negri bodies have been observed only the

Ernest M. Watson. 39

nucleus, chromatin granules, and possibly occasionally a vacuole or
two. In the tissue-infecting forms, the pseudopodia are usually
small, of ectoplasmal origin, and cannot be observed in the stained
preparations. In the Negri bodies no pseudopodia have been recog-
nized. Here perhaps we have, as in other well known cases, the
protective function of the ectoplasm developed at the expense of the
motility. The youngest trophozoites have but one nucleus, the
endoplasm increases continually by division until there are many
present in the full grown forms, often ten or more, while definite
nucleoli or karyosomes have not been recognized except in a few
instances.

Spore Stage.—Spores are seen to develop in a very early stage of
the trophozoite and sometimes they continue to be formed until the
whole endoplasm of the trophozoite is used up in its production. In
other cases, however, there is a small amount of residual protoplasm.
All the stages of spore development are commonly found at the
same time in a given individual. The first sign of spore formation
is the gathering of protoplasm around one of the nuclei in the endo-
plasm. This comes to form the sporoblasts and later the spherical
corpuscles, or pansporoblasts. All nuclei may not be used up in
this manner, but may remain as residual nuclei (plate 2, figures
i to xill).

Among the Cryptocysts the pansporoblasts always give rise to
more than two spores, 7. e., four in Gurleya, eight in Thelohania, and
to a large number in Pleistophera and Glugea. One or more pan-
sporoblasts may be formed from each trophozoite, the Oligo-
sporogenea under this order produce but one, however, and here the
condition simulates greatly the development of the Coccidia except
that with them we have an exogenous development instead of an
endogenous one. In general we may say that the Myxosporidia
represent an alternation of generations between trophic and repro-
ductive individuals.

The spores of the Myxosporidia are rather variable in their ex-
ternal structural.details. The spores of the Cryptocysts are for the
most part minute, measuring 3 to 4 microns in Glugea anomala.
and 1.5 to 2.5 microns in Glugea ovidea, and are usually pear shaped
or oval. The Phzenocysts have much larger spores, some 100 by
I2 microns (in Cestomyxa sperulosa).

40 The Negri Bodies in Rabies.

The capsule of the spore is usually situated at one end and when
this is the case, that end is termed the anterior pole. In the
Glugeidz the spore capsule is invisible in the fresh condition. These
spore capsules are a distinctive feature of the Myxosporidia and by
some it is believed that they represent sporozoites that have become
specialized in nature. The developmental period which intervenes
between the ripe spore and the youngest trophic stages is the least
known period in the life cycle of this group of organisms. The
microscopic parasite often migrates a great distance in the body of
the host before it gets to the organ or tissue which is its final field
for growth. This is well illustrated in the rabic infecting agent
which, from a cutaneous bite or abrasion, must travel until it
reaches the central nervous system before it finds a suitable field for
growth. This stage shows that undoubtedly at one time in its life
the parasite is able to select, and, furthermore, to seek out its
specific tissue which may, in some instances, be at a great distance
from the original seat of infection.

Schizogony, or Multiplicative Process—When the amebula
reaches its special tissue it can grow endogenously with great
rapidity. The two processes of growth are called multiplicative,
like schizogony, and reproductive, like sporogony. Multiplicative
growth has always been attributed to the Negri bodies on account
of the vast numbers of parasites that may be present in a given host,
and the difficulty of supposing that each parasite could have orig-
inated from a separate and distinct spore infection. The multipli-
cative reproduction in the full grown trophozoite takes place in one
of two ways, by simple fission, or by budding. This plasmotomous
division has been termed, in the first case, simple, and in the latter,
multiple amitosis (plate 2, figures I to 18).

The youngest forms divide by nuclear fragmentation and mul-
tiple amitosis following a breaking up of the protoplasm into minute
uninucleate swarm spores, and in this way the tissue becomes
diffusely infected.

CONCLUSION.

1. The Negri bodies, as the etiological agent in rabies, present
two general types or phases in morphology, in growth, and in
reproduction.

Ernest M. Watson. 41

2. These two phases are constantly cyclic in their development
and correspond (1) to a multiplicative, or schizogonous, and (2) to
a reproductive, or sporogonous, life cycle.

3. By the detailed study of these forms and their succeeding
stages we are inclined to believe that the Negri bodies are definite
protozoan parasites, and from a study of their life history we are
led to place them in the suborder of Cryptocysts, or Microsporidia,
of the Sporozoa, and more definitely among the Oligosporogenea of
the Glugeidz family, which forms produce but one pansporoblast.

With the recent advances in the growth of tissues outside the
body, particularly the work of Carrel, Burrows, Harrison, Lewis and
Lewis, and Rous upon the infectiousness of tumor extracts, with
the results of Noguchi in cultivating pathogenic Treponema pal-
lidum, important discoveries concerning the Negri bodies will un-
doubtedly be made in the near future.

I wish to extend my thanks to Dr. Charles V. Chapin, Superin-
tendent of Health of the City of Providence, for the material used
in this work, and to Prof. F. P. Gorham, of Brown University, for
his valuable suggestions.

BIBLIOGRAPHY.

Babes, V., Ztschr. f. Hyg. u. Infectionsk., 1907, lvi, 435.

Calkins, G. N., Protozodlogy, Philadelphia and New York, 1900.

Fremi, C., Centralbl. f. Bacteriol., tte Abt., Orig., 1907, xliv, 502.

Frothingham, L., The History, Prevalence, and Prevention of Rabies and Its
Relation to Animal Experimentation, American Medical Association, De-
fence of Research Pamphlet, 1910, No. 7.

Hadley, P. B., Providence Med. Jour., 1907, viii, 25.

Lankester, E. R., A Treatise on Zodlogy, London, 1909, pt. 1.

Negri, A., Boll. d. Soc. med.-chir. di Pavia, 1903, 88; 1904, 22; 1905, 321; Speri-
mentale, 1904, lvili, 273; Ztschr. f. Hyg. u. Infectionsk., 1903, xliii, 507;
1903, xliv, 519.

Poor, D. W., Proc. N. Y. Path. Soc., 1904, iv, rot.

Ravenel, M. P., Bulletin of the Pennsylvania Department of Agriculture, 1901,
No. 79.

Reichel, J.. Am. Vet. Rev., 1910-11, xxxviii, 447.

Remlinger, P., Bull. Soc. cent. méd. et vet., 1907, lxxxiv, 269; Compt. rend. Soc.
de biol., 1907, \xii, 800.

Stefanescu, E., Compt. rend. Soc. de biol., 1907, |xii, 886.

Stimson, A. M., Bull. Hyg. Lab., U. S. P. H. and M.-H. S., 1910, No. 65.

Sime, D., Rabies,—Its Place amongst Germ-Diseases, and Its Origin in the
Animal Kingdom, Cambridge, 1903.

Williams, A. W., Woman’s Med. Jour., 1908, xviii, 113.

Williams, A. W., and Lowden, M. M., Jour. Infect. Dis., 1906, iii, 452

The Negri Bodies in Rabies.

EXPLANATION OF PLATES.

PLATE I.

Fic. 1. Two nerve cells from hippocampus major in a smear preparation.
. Deeper staining nuclei of the two nerve cells.
. A large Negri body of the sexual type and pansporoblast stage, without

a surrounding membrane and containing many individual spores.

C. Spores of the sexual type lying free in the protoplasm of the nerve cell.

D. A Negri body of the asexual type or multiplicative stage, containing
the characteristic chromatin granules.

E. A small sexual pansporoblast containing several spores.

F. A small sexual pansporoblast surrounded by a membrane and contain-
ing several spores.

Fic. 2. Two nerve cells in a smear preparation.

A and B. Asexual type of Negri bodies with chromatin granules.

C and D. Nucleus of one of the nerve cells.

E. Sexual type of Negri body containing spores.

F. Smaller sexual type of a Negri body containing spores.

G. Proliferating endothelial cell.

H. A small sexual type of a Negri body containing spores.

I. A large pansporoblast containing many spores which have begun to
leave the center of the Negri body preparatory to being dissemi-
nated through the protoplasm of the nerve cell. The membrane is
lacking.

J. Small sexual type of a Negri body with membrane and spores.

Fic. 3. A. A small asexual trophozoite with chromatin granules.

B. Nucleus of the nerve cell.

C. A small asexual type of body.

D. A large pansporoblast pressed out of the nerve cell, showing absence
of the membrane and the characteristic arrangement of the spores
about the periphery.

Fic. 4. A. Spores liberated from the pansporoblast and lying free in the pro-
toplasm of the nerve cell.

B. Nucleus of the cell.

ww >

PLATE 2.

Sexual and asexual life cycle.

Fics. 1 to 18. Asexual or multiplicative cycle of the Negri body, showing
the development from the spore and trophozoite through the various
division forms, all containing the characteristic chromatin granules.

Fics. i to xiii. Sexual or sporogonous cycle, showing the development from
the original spore to the pansporoblast with the surrounding mem-
brane and endogenous spores; later, without the membrane and
with the peripheral arrangement of the contained spores; and finally,
the disseminated spores throughout the protoplasm of the nerve cell.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVII. PLATE 1.

FIG. I. FIG. 2.

FIG. 3. FIG. 4.

(Watson: The Negri Bodies in Rabies.)

=
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7
a

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVII. PLATE 2.

(Watson: The Negri Bodies in Rabies.)

122

THE PRODUCTION OF ACID BY THE BACILLUS
COLI GROUP.
BY WILLIAM W. BROWNE.
Journal of Infectious Diseases, Vol. XV, No. 3, November 1914,
pp 580-604.

THE PRODUGTION-OF ACID BY THE
BACILEUS COEF GROUP

WiDLIAM, Wa BROW NE

Reprinted from
THE JOURNAL oF INFECTIOUS DISEASES, Vol. 15, No. 3, Nov. 1914, pp. 580-604

=
"

THE PRODUCTION OF ACID BY THE BACILLUS
COLI. GROUP

WILLIAM W. BROWNE

(From the Biological Laboratory of the Brown University, Providence, R. I.)

The year 1885 marks the beginning of the study of the bacteria of
the intestinal tract in relation to their action on the various carbohy-
drates. In that year, Buchner, working with his “Darmbacillus [&,”
which, in all probability, was a member of the bacillus coli group,
found that growth in a medium, consisting of meat infusion, peptone,
and sugar, was accompanied by the formation of acid and gas. The
acid was demonstrated by the addition of litmus to the medium. The
production of acid and gas, according to Buchner, was due to the
breaking down, or decomposition, of the sugar brought about by the
action of the bacteria. The evolved gas was found to be carbon dioxid,
and the acids were defined as members of the fatty acid series. The
work of Buchner may be considered as the starting point of all the
work done on the relation of bacteria to the decomposition of carbo-
hydrates with the production of acid and gas.

In connection with the production of acid in carbohydrate solutions
by bacteria, progress has been particularly active along two distinct
lines: (1) The differentiation of the colon group from the typhoid
group and other alkali producers, and (2) the classification of the
bacilius coli group, according to the ability of its members to ferment
the various carbohydrates.

Petruschky’s litmus whey medium, Beijerinck’s calcium carbonate medium,
Wurty’s litmus lactose agar, Kaufmann’s jequirity solution, and Hanna’s pro-
teid medium were important steps in the investigations along the former line.
Among the workers of this period, who have contributed to our knowledge of
carbohydrate fermentation, are Beginsky (1888), Lembke (1896), Von Som-
moruga (1892), Capaldi and Proskauer (1897), Ziellecvky (1902) and Segin
(1903). These results were augmented by Theobald Smith by use of the fer-
mentation tube.

Beginning with the work of Durham in 1900, nearly all the investigations
along the line of fermentation of carbohydrates by the bacillus coli group have
been done with the idea of classifying the members of the group by means of
their fermentative reactions on the various carbohydrates. The work of Dur-

4 WILLIAM W. BROWNE

ham has been extended and augmented by the notable researches of Houston
(1902), MacConkey (1905), Winslow and Walker (1907), Graham Smith (1909),
and Jackson (1911).

In spite of the importance of this fermentative reaction, however,
there are many of the finer points of technic which have never been
fully and conclusively worked out. Acid production, like all other
biochemical reactions, is a function of the reacting organism, the sub-
stance decomposed, the end products formed, the time, the tempera-
ture, and all conditions which may check or favor the vital process.
In the present investigation, ] have attempted to isolate these various
factors and study them, one by one, with the idea of gaining a clear
idea of the quantitative significance of each. No startlingly novel con-
clusions could be expected, but it is hoped that the data obtained may
furnish a surer basis for comparative studies of fermentative power
in carbohydrate media than has been available in the past.

ORGANISMS USED IN THE INVESTIGATION

The organisms used in this work were all members of the bacillus
coli group. Altho the identification of this group has been a subject
of dispute for over a quarter of a century, it is now generally conceded
that the following characteristics may be considered criteria of mem-
bership: (1) Short bacillus with rounded ends; (2) gram-negative;
(3) non-liquefaction of gelatin in 16 days; (4) fermentation of dex-
trose and lactose with the production of acid and gas; (5) non-spore-
forming; (6) facultative anaerobe; (7) gas in lactose-peptone-bile ;
(8) grayish white growth on agar at 20 C. and 37 C.

Other characteristic properties of this group, such as the reduction
of nitrates to nitrites, production of indol, motility, and style of growth
on various media, are valuable, if at all, merely for establishing minor
subdivisions.

The organisms used in this investigation all conformed to the
general characteristics of the bacillus coli group, as noted. In the
early part of the work, no particular attention was paid to the sub-
divisions of the group, and, as a result, organisms were used which
were identified only by the general group characteristics. As the work
developed and the number of carbohydrates in use increased, the sub-
divisions of the group began to manifest themselves. A clean-cut
differentiation into various species was obtainable by the production

Acip PropucTION BY COLI GROUP 5

or non-production of acid in the carbohydrate solutions. As the writer
had no dulcite at hand, a subdivision of the group into species, as
recommended by Jackson, was impossible, and, as a result, all the
strains used throughout the work have been designated as the bacillus
coli, altho it is certain that a number of species were present. As far
as possible, those organisms which gave similar fermentative reactions
have been grouped in the same table.

The cultures were obtained from two sources; either from oysters grown in
contaminated water, or directly from feces. The oysters, from which the
organisms were isolated, were taken from 242 different stations in Narragansett
Bay during an investigation to determine the amount of pollution of the oyster-
beds of the State of Rhode Island. The area from which the oysters were
taken included localities, varying from those extremely polluted near sewer out-
lets to the less polluted areas, which were sometimes coli-positive and other
times coli-negative.

One series of the cultures, isolated from feces, was obtained from normal
stools of people in and about the laboratory at Brown University, while the
other series was obtained from the stools of Italian immigrants, quarantined
aboard the S. S. Roma, who were undergoing examinations for the cholera
vibrio. These two series were kept entirely distinct and will be so designated
throughout the work.

MeEtTHops

Preparation of Culture Media—The media were prepared after the methods
proposed in the Report of the Committee on Standard Methods of Water Anal-
ysis to the Laboratory Section of the American Public Health Association, Jan-
uary 5, 1905, Liebig’s meat extract was used, 3 grams to the liter. Ease of
preparation rendered it more suitable for the work of this kind than a medium
made up with meat. Great care was taken in the preparation of the media to
make all the lots as uniform as possible. For this reason, all the constituents
were taken from the same lot of materials during the entire course of the
experiments.

The carbohydrate solutions were made by the addition of 1 percent of the
various carbohydrates to neutral nutrient broth. Twenty-five cubic centimeters
of the broth were placed in regular laboratory test tubes of large size, and
sterilized on three successive days in streaming steam at 100 C.

During the course of the experiments, the production of acid was deter-
mined in the following carbohydrates :

I. I. Monosaccharids:
A. Hexoses.
1. Dextrose (Merck).
2. Galactose (Merck).
3. Levulose (Kahlbaum).
B. Pentoses.
1. Arabinose (Kahlbaum).
2. Xylose (Kahlbaum).

6 WILLIAM W. BROWNE

II. Disaccharids:

1. Lactose (Merck).

2. Maltose (Merck).

3. Saccharose (Merck).
ILI. Trisaccharid:

1. Raffinose (Kahlbaum).
IV. Hexatomic alcohols:

1. Iso-dulcite (Kahlbaum).

2. Mannite (Merck).

All cultures used in the determination of the production of acid in the carbo-
hydrate solutions were grown at 37 C. for 24 hours, unless otherwise stated.
The titrations were made with N/20 sodium hydroxid at the twenty-fourth
hour, and results tabulated in direct percentages of normal sodium hydroxid.

Method of determining amount of acid produced—From a twenty-four-
hour agar slant culture of the organism, which had been identified as a mem-
ber of the bacillus coli group, inoculations were made into peptone solutions,
which were incubated for 24 hours at 37 C. At the end of 24 hours, the tubes
of carbohydrate solution were inoculated by the addition of 0.5 c.c. of the twenty-
four-hour peptone culture. By the addition of a definite amount of the twenty-
four-hour culture, more consistent results were obtained than by the direct
inoculation from the agar slant culture.

The inoculated carbohydrate solutions were incubated for 24 hours and
were then ready for titration. The cultures were titrated as soon as possible
after their removal from the incubator."

Five cubic centimeters of the culture and 45 c.c. of distilled water were
placed in a caserole and boiled briskly for one minute. One cubic centimeter
of phenolphthalien, which consisted of 5 gm. of the commercial salt dissolved
in one liter of 50 percent alcohol, was added as an indicator. Titrations were
made into the hot solution until a faint, but permanent, pink color was obtained
with N/20 sodium hydroxid solution.

Tue RELATION OF TEMPERATURE TO THE AMOUNT OF AcrID PRODUCED

Twenty-five cubic centimeters of the various sterilized carbohydrate solu-
tions were inoculated with 0.5 c.c. of a twenty-four-hour peptone culture of the
identified organisms. The cultures and their controls were kept for 24 hours
at the following temperatures: Ice-box temperature, 3 C.; room temperature,
16 C.; incubator temperature, 28 C.; incubator temperature, 37 C.; incubator
temperature, 45 C.

At the end of the twenty-fourth hour the cultures and their controls were
titrated as soon as accuracy would allow. All titrations were made with N/20
sodium hydroxid, and all results are expressed in direct percentages of normal
sodium hydroxid.

The results obtained in these experiments show that cultures of
the bacillus coli group, grown at a temperature of 37 C., tend to pro-
duce more acid in carbohydrate solutions, in a given time, than cultures

grown at other temperatures. Cultures grown at 3 C. show almost no
acid production in 24 hours. As we approach 37 C., either from below

1. The method of titration followed was the one outlined in Standard Methods of Water
Analysis, 1905, p. 106.

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or above, the amount of acid increases until the maximum amount of
acid is produced at 37 C. We should expect this, since 37 C. is the
optimum temperature for the growth of the bacillus coli group. If the
temperature is too low or too high, we get less growth, and since acid
production is dependent on the growth of the organism, we must surely
find the optimum temperature for the production of acid identical with
the optimum temperature for growth. This was found to be true in
all the carbohydrate solutions used.

THE RELATION OF TIME TO THE AMOUNT OF ACID PRODUCED IN VARIOUS
CARBOHYDRATE MEDIA

The general method of these experiments consisted in the inoculation of a
large number of tubes of the 1 percent carbohydrate media with various mem-
bers of the coli group. At definite intervals, a certain number of the tubes were
removed from the incubator and titrated with N/20 sodium hydroxid. These
experiments include 10 carbohydrates using various members of the bacillus
coli group. Fifteen tubes of the 10 carbohydrates noted below, each contain-
ing 25 c.c. of the medium, were inoculated with 0.5 cc. of a twenty-four-hour
peptone culture of the bacillus coli. The tubes were incubated at 37 C., and
were removed from the incubator and titrated at intervals of three hours.

From the results obtained in these experiments it is seen that, by
the end of the twenty-fourth hour, the members of the bacillus coli
group produce their maximum amount of acid when grown at 37 C.,
after the inoculation of a sugar medium with 0.5 c.c. of a twenty-four-
hour peptone culture. In the case of some carbohydrates, such as
dextrose or lactose, the maximum production of acid occurs in the
eighteenth hour. The organisms used in this experiment produced
their maximum amount of acid either before or at the twenty-fourth
hour. Cultures, which were grown for 48 and 72 hours at 37 C.,,
showed no increase over the amount of acid produced at the end of
the twenty-fourth hour. The maximum amount of acid was produced
by the organisms by the end of the twenty-fourth hour.

THE RELATION OF THE AMOUNT OF ACID PRODUCED TO THE AMOUNT OF
MEDIUM INOCULATED
Erlenmeyer flasks, containing 25, 50, 100, 200, 300, 400, and 500 c.c.
of dextrose and lactose broths, were inoculated with 0.5 c.c. of a
twenty-four-hour culture of the coli bacillus. The flasks were incu-
bated for 24 hours at 37 C., and then titrated with N/20 sodium
hydroxid.

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12 WILLIAM W. BROWNE

The results obtained show that no matter how much carbohydrate
media is inoculated, up to 500 c.c., the same percentage of acidity is
produced by the bacillus coli when grown at 37 C. for 24 hours after
inoculation with 0.5 c.c. of a twenty-four-hour peptone culture.

THE RELATION OF THE CONCENTRATION OF THE CARBOHYDRATE MEDIUM
TO THE AMOUNT OF ACID PRODUCED

Tubes of neutral nutrient broth, to which were added various per-
centages of dextrose and lactose, were inoculated with 0.5 c.c. of a
twenty-four-hour peptone culture of the bacillus coli. The tubes were
incubated for 24 hours at 37 C., and at the twenty-fourth hour the
tubes were titrated with N/20 sodium hydroxid. .

TABLE 5

AMOUNT OF ACID, IN PERCENT NORMAL, PRoDUCED BY THE BaciLLus Cort IsoLaTED FROM
Feces, In NEuTRAL BrotH CONTAINING VARYING PERCENTAGES OF DEXTROSE AND LACTOSE

Amount of Acid Amount of Acid

Percentage Percentage
of Dextrose Produced of Lactose Produced

0.125 0.4 0.125 0.35
0.25 0.5 0.25 0.5
0.5 201 0.5 y/
0.1 2.2 1 1.8
1.5 2 155 1.8
2 2n3, 2 1.8
2.5 272 2.5 1.75
3 2.4 3 1.8
3.5 223 3.5 1.8
4 Be) 4 1.8
4.5 2.3 4.5 1.8
5 2.4 5 1.8
ile) 2:35 Hes 1E7,

10 AS) 10 1.8

15 2.3

20 223

25 253

30 1.8

35 0.9

40 0.8

45 0.6

50 0.3

The results are the average of the titrations of two cultures.
Temperature at 37 C
From these experiments it appears that the concentration of the
carbohydrates (dextrose and lactose) has little effect on the amount of
acid produced, within certain limits, by the bacillus coli. Between
1 percent and 25 percent concentration, the amount of acid produced
is nearly constant. Below 1 percent and above 25 percent there is less
acid produced. A 1 percent solution gives the maximum amount of
acid from the least amount of the carbohydrate.

*

Actp PRODUCTION BY COLI GROUP 13

THE RELATION OF THE INITIAL REACTION OF THE CARBOHYDRATE
MEDIUM TO THE AMOUNT OF ACID PRODUCED

The purpose of these experiments was to determine whether or not
the initial reaction of the carbohydrate medium had any influence upon
the production of acid by the organisms.

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Time in Hovurs

Chart 3.—The relation of time to the amount of acid produced by the bacillus coli,
isolated from feces.

Tubes of dextrose broth, to which varying amounts of sterile acid and
alkali were added after sterilization, were inoculated with 0.5 c.c. of a twenty-
four-hour peptone culture of the bacillus coli. These tubes were incubated
for 24 hours at 37 C. At the end of 24 hours the cultures were titrated with
N/20 sodium hydroxid.

The amount of acid produced by the bacillus coli group, in various
carbohydrate media, depends, in great part, upon the initial reaction of
the medium. The maximum acid production of an organism is the

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Acip PropuCcTION By CoLI GROUP 15

amount of acid necessary to prevent further growth. The farther
away the initial reaction is from the maximum acid production, the
more acid an organism can produce until it reaches the maximum.
The limiting acidity of the bacillus coli group is about 2.4 percent
normal sodium hydroxid. A medium with a slightly acid reaction
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Chart 4.—The relation of time to the amount of acid produced by the bacillus coli,
isolated from oysters.

with a neutral or slightly alkaline reaction. Hence, in a medium which
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medium which is slightly acid, but in the end they will both reach the
same goal— the maximum acidity of the organism. No acid is pro-
. duced in a medium with over 2.4 percent acidity. The limit of alka-
linity from which the organisms will produce acidity, was not deter-

mined.

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Acip PropucTion By CoLt GRrouP 17

Acrip PropucTION IN A MeEpDIuM PERIODICALLY NEUTRALIZED TO REMOVE THE
Acips ForMED

Erlenmeyer flasks containing 100 c.c. of the various carbohydrates (1 percent
concentration) were inoculated with 0.5 cc. of a twenty-four-hour peptone
culture of the different members of the bacillus coli group. Before inoculations
were made, 0.5 cc. of phenolphthalein (5 gm. of the commercial salt to one
liter of 5 percent alcohol) was added as an indicator. The flasks were incu-
bated at 37 C. and at intervals of 24 hours, the flasks were removed and
sterile normal sodium hydroxid was added from a burette, until a faint pink
color was obtained. The cultures were replaced in the incubator and the process
was repeated at the end of every 24 hours, until no more acid was produced
by the organisms.

Table 8 shows that the maximum acid production of 24 hours,
which is, as previously proved, the greatest amount which the organism
can produce at any one time, is limited by the excess of acid formed,
and can be greatly increased by periodical neutralization.

The maximum amount of acid, which the bacillus coli group is able
to produce in 1 percent carbohydrate solutions in 24 hours can be
greatly increased if the excess of acid formed is neutralized. In all
carbohydrates of 1 percent concentration, the total acid production is
from 3 to 6 times as large as the maximum twenty-four-hour produc-
tion which, as previously proved, is the same for 48 and 72 hours under
ordinary conditions. From these experiments it may be assumed that
acid production goes on until a maximum is reached (24 hours) and
then ceases until some of the acid present is neutralized, when acid is
again produced until the maximum is reached. This same thing occurs
until all the carbohydrates are used. None of the cultures on the
twelfth day gave a positive reaction for sugar, showing that the carbo-
hydrates had been used entirely by the organisms in the production of
acid. The total amount of acid an organism is able to produce, if the
excess acid is neutralized, depends, in a large part, on how much fer-
mentable carbohydrate there is present in the medium. The maximum
twenty-four-hour production on the other hand, will use only that
amount which is necessary to produce the maximum amount of acid
which can be tolerated.

THE AMOUNT OF ACID PRODUCED IN VARIOUS CARBOHYDRATES

An attempt has been made to draw a comparison between the
various carbohydrates by the amount of acid produced from them by
different members of the bacillus coli group.

?

BROWNI

WILLIAM W.

Tr-
SAcCHARIDE

DisaccHaRiogs

HEXITES

MonosaccHARI DES

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14

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Acip PropuUCTION BY COLI GROUP 19

Tubes containing 25 c.c., of the various carbohydrates, were inocu-
lated with 0.5 c.c. of a twenty-four-hour culture of the different mem-
bers of the bacillus coli group. After 24 hours incubation at 37 C., the
cultures were titrated with N/20 sodium hydroxid. In each case
strains were selected which could ferment the carbohyrate in question.

Tables 9 and 10 show that the colon bacillus, whether isolated from
feees or oysters, produces the maximum amount of acid inthe mono-
saccharids, less in the disaccharids, and the least in the trisaccharid.

The results obtained in this series of experiments show that the
various members of the bacillus coli group are able to produce more
acid in solutions containing carbohydrates of simple chemical structure
than in solutions containing carbohydrates of complex chemical struc-
ture. For instance, more acid is produced in solutions containing the
monosaccharids and hexites, such as dextrose, levulose, galactose,
xylose, arabinose, mannite, and isodulcite than in solutions containing
the disaccharids, lactose, maltose, and saccharose. The disaccharids
are, in turn, more easily fermented by the bacillus coli group than the
trisaccharid raffinose.

The polysaccharid starch is not fermented by any of the members
of the coli group. The trisaccharid raffinose, while not so complex a
molecule as starch, appears to offer chemically greater difficulties to
the organisms in the carrying out of their oxidative processes in the
formation of the various acids from the sugars than the organisms
would encounter when fermenting, for instance, a disaccharid. In
fact, there are members of this group which are unable to ferment
raffinose at all.

The arrangement of the carbohydrates, according to the amounts
of acid which the members of the bacillus coli group are able to pro-
duce from them, agrees somewhat with an arrangement made accord-
ing to their chemical complexity of structure, as may be seen by the
following:

Monosaccharids, 2.03, 1.82.

Disaccharids, 1.74, 1.61.

Trisaccharid, 1.56, 1.38.

Polysaccharid (Starch), 0, 0.

Among the monosaccharids, the hexoses, dextrose, and levulose,
seem to be most easily oxidized by the organism with the formation of
acid. No distinct difference could be noted in the amounts of acid
produced in the levulose and dextrose by the methods used in this

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Acip PropuctTion By CoLt GROUP

investigation. The amount of acid produced for these sugars also was
very constant. Galactose, while not forming so much acid as dextrose
and levulose, showed a greater variance in the amount of acid pro-
duced. All organisms investigated produced acid in the monosacchar-

ids mentioned.

TABLE 10

21

Acrp, In Percent NorMAL, PropuceD IN VARIOUS CARBOHYDRATES BY BacriLLus Cot, ISOLATED FROM OYSTERS

Cultures
Carbohydrates
1 2 3 4 5 6 Average
Mlextrose ..........- a 1.9 2.0 2.2 <2 ie
Levulose 1 1.6 1.6 2.2 2.1 ea
Galactose Bi 1.9 : 2.0 1.9 1.8 ee
Arabinose ae ioe . 1.8 1.9 1.5 oe
1.8 cm 1.8 1.8
16 1.8 Ae, 1.85
1.6 n bape n be} 1.63
1.82
1.8 1.3 1.66
WG 73 1.61
18 1.6 1.56
1.61
ee te ee id ee ie et ss eee
| 1.2 1.4 1.38
Average of Trisaccharid. 128
Tables 9 and 10 show that the colon bacillus, whether isolated from feces or oysters, produces the maximum

amount of acid in the monosaccharids, less in the disaccharids, and least in the trisaccharid.

The pentoses, xylose and arabinose, seem to offer greater resist-
ance to the bacterial oxidation processes, as we find less acid produced
from the pentoses than from the hexoses. Throughout the work, these
two sugars behaved exactly alike, and no differentiation was possible by

the amount of acid produced from them by the group.

The hexites, mannite and isodulcite, showed less acid than the
hexoses, and at times the amount of acid produced from them exceeded
that produced from the pentoses. Many strains of the bacillus coli
group were found, which were unable to ferment mannite and iso-

dulcite.

Pap WILLIAM W. BROWNE

Of the three disaccharids used, saccharose seems to offer greater
resistance to the oxidative processes of the colon group than either
lactose or maltose. In fact, many members of the group are unable to
ferment it. This seems to be in accord with the chemical structure of
the sugar, since we know that saccharose is the only one of the three
mentioned disaccharids which cannot be oxidized. At times, the
amount of acid produced in lactose and maltose equals or even exceeds
the amount of acid produced in galactose, a monosaccharid.

The more complicated raffinose offers great difficulties to the fer-
mentative processes of the bacillus coli group. Some strains failed to
produce any acid at all from this sugar. As already mentioned, starch
was not fermented by any strains used in this investigation.

It should not be understood, of course, that the size of the molecule
is the only factor involved, for its configuration is also important.
Winslow and Walker (1907) have shown that bacilli of the colon
group, which ferment saccharose, usually ferment raffinose as well.
The same thing appears in my results as to the amount of acid formed,
saccharose results being lower than lactose results tho not quite so low
as those obtained in raffinose.

There is an important distinction to be drawn between the power
to ferment a given sugar and the amount of acid formed when it is
fermented. In my work I have used only strains capable of attacking
the sugar in question. When a low result is obtained it may be due to
a slow action upon the sugar, or, in view of the evidence that it is the
amount of end product formed which usually stops the reaction, it
may be that the lower acidity produced in more complex sugars is the
result of some other decomposition products, which accompany the
acids, and, in connection with them, are able to inhibit growth.

A COMPARISON BETWEEN THE AMOUNT OF ACID PRODUCED BY VARIOUS
MEMBERS OF THE BACILLUS COLI GROUP ISOLATED FROM
DIFFERENT SOURCES

In this series of experiments, a comparison has been made between
the amounts of acid produced in various carbohydrates by the different
members of the bacillus coli isolated from three distinct sources: (1)
from stools of healthy individuals in and about the laboratory; (2)
from stools of Italian immigrants quarantined on board the S. S. Roma;
(3) from oysters taken from different locations in Narragansett Bay,
representing areas of widely diverse characters.

sb bien

Numser oF Strains

Acip PrRopDUCTION By COLI GROUP 23

The organisms were inoculated into tubes of peptone broth which
were incubated at 37 C. for 24 hours. The various carbohydrates
were inoculated by the addition of 0.5 c.c. of this twenty-four-hour
culture. At the end of 24 hours’ incubation at 37 C. the cultures were
titrated with N/20 sodium hydroxid.

zo

———————————————
A=B.covi From OYSTERS
B=B cov: FROM LaBoRATORY AssiSTANTs
C =B cou: FROM ITALIAN IMMIGRANTS

eo

Acio Proouction iw Percentaces oF Nokmar Sodium Hv OROXI02

Chart 6.—The amount of acid produced by the bacillus coli in dextrose (upper curves)
and lactose broth (lower curves).

The results obtained in this series of experiments seem to show
that the source from which the members of the bacillus coli group
were isolated has a direct effect upon the ability of the organisms to
ferment carbohydrates with the production of acid.

24 WILLIAM W. BROWNE

A comparison between the amounts of acid produced in lactose and
dextrose broths by members of the bacillus coli group gives the follow-
ing results:

From Laboratory From From

Assistants Immigrants Oysters
Percentage in dextrose ...... 2.28 2.06 1.89
Percentage in lactose ........ 1.79 75 L772

The bacillus coli isolated from feces, both from laboratory assist-
ants and from the immigrants of the S. S. Roma, produced more acid
in dextrose and lactose broth than the colon bacillus isolated from
oysters. This seems to indicate that bacillus coli loses some of its
ability to ferment carbohydrate with the production of acid during the
journey from the intestinal tract to the oysters. Bacillus coli, isolated
from the stools of laboratory assistants, produced more acid in dex-
trose and lactose broths than similar organisms isolated from the stools
of Italian immigrants. The significance of this difference in the ability
of the organisms, isolated from the above-named sources, to produce
acid from carbohydrates means, is impossible to explain except that
the general character of the diet may have had some effect on the
ability of the organism to ferment carbohydrates with the production
of acid.

EFFECT OF IMMERSION IN SEA WATER ON ABILITY TO PRODUCE ACID

The purpose of this experiment was to determine if the members
of the bacillus coli group, after being kept for various periods in sea
water, were affected in regard to their ability to produce acid in various
carbohydrates.

In these experiments bottles of sea water, which gave two negative
tests for the presence of the members of the bacillus coli group with
lactose peptone bile, were inoculated with known cultures of the coli
group and kept at the following temperatures: ice-box, 4 C.; room,
ZONE

At intervals of a week, portions of the sea water were inoculated
into lactose peptone bile tubes, from which the members of the bacillus
coli group were isolated and identified. From twenty-four-hour pep-
tone cultures of these organisms, inoculations were made into the
various carbohydrate media and incubated 24 hours at 37 C. At the
end of that period titrations were made with N/20 sodium hydroxid.

Acip PRODUCTION By COLI GROUP

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26 WILLIAM W. BROWNE

These experiments show that the various members of the bacillus
coli group, after remaining for 8 weeks in bottles of sea water kept at
4+ C. and 20 C., were able to produce the same amount of acid in the
various carbohydrates as when first added.

SUMMARY

The optimum temperature for the maximum production of acid in
24 hours by the members of the bacillus coli group, in a medium con-
taining a fermentable carbohydrate, is 37 C. Acid production in this
time is almost nil at 3 C., rises rapidly to 37 C., and falls as rapidly
above that point, ceasing between 50 C. and 60 C.

Twenty-four hours’ incubation at 37 C. is a sufficient period for the
maximum production of acid by members of the coli group in a
medium containing a fermentable carbohydrate, when 0.5 c.c. of a
twenty-four-hour peptone culture is used as an inoculum. Under these
conditions no further increase in acidity occurs after 20 hours.

The amount of medium, up to 500 c.c., inoculated with the members
of the coli group, has no effect upon the percentage of acidity produced
by the organisms, when incubated at 37 C. for 24 hours after inocula-
tion with 0.5 c.c. of a twenty-four-hour peptone culture.

A medium containing 1 percent of fermentable carbohydrate offers
a suitable amount of carbohydrate for the maximum acid production
by members of the coli group. A medium of much less than 1 percent
concentration does not contain enough carbohydrate to bring about the
maximum reaction, while a medium of more than 1 percent concentra-
tion serves no useful purpose. A high concentration of carbohydrates
(over 25 percent) will not only hinder but will prevent the production
of acid, but between 1 and 25 percent the twenty-four-hour acid pro-
duction is the same.

The more distant the initial reaction of the medium is from the
maximum acidity necessary to inhibit production of acid, the greater
amount of acid the organism can produce before that maximum is
reached. The extreme limit of alkalinity, in which the organism will
produce acid, was not determined.

The maximum amount of acid, which bacilli of the colon group will
produce in ordinary carbohydrate media, is fixed by the tolerance of
the bacteria to the acid itself. If the acid formed is neutralized daily
by the addition of free alkaii, the acid production will go on until all
the carbohydrate present has been consumed. With 1 percent carbo-

a tee

Acip PropucTION By COLI GROUP 27

hydrate in the medium, there will be 4 or 5 times as much acid formed
as ordinarily.

The members of the coli group produce the greatest amount of acid
in media containing the monosaccharids and hexites, dextrose, levu-
lose, galactose, arabinose, xylose, mannite, and isodulcite; less in the
disaccharids, maltose, lactose, and saccharose; and least in the tra-
saccharid, raffinose.

Members of the bacillus coli group, isolated from feces, produce
more acid in media containing fermentable carbohydrates than strains
of the bacillus coli group, isolated from oysters taken from different
portions of Narragansett Bay. Of the members of the bacillus coli
group isolated from feces, strains which were obtained from the stools
of laboratory assistants produced more acid in media containing fer-
mentable carbohydrates than strains isolated from the stools of Italian
immigrants quarantined aboard the S. S. Roma.

The amount of acid produced in various carbohydrate media by the
bacilli of the colon group is unaffected by storage in unsterilized sea
water for a period of 8 weeks at temperatures of 20 C. and 40 C.

123

ON THE CLASSIFICATION OF STREPTOCOCCI.

OBSERVATIONS ON HEMOLYSIN PRODUCTION
BY THE STREPTOCOCCI.
Reprints Not Available.
BY HAROLD W. LYALL.
Journal of Medical Research. Vol. XXX, pp 487-532.

124

THE STRUCTURE OF THE COTTON FIBRE.
BY BENJAMIN §S. LEVINE.
Science,*N. S., Vol. XL, No. 1042, p 906, December 18, 1914.

[ Reprinted from SciEncE, N. S., Vol. XL., No. 1042,
Page 906, December 18, 1914]

THE STRUCTURE OF THE COTTON FIBER

In any kind of cotton the typical fiber, that
is the one in which all the essential parts may
be determined, can be found in rare cases. For
this reason the structure of an ideal fiber can
be inferred only from a series of studies of
fibers in successive stages of development.

By subjecting such fibers to certain chem-
ical and bacteriological treatments and then
studying them under the microscope, we found
that the typical cotton fiber consists of the
following parts:

1. The outer layer or the integument.
2. The outer cellulose layer.

3. The layer of secondary deposits.

4. The walls of the lumen.

5. The substance in the lumen.

1. The outer layer or the integument is the
incrusting layer and forms the cementing
material of the fiber. Its chemical structure
is not an homologous one, but is a mixture of
components, some soluble in alcohol, some in
ether, and some in water. The components are
cutinous, pectinous, gummy, fatty and other
unidentified bodies,

2. The outer cellulose layer is in its struc-
ture a distinct spiral, consisting of a limited
number of component fibers, perhaps of one or
of two. The structure of this layer is deter-
mined under the microscope from a longi-
tudinal section of the fiber after the latter has
been subjected to a series of chemica] and
bacteriological treatments. Careful treatment
of some of the fibers by cuprammonia will
show under the microscope this spiral. There

2

is some evidence to show that this spiral con-
sists of impure cellulose.

3. The layer of secondary deposits seems to
be made up of component fibers which in no
ease have shown a spiral structure. Unlike
the fibers of the above described layer, these
components are from about five to ten in num-
ber and run with some irregularities along
the length of the fiber.

4, The structure of the layer forming the
walls of the lumen is a spiral much the same
as the outer spiral, but differs from it greatly
in its chemical composition. This is deter-
mined from a microscopical study of the fiber
while under a cuprammonia treatment.

5. The substance in the lumen is structure-
less and, as is proven by a microscopical test,
is of a nitrogenous nature.

B. S. Levine

THE BIOLOGICAL LABORATORY,
BrowN UNIVERSITY

125

THE EFFECT OF WATER-GAS TAR ON OYSTERS.
BY PHILIP H. MITCHELL.

From Bulletin of the United States Bureau of Fisheries, Vol. XXXII,
1912. Document 786. Issued February 18, 1914.

‘

FROM BULLETIN OF THE UNITED STATES BUREAU OF FISHERIES : VOLUME XXXII, 1912
Document 786 - : é , é : :

Issued February 18, 1914,

THE EFFECT OF WATER-GAS TAR ON OYSTERS
&

By Philip H. Mitchell

15203°—14

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THE EFFECT OF WATER-GAS TAR ON OYSTERS.

&
By PHILIP H. MITCHELL.

&

For a number of reasons it has become desirable to know the effects of oily and
tar-like wastes on marine life of economic importance. Damages have been claimed
for pollution of oyster beds by wastes from the manufacture of gas. A report to the
Rhode Island shellfish commission has attributed to water-gas tar harmful effects to
oysters in Narragansett Bay.? Tar of various sorts is used for coating piles or stakes,
which might be in proximity to shellfish. Oily wastes are constantly escaping from
passing craft in inland waters.

The present investigation does not cover the entire subject, but is confined to one
important phase—the effect of water-gas tar on oysters. The experiments made
during the summer of 1912 at the laboratory of the Bureau of Fisheries at Woods Hole
could not then be carried further.

The tar was obtained from the separator at the works of the Providence Gas Co.
on October 25, 1910, and was a mixture of the heavier and lighter tars as obtained
in the manufacture of water gas, using an average temperature of 1,450° F. An analysis
of tar taken under comparable conditions from the same separator at another time
showed, according to the records of the Providence Gas Co., the following analysis:

Per cent

Specific gravity at 65.5° F., 1. 050. by volume.
WV AEC td sos Serine tts esis PIAS didss tae Tere oan eee aya hee = cre ae Ge
| REEL CHIRAL ROG SE areata Shinn 5 0 2 vi ta eS eae Pand «eines aie ae Se
; DEN atoh al tay Kove hae! aut. eer noaeltnn Ae eo tt OE AR tet Ne Sole ala ON, oA Lit.
Mediums pitchy7oo. 25: T2928. 32). DRE ae ee Sea. DOSE. Bases 36. 20
. Bree entbonu:) Fyscyreh cbse Sette ae). ae. Pees er eee tis. Sgety. REE “2270

_ Three series of experiments were made. In the first, oysters were exposed to

_ water-gas tar in stagnant sea water; in the second series they were exposed to the

_ tar in running sea water; while in the third small amounts of water-gas tar were intro-
duced inside the shells of oysters.

SERIES I.

Experiment 1.—An oyster, marked with a file for identification, as were ail the
oysters used in these experiments, and weighing 83.2 grams, was put into a battery
jar with 4o c. c. of water-gas tar and 2,500 c. c. of sea water. On the following day
the water was changed by the method used in all the experiments of this series. A
siphon delivered sea water from an aquarium to a point about 2 inches from the bottom
of the jar, while another siphon at the same time drew off water from the middle of the
jar. Water was allowed to run thus 10 to 15 minutes. As the tar stuck to the sides

@ Field, G. W. In Annual Report of the Commissioners of Shell Fisheries, Rhode Island, 1906, appendix D, p. 46-64.

201

202 BULLETIN OF THE BUREAU OF FISHERIES.

and bottom of the jar, or, in the case of the lighter more oily portions, floated on the
surface of the water, none, or very little of it, was removed while the water flowed through
the siphons. The water, therefore, was almost completely changed without removing
the tar. Shortly after the water had been changed the oyster was seen to be slightly
open, but could close at the slightest jar. The water was changed in this manner once
or twice daily for 10 days, The oyster was often seen partly open during this time,
but would always respond to a mechanical stimulus by closing. After 10 days the
water was no longer changed, but was left entirely stagnant. At the end of two weeks
under these conditions the oyster seemed to be affected. It now remained continuously
open, and appeared unable to close when stimulated. A few days later it showed signs
of putrefactive disintegration.

Expervment 2.—An oyster weighing 63.7 grams was treated exactly as in experi-
ment 1. The results were quite the same, except that when the water was left entirely
stagnant disintegration set in after five days.

Experiment 3.—An oyster weighing 71.6 grams was treated as in the preceding
experiments, except that the tar was smeared all over the inside of the jar and on the
oysters. After 24 hours the oyster was found wide open and unable to close when
stimulated. It was removed from the jar, and was seen to have its mantle greatly
retracted. It would spring open when closed by hand. It was washed and put into an
aquarium in running sea water. Two days later it appeared to be entirely recovered,
as it would close normally when stimulated. It was then put back into the jar of sea
water and tar in which it had been at first. It was now kept in this jar during six days,
with the water changed once or twice daily. At the end of this time it had again lost
the ability to close normally, but when put in the aquarium once more it again appar-
ently recovered. About three weeks later, however, it became disintegrated.

Expervment 4.—An oyster weighing 95 grams was arranged exactly as in experi-
ment 3. The results were quite the same, that is, after 24 hours it refused to close,
but recovered when put in the aquarium of running sea water. Some weeks later,
however, it died. That its death was due to the tar is not certain, because at that time
other oysters in the same aquarium died without any previous exposure to tar.

Experiment 5.—An oyster weighing 78.7 grams was treated exactly as in the preced-
ing experiment. It, too, became unable to close after 24 hours, and when put in run-
ning sea water entirely recovered. At the end of the summer, nine weeks later, it
seemed entirely normal and had a normal appearance when opened.

Experiment 6.—An oyster weighing 62 grams was treated exactly as in the preced-
ing experiment. The result was slightly different in that the oyster did not begin to
show a tendency to remain open until after three days, and became entirely unable to
close after five days in the tarry water with daily changing of the water. It was then
put in running sea water and began to disintegrate a few days later.

Experiment 7.—An oyster weighing 70.8 grams was put into a battery jar with 20
c. c. of the tar not in contact with it and 2,500 c. c. of sea water. The water was changed
daily during the next 10 days. It was then left stagnant during 8 weeks. The oyster
was sometimes observed to be open, but would then close if jarred. At the end of that

i

EFFECT OF WATER-GAS TAR ON OYSTERS. . 203

time it was cleaned and dried and found to weigh 71.6 grams. It had formed new shell
all around the edge. Opened it gave no smell of tar, the heart was beating and the
mantle was normally sensitive to mechanical stimuli. Part of the heart and portions
of the gills were discolored.

Control experiment.—An oyster weighing 72.4 grams was put in a battery jar with
2,500 c. c. of sea water. The water was changed during the next 10 days as in the pre-
ceding experiment and was then left stagnant during 8 weeks. Examination then
showed no noticeable new shell, but the heart, gills, and mantle were quite normal.

The experiments of this series indicate that when considerable quantities of
water-gas tar are in intimate contact with oysters in stagnant water serious or fatal
effects are produced. Under these circumstances the oyster can not use its natural
defense against a relatively or entirely insoluble substance. When the water is stag-
nant, there is little opportunity to eject such substances and free the organism from
them. As will be shown later, the oyster can rid itself of water-gas tar when in running
sea water. When the tar can not be ejected it seems to produce an effect similar to
paralysis, so that the initial symptom is a failure of the adductor muscle to respond
to stimulation of the sensory nerves. No conclusions as to the structures specifically
affected can be drawn from these experiments. Whether the fatal effects produced in
five of the above experiments were due to a direct toxic effect of water-gas tar, or to
some indirect effect also, does not appear from these experiments.

SERIES Il.

, Method.—Two oysters were put in each of four battery jars. Each jar was arranged
_ with two siphons, one bringing sea water from an aquarium to the jar with the lower
end of the siphon about 2 inches below the level of water maintained by the other siphon,

which carried water from the jar to a sink. The running water therefore tended to
carry off the light floating oils but left the heavier tar sticking to the bottom and sides
of the jar. The siphons were so arranged that each jar contained constantly about
2,500 c. c. of sea water. Into each jar there were put 30 c. c. of water-gas tar mixed
with sand and thoroughly smeared over the bottom and sides of the jar and on the
shells of the two oysters. From time to time during the following weeks small amounts
of tar were added to replace that carried away by the siphons. After remaining in
the jars as described during nine weeks the oysters were cleaned, weighed, and examined.
Comparison of their weights at the beginning and at the end of the experiments is given
in tabular form:

|
| Jar | Initial | Final
: weight. weight. |
ei |
{
| Grams. Grams
ds 98. 0 99-9
1 47-8 47-8
{ 92.0 92.90
52.4 52-3
¥ { 96. 8 94-0
| 61.8 62
i { IOI. I 100. 7
45-2 44-3

204 BULLETIN OF THE BUREAU OF FISHERIES.

That these variations in weight have no special significance is indicated by com-
parison with the variations in the weights of oysters kept in the aquarium during the
summer but not exposed to tar.

Initial Weight after
weight. five weeks.
Grams. Grams.
66. 5 66.0
76.0 70.5
| 244.0 243-0 |
147-0 I5t-0 |
|

When opened all were found to be normal in appearance and function. No odor
of tar was detectable in the shell contents of the oysters, although an abundance of tar
was left in the jars at the end of the experiment. Smears of.tar were also still present
on the outside of the oyster shells.

These experiments indicate that even intimate contact with water-gas tar does not
injure oysters in the course of nine weeks, provided facilities for defense in the form of
moving water frequently renewed are available.

SERIES III.

Experiment 1.—A small oyster was first pried open and injected with 0.5 c.c. of water-
gas tar. It was then put in a jar of running sea water. It remained tightly closed
during the next two hours. On the next day it was found to be quite normal. It was
open and apparently feeding, but closed when stimulated. Drops of tar near it indicated
that the foreign material had been ejected. One week later it still appeared entirely
normal. It was then again injected with 0.5. c. of tar, and was now put in stagnant
sea water. Four days later it did not, when stimulated, close as readily and tightly as a
normal oyster. It was then opened and found normal in its heartbeat and in con-
tractility of the bivalve muscle, but the mantie was not normally responsive to mechanical
stimulus.

This experiment indicates that when an oyster ingests tar and can not get rid of it
because the surrounding water is stagnant, some impairment of the sensory apparatus
in the mantle results. This interferes with certain activities of the oyster, prevents
normal closure, and eventually causes degeneration of muscular and other tissues.

Experiment 2.—Three medium-sized oysters were each injected with about 1.5 c. c.
of water-gas tar and then put in separate jars of running sea water. Some time after
they were seen in each case to open slightly and in a few minutes close violently so as to
eject masses of tar. This process was repeated a number of times in the course of one
to three hours after injection. They then remained constantly closed for some time, but
were found normally open on the following day. They were left in the running sea
water for a period of eight weeks and behaved throughout that time like control oysters
inthe aquarium. As it was then necessary to terminate the experiments, the oysters were

EFFECT OF WATER-GAS TAR ON OYSTERS. 205

opened and carefully examined. They were found to be normal in color, odor, heartbeat,
responsiveness of the mantle, ciliary movement, and in short in every respect.

This experiment distinctly indicates that water-gas tar in considerable doses is
harmless to an oyster in running sea water. The conditions of this experiment more
closely resemble those of the native habitat of the oyster than do those of the preceding
experiment, because tides and other currents over oyster beds maintain a constant
movement and a continuous changing of the surrounding medium.

Experiment 3.—A medium-sized oyster was injected with 1 c. c. of water-gas tar,
which was distributed all around the mantle. It was then put into about 1,500 c. c.
of sea water and carefully observed. During the next five hours it did not visibly open
and no tar escaped from it. On the following day, however, a few drops of tar were
floating on the surface of the water. During the next two days the oyster was only
infrequently observed and was not seen open, but on the third day it was found nor-
mally opened and able to close when stimulated. It was left in the same sea water
during the next two weeks. It had then developed the usual symptoms of imperfect
closure and when opened did not show a normally beating heart or a responsive mantle.
This experiment confirms the first one of this series.

EFFECT OF WATER-GAS TAR ON THE DISSOLVED OXYGEN OF SEA WATER.

It seemed possible that tar and similar substances might in a measure reduce the
oxygen content of water so as to affect shellfish. Mixtures of tar and sea water were,
therefore, allowed to stand for varying periods of time and then tested by Winkler’s
titration method to measure the quantity of dissolved oxygen in the water. The
_ experiments are summarized in the following table. Three liters of sea water were used
_ in each case.

Time mixture Oxygen re-

ase oes was allowed to| maining in
friscd stand at room | water at end
: temperature. of the time.
=
Cubic Parts per
cenlimeters. Hours. million.
200 | 20 6. 61
200 24 6. 10
200 45 - 00
200 130 - 08
5° | 40 2. 71
50 45 J.10
50 130 ;
None. 40 a@7.10
None. 45 @ 8.09
None. 130 48.26

4 Control, sea water alone.

These experiments show that the tar can cause the disappearance of dissolved
oxygen in sea water. How potent a factor this may be in causing the effects of the
tar on oysters in stagnant water it is not, however, safe to say. Ovysters, as the author
has shown, are remarkably resistant to lack of oxygen and do not when deprived of it

206 BULLETIN OF THE BUREAU OF FISHERIES.

show inability to close except in the advanced stages of oxygen starvation. That oxygen
consumption by tar may help to account for the fact that oysters are injured by stag-
nant tarry water, while they are uninjured by the tar in running sea water, is quite
probable. In the natural habitat of the oyster, however, it seems quite impossible
that the slight reduction of dissolved oxygen which small amounts of tar could effect
would alter the results of oyster culture.

CONCLUSION.

These experiments show no noticeable effects of water-gas tar on oysters in con-
stantly renewed sea water. ‘This is true in spite of the fact that large amounts of tar
mixed with stagnant sea water, or small amounts injected into oysters which are kept
in stagnant water, do cause serious or fatal effects. Considerable quantities (1.5 ¢. c.)
may be put inside the shell of an oyster kept under conditions resembling those of its
natural habitat without causing any effect. The harmlessness of the tar under these
circumstances is due apparently to the ability of the oyster to rid itself of such foreign
matter. In stagnant water the organism can not be effectively washed out, and effects
involving a loss of sensitiveness in the mantle result. That consumption of the dis-
solved oxygen in the stagnant water by tar may have some effect on oysters is a
possibility.

126

THE OXYGEN REQUIREMENTS OF SHELLFISH.
BY PHILIP H. MITCHELL.

From Bulletin of the’ United States Bureau of Fisheries, Vol. XXXII,
1912. Document 787. - Issued February 18, 1914.

FROM BULLETIN OF THE UNITED STATES BUREAU OF FISHERIES : VOLUME XXXII, 1912
Document 787 : : : : : : : : : : : : : Issued February 18, 1914.

THE OXYGEN REQUIREMENTS OF SHELLFISH
&

By Philip H. Mitchell

16284°—14 207

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THE OXYGEN REQUIREMENTS OF SHELLFISH.

&*
By PHILIP H. MITCHELL.
&*

The respiratory exchanges in lamellibranchs seem not to have been investigated.
Probably the most notable work related to it is that of Vernon.* He measured the oxy-
gen utilization and carbon dioxide emission in a large number of marine forms, including
certain Mollusca but no lamellibranchs. He showed that in the lower marine forms
investigated, including Coelenterata, Tunicata, and Mollusca, the respiratory exchange
was very small compared to the higher ones, for example, teleosts. There were, however,

_ certain exceptions to this rule, notably the protozoan Collozoum inerme, which showed
_ nearly as high a respiratory exchange as the fishes. He found also that, in general, the
respiratory activity was more readily responsive to temperature in the lower than in
the higher forms. He further showed that the gaseous exchange was relatively greater
_ in the small than in the large individuals of the same species and found that, in general,
the same distinction held between small and large species. The transparent pelagic
animals were shown to have a very small proportion of solid organic matter in their
_ tissues, so that calculated on that basis their respiratory activity was very large, greater
_ indeed than fishes, amphibians, or even mammals.

In the present work some of those findings have been confirmed for the lamelli-
branchs. They show a ready responsiveness to temperature changes, a smaller utili-
_ zation of oxygen in proportion to the body weight with increase in size, and those forms
which showed a low oxygen requirement in relation to their entire weight showed a
higher utilization in proportion to their dried weight.

The resistance to lack of oxygen in forms which have no power of locomotion is
an important factor in adverse conditions. This is especially true of the edible shellfish,

which, because of enforced closure during cold weather, or in the presence of polluted
or roily water, or in water whose oxygen has been lowered by the presence of certain
wastes or an abundance of life, must at times be deprived of their normal supply of
oxygen. The subject therefore possesses an economic significance, and it was, in fact,
the possibility that certain manufacturing wastes, removing oxygen from sea water,
might therefore cause the death of oysters and clams which first directed the writer’s
attention to the subject. The particular wastes involved were those of gas works con-

@ Vernon, H. M.: Respiration in marine forms, Journal of Physiology, vol. x1x, 1895, p. 18.
209

210 BULLETIN OF THE BUREAU OF FISHERIES.

taining at least traces and sometimes significant quantities of water-gas tar and other
oily matter. The results of this investigation indicate, in a word, that the tar or oily
wastes could have no effect on shellfish in this way. The details of this part of the work,
however, are reserved for a report dealing specifically with the effect of water-gas tar on
oysters. This paper is confined to oxygen requirements of shellfish and their resistance
to lack of oxygen.

METHOD OF EXPERIMENT.

The method of manipulation was, in brief, to place the shellfish in a desiccator
completely filled with sea water of known content of dissolved oxygen, leave the apparatus
at some constant temperature during a definite period, and then to sample the water
in the desiccator so as to compute its decrease in dissolved oxygen. Winkler’s well-
known titration method was used to measure the oxygen both at the beginning and end
of each experiment. A vacuum desiccator was used for the containing vessel because
it could easily be closed water-tight, could accommodate almost any size of shellfish,
and enabled one to take through the side opening with glass stopcock a fair sample of
the contents.

In practice a number of precautions were found necessary. The hollow dome of
the desiccator cover was entirely filled with paraffin to exclude air from entrapment
init. A glass tube within the desiccator was connected to its side stopcock and reached
nearly to the bottom. When, therefore, the filled desiccator was opened at the top
water could be sucked off through the opened side cock into the sampling bottle so that
the sample would come from a point well below the surface of the water, where the oxy-
gen content would be fairly constant. The sea water % used in each experiment was
brought to the required. temperature and placed in a large reservoir jar on a shelf, from
which it could be siphoned without bubbling into the desiccator. When it was thus
nearly filled, an oyster was gently placed on a glass tripod, where it would rest near the
center of the desiccator. With clams it was found necessary to leave them in the
desiccator submerged in water for a few hours before the experiment, because, unlike
oysters, they would not open quickly after they had been handled. In either case,
though, water from the shelf reservoir was siphoned into the overflowing desiccator for
sufficient time to bring the oxygen content to approximate constancy and then the
cover was put on, leaving just opening enough to admit the siphon tube.

The side cock of the desiccator was then connected by rubber tubing to the sampling
bottle, and after starting by suction the water was allowed to siphon through the bottle
two to three minutes. ‘This period was found quite sufficient to give reliable duplicate
results for water containing any percentage of oxygen measured in these experiments.
The water running into the desiccator meantime was in excess of that running into the
bottle, so that an overflow was maintained from the top of the desiccator and the sample
rendered as fair as possible. At the exact second recorded as the beginning of the experi-
ment the side stopcock was closed, the siphon quickly withdrawn from the desiccator,

a From aquarium of running sea water, a part of the aquarium system of the laboratory.

OXYGEN REQUIREMENTS OF SHELLFISH. 211

and its lid put completely on, excluding bubbles. The entire apparatus was then kept
in a bath of sea water whose temperature was maintained approximately the same as the
water within the desiccator. The oxygen in the sample was meantime measured by
titration.

After a period, usually about an hour in length, the desiccator was two or three times
inverted to render its contents uniform and to stimulate the shellfish to close and thus
stop using oxygen. The exact time of the first rough movement of the apparatus was
recorded as the closing time of the experiment. The outside of the desiccator was then
dried with a towel and the entire apparatus weighed. When currents in the water had
come practically to rest, the side stopcock was again connected to the same sampling
bottle used at the beginning of the experiment and a sample taken as before. Since
the capacity of the desiccators was sufficient for 1,200 grams of water, even with the
large shellfish, and as the sampling bottles held approximately 300 grams, it is plain
that water could run through the sampling bottle some time without emptying the
desiccator. The stream indeed was allowed to run at least two minutes. The capacity
of the desiccators was not enough to permit of taking a duplicate sample that would be
reliable, but this was not necessary because there is little chance of error in the Winkler’s
titration.

The last step in the process was to empty the desiccator completely, dry it and the
shellfish, and weigh them together. This gave a means of determining by difference the
weight of the water. It was probably accurate to within 2 grams. As the dissolved
oxygen of the water both at the beginning and the end of the experiment was calculated
in parts per million, it was only necessary to multiply the weight of water used by the
decrease in its oxygen content expressed in parts per million and divide the result by
100 to find the decimilligrams of oxygen used by the shellfish. Experiment showed that
no correction for change in the oxygen content of the water due to microorganisms or
physical factors was, under the circumstances of constant temperature, etc., necessary.
Results were expressed in decimilligrams of oxygen per hour and in most cases also com-
puted in decimilligrams per hour per 100 grams of shellfish and in some cases in decimil-
ligrams per hour per 1 gram of the dried weight of the total shell contents of the organism.

A formula for the first of these three computations might be expressed, therefore, as
follows:

a’—a"’ 60
Toa}! ¢.

A=W

where A is the decimilligrams of O used per hour, W the weight, in grams, of water in the
desiccator, a’ the parts per million of oxygen in the first sample and a’’ in the second
sample, and ¢ is the time of the experiment in minutes.

Throughout the experiments it was found necessary to observe the animal at fre-
quent intervals, and if the shells closed up to discard the experiment, or if the shells
closed only after a considerable number of minutes had elapsed to terminate the experi-
ment immediately. This precaution was necessary because, as is well known, certain
of the shellfish used in this work can close water tight and in that condition take from the

212 BULLETIN OF THE BUREAU OF FISHERIES.

water, as will be shown below, no oxygen except the very small amount absorbed by
their shells.

It appeared early in the work that constancy of results was exceedingly difficult to
obtain. ‘This was due to a variety of causes, chief of which was the great variability in
the openness of the shells, for not only could the oyster or clam entirely or at least
obviously close so that the experiment had to be terminated, but it could partially and
unnoticeably close or indeed fail to open wide even from the beginning of the experiment.
For the oyster, at least, the author succeeded in demonstrating this by graphic records.
One shell of an oyster was connected by tying a string to the projections with a lightly
balanced lever recording on a slow kymograph. As soon as the oyster in a water bath at
a temperature between 18° and 20° C. had opened somewhat, the kymograph was started
and the temperature of the bath raised at the rate of 1° C. in about eight minutes. The
oysters remained fairly well open with brief periods of partial closure as the temperature
increased to about 22° C. At that point there invariably appeared in the three individ-
uals observed periods of maximum openness lasting as long as no disturbing factor inter-
vened. Sufficient stimulus for partial closure, however, was likely to occur frequently.
A light tap on the table or water bath, a heavy step in the room, the slamming of a door
in a neighboring part of the building, or, indeed, any slight jar was surely registered by
some movement of the shell. As the temperature increased up to about 26° or 27° C.,
the effect of these stimuli was much less marked. ‘The oysters then maintained their
maximum openness very persistently. Between 27° and 30° a tendency to very slow
and incomplete opening after closure was noticeable, indeed no maximum openness was
seen. At about 30° or 31° C., the oysters closed tightly, even if no mechanical stimulus
was given.

As the kymograph method served to detect movements of the shell not noticeabie
to unaided observation and also slight openings of the shell not otherwise visible, these
experiments were an aid to planning and interpreting measurements of oxygen utiliza-
tion. They showed that below 19° C. and above 26° C. observations on the opened
oyster were impossible, that temperature must be maintained constant throughout the
experiment, and mechanical disturbances must be avoided as far as possible. A further
source of difficulty had also to be overcome. When the oyster excreted it closed vio-
lently, to drive the fecal matter out of the shell. If the position of the animal rendered
complete excretion difficult, closures were frequently repeated, and sometimes the oyster
shut up tightly. It was necessary, therefore, to lay the oyster in the desiccator tipped
so that the more concave side of the shell, where excretion occurs, would be lower than
the other.

In spite of all precautions, however, perfectly consistent results could not always
be obtained. Under the same or comparable conditions of temperature, oxygen content
of the water, and physical conditions, the same individual would sometimes in different
experiments give results disagreeing beyond the limits of the calculated, probable, experi-
mental error. Various observations make it seem likely that the nutritive condition
of the individual could account for some, at least, of these discrepancies. Thus, after

OXYGEN REQUIREMENTS OF SHELLFISH. 213

remaining at a high temperature (e. g., 24°-26° C.) for some time, the oxygen require-
ment at a slightly lower temperature would tend to be greater than if the shellfish had
been at a lower temperature before the experiment. If an oyster had been out of water
for some time before the measurement, more oxygen would be used, generally, than if
the specimen were left in the aquarium until the time of the experiment. After the
shellfish had been kept in the aquarium some weeks they tended to use less oxygen
than when taken recently from their native environment. Exceptionally an individual
would show a high utilization of oxygen out of proportion to previous measurements
and lasting for several days. As many of the interfering conditions as possible were,
of course, eliminated, but still it seemed necessary to make a considerable number of
experiments and draw conclusions only from averages. More than 350 measurements
were made under various conditions on three types of lamellibranchs—the oyster (Ostrea
virginica), the soft-shell or long clam (Mya arenaria), and the quahog or round clam
(Venus mercenaria).
RESULTS.

OXYGEN REQUIREMENTS OF THE OYSTER.

Three series of experiments, each made on a limited number of individuals, are sub-
mitted. Although the results can scarcely be taken to show any seasonal variation,
they are grouped, for convenience, according to the time they were carried out. In
table I are the results of measurements taken during July, 1911; in table m1, those of
July and August, 1912; and in table m1 are results obtained during the latter part of
August, 1912.

In the experiments of table 1 definite temperatures were not previously chosen and
carefully adhered to as in the later work. The results, therefore, are here grouped for
comparison as follows: All measurements taken at temperatures between 20° and 21.3°C.
appear in one column, those at 21.5° to 23.5° C. in another, and those between 26° and
28° C. in a third. Where two or more experiments were made with one oyster at tem-
peratures within a given range the average of the results is placed in parentheses. In
the last three columns are the averages of comparable experiments computed as the .
decimilligrams of oxygen used per hour per 100 grams of oyster. By weight of oyster
is meant in this and other tables not otherwise specified the weight in grams taken after
it was closed under water, wiped as dry as possible with a towel, and left to dry in the air
not more than half an hour. Such weighings were shown by duplication to be accurate
to within two-tenths of a gram.

In table 11 are the results of experiments performed at definite chosen temperatures
so controlled that the variation was not more than half a degree centigrade in any single
experiment. The first five columns of results show the decimilligrams of oxygen used
per hour at five temperatures by nine oysters. The averages of all comparable experi-
ments are put in parentheses. The last five columns contain the averages, expressed
in decimilligrams, of the oxygen used per hour per 100 grams of oysters.

214 BULLETIN OF THE BUREAU OF FISHERIES.

TABLE I.—OxyYGEN USED BY OYSTERS.

A, © oe

Nore.—Figures in parentheses are averages of experiments under approximately uniform conditions,

|

Average decimilligrams of oxygen
per hour per roo grams of oyster.

Decmlhaan oxygen used
r hour.
Weight me
of whole
oyster. At 20° to | At 21.5°to| At 26°to | At 20° to
areas 23.5° C. 28°C. Pac pe kh
Grams.
2nOl wi ale olefexcstels Koi tats CibP © less gepaasclaaas coe ago.
9.0
7-8
6.8
(7-3)
Reso) learsocourcde TU oS hat | areas seyetrcheas te tell sien cisvenreree
7-6
(9-7)
85.0 Go Raa ye San codesan 19-3 14-9
14-7 18.8
14.0 (19. 1)
(12. 7)
106.0 10.1 19-4 22-9 12.1
15-6 22.9
(12. 8) 26.2
{ (24-0)
113-0 14-6 2 Se eS ES Reins 12-9
16.1
(17-1)
127-0 I2.7 BUG es interes ers Tee
13-6 21.5
15-7 19.0
(14.0) 21.2
‘ (20. 9)
DAT AO She et ceetererstsavelars TQM ght |e ratoneerslevavatsll le venctecaretebelokeks
| 18.0
| (18. 5)

At 21.5° to
23.5 'C.

17-4

17-2

18.3

15-1

I3-1

At 26° to
28° Cc.

22-4

22.6

OXYGEN REQUIREMENTS OF SHELLFISH. 215

TaBLE II.—OxyGEN USED By OYSTERS.

Nore.—Figures in parentheses are averages of measurements under approximately uniform conditions.

Decimilligrams of oxygen used per hour per 100

Decimilligrams of oxygen used per hour. grams.

Weight
of whole |———

oyster. | atro.5° | At 21° to| At22°to| At 24°to| At 26°to| At 19.s° | At21°to| At 22°to| At 24°to| At 26° to
HOO ues oats Ge | aase Gel ads Ge | aOee Co ltooe Calas Gy Wage @.0) eae. lo26. 221s

Grams.
42-0 7.6 12.3 10.3 14-6 13-4 20.7 31-0 29-1 34-8 31-3
9-8 13-8 13-9 14-9
(8. 7) (13-1) (12. 2) Ir.2
(13-1)
51-0 11.6 9-3 II.7 12.6 13-6 22.2 17-9 22.8 24.8 252.7
10. 7 II. 4 II.5 12.7
11.8 9-6 (11. 6) (12. 65)
(11.3) 6.2
(9. 6)
66.5 15-1 16.0 15-5 16.0 18.6 16.9 21.0 20.6 24-1 26.9
9-4 9-9 11.8 17-2
10. 5 16.1 (13-7) (17.9)
9-1 14-0
(11. 2) (14. 0)
76.0 Il. 5 13-9 15-3 15-8 23-5 16.7 19-0 18.5 23-0 29-5
14-0 15-3 12.8 20.5 20-9
(12. 7) 14.2 (14.05) 16.2 24-5
(14. 5) (17-5) 20-7
(22. 4)
97-4 13-4 TAI: | lic pecs 18.0 22.1 13-8 THOS Wit aloleralosiek 18. 5 22.7
128.0 Teen elise are cxeseiatcre 17-9 25-9 24.8 Teg. hth datecera oe 15:7 17-8 19-4
17-8 25-9 19-8 :
(15. 75) 18.6 (22.8)
18.4 }
(20. 2)
147-0 16.0 18.4 20.4 20.8 26.6 12.1 12.5 Tat5 14-5 17-9
19-4 18.2 19-3 21-9 26.0
(17-7) (18.3) | (19.8) (21.3) (26.3)
244-0 18.1 23-5 34-6 30-0 35-1 8.3 10.1 12.3 II.o 14-4
22.3 25-7 25.7 26.1
(20. 2) (24. 6) (30. 1) 24-0
(26. 7) |
262.0 19-0 29.1 27-4 28.7 37-0 9.3 9-6 10.0 II.3 4.x
29-5 24-8 30-7
21.4 (26. 1) (29. 7)
20.8
(25.2)
2ANGEIC (S96 Oormanieidoia] PEEPS ap yey Negi ad | Sek nh eS | ail 14-5 17-0 17.8 , 20-0 22-5

In table 1m are the results of measurements at four chosen temperatures on four
oysters. These experiments were all done within a few days after the oysters were
_ taken from the beds and therefore serve to some extent as a control on the condition
_ of the oysters used in the other series. The first columns of results show the decimilli-
_ grams of oxygen used per hour at the designated temperatures. The averages of com-
_ parable experiments are put in parentheses. The figures in the next four columns are
_ obtained by computing the averages of the oxygen utilization per 100 grams of oyster.
In the next column is the weight, in grams, of the shell contents of each oyster when
_ dried to constant weight. The last four columns show average decimilligrams of oxygen
used per gram of dried substance. ‘The figures in these columns were corrected for the

216 BULLETIN OF THE BUREAU OF FISHERIES.

oxygen used by the shells. The shells of each oyster were at the end of the observations
carefully cleaned in sea water and then used for two or three measurements of their
oxygen-absorbing power at different temperatures. As these results were obtained
under the same conditions as those expressed in table 1, they represent a fair estimate
of the oxygen absorbed by the shells during measurements with the intact oyster. There
is reason, as will be shown below, to believe that oxygen removed from the water in this
way is not utilized by the active tissues of the oyster. It seems reasonable, therefore,
to subtract the amount (measured or computed) of oxygen utilized by the shells from
that used by the whole oyster before computing the oxygen requirements per gram of
dried tissue.
TABLE III.—OxyGEN USED By OYSTERS.

Nore.—Figures in parentheses are averages of measurements under approximately uniform conditions.

Decimilligrams of oxygen used | Averages of same per hour per Average decimilligrams used el

| per hour. 100 grams. hour per gram of dried weight. |
Weight, |_ Weight,
whole dried | |
| oyster. At At At At At At At At oyster. At Bots} AGE At
20° to | 22° to | 24° to | 26° to | 20° to | 22° to | 24° to | 26° to 20° to | 22° to | 24° to | 26° te
20.5°C. |22.5°C. |24.5°C. |26.5°C.]20.5°C. | 22.5°C. | 24.5°C. | 26.5°C. 20.5°C. 22:5) CaS Asp (0
| Grams. Grams. |
47-8 10. 6 12.2 14-2 ry. 5 22.2 27-4 29-7 35a Kens 7-92 9-74 | 10.40 I2.g0
14-1 |
(13- 1) |
|
73-5 16.6 20.0 23-1 22.0 22.6 25.0 28. 2 30.0 1. 66 8. 20 8.61 | 9.43 9. 56
17-5 18. 3 |
(18.3) | (20. 7)
I53-0 19-4 22.4 21.4 25-2 12.7 14-6 14. 7 16. 6 2.36 5-95 6.78 j}\ 16756 Ye
21.7 25 ~
24-5 | (25-5)
(22. 5)
|
173-0 23-5 | 24.5 29. 2 39-0 13-6 14-2 sd Be) 21.2 3-64 3-49 4-00 | 4.53 6. 10
| 24s: 30. 8 3 ;
(24.6) | (30.0) | (36. 6) | |
|
| |
Average.| 17.5 19-7 21.9 25-3 17-8 20. 3 23-5 AY WeGodod se 6. 41 7.28 alg 8.94

A graphic representation of the oxygen utilization of oysters at different tempera-
tures is given on page 217. The ordinates represent decimilligrams of oxygen used
per hour and the abscisse are degrees Centigrade. The full lines represent measure-
ments on intact oysters. Curve 1 is constructed from the averages of the oxygen utiliza-
tion per hour per 100 grams in experiments on nine oysters as summarized in table 1.
Curve 0 is constructed from similar averages of experimental results on four oysters as
shown in table mz. Curve m1 is based on the same results used for curve 1, but repre-
sents the averages of oxygen used per hour per gram of dried shell contents. The rela- iy
tionship between oxygen and temperature is apparently a simple one. The interrupted
lines represent measurements on empty oyster shells showing the decimilligrams of
oxygen used per hour by shells as recorded in table 1v. Curves a, 6, c, and d refer,
respectively, to shells weighing 47.8, 73.4, 153, and 161 grams. ‘The effect of tempera-
ture on the three larger shells seems to be fairly constant; their curves are parallel.
The discrepancy in the smallest shell may be due to experimental error as so few deter- .
minations were made.

Io per hour

OXYGEN REQUIREMENTS OF SHELLFISH.

217

in the text on page 216.

e. Explanations

in the oyster to temperatur

Relation of oxygen ut

218 BULLETIN OF THE BUREAU OF FISHERIES.
NONUTILIZATION OF OXYGEN BY CLOSED OYSTERS.

Since oysters can close absolutely water tight, it seemed possible that under such
conditions they would take in no oxygen. The very small oxygen requirements of
voluntarily closed individuals could not, however, be interpreted as proof of this because
when the oyster appears to be closed it may be slightly and invisibly open. This was
proved by graphic records. To insure closure throughout an observation it was neces-
sary, therefore, to use some artificial closing device. Several clamps were tried. An
ordinary surgeon’s artery clamp was found to be most satisfactory. If clamps without
imperfections, carefully vaselined, were used, they did not appreciably rust during the
experiment and withdrew from the water only a small and constant quantity of oxygen.
As control experiments the oxygen absorption of empty shells of oysters about the same
size as the one observed was measured. The control shells were fastened together by a
clamp duplicating the one used on the oyster. Water had some access to the interior
of the empty shells through nicks in their edges. On their immersion in water all air
was driven from them. In many experiments controls were deferred until the same
oyster could be emptied and its shells used for control measurements.

The results of a number of determinations are given in table v. It is seen that in
every case the experiment and its control are in close agreement, as the difference is
within the limit of experimental error except in the case of the largest oyster used.
Even these differences, however, show a larger oxygen absorption by the empty shells
than by the closed intact oysters. That the slight oxygen disappearance does not repre-
sent a respiratory intake by diffusion through the shells is indicated not only by the
controls but by the observation that a two hours’ exposure of the clamped oyster does
not cause twice the oxygen absorption observed during one hour. Thus in one experi-
ment a clamped oyster used 1.83 decimilligrams of oxygen in one hour, but 3.01 in two
hours, while another, which used 1.70 decimilligrams in one hour, required 2.80 for two
hours.

The explanation of the constant slight absorption of oxygen by clamped oysters
and empty shells was not positively determined, but would seem to lie in several
causes. Bacteria and various marine vegetative growths on the shells were first con-
sidered as possible oxygen users. Oyster shells that had been soaked 16 hours in 80
per cent alcohol and then carefully scrubbed and dried, did, indeed, show a diminished
oxygen absorbing capacity, in one case lowered from 5.25 decimilligrams per hour before
the alcohol treatment to 4.68 after it, and in another, from 3.08 to 2.34. This indicates
that foreign growths do not account for all the oxygen absorption, and, indeed, it was
found that the cleanest and most carefully sterilized shells took oxygen from the water.

An adsorption effect of porous substances on dissolved oxygen suggested itself as
another possibility. It was found that porcelain evaporating dishes about the size
of oysters showed equivalent oxygen absorbing powers. Corks had the same capacity.
Water containing medium sized corks lost 2.34 decimilligrams of oxygen per hour, while
in a control experiment water alone lost only 0.26 decimilligram, which is within the
limit of experimental error.

OXYGEN REQUIREMENTS OF SHELLFISH. 219

TABLE I1V.—OxYGEN ABSORBED BY Empty OYSTER SHELLS.

Weight of whole oyster in grams............. 47-8 | 47-8 | 73-4 | 73-4 | 73+4 | 153 | 153 161 161
Temperature of experiment in degrees centigrade. . 22-4 25-8 21.7 22.3 26.1 22.0 25-8 21.5 26.5
Oxygen absorbed per hour in mgr/1o............... I-9 2-4 3-6 4-1 6.3 6.1 8.3 Tiss 14-4

TABLE V.—OxyYGEN ABSORBED BY CLOSED OYSTERS.

Tempera-
. Oxygen
Weight of | ture of used per Description of control experiment.
oyster. experi- Rone
ment. ;
bi Bs Mor|r10.

45 grams... 22.0 I-70
Control.... 22-0 1-70 | Empty shells of same oyster with artery clamp.
50 grams... 22-0 1.83
Control..... 22.0 1.83 Do.
66.5 grams 23-2 2.04
Control..... 21.0 I.92 Do.
Control..... 23-2 2.58 | Empty shells of oyster weighing 45 grams with clamp.
66.5 grams.. 20. 5 I-41
Control..... 20.5 1.48 | Empty shells of oyster weighing 51 grams with clamp.
128 grams.. 20.5 I-79
Control..... 20.5 1.92 | Empty shells of same oyster with artery clamp.
147 gfams.. 20.5 I-74
Control..... 20.5 1.62 Do
244 grams 20-5 I-70
Control..... 21.0 2-34 Do.
262 grams.. 20.5 3-39
Control..... 21.0 4-68 Do.
244 grams 27-5 3-47
128 grams 27-5 2-36 |\control experiments.not made.
66.5 grams 27-5 2.49
76 grams 27-5 2-80

RESISTANCE OF OYSTERS TO LACK OF OXYGEN.

A series of experiments was undertaken to find out the minimum oxygen supply
that would maintain an oyster alive. The sea water surrounding an oyster in a vacuum
desiccator was as far as possible rendered oxygen free by boiling at room temperature
under diminished pressure for half an hour. It was found in two trials that sea water
alone when so treated and kept twelve hours in the closed desiccator still had an oxygen
content less than 0.5 of a part per million. An oyster kept under these circumstances
four days showed no ill effects. Another was kept thus three days, transferred to a
second desiccator of exhausted sea water, which was quickly again pumped out, and
was then kept four days further in the oxygen poor medium. The shells then had some
black deposits on them indicative of incipient anaerobic putrefaction. The sea water
on opening the desiccator was found absolutely oxygen free. The oyster, however,
seemed unimpaired, and after remaining in the aquarium some time was opened and
found apparently entirely normal. As each desiccator held about 1,200 c. c. and the
oxygen content of the water in each case was at the most o.5 of a part per million, the
- oxygen available to the oyster might be estimated as 1.2 milligrams plus the small
i amount obtained during the transfer from one desiccator to the other. If we disregard
_ the latter source because the oyster was tight closed at the time, only 1.2 milligrams of
_ oxygen were used during seven days.

220 BULLETIN OF THE BUREAU OF FISHERIES.

In another experiment an oyster was kept three days in the first desiccator full of
pumped-out water, three days in the second, two days in the third, two days in the fourth,
and two days in the fifth. By that time it was black and unable to close properiy.
After a few hours in the aquarium it showed disintegration, so that twelve days of life
in oxygen-poor water proved fatal. ‘Time and facilities for carrying out many of these
experiments were missing, although the seven-day experiment was practically dupli-
cated by one in which an oyster was kept in the same desiccator full of water six days
with no ill effects. That the fatal effect of a twelve days’ exposure was not due to
insufficient renewal of the water was shown by control experiments in which several
oysters were kept in the same water during fourteen days while air was bubbled through
it. The oysters survived this treatment uninjured.

OXYGEN REQUIREMENTS OF CLAMS.

Measurements on clams were made by the same method used for oysters. The
results of successful experiments are embodied in table v1. They show a general agree-
ment with measurements on oysters. The amount of oxygen used by ciams weighing
about 60 grams is somewhat higher per 100 grams than that used by an oyster of about
the same weight and observed at the same temperature. Computed per gram of dried
shell contents, however, the oxygen requirements of the clam seem somewhat smaller
than those of the oyster. Here, as with oysters, the amount of oxygen used is more
or less proportional to increase in the temperature, though sufficient data to show a sim-
ple relationship were not obtained. In this case, also, the oxygen requirement per gram
is less in the larger individual than in the smaller, again like the oyster.

TABLE VI.—OxyYGEN USED BY CLAMS.

Note.—Figures in parentheses are averages of measurements under approximately uniform conditions.

Decimilligrams of oxygen used | Averages of same per hour per | Average mgr/ro used per hour
mas per hour. roo grams. ; | per gram, dried weight.
Weight Weight |
whole |—— = PYEANIEY | dried _ |-
clam. At 20° to| At 22° to} At 24° to| At 20° to} At 22° to} At 24° to clam. At 20° to| At 22° to} At 24° to
are Cy ance Cau aa eee amici, AA Gy Wey ec (Ce Zia 22.5 (Ci || Vanes
Grams. Grams.
7-0 TOD) Metatasel stata: e\ ala inc oat! staid aie PICO I BRO OOOG ODO Ooo GovOr Ors cs Oooo omits Cticar cocc hy) Sagoo ocn.
3-0
(2.1)
19-5 5-19 9-7 I5-t 26.7 49-7 77-4 I-32 3-93 7°35 TI.4
21-0 Che aN BENE onacl Acrero soso AGO Pl |\ucteetere.<.o b1sil elelolsieleisre, ©-sillo\oisie\ diataye)el=illclefsiwfelefstels's [prs a¥a(e lure latetell tataietetenateiete
6.7
(7-9)
28.0 Bria APR tele: ota lO elk Se letatevard AOS BU Til vinistals) aleicie loll sputetelclelele’s/al|/u(biate: o'syelaie ie iaVorsiahalelolefels llelelelezeia eietelal| Mieetete tee tatane
57-0 Ls, O}iy 8 zien a Arita evetece eletateret BOs 7s Ullcihrs, oi devote soyeilla o16/stoisi'es e/a 0} stoveletaie otlels | elotebs wtoiplele’s java nloial fefetate ie tetateis amen
12. |
(21.7) |
Ie lo a Uieuc Oe 4 bc I7.2 30-0 | ee veins 30-0 52.2 ZalOTp illeasateveteyeveyeye 4-34 7°55
BOLO wiltal Megs ci sials OP Ne en Aer yA ring Dood BESO vis |laieyats & Sleveuetol|tofat olay lay slajal| iv ete iajefofefeve!el| afetuys a efayalanal steers eistata
63-0 17-7 CY Bto tae | WeRSE: each seth) (Gor IAtS AC LOU UMaD ICC FOO Ir ct endo soca ie Qe aan
15-2
(16. 4)

OXYGEN REQUIREMENTS OF SHELLFISH. 221

TABLE VII.—OxyGEN ABSORBED BY CLOSED QUAHOGS.

|
Weight of quahog in grams... ....eessscw esse 150 Iso 9r gt 246 246 @ 504 1505 gtd
‘Temperature in degrees centigrade................. 24-0 24.0 24.0 23-0 24-3 23-0 23:0 | 23-6 23-5
Peeoeen per POUL in! MET/IO. <5.<..2.0060ce ee wanna eons -37 +25 238 +25 +23 4-60 30 | 2+ 43 2-95
@ Voluntarily closed but not clamped. b Measurements on empty shells alone.

OXYGEN ABSORPTION OF CLOSED CLAMS.

Only one experiment was made in this connection. A medium-sized clam was
closed by an artery clamp over the siphon end of the shells. As a control, empty shells
of an oyster of the same size similariy clamped were used. ‘The live clam absorbed 2.68
decimilligrams of oxygen per hour at a temperature of 23.5° C., while the shell took 2.58.
This indicates that the clam, like the oyster, has little or no opportunity to obtain oxygen
when the shell is not open. Further observations incidentally made confirm this con-
clusion. A medium-sized clam which visibly failed to open throughout an experiment
to determine its oxygen requirements took only 2.44 decimilligrams of oxygen from the
water in an hour. Other similar results were obtained.

OXYGEN REQUIREMENTS OF QUAHOGS.

Great difficulty was experienced in measuring the oxygen utilization of quahogs,
because they seldom opened for any length of time under the conditions of experimen-
tation. Asa result of this, only a few measurements that could be considered reliable
were obtained. It was found necessary to place the specimen in sea water in the desic-
cator a long time before the experiment, usually the night before, in order to have it
open at the time of observation. Handling or moving caused it to close and stay closed
for some hours.

The few successful observations showed a rather low oxygen utilization. One
specimen weighing 91 grams used, at 24° C., 10.1 decimilligrams of oxygen in one hour,
10.8 in another measurement, and 6.4 in a third. Another quahog, weighing 150 grams,
used, at 24° C., 7.8 decimilligrams per hour, and a large one (470 grams) used 22.4 deci-
milligrams per hour. Many other measurements were attempted, but owing to closure
soon after the beginning of observation were unreliable. The oxygen requirements in
proportion to the dried weight showed a still greater discrepancy in comparison with
similar computations for the oyster. The dried weights of the shell contents of the
first two quahogs observed were, respectively, 2.22 grams and 3.86 grams. The oxygen
used per hour and per gram of dried weight, then, was 4.10 decimilligrams for the first
and 2.21 for the second. This is less than half the amount of oxygen used by oysters
of comparable weight observed at the same temperature. To prop the shells open
seemed hardly worth while, because the oxygen utilization under such circumstances
would be abnormal on account of the resulting violent contractions of adductor muscles.
_ It seemed best, therefore, to be content with the conclusion that under the circumstances
] of these experiments quahogs used only a small quantity of oxygen.

202 BULLETIN OF THE BUREAU OF FISHERIES.
UTILIZATION OF OXYGEN BY CLOSED OUAHOGS.

Clamping these shells as in the experiments described for oysters showed that closed
quahogs used no oxygen. ‘Their very smooth shells apparently took almost no oxygen
from the water. With various sizes of quahogs clamped and observed at 24° C., results
were obtained as follows: 0.37, 0.25, 0.38, 0.23, and 0.25 decimilligram of oxygen per
hour. The empty shells, considerably broken in the process of opening, used a
distinctly larger amount of oxygen than did the closed intact animal. The small amounts
of oxygen taken up under these conditions are no more than would disappear from the
sea water with a clamp in it.

It was clearly shown, however, that voluntarily closed quahogs did take up appre-
ciable quantities of oxygen. In observations where the shells were apparently quite
closed, various medium and large sized specimens took up, at 24° C., 2.9, 6.1, 4.6, and
4.3 decimilligrams of oxygen per hour. It would seem, then, that when voluntarily
closed they do not remain shut absolutely tight, but take in small amounts of water
through an aperture too narrow to be visible to the naked eye. The results are sum-
marized in table vit.

CONCLUSIONS.

1. Oysters of medium sizes, at temperatures between 19° and 28° C., used from 7 to
35 decimilligrams of oxygen per hour per 100 grams of entire weight. "The amount vayies
with the temperature, so far as experiments show, according to simple relationship, so
that the curve approximates a straight line. It is proportionally less for larger speci-
mens. The oxygen utilization is, however, exceedingly variable, depending on a variety
of conditions, most of which probably affect the openness of the shell.

2. Oysters when tightly closed take no oxygen from the surrounding water; at
least, no more than is taken by empty shells.

3. Oysters show considerable resistance to lack of oxygen. Only exposure for more
than a week to water containing very small quantities of oxygen proved fatal. This —
indicates that any conditions causing temporary decrease in the available oxygen are not
a significant factor in oyster culture.

4. The common clam (Mya arenaria) shows a higher oxygen requirement than the
oyster. This seems surprising, in view of the fact that clams so often exist in muds
where oxygen-consuming putrefactions are going on. The oxygen requirements of
clams and oysters in proportion to their dried weights are about equal.

5. Both clams and quahogs (Venus mercenaria) use no oxygen from the water when
tightly closed, but the quahog takes up oxygen while slightly and invisibly open.

6. The oxygen requirements of the quahog are, under the conditions of these experi-
ments, conspicuously low.

127

CONTRIBUTIONS TO THE BACTERIOLOGY OF THE
OYSTER.
BY LESTER A. ROUND.

The Results of Experiments and Observations made while Conducting an
Investigation Directed and Authorized by the Commissioners of
Shell Fisheries of the State of Rhode Island, 1914.

State of Rhode Island and Providence Plantations

CONTRIBUTIONS

TOL DEE

bacteriolosy: of the Oyster

THE RESULTS OF EXPERIMENTS AND OBSER-
VATIONS MADE WHILE CONDUCTING
AN SINVESTIGATION DIRECTED
ANDY AUTHORIZED (BY (CEE
COMMISSIONERS OF SHELL
PIS ELE, RES: Oo aera,
STATE..OR, sREODE
ISLAND:

BY

LESTER A; jSROUND, «PH.D.

PROVIDENCE:
E. L. FREEMAN CO., STATE PRINTERS,
1914

x oN p

*,
wt

TABLE OF CONTENTS.

PYGLA COs ccc eee eee ie et es ie eat Se See MRO lee on ea
Bacteriology of the Branchial and Cloacal Chambers of the Oyster..... 4
Review of Methods of Shellfish Examination. ......-....---+++-+++++- 11
Bacteriology of the Shell Liquor and ‘‘Washings” from the Body of

the ‘OYVSter. oo. «exe essence eke 8 om ose seks eee enete ae 22
Comparison of the Bacterial Contents of the Shell Liquor and Stomach

Contents of the Oyster: 2 io. aoa 0 tk = here - e ke hs tee 46
Length of Time Necessary for Bacteria to Pass through the Intestinal

Tract of the Oyster... secs. : sei ccc see hs os nik Sie 49
Bacterial Content of Oysters During Storage. ......-.-.-.++++ss0:: 51
Cleansing of Polluted Oysters. ......--. 2+. 2.2) s segs sae ae 63
Experiments on the Hibernation of the Oyster. .......-+--.---++->: 78

Changes Suggested in ‘Standard Methods of Shellfish Examination”... i15

PREFACE.

In March, 1910, the Commissioners of Shell Fisheries authorized
an examination of the sanitary condition of the waters of Narragansett
Bay and its tributaries, relative to the growing of oysters. They
placed Prof. F. P. Gorham, head of the Department of Bacterio-
logy of Brown University, in charge of this work, with the writer as
an assistant.

On beginning this work it was found that there were many problems
that would require more study than could be given while performing
the routine bacteriological examinations of water, mud, shellfish, ete.
To a great extent this work was new and the method of procedure had
to be worked out as the investigation progressed. While there had
been much work performed upon shellfish examinations, both abroad
and in this country, there were still many problems which had not
been solved and it was deemed advisable by the Commission that
some of these problems which were of great importance to the shellfish
industry should be given special attention.

The writer was early assigned to conduct a series of experiments and
investigations along the lines that had been found would apparently
prove of the greatest advantage to the oyster industry. The results
of some of these investigations is published in this booklet.

The writer wishes to take this opportunity to express his sincerest
thanks to Prof. F. P. Gorham, head of the Bepartment of Dacterio-
logy of Brown University, whose valuable advice and criticisms have
_ been exceedingly helpful and under whose direction the work herein
reported has been done; to Drs. A. D. Mead, H. E. Walter and P. H.
Mitchell, who have made valuable suggestions and criticisms on differ-
ent points in the work; to the members of the Narragansett Bay
Oyster Company, the American Oyster Company, the Wickford
Oyster Company and the Beacon Oyster Company, and to Captain
William B. Welden, all of whom have rendered valuable aid in carrying
out many of the experiments; also to Mr. W. B. Mason of The Mer-
chants’ Cold Storage and Warehouse Company who has given free
use of the company’s cold storage rooms for the experiments on
hibernation.

We As he.

Brown UNIVERSITY,

May 1, 1914.

THE BACTERIOLOGY OF THE CLOACAL AND GILLS
CHAMBERS OF THE OYSTER.

In describing the anatomy of the gills of the oyster, Kellogg in his
book on ‘Shellfish Industries’ makes the following statement:
‘Behind the body the four gills unite so as to separate a space above
the cloacal chamber, from the large mantle chamber below.”’ From
this statement it has been assumed at times that the two chambers
were entirely distinct and so constructed that bacteria could not pass
from one chamber to the other, and that for this reason the bacterial
content of the two chambers would differ. Anyone familar with the
anatomy of the oyster knows that every day several gallons of water
are filtered through the gills into the cloacal chamber. While it is
probable that most of the protozoa and alge are caught in the mucus
which the gills secrete, it is also probable that a great many of the
bacteria escape, being entrapped by the mucus and pass on into the
cloacal chamber. But even though the gill-filter were proven to be
bacteria-proof no one has demonstrated that bacteria cannot pass along
the space between the mantle and the shell, or around the edge of the
shell, between the flaps of the mantle and so pass from one chamber
to the other. While it seems very probable from the structure of the
oyster that bacteria can pass fron one chamber to the other without
difficulty, properly conducted experiments are necessary to prove it.
In order thus to prove that bacteria can and do pass, from one
chamber to the other, the following experiments were tried.

SERIES 1.

Exp. 1. A well shaped mature oyster about four inches long and
two broad was selected. Care was taken to obtain an oyster with a
flat right valve. The oyster was placed in a frame with the left
valve down so that the right valve was level and was then clamped
firmly to the table. A hole was bored through the right valve into
the gill chamber quite close to the edge and another into the cloacal

tt ee a a

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So X Xn ne :

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DNIMOHY SMOUUW AONV UILSAQ JO TITHY NI CANO SATOH AO NOMWISOd DNIMOHS WLW

BACTERIOLOGY OF THE OYSTER. 5

chamber, at the anal orifice. This latter opening was 1% to 34 of an
inch above the lower end of the partition which separated the two
chambers. A loopful of B. prodigiosus was then placed in the gill
chamber, and loopfuls were removed from the cloacal chamber and
plated at intervals of ten minutes for one hour, and then at the end of
two hours and three hours. B. prodigiosus is a non-motile organism
and was chosen because of its ease of identification and because in all
our work extending over four years we have never isolated it from
oysters.

No red colonies were found in the two control samples from the
two chambers, but every plate made from the cloacal chamber after
the introduction of the B. prodigiosus into the gill chamber showed
colonies of B. prodigiosus.

Exp. 2. The above experiment was repeated with another
oyster and B. prodigiosus was again found in the cloacal chamber ten
minutes after its introduction into the gill chamber.

Series II.

Exp. 1. In another set of experiments four oysters were used.
In these oysters five holes were bored as indicated in the plate
shown on opposite page. Three of these holes opened into the gill
chamber (1, 4, 5). Another hole (2) was made into the cloacal
chamber near the anal orifice, and the last hole (3) opened on the edge
of the mantle about an inch above the anal orifice.

All four of these oysters were inoculated in hole No. 5 with a loopful
of B. prodigiosus. Loopfuls from the other holes were inoculated
upon agar slants at two minute intervals, for ten minutes and then
every five minutes, for twenty minutes, making a total of 30 minutes
in each case. The result is seen in the following table:

6 BACTERIOLOGY OF THE OYSTER.

TABLD INO, 1,

Showing the time at which B. prodigiosus was isolated from the
different holes after inoculation of the gill chamber at hole No. 6.

Oyster No. | 1 2 3 4

Hole No...) Ly 220 sot ee 4 a 2S All I ee oe
Control. ..... 0.0) OF OO. O08 0) 0 Oy 0! 0) 0 SO omer
2 TN. 5... 0) =e OP SION SO OMe IkO” =e 0! OF OR VO enone
4b + + +4 + + 4/0 + + H+ 4°20 4
6 ft of + F]t + + +4 + + 4/4 + °=0 4
ge ib oF + +4 + 4+ H+ 4 + +14 + =«20 +
10 ib + + +4 + + Ht 4+ + $14 4+ =«20 4
15 | bot + +4 + 4 $+ 4 + +4 4+ «+ «+
20 iF ot + 44 + + $+ 4+ + +4 + =«20 +
25 Jt + + Ft + + 44 4 + H+ 4+ 4+ «4
30 + + 4.4/4 4 + +4 +: + 414+ + =20 4

-++ = presence of B. prodigiosus. 0 absence of B. prodigiosus.

From this table it is seen that in oysters Nos. 1 and 2 B. prodigiosus
was isolated from all the holes at the end of four minutes; in oyster
No. 3 at the end of six minutes, and in oyster No. 4 not until the end
of fifteen minutes. Oyster No. 4 was a long narrow oyster and hole
No. 3 in this case was necessarily moved further into the middle of
the oysters, so that the opening came nearly over the stomach, and so
did not reach the cloacal chamber. In the other cases, hole No 3 was
made nearer the edge of the oyster and close to the free edge of the
mantle, so that there was much greater chance of bacteria reaching
the hole from the liquor between the free edges of the mantle, for
the edges of the mantle are everywhere free except, at the ‘‘head” end,
where the the edges fuse and form a hood, which is attached to the
body by a flap of tissue. Between the free edges of the mantle and
between the hood and the body there is a space which extends around
the whole oyster and forms a kind of moat or trench filled with the
liquor in which bacteria can and do move by the currents set up by the
ciliary mechanism of the oyster, which will be described a little later.
It will also be noticed that in every oyster of this series B. prodigiosus
was isolated from hole No. 4 before they were from hole No. 1. although
the distance between holes Nos. 4 and 5 were nearly twice as far as

BACTERIOLOGY OF THE OYSTER. 7

between Nos. 1 and 5. It will further be noted that in oysters Nos. 1
and 3 B. prodigiosus was isolated from the cloacal chamber (hole No.
2) before it was found in hole No. 1, although the distance between
holes Nos. 5 and 2 was also about twice as faras between 5and 1. In
no case did B. prodigiosus appear at hole No. 1 before it did at No. 2.

Series III.

A third set of experiments were now performed in order to show
that bacteria can pass from the cloacal chamber to the gill chamber
and to ascertain, if possible, the avenue through which this takes
place. In this experiment two oysters were used and secured to the
bench in the same manner as in the previous experiments. Three
holes were bored into the branchial chamber as indicated by the
Nos. 1, 5 and 4 in the plate. One hole was bored into the
cloacal chamber as indicated by No. 2 in the plate. Control
inoculations were made as before from these four holes. These
showed no colonies of B. prodigiosus. A loopful of B. prodig-
iosus was placed in hole No. 2, and loopfuls were taken from holes
Nos. 1, 5 and 4 at intervals of two minutes for fourteen minutes.
The results are shown in table No. 2.

TABLE No. 2.

Showing the time at which B. prodigiosus was recovered from holes
Nos. 1, 5 and 4 after inoculation of the cloacal chamber at hole No. 2.

Oyster No. 1 Wy
EROS UNOS: ol: Bakes ee eds Seine: lh ceed 64 YW SrA
‘Clot Ee een reese, os ae eee 0 O OF 1040
PRUNES, Gs sates TRS Alt eer ne fics ss 0: Oh" 0 + 0 O
BM ris: 5.0 Snr Kesey «a 1 SRM aS 00 0; + + +
6 i gals RUN nara be BB Adie 2 co) + 0 0 + + +
8 pe) OA ais SPAM Se ee dere + + 0 + + +
10 Ce Vege cl ca. eo Re UU a te aE + + + + + +
12 Oey il a ot, ate oes ee Ee. ot + + + =the =f AT
lay SR Wie es i ee A + + +/+ + +

+ — presence of B. prodigiosus. (0 — absence of B. prodigiosus.

8 BACTERIOLOGY OF THE OYSTER.

From this table it can be seen that B. prodigiosus was isolated from
all the holes in oyster No. 1 at the end of ten minutes and in oyster
No. 2 at the end of four minutes. It is further seen that in oyster
No. | B. prodigiosus appeared first at hole No. 1, two minutes
later at hole No. 5, and after another interval of two minutes
at hole No. 4. In oyster No. 2 the bacillus appeared first at
hole No. 1 and two minutes later at holes Nos. 5 and 4. In neither
case did B. prodigiosus appear at holes 5 or 4 before it appeared at hole
No. 1, nor in either case at hole No. 4 before hole No. 5.

To understand the reason for these results a description of the
ciliary mechanism of the oyster is necessary.

When one opens an oyster without mutilating it, there is found
between the two flaps of the mantle four folds of tissue which are the
gills. These folds appear solid, but are really flaps folded back upon
themselves and attached by the edges to the body so that really each
gill is V shaped in cross section and the four gills form a double W
(WW). With the unaided eye it can be seen that there are fine stria-
tions running vertically across each gill. These are the gill filaments.
If we examine these filaments with a microscope we will see innumera-
able hairs or cilia about 1—-500th of an inch long or less, waving vigor-
ously back and forth. If we examine the cilia closely we find that
they lash vigorously in one direction, recover themselves slowly and
‘repeat the vigorous stroke. The movement is quite comparable
to a man rowing a boat. He pulls vigorously in one direction,
recovers himself and repeats the stroke. Now if we consider the boat
fastened so that it could not move, the oarsman’s efforts would send
the water past the boat instead of propelling the boat through the
water. Here we have the exact condition in the oyster. As Brooks
(The Oyster, 1906) says, these little hairs “set up a current in the
water. Each one is so small that its individual effect is inconceivably
minute, but the innumerable multitude causes a vigorous circulation
and each one is set at such a position that it drives the water before
it from the gill chamber into one of the water pores and so into one
of the water tubes inside the gill. As these are filled they overflow
into the cloacal chamber and fill that.’’ This set of cilia are located
on the edges of the filaments and force the water through the gills
from the branchial into the cloacal chamber. There is another set
of cilia which wave in the opposite direction and by means of the
mucus which is secreted by the mucus cells, they collect and entangle
the micro-organisms and carry them over to the free edge of the gill

BACTERIOLOGY OF THE OYSTER. 9

where a third set of cilia located on the very edge of the gill conveys
the entangled organisms on the mouth. The arrangement of these
last two sets of cilia can be seen in the plate. In this diagram
the bent arrows show the course of the water through the gills into
the cloacal chamber. The straight arrows indicate the course of the
mucus and the entangled micro-organisms to the mouth.

When the valves of the oyster are open the current induced by the
cilia is carried out of the oyster between the points “A” and “‘B.”
When the valves of the oyster are closed, however, the cilia keep
waving as vigorously as before, because the oyster has no control over
their movement, but in this case the current cannot pass out between
the valves and we have what might be called a closed circulation.
Instead of going out between the points “‘A’”’ and “B,’’ as is the case
when the valves are open, the current must neccessarily return to the
gill chamber around point “A,” for a study of the currents induced by
the cilia and taking the direction indicated by the arrows shows that no
other course is possible. All the cilia of the cloacal chamber direct
their motion towards point ‘‘A”’ and ‘‘B.”’ All the currents in the
branchial chamber are either through the gills into the cloacal chamber
or along the edge of the gills to the mouth. As water is driven through
the gills to the cloacal chamber water from the cloacal chamber must
necessarily take its place. As point ‘‘A”’ is the point of least resistance
the water necessarily passes from the cloacal chamber to the gill
chamber around that point and further not only is there nothing to
obstruct this current, but the current induced by the cilia on the edge
_of the gills is such that it would draw the water from the cloacal
chamber into the gill chamber around this point. Hence we see that
in the oyster we have a complete cycle of currents induced by ciliary
motion. The result is that all the water in the oyster is filtered
through the gills many times in an hour and the process is repeated
every few minutes.

It happens that when bacteria enter the gill or branchial chamber,
two courses are open. They may follow the currents through the
gills into the cloacal chamber or they may become entangled in the
mucus of the gills and be conveyed along the edge of the gills to the
mouth. The chances of a bacterium going in either of these courses
are about equal and if many bacteria are present some may go by one
course and some by the other.
® A study of table No. 1 will show that the B. prodigiosus followed

both of these courses, some were entangled in the mucus and were
2

10 BACTERIOLOGY OF THE OYSTER.

carried to the mouth (hole No. 4) while others escaped the mucus
and passed through the gills into the cloacal chamber (hole No. 2).
A further study of table No. 1 will show that the bacteria passed with
the currents for this particular bacterium was non-motile and so
could not have reached the different points by its own activity.
Moreover, the interval of time which separated the inoculation of the
branchial chamber and the subsequent recovery of the bacterium
from the different holes in series II was only 4 minutes in all, except
two cases when it was six and fifteen minutes. The distance between
holes 5 and 4, 5 and 2, and 5 and 3, in all cases was at least an inch,
in most cases, more. In series III the bacterium was recovered from
all the holes in four minutes in one case and ten minutes in the other.
The rate of travel of bacteria varies with the species, temperature,
etc., but it is inconceivable that a bacterium of the speediest variety
could move a distance of over an inch in four minutes by its own activ-
ity. In the case in hand, 2. e. a non-motile bacterium, it is out of
the question.

It is also seen in table No. 1 that in two cases B. prodigiosus was
isolated from hole No. 2 before it was recovered from hole No. 1. In
the two other cases they were recovered at the same time. While this
is not conclusive it leads the writer to believe that the bacteria
isolated at hole No. 1 had previously passed through the gills and the
cloacal chamber and back into the branchial chamber by the return
current. The results of the experiments in series III lend support to
this view. The bacteria did not go directly from hole 5 to hole 1
because the currents along the edge of the gills is too strong to allow
a bacterium to pass in that direction. An examination of this current
under the microscope will convince anyone that a bacterium could not
travel in that direction.

A study of table No. 2, which shows the appearance of B. prodigiosus
in the gill chamber after the inoculation of the cloacal chamber,
shows that the organisms appeared at hole No. 1 and later at hole Nos.
5and4. This is the order of time in which a current from the cloacal
chamber and taking the direction of the arrows of the edges of the
gills would appear at holes Nos. 1, 5 and 4, in the branchial chamber.

From the foregoing facts it is plain that the gills are not bacteria
proof; that bacteria can and do pass from the gill chamber to the
cloacal chamber through the gills and moreover, that bacteria may
pass from the cloacal chamber to the gill chamber without passing
through the gills. It is seen that we have a complete circle of currents

BACTERIOLOGY OF THE OYSTER. 11

within the closed shell of the oyster which, under the conditions of the
experiments, makes a complete circuit several times in an hour,
and thus ensures a thorough mixing of the water and the bacterial
content of the two chambers. In the conditions of the experiments
the complete circuit was made in at least six minutes and in three
cases in so short a period as four minutes. It naturally follows that
any difference of bacterial count between the two chambers is not to be
expected and such differences as are observed are within the limits of
experimental error.

METHODS OF SHELLFISH EXAMINATION.

As soon as sufficient epidemiological evidence had been accumulated
to show conclusively that oysters are under certain circumstances
contributing factors in the spread of typhoid, Asiatic cholera and other
gastro-enteric disturbances, it was but natural that bacteriologists
should look for the specific cause of these diseases in the oysters
themselves. If the typhoid bacillus and the spirdlum of Asiatic
cholera could be found in oysters, that would be evidence which no
one could dispute. Although diligent search has been made for the
typhoid bacillus in oysters on numerous occasions since 1893, it is
interesting to note that there are on record four instances only in
which B. typhosus has been reported to have been isolated from
oysters. The first instance was reported by Klein.’ Regarding this
finding Klein says:

“In view of the importance likely to be attached to the finding
of this bacillus in such numbers in one of these East Coast oysters,
particular care has been exercised in subjecting it to every possible
test . . . Asa result, in all and every one of its characters it
coincides with the typhoid bacillus obtained from the spleen of a
typical case of typhoid fever, and for this reason I am prepared to
affirm that this bacillus obtained from the “Deep Sea’’ oyster is the
typhoid bacillus.’’ Besides the cultural tests used, the Bordet-
Durham reaction (macroscopic agglutination with immune serum
1:100) and Pfeiffer’s phenomenon were also used and both proved
positive while the controls in both instances were negative. In
this instance the evidence seems quite sufficient to support Klein’s
assertion.

lReport of the Medical Officer to the Local Government Board, 1894-5. Supplement, Appendix
No. 2, p. 115.

12 BACTERIOLOGY OF THE OYSTER.

The second instance is cited by Fuller:' In 1902 at a meet-
ing of physicians at Pera, Turkey, it was reported that a large
percentage of the typhoid cases occurring in Constantinople could be
traced to the consumption of polluted oysters and an examination
of the oysters in a few instances showed the presence of B. typhosus.
The writer has not been able to obtain the reference to this paper and
the characteristics of the species have not been studied. As a result
no definite comment can be made upon the findings in this instance.

The third instance is reported by Johnstone.” There is not so
good evidence to support the identity of this bacillus as in the case
reported by Klein. Johnstone’s bacillus “formed acid and gas in
bile salt glucose broth” and a ‘‘a slight discoloration in lactose
litmus broth” and “agglutinated—in a dilution of one to thirty—
in a serum which gave a positive reaction with a known strain of
bacillus typhosus.’’ All authorities are agreed that the typhoid
bacillus produces no gas in any sugar medium. In regard to the
agglutination in a dilution of one to thirty, the writer is inclined to
question the specificity of so low a dilution. The report referred to
above does not say what the titre of the serum was with any known
strain of typhoid, nor whether one to thirty was the highest dilution
that would give a positive reaction, though we are led to suspect that
this was the case. A dilution of one to thirty cannot be relied
upon explicitly, for other organisms closely related to typhoid as
some strains of B. coli will agglutinate in a dilution of one to thirty
and in the case of a strong serum in one to one hundred.®

In 1908 Stiles* isolated four organisms from oysters obtained from
Jamaica Bay, Long Island which ‘‘resembled B. typhosus biologi-
cally, but did not agglutinate typhoid immune serum.” In 1911,
while investigating an epidemic of typhoid following a banquet given
October 5, 1911, at the Music Hall, Goshen, N. Y., Stiles again
examined oysters from Jamaica Bay, where the oysters were obtained -
for the banquet and in this instance he was able to isolate two
strains of B. typhosus from oysters “which had been allowed to
‘drink’ under an oyster house at Inwood, Long Island.” Besides

1The Distribution of Sewage in the waters of Narragansett Bay, with Especial Reference to the
Contamination of the Oyster Beds, App. to Rep. of Commissioner of Fisheries for year ending
June 30, 1904.

2Routine methods of Shellfish Examination with Reference to Sewage Pollution, Journal of
Hygiene, IX. 1909, 433.

3Hiss & Zinsser; Text Book of Bacteriology, 1912, p. 42.

4Bureau of Chemistry, Bulletin No. 136.

BACTERIOLOGY OF THE OYSTER. 13

showing all the cultural characteristics of the typhoid bacillus, it
also agglutinated in five minutes in a 1:1000 dilution of typhoid
immune serum. This organism was isolated from the oysters seven
days after they were taken from the water. Later oysters from the
same lot were examined after they had been out of the water twenty-
one days and kept at 39° F. An organism was isolated which
resembled typhoid in all its cultural characteristics and agglutinated
macroscopically in a dilution of 1:1000. This test was confirmed by
hanging drop preparations in dilutions of 1: 200.

There can be no possible doubt that the organisms isolated by Stiles
are true typhoid bacilli, while little can be desired to confirm the
identity of the organism isolated by Klein.

An interesting feature of the work of Stiles is that he demon-
strated the typhoid bacillus in oysters which had been infected
under natural condition and which had been kept out of water
for three weeks. Klein,’ Foote? Herdman and Boyce,’® and
others have reported instances in which typhoid bacilli have been
isolated after varying lengths of time up to 18 days after infection
from oysters artificially infected with large numbers of typhoid bacilli
in pure cultures or from typhoid stools and kept in sea water in the
laboratory. So far as the writer is aware Stiles is the first one to show
that oysters infected under normal circumstances with sewage con-
taining typhoid bacilli and kept under favorable conditions can still
harbor B. typhosus after 21 days. The condition here are somewhat
different from laboratory experiments in that in sewage along with the
- typhoid bacilli are other bacteria whose influence is exceedingly
hostile to the growth of B. typhosus.*

It is interesting to see that this organism has been isolated so few
times, in spite of the abundant epidemological evidence in so many
instances which points conclusively to the infection of oysters and
other shellfish with typhoid bacilli. The reason for this, however,
is quite readily understandable when we consider the number of
typhoid bacilli which could be found in the sewage of any town or
city in comparison with the number of other organisms found in
that same sewage. It would be a case of searching for the proverbial

Loc. cit.

24 Bacteriological study of Oysters, with Special Reference to them as a source of Typhoid
Infection,’”’ 18th Ann. Report Com. State Board of Health.

3Oysters and Disease, Thompson Yates Laboratory Report, 1-2.
4Jordan, Russell & Zeit, Journal of Infectious Diseases 1, 1904, 641.

14 BACTERIOLOGY OF THE OYSTER.

needle in the hay stack. Moreover the incubation period of typhoid
varies from two to three weeks and this would make the period of
infection two or three weeks before suspicion would be thrown on the
oysters. That is, two or three weeks would elapse after infection,
before we began to look for the organism. During this space of time
the other oysters of the same laying would, in all probability, have
time to rid themselves of the organisms, provided they too were
infected. In the case of an epidemic of typhoid due to eating raw
oysters, the time to examine the oysters for typhoid bacilli would be
at the moment they were eaten. We may be quite sure from the
history of the cases that the oysters which were consumed did
contain B. typhosus, but we have no assurance that all the oysters
of that particular bed contained the organism. There is great
variation in the number of sewage organisms contained in the indi-
vidual oysters of the same bed. ‘This individual variation will be still
greater if the bed is large and the amount of sewage small, tho highly
infected with B. typhosus and other sewage organisms. Sewage
does not ordinarily contain typhoid bacilli in constant numbers at
any time and unless there is an extensive epidemic, B. typhosus would
appear only intermittently and then in comparatively small numbers.
In view of these facts the wonder is, considering that B. typhosus
die off rapidly, both in sea water and in oysters, that typhoid bacilli
have ever been found at all.

The spread of cholera through infected oysters has not attached so
much attention as the transmission of typhoid. The latter is distribu-
ted much more widely throughout the world and the opportunity
for such transmission is much greater. Occasionally, however, there
has appeared references to the spread of cholera through infected
oysters. In 1849 there was a small epidemic of cholera in England
which was attributed to eating oysters. In 1893 Sir Richard Thorne
attributed a number of scattered cases of cholera in England to the
consumption of oysters. Recently it has been reported that a large
extent of oyster beds in Italy have been destroyed because they were
thought to be a menace to the public health on account of the danger
of the cholera infection.

In most, if not all epidemics of typhoid from infected oysters or
other articles of food, there have been a greater number of cases of
gastro intestinal disturbances which have not developed into

* --

BACTERIOLOGY OF THE OYSTER. 15

typhoid.1 We cannot tell the exact cause of these intestinal
upsets. It may be due to bacteria other than typhoid or it may be
due to chemical or ptomaine poisons which appear in the sewage as
the end products of bacterial metabolism. Whatever the cause we
are led to expect these disturbances as concomitants of any outbreak
of typhoid due to an infected food.

Since one can rely so little upon the finding of the specific disease
organism in sewage and in oysters, it was but natural that an index
of greater reliability should be sought. Klein? at the begin-
ning of his experimental work as well as in some previous investigations
ascertained that B. coli and other intestinal bacteria form no part
of the flora of oysters grown in non-polluted water and for this reason
used B. coli as an index of pollution. Klein’s observations in regard
to the bacterial content of oysters grown in water free from sewage
has been confirmed by Houston,® Ferguson,* Fuller® and others. The
presence of B. coli as an indication of sewage pollution has been
adopted by all workers in this field and is the index used to-day to
determine bacteriologically the presence of fecal matter.

In examining oysters, however, we have quite a different problem
from the examination of water, for we have not only the juice, but the
body of the oyster, the mucus covering the body, the alimentary
canal, etc., to consider. It is interesting to see how the methods of
examination have changed as our knowledge of the bacteriology of
the different parts of the oyster has increased.

Perhaps the first person to make an extended study of the bacteri-
ology of the oyster was Klein, who in 1893,° made a study of the
“Relation of Oysters and Disease” for the Local Government Board.
Klein describes his method of analysis as follows:—

“Hach oyster was carefully washed and brushed in a small quantity
of sterile water, with a view to collect therein any microbes adhering
to its shell. Next, the oyster, after a further cleansing under a water
tap and drying with a clean cloth was opened with a sterile knife,

14s an illustration, the reader is referred to the following reports: H. T. Bulstrode, in local
Government Board, 32d Annua! Report, 1902-1903, Suppl. App. A. pp. 129-189; H. W. Conn, The
“Oyster Epidemic”’ of typhoid fever at Wesleyan University, Medical Record, 46,1894, 743-6;
G. W. Stiles, Sewage Polluted Oysters as a Cause of Typhoid and other Gastro-intestinal Disturb-
ances, Bureau of Chemistry, Bulletin 136, 1912.

2Loe. cit.

34th Rep. Royal Sewage Commission, 1904.

4Bull. Virginia State Board of Health, May, 1909.

*|Loc. cit.

®Supplement to Report of Medica! Officer to Local Government Board, Appendix No. 2, pp. 109
and 117.

16 BACTERIOLOGY OF THE OYSTER.

and its body mashed up with the liquor contained inthe shell . . .
and about 14 to % c.c. of the liquor and the oyster tissue was removed
by means of a freshly made capillary pipette and introduced into a
phenolated broth tube which was incubated at 37° C for 24 hours.’’
If growth occurred the culture was plated and the suspicious colonies
fished and studied in pure culture. This method allowed no compari-
son between the bacterial content of the shell liquor and the “oyster
tissue.”’ Besides it did not allow a determination of the number of
colon bacilli in the whole oyster nor per unit volume. Moreover,
we have no evidence that any part of the oyster tissue except the
epithelium of the outside of the body and the lining of the alimentary
tract contain bacteria and this large amount (in comparison to the
amount of shell liquor) of finely divided tissue—for it must have been
finely divided to have been taken up in a capillary pipette—would
interfere greatly, if one tried to obtain an accurate count.

Chantemesse, in June, 1896, reported to the Académie de Medicine,
Paris, his observations on the relation of oysters to disease. In the
article presented at this meeting he does not give the details of his
technique, but says the shell liquor and the bodies of the oysters were
submitted to a bacteriological examination and B. coli were found.

The next important investigation after that of Klein is the work of
Herdmann and Boyce.! A great number of experiments were per-
formed on the chemistry and biology and also on the bacteriology
of the oyster. Only a small part of their work related to the presence
of B. coli in normal oysters. For this work the stomach contents
were used. The following is quoted from their report :—

“The method of analysis consisted in first cauterizing the mantle
over the region of the stomach and then inserting a fine sterilized
glass pipette, the pipette was moved about and when sufficient of
the contents of the stomach and the juice had risen in the pipette,
the latter was removed and its contents transferred to liquified agar,
ordinary gelatine or sea-water gelatine and plate cultivations made.”

Apparently no attempt was made to determine the number of colon
bacilli either per unit quantity or in the contents of the stomach as a
whole.

The next important investigation we have noted is the work of Dr.
Houston.” Dr. Houston’s method of analysis is as follows:—

1(1) Lancashire Sea Fisheries Memoir No. 1. (2) Proceedings of Royal Society, 1899. (3)
Thompson Yates Lab. Rpt. 1-2.
2Fourth Report of Royal Sewage Commission. Vol. III, 1904

BACTERIOLOGY OF THE OYSTER. 17

Cleaning of Oysters :—

“The outside of the oyster shells was well scrubbed with soap and
water, and cleansed as thoroughly as possible under running water;
the shells were then well washed in running main water, and finally
with sterile water.

Cleansing of the Hands :—

“The hands of the experimenter were thoroughly cleansed with a
hard scrubbing brush, soap and water, then rinsed first with 1:1000
corrosive sublimate solution, and finally with sterile water.

Subsequent Procedure :—

“The oysters were laid out upon a sterile towel, the flat shell
uppermost. They were opened in this position with a sterile knife,
held in the right hand, while they were held in this position with a
corner of the sterile towel grasped in the left hand. Great care was
taken to avoid any loss of liquor in the shell. This liquor was poured
into a sterile 100 ¢.c. cylinder, the oyster was then partly cut with
sterile scissors and the liquor thus freed allowed to run into the
cylinder. Ten oysters were thus treated in each experiment. The
volume of the oyster plus the oyster liquor was read off, and usually
varied bewteen 80 and 120 c¢.c.. so that the oysters, being of medium
size and containing a medium amount of liquor, 100 ¢.c. might be
considered a fair average of the total shell contents of the ten oysters.
- Sterile water was then poured into the cylinder up to the 1,000 e.c.
mark, and the whole well stirred with a sterile rod.

“An Alternative Quantative Method for the Bacteriological
Examination of Oysters.

“An alternative method for the bacteriological examination of
oysters may be given here, although the routine work, except where
otherwise stated, has been carried out by the foregoing method.

“The oysters are cleansed and opened, with the same precautions
already noted. Then the body of the oyster is cut into small pieces
with sterile scissors: this process should be carried out in such a way
as to insure the thorough mixture of the gastric juice of the oyster
and the liquor. The oyster, meanwhile, is carefully held with the
concave shell downwards and the flat shell bent back or altogether

removed. To examine the liquid contents of the shell without this
3

18 BACTERIOLOGY OF THE OYS TER.

preliminary step may partake of the nature of the examination of
the last sample of sea water imbibed by the oyster before finally
closing the shell. Indeed, the experiments detailed elsewhere seem
to indicate that per unit volume the gastric juice of the oyster may be
more impure bacteriologically than the oyster liquor.

“For cultural purposes the following quantities were made by proper
dilutions:—100 ¢c.c., 10 ¢.c., 1 ¢.c.. 1-10 c.c., 1-100 e.c., 1-1000 ¢.c.”

It appears that this was the first attempt to determine the number
of B. coil or coli-like organisms within the oyster. The supposition
was that the supernatant liquid above the oysters contained in an
even distribution all the bacteria that were present in the shell liquor,
the juices of the body, and on the outside of the oyster. Whether
this assumption is true or not will be discussed later when the writer
takes up his own experiments. Houston also performed “‘a series of
experiments to ascertain the relation between the biological (bacterio-
logical L. A. R.) composition of (1) the shell liquor and surface
“washings”’ of the oyster, and (2) the “‘ washed bodies of the oysters.’
In this series of experiments, four in number, by rapid fire calculation
and assumptions, Houston arrives at some very startling conclusions.?
From these experiments he states that volume for volume the stomach
of the oyster contains more bacteria than any other part of the
oyster. The method of conducting the experiments and the premises
assumed and conclusions drawn will be discussed more at length
when the writer takes up similar experiments of his own.

Fuller in the article cited above describes his method as follows :-—

“In the examination, inoculations were made from the liquor
contained between the shells, from the contents of the intestines,
stomach, and rectum, and in some cases from portions of the visceral
mass. In order to obtain samples of the juice from an oyster under
aseptic conditions, the speciments to be examined were scrubbed’
thoroughly in tap water with a stiff brush, washed off in running
sterile water, and dried on a sterile towel, after which they were
opened with a sterile knife. To obtain cultures from the stomach,
the top of the mantle covering the interior end of the oyster was slit
open and the large palps on either dide of the mouth pushed aside;
the mouth region was sterilized by passing a hot scalpel over these
parts and a portion of the stomach contents was drawn out by means
of a fine pipette or platinum loop introduced through the mouth
opening. Cultures from the intestines were made in the following

ISee page 27.

yt

BACTERIOLOGY OF THE OYSTER. 19

manner: After opening the shell, the oyster was removed from the
shell and dried between filter papers. A hot spatula was then passed
upon the surface of the mollusk directly over that portion of the in-
testine which it was desired to reach, and the tube was then opened
with a sterile scalpel. Through this opening a portion of the contents
was drawn out by means of a pipette or platinum loop. Portions
of the visceral mass were obtained by cutting out cubes of flesh from
that portion of the body after sterilizing the surface with a hot
sealpel.”’

MeWeeney’ in his examination of oysters on the Irish Coast used
the shell liquor alone, if abundant. But in cases where the amount
of shell liquor was small he supplemented the small quantity of
liquid ‘with a block of tissue cut from the animal itself so as to
include portion of the alimentary canal.”

The next worker to do a great deal of routine and experimental

work in the examination of shellfish was H. W. Clark. In a prelimi-

nary report published in 1902? Clark describes his method of analysis
as follows :—

“To determine the presence of B. coli in the juice on the shell, the
clams, oysters, etc., were washed with sterilie water, then opened,
and this juice inoculated into bouillon.”

“To determine whether the germ was present in the bodies of the
clams, oysters, etc., they were opened after washing with sterile
water, and the intestine, after maceration with sterile water, was
inoculated into phenol dextrose bouillon.

In 1905, Clark? in a report covering his experimental work for the
previous five and one-half years makes the following statement in
regard to the “Examination of Raw Oysters:’’—‘‘The shell liquor
and the crushed body of the oyster were examined together by insert-
ing the entire mass in a fermentation tube, and if fermentation was
obtained, carrying out the cultural tests.”

In determining the presence of B. coli in the body of the oyster as
detailed in his first report it appears that Clark disected out the ali-
mentary tract. This is not stated as part of the procedure, but it is
implied from the above quotation. This procedure would be rather
cumbersome if one attempted to use it on a large scale in routine exam-

1Report on the Bacteriosecopic Examination of Samp!es taken from Shellfish Layings on the Irish
Coast, Local Government Board for Ireland, 1904

2Senate Document 336, State of Mass., 1902.

3Report Mass. State Board of Health, 1905, 427.

20, BACTERIOLOGY OF THE OYSTER.

inations. Moreover, Clark apparently assumes that the bacteria
isolated in this manner all came from the intestinal tract and that no
contaminating organisms from the mucus on the outside of the body
entered into the bacterial flora of the macerated intestine. The writer
in some experiments to be given in detail later has shown that on the
average there are more—often many times more—bacteria in the
mucus on the body of the oyster than in the total amount of shell
liquor and further that volume for volume the contents of the stomach
do not contain so many bacteria as the shell liquor. Since the stomach
contains more liquid on the whole than the rest of the intestinal tract,
it is but natural that it should contain more B. coli than the remainder
of the intestinal tract. This would be all the more evident when it is
understood that B. coli do not grow in oysters, but probably diminish
as they pass through the intestinal tract.’

In his second article cited above, the whole contents of the oyster
shell, ‘‘the shell water and the crushed body of the oyster were ex-
amined together by inserting the entire mass in a fermentation tube.”’
Obviously this would allow of no comparison between the bacterial
flora of the shell liquor and the body of the oyster. Yet in a following
paragraph and also in a table he gives the results of the analysis in
“Per cent. of Samples Giving Positive Tests,’’ in “Shell Water,
Intestine” and ‘‘Mash.”’ Obviously there is some discrepancy, for
if he followed out the method described it would be impossible to
make such a differentiation. It is possible, however, that Clark was
using a combination of the technique as stated in the two reports.
The shell water and the “intestinal content’’ were examined as stated
in his report of 1902, and his ‘‘mash”’ consisted of the shell liquor
and crushed body, the entire mass of which was inserted into the fer-
mentation tube. It would appear, however, that in order to carry
out a combination of these two pieces of technique, two oysters would
be necessary, one for the shell liquor and intestine and another for
the “shell water and the crushed body.” If this were true the
individual variation of course, would allow of no definite comparison
between all the parts tested. It may mean that the remains of the
body tissue after dissecting out the intestine and the unused portion
of the shell liquor were mixed and constituted the shell water and
crushed body. But, in whatever manner we try to explain the matter,
the fact remains that the method as described is insufficient to account

Hardman & Boyce, !oc. cit.

BACTERIOLOGY OF THE OYSTER. 21

for the results obtained. But, as the results are expressed in the text
and again in more detail in a table, we can feel quite certain that the
method of analysis in the second report is not given in sufficient detail
and the results expressed in the table are accurate so far as his
methods would allow.

From the table referred to above it is seen that in the examination
of one hundred and forty-five oysters approximately fifty per. cent of
them gave positive tests for B. coli in the shell liquor, seventeen
per cent in the “mash” and between seven and eight per cent. in
intestine.

In three following tables is given the results of the analysis of shell
liquor and intestine of 265 other oysters, making a total of 410 oysters
examined in all. A comparison of the percentage of positive results
in the shell liquor and intestine shows that B. coli were found nearly
four times—50 to 14— as often in the shell liquor as in the intestine.
From these experiments it seems apparently beyond question that
the greatest number of B. coli are in the shell liquor of the oyster
and that the body of the oyster should be disregarded in our search
for the colon bacillus.

Stiles! describes his method of analysis as follows:

“The examination of composite samples of five or more oysters was
supplemented by inoculating media with the liquor from single oysters
to determine the presence of Bacillus coliin each. It was also decided
to use only the liquor bathing the oysters, instead of both meat and
_liquor, as the latter represents the character of the whole contents
of the shell sufficiently well to determine the presence of pollution.’’

Gage” discribes his methods as follows :—

“The upper shell being removed, a portion of the liquor in the
lower shell is now transferred to a fermentation tube with a sterile
pipette, or a portion of this shell-water may be carefully poured
directly from the shell into the tube. The latter method is much
simpler than the use of pipettes, but requires that the shell be so
handled in the previous operation that the lip over which the liquor
is poured has not been contaminated. The body is now washed with
sterile water, then while held with the fingers of the left hand, an
incision is made with a sterile scalpel and a portion of the intestine

Shellfish Contamination from Sewage-Polluted Waters and from other Sources, Bureau of
Chemistry, Bulletin 136, Apri! 11, 1911.
2Methods of Testing Shellfish for Pollution, Jour. or Infectious Deseases, 1910, VII, 78.

22 BACTERIOLOGY OF THE OYSTER.

transferred with sterile forceps to another fermentation tube, care
being taken not to touch the parts where the incision is made with
the fingers or to contaminate it in any way. This procedure is
repeated until 10 individuals have been tested from each sample jar.’”’

It would appear that the work of Clark has had wide influence in
determining the method of shellfish analysis now in use in this country.
So far as the writer is aware and so far as the literature at hand shows,
the only part of the oyster used for bacteriological analysis for some
years has been the shell liquor. The ‘Committee on Standard
Methods of Shellfish examination’? appointed by the American
Public Health Association has recommended the use of the shell
liquor only. So far as a perusal of the recent literature is concerned
no one has questioned the advisability and propriety of using the shell
liquor alone for analytical purposes except Gorham’ upon results
obtained by the writer in the laboratory of Brown University.

It will be noticed in all the work cited in which parts of the intestine
have been used for analysis, except in the case of Fuller, no mention
has been made of trying to avoid taking bacteria from the outside
of the oyster as well. In the writer’s opinion a great many of the
bacteria alleged to have been found in the intestinal tract have come
from the mucus on the outside of the body. There is no doubt that
the’ intestine of the oyster does contain bacteria of sewage origin,
but the mucus on the outside of the body is much more likely to
contain such bacteria.

BACTERIOLOGY OF THE SHELL LIQUOR AND “WASHINGS”
FROM THE BODY OF THE OYSTER.

A matter of great interest to the writer is that in all the work done
upon oysters experimentally and otherwise no one has mentioned the
mucus of the oyster or apparently realized that it plays any part in the
bacteriology of the oyster.

The matter of the mucus in the oyster Juice and on the oyster’s body
appears so self-evident that it seems impossible that it should have
been entirely neglected. This mucus serves at least two purposes.
(1) It acts as a protection to the body of the oyster and protects it
from the deleterious effects of sea water in just the same way as the
mucus of the dog fish and other selachians protects their skin from

1Report of Commissioners of Shell Fisheries, State of R. I., 1914.

BACTERIOLOGY OF THE OYSTER. 23

the action of the sea water. (2) The other and more important
function from the bacteriological point of view is that it serves as a
net for the entrapping of the food of the oyster which consists largely
of diatoms and alge, but is made up of all sorts of microscopic
particles, living or dead, organic or inorganic. As a consequence, the
bacteria as well as the other microscopic organisms get entangled in
this mucus.

When one opens an oyster and collects the juice, usually a great
many particles of mucus, some particles very large comparatively
speaking, are seen in the liquid. If one handles an oyster after
opening, it is found covered with a vicid, slimy substance which does
not wash off the hands easily If the bodies of the opened oysters
are allowed to stand for sometime there rises to the surface long
strings and flakes of this greenish yellow mucus. In shucking houses
it is customary to allow opened oysters to lie for some time in large
vats filled with water and with occasional stirring allow the mucus
to rise to the surface of-the water and run over the edge, if running
water is used, or if not, it is skimmed off with a perforated dipper.
This mucus often collects in “ropes” two, three, or more inches long
and sometimes in large flakes the size of a half dollar.

If one examines the liquor of the oyster he has just opened, it usually
contains a great number of particles of mucus, some large, some small.
If one collects the liquor in a bottle and allows it to stand over night
it will be found to have separated into two distinct layers, a heavy,
thick, viscous layer on the bottom and a clear, more limpid layer on
the top. The bottom layer is the mucus which has precipitated out.
Standard Methods of Water Analysis requires a water sample to be
shaken twenty-five times before the analysis commences, in order to
break up any clumps of bacteria. The second Progress Report of the
Committee on Standard Methods of Shellfish Examination’ recom-
mends that “bacterial counts shall be made of a composite sample
of each lot obtained by mixing the shell liquor of five oysters. Agar
shall be used for the culture medium and in general the procedure
shall be in accordance with the method recommended by the com-
mittee on Standard Methods of Water Analysis of the American
Public Health Association.”’ It can be inferred from the last sentence
of the quotation that it includes shaking the sample. In draining
the liquid from the oyster the water runs out of the shell not at a
single point, but over a considerable part of the edge of the shell.

1Jour. Am. Pub. Health, IT, 1912, 34.

24 BACTERIOLOGY OF THE OYSTER.

For this reason the mouth of the ordinary water sample bottle is not
large enough to collect all the juice and so in most laboratories a
sterile petri dish is used for the purpose. This would preclude the
possibility of shaking. Now if shaking of a water sample, which
to the eye is perfectly clear, is advisable to break up the clumps of
bacteria and give a more even distribution of bacteria, what can be
said of the juice of the oyster which has a decided milky appearance
and which usually contains strings and flakes of mucus large enough to
be seen several feet away? If one plates a cubic centimeter of this
mixture without shaking, the flakes will appear in the solid medium
as irregular, opaque particles. The probabilities are from the
writer’s experience that the flakes of mucus carry a large number of
bacteria and we have a large confluent mass of colonies developing
around each mucus flake. Even if flakes are not present, large
confluent masses of colonies from the size of a penny to the size of a
quarter develop, which render counting impossible. Usually, how-
ever, only bile tubes are used for the presumptive test for B. coli and
no plates made so that this clumping is not noticeable except where
bile tubes do not duplicate or where one gets a positive test in the
1—10th c.e. or 1-100th ec.c. dilution and not in the 1 c.e. or a positive
presumptive test in the 1—100th c.c. dilution and not in the 1 e.e.
or 1—10th c.c. dilution. In a study of about 2,000 tubes in the pre-
sumptive test the writer found that they duplicated only about two-
thirds of the time and that in one set one might get a positive presump-
tive test in the 1—-100th c.c. dilution and in the duplicate set only in the
1 c.c. dilution or not at all.

Aside from the part played by the mucus in oyster juice the part
played by the mucus left upon the body of the oyster is, generally
speaking, much more important. Often much more mucus is left
upon the body of the oyster than is found in the oyster juice. As the
mucus is the part which catches the bacteria and holds them, it follows
that often more bacteria are left upon the oyster’s body than are
found in the oyster juice. Hence, it follows that, if we only examine
the juice of the oyster we are only finding a fraction of the bacteria
really present in the oyster. These facts will be brought out more
clearly when the experimental work upon which these statements
are based, is taken up.

The idea of comparing the number of bacteria found in the shell
liquor with the number that can be ‘‘washed” from the body of the

BACTERIOLOGY OF THE OYSTER. 2d

oyster is not new. ‘Houston performed a series of experiments
on this point and his technique and results are given in some
detail.

EXPERIMENT “A.”

September 9th, 1903.

“The oysters utilized for this experiment were gathered in the Helford
River, at low tide, on September 8th. They were cleansed in the
manner described elsewhere, before being opened with a sterile knife.
Each oyster was carefully detached from the two valves of its shell,
with as little injury as possible, and washed in the manner about to
be described.

“A sterilized funnel was placed in a sterile, 1,000 c.c. measuring
cylinder as shown in the accompanying figure. The liquor in the
oyster shell was poured into the cylinder before the oyster was
completely detached, and then the oyster was removed from the shell
with sterile forceps, held over the funnel, well washed with sterile
water, and allowed to rest in the funnel. Ten oysters were treated
severally in this manner, and then allowed to drain in the funnel.

ft. LIQUOR.

“The total amount of sterile water employed for washing
Peers ees owes Jasreiry Jet arde -hos (eral Siaioih More eae oe 810 c.c.
The total volume of liquid (oyster liquor and “washings’’)

Mm tne measuring cylinder was. =)... 5) oS. 2 Pee. 840 c.c.
Therefore the volume of oyster liquor for 10 oysters was...... 30 c.c.
or 3 ¢.c. liquor per oyster.

“The funnel containing the oysters was then lifted into a second
sterile cylinder, and sterile water was poured into the first cylinder
up to the 1,000 ¢.c. mark.

“The cultures were then carried out in the ordinary way described
elsewhere.

Results of the Examination of the Liquor.

“ Coli-like microbes were isolated in pure culture from 1 c.c. and 0.1
c.c. of the litre of mixed oyster liquor and sterile water.

Loc. cit.

4

26 BACTERIOLOGY OF THE OYSTER.

This result indicates that the litre consisting of oyster liquor
+ sterile water contained coli-like (apart from slow liquefaction of
gelatine) microbes in amount corresponding to about 10 per e.e.
The whole litre could thus be considered to contain about 10,000 coli-
like microbes derived from 30 c.c. of oyster liquor.

Hence, if 10 oysters yield 30 c.c. of liquor containing 10,000 coli-
like microbes, taking the average liquid contents of each oyster as
3 ¢.c., this works out at 1,000 coli-like microbes in the liquid contents
of each oyster, or about 330 coli-like microbes per c.c. of oyster liquor.

II. OYSTERS.

‘The oysters were one by one removed from the funnel, cut up with
sterile scissors, and placed in the second sterile cylinder. A known
quantity of sterile water (100 ¢.c.) was then added, the total volume
read off, and hence after deducting 100 c.c. the volume of the oysters
was obtained. It was found to be 90 c.c. Sterile water was then
added to the cylinder until the volume of the liquid was equal to
O00 Ge:

“The cultures were then carried out in the ordinary way described
elsewhere.

Results of the Examinations of the Oysters’ Bodies.

“ Coli-like microbes were respectively isolated from 10 c.c. and 1 ¢.e.
of the litre consisting of a mixture of washed oyster bodies and sterile
water.

‘ The litre consisting of sterile water-++macerated oysters might be
considered to contain about 1,000 coli-like microbes derived from the
bodies of 10 oysters. Therefore, each oyster body (deprived as far
as possible of its natural liquor) would contain coli-like microbes
corresponding in number to about 100.

“ The total volume of oyster bodies being 90 c.c., the volume of each
of the 10 oysters averaged 9 ¢.c.; each 9 c.c. of oyster body could be
‘considered to contain 100 coli-like microbes, or, roughly speaking,
11 coli-like microbes per c.c. of body bulk. The contrast is very
striking when this number is compared with that of 330 coli-like
microbes per c.c. of oyster liquor, ¢. e., volume for volume the oyster
liquor contains about 30 times as many coli-like microbes as the oyster
body.

BACTERIOLOGY OF THE OYSTER. 27

“ But the liquid contents of the oyster’s stomach are certainly much
less than 1 ¢.c., probably about 0.1 ¢.c. It is probably that the coli-
like microbes isolated from the macerated oyster bodies in the fore-
going experiment were totally, or in great part, derived from the
contents of the stomach and intestinal tract. In fact, it is conceivable
that the 100 coli-like microbes, which each washed oyster was found
to contain, were all, or to a great extent, derived from the stomach
juice which, for comparative purposes, may be assumed to be about
0.1¢.c. But if the body volume of each oyster be taken as 9 ¢.c., the
volume of the stomach contents on the above assumption is only about
one-ninetieth of the total bulk.

‘This view alters considerably the complexion of affairs. For the
ratio between the number, per unit of volume, of coli-like microbes
present, respectively, in the oyster liquor and stomach juice, would
then be 33:100. In other words, acting on this assumption the coli-
like microbes were three times more numerous per unit of volume in
the stomach or intestinal juice then in the oyster liquor.

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*On the assumption that all the coli-like microbes obtained from the macerated bodies of the
oysters were derived from the stomach juice (taking the volume of the stomach juice as 0.1c.¢,
and the volume of the oyster apart from its liquor as 10c.c.).’’

bo
CO

BACTERIOLOGY OF THE OYSTER.

From these experiments and the conclusions drawn it is clear that
Dr. Houston thought that all the bacteria on the outside of the oysters
were washed off with the sterile water used to wash the bodies of the
oysters and that all the organisms found in the minced oysters came
from the stomach. Whether we can accept Dr. Houston’s supposition
ornot will be discussed later under the writer’s own experiments in
this connection.

Stiles in the bulletin referred to above made some analyses showing
the relative numbers of bacteria in the shell liquor and meat of
oysters. He concludes: ‘‘The results show that the oyster liquor in
these samples contained more than seven times as many organisms per
given volume as did the minced meat and body contents of the same
oysters. The results further show that the liquor contained eight
times as many B. coli per cubid centimeter as the minced meat.”

Stiles does not give his method of determining the number of bacteria
in the minced body of the oyster. It may well be that his results
actually do show the relative numbers of bacteria in the two parts of
the oyster. His experiments included the results of only fifteen
analyses, and the results uniformally show a greater number of
bacteria in the shell liquor than in the minced body meat. In the
light of the writer’s results of similar analyses, however, we are led
to believe that the method of analysis is not adequate to demonstrate
the relative number of bacteria in the two parts of the oyster. It is
conceded by all that the tissues of the oyster are sterile. It is only
the outside of the body and the alimentary tract which normally
harbor bacteria. It is easy to understand how so much minced
tissue will interfere with accurate results. Secondly no mention is
made of how the bacteria were separated from the minced meat. An
immense amount of shaking would be necessary to make an even
suspension of bacteria if one tried to wash them from the minced
particles of the oyster meat. The bacteria are attached to the body
of the oyster by the mucus which is not easily removed. Even though
the minced oysters were shaken vigorously in water or salt solution,
the particles would quickly settle out and being more or less entangled
in the mucus a coagulum would be formed which settling out rapidly
would take a great many if not most of the bacteria out of suspension.
This is purely suppositional since the method of analysis is not given,
but this is a perfectly logical method of procedure and a very probable
explanation of the results. The temperature of the water from which
the oysters were taken is not given. In the writer’s opinion this is

=

BACTERIOLOGY OF THE OYSTER. 29

an important matter, for the temperature of the water will influence
the metabolism of the mucus secreting cells and will determine the
amount of mucus present on the body of the oyster. This matter will
be discussed further in another connection.

When the writer began his experiments, he did not know of Hous-
ton’s work and so the experiments were not carried out in exactly
the same manner, but, nevertheless, the experiments throw consider-
able light on the work just cited. The idea that the mucus of the
oyster played a part as yet unappreciated led the writer to perform
the following series of experiments.

Experiment I.

September 29, 1913, ten oysters were taken to the laboratory and
analyzed as follows: The oysters were opened according to
“Standard Methods” and the liquor drained into a small bottle
graduated in two cubic centimeter divisions. The oysters were
allowed to drain until a drop would not come away at least every five
seconds. The amount of liquor was then read off and an equal
volume of sterile salt solution added and the whole shaken vigorously
one hundred times. The body of the oyster was removed from the
shell and placed in a sterile jar and a quantity of sterile salt solution
added equal to the volume of the shell liquor. The jars were covered
and allowed to stand for a short time while the oyster juice was being
inoculated into plates and bile tubes. The jars containing salt
solution and oyster meat were then stirred vigorously with a sterile
- pipette and an attempt made to remove with the pipette as much
mucus as possible from the body of the oyster. Then one cubic
centimeter of the solution and dilutions thereof were inoculated into
plain agar plates and lactose-peptone-bile in the same manner as in
the case of oyster juice. A careful record was kept of the number of
cubic centimeters of juice obtained from each oyster and the amount
of salt solution used in washing each oyster in order to make a com-
parison of the bacterial content of all the shell liquor with the total
number of bacteria washed from the oyster. This would show which
part contained the greater number of bacteria.

Experiment II.

The above experiment was repeated on oysters obtained October 7,
1913. The total number of bacteria found in the shell liquor and the

30

BACTERIOLOGY OF

THE OYSTER.

washings from the bodies of the oysters in each of the two experiments
is shown in the following table:

Table Showing the Total Number of Bacteria in the Shell Liquor of each Sample
and the Total Number Washed from the Bodies of the Oysters Without Shaking.

Date. | 20 Count. B. coli Count.
|| —_——-.
Septe2Osmshellsichorsea ar 6 eee nee 330,000 7,400
SONMaS in 0624 5 Mh aeanter chon cise meter Ae 48,000 1,700
Octipenicens bellUlbiquones cei cece are 480,000 5,900
Washines! st Palsy Wee amen ne | 50,000 850
The detailed results are shown in the two following tables:
z | 20°C Count. B. coli Count. Score.
| &
[a
DATE. g = Number of S Based
S) & “iiera'| Racers | Nua | Wanner | Bose! | on Shel
= | % | in Shell fork Shell from Shell aaa
3 Es | Liquor. Oyster. Liquor. Oyster. | Liqour. Washings
Zi iow, |
Sept. 29, 1918..; 1]| 8) 27,000 1,900 1,600 800 200 300
He a Zee 4,000 1,600 80 400 20 120
oa " ial f3l fy 223000 2,400 60 30 20 30
ee ee 41 10 51,000 2,400 200 10 20 21
sf a 5| 11) 138,000 5,500 220 11 20 21
i ve 6; 10, 14,000 4,800 200 100 20 30
sf a 7| 9} 76,000) 14,000 1,800 90 200 210
4 ~ 8 13) 14,000 4,700 260 130 20 30
s i 9} 5) 75,000 6,000 1,000 50 200 210
o 10) 10, 58,000 4,600 2,000 100 200 210
Totals... .|...| 838 334,900) 47,900 7,420 1,721 920 1,182

nai ae

BACTERIOLOGY OF THE OYSTER. 31

i 20°C Count. B. coli Count. Score.
2 “a
4 |
Z| 8 : Based
Date. = 2 Number of Amare ot Number Number | Based ‘on, Shell
@ , r ak |
6 |° Bacteria | Washed in Washed on Liquor
alee in Shell ae shell from Shell and
° 3 Liquor. Oyster. Liquor. Oyster. | Liquor. | Wash-
6 | 0 ings.
a | ©
October 7,1913..| 1]| 10] 40,000] 1,000 200 100|,.. 20), 30
ss id .| 2] 20) 20,000 3,700 2,000 10 100 101
43 S 3| 12} 54,000) 20,000 240 10 20 21
. 7 4/10} 70,000 300 20 10 2 3
sy es 5] 18} 138,500 1,200 18 100 1 61
ne oe 6) 14) 126,000) 12,000 2,800 200 200 214
“ se 7| 18} 73,800 5,000 180 200 10 21
et 8} 10; 12,000 3,200 200 200 20 20
F fe 9} 12} 72,100 3,400 240 0 20 20
es a Pl) A Reppem alt 28) TES LLEE! Sb RT ORE PRED A ROeRO RO Ay, pd
Metal sig te arr’ us ...|...| 481,800) 49,800 5,898 830 393} ouletl

*Not examined.

It will be noticed that in the last two columns of the table is given
the score based upon the shell liquor alone and upon the shell liquor
and the “‘washings” from the oyster combined. The method of
scoring is based upon the same principal as the method of scoring

- recommended by ‘Standard Methods,”’ but it works out a little

differently for the method of analysis followed by the writer is not
strictly in accordance with ‘‘Standard Methods.” In the latter
method 1 c.c., 1-10 ¢.c. and 1-100 c.c. quantities of the shell liquor
are inoculated into lactose-peptone-bile. If the presumptive test
shows B. coli in 1 c.c. dilution and not in the 1-10 ¢.c. and the 1—100
c.c. then the score of this oyster is one. If it shows B. coli in 1-10 ¢.e.
and not in 1-100 c.c., the score is ten; if in 1-100 e.e. the score is 100.
In other words, the score of the oyster equals the number of B. coli
found in one cubic centimeter of the shell liquor. In the writer’s
experiments the shell liquor was carefully measured and diluted with
an equal volume of one per cent. NaCl solution. One cubic centi-
meter of this mixture was used to make the various dilutions. The
result is that the various dilutions contained 1% e.c., 20 ¢.c. and

32 BACTERIOLOGY OF THE OYSTER.

Vooo c.c. of the shell liquor. Gas appearing in these respective
dilutions would indicate two, twenty and two hundred B. coli per cubie
centimeter instead of one, ten and one hundred as in the procedure
of “Standard Methods.”

The last column gives the combined score, in other words, the score
based upon the number of B. coli in both the shell liquor and in the
washings. The number of B. coli in each is added together and
divided by the number of cubic centimeters of shell liquor. This
method makes no allowance for the amount of mucus present on the
body of the oyster. This quantity would not exceed one cubic
centimeter on the average, for many of the oysters were small. It is
more convenient and just as accurate for comparative purposes to
ignore this quantity, while it is much more convenient in dividing the
total number of B. coli found in the oyster by the quantity of shell
liquor. It avoids fractions much more often than would be the case
if we added one to the number of cubic centimeters of shell liquor.
Occasionally, however, in the combined score, the quotient is not an
even number and so the score is made the whole number next above
or below depending whether the fraction was less than or more than
one-half. Thus if the score came 20.4 it would be called 20; if the
fraciton were .5 or more it would be called 21.

It would seem from these experiments that there is no question that
the shell liquor contains many more bacteria than are left on the body
of the oyster and that in analysis we could ignore entirely the bacteria
left on the body of the oyster.

These results did not equal the writer’s expectation and it was
thought that perhaps the treatment of the oyster’s body was not suf-
ficient to remove all the mucus and bacteria present. Accordingly the
following method of analysis was adopted for the subsequent experi-
ments: The oyster liquor was collected and diluted in the same manner
as before. It was shaken vigorously one hundred times before inocu-
lating into agar and bile. The body of the oyster after draining was
transferred to a sterile large mouthed, glass stoppered bottle and
covered with twenty cubic centimeters of one per cent. NaCl solution.
The oyster and salt solution were shaken fairly vigorously one hundred
times and the solution of salt and mucus was removed by the pipette
or poured into a smaller glass bottle and again shaken vigorously
one hundred times. This mixture was then inoculated into the bile
tubes and the agar plates. At first one per cent. sodium carbonate

BACTERIOLOGY OF THE OYSTER. 33

solution was used with the hope that it would cut the mucus more
readily, but later the salt solution was found just as effective. The
shaking appeared to be the important feature.

It was found that a great deal of shaking was necessary to break
up the clumps of bacteria and separate them from the mucus. If
not thoroughly shaken the resulting plates would be found to contain
large areas of confluent colonies which rendered counting impossible.
Every bit of mucus would be found to be a nucleus around which
would be a large confluent ring of colonies. After a thorough shaking,
however, the flakes of mucus would in nearly all cases remain sterile
and the bacteria would be found in well separated colonies evenly
distributed in the medium.

5

THE OYSTER.

BACTERIOLOGY OF

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BACTERIOLOGY OF THE OYSTER.

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BACTERIOLOGY OF THE OYSTER.

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OYSTER.

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BACTERIOLOGY OF THE OYSTER. 43

In comparing the total number of bacteria in the shell liquor of all
the oysters in each of the experiments with the total number washed
from the bodies of these oysters it is seen that the total number of
bacteria in the shell liquor of all the oysters was greater than the
number washed from the bodies of the oysters. In the first experi-
ment the numbers are nearly equal, but in the subsequent experiments
there is a great difference. If we consider the individual oysters in
all the experiments, we find that in only ten of the oysters out of
seventy-seven was there a greater number of bacteria washed from
the body than was found in the shell liquor of the corresponding
oyster. In one instance the numbers were equal. In the remaining
sixty-six oysters there were more bacteria in the shell liquor that were
washed from the bodies of the oysters. The 37° C. count and the
“red count’? were made on only seventeen oysters and in only two
instances did the number of bacteria washed from the bodies of the
oysters exceed the number found in the shell liquor, while the total
number from all the oysters of the two experiments showed that there
were on the average a great many more in the shell liquor than were
washed from the bodies of the oysters.

When we consider the number of B. coli found in the shell liquor
and the number washed from the body of the same oyster we find the
relative numbers quite different. It will be seen in six out of the eight
experiments the total number of B. coli washed from the bodies of all
the oysters of the experiment exceeded the total number in the shell
liquor. In the first two experiments the difference is especially
marked. If we consider the individual oysters we find that in thirty-
three instances there were more B. coli on the body of the oyster than
were in the shell liquor; in thirty oysters the number in the shell
liquor exceeded the number washed from the body; in fourteen
instances the numbers were equal. But if we consider the total
number of B. coli found in the ‘‘washings”’ with the total number
found in the shell liquor of all the oysters examined in this series of
experiments we find there were on the average more B. coli in the
“washings”’ than there were in the shell liquor.

We have no reason at present to suppose that B. coli should be
distributed other than equally among the other bacteria in the oyster,
yet there seems to be a concentration of B. coli in the mucus on the
outside of the body of the oyster. The amount of shell liquor in the
oysters averaged about ten cubic centimeters. If we consider that
there was left upon the body of the oyster one cubic centimeter of

At BACTERIOLOGY OF THE OYSTER.
mucus, we find that there were volume for volume more than ten
times as many B. coli on the body of the oyster as there were in the
shell liquor. The question arises at once as to whether this unequal
distribution of B. coli among the other bacteria in these two parts
of the oyster is real or only apparent. It may be due to the difference
in methods of analysis. With our present knowledge of the bacterio-
logy of the oyster the writer is led to believe that this relation does
not actually exist, but is due to the difference in methods used to
determine the total number of bacteria and the number of B. coli.
Another point which appears interesting to the writer is that there
is apparently a direct relation between the temperature of the water
from which the oysters are taken and the relative number of B. coli
found in the shell liquor and on the body of the oyster. It will be
noticed that in the first three experiments there were a great many
more B. coli on the body of the oyster than in the shell liquor, but
this proportion is gradually reduced and in the sixth and eighth experi-
ments there were more B. coli in the shell liquor than were found on
the bodies of the oysters. The ratio of the total number of bacteria
in the shell liquor to the total number washed from the bodies of the

oysters in each sample is shown in the following table:

Table Arranged According to Temperature Showing the Approximate Ratio of the
Total Number of Bacteria in the Shell Liquor to the Number in the Wash-
ings from the Bodies of the Oysters in each Sample.

202; 37°C. *aRedi B. coli
TEMPERATURE. Date. Count. Count Count. Count.
16S Gas oer ae Octrals a Dro eal Way 8 eseae ie ee Bd ED BR ono 35 Oh 1:3
Ger Cees eee Pe DE Deh Ie) Saale e eect || ane eee 1:18
Tey onl Gite en aa wapea See aR oa AA er eR ee IE Soi cogs: Sic 1:2..8
SS? Gy CoN Le BE Sh ye iad Nae ge VI NY aN Be Ibi ES}
PAS CLE petal Bry, Nov. 3 DeZi lbs a) Ae es See Ana eee Deel
LDS OCHS PaaS RR =f 8 eT dl Aes 8 FE eee PM
Srl iC dae. ee eee Dec. 6 GROr 6:1 (fal seal
(ahr es Cree aha ramet Sa ha io) Sis 10:1 AN weil Sell

A long series of experiments necessitating the examination of a
great number of oysters and extending over a whole year would be

required to establish this relationship.

However, this supposition

is not so different from what we might expect when we consider the

me

BACTERIOLOGY OF THE OYSTER. 45

biology of the oyster. The optimum temperature for the growth of
the oyster is, probably between 20° C. and 25° C. At this temperature
the cells of the oyster are most active. The mucus sells will secrete
a larger amount of mucus than at decidedly lower temperatures.
The more mucus secreted the more will remain clinging to the body
of the oyster. Generally speaking the greater the amount of mucus
the greater the number of bacteria we would expect to find in the
mucus on the outside of the body. As the temperature of the water
lowers, the metabolic processes of the oysters are correspondingly
slowed and a smaller amount of mucus and for this reason fewer
bacteria will be found on the body of the oyster. For this reason it
seems fair to assume that the apparent relation between the tempera-
ture of the water and the proportion of B. coli on the outside of the
oyster and the shell liquor is real and not accidental.

These two sets of experiments throw light on the findings of Houston
cited above. It is easily seen that simply pouring water over the
body of the oyster is not sufficient to remove all the bacteria. The
experiments of the writer on the comparison of the bacterial content
of the stomach and shell liquor shows that per unit volume the shell
liquor contains on the average over twenty times as many bacteria
as the stomach juices. Evidence from all sides shows that Houston’s
assumption that all the bacteria were washed from the body of the
oyster by simply pouring water over the oyster and further that the
bacteria found in the minced meat of the oysters so treated came
entirely from the stomach are not in accordance with the facts.

These experiments show the necessity of examining not only the
shell liquor, but also the mucus on the outside of the body of the oyster.
This is especially true during the warmer months. At this time
there are on the average many more B. coli on the body of the oyster
than is contained in the shell liquor. It is perfectly legitimate to
consider the mucus on the body of the oyster as part of the oyster
juice. If we so consider the mucus, it makes a very decided difference
in the score of the oyster. In one instance the combined score of one
oyster was ninety-six times the score based upon the shell liquor alone.
The combined score is never less and often many times more than the
score based upon the shell liquor. If there were any constant
relation between the B. coli content of the shell liquor and the mucus
removed from the body of the oyster, the examination of the shell
liquor alone would be sufficient. But as no such relation exists the
necessity of examining both the shell liquor and the mucus is at once
apparent.

46 BACTERIOLOGY OF THE OYSTER.

COMPARISON OF THE BACTERIAL CONTENT OF THE
STOMACH AND OF THE SHELL LIQUOR OF OYSTERS.

Houston in his report to the local Government Board, 1904, makes
the following statement: ‘‘The experiments detailed elsewhere seem
to indicate that per unit of volume the gastric juice of the oyster is
more impure bacteriologically than the oyster liquor.’’ The experi-
ments upon which this statement is based are taken up in some detail
under “Bacteriology of the Shell Liquor and ‘Washings’ from the
Body of the Oyster,” and so it is not necessary to take up these
experiments in this connection. The writer has shown that these
experiments and the conclusions drawn are not based upon sound
assumptions and so these results are not to be relied upon.

Clark,’ in along series of experiments has shown that in both
clams and oysters the shell liquor is much more likely to yield B. coli
or sewage streptococci than either the stomach, intestine or rectum.

Both of these workers studied the B. coli content of the different
parts of the oyster. The writer could not obtain any badly polluted
oysters at the time of year during which the experiments were con-
ducted and so he examined the shell liquor and stomach contents
for the total number of bacteria which each part contained. It
would have been possible to infect oysters artificially with the colon
bacillus, but it is not certain that one could simulate natural conditions —
exactly and consequently wrong conclusions might be drawn. We
have no reason to suppose that B. coli are distributed other than
equally among the other bacteria in the oyster and so a comparison
of the total quantity per unit volume ought to show the relative
frequency with which one would expect to find any particular bacter-
ium in either part of the oyster.

In this series of experiments forty-one oysters were used. The
method of examination was as follows:—The juice of each oyster was
collected in a small glass-stoppered bottle which was calibrated in two
cubic centimeter divisions. The amount of shell liquor was read off
in cubic centimeters and diluted with an equal amount of one per cent.
sodium chloride solution. The shell liquor and the sodium chloride
solution were shaken vigorously one hundred times and one cubic
centimeter of this mixture was transferred to a tube containing nine
cubic centimeters of one per cent. sodium chloride solution and

1Report State Board of Health of Mass. 1905, 428.

BACTERIOLOGY OF THE OYSTER. 47

one cubic centimeter of this dilution was plated in agar. Also a
cubic centimeter from this tube was transferred to another tube in
nine cubic centimeters of salt solution. A cubic centimeter of this
mixture was also plated. By this method of dilution the plates
contained respectfully one twentieth and one two hundredth of a cubic
centimeter of the original shell liquor. The plates were made in
duplicate. After the oyster had been drained of its liquor the flat
valve was removed and the other valve containing the body of the
oyster was set on the edge and allowed to drain for several minutes.
The excess of liquor was then removed with a piece of blotting paper
and the region over the stomach was seared with a hot spatula and an
incision made into the stomach with a sterile scalpel. With a gradu-
ated pipette one-twentieth of a cubic centimeter of the stomach
contents was removed and plated another twentieth of a cubic centi-
meter was transferred to a tube containing nine cubic centimeters
of salt solution and 1 cubic centimeter of this mixture plated. These
plates contained respectfully one-twentieth and one-two hundredth
of a cubic centimeter. The plates were also made in duplicate and
in all cases the average of the two plates was taken as the count for
each oyster. The counts given in the table below are for one-twentieth
of a cubic centimeter of the oyster juice and the stomach contents.

48 BACTERIOLOGY OF THE OYSTER,

Table Comparing the Number of Bacteria in One-Twentieth of a Cubic Centimeter
of the Shell Liquor with the Number of an Equal Quantity of the
Stomach Contents.

| No. ot bacteria | No. of bacteria

No. or Oyster. | in shell liquor | in stomach con-

| of oyster. tents of oyster.
EL. Co bs PAS REO Ce eee ee ede BP trod 20 1
Dre Ny eb res: hte: Sek OCC Eee 220 5
SGN LA. ASSES ERR ee er bebe Ae | 8 1
yt Re MCR the hl aaa owed aan et | 130 6
Fae Sk Re RS ONT aL A eer a Ma, Mics Ree (ie core 16 1
Ge ti heth GF Ake A RL te Seite Ea er opt saeete 1 11
Tee SS AEE E ET a. Bet Pe OER ED e-bae eeA 50 3
SNe eae. ede TORO Ge ER a Eres cye are 90 2
Od ae et TO Fan Bet oe 2a eee eng Mo oe 20 8
VO) ne ak SL Oe CE Ie oes 110 13
MD ee Ss oe. cece Re ete Cty ete nee aera eer 3 9
UDR so cc te ee ere ee Ata Cn ER RES Seta | 85 6
US iscpa fortes, seh Se any se cles PONE Rt de | 12 0
1: ae ae ee ers Oe Ie lol Wa ae ae 17 4
1c AL RMA oe ame ast BGA Ie SE ces 430 3
1 ea ee RAP ATEN acy Sc, bao tO .b Miche oF 55 4
(2 tec opts Soe ck ee Oe RRR OOM eas | 1,000 1
CPA Re A APL OM eee Sao a) SPER OB ee ra ticats Ae 95 1
1 0 ear A Pees: eyeee eer oki EU EE ES re ante 500 2
=A 0 tests aA ard hehe Fahd seta esata men noe Ee 260 1
Dia eek Se SAI tok SIRE To LR AYR ame tote io dewes Sel are 340 il
DOG MAIR AMEE POET TEE OS 6 toh 120 1
A ae oP Se RE, Bee ge Deanne Serene cae Mercere 340 1
PLE Fe CORRE Grr eer Cate Geen CEA AIO ee 155 0
DD ei Rae aheptaehs are TER a Soe a ner sie ee 1,000 2
PPA SWINE ite Bites a He ead deme el ele ae Avge 725 1
Dil Sa Rae GRATE MN bees te cee ene ghee 38 6
PAPE, cs Bites Pa dito eS A GS Ne ECR Ome 2 2
DO Ne RPE Os Entire epee rerun esata cts To 1,000 20
OO): Ace ae OR eae oh Mates eee 1,000 3
Oe SP boast LEE Pees, eet oe oii Ni gees 90 12
ACR PR be Beton ante ua Sey ote eae ana er A “19 14
er eS tes Sr COG Ty ho 5 ee An EA on 50,000 470
Oe EERE che Saat aa LAR ERS Merona cate tc 10,000 280
2339 PMP Ie Rees ts Pan de Ars eRe Go G.o8h 100,000 150
2 eR RRR ET oc aia ig Beat 7 5 15,000 10,000
2ST Rr aetna PONG Vr ts hls cry ed Rh Ae Se EE EC 10,000 10
ao alsa LRP ERDE AES casi ccilarhine tcf Poe ce etme ce eh 24,000 30
DOA SORE RAR Fs CRMSE ee Oc Ce cee eye 10,000 10
AO) pees RE te TO Oe ee 50,000 2,500
cE ROR rae le Rr RE, Ee Ar rake As eA eC Re tar 50,000 25
Eotals.ei cro con nee a eae ee rare 304,351 13,620

*

BACTERIOLOGY OF THE OYSTER. 49

From the table it is seen that in only two oysters, numbers six and
eleven, out of the forty-one examined, was the number of bacteria
per unit volume greater in the stomach contents than in the shell
liquor. In oyster, twenty-eight the numbers were equal. In the
remaining thirty-eight oysters there were more bacteria per unit
volume in the shell liquor than in the stomach contents. In these
thirty-eight oysters the ratio per unit quantity of the number of
bacteria in the shell liquor to the number in the contents of the
stomach varied from 3 to 2 in oyster Nos. 37 to 2000 to | in oyster
No. 41. The ratio of the total number of bacteria per unit volume
in the shell liquor of the forty-one oysters to the total number of
bacteria in an equal quantity of the stomach contents was as 21.6 to 1.
That is, a comparison of the average number of bacteria found in shell
liquor with the number of bacteria in the stomach contents shows that
per unit quantity there were more than twenty times as many bacteria
in the shell liquor as in the stomach juice.

LENGTH OF TIME NECESSARY FOR BACTERIA TO PASS
THROUGH THE INTESTINAL TRACT OF THE OYSTER.

So far as the writer is aware no one has ever made any determination
of the rate at which food passess through the alimentary tract of the
oyster. While it is difficult to determine this matter directly, it
seemed possible to inoculate the shell liquor of oysters with some
bacterium not found in oysters and trace its progress through the
intestinal canal. B. prodigiosus was chosen because of its ease of
identification and because the writer has never found it in oysters,
and so far as he is aware it has never been reported as occurring in
oysters.

In the first experiment twelve oysters were inoculated by sawing off
a piece of the lip of the shell and inserting a loopful of a culture of B.
prodigiosus into the branchial chamber. The oysters were layed very
carefully upon cotton thoroughly saturated with water and covered
with a glass dish to prevent evaporation. They were kept at the
laboratory temperature which is about 20°C. At various intervals,
as shown in the tables, three oysters were removed and examined. The
examination was made as follows:—The right valve of the oyster
was removed and the gills and mantle carefully dissected away. The
remaining part of the body was then washed for several minutes in

running tap water. The left valve containing the oyster was then
7

50 BACTERIOLOGY OF THE OYSTER.

set on edge and allowed to drain thoroughly. The surplus water was
removed with filter paper. The oyster was then seared with a hot
spatula over the stomach, over the intestine, where it bends sharply
upon itself on the ventral side and on the rectum just above the anus.
An incision was then made into these three parts of the alimentary
tract with sterile scalpels and a sterile capillary pipette inserted and a
portion of the contents removed and plated upon agar which was
grown at room temperature for two days and examined for red colo-
nies. Control samples of the shell liquor were plated before the inocu-
lation with B. prodigiosus and these were negative in all cases. In
the first experiment the time of examination after inoculation ranged
from thirteen hours to twenty-seven hours. In the second experiment
the time varies from five hours to seventy-four hours.

Table Showing Length of Time at which B. Prodigiosus was Isolated from Different
Parts of the Alimentary Tract after the Inoculation of the Gill Chamber.

No. or Oyster. Hours after Stomach. Intestine. Rectum.
inoculation.
EXPERIMENT I.
DPE AN bean Piprser eben ir meth anueree S ie 13 + 0 0
2 13 + 0) 0
3 13 0 + 0
ANG MIN FE Ae Lae tk SEN: Ne 18 0 0) 0)
D.. Messe esa od. Resor peers sine are 18 + 0 0
Gilepeshink Seis oRneee saa eptacueneee cee ite 18 0 + a
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8 23 0 0 0
Om. 23 0) (0) =f
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We 27 0) 0 0
Ta > Sea Bexe ist pee Meus Eat ame SER 27 + 0) a
EXPERIMENT I].
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De: 5 0 0) 0)
Se 5 a 0) 0
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De 22 0) 0 0
Gis u ce eee aa eS ence Daren ee 22 + 0 0
en 48 0 0 0
Sue 48 0 0) 0
Jerr 48 0 0 +
NO hatrd var. Si Bena cetaet Gare ace 74 0 0 -|-
11 74 0) sr aE
12 74 0 0 0

BACTERIOLOGY OF THE OYSTER. 51

From these tables it can be seen that the first appearance of the
bacteria in the intestine was thirteen hours after inoculation and in the
rectum five hours later. We would expect to find the organisms in
the stomach within a very short time after inoculation of the shell
liquor. Since these experiments are few in number one must neces-
sarily be conservative in the conclusion drawn.

THE BACTERIAL CONTENT OF OYSTERS DURING STORAGE.

The change in the bacterial content of oysters during storage at a
temperature at which they are kept in oyster houses and during
transportation is a matter of very great importance from the point
of view of the public health. The oyster is a living organism capable
of maintaining itself for a long period when removed from its natural
element. It is possible that the digestive juices or the phagocytic
cells of the oyster might materially decrease the number of bacteria
in the oyster. On the other hand, even if the digestive secretions and
the phagocytic cells were bactericidal, it is possible that the rapid
multiplication of the bacteria in the shell liquor might be sufficient
to maintain or increase the number of bacteria in the oyster as a whole.
In order to observe the change in the bacterial content of oysters
during storage the writer carried out the following experiment:

About a bushel of polluted oysters were taken from the Providence
River December 5, 1913. and put into storage in the Laboratory at an
average temperature of 10°C. The temperature was fairly constant
and did not rise above 11°C., although for a short time during a period
_ of exceptionally cold weather the temperature fell to 8°C., but it
soon rose again to 10°C. The oysters were put into storage in the
bag just as they were brought to the laboratory. No attempt was
made to clean them in any way. As soon as they arrived a sample
of ten oysters was taken from the bag and put on ice and examined
the following day. At intervals other samples of ten oysters were
removed and examined. The method of examination was the same
as that described under ‘‘The Bacteriology of the Shell Liquors and
the Washings from the Bodies of the Oysters.’’ In all except two
instances a 20°C. count, a 37°C. count, a “‘red’’ count, and a B. coli
count were made. The detailed analysis of each oyster and the
bacterial content of each sample as a whole is shown in the following
table:

THE OYSTER.

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61

BACTERIOLOGY OF THE OYSTER.

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62 BACTERIOLOGY OF THE OYSTER.

The results are so irregular that we can draw no very specific
conclusions. It appears that in the first two weeks there is no initial
decrease but rather a steady increase in the total number of bacteria
present. ‘This increase is also apparent in the 37°C. count and the
“red”? count. On January 27, fifty-three days after the beginning
of the experiment there was a remarkable change in the proportion
of bacteria in the “washing”? as compared with the shell liquor in
all except the B. coli count. The detailed analsyis for this date
shows that oyster number nine is responsible for this marked change.
It is very probably that this oyster had died and decomposition was
taking place.

The B. coli count shows a decrease on the fourth day, but this
decrease is not particularly marked and may well be due to variations
in the oysters and not to an actual decrease. This is all the more
likely when it is found that on the seventh day the number of B. coli
is approximately the same as on the first day. The subsequent
examinations show that the number of B. coli is about one-half the
initial number and remains fairly constant throughout the experi-
ment. In the last analysis made, eighty days after the beginning of
the experiment, all the bile tubes showing gas after twenty-four hours
incubation were tested for B. welchii by inoculating a cubic centimeter
of the bile into freshly sterilized milk tubes and incubating anzrobi-
cally. No visible change took place in the milk after eighteen hours
incubation. It was a noticeable fact that not over ten per cent. of the
tubes showing gas after twenty-four hours incubation had one
hundred per cent. of gas, the amount said to be characteristic of
B. welchii. Most of the tubes had about fifty per cent. gas. From
these facts it appears that the fermentation was caused by some mem-
ber of the B. coli group and not by B. welchii. It is not surprising
to find that B. coli should live eighty days in oysters under such
conditions for Clark’ has shown that B. coli will live in ten per cent.
sewage eighty-four days and in fifty per cent. sewage one hundred and
sixty-six days. Unpublished results from this laboratory show that
B. coli will live in sea water for one hundred and eighty days. The
writer has shown in the experiments on the hibernation of the oyster
that B. coli will live in oysters kept at 1.5°C. for at least one hundred
days.

1Report of State Board of Health of Mass., 1905, p. 455.

BACTERIOLOGY OF THE OYSTER. 63

The important conclusion to be drawn from this series of experi-
ments is that under the conditions of the experiment bacteria of the
B. coli group do not materially increase or decrease in oysters in the
shell during storage.

CLEANSING OF POLLUTED OYSTERS.

As soon as the etiological connection between oysters and certain
epidemics of typhoid and gastro-enteritis was firmly established, the
question at once arose as to how long a time it would take oysters
known to be polluted to free themselves from sewage organisms after
they had been removed to water free from sewage contamination.

Klein’ put oysters into tanks in the laboratory and infected them
with B. typhosus. About one-third of the water was removed every
day and replaced with clean sea water. Oysters were removed at
various intervals and examined for B. typhosus. The experiments
were repeated several times and B. typhosus was isolated at the end
of the experiment in every case. The various experiments were con-
cluded on the seventh, ninth, fourteenth, sixteenth, seventeenth and
eighteenth day after infection. The bacilli were isolated from the
sea water twenty-one days after the beginning of the experiment.
Of course, these experiments did not approximate natural con-
ditions and so we can draw no definite conclusions from them regard-
ing the length of time necessary for oysters to rid themselves of these
bacteria when taken from polluted areas and re-layed in water free
from pollution.

Herdmann and Boyce? tried the experiment of infecting oysters
artifically with large numbers of B. typhosus and then subject-
ing them “to a running stream of pure clean sea water.”’
Eighteen oysters were infected and examined at different intervals
varying from one to seven days. Only the stomach contents were
examined and considerable allowance must be made for this, for the
writer has shown in another part of this paper that the number of
bacteria contained in the stomach are quite insignificant compared
with the number in the shell liquor and on the body of the oyster.

In three of the eighteen oysters examined which had washed for
three, five and seven days, respectfully, no typhoid bacilli were
found. In the other fifteen oysters examined B. typhosus was

lRelation of Oysters and Disease, Supplement to the Report of the Medical Officer to the Local

Government Board, 1893.
2Loe. cit.

64 BACTERIOLOGY OF THE OYSTER.

found in varying numbers. Herdmann and Boyce sum up the
matter as follows: ‘The result was definite and uniform; there was
a great diminution or total disappearance of B. typhosus in from one
to seven days.”

Johnstone’ took oysters known to be polluted and transferred
to the purest water available. He found under the conditions of
the experiment that four days was a sufficient period of quarantine,
since after that time no further cleansing took place, because the
water of the locality was not entirely free from sewage contamination.

Phelps in this country” found that only two to four days was
necessary for polluted oysters to cleanse themselves when transferred
to clean water.

In 1913, Fabre-Domergue® read a paper before the Académie de
Medicine in which he recommended the placing of polluted oysters in
basins fed by filtered water and removed often enough to insure com-
plete evacuation of the liquid contained in the shells and in the
digestive tract. From his results he considers it an established fact
that this procedure eliminates all pathogenic bacteria from the mol-
luses in six or seven days.

Field* says: ‘‘These (oysters) get bacteria from the waters filled
with waste and sewage, and it takes them at least seventy-two
hours to free themselves from these impurities that they have taken
in from the waters of the different harbors.’’ Field does not say upon
what evidence, if any, this statement is based. But he adds that
in Massachusetts a law has been passed requiring such polluted
oysters to be transferred to clean water and allowed to remain for
four weeks before offered for sale.

The writer’s own experiments on the cleansing of polluted oysters
confirm in part the work cited above. It appears that the rapidity
with which sewage bacteria are eliminated is influenced to quite a
large extent by the temperature. If the water is warm, say around
20°—25 C. the oysters remain open probably most of the time.
As this is about the optimum temperature for the most rapid growth
and development of the oyster, it is also the temperature at which
the oyster is most active. The ciliary motion is more rapid than at
lower temperatures which would increase the amount of water

lJour. of Hyg. IX, 1909.

2Jour. Am. Public Health Assn., Vol. 1, 1911, 305.

3Cited in Jour. Am. Med. Asso., LXI, 1913, 134.

4Report of Proceedings of 3rd Am. Convention of Nat. Asso. of Shellfish Commission, p. 34.

BACTERIOLOGY OF THE OYSTER. 65

filtered through the gills and so increase the amount of “wash water’’
for carrying away the bacteria. Also the ciliary motion in the
alimentary canal would be hastened and so the organisms contained
therein would be more quickly disposed of. Further, the capacity of
the oyster to digest and assimulate bacteria would be at its height at a
temperature at which the cells are most active. Hence, the optimum
temperature for the growth and development of the oyster we would
expect to be the period at which all contaminating organisms would
be eliminated most rapidly. As the temperature lowers the activities
of the oyster lessen accordingly. Further, while above 20°C. the
oyster has its valves open most of the time, as the temperature is
lowered the oyster is more and more inclined to keep its valves closed
for longer and longer periods. This would prevent the mechanical
effect of the filtered water in carrying away the bacteria. This
mechanical effect is very important for the writer has shown in
another part of this paper that bacteria pass through the gills with
the filtered water very rapidly. Further, the activity of the cells
concerned in the digestion of bacteria would also be less active and
also the antagonism between different species of bacteria would be
lessened. So it is seen that at lower temperature the tendency would
be for oysters to eliminate bacteria more slowly than at higher
temperatures. Various opinions have been expressed regarding the
temperature at which oysters ‘‘hibernate”’ or close their shells and
remain closed due to the low temperature of the water. The theory
of “hibernation” of the oyster was first proposed by Gorham? to
explain the results obtained in his investigation of the sanitary con-
ditions of the oyster beds of Narragansett Bay. The temperature
at which this phenomenon is supposed to take place is a little above
0°C. So far as the writer is aware no experimental work of an exact
nature has been done to substantiate or disprove this theory, but
from personal observation the writer is led to suspect that the
temperature at which the oyster closes its shell for a relatively
long period is considerably higher as will appear from one of the
experiments detailed below.

On the other hand, experiments to be detailed later under the
hibernation of oysters seem to show that oysters do open and are
active at temperatures only one to two degrees above 0°C. It
appears that when the temperature is low oysters will close their shell

1(1) Rep. of Commissioners of Shell Fisheries of R.I.,1910. (2) Seasonal Variation in the
Bacterial Content of Oysters, Jour. Am. Pub. Health, Jan., 1912.
9

66 BACTERIOLOGY OF THE OYSTER.

for sometime, but not for indefinite periods. It also appears that
the closure of the shell is not due to cold rigor or loss of control of the
adductor muscle, for at a temperature of 1.5°C. the oyster can open
and close its shell with the same ease as at higher temperatures.

In the following experiments only the shell liquor was used. The
method of examination of the oysters was the procedure recom-
mended by the Second Progress Report of the Committee on Standard
Methods of Shellfish Examination of the American Public Health
Association. The medium used was lactose-peptone bile and the
tubes were inoculated in duplicate. The tubes were examined every
twenty-four hours for three days. If ten per cent. or more of gas
appeared during this time, it was considered to show the presence of
intestinal bacteria. Unfortunately in the first experiment the
investigation had to be discontinued after November 29th, so that
we have only the results extending over 12 days.

Experiment J.

November 16, 1912, about a bushel of polluted oysters were taken
from the Providence River and the following day were transferred to
Wickford Harbor. They were laid upon clean sandy bottom on the
edge of the channel and were well separated to allow free access of
water. The temperature of the water at the time of taking the
oysters was 14°C. The average of the maximum and minimum
temperature at Wickford for November 16 and 17 was 6.5°C.
A sample of the oysters was taken at the time they were placed in
Wickford Harbor and the analysis showed a score on fifteen oysters of
870. Samples were shipped to the laboratory every day until
November 29th. These were analyzed immediately so that only
three or four hours elapsed between the time of collecting the sample
and the time of analysis. The following table shows the results of
the analysis of fifteen oysters on the different days. The temperature
is the average of the maximum and minimum temperature as recorded
at the lobster hatchery of the Inland Fish Commission which was
located nearby.

67

BACTERIOLOGY OF THE OYSTER.

069 ‘a1099
|
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BACTERIOLOGY OF THE OYSTER,

68

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TIGHY sO NOIATIg

Date, November 24, 1912.

AVERAGE TEMPERATURE 6.1°C.

No. of Oyster.

DILUTION OF :
Liquor

wn
onl

14

13

12

10

BACTERIOLOGY OF THE OYSTER.

5 tae

“pa ah
Si

+++
++¢

+++
++

a gS
ry ore

maT 2
bare

aie ai
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a ae
hee

+++
+++

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+++

ed
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cli
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2

+++
+++

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feet

Ota: tence
=
DOO NCe ee

: 1,320.

Date, November 25, 1912.

AVERAGE TEMPERATURE 6,.1°C.

No. of Oyster.

DiLutTion or SHELL
Liquor

12

11

tao

+++

++¢e

+++

5 SaaS

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10

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a

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Score, 420.

69

BACTERIOLOGY OF THE OYSTER.

70

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71

BACTERIOLOGY OF THE OYSTER.

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‘TOO “gq 10F 480} oaTyduinsoid earytsod = +

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0 DOT 00> O 0. “O10 = OlO- 00 Tah) 10m 2S V0r- SOKO O1KO™ O10 O10) = 0" =F 2 °9 OOI-T
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to

BACTERIOLOGY OF THE OYSTER.

Unfortunately the experiments could not be continued and so we
‘cannot say whether the apparent cleansing, which appeared on the
last two days, especially on the last day, was due to a fortunate
selection of oysters or was the indication of a real elimination of the
intestinal bacteria. The writer is led to believe that the oysters
had just begun to open and so allowed the bacteria to be washed out.
The low temperature of the water slowed the metabolic processes of
the oyster and so, as food and oxygen were not needed in so great
quantities, an oyster could maintain itself for sometime without
renewing its supply. As soon as the supply was exhausted, however,
the oyster opened its shell.

This investigation shows that under the conditions of the experi-
ment with a temperature between 7.2°C. and 5°C. a period of twelve
days is not sufficient to allow oysters to free themselves from intestinal
bacteria.

Experiment II.

May 18, 1913, about a bushel of polluted oysters were taken
from Providence River and transferred to the same location in
Wickford Harbor as in the previous experiment.

The water of Wickford Harbor at the place where the oysters were
put down was tested by the lactose-peptone-bile presumptive test and
no sewage organisms were found. The methods and conditions of
the experiment were the same as in the previous experiment except
that ten oysters were used instead of fifteen. The sanitary condition
of the oysters at the time of transplantation and on two subsequent
occasions is shown in the following table:

BACTERIOLOGY OF THE OYSTER. 73

Tables Showing Results of Analysis of Ten Oysters from a lot of Polluted Oysters which had
been put into Relatively Unpolluted Water at Wickford, May 13, 1913.

Date, May 13, 1913.

AVERAGE TEMPERATURE.
“hse No. of Oyster.
1 2 3 + 5 6 7 8 9 10
Mee. eee EE ae eee) eee peer iee ere en eevee
1-10¢.c...... “0 EE Bie EO Ole eee ieee 6-FI-F °070°° «0
1-100e.c...... 0 O|j+ +/+ OO 0Oj/+ O/]+ O/]+ +130 0/0 O/]0 O
Score, 460.
Date, May 17, 1913.
AVERAGE TEMPERATURE, 12.7°C.
Boor Piacon. No. of Oyster.
1 | 2 3 4 5 6 @ 8 9 10
eee a +|+ ee HE Se sie ole OFF ie Fie +
1-10e.c...... + 0'0 O;+ +/+ +/0 O;\+ O;0 O|]|+ O|};+ +/0 0
1-100e.c...... 0 0/0 0/0 0'0 0;0 O|}0 0;0 O;O0 O}+ O;0 O
Score, 73.
Date, May 22, 1913.
AVERAGE TEMPERATURE 14.7°C,
Beat tehoon No. of Oyster.
1 : 2 } 3 4 5 | 6 iy 8 | 9 10
lee... + OF ++ ++ 4/4 44 $0 OF H+ 44 4
1-10c.c...... _ od OF On FO: Oe OK SAE OyecO0r 4.0/0) 4-0). 0&1 OO; 0
P100c.c..:... 0 aN OVO R50 OFS OOe VOLO) F ONO: 20)).0.) .OrO 10.0, 6
Score, 28.

10

“I
SS

BACTERIOLOGY OF THE OYSTER,

From the table it is seen ‘that at the beginning of the experiment
there were on the average forty-six B. coli per cubic centimeter of
oyster juice. After a period of four days this number had dropped to
an average of 7.8 per cubic centimeter and after a period of nine days
the number had still further decreased so that there were on the average
only 2.8 B. coli per c.c. of the oyster juice. This shows that under the
conditions of the experiment, oysters which contained 46 B. coli per
cubic centimeter can in nine days free themselves from B. coli to
such an extent that there remains only 2.8 B. coli pr cubic centimeter.
This is well within the standard adopted by the Bureau of Chemistry
which allows oysters to be shipped in interstate commerce which
contain 4.6 B. coli per cubic centimeter of shell liquor.

Experiment III.

On November 8, 1918 a bushel of oysters were taken from Provi-
dence River and transplanted to Wickford. These oysters were put
into two galvanized iron baskets and hung into the water from the
floor of the Beacon Oyster Co. These oysters were suspended in the
water near the edge of the channel and located only a few yards from
the place where the oysters in the two previous experiments were
placed. A sample of ten oysters was taken from this lot and carried
to the laboratory for analysis. These ten oysters were found to be
badly polluted and had a score of 640. Samples were sent to the
laboratory and analyzed on November 10, 12, 14, 17, 19, 21 and 24.
The methods of analysis were the same as in the previous experi-
ments with two exceptions. The 1-10 c.c. and 1-100 c.c. dilutions
were made in duplicate, while the one cubic centimeter samples were
only inoculated singly. The oyster liquor was drained into glass-
stoppered bottles which were graduated so that the amount of liquor
could be read off in cubic centimeters. An equal amount of sterile
one per cent. sodium chloride solution was added and the bottle
shaken vigorously one hundred times. One cubic centimeter of this
mixture was used for the first inoculation and to make the proper
dilutions. Asa result the quantities as given in the table are for the
mixture of shell liquor and salt solution. The amount of shell liquor
in the dilutions is not 1 c.c., 1-10 c.c. and 1-100 c.c., but % e.c.,
1-20 c.c. and 1-200 c.c. But as ten oysters were used the result
equals an analysis of five oysters where 1 ¢c.c., 1-10 ¢.c. and 1-100 c.e.
samples of the shell liquor were used. For comparative results,

ee

eTr

BACTERIOLOGY OF THE OYSTER. 75

however, it does not matter what quantity we use provided we use

the same amount every time.

The following table shows the results

of the examination on the different days.

Table Showing the Results of Analysis of Polluted Oysters which were put into Compar-

atively Uncontaminated Water at Wickford, November 8, 1918.

Date, November 8, 1913.

QUANTITY OF
SHELL Liquor
AND Sart SoLvu-

TION.

AVERAGE TEMPERATURE.

No. of Oyster.

1 2 3 4 5 6 7 8 9 10
toe ae x oi he == he a8 ae +
Pldec...... + ++ ++ +/+ t/t +4 4)4+ 4/4 4/4 4/4 4+
1-100e.c...... 0 O/|+ +/+ +14 O|4 O}F +/+ O];0O O]}F+ O70 O
Score, 730.
Date, November 10, 1913.
AVERAGE TEMPERATURE 11.1°C.
QUANTITY OF
aye el No. of Oyster.
TION.
1 2 3 4 5 6 é 8 9 10
GC ae a 0 + + + + + + + +
1-10ec.c...... + O|}+ O|;+ O|}O0 Olt +/+ 4/4 474+ 4/+ Oi+ O
1-100c.c...... OP OVOF, OO OOS OVERS ONOr OO: TORO “OO O-R O
Score, 190.
Date, November 12, 1913.
AVERAGE TEMPERATURE 8.8°C.
QUANTITY OF
ge ed No. of Oyster.
TION.
1 2 3 | 4 5 6 7 8 9 10
Be tce oc... ) 0 0 | 0 + + + + + ==
PVOCiC. 5. 0 O10 OVO OF SOLO OO OF O10) OF O|F 0
1-100c.c...... O. Ore Ore O10 Cth Ore O70 00 Oj;-— \O|0 0
Score, 37.

76 BACTERIOLOGY OF THE OYSTER.

Date, November 4, 1913.

AVERAGE TEMPERATURE 9,7°C,

QUANTITY OF
SHELL Liquor
AND SALT SOLv-

No. of Oyster.

TION, = = = = ——
1 2 3 4 5 6 Ub 8 | 9 | 10
NOHO ee ar erete + + + 0 0 + -/- -}- -{- +
OCR ae 0 Oj+ O1;0 O;O0 O|4 O10 OO) OO VON ORO} Oma
1-100e.c...... OO} OF OOOO ONO a0) 0 010: OF) :0> (010) S40 0a
Score, 10.

Date, November 17, 1913.

AVERAGE TEMPERATURE 7.7°C.
QUANTITY OF |——
ore aee. No. of Oyster
TION.
1 2 3 4 5 5 a 8 9 10
TerG ache = ap =F =P le “lf ah ap + +
1-10eic.. os. 0 OO" 200° SOE 010) Ole O10) 100) SOO RRORO 0
1 lO0GG ee OOOO) ao So SOO Ord OlO. ©
Score, 10.

Date, November 19, 1913.

AVERAGE TEMPERATURE 7.3°C.

QUANTITY OF
SHELL Liquor

AND Sat Souv- No. of Oyster.

TION.
1 2 3 4 5 6 7 8 9 10
NGO a'5 15-6 a SF =F st 0 ++ = 0) aR 0
1=lOcie a. + 0/0", O|+ OFO OO OF +/0 © 0/0" O08 90) ae
1 =100cCie ee 0..010 0/0 0/0 0/0 » 0)0...0)0 (00) (OOS sO) tsa
Score, 19.

SHELL Liquor |
AND SALT Souv- |

BACTERIOLOGY OF THE OYSTER. as

Date, November 21, 1913.

AVERAGE TEMPERATURE 10.5°C.

QUANTITY OF] A —

No. of Oyster.
TION. SS

-+— positive presumptive test for B. coli.

0 —negative presumptive test for B. coli.

It is seen from the tables that the oysters cleaned themselves as
much in six days as at any time. The samples taken on the day of
transporting showed on the average twenty-three B. coli per cubic
centimeter of oyster juice. Six days later the sample showed only one
B. coli per cubie centimeter and they showed no further cleansing
after ten more days. The fact that the water at this place is not
entirely free from sewage contamination probably explains why the
oysters did not show any further elimination of B. coli, apparently
there were enough B. coli in the water to maintain a small number in
the oyster at all times.

Conclusions.

In one experiment with a temperature averaging 9.7°C. over the
period of the investigation the oysters showed an elimination of B.

1 2 3 4 5 6 7 8 9 | 10
SOM ice a a ie a ce a
1-10e.c...... + 0/0 0|0 0|0 0/0 O\+ 0/0 0/0 0\0 0)0
1-100e.c...... 0 0/0 0/0 0|0 0\0 0|\0 0|0 0/0 0/0 0) 0
Score, 19.
Date, November 24, 1913.
AVERAGE TEMPERATURE 10.5°C,
QUANTITY OF
eee No. of Oyster.
TION.
1 2 3 4 5 6 Tf 8 9 10
ee... + | oO} + ue se + (ae hae 42 as
1-10e.c...... 0 0.0 O;+ +10 O;\+ 0O/}0 0/0 O;\+ +/0 0O}0
1-100c.c...... 0 ab 0|0 0/0 0/0 0\0 0/0 O;j+ 0/0 0/0
Score, 28.

78 . BACTERIOLOGY OF THE OYSTER.

coli from 73 per cubic centimeter to one per cubie centimeter in six
days. In another experiment with an average temperature of 13°C.
during the period of investigation the oysters showed an elimination
of B. coli from an average of 46 B: coli per cubic centimeter to 7.3 B.
coli per cubic centimeter in four days and to 2.8 per cubic centimeter
of shell liquor in nine days. As no examination was made between the
fourth and ninth day, it is quite possible that the limit of possible
elimination was reached sometime before the ninth day. No doubt
an examination on the sixth or seventh day would have shown a B.
coli content sufficiently low to pass the standard set by the Bureau of
Chemistry of the Federal Government.

In another experiment in November, 1912, with an average tem-
perature 5.4°C. twelve days was not sufficient to eliminate B. coli to
any appreciable extent. The examination on the twelfth day showed
a very marked decrease in the number of B. coli, but as no subsequent
examinations were made it is not possible to say with authority
whether this was the beginning of an elimination process or not,
though the writer is led to believe such was the case. The interesting
feature of this experiment is that no elimination took place in nine
days, while in the other two experiments a very marked reduction
took place in six days in one ease and in five and nine days in the other
case.

These sets of experiments seem to throw some light upon the so-
called hibernation of the oyster. With an average temperature of
13°C. in one case and 9.7°C. in the other the oysters opened and began
to eliminate B. coli almost immediately, but in the first experiment
with an average temperature of 5.4°C. no reduction in B. coli was
found until the twelfth day. These experiments lead the writer to
believe that when the temperature of the water is somewhere between
9°C. and 5°C. oysters close their shells for a longer or shorter period.
But from experiments detailed elsewhere, the writer believes that
there is no time above O°C. when oysters close their shells for an
indefinite period. The length of time that oysters remain closed is in
inverse proportion to the temperature which determines the rapidity
of the metabolic processes going on within the oyster.

EXPERIMENTS ON THE HIBERNATION OF THE OYSTER.

The so-called hibernation of oysters has attracted much attention
during the last four years. The theory that oysters close their shells

*~y

BACTERIOLOGY OF THE OYSTER. 79

when the temperature of the water approaches 0°C. was first put
forward by Gorham in 1910;' to explain certain bacteriological find-
ings in Providence River oysters. It was found that during the
warmer months the oysters in certain parts of the river were badly
polluted, but in January, with the temperature of the water around
0°C., the oysters were found free from colon bacilli. In order to
explain this phenomenon Gorham advanced the theory that when the
temperature of the water approaches 0°C. the oyster closes its shell
and remains closed until the temperature of the water begins to rise
and then it opens its shell and resumes its normal activity. This
period was called its “Hibernation Period.” A little later Pease,”
Field, of the Massachusetts Fish and Game Commission, and others
advanced asimilar idea. So far as the writer is aware, however, no
experiments have been tried to confirm or deny this theory. The
experiments of the writer cited elsewhere on the cleansing of polluted
oysters seem to show that oysters do remain closed for several days
with a temperature of about 5°C. But in order to throw further
light upon the matter the following experiments were tried.

Experiment I.

January 12, fourteen oysters were placed in sea water which had
been inoculated with a pure culture of B. coli. The oysters were
left in the sea water a day and anight. They were removed January
13th, and the outside of the shells scrubbed thoroughly with a stiff
brush and running tap water and were then put into a strong solution
of calcium hypochlorite for one-half hour and stirred up about once
aminute. They were then put into 7% formalin for the same length
of time and stirred with a glass rod for a few seconds at about one
minute intervals. They were then washed for a considerable time in
fast running tap water, temperatures between 7°C. and 8°C., and
stirred at intervals of two or three minutes. The oysters were then
taken (Jan. 13), to a cold storage room of the Merchant’s Cold
Storage and Warehouse Co., Providence, and put into storage at
34°F. (about 1.1°C.) The temperature of. the room is maintained
constant throughout the year and is never allowed to vary more than
.5°F. The next day sterile sea water which had been kept in the

1(1) Report of Commissioners of Shell Fisheries of R.I., 1910. (2) Seasonal Variation in the
Bacterial Content of Oysters, Am. Jour. Pub. Health, II, 1910, 24.

2Some Bacteriological Problems in the Oyster Industry, The Fishing Gazette, 28, 1911, 865.
July 15.

80 BACTERIOLOGY OF THE OYSTER,

room for several days was poured into the dishes until it covered the
oysters. Immediately after the oysters were covered five samples
of two cubic centimeters each were taken from each dish and inocu-
lated into bile tubes and incubated at 37°C. for 18 hours. Every tube
showed gas. January 22, the oysters were examined in the dishes
and it was found that four were closed tightly, five were open widely
enough to be seen as they lay in the dishes and the other seven were
found to be slightly open. The opening of these last seven was not
perceptible to the eye, but upon taking them out and squeezing them
one could hear a ‘‘squashy”’ sound, showing that they were not firmly
closed. Apparently the five oysters that were open had lost their
sensitiveness, for they would not remain closed when the valves were
pressed together. The mechanical stimulation of the gills and mantle
was not tried. The oysters were observed on several days until
February 2nd and it was found that some of the oysters that had been
firmly closed at first had opened and vice versa.

The oysters were not observed again until March 23. It was found
that two of the oysters in one dish were open and dead. Two others
were wide open but closed immediately when touched. These two
oysters were brought to the laboratory and put into a dish of sterile
sea water and observed for several days. They were just as active
as oysters freshly brought from the beds. They were then tested for
B. coli. Both oysters showed gas in 1-100 ¢.c. of shell liquor. These
tubes were plated in litmus-lactose-agar and typical colon colonies
were found in the plates from one oyster, but not from the other.
This showed that B. coli can live under such condition for at least
sixty-nine days.

The remaining oysters were again examined April 24, one hundred
days after they were put into storage. Five of the oysters were
apparently living, while the others were dead. These five were
brought to the laboratory and examined. It was found that three were
closed tightly, while the other two appeared a little “weak.” One
of the tightly closed oysters was put into a dish of sea water and it
soon opened like an oyster removed only recently from its natural
element. When the shell was touched it would close immediately,
though its movements were not so vigorous as those of an oyster
taken directly from the water. When the gills and mantle were
touched with a wire it did not respond readily. Apparently its
tactile sensations were not very acute, although after repeated
stimulations it closed and gripped the wire so that it took considerable

BACTERIOLOGY OF THE OYSTER. 81

strength to pull it out. The writer has noticed that oysters which
have been removed from sea water for some time require a great deal
of stimulation to make them close again, though after they have been
open for a time they react immediately. It may be that the tango-
receptors are very much dulled or that the desire for oxygen is stronger
than the sense of self-protection.

The other four oysters were opened with the proper precautions
and the mixed shell liquor and the “washings” from the body were
inoculated into bile tubes. Two of the oysters were normal in
appearance and exceptionally plump. The other two showed slight
evidences of decomposition. All the tubes from three of the oysters
showed gas and typical B. coli colonies were isolated on litmus-lactose-
agar plates. Further identification was not regarded as necessary.
The tubes inoculated from the third oyster showed no gas after three
days incubation.

Experiment II.

Seven oysters were obtained fresh from the water and impregnated
with a solution of azolitmin in sea water. They were then washed
thoroughly with a stiff brush in running water and immersed in
chromic acid for a few seconds and then washed again. All the
color was removed in this manner. January 29 they were placed
in tumblers and put into cold storage at 34°F. They were left over
night to acquire the same temperature as the room and then the
tumblers were filled with sea water. The dishes were watched to see
if any color had escaped from the oysters. February 2 a slight
coloration was found in the bottom of two of the tumblers, but this
did not appear to increase for several days. The oysters were not
examined again until March 23rd. The color had disappeared from
the two tumblers that had previously been discolored. It was
observed, however, that the water in the tumblers was not entirely
clear. There was a sediment in the bottom of the tumblers that
resembled the bits of mucus thrown off by oysters. One of the
oysters was taken to the laboratory and placed in sea water. It
soon opened, but did not contain any color. It was as active as a
normal oyster. Some of the mucus thrown out by the oyster had a
purplish color which had been stained with azolitmin.

April 17 the remaining six oysters were examined. It was found

that three of the oysters were open and the other three closed. Covers
11

82 BACTERIOLOGY OF THE OYSTER.

were fitted to all the dishes and during the process two of the oysters
closed. The other one remained open even. after reaching the
laboratory. After the cover was removed and the shell touched with
a glass rod it closed immediately. A heavy precipitate was found on
the bottom of each of the dishes and a great deal of mucus was seen
in suspension. This matter could not have come from the outside
of the oyster, because they were thoroughly cleaned before the
experiment began. There is no question but what the oyster had
opened; three were found open and two of them closed immediately
upon being agitated. The other three must have opened in order
to discharge so much mucus, but had closed again of their own
accord at 34°F.

Two oysters were infected with B. coli and put into dishes in cold
storage January 20. The dishes were later found to be cracked and
the water leaked out. These two oysters were brought to the
laboratory April 17. One was put into a dish of sea water, while
the other was opened and two cubic centimeters of the Juice was
inoculated into each of four bile tubes. Gas appeared in each tube
and typical B. coli was isolated on litmus-lactose-agar plates. This
was eighty-seven days after infection. The other oyster opened
before morning, but was apparently dead for it would not respond to a
mechanical stimulation of its gills and mantle. When opened both
oysters appeared plump and in prime condition. From their appear-
ance they could not have been told from oysters freshly caught.

From these experiments the writer believes that oysters do close
their shells for varying periods, depending upon the temperature.
Whether they close their shells under natural conditions when the
temperature falls around 0°C. no one has determined. That they do
not lose control of their adductor muscles is demonstrated in both
experiments. The writer is lead to believe that there is no definite
period at which this phenomenon can be said to begin. Mitchell
in an unpublished observation states that with a temperature below
20°C. oysters get ‘‘nervous”’ and will close upon the slightest provo-
cation and remain closed for fairly long periods. It appears that at
this temperature the irritability of the oyster is much increased.

These experiments lead one to conclude that the so-called period
of hibernation of the oyster is a relative term. The length of time
that they remain closed depends upon the temperature which deter-
mines the rapidity of the oxidative and other metabolic processes of
the oyster. An oyster will remain closed as long as its supply of

BACTERIOLOGY OF THE OYSTER. 83

food and oxygen remains sufficient and the lower the temperature
the longer this period will be. The oyster does not close on account
of “rigor frigoris,” for the control of the adductor muscle is still very
marked at a temperature of 1.1°C., and is searcely distinguishable
from normal.

Mitchell, in an extended study of the oxygen requirements of
shellfish states as one of his conclusions that ‘oysters of medium
sizes, at temperatures between 19° and 28°C., used from 7 to 35 deci-
milligrams of oxygen per hour per 100 grams of entire weight. The
amount varies with the temperature, so far as experiments show,
according to simple relationship, so that the curve approximates
a straight line.” . . . “The common clam (Mya Arenaria)
shows a higher oxygen requirement than the oyster.”’

The theory of hibernation which the writer has advanced appears
to be in harmony with the experiments of Mitchell on the oxygen
requirements of oysters. The lower the temperature the less the
amount of oxygen used. But no matter what the temperature so
long as the oyster is living it needs a certain amount of oxygen to
carry on its oxidative processes. When the amount available within
its shell is exhausted, it will open to renew its supply.

The statements of practical oyster growers also leads to the same
conclusion. It is said that oysters from Narragansett Bay in February
cannot be shipped very far in the shell, because, as the oyster men say,
they will ‘“cluck,” that is, open their shells and allow the shell liquor
to run out. The explanation no doubt is that during the ‘zero
weather” of January, the oysters are closed and as their oxygen
requirements under the circumstances are small they can remain
closed for sometime without exhausting the supply available in the
shell liquor. The period of cold weather, however, is sufficiently long
perhaps to allow the oysters, even with their small requirements, to
nearly, if not quite exhaust the available supply of oxygen within
their closed shells. The result is that in February when they are
removed to the opening house or express car which has relatively a
much higher temperature than the water from which they were taken,
the metabolic processes of the oyster are greatly increased and
there is a demand for more oxygen. The supply within the shell,
which has already been greatly reduced, is quickly used up, and
consequently the oyster opens to renew its supply.

I1The Oxygen Requirements of Shellfish, Bull. U. S. Bureau of Fisheries, XXXII, 1912, 209.

S84 BACTERIOLOGY OF THE OYSTER.

It is said that the soft-shelled clam does not hibernate during the
winter. The second quotation from Mitchell’s paper, namely, that
the oxygen requirement in the common clam is higher than in the
oyster may account for this phenomenon. The sooner the available
quantity of oxygen is used up, the more quickly will the molluse open
to renew its supply.

SUGGESTED CHANGES IN STANDARD METHODS OF
SHELLFISH EXAMINATION.

The Second Progress Report of the Committee on Standard
Methods of Shellfish Examination recommends that ‘‘twelve oysters
of the average size of the lot under examination, with deep bowls,
short lips and shells tightly closed, shall be eee out by hand and
prepared for transportation to the laboratory.”

“Bacterial counts shall be made of a composite sample of each lot
obtained by mixing the shell liquor of five oysters.” . .

Under the heading of “Methods of Rating Oysters for B. coli,”
the following statement is made:. “The following values shall be
assigned to the presence of bacteria of the B. coli group in each of the
five oysters examined.”’ Then follows a statement and illustration
of the method of scoring as adopted by the American Public Health
Association. It is clear at once that if we mixed the shell liquor of
the five oysters and examined it as a composite sample, it would be
impossible to assign values “‘to the presence of bacteria of the B. coli
group in each of the five oysters examined,” for the composite sample
must be treated as the juice of a single oyster. It is evident that a
composite sample is not what is intended, but rather that each oyster
shall be examined separately.

Some workers have based their analysis upon several composite
samples of five oysters each, while others have used five, ten or fifteen
oysters separately. There is great variation in the bacterial content
of oysters from the same lot. In one oyster there may be one
hundred B. coli per cubie centimeter of the shell liquor, while in
another oyster from the same sample they may be entirely absent.
The important consideration in the examination of oysters is the
average number of B. coli in the oysters as a whole and not the number
in any individual oyster. For this reason the larger the sample,
within reasonable limits, the more accurate the results as an indication

BACTERIOLOGY OF THE OYSTER. 85

of the B. coli content of the oysters of any particular area. Smith,’
in the analysis of one hundred and twenty-five oysters in each of a
series of samples, came to the conclusion that not less than fifteen
oysters should be used. The use of too small a sample may account
in part for the wide variation in results obtained by different analysts
in the examination of the same oyster bed at approximately the same
time. In the writer’s opinion twenty-five oysters is not too large a
sample to be used in any analysis.

The changes which the writer would suggest in “Standard Methods
of Shellfish Examination,” are as follows:

The size of the sample should be at least twenty-five oysters.
After reaching the laboratory the oysters should be scrubbed
thoroughly with a stiff brush in water free from B. coli and dried.
When ready for examination the oyster should be held between the
thumb and the fore-finger and the lip of the shell flamed in the bunsen
burner or burned off with aleohol. The opening should be done with
an oyster knife which has previously been burned with alcohol. The
method of drilling a hole through the shell and pipetting out the
oyster juice should never be substituted as an alternative method.

The shell liquor of the five oysters of each of the five composite
samples should be collected in sterile, graduated, glass-stoppered
bottles and the bodies of the five oysters should be placed in a wide-
mouth, glass-stoppered bottle. The amount of shell liquor should
be read off and an equal amount of sterile one per cent. salt solution or
sea water added to the bottle containing the bodies of the oysters.
The stopper should be replaced and the bottle shaken at least one
hundred times. (The writer’s experience has been that, if the oysters
are opened carefully so as to avoid mutilation, the bodies of the oysters
are damaged but very little by this procedure unless the shaking is
especially vigorous.) The salt solution and mucus should then be
decanted into the bottle containing the shell liquor and the whole
shaken vigorously one hundred times to break up any clumps of
bacteria and to separate as far as possible the bacteria from the bits
of mucus. ‘The five sets of oysters should be treated in this manner,
making five samples of five oysters each. If the operation is conducted
properly there should be an equal quantity of shell liquor and salt
solution in each of the five composite samples.

lSize of the Sample Necessary for the Accurate Determination of the Sanitary Quality of Shell
Oysters, American Journal of Public Health, III, 1913, 705.

86 BACTERIOLOGY OF THE OYSTER.

The subsequent procedure should be the same as that recom-
mended by “Standard Methods” and the method of scoring should
be the same except that the score as obtained by this method should
be multiplied by two, because we are using 1% c.c. 1-20 e.c. and 1-200
c.c. of the original shell liquor instead of 1 e.ec., 1-10 ¢.c. and 1-100 ¢.e.
as recommended by ‘Standard Methods.”

The advantages of this method are that we are basing our exami-
nation upon twenty-five oysters instead of five and the result will be
much nearer the true bacterial content of the sample.

Another point worthy of consideration by the Committee on
“Standard Methods” is the number of bile tubes to be used in the
different dilutions. The writer in all the work reported in this paper
and for a long time previous has used duplicate tubes. An interesting
feature of this method is that both tubes from each dilution show gas
only approximately two-thirds of the time. The writer has regarded
gas in either of the two duplicate tubes as positive for the dilution and
has assigned it the value as recommended by ‘‘Standard Methods.”’
By using this method approximately thirty-three per cent. more B.
coli are found than would be the case if only one tube were used.

“Standard Methods” under “Illustration of the Application of the
Method of Rating Oysters for B. coli’? recommends the transferring
of a positive result in a high dilution in one oyster to a lower dilution
in another oyster, if in the latter oyster the B. coli test is negative

in the lower dilution. Below is an illustrated case from “Standard
Methods:”’

Case C. Results of B. Coli Tests in Dilutions Indicated.

OysTERr, 1.0c.¢ 0.1e.¢ 0.11e.¢ piace
1 + + 0 10 (not 100)
2 + ae 0) 10
3 => =| + 100
Ae + + + 10 (not 100)
5 egg See dieanae re Ns (ea Ae + 0 0 10 (not 1)
140 = rating.

But suppose in this sample the tubes have been inoculated in dupli-
cate instead of one tube for each dilution. The following table shows
a not unexpected result:

BACTERIOLOGY OF THE OYSTER. 87

Results of B. Coli Test in, Duplicate Tubes in Dilutions Indicated.

OysTER 1 cre: 0.1c.¢ 0.001c.¢
1s aol hepa BEERS eee earn a oot = 0 0 0
oy oy AERO OIG CaO + + + 0 0 0
3... oe ch + 0
Sere ACE Ms AE ne 950 TY Jeo ae BG) sey MG)
15) 0 hp ReneS Oe ER DR AA Be + 0 OO 0 0

The question now arises as to what numerical value we shall assign
to the dilutions which are positive in one tube and not in the other.
Is it proper to assign full value to these dilutions? Would it not be
fairer to assign one-half the value recommended by “Standard
Methods” to these dilutions, because basing our calculation upon both
tubes there are only one-half the B. coli present that would be indicated
by the positive result alone?

Suppose now we wanted to transfer the positive result in the 1-100
ce.c. dilution of oyster No. 4. Should it be transferred to the negative
tube in oyster No. 5, or to the 1-10 c.c. dilution of the same oyster?
Further, what shall we do with the positive result in the 1-100 c.c.
dilution of oyster No. 3. Shall it remain where it is or shall it be
transferred to the negative tube in the 1-10 c.c. dilution of oyster
No. 1 or 2? Again suppose in oyster No. 3 in the 1—100 c.e. dilution
both the tubes should be positive and there were only one tube negative
in the 1-10 c.c. dilution of any of the oysters, should these two positive
tubes be separated and transferred to the negative tubes in two of the
other oysters?

But whatever method we use for transferring, shall we assign the
full value recommended by ‘‘Standard Methods” to the dilutions
which are positive in one dilution and negative in the other, or shall
we assign the better value, 7. e., one-half the value recommended by
“Standard Methods?” By taking advantage of the various possibili-
ties we can obtain ratings varying between thirty, the lowest possible,
and one hundred and forty, the highest possible. In the first case
the oysters would be very near the permissible standard, while in the
other, the oysters would be considered badly polluted. If we use

- three tubes in each dilution the matter is still further complicated.

t

88 BACTERIOLOGY OF THE OYSTER.

Another possibility would be to regard each set of tubes separately
and average the results. This would be the simplest method, but
it would not give so low a result as would be possible by one of the
other methods. In the case in hand the rating would be seventy-
seven as against thirty, the rating obtained by one of the other
methods.

The writer has a case in mind in which the rating on one set of
tubes was three, which showed the oysters to be in a high state of
purity, while the duplicate set showed a rating of thirty-two, which
would condemn the oysters on the strict application of the standard
set by the Bureau of Chemistry. Obviously it would be unjust to
base our rating on either of the two sets of tubes alone.

In the writer’s opinion the standard set by the Bureau of Chemistry
of twenty-three as the highest permissible rating is very stringent
and every opportunity should be given the oyster growers to avail
themselves of a method of oyster analysis which will be more accurate
in its results and a method of rating that will more nearly represent
the sanitary condition of their product.

REPORT ON THE INSPECTION OF CAMPS, 1913.
BY LESTER A. ROUND.

Health Bulletin of the Rhode Island State Board of Health for
February, 1914.

REPORT

ON THE

INSPECTION OF CAMPS

IIIS

LESTER A. ROUND

Reprinted from the HeaurnH BuLuLerin of the Rhode Island
State Board of Health, for February, 1914

PROVIDENCE, R. I
E. L. FREEMAN COMPANY, PRINTERS

IQI4

REPORT ON THE INSPECTION OF CAMPS, 1913.

The following is a report of the work on the inspection of
camps during the summer of 19138. The work occupied parts of
four days and included the inspection of four camps, two camps
on Narrow River, west of Saunderstown, Crescent Heights, near
Riverside, and the camp ground at Rocky Point. Owing to
the fact that the summer was far gone when the inspection was
commenced and to the interruption during the epidemic of
infantile paralysis the work was necessarily incomplete.

TRINITY CHuRCH CLUB OF NEWPORT.

This camp was situated on Narrow River, back of Saunders-
town, and was in charge of Mr. Newcome, a student at Brown
University. The camp was located on a knoll on the west bank
of the river on land belonging to Mr. A. F. Carson, and con-
sisted of three tents and one building. The latter was divided
into a cook house and a sort of club room. On the west side
of the building was a wide piazza which protected the mess
table. The tents were large and roomy and were used only
for sleeping.

The camp was under the administration of the Trinity Church
Club of Newport, and consisted of boys belonging to the club.
At the time of inspection there were twenty-eight persons in
the camp, twenty-two of whom were boys under the age of
fifteen. The number varied from week to week as the boys
came and went. So far as I could learn there were no cases of
sickness among the boys before coming. Mr. Newcome’s
attention was called to the dangers which might arise upon
admitting to the camp a typhoid convalescent or any one who
had recently suffered from any infectious disease. He was

advised not to admit any such person without a physician’s
certificate. He promised to give the matter careful attention.

The water supply was derived from a spring nearby and
judging from its physical qualities appeared to be of excellent
character. The milk used in the camp came from Mr. Carson’s
farm, the owner of the land upon which the camp was located,
and who has the reputation of having an excellent dairy.

The privy vault was located on the same lot about forty
yards northwest of the camp and consisted of an unscreened
pit-vault. An attempt was made to cover the faeces, but was
unsuccessful. The copious use of bleaching powder, however,
protected the fecal matter from flies which were not numerous
in any part of the camp, not even in the cook house. All kitchen
wastes were thrown into a pit and covered with earth. The
bathing beach was located at the foot of the knoll on which
the camp was located, and was free from any drains or sewage
outfalls. Although marshy land was near, the campers were
troubled with mosquitoes only when the wind was south or
southeast, in which case the mosquitoes were brought from
the marshes situated below on the river.

St. JoHN’s CHuRCcH OF NEWPORT.

This camp was also located on Narrow River, about two
hundred yards north of the camp of the Trinity Church Club.

It was situated on a knoll on the west bank of the river. The
camp was in charge of Mr. J. A. McNulty, and consisted of
four tents and a cook house. The location of the camp was new
and the boys had put in most of their time clearing away the
brush and making a clearing in the woods. At the time of
visitation there were fifteen boys in the camp, eight being
under fifteen years of age. As in the case of the Trinity Church
Camp, however, the number varied, for some of the boys left
each week while others came to take their places as the accommo-
dations of the camp permitted. So far as I could ascertain,
no one had suffered with any infectious disease while in camp
or for a considerable time before coming.

5

The water supply was derived from the same spring as in
the case of the Trinity Church Camp, and the milk supply was
derived from the same dairy. All kitchen wastes, garbage,
swill, etc., were disposed of by burial.

The privy was located about fifty yards northwest of the
camp and consisted of a pit-vault which was tightly boarded.
No screens were used, but a plentiful supply of bleaching powder
prevented flies from breeding in the fecal matter or from being
attracted to it. Almost no flies were seen in any part of the
camp nor had the campers been troubled with mosquitoes.

The sanitary aspects of both the camps on Narrow River
were very good and left but little to be suggested by the
inspector.

CRESCENT HEIGHTS.

Crescent Heights is the name now given to what was formerly
known as Camp White, and is situated at the south end of
Riverside, just north of Crescent Park. The camp was situated
on land owned by the Hope Land Co., and was in charge of
Mr. Looff, manager of Crescent Park. At the time of inspec-
tion there were twenty-eight tents and seventy-four persons in
camp, though the number fluctuated as campers were coming
and going continuously. Some of the campers had tents on
the grounds that were occupied only during holidays and week-
ends. On the day of inspection there were thirty-five adults
and thirty-nine children in the camp.

No eases of sickness of any kind were found on the grounds,
nor had any of the people suffered from any infectious disease
for some considerable time before coming to camp.

The water supply was derived from the East Providence
system, and the milk from the Broadway Dairy Co., and from
several farmers whose names were not known. The garbage
and kitchen refuse was placed in kegs and these were emptied
every day. Refuse matter, such as paper and other combustible
material, was burned by the campers themselves, while tin cans,
cornhusks, etc., were put into large bins which were emptied
once a week.

6

The toilets were located at either end of the camp ground
and were pit-vaults in which no attempt was made to screen
or cover the feces. Yet in spite of this fact there were very few
flies around the buildings, though in other parts of the camp they
were very numerous. So far as I could learn no disinfectant,
such as bleaching powder or some coal-tar derivitive, was used
in the privy vaults. Some method of protecting the fzeces
from flies should be provided, however, since many of the
tents are located very near. As the inspection was made the
Saturday before Labor Day, and most of the campers were
intending to leave the following Monday or Tuesday, the matter
was not brought to Mr. Looff’s attention. This was the only
glaring deficiency that I could find in the camp. This matter
should be called to the attention of Mr. Looff at the opening of
the next season, in order that proper protection be provided.

An inspection of the shore from the camp grounds north
showed nothing tnusual. Some seaweed and other marine
growths were scattered along the beach. Evidently the con-
dition which caused the complaint earlier in the season had
been removed.

Rocky Point.

This camp was situated just north of the amusement park
in the same place it has been for a number of years. At the
time of inspection it consisted of sixty-eight cottages and forty-
four tents. In the tents were found eighty-two persons, twenty-
five of whom were children under fifteen years of age. The
camp was in charge of Mr. R. A. Harrington, who also controls
the amusement park.

So far as I could learn, no one had been sick in spite of the
unsanitary conditions that existed. The water supply was
derived from a spring on the shore some distance from the
camp ground, and necessitated considerable inconvenience to
the campers in obtaining their water supply. This spring is
the only water supply available on the camp grounds and
furnishes the water for from three hundred to five hundred
persons throughout the season. The location of the spring

7

is very bad. It is just level with the beach and is wholly
unprotected, while a quahaug shell or an old tin can serves
as a “common drinking cup.” The fact that the spring is
perfectly unprotected in such an open spot gives abundant
opportunity for gross pollution of all kinds. The milk supply
is obtained from various farmers.

The privy system and the method of garbage disposal found
on the camp grounds would be an abomination to any civilized
community. Four barrels were placed at different corners of
the lot and in these the swill, garbage and kitchen wastes were
placed by the cottages and campers. It was the intention of
the man in charge of this work to empty these barrels once every
week. But I was informed by several campers independently

This picture shows two of the swill barrels and a privy and the proximity
to some of the tents. These barrels were only emptied once each week
and on one occasion were not emptied for three weeks. At this time the
barrels were not only filled, but the garbage completely covered the barrels
and filled the space between them.

8

that on one occasion, these barrels were not emptied for
three weeks. At this time they were not only filled, but the
garbage formed a pile around the barrels large enough to com-
pletely hide them, and in the picture shown on this page the
space between the two was filled according to the statement of
several of the campers. As is seen in the picture these barrels
were sometimes located very close to the camps, only twenty-
five or thirty feet in some cases. One can easily imagine the
odor and stench arising from a pile of garbage of such dimen-
sions which had been collecting for three weeks during the hot-
test days of our summer months. The description of the odors
by the campers in all probability did not exaggerate the matter
in the least. The number or flies attracted, plus the number
bred during this time, would be almost inconceivable. But
during the other periods when the garbage was collected every
week the conditions must have been very bad, especially during
a period of the warmest weather of the year. The amount of
garbage collected from three to five hundred people during a
week would be considerable, and would furnish one of the best
baits for flies imaginable. This would be especially true when
the garbage was unprotected. Not only that, but it would
furnish an excellent breeding place for flies, certain species of
which could pass through their Javal and pupal stages during
this short period. Moreover, it would give sufficient time for
the ordinary house fly—musea domestica—to pass through its
laval stage and then bore into the ground and complete its
metamorphasis. At both visits the barrels had just been
cleaned, but in no very cleanly manner, and swarms of pseudopy-
rellia cornicina were seen in and around the barrels. Inacamp
of the size of Rocky Point, with conditions such as they are,
there can be no reasonable excuse for not collecting the swill
and garbage every day, especially when we consider the revenue
which is obtained from this small plot of ground. This revenue
is estimated at $1,500 to $2,000 annually, with almost no cost
of maintenance except taxes.

The toilets were arranged in long rows on the north and
south ends of the lot. The construction of the houses and the

9

vaults was exceedingly flimsy. The latter were simply pits
unprotected from flies. As can be seen from the picture on this
page and the ones following, the buildings were well ventilated,
both under the bottom—into the vaults—and over the doors or
on the sides. The purpose of the holes no doubt was for light
alone, but as they were unscreened they afforded an excellent

avenue of entrance and exit for flies which were very numerous,
not only around the privies, but in other parts of the camp as
well. As far as I could learn no repellent as chloride of lime was
used except in a few instances when it was furnished by the
tenant. These buildings were often located within a few feet
of the camps as can be seen from the pictures.

Showing the construction and “‘ventilation” of the privies. Note the
wide open spaces under the bottom and over the tops of the doors.

10

tf

Two more views showing the construction of the privies and the prox-
imity to the tents and cottages. (The distance is much shorter than it
would seem from the pictures.)

Another view showing the arrangement of swill barrels, privy and
camps.

Before the opening of next season Mr. Harrington should be
informed of the conditions which he allows to exist upon his
‘amp ground and the remedies pointed out, and the State Board
of Health should see to it that such improvements be made as
to ensure the campers against a nuisance such as existed at
Rocky Point during the last summer.

Respectfully submitted,

LesteER A. ROUND.

129

THE IMPORTANCE OF A CONSIDERATION OF THE
FIBER PROTEINS IN THE PROCESS OF
BLEACHING COTTON.

BY BENJAMIN 8. LEVINE.

Science, N. S., Vol. XLI, No. 1058, April 9, 1915, pp 543-545.

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[Reprinted from Science, N. S., Vol. XLI., No. 1058, Pages 548-545, April 9, 1915]

THE IMPORTANCE OF A CONSIDERATION OF THE FIBER PROTEINS
IN THE PROCESS OF BLEACHING COTTON

Tue nitrogen which is found in the ripe
cotton fiber seems to have some bearing upon
the yellowing of bleached cotton cloth, as was
pointed out by J. C. Hebden in his paper read
in Troy before the American Institute of
Chemical Engineers.1 He showed that in the
process of bleaching cotton cloth after the first
caustic boil 91.5 per cent. of the proteins were
removed from the fiber, whereas of the fats
and waxes only 20.4 per cent. were removed;
and after the second caustic boil 91.7 per cent.
of the proteins and only 64 per cent. of the
fats and waxes were eliminated; the “ chemick ”
and the “sour” together, he showed, removed
12.05 per cent. of the remaining protein im-
purities and 10.23 per cent. of the remaining
fats and waxes. According to his analysis,
after all the bleaching operations there were
still left on the fiber 30.4 per cent. of the total
fatty and waxy impurities, whereas of the
total proteins there were left only 7.3 per cent.,
and as the cloth which he analyzed had under-
-gone a “ good bleach,” he felt safe in inferring
that it is the failure to remove the protein
impurities from the cotton that results in a
“bad bleach ” or causes the yellowing of cloth
in steaming or during storage.

So far as we know, the investigator above
referred to was the first to point out the pos-
sibility that the proteins of the fiber played
such a part in the bleaching of the cloth.
Previous to this it has been believed that the
fatty and waxy matters and especially the
pectins were chiefly responsible for the yellow-
ing of the fiber, since they formed water in-
soluble compounds, which remained on the
fiber. The analysis of Hebden, however,
showed that the calcium fixed on the fiber in
the form of calcium salts was decomposed by

1 Journal of Industrial and Engineering Chemis-
try, September, 1914, Volume 6, No. 9, page 714.

the following acid treatments, and he explained
the presence of the calcium on the fiber after
the sour by the formation of a calcium cellu-
lose similar to that of soda cellulose. The pos-
sibility of the formation of such a cellulose,
he believed, was supported by the fact that cot-
ton cloth which has been boiled and bleached
did not produce as clear and as brilliant a
turkey-red as cloth which had been simply
boiled, because the former was not in a con-
dition to fix calcium.

As the result of investigation in this line
on cloth from different bleacheries, it occurred
to us that an analysis of the growing cotton
fiber with a view of determining the nitrogen
and the fat and wax factors might reveal some
points of importance. The determinations
were carried out on Durango cotton raised on
the United States Experiment Farm, San
Antonio, Texas.?

The nitrogen factors were determined by the
Kjeldhal-Gunning method, and the fat and
wax factors by extracting samples of the fibers
first by ether and then by alcohol. Some of
the experiments were carried out in duplicates
and some in triplicates, and the averages of
the determinations were recorded as the final
results. The figures given in the table below
can only be regarded as approximating the
absolute values of the nitrogenous and fatty
and waxy constituents of the fiber; for the
determination of exact values a much larger
number of experiments should be performed.
Nevertheless, they show the tendencies of the
two factors and have, therefore, significance.
The nitrogen determinations were made and
recorded beginnings from the 14-16-day stage

2 We wish to thank Mr. Rowland D. Mead, of
the United States Department of Agriculture, for
supplying us with the necessary samples of the
cotton fibers.

2 SCIENCE

up to the 86-38-day stage, whereas the ether
and the alcohol extracts were recorded only
beginning with the 22-24-day stage, because
in the stages previous to this the amount of
tannins extracted by both ether and alcohol
were much higher than the fats and waxes.

|
Aleohol |

Agein |

Days Nitrogen | Protein Ether Fat and

from in Per jn x 6.25 Extr.in | Extr.in | Wax in
Flower- Cent. |~°“. ~*~ |Per Cent.|/Per Cent./Per Cent.
ing |
PANG 3|422800. Mk TOSS shar.” a
16-18 | 1.9480 | 12.175 |
18-20 | 1.4250 | 8.907
20-22 | 1.1820 | 7.388 doiege slacks ||): eer.
22-24 svete wicks 4.405 2.819 | 7.225
24-26 | .3760 | 2.350 | 1.745 775 | 2.418
26-28 .3195 W997 | 1.398 713 2.111
28-30 3123 1.952 | 1.415 -800 2.215
30-32 2657 1.661 1.522 782 2.304
32-34 | -2590 1.619 | 1.536 -709 2.245
34-36 2503 1.564 | 1.403 -802 2.205
36-38 | .1815 1.134 | 1.409 791 2.200

From the above table it may be seen that
the fats and waxes showed neither a gradual
increase nor a gradual decrease in their per-
centages, and in view of the fact that the fiber
was growing, it seems reasonable to suppose
that the fatty and waxy substances increased
proportionally as did the fiber. We believe
that were the numbers of the experiments large
enough to give averages approximating the
absolute values of the fatty and waxy factors,
this point would have been brought out much
clearer. But even from the determinations
which we can report, it appears that the fats
and waxes extended in an even and constant
thickness over the fiber. If we accept the
view that the function of these substances is
to protect the fiber from external influences
of weather and disease, that is that they are
merely external coats of the fiber, the signif-
icance of such a proportional growth of these
constituents becomes clear. If, however, the
fats and waxes are phosphotides taking part
in the growth, there would also be a propor-
tional increase. The nitrogen figures, on the
other hand, show gradual decrease in percent-
age with the increase of the age of the fiber.
The sudden increase of the factor at the 20-22-
day stage as compared with that of the 24-26-
day stage may be due either to a rapid growth

of the nitrogenous constituents of the fiber or
to the adhering nitrogenous coloring matters
of the parts of the boll which surrounded the
fibers. If we limit ourselves to a consideration
of the nitrogen figures of the samples repre-
senting only the higher stages of development
of the cotton fiber even then we are per-
mitted to assume that the nitrogen was de-
posited early in the lumen of the fiber and its
absolute value remained constant. This as-
sumption becomes more plausible when the
nitrogen figures are multiplied by 6.25 to .ex-
press the percentage of proteins present in the
fiber. Most of this early and constant protein
deposit remains in the lumen in the form of
insoluble albuminoids and in the form of
alcohol soluble proteins; some of it is utilized
by the growing fiber, probably by the spiral
forming the walls of the lumen. That the pro-
teins of the fiber are of an insoluble nature
is shown by the fact that the percentage of
nitrogen of gray cloth as obtained by Hebden
(0.191 per cent.) remained practically un-
changed after the “steep” (0.192 per cent.),
and that some of it exists in the fiber in the
form of alcohol soluble proteins, is shown by
the number which he obtained for nitrogen
after extracting the cloth by ether and alcohol.
The percentage, as shown in his table, was
reduced from 0.191 per cent. to 0.161 per cent.
The fact that the first caustic boil removed
91.5 per cent. of the protein content clearly
points to the decomposing action of boiling
alkali upon the albuminoids.

The 7.8 per cent. of total protein content
remaining in the fiber after all the operations
of the bleaching process can be considered as
that part of the fiber proteins which has be-
come an inseparable part of the wall of the
lumen. The lowest percentage for fats and
waxes (2.200 per cent.) obtained by us for the
fiber taken directly from the field was con-
siderable higher than that obtained by Hebden
for fibers which were ginned, carded, spun and
woven (1.405 per cent.). The removal of a
large part of the fats and waxes by mechanical
means during ginning, carding, spinning and
weaving proves that these constituents form
the outside cover of the fiber, and it is reason-
able to suppose, therefore, that they do not

SCIENCE 3

play as important a part in bleaching ag is
ascribed to them. The percentage of nitrogen
in our experiment (0.1815 per cent.) was some-
what smaller than that obtained by Hebden
for cotton in the form of cloth (0.191 per
cent.) and points to the fact that, unlike the
fats and waxes, the proteins of the fiber are
not adventitious nor coating factors, but that
they are within the lumen or are in part
intimately bound to the fiber. As the proteins
are of the insoluble kind, the above seems to
justify the assumption of Hebden that in

bleaching the removal of the proteins may
be of more importance than that of the fats
and waxes.

These results and the results of Hebden
show the necessity of a careful investigation
of the chemical nature of the fatty and waxy
substance as well as of a further study of the
effect of growth on these constituents of the
cotton fiber. B. S. Levine

BIOLOGICAL LABORATORY,

BROWN UNIVERSITY,
PROVIDENCE, R. I.

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130

AN EPIDEMIC, SIMULATING TYPHOID, CAUSED BY
A PARAGAERTNER ORGANISM.
BY GEORGE H. ROBINSON.
Journal of Infectious Diseases, Vol. XVI, No. 3, May, 1915, pp 448-455.

AN EPIDEMIC, SIMULATING TYPHOID, CAUSED BY A
PARAGAERTNER ORGANISM

GEORGE H. ROBINSON

(From the Bacteriological Laboratory of Brown University and the Providence Health
Department, Providence, R. I.)

Epidemics of typhoid fever due to the drinking of sewage-polluted
water show a marked similarity in the ratio of those affected with
gastro-enteritis to the cases of clinical typhoid fever. Comparison of
several epidemics of this nature indicate that 40-60 percent of those
exposed to infection contract gastro-enteritis and 2-8 percent develop
clinical typhoid. In epidemics in which food or milk, infected by
typhoid carriers or incipient cases of typhoid, are the vehicles of
infection, the incidence of typhoid is much higher and the accompany-
ing gastro-enteritis is generally absent. During the investigation of
the following epidemic a study was made of the cases of enteritis as
well as of those pronounced as typhoid.

In the fall of 1913 there occurred an epidemic simulating typhoid
among the Rhode Island party attending the Perry Centennial at
Put-In-Bay, Lake Erie. The itinerary of the party and the sanitary
aspect of the epidemic are thoroughly discussed in De Valin’s report*
and that of Swarts.? The party, consisting of 422 members, left Provi-
dence September 8 and upon reaching Buffalo was quartered on two
steamers, the Rochester and the Greyhound, 300 being accommodated
on the former and 122 on the latter. The courses of both boats were
practically the same and both parties attended the same functions. The
delegation returned to Providence on September 14.

Reports from 235 members of the party on the Rochester showed
122 cases, or 52 percent, of gastro-enteritis. Of the total 300 on board
42, or 14 percent, developed clinical typhoid, including 6 deaths. The
party on the Greyhound yielded upon inquiry 12 mild and temporary
attacks of diarrhea” such as any change of water or diet might cause.
In many ways this epidemic resembled others of infected-water origin,
except that the number of typhoid cases was larger than the average.

1. Publ. Health Rep., 1913, 28, p. 2761.
2. Health Bull. Rhode Island State Board of Health, 1914, 3, p. 1.

bdo

GEORGE HH. Ropinson

All possible sources of infection were carefully investigated by
Dr. C. V. Chapin of the Providence Health Department, Dr. G. T.
Swarts of the State Board of Health, and Dr. de Valin of the U. S.
Public Health Service. The most salient points of the inquiry were:
On September 11 the Rochester pumped water for drinking purposes
from Lake Erie at points “considered decidedly unsafe; during the
trip a cook had performed his duties while suffering from typhoid
fever; an electrician on the Rochester was taken sick with typhoid
August 16; a maid and a water-tender both left the boat on September
20 feeling unwell and were later diagnosed as cases of typhoid. The
maid died without giving a positive agglutination test with typhoid
bacilli.

A check on the manner of infection is supplied in the case of mem-
bers of the Newport Artillery, one of the organizations taking passage
on the Rochester. The 83 members of this company were previously
warned against the dangers of drinking water and so drank sparingly.
None of them was affected with enteritis, but twenty-two of them
developed typhoid symptoms about two weeks after their return.
Eliminating those who drank little water, we find at least 122 cases
of enteritis, or 80 percent, of the 152 members of the party who did
drink freely of the water.

SYMPTOMS
The most common symptoms were enteritis, diarrhea, malaise, and
fever. “Forty-three presented symptoms varying from a mild para-
typhoid, to a perfect clinical, typhoid character.” [Four of the patients
were admitted to the Rhode Island Hospital, extracts from whose
records follow.

Case 16—Admitted Oct. 4. Diagnosis, typhoid fever with lobar pneumonia
complication.

Patient began to complain of headache, abdominal distress, pain in back,
and fever, September 25. Went to bed September 28 because he felt weak.
Appetite poor, bowels regular. No nose bleed. No cough. Stated that he had
had a similar attack six years before. Malaria four years before.

Well developed and well nourished. Face flushed, lips cracked. Tongue
thickly coated on dorsum, red and moist at tips and edges. On chest and abdo-
men are a few circumscribed papules varying from the size of a pin to half
the size of a pea, which disappear on pressure. No rales in chest. No mur-
murs cf heart. Leukocytes 14,000, polymorphonuclears 81 percent. No agglu-
tination of typhoid bacilli.

Oct. 6.—Irrational, tosses about in bed, gets up if not watched. Spleen not

palpable.

TyrpHoin EpIpEMIc CAUSED BY PARAGAERTNER ORGANISM 3

Oct. 7—More delirious, temperature 103 F.

Oct. 8—Temperature 101 F. yesterday, during the night rose to 104 F.;
pulse 130. Abdomen soft. No blood in stools. Impaired resonance in right
lower lobe of lung. Died.

Case 21.—Admitted Oct. 8. Diagnosis, typhoid fever.

Patient had headache and diarrhea with cramps, Sept. 14. This lasted four
days after which he felt as well as ever except that he became tired easily. On
Oct. 4, he went on a canoe trip and became thoroughly exhausted. Since then
has had severe headache. No chills and no fever. Had typhoid fever with
a relapse nine years ago.

No rales, no heart murmurs, no tenderness on abdomen, no glandular
enlargement. Leukocytes 5,600, polymorphonuclears 63 percent, mononuclears 33.

Oct. 10.—Temperature reached highest point, 103.5 F.

Oct. 15—Many typhoid bacilli in blood culture.

Oct. 20.—Temperature normal.

Nov. 4.—Discharged.

Case F.—Admitted Oct. 4. Diagnosis, typhoid fever.

Felt sick on return trip Sept. 13. Headache, and later in the day watery
and frequent bowel movements and vomiting. Had profuse diarrhea up to
Oct. 4. Has cramp in abdomen before every bowel movement and pain in the
middle of the back almost constantly. No blood in stools. Has lost about 8
pounds. No nose bleed. Bad taste, appetite poor. High fever.

No tenderness in abdomen. Spleen palpable. No rose spots. Leukocytes
6,400, polymorphonuclears 70.5 percent, mononuclears 28.

Oct. 7—Only complaint is dizziness. Spleen greatly increased in size.
Serum agglutinates typhoid bacilli.

Oct. 10.—General condition good. Feyer declining.

Oct. 13—Patient at times irrational and slightly delirious. Temperature
again elevated. No sign of perforation or hemorrhage.

Oct. 16.—During last three days has been profoundly toxic. During last
twénty-four hours heart has been failing. Died.

Case 40.—Admitted Oct. 29. Diagnosis, typhoid fever.

Symptoms began Oct. 22 with headache and loss of appetite, slight cough,
but no nose bleed; slight fever, no chills, no vomiting, no pain in abdomen or
chest or joints or back, no night sweats; bowels moved frequently last four
days. Headache and malaise. Took complete typhoid inoculation one year ago.

Abdomen has suggestive rose spots. No tenderness or rigidity or spasms.
Lower end of spleen felt. Leukocytes 11,000. Serum agglutinates typhoid
bacilli.

Nov. 2—Blood culture negative. Comfortable. Diarrhea stopped.

Nov, 5.—Feeling well but temperature is again 103 F. at evening.

Nov. 8—Is beginning to feel listless and a little weak; no headache and no
abdominal pain.

Nov. 11—Temperature up. Weak.

Nov. 21.—Temperature normal.

Dec. 11.—Uneventful recovery. Discharged.

+ GEORGE HH. RoBinson

AGGLUTINATION

The first agglutination of typhoid bacilli by the serum of any of
the party was obtained September 28. This patient, however, had
received prophylactic treatment for typhoid a few months previously,
but during the investigation of his case it became evident that there
was an infection of some kind among the members of the party. The
last specimen of blood from this epidemic was sent in November
9. During this time 39 specimens of blood were received at the city and
state health laboratories from those residing in or near Providence
who attended the Perry Centennial.

These yielded the following results:

City State
Laboratory Laboratory
IPOSitive tO) EV DHOSUS alONE wyarrerereterelel cle ele leis) elojeroieters 4
Partial) tol ebostypnosus alone. cle cicieleielete sie leleioielole 2 0
Positive to B. paratyphests. (B) alone............. 0 1
I OSIELV.E st OL IDOLM ecco derstar-letotstletistetolel terete loresste sMeretomeretone 2 4
Partial to B. typhosus and suspicious to B. para-
LN quieorecy (si) iM iohdoodossdosdooe doo doansopacoacac 0 1
Negative tonDOtH cyclers er ttersiotcieeeteteeinetelatekateletcialcte elavatare 8 11
MLO tal) Waicteselctevctencrerstoforsieuekencicle ciclo teleaers eketonepetereners 16 23

The large percentage of the cases which showed a reaction with B.
paratyphosus (B) was noticeable throughout the epidemic.

EXAMINATIONS OF FECES

Specimens of feces were obtained from nineteen of the men in
Providence. Through the efforts of Dr. C. V. Chapin specimens of
feces and blood were obtained from the cook on the Rochester. About
two months after the infection, through the kindness of Dr. Swarts,
specimens were obtained from the convalescents in the Newport Hos-
pital. A technic which had proven very successful was used in the
examination of the feces. Small portions of the material were incu-
bated in lactose peptone bile for six to twelve hours and then the
bile culture was plated on Endo’s medium. No typhoid organisms
were isolated from any of the specimens. An organism was found in
ten of the specimens, however, which resembled both typhoid and
paratyphoid types in some respects, and differed in others.

This organism was obtained from feces of the cook of the
Rochester and also from Cases 16 and 21.

It will be seen that the organism is similar to typhoid in that it
does not ferment dulcite and does ferment dextrin. Like B. para-
typhosus (B) it produces visible amounts of gas and ferments arab-
inose.

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tT aiavl

6 GEORGE HH. Ropinson

It is about the size of the typhoid bacillus, actively motile, and is
gram-negative. According to its cultural characters and carbohydrate
reactions, this organism would be classified as a member of the inter-
mediate group. It could not be reckoned as a true gaertner organism
on account of its production of permanent acidity in litmus milk. Evi-
dently, it is one of the paragaertner organisms.

Serum Reactions.—These cultures were tested with the serum of
the patients from whom they were isolated, as well as with known
typhoid and paratyphoid (A) and (B) sera. No reaction was obtained
except in the case of the cook (Case 33). Cultures obtained from him
agglutinated with his serum at a dilution of 1:60. Two rabbits were
immunized against Culture 16.3 obtained from a fatal case. Five
inoculations of 1 c.c. of killed twenty-four-hour broth cultures at inter-
vals of 7 days in one rabbit and 5 days in the other failed to produce
a serum reacting strongly with the organism injected or any of the
other organisms isolated during the epidemic. One of the sera did
produce an agglutination at a dilution of 1:10. Both sera, however,
gave a positive reaction with B. typhosus at a dilution of 1:60. This
would account for the positive agglutination tests among those of the
party who were infected. The organism is evidently one which stimu-
lates the formation of agglutinins slowly. Those patients who did not
give positive agglutination tests had probably not been infected long
enough.

PATHOGENICITY FOR Lasoratory ANIMALS.—The pathogenicity of
the different strains evidently varied. Each strain was inoculated into
a mouse but several of them did not succumb. Culture 16.3 isolated
from one of the fatal cases seemed to be most pathogenic.

Mouse 2—Weight, 20 gm. Inoculated subcutaneously with 0.2 c.c. twenty-
four-hour broth culture of 16.3; death in 12-18 hours; congestion about point
of inoculation, lungs somewhat congested. Organism isolated in pure culture
from liver, lungs, heart, and kidneys.

Mouse 21.—Weight, 25 gm. Inoculated subcutaneously with 0.25 c.c. twenty-
four-hour broth culture of 16.3; evidently sick and feverish after forty-eight
hours; diarrhea; died after fifty-five hours; lymph glands hemorrhagic, con-
gestion at point of inoculation. Isolated organism from heart, liver, lung,
stomach, and feces.

Mouse 12.—Jan. 20, fed bread soaked in twenty-four-hour broth culture of
16.3; Jan. 26, fed bread soaked in twenty-four-hour broth culture 16.3; Jan. 30,
drank about .3 c.c. of twelve-hour-broth culture; Feb. 3, drank about 1 c.c. of
twenty-four-hour broth culture; Feb. 6, looked sick; Feb. 7, died, eight days
after drinking broth culture. Axillary and lumbar lymph glands swollen and

TypHoIp EPIDEMIC CAUSED BY PARAGAERTNER ORGANISM f

congested, gall-bladder very full, spleen apparently normal, diarrhea. Serum
would not agglutinate culture. Organisms isolated from gall-bladder, small
intestine, urinary bladder, and kidney. Heart and liver sterile.

Mouse 15——Drank 2 drops twenty-four-hour broth culture of 16.3; died ten
days later; appearance of organs normal. Organism isolated from heart and
liver.

Guinea-pig 15.—Drank 2 c.c. twelve-hour broth culture of 16.3; died six days
later; gall-bladder much enlarged, suprarenals enlarged, abundant peritoneal
fluid, spleen normal, other organs apparently normal. Organism isolated from
heart, liver, kidney, peritoneal fluid, gall-biladder, urinary bladder, and small
intestine.

Guinea-pig 16—Weight, 305 gm. Inoculated intraperitoneally, 1 c.c., twelve-
hour broth culture of 16.3; died four days later; abscess at point of inoculation,
peritoneal cavity full of a white fluid, intestine and mesenteries inflamed, spleen
apparently normal. Organism isolated from heart, liver, and peritoneal fluid.

Mouse 7.—Weight, 22 gm. Inoculated subcutaneously with 0.25 c.c. twenty-
four-hour culture of 54.2; died after five days; congestion at point of inocula-
tion; lymph glands swollen. Organism isolated from heart, liver, lungs, stom-
ach, large intestine, and point of inoculation.

Mouse 14.—Drank 0.5 c.c. twenty-four-hour broth culture of 54.2; a week
later refused to eat, evidently sick; died next day; appearance of organs normal.
Organism isolated from stomach, small intestine, and large intestine. Heart,
liver, and kidneys sterile.

Mouse 6—Weight 22 gm. Inoculated subcutaneously with 0.25 c.c. twenty-
four-hour culture of 33.29; died after fifteen days; spleen swollen; point of
inoculation congested. Organism could be isolated only from point of inocula-
tion.

Mouse 19.—Weight 20 gm. Inoculated subcutaneously with 0.2 c.c. twenty-
four-hour broth culture of B. paratyphosus (B); died in forty hours; appear-
ance of organs normal; lymph nodes congested; mesenteries congested. Organ-
ism isolated from heart, liver, kidneys, small intestine, and large intestine.

Examination of the organs in fatal cases showed no marked patho-
logical changes. When infection occurs through the digestive tract,
it is capable of producing enteritis and diarrhea in mice and guinea-
pigs.

Mouse 12 was apparently unaffected ten days after eating solid
food infected with culture of 16.3 but died on the eighth day after
taking the organism in a liquid. Two other mice (Mice 13 and 15)
-died seven and ten days, respectively, after drinking liquid culture.
Infection through the intestine is best accomplished by means of liquid
rather than solid food.

PROPHYLACTIC TREATMENT OF MEMBERS OF THE PARTY
Sixteen members of the party had taken typhoid immunizing treat-
ment less than a year before this epidemic. Of these, five were affected
with enteritis and diarrhea and one (Case 40) was considered at the

8 GEORGE H. Ropinson

hospital as having typhoid. Two other cases considered as typhoid
fever (one of them Case 21) gave definite histories of having had it
before. In view of the opinion among medical men that one attack
of typhoid protects against another and that the prophylactic treat-
ment protects for at least two years, these data would confirm our
findings that it was not the typhoid bacillus with which the members
of the party were infected.

SUMMARY
An epidemic has occurred resembling in many ways a water-borne
typhoid epidemic. A careful bacteriological examination of feces of
patients was negative for typhoid, but an organism was found which
seemed to have the cultural characteristics of the paragaertner group.

Reprinted from
Tue JourNat oF INFecTIous Diseases, Vol. 16, No. 3, May, 1915. pp. 448-455

131

ISOLATION, IDENTIFICATION, AND SERUM REACTIONS
OF TYPHOID AND PARATYPHOID BACILLI.
BY GEORGE H. ROBINSON.
The Journal of Medical Research, Vol. XXXII, No. 3, July, 1915.

ISOLATION, IDENTIFICATION, AND SERUM REACTIONS OF
TYPHOID AND PARATYPHOID BACILLI.*

GEORGE H. ROBINSON.
(from the Bacteriological Laboratory of Brown University and the Providence
FHlealth Department, Providence, R./.)
Introduction: Statement of Problem.

I. Methods of isolation of typhoid and paratyphoid bacilli: (a) Enrichment
media; (4) Differential media; (c) Comparison of different methods.

II. Methods of identification: (@) By carbohydrate media; Preparation of
media. (4) By serum reactions: 1, Specific agglutinins; 2, Group
agglutinins. (c) Classification of organisms.

III. Diagnosis by serum reactions: (@) Reaction of normal blood; (4) Par-
tial reactions; (c) Mixed infections.

IV. Serum reactions during vaccine treatment.

It has been our experience in the routine diagnosis of
typhoid fever that problems are constantly arising which
need more experimental data. In order to gain more
information on some of these questions the work dealt with
in this paper was undertaken. The following subjects were
considered :

I. The development of an accurate, quick, and easy
method of isolating typhoid and paratyphoid organisms.

Il. The value of cultural and serum reactions in the
identification of these cultures.

III. The proper interpretation of serum reactions obtained
in the routine examination of suspected blood specimens.

IV. A comparison between the serum reactions produced
as the result of vaccination and those in clinical cases.

' I. METHODS OF ISOLATION.

Considering the fact that both typhoid and intermediate
organisms grow readily upon artificial media, methods
devised for the isolation of typhoid are equally applicable to
the intermediate group. Hurler (1912) has given a detailed

* Received for publication March 29, IgI5s.

(399)

400 ROBINSON,

description of the members of the intermediate group upon
special media.

As any material, whether feces, water, sewage, or food
likely to contain typhoid bacilli, also contains a vastly greater
number of other organisms, the isolation of the desired
organism much resembles the proverbial finding of a needle
in a haystack. A great many methods and media have been
devised, some of them giving fair results but more of them
not attaining the degree of perfection desired. In general,
the media prepared for the isolation of typhoid aims at one
or both of two ends: isolation by inhibition of other organ-
isms or isolation by favoring the typhoid in some cultural
character.

(a.) Enrichment media. — By the first method, inhibi-
tion is brought about by means of some weakly bactericidal
substance. Carbolic acid was first used but this was found
to inhibit the typhoid bacilli almost as much as the others.
A method suggested by Elsner (1889) was the addition of
one per cent potassium iodide. This method has been dis-
carded. Drigalski and Conradi (1902) prepared a medium
containing crystal violet as an inhibiting agent. Loeffler
(1906) devised a plate medium containing malachite green.
Peabody and Pratt (1908) used malachite green in an enrich-
ment broth before plating upon Conradi-Drigalski medium.
Brilliant green was recently suggested by Browning, Gilmour,
and Mackie (1913). Jackson and Melia (1909) recommend
the use of lactose bile as an enrichment broth. The authors
say that it will inhibit all organisms except B. coli and B.
typhosus and that eventually the typhoid will overgrow the
colon organisms.

(6.) Differential media. — Of the media differentiating
typhoid by favoring some cultural characteristic, there are
several worthy of note. Hiss (1897) advocated the use of a
gelatin-agar medium upon which the typhoid grew in small
colonies having fringes of thread-like projections. Endo’s
(1904) medium contains fuchsin and lactose. Fermentation

TYPHOID AND PARATYPHOID BACILLI. 401

of the lactose turns the fuchsin red, thus distinguishing the
colon from the non-fermenting typhoid. Hesse (1908), rely-
ing on the active motility of typhoid organisms, prepared a
semi-liquid medium containing only .5 per cent agar. The
typhoid present a large ringed colony; colon a smaller
Opaque appearance.

For confirming a suspicious colony, Hiss (1902) has also
devised a tube medium containing .5 per cent agar, eight per
cent gelatin and one per cent dextrose. Typhoid bacilli
give a characteristic growth along the stab, whereas colon
will form gas bubbles along the line of inoculation. Russell
(1911) has described a medium much better than that of
Hiss for general work, containing one per cent litmus, one
per cent lactose, and .1 per cent dextrose, added to 1.5
per cent agar. Typhoid organisms produce a characteristic
coloring.

(c.) Comparison of different methods. — Most investi-
gators agree that some of these media are no better than
others, but that the advantage lies wholly in the familiarity of
the worker with one particular medium. Thus, in selecting
a method of procedure for a routine examination of feces
regard should be taken for speed, accuracy and ease of
preparation. Owing to the fact that the typhoid bacilli are
few in number in comparison with other intestinal organisms,
an enrichment medium seems desirable before plating.

Enrichment media: The brilliant green medium sug-
gested by Browning, Gilmour and Mackie was tried but
without success. A concentration of brilliant green suffi-
cient to inhibit B. coli and other forms would also inhibit
typhoid. Conversely, a dilution permitting the growth of
typhoid also allowed B. coli to flourish.

402 ROBINSON.

ENRICHMENT IN BRILLIANT GREEN BROTH.

B.G' Solution, | TYPhoid. | Para A. | ParaB. | B. Coli. | SihRionot BG,
SOANCCrtetersttenetet O O Inf. | 9 I: 1,250,000
OGeme rrr cherie | O O Inf. O | 1: 625,000
| Oe MS ge I Se O O Inf. O I : 416,000
AOE Gao dad Go O O Inf. O I : 310,000
OL IS SS a VOT OO O 100 O I : 230,000
eit ce a atate pons oc O 50 O I: 170,000

The brilliant green solution was made at a dilution of
I: 10,000. Amounts indicated were added to broth (reac-
tion-+ 0.5) tubes containing five cubic centimeters. A
loopful of twenty-four-hour broth culture of the organisms
to be tested was added to each tube. After twenty-four
hours’ incubation a loopful was removed from each of the
tubes and plated on Endo’s medium. When possible the
number of colonies was indicated; otherwise the number
was expressed as infinity (Inf.).

B/G. Solution, | TYPhoid. | ParaA. | Para B, | B. Coli, | pittionof BAG.
TAC Cofueie resets le ssierats + + + + en I : 5,000,000
73) Cy MR SRE et i. oh + + ++ I : 2,500,000
BYE SANSA Se + of ++) ++ I: 1,500,000
SAREE ANS Sentara ate —_ + ++ + + I : 1,250,000
Bint tala ote. cots ferctane — — ++ 4 I : 1,000,000
26) Sts ure heReuaiers + — + + — I : 700,000

TYPHOID AND PARATYPHOID BACILLI. 403

The brilliant green solution was made at a dilution of
1: 100,000. The amount of growth was judged by the
clouding of the medium. Grubler’s ‘“ Brilliantgriin’’ was
used.

The lactose bile medium of Jackson and Melia was tried
and gave very encouraging results, although nothing like the
claims of the authors could be realized. A loopful of
normal feces and a loopful of a twenty-four-hour broth
culture of typhoid were inoculated into a lactose bile tube
and thoroughly shaken. After different intervals of time a
loopful of the bile culture was removed and plated on
Endo’s medium and litmus-lactose agar. The lactose agar
was used in order to determine whether or not any of the
constituents of the Endo’s medium inhibited growth. Counts
were practically the same on both media. In the first three
readings the proportion of the lactose fermenting to the
non-lactose fermenting organisms is given. Where the
number of colonies was too great to count it is expressed as
infinity (Inf.). The non-lactose fermenters were found by
agglutination tests to be typhoid. Time is expressed as the
number of hours from inoculation into bile to time of
plating. Duplicate experiments (A and B) were recorded.
Cultures were kept at 37° C.

404 ROBINSON.

ENRICHMENT IN LACTOSE BILE.

A. B
Medium, F Feein, 7 i Time.
| germentera,) || TYPROUG,, | "peeetatters’ | EypROIa:
Endolsin. steels I I | 5 I 3 hours,
I UHI Sisal: I I 5 I 3 ae
End Gis ais orcte 5 I Inf. O Opa
BDA. 8. hehe Allene de (NO Inf. O 6 «
Endoisi. sexist | Inf. O Inf. O PQ
Bo Clee Renmin 65h 7 ) lee 2
EndOiSitsisrckeresers | Inf. O Inf. O LShe vss
TariAPe yess Ine hea Inf O ioe
WG O28 kicsetorel es 100 O 100 O ZA eSs
Te STE SAG svoneitel es 100 O 100 O 2A an oe
Find oisy asteraeies 30 3 100 I eto)
JDeCO Siacorac 150 O 200 O Gol mass
Bndorsii ee ane 200 O 200 15 Saunas
FinGo?sifrcrcreneors 200 O 12 O OGite
FENG OS Haeleyeieiere 150 O 50 | O 108.5
|

It was found that in from three to twelve hours a loopful
of the surface liquid contained a greater proportion of
typhoid organisms than at any other time. After twenty-
four hours the number of all organisms began to decrease
and the typhoid also disappeared, contrary to the claim of
the authors.

Plate media: The ideal plate medium is one which will
permit the distinction of the greatest number of colonies and
will not inhibit any of the desired organisms. Hiss’ plate
medium is undoubtedly excellent for growth, but various
investigators have found that the distinguishing thread forma-
tion of typhoid is variable and careful search must be made

TYPHOID AND PARATYPHOID BACILLI. 405

with a microscope for suspicious colonies. Hesse’s medium
is very favorable for growth of typhoid and was given an
extensive trial in this work and previously. It is easy to
prepare and easy to manipulate. It does not, however, seem
to be particularly valuable in the isolation of typhoid from
feces. Owing to its semi-liquid consistency all colonies are
of large size, allowing in a thickly seeded culture only the
last few dilutions to be clearly distinct. If the typhoid
bacilli are greatly outnumbered the chances of finding them
in the higher dilutions are small. When urine or blood con-
taining a pure culture of typhoid was examined, Hesse’s
proved to be a most efficient and satisfactory medium.
Litmus lactose agar gave poor results owing to the spread of
the acidity throughout the medium. Endo’s fuchsin agar,
made according to the method described in Standard
Methods of Water Analysis (1912), gave very satisfactory
results. By the method of inoculating with the bent rod a
large number of organisms can be spread upon a plate.
The high percentage of agar (three per cent) prevents
spread of the acid and the colorless typhoid colonies are
easily distinguished by the naked eye. On this medium it is
quite possible to detect typhoid colonies when outnumbered
a thousand to one. Endo’s medium used in connection with
bile constituted the most efficient and expedient method of
isolation. Enrichment in bile for six hours and plating on
Endo’s for eighteen to twenty-four hours was sufficient time
to establish the presence or absence of typhoid. Inoculation
of suspicious colonies into Russell’s medium was found
valuable as a check on the Endo’s medium.

Eight specimens of feces from persons giving positive
Widal reactions were inoculated into bile and after three, six,
twelve, eighteen, and twenty-four hours portions of the bile
culture were inoculated into Endo’s medium, Hesse’s medium,
and litmus lactose agar. Three yielded cultures of typhoid
on Endo’s medium after six hours and were negative on the
other media. One was positive on litmus lactose agar after
six hours and on Endo’s medium after twelve hours. The
urine from one case gave a pure culture of typhoid bacilli,

406 ROBINSON.

while the feces were negative. In the other three cases no
typhoid bacilli could be found.

II. METHODS OF IDENTIFICATION.

At present we have but two convenient methods of identi-
fying isolated organisms of the typboid-colon group; by
means of their cultural characteristics in carbohydrate media
and by theirserum reactions. The acid agglutination method
of Michaelis (1911) may have possibilities but at present it
seems unsuited to routine work.

(a.) By carbohydrate media. — The power of carbohy-
drate fermentation is considered by some to be one of the
most constant means of identification. Typhoid is charac-
terized by acid production without visible evolution of gas in’
dextrose, mannite, maltose, levulose, galactose, dextrin, and
sorbite, with no change in lactose, saccharose, dulcite, salicin,
and arabinose. MacConkey (1905) states that typhoid fer-
ments raffinose while others have found that there is no
change. it has been our experience, however, that the sugar
is not affected. |

Penfold (1911) and Twort (1907) show that a typical strain
of typhoid can be induced to produce variations under cul-
tural conditions. Twort, after two years of cultivation on
lactose media, was able to produce a lactose fermenting
strain. Penfold with less difficulty was able to produce
mutants toward dulcite, isodulcite, and arabinose. How fre-
quently these variations occur in nature there is no record,
but since strains differing in their reaction towards glycerin
occur it is quite possible that variations in other directions
exist. Ifa lactose fermenting strain did occur under natural
conditions its presence would never be suspected from the
isolation plates. The mere fact that these mutants are pos-
sible might tend to make one skeptical of slightly discordant
results.

The intermediate group of organisms produce acid and gas
from dextrose, mannite, dulcite, maltose, levulose, galactose,

TYPHOID AND PARATYPHOID BACILLI. 407

and sorbite. There is no change in lactose, saccharose, raf-
finose, salicin, glycerin, erythrite, and inulin. These reactions
refer only to the paratyphoid and Gaertner strains.

Paratyphoid organisms were induced by Penfold to form
variants from some of the usual types of carbohydrate
reactions. If these bacilli are, as some investigators believe,
off-shoots of either the typhoid or colon types they should
be particularly liable to mutation. The ‘“ gas-los”’ strain
isolated by Wagner (1913) from the blood was no doubt a
mutant occurring under natural conditions. Cultural charac-
teristics alone indicated that this strain was neither typhoid
nor paratyphoid. In spite of the fact that there are varia-
tions from the usual type of carbohydrate fermentation, these
reactions must be relied upon to a great extent for want of a
better method.

Preparation of sugar media: Whether or not sugars are
broken down during sterilization under steam pressure has
not been satisfactorily determined. In order to avoid any
complications on this score the carbohydrate media employed
in this work were filtered through Berkefeld candles. A two
per cent solution of Witte’s Peptone having a reaction of
+ 0.6 to phenolphthalein was made and sterilized in the auto-
clave. A two per cent sugar solution was made and filtered
into sterile flasks. The two solutions were mixed in equal
parts and .05 per cent of azolitmin added. The resulting
sugar broth was placed in sterile tubes (10x 43 millimeters)
and incubated twenty-four hours to insure sterility.

The organisms to be tested were inoculated into nutrient
broth and a loopful of twenty-four-hour culture was trans-
ferred into the sugar broth tubes. This method insured the
inoculation of an approximately equal number of organisms
into each tube, which cannot be done when inoculating
directly from solid media into broth. Growth in this medium
took place readily and abundantly. Acid production could
be easily detected by the reddening of the medium. Gas
production was indicated by a ring of foam around the sur-
face of the liquid if care was taken not to violently agitate
the tubes. A test of all cultures for gas production was

408 ROBINSON.

made in dextrose broth with the inverted vials. The results
with media prepared in this manner corresponded with the
reactions obtained by others with the fractional sterilization
which they probably used. Liquefaction of gelatin and indol
production was tested with all cultures.

(6.) By serum reactions. — Specific agglutinins: The
agglutination of typhoid bacilli is, theoretically, a specific
reaction. The organism is, as a rule, very sensitive to
agglutinin; the reaction with immune serum taking place
often in a dilution of one to a hundred thousand and in some
cases at one to several million. There is said to be but little
difference in the agglutinability of different strains which
agglutinate at all. Several hundred tests during the routine
work in this laboratory showed no difference in two strains:
one Worcester, the other Rawlings. In work to be referred
to later no difference was noted in the agglutination of
homologous and heterologous strains. Strains are frequently
isolated which agglutinate only after repeated transfers on
artificial media. Recently McIntosh and McQueen (1914)
have reported work with a strain which was incapable of
agglutination. When an animal was immunized with this
strain the serum failed to react with it, yet a strong reaction .
was obtained with other typhoid cultures. Strains of the
intermediate group have been found which failed to agglu-
tinate. Torrey (1913) reports such strains. No specific
agglutinins were found in the blood of the dogs from which
the organisms were isolated. These mutants from the
prevailing type seem to be observed only incidentally.
Undoubtedly a great number of these atypical strains escape
unnoticed,

Group agglutinins: In agglutinating powers the members
of the intermediate group are similarto typhoid. The speci-
ficity of the reaction is not absolute with these organisms
owing to the interaction of the group. Boycott (1906)
reports that out of eighty-six typhoid immune sera fifty-nine
per cent showed agglutinating power towards members of
the intermediate group, fifty-five per cent toward Gaertner,

TYPHOID AND PARATYPHOID BACILLI. 409

forty-one per cent toward B. paratyphosus B, and twelve per
cent toward B. paratyphosus A. Ten of these (or nine per
cent) showed a reaction toward all four strains. Sera immune
to one of the members of the group often possess secondary
agglutinins for several of the others. It was not usual, how-
ever, for the secondary agglutinins to reach more than fifty
per cent of the primary.

Castellani (1902) has developed a method for separating
the primary from the secondary agglutinins. He found that
if a typhoid immune serum, which would agglutinate also a
member of the intermediate group, was saturated with typhoid
organisms, not only were the typhoid agglutinins removed but
also the secondary ones. On the other hand, if the serum
was made immune against two organisms, then absorption with
one of them would not remove the agglutinins from the other.
To express the reaction graphically in the terms of Durham
(1901): let A+B+C+D+E represent the agglutinins
for the typhoid bacillus and C+D-+E-+F that for an
intermediate organism. Then after absorption with typhoid
the mechanism for a reaction with an intermediate organism
would be incomplete if the serum was immune to typhoid
only. After the serum was immunized against two organisms
as in a mixed infection then there would bea double quantity
of C+D+E and, after absorption with typhoid, agglu-
tinins for the intermediate organism would be intact. We
might expect organisms culturally and morphologically
similar to show inter-reactions, but we are at a loss to explain
the inter-reactions of widely different organisms. Frost
(1910) reports strains of Pseudomona proteus which will
agglutinate at higher dilutions with typhoid immune serum
than will the typhoid bacillus itself. Boycott (1906) de-
scribes an organism “ Valerie 21” culturally quite distinct
from any members of the typhoid-intermediate group and
yet agglutinating at high dilutions with both typhoid and
intermediate sera. Group agglutinations, therefore, although
they may have some significance are uncertain and unreliable.

Cultures obtained in the routine examination of feces

410 ROBINSON.

which conform to the cultural reactions of typhoid or paraty-
phoid are tested for their agglutinating powers with a known
serum. The serum used is of high titer obtained from the
Mulford Company. The serum is dissolved in normal saline :
one part of serum to fifty parts of saline. Tests are made
microscopically using a dilution of 1: 100. Results are
observed at the end of one hour.

(c.) Classification of organisms. — With so many varia-
tions from the type organisms the question of differentiating
species is an important one. The typhoid bacillus, as we
know it, is characteristic. Its failure to produce gas during
carbohydrate fermentation, the carbohydrates upon which it
acts, and the specificity of the serum reactions, are distinctive
enough for practical purposes. A strain varying in one
respect is likely to conform to type in other respects. This
assumption is made in spite of the fact that variability is one
of the characteristics of bacteria and also ignoring the possi-
bility of several mutants occurring at unce. Few if any
bacterial strains have been found closely resembling the
typhoid type, hence cultural and serum reactions are of
especial value in the classification of the typhoid organisms.

The classification of the intermediate group is more com-
plex. The criteria for placing an organism in this group are
vague and ill-defined. The cultural characteristics of the
Gaertner organism are generally considered as the type for
this group. Savage (1913) would divide the group into the
true Gaertner and paragaertner types by an extended set of
carbohydrate tests. He also states that the paragaertner
organisms are for the most part non-pathogenic. Para-
typhoid A would thus be considered in the paragaertner
sub-group, while ParatyphoidB would be a species of true
Gaertner. Bradley (1912) claims that ‘‘ the sub-division of
A and B types cannot be strictly maintained biochemically
as linking types are found.” Henderson Smith (1913)
would classify the group primarily by cultural character-
istics. The true’ type organisms are then divided by agglu-
tination tests into three groups: one containing B. gaertner,

TYPHOID AND PARATYPHOID BACILLI. ALE

a second B. paratyphosus B, and a third B. suipestifer.
Bainbridge (1911) would identify and classify the organisms
conforming culturally to the Gaertner type by agglutination
and absorption tests.

Since the agglutination reaction is not specific among the
members of the intermediate group this test alone cannot
be relied upon. Thus, in this group, classification must
depend largely upon the carbohydrate reactions. It is the
most practical method available at present. It need not be
an arbitrary method but is elastic enough to allow the
addition of new species which undoubtedly exist.

III. DIAGNOSIS BY SERUM REACTION.

In the routine diagnosis of suspected blood we use three
strains of organisms, viz.: ‘‘ Worcester” Typhoid, Para-
typhoid A (Army), and Paratyphoid B (Army). All are
actively motile and free from spontaneous agglutination.
Three reports are given: positive, the complete, closely

?

clumping, ‘‘windrow formation;” partial, in which the
organisms show some tendency towards agglutination, and
negative, being the same as the control test. Specimens to
be tested are drops of patient’s blood dried on a glass slide.
The blood is suspended in normal saline and made up to
match the color of a known suspension of normal blood.
A dilution of I to 60 was employed in the tests and a test
showing complete reaction within an hour was considered
positive. ;

During the six months July-December, 1913, two hundred
and forty-five specimens of blood from suspected typhoid
patients were examined. These were reported as follows:

Positive to Bacillus typhosus alone..............-.00- 47 19%,
Partial Bs ce SOON Ret ot ag sieictavsce, cis, aria ate 40 15%
Positive to Bacillus paratyphosus B alone............. 5 2%
Partial “ se u SE se redo os ccisy ev eteVe, aver > 4 2%
ROSHI GENO OEE misiare nls aisles cates eller tea. :3. ale lay “nieeayevala 8 4%
Positive to B. typhosus and partial to B. paratyphosus B, 2 1%

PMG PApLV CALOMBOEN stot sted srors] evecare teens Sracieiaresere sie cravee\s 139 57%

412 ROBINSON,

Most of the cases reported positive were confirmed by
the physician from the clinical symptoms. Occasionally a
complete or partial reaction would be obtained with blood
from a person who was either not sick or developed some
other disease. The specimens of blood were taken very
early in the course of the disease in most cases, which seemed
a plausible reason for getting partial or incomplete reactions.

A number of positive bloods sent in by physicians were
tested as to the limits of the agglutinating power. It must
be remembered that the majority of them were taken during
the first week of illness. It was found from the history of
the cases that those having the highest titer had been sick
longest. The highest titer of those tested was 1200 and
from that down to those which gave a partial reaction at
1:60. The greater number of those examined were found
to have a titer between 100 and 200. These results would
confirm the use of 1: 100 as the most favorable dilution.

(a.) Reaction of normal blood. — In order to get a suit-
able check on the reaction it seemed best to examine speci-
mens of normal blood. Specimens were obtained from
University students according to Wright's method in capillary
pipettes. The blood was allowed to stand until the clot
formed. Then the clear serum was removed and accurate
dilutions were made with capillary tubes. Tests were first
made at a dilution of 1:10 and if positive the dilution was
increased until the agglutinating limit was reached. All
tests were made microscopically and results observed at the
end of one hour. Results were as follows:

Number of specimens examined 225.0). .0/<e1\0 00s)» sleere si == 103
Positivelatet: £0 for Bs ty Phosusy sj cree slertdarererenetetelete totes 29
os Hie LG ho} Sip ae 4h pooduaodcdpodD oo CO ODOnc Il
So age lehq Ona” mit Ge sa0bbanbOHOoOOD0N aan “(8
ee sre OOlnees ae G “neanodos0oDdKon0bsooDGDC fe)
cr ro) °° B; paratyphosus) Bove. -jev-)e1eye4s)a¥e/a'=r= 6
yp
< SST ZO Wee Line ss UA Soanooroopene noe 2
ss SES iy Koy aa <§ ST epodoabDoGasabca oO

TYPHOID AND PARATYPHOID BACILLI. 413

Only one reported a previous attack of typhoid fever five
years ago and this blood was only positive at 1:10. Another
had taken one inoculation of typhoid vaccine and his blood
was negative. From these results it was evident that no test
should be made at a dilution of less than 1:60 and that
probably 1: 100 would be less prone to complications with
natural agglutinins. Park (1910) reports that in the New
York Health Department a dilution of 1:10 is used and an
immediate reaction only is considered positive. It has been
our experience, however, that specimens of dried blood
cannot be conveniently diagnosed at such a low dilution.

(6.) Partial reactions. — The partial reaction which it
was found necessary to report at times is frequently ques-
tioned by the physician. Wilson (1901) in the examination
of one thousand six hundred and fifty blood specimens found
that one hundred and sixty or about ten per cent gave a
partial reaction. He also had eight cases giving a positive
reaction which were not diagnosed as typhoid. From his
work he concludes that the partial reaction is valueless as far
as a diagnosis is concerned. With such a report the
physician should judge by the clinical symptoms during the
course of the illness. Of the forty partial reactions reported,
further inquiry elicited the following results:

Slinicallya hyp Voter parece stevs\areleherersie share cialetecsie; eras s+ se 14
a NOt LY PHO sc). c's os le viele AbpEdeo ac moeGtsC 13

Ph ysichanidtd mob eno wis: eciaicid ofl cta« «zis aisha < aie ei acese 5 ote Z
1s OY TOT? SSS ES ape co J ect oo Been ne Oe AI ace eroe Sern 5
Batersiotindane Satlverrsre ar eteteicysrctst cial fae e-(a03 nis ats sve c.<.> ole 2
3 eel POSIELV Era rereteteren el ot “60 gdeecsubontboonce 2

PC OUMLCERE UIC Copter tatna date Nel orsia eee tol Jace etch alot wotalate. ate 6

Two of these cases had suffered an attack of typhoid three
or four years previously; one the physician considered a
mild case at the time of the partial Widal. Two of those in
which the partial reaction persisted were considered as _posi-
tive cases and two were not typhoid. From these results it
is evident that the final diagnosis of a partial Widal reaction
must be made on the clinical symptoms of the patient.

414 ROBINSON.

(c.) Mixed infections. — Unfortunately it was not possi-
ble to make a thorough study of all of the eight cases showing
a positive reaction to both B. typhosus and B. paratyphosus
B. In only two of these specimens was the exact titer deter-
mined, and in both instances were positive to B. typhosus at
1: 200 and B. paratyphosus B at 1:100. Specimens of feces
yielded in one case typhoid and the other paratyphoid organ-
isms. Whether or not these were mixed infections we do
not know, yet it seems probable that they were.

IV. SERUM REACTIONS DURING VACCINE TREATMENT.

Just how far a comparison may be drawn between the
serum reactions produced by vaccination and those in clinical
cases of typhoid, there may be considerable doubt. In the
case of infection the number of organisms probably increases
gradually from comparatively few toavast number. In vac-
cination a known number of dead organisms are given at
definite intervals. There are several points upon which
comparative data are of interest.

(1.) The length of time from infection to the appearance
of agglutinins in the blood.

(2.) The rate of production of agglutinins.

(3.) Whether or not secondary agglutinins appear.

(4.) Whether there is variation in different individuals.

In order to study the serum reactions during typhoid vac-
cination fourteen of the students in the Brown University
Biological Laboratory were given the treatment. The first
four were given the typhoid vaccine and ten the mixed.
Commercial bacterins were used. The pure typhoid bacterin
contains five hundred million bacilli for the first dose and
one billion for each of the second and third doses. The
mixed bacterin contains the same number of typhoid bacilli
with the addition of two hundred and fifty million each of
paratyphoid A and B for the first dose and five hundred
million for the second and third. Injections were made at
intervals of a week except in case of five of those taking the
mixed vaccine when the interval was ten days. Blood was

TYPHOID AND PARATYPHOID BACILLI. 415

taken every other day as nearly as possible. The clear
serum was used for tests made according to methods pre-
viously outlined. In each case the titer recorded was that at
which a complete reaction took place.

SERUM REACTIONS DURING TYPHOID VACCINATION.

= = = = ——— ——— —————
Agglutinins Agglutination Maximum Titer of

‘Gs: Increase After — at 1:60 After — Reaction After — | Maximum Reaction,

Pypia |) An cB. \eryph. |! AL tie. Vryphs | A. '| B: | Typ. | Ay |B:

|

Days. Bags \ Date Days. |Days.|\Days.| Days. |Days.| Days |
I 4 OF | 70 uC, Oo 10 @) Oo 100 Oo oO
2 18 oO | oO 18 | oO Oo 30 0) O | 1,000 Oo oO
3 SiGe eo au Omori’ | io Mo 80 | ‘oe |-o
4 4 o | 0 6 GeO 12 | OF. 80 ‘tom |hO
5 20 26 12 26 | 28 26 30 30 30 1,000 | 1,000 | 1,000
6 8 8 $ 14 | 14 14 20 18 18 6,000 | 4,000 | 4,000
” 8 14 12 16 16 12 1S 18 28 1,600 | 1,600} Soo
S 10 18 18 18 | 18 18 18 18 18 2,000 Soo | 600
9 12 12 12) eZ Tm wad 19 16 16 1,200 | 1,200! 1,200
10 20 20 20 20 20 20 22 22 22 600 600 | 600
II 18 18 18 1§ 18 18 1S 18 18 400 400 | 400
12 20 20 20 20 20 20 24 24 24 1,000 | 1,000 | 1,000
13 20 20 20 22 22 22 24 24 24 sfele) S00 | 800
14 16 16 16 24 24 24 26 26 26 400 400 | 400

|

It must be observed at first glance that there is a great
individual variation in all respects. The period from the
first inoculation to the appearance of an increase in agglu-
tinins varied from four days to eighteen days. The average
length of time from the first inoculation until the blood
agglutinated in a dilution of 1:60 was sixteen days, and
varied from six to twenty-eight days. Park (1910) states
that twenty per cent of the clinical cases give positive results
the first week, sixty per cent the second, eighty per cent the
third, and ninety per cent the fourth. If ten to fourteen
days are allowed for an incubation period in clinical cases,
the average length of time from infection until a positive
reaction is obtained is probably slightly longer than the time

416 ROBINSON.

from the first inoculation of a vaccine until a positive reaction
appears.

The agglutinins are not produced in gradually increasing
amounts. Castellani (1914) reports that, although the cases
which he vaccinated showed no agglutinating power the first
week after inoculation, at the end of the second the maximum
agelutinating power was reached. The writer’s results corre-
spond very nearly to those of Castellani. There seemed to
be a general tendency towards a marked rise in agglutinating
power during the first part of the third week. That this
also occurs in infection seems probable. One clinical case
which came under our observation was negative twenty-four
days after infection but was strongly positive on the twenty-
seventh day.. Another which was partial on the twenty-eighth
was strongly positive on the twenty-ninth. After the maxi-
mum agglutinating power is reached, the amount may fluctu-
ate but the tendency is to diminish.

The typhoid bacterin was used chiefly to observe whether
or not secondary agglutinins were produced. The bacterin
was used almost at the expiration of the period for which it
is guaranteed, which probably accounts for the low titer of
the sera. There were, however, no secondary agglutinins
observed. In the sera of those immunized against three
strains the agglutinins were practically the same for each
one, although the number of paratyphoid organisms was
only half that of the typhoid. Wherever there was a differ-
ence the typhoid antibodies exceeded those for paratyphoid.
Two typhoid cultures were used in all of the tests, the Raw-
ling’s strain, of which the bacterin was made, and the
Worcester strain, but no difference in agglutinating power
was noted. The same strains of paratyphoid were used as
were employed in the bacterin.

These observations upon the production of agglutinins
during immunizing treatment confirm the experience of
routine diagnosis. The physician should not consider a
negative Widal as final. If the suspicious clinical symptoms
persist the Widal should be repeated at intervals. Whereas
some individuals would show a reaction early in the disease,

TYPHOID AND PARATYPHOID BACILLI. 417

others would not show it for some time. The agglutinins
should appear during the third week after infection although
individual differences occur. A serum which reacts to both
typhoid and paratyphoid seems to indicate an infection by
both of these organisms.

SUMMARY.

1. The most satisfactory method of isolating typhoid and
paratyphoid organisms from feces is by incubation in pep-
tone lactose bile for six to twelve hours and then plating
upon Endo’s medium.

2. Identification of organisms is best accomplished by
reactions in carbohydrate media. Serum reactions are of
more value with typhoid than with intermediate group
organisms.

3. Normal blood is frequently capable of agglutinating
typhoid and paratyphoid bacilli.

4. A partial Widal reaction has no diagnostic significance.
The test should be repeated at intervals until a positive or
negative result is obtained or else the case should be diag-
nosed by its clinical symptoms.

5. A-serum agglutinating both B. typhosus and B. para-
typhosus A or B indicates a mixed infection.

6. A study of the serum reactions during typhoid
immunization demonstrated that —

(a) Great individual differences occur.

(6) Agglutinins begin to increase in the blood from four
to eighteen days after the first inoculation.

(c) The blood gives a positive agglutination at 1: 60, on
the average, sixteen days after the first inoculation.

[Acknowledgment of the advice and assistance in the preparation of
this paper is gladly given Professor Frederic P. Gorham of Brown Uni-
versity and Doctor Charles V. Chapin and Doctor Eugene P. King of the
Providence Health Department. ]

418 ROBINSON,

BIBLIOGRAPHY.

Bainbridge and O’Brien, tg11. Jour. of Hygiene, xi, 68.

Boycott, 1906. Jour. of Hygiene, vi, 33.

Bradley, 1912. Second Report of the Gov. Bureau of Microbiology,
New South Wales, 159.

Browning, Gilmour, and Mackie, 1913. Jour. of Hygiene, xiii, 335.

Castellani, 1902. Zeitsch. f. Hyg. u. Infekt., xlii, 1.

Castellani, 1914. Centralbl. f. Bakt., 1 Abt. Orig., Ixxii, 536.

Drigalski und Conradi, 1902. Zeitsch. f. Hyg. u. Infekt., xxxix, 289.

Durham, 1go1. Jour. of Exp. Med., v, 353.

Elsner, 1895. Zeitsch. f. Hyg. u. Infekt., xxi, 25.

Endo, 1904. Centralbl. f. Bakt., 1 Abt. Orig., xxxv, 109.

Frost, 1910. Hygienic Laboratory Bulletin, Ixvi, 27.

Hesse, 1908. Zeitsch. f. Hyg. u. Infekt., Iviii, 441.

Hiss, 1897. Journ. of Exp. Med., ii, 677.

Hiss, 1902. Jour. of Med. Research, viii, 148.

Hurler, 1912. Centralbl. f. Bakt., 1 Abt. Orig., Ixiii, 341.

Jackson and Melia, 1909 Jour. of Infect. Diseases, vi, 194.

Loeffler, 1906. Deutsch. Med Woch., xxxii, 289.

MacConkey, 1905. Jour. of Hygiene, v, 333.

McIntosh and McQueen, 1914. Jour. of Hygiene, xiii, 409.

Michaelis, t911. Deutsch. Med. Woch., xxxvii, 969.

Park, 1910. Pathogenic Bacteria and Protozoa, 297.

Peabody and Pratt, 1908. Boston Med. and Surg. Journal, clviii, 213.

Penfold, 1911. Jour. of Hygiene, x1, 30.

Russell, rgtt. Jour. of Med. Research, xxv, 217.

Savage, 1913. Report to the Local Government Board, New Series 77,
Food Reports No. 18.

Smith, 1913. Fifteenth International Congress on Hygiene and Dem-
ography, Sept. 23-28, 1912. Gov. Printing Office, gg.

Standard methods of water analysis, 1912. American Public Health
Association, 133.

Torrey, 1913. Fifteenth International Congress on Hygiene and Dem-
ography, Sept. 23-28, 1912. Gov. Printing Office, 127.

Twort, 1907. Proc. of the Royal Society, Series B, xxix, 329.

Wagner, 1913. Centralbl. f. Bakt., Abt. 1, Orig., Ixxi, 25.

Wilson, 1r901. Medical News, Ixxix, 81.

THE JOURNAL OF MEDICAL RESEARCH, Vol. XXXII., No. 3, July, 1915.

132

THE OLD MEDICAL SCHOOL IN BROWN UNIVERSITY.
BY FREDERIC P. GORHAM.
The Providence Medical Journal, Vol. XVI, July, 1915.

The Old Medical School in Brown University*

By FREDERIC P. GORHAM,

PROFESSOR OF BACTERIOLOGY, BROWN UNIVERSITY.

Medical instruction in colonial times consisted usually of an
apprenticeship to some retired army surgeon or physician of renown
for a term of three or four years, after which the student was certified
for practice not by receiving the degree of M. D. but by a license to
practice from some examining board. One of the first acts of the
General Assembly of the Colony of Rhode Island after receiving the
charter of 1663 was to confer upon Capt. John Cranston a license
“to administer physic and practice chirurgery” and also the degree of
“Doctor of Phissick and Chirurgery.”

The first practical instruction in anatomy given in Providence was
that imparted by Dr. David Vandelight to a few students in his own
house. Dr. Vandelight was a graduate of Leyden. He was attracted
to Providence in all probability by its commercial possibilities for
besides his duties as physician and apothecary he was skilled in the
chemistry of the day and introduced here the Dutch method of separat-
ing spermaceti from its oil, a process which brought much wealth to
the doctor and to the Plantations. At his death on February 14, 1755,
the value of his drugs and instruments was inventoried at over four
thousand pounds. His house is still standing on South Main Street
between College and Hopkins Streets. Dr. Vandelight died fifteen
years before Rhode Island College moved to Providence and as far as
we know he had no connection with that institution, though he was a
brother-in-law of Nicolas, John and Moses Brown whose name was
later to adorn the University.

Until 1811 only two medical schools existed in New England,
that at Harvard, founded mainly through the efforts of Dr. John

*Read at the annual meeting of the Rhode Island Medical Society, June 3, 1915.

Reprinted from THE PROVIDENCE MEDICAL JOURNAL, July, 1915

NO

Warren about the close of the Revolutionary. War; the other at Dart-
mouth, created in 1798 by the persistence of Dr. Nathan Smith, a na-
tive of our neighboring town of Rehoboth, ‘a great organizer and
very eminent medical teacher and writer, who for some years was
its only professor.”

In September, 1811, three medical professors were appointed in
Brown University. Dr. William Ingalls of anatomy and surgery, Dr.
Solomon Drowne of materia medica and botany and Dr. William
Corlis Bowen of “chymistry,” and a committee of the Corporation
was appointed to procure a suitable person to give lectures on the
theory and practice of physic.

Previous to this time the degree of M. D. had been conferred
by the university only as an honorary degree upon persons already
eminent in the profession. With the appointment of these professors .
the university was now equipped to give the degree in course, and in
the year 1814 we find the first graduates with the degree of M. D.
appearing on the record. At the time of the appointment of these men
the university was pursuing its ordinary courses of academic train-
ing with about one hundred students. It had moved from Warren in
1770, had changed its name from Rhode Island College to Brown
University in 1804. Dr. Messer had been President since 1804. Dr.
Solomon Drowne of the class of 1773 had become a Fellow of the
college in 1783, and was given the honorary degree of M. D. in 1804,
which he had previously earned in course at the University of Penn-
sylvania. As the only physician on the Board of Fellows when the
Medical School was created, he probably had a large part to do in
suggesting and promoting its establishment.

Dr. Solomon Drowne was an interesting character. Born in
Providence, March 11, 1753, “he graduated in the fifth class at Rhode
Island College in 1773. He studied medicine with Dr. William Bowen
and at the University of Pennsylvania, then the only organized
medical school in the country. He was in the service of the colonies
as surgeon, was in General Sullivan’s expedition on Rhode Island,
served at different points in Connecticut and New York and witnessed
the evacuation of New York City by the British troops. In the fall
of 1780 he went on a cruise as surgeon in the private sloop-of-war
Hope, his journal of which has been printed. He won the regard of
Lafayette, the Counts de Rochambeau and d’Fstaing, as well as of
other French officers, to stich a degree by his medical ability and skill

3

as a surgeon that the chief of the medical staff entrusted their invalid
soldiers to his care when they left for home.” In 1784 he went to
Europe, attended hospitals and lectures in London and Paris. Later
he moved to Ohio, removed to Virginia and then for several years
lived in Pennsylvania. In 1801 he came back to Providence and
bought an estate in the town of Foster, “where he could indulge his
love for country life, his fondness for gardening, for botany and
varied reading. He built his house on a hill, which he named Mount
Hygeia. He founded a botanical garden, and from his professional
tours, which extended to Providence, and not infrequently to other
states, he always returned with seeds or plants to enrich his collection.
He also sent abroad for plants and was the first to introduce many
species into our country which have since become common. People
came from long distances to see his famous garden. He is said to have
raised and prepared his own opium. In medical practice he appears
to have tended to simplicity and to a greater trust in the powers of
nature than was common in his time. By simplicity he meant an
avoidance of officious interference, and in particular of uniting many
remedies in one prescription. Butternut pills, decoction of mallows
and pussy-willow tea were among his favorite medicines.’”’ Last spring
the graduate students and faculty of the Biological Department of the
university made a pilgrimage to his old home in Foster, visited his
grave, wandered through the remains of his botanic garden, where
still many unusual herbs and shrubs run wild, examined his library,
which is still intact, and saw many relics of the old doctor, such as
wearing apparel, diplomas, foreign decorations, mortars and pestles,
medicine chests, bottles and jars, many still containing the curious
medicines which he compounded. In all probability the medical
school in Brown University had its origin in the mind of Dr. Solomon
Drowne.

Of the other members of this first medical faculty just a word.
Dr. Ingalis was the least known in Rhode Island. He was born in
Newburyport, Mass., May 3, 1769, graduated at Harvard in 1790 and
took his degree of Doctor of Medicine at Harvard in 1801. He lived
in Boston throughout his professional life and became prominent in
practice, especially in surgery, and as an instructor in anatomy. He
began to give lectures in Brown University soon after his appoint
ment as Professor of Anatomy and Surgery, in 1811, and continued
to lecture there until 1816, when he resigned. His resignation was

4

not accepted, however, although he did not thereafter teach in Provi-
dence, but students of the university continued to attend his lectures
in Boston, at least until 1820, as a part of their course for the medical
degree at Brown. Dr. Ingalls died in 1851.

In 1815, just before Dr. Ingalls resigned, Dr. John Mathewson
Eddy of Providence was appointed adjunct or assistant professor to
Dr. Ingalls. Dr. Eddy gave little instruction and died early in the
year 1817. He was one of the forty-nine physicians who petitioned
for an act of incorporation for the Rhode Island Medical Society and
who were named in it as the original fellows in 1812.

Dr. William Corlis Bowen, professor of ‘“chymistry,” belonged
to a family eminent in medical practice. Indeed, long before the town
records of Providence mention the name of any physician, during the
early decades of the eighteenth century, whoever desired the best
medical or surgical aid sent to Rehoboth for Dr. Jabez Bowen. Later
Dr. Bowen moved to Providence and was the first regularly educated
practitioner of medicine and surgery who permanently resided in the
town. Dr. William Corlis Bowen, the professor, was his son, born
June 2, 1785, entered Rhode Island College, but went with President
Maxcy to Union College, where he graduated in 1803. “After a few
years of practice in Providence he went to Europe and studied at the
University of Edinburgh, where he took his medical degree in 1809.
He studied also at Paris and London, where he was a private pupil
of Sir Astley Cooper.” He resigned his professorship in 1813 and in
1817 his successor, Professor John DeWolf, Jr., of Bristol, was ap-
pointed.

In September, 1815, the chair of theory and practice of physic
was filled by the appointment of Dr. Levi Wheaton, a native of Provi-
dence and a graduate of Rhode Island College in 1782. In 1778 he
was attached to a military hospital in Providence; in the summer of
1779 he studied medicine with Dr. Joshua Babcock of Westerly. He
was afterwards surgeon of a privateer and later had charge of the
prison-ship Falmouth at New York. At the close of the war he
settled in Hudson, N. Y., where he practiced ten years, and later re-
moved to his native town, where he lived for over half a century,
dying August 29, 1852.

In September, 1823, Dr. Usher Parsons of Providence was ap-
pointed adjunct professor of anatomy and surgery and one year later
was chosen professor in place of Dr. Ingalls. Dr. Parsons was born

5

in Alfred, Maine, August 18, 1788. He studied medicine in his na-
tive town and afterwards at Boston under the tuition of Dr. John
Warren. In July, 1812, he was commissioned surgeon’s mate in the
navy and served under Commodore Perry on the Great Lakes and in
the battle of Lake Erie. He received the degree of M. D. at Harvard
in 1818. During his service in the navy he visited the medical and
scientific institutions of Naples, Palermo, Rome, and Florence and
spent several months in Paris and London, attending the hospitals
and gaining the acquaintance of eminent surgeons and _ naturalists.
He was professor of anatomy and surgery at Dartmouth College in
1821 and took up his residence in Providence in 1822. He wrote that
his “motive for engaging in the business of lecturing was a desire
to establish a museum of anatomy, human and comparative, on the
plan of the late John Hunter’s.” In Brown University he gave lec-
tures both to the medical class and to the college students till 1826,
and it is said that the lectures of Dr. Parsons gave new life to the
institution. Dr. Parsons subsequently became very prominent in the
practice of his profession, especially in surgery and as a consulting
physician. He contributed largely to medical journals, he served as
Vice-President and acting President of the American Medical Asso-
ciation and took an active part in the founding of the Rhode Island
Hospital. He died in Providence, December 109, 1868.

This was the distinguished faculty of the Brown University
Medical School. What of the students and graduates? During the
period from 1814 to 1828, ninety-three men received the medical de-
gree in course. It would take too long to mention even the names of
these Brown doctors. Four of them became distinguished practi-
tioners in this State, Dr. Lewis J. Miller of the class of 1820, Dr.
George Capron of the class of 1823, Dr. Hiram Allen of Woonsocket
of the class of 1825 and Dr. Francis L. Wheaton of the class of 1828.

Dr. Jerome V. C. Smith of the class of 1818 was a well-known
author and for many years editor of the Boston Medical and Sur-
gical Journal and Mayor of Boston in 1854. Dr. Alden March of the
class of 1820 practiced many years in Albany, N. Y., going there di-
rectly after graduation, was founder and President and for many
years leading instructor of the Albany Medical College, was Presi-
dent and one of the founders of the American Medical Association.
Dr. Elisha Bartlett was a member of the class of 1826, a professor
of Dartmouth College and later of the College of Physicians and

Surgeons.

6

Besides the ninety-three graduates the names of twenty non-
graduate medical students are known. Thacher’s American Medical
Biography, published in 1828, gives the number of students in the
Medical Department of the university, 1825-’26, as forty. In the
years 1818-’20 there were sixteen graduates in course; 1821-’23,
twenty-two, and in 1824-26, twenty-eight. The number appeared to
be on the increase and promised well for the development of a strong
Medical School at the university.

But its death blow fell suddenly and unexpectedly. At a special
meeting of the Corporation held on December 13, 1826, Dr. Messer
resigned the presidency and the Rev. Francis Wayland, Jr., of Boston
was unanimously chosen to fill that office. Dr. Wayland had very
definite and pronounced views as to the requirements of college dis-
cipline. Early in 1827 the Corporation voted “that salaries shall be
paid only to such professors, tutors, or other officers as shall devote
themselves during term-time exclusively to the instruction and dis-
cipline of the institution, and shall occupy rooms in college during
study-hours, and attend in their several departments such recitations
as the President may direct, not exceeding three recitations of one
hour each in every day.” Such regulations were impossible for a medi-
cal school. Physicians in active practice could not serve as officers of
discipline as thus strictly defined.

The names of the medical professors appear in the catalog of 1827
with no special remark. But in 1828 an asterisk is prefixed to the
names of Drowne, Wheaton, DeWolf and Parsons with the following
note: “The gentlemen to whose names the asterisks are affixed are not
of the immediate government and do not at present give any instruction
in the University.” In 1829 Dr. Wheaton’s name is omitted, but the
same asterisks and note mark the others. In the catalogs of 1830 and
1831 the note is changed to read thus: “The gentlemen to whose names
the asterisk is prefixed give no instruction in the University and have
no concern in its government.” In succeeding issues the names of these
eminent men drop out altogether.

The downfall of the medical school was not due to any hostile
feeling on the part of the Corporation and President, but was appar-
ently an incidental result of President Wayland’s unswerving con-
viction and policy in regard to college discipline and government. In-
deed, as far as we can learn, President Wayland was particularly in-
terested in this department of the university, for he himself had

7

studied medicine in his youth, and for some years after the close of
the medical school, he taught anatomy and physiology along with a
host of other subjects.

But for fifty years after President Wayland came, not a man hold-
ing a medical degree taught upon the faculty. In 1834 Professor Chase
took the title of professor of chemistry, physiology and geology, but
it was not until 1867 that we find the name of Dr. Charles W. Parsons
appearing, first as lecturer in 1874 and then as professor of physi-
ology in 1878. In 1872 Dr. C. L. Nichols was assistant in analytical
chemistry, in 1878 Dr. A. S. Packard became professor of zoology
and geology and in 1882 Dr. C. V. Chapin became instructor in physi-
ology and in 1886 professor of physiology. These are all the medical
men who have served upon the faculty until quite recent years. Since
the death of Dr. Packard in 1905 not a man holding the medical de-
gree has served the university as professor.

But this does not mean that medical science has received no at-
tention during this time. Physiology was regularly taught from 1867
to 1895 under the able instruction of Drs. Parsons and Chapin. But
it was not until the advent of Dr. Bumpus in 1890 that other depart-
ments of medical instruction received adequate attention.

This date, 1890, marks the beginning of the present Biological
Department of the university. Starting with a single course in com-
parative anatomy with ten or a dozen students, under Dr. Bumpus,
this department now numbers six instructors and a total enrollment
of 424 undergraduates and fifteen graduates. Courses in general
biology, comparative anatomy, physiology, bacteriology, histology,
pathology, neurology are regularly given. Although the names of
these courses suggest the medical school, the work of the department
1s carried on with no idea of developing at some time into a new med-
ical school at Brown University, but with three very definite objects
in view.

First, to provide a broad biological training in the principles of
life and healthful living for the young men and women of Brown.
That this is being accomplished at the present time may best be at-
tested by the fact that only recently the faculty and corporation have
made the general biology course required for all candidates for the
degree of Bachelor of Philosophy, and further by the fact that in this
course at present there are registered over one hundred and fifty men
and sixty women.

Second, to provide a sound preliminary training for those who
intend to enter the medical profession, giving instruction in matters
which in no way duplicate the work which they will later do in med-
ical school, but giving them a broad foundation upon which to build
their future medical education. That this is being done at present may
be fully demonstrated by pointing to the records in medical schools
and hospitals of the graduates of the department. At Harvard, Yale,
Columbia, Cornell and Johns Hopkins, Brown men are winning
honors for themselves and their Alma Mater. I have only to glance
about me in this room to see men ranking high in the medical work
of this city and state who are building on a foundation laid in the
biological department of Brown University.

Third, and to this function of the university I wish to call your
particular attention, as I believe it marks a new line of development
for the future, one of great usefulness to the community which the
university serves, and one which will require your active assistance
and codperation,—the development of a school of health officers.

We are at the present time in Brown University actively en-
gaged in devoloping the instruction in sanitary science for the pur-
pose of training men competent to serve their communities as health
officers, sanitary officials, men in charge of health department labora-
tories, water works, sewage-disposal plants and men trained in the
work of the sanitary engineer. I do not need to call your attention to
the rapid development of the science of sanitation in the last few
years. There was a time when the doctor of medicine was fully com-
petent to serve as health officer in a community. That time has
passed. The protection of the health of a community, the prevention
of disease rather than its cure, has developed to a point at present
where men equipped to serve as sanatarians must be especially trained
along lines but little taught in medical schools. The science of bacteri-
ology, the laboratory diagnosis of disease, the application of chemistry
and bacteriology and engineering to the purification of water supplies,
the disposal of garbage and sewage, the purity of foods, the bacterio-
logical and chemical control of the milk supply, mosquito extermina-
tion, the preparation of vaccines and sera for the treatment of disease
or for immunization, are becoming increasingly more important and
are demanding specially trained workers.

Brown University is ideally situated for the training of such men.
The present affiliations of the biological department of the university

9

with the Health Department of Providence, the State Board of
Health, the Rhode Island Hospital, the Providence City Hospital, the
State Sanatorium, the State and Federal Fish Commissions, the Fed-
eral Bureau of Chemistry, the Providence Milk Department, make our
opportunities unique. After undergraduate training in biology, an-
atomy, physiology, histology, pathology and bacteriology our students
are ready as graduate students to begin actual experience in practical
work under the guidance of men of national reputation in sanitary
matters. Graduate students in the department are at present and for
sometime have been actively engaged in the work of the Health De-
partment of Providence under Dr Chapin. Here they may learn the
methods of laboratory diagnosis of diphtheria, typhoid, gonnorhea,
syphilis, malaria and rabies, the methods of keeping vital statistics,
the actual operation of a modern health department. At the Rhode
Island Hospital, at the City Hospital and at the State Sanatorium,
they may engage in a study of the diagnosis of contagious diseases,
the methods of serum and vaccine treatment and such other activities
as belong to the operation of such hospitals. In the milk department
of Providence they may learn the chemical and_ bacteriological
methods of the sanitary control of a milk supply. Through affiliation
with the United States Bureau of Chemistry, (Dr. Mitchell of the
department staff is collaborating chemist of the Bureau), they may
learn the methods of sanitary food analysis and control. Through
affiliation with the Rhode Island Shellfish Commission they may learn
the methods of sanitary analysis and control of oysters and other
shellfish. Through codperation with the engineering department of
the University they may become trained sanitary engineers, competent
to serve as chemist, bacteriologist or engineer in charge of water-
works, sewage plants, etc. Two or three years of graduate work
after preliminary undergraduate training will fit a man for any of
these lines of work.

It is but necessary to mention some of the positions graduates
of the department are filling at present to show that the training is
efficient: Pathologist of Otisville Sanatorium, city of New York;
bacteriologist, Hudson County, New Jersey; associates in Rocke-
feller Institute and Hospital; chemists and _ bacteriologists with
various commercial houses, city departments and in Government po-
sitions; professor of bacteriology in the University of Wisconsin;

10

professor of sanitary science, College of the City of New York; in-
structor in sanitary science, Harvard University.

With a grand new laboratory just finished for the department
through the generosity of Dr. Oliver Henry Arnold, completely
equipped through the untiring efforts of certain members of this
Society, to be dedicated on Tuesday, June 15, we have the material
equipment necessary for this work. With such men as Dr. Chapin
of the City Health Department, Dr. Swarts of the State Board of
Health, Dr. Richardson of the City Hospital, Dr. Barnes of the State
Sanatorium, the officers of the Rhode Island Hospital and the various
health officials throughout the State, ready to codperate, we could
establish a school of health officers second to none in the country.

I believe it is time to resuscitate the old medical school, not for
the purpose of providing medical instruction in competition with
other well-founded and endowed medical schools, but for the purpose
of providing adequate, modern instruction in preventive medicine.

Let us not look forward to the time when the degree of doctor
of medicine shall be given again by the university on the hill, but
rather, in answer to the insistent call from a new field of medical
science, let us plan to give the new degree of doctor of public health. .

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133

THE AIM AND CONTENT OF HIGH SCHOOL BIOLOGY.
BY HERBERT E. WALTER.
School and Society, Vol. II, No. 48, November 27, 1915, pp 757-762.

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[Reprinted from Scuoou anv Soctrgty, Vol. II., No. 48, Pages 757-762, November 27, 1916)

THE AIM AND CONTENT OF HIGH-SCHOOL BIOLOGY!

In reconstructing our educational ma-
chinery at least four variable factors must
be considered, viz.: equipment, pupil,
teacher and plan.

By equipment is meant all sorts of ped-
agogical material, both animate and inani-
mate, such as school buildings and furni-
ture, library and laboratory facilities,
janitors and superintendents.

Good educational results can be, and
often are, attained in spite of a miserably
poor outfit, and, on the other hand, it is
possible to be so over-equipped that degen-
eration of initiative and resource is the re-
sult. In most instances there is provided
for us an outfit to work with and it is our
proper function to make the best of what
we have rather than to attempt to change it
in any fundamental way. At any rate
there are more urgent and modifiable
phases of our work than this. On a basis
of 100 I should evaluate the equipment
factor at about 10 per cent.

The pupil is at least four times as im-
portant as the schoolhouse with all its
equipment. In the final issue the unfold-
ing process of education can be successful
only when there is something worth while
to unfold.

Here again the teacher can not choose
his material. He must take the raw human
stuff as it comes to him to make the best of

1 An address given before the science section of

the Worcester County Teachers’ Association at
Worcester, November 5, 1915.

it. I thoroughly believe, however, that we
shall soon come to recognize more than we
do now the fact that the far-sighted way to
get the best educational results is to provide
the teacher with better pupils at the start,
or, in other words, with pupils having
greater inborn capacities. This, of course,
is applied eugenics. At present the aver-
age educator is swamped so immediately
with boys and girls already born and thus
having their innate capacities irretrievably
determined, that he hardly has time or op-
portunity to raise his eyes to that larger
ideal of theoretical unborn children which
so stirs the imagination of the eugenist.
Granting that the ordinary pupil reckons
at 40 per cent., surely the teacher should be
valued at, at least, 30 per cent. of the edu-
cational complex, that is, at three times the
value of the material equipment with its
administration.

The value of the teacher is particularly
emphasized in our American schools where
educational schemes are so varied and em-
pirical and where so much depends upon
the execution of the scheme. A _ good
teacher can make even a faulty scheme
sueceed, but a poor techer may confuse and
upset things generally, although provided
with the best of schemes.

It is not proposed to-day, however, to
open the embarrassing personal question of
how better teachers may be obtained, but,
having recognized their importance in the
educational system, to pass on to the fourth

2 SCHOOL AND SOCIETY

phase of our common problem, the plan, or
content of our work. It is a good thing that
our plans are not fixed forms from the out.
side and that we teachers repeatedly apply
ourselves with diligence to their revision.

So far as the subject of biology in the
high schools is concerned, in spite of its
prime importance, its status, particularly in
conservative New England, is by no means
clear.

At the present time it is frequently true
that the biological course of the average
high school has a strong resemblance to
hash. To speak plainly, it too often con-
sists of warmed-up left-overs, for example,
a little nature study from the grades, some
large indigestible chunks of college zoology,
a dash of elementary physics and chemis-
try, a little botany and hygiene, and, in cer-
tain states where required by law, an intem-
perate dose of temperance.

Now hash, correctly compounded and
browned in the pan, requires no apology,
but we all have a pardonable interest in
what goes into it, hence this inquiry into
the content of high-school biology.

At the July meeting of the National Edu-
eation Association in 1913 with the coopera-
tion of Mr. Claxton, the U. S. Commissioner
of Education, an important national com-
mission on the reorganization of secondary
education was appointed, in the hope that
it would, among other things, ‘‘formulate
statements of valid aims, efficient methods
and kinds of material whereby each sub-
ject may best serve the needs of high-school
pupils.’’ This commission was organized
into 14 committees and numerous subcom-
mittees. Mr. William Orr, the deputy
state commissioner of education of Massa-
chusetts, was made chairman of the com-
mittee on natural science and he very soon
organized his department into five sub-
committees, one of which, that of biology,
had in its personnel ten high-school teach-
ers, three university professors, three

normal-school instructors and one physi-
cian. The seventeen members comprising
this committee, coming as they do from New
York and vicinity, from Chicago and the
middle west, from the Pacific coast and
from New England, are fairly representa-
tive of the country at large.

Under the leadership of Mr. James E.
Peabody, chairman, of the Morris High
School, New York City, this subcommittee
on biology has had numerous conferences
during the last two years and is still wres-
tling with its uncompleted task. A final
report is not yet ready, but a preliminary
draft has been published in which certain
conclusions have been reached, and I wish
to present to you some features of this ten-
tative statement in the hope that construc-
tive criticism may be called forth which
may aid in the formulation of the final re-
port as well as in clarifying our ideas gen-
erally about the aims and content of high-
school biology.

First. ‘‘Fundamental Principles.’’
quote from the preliminary report,

To

The committee maintains that unity of subject
matter in any course in science is of first wmpor-
tance, by which is meant that the subject matter
should be so organized that appreciation of wnder-
lying principles shall form the foundation of the
student’s knowledge, thus giving him a scientific
basis for the organization of his knowledge.

This concept of ‘‘unity of subject-
matter’? immediately differentiates biolog-
ical science of the secondary school from the
nature-study which is supposed to precede
it in the grades.

Hitherto the attention of the well-trained
child has been engaged, and diverted with
many varied aspects of the living things
more or less unrelated as they may happen
to be incidentally encountered. Now
Mother Nature is an exceedingly entertain-
ing, instructive and edifying companion, as
Gilbert White well knew, but we must all
confess that she is hopelessly illogical in

_—

SCHOOL AND SOCIETY 3

the way that her materials are distributed
throughout the earth and that she almost
entirely lacks any bump of order. When
the pupil reaches high-school age, his mind
begins to crave and appreciate the orderly
sequence of facts and ideas which consti-
tutes the science of biology as contrasted
with the hit-or-miss art of nature study.
It is a keen intellectual delight for him to
ereate out of the scattered blocks of famil-
iar facts a building of ideas whose archi-
tecture gives mental satisfaction. This is
why ‘‘underlying principles’’ and ‘‘unity
of subject-matter’? begin to make their
definite appeal to the unfolding mind at the
high-school age. The psychological moment
has arrived for replacing disconnected ex-
cursions and picnics in the pleasant fields
of natural phenomena with the laborious
straight furrows of scientific husbandry,
hence the committee stands, first of all, for
fundamental principles and for a unity of
subject-matter. *
It follows from the foregoing that courses
in so-called ‘‘general science’’ for the first
year in high school would not receive the
indorsement of this committee since, in the
very nature of the case, such courses can
not deal with a unified subject-matter.
They must consist necessarily of fragments
of various sciences superficially treated,
with the result that the furrows thus plowed
in the field of knowledge are all so short,
divergent and interrupted that they lead
nowhere with definiteness. The committee’s
report formulates the aims of teaching biol-
ogy as follows: ‘‘(1) to train the pupil in
- observation and reasoning; (2) to acquaint
him with his environment and with common
forms of plant and animal life, and espe-
cially with the structure, functions and
care of his own body, together with the gen-
eral biological principles derived from this
study; and (3) to show him his place in
nature and his share of responsibility for
the present and future of human society.”’

These aims may be ticketed under five

heads:

1. Research

2. Information

3. Prophylaxis

4, Orientation

5. Application
In review I wish to pass briefly these five
aims for teaching high-school biology.

I. Research, or ‘‘training in observation
and reasoning,’’ is a method of procedure
that has become one of the greatest instru-
ments at the command of the human mind.
President Bumpus, of Tufts College, has
recently defined research as ‘‘finding out
things for yourself at first hand.’’ There
is nothing obscure or uncanny about the
scientific method of research. Anybody
can engage in it, but not every one habit-
ually does so. It is often easier, if one has
a slothful mental attitude, to rely upon the
authority of others in getting the answer to
life’s problems, than to replace the passive
acceptance of ready-made ideas with inde-
pendent and resourceful conclusions based
upon research.

II. The second aim, information, is the
necessary ballast in any course of study. I
believe that young minds require a whole
lot of it, Just as young robins will thrive
best when they have literally yards of
worms inserted into their cavernous throats.
What if the pupil does forget a large part
of the information that comes his way?
It is a good thing to do so and it may be
reasonably expected to occur with the aver-
age pupil. The young robins just cited do
not retain the great amount of worms they
devour, but in the process of getting rid of
them they grow visibly. So a pupil will
grow in intellectual capacity and power in
the process of passing information through
his central nervous system, and it is well
for him to cultivate the habit of doing so.
A large part of the high-school course in
biology, consequently, should be informa-

4 SCHOOL AND SOCIETY

tional in character and administered in
good, stiff, honest doses with a minimum
penalty applied if inquisitorial tests and
examinations reveal that much of the in-
formation engulfed refuses to emerge.

Ill. By prophylaxis is meant personal
application to the pupil’s own body and life
of those hygienic measures taught by biol-
ogy which will prevent inefficient living
on his part. The importance of this aim is
so obvious that it calls for no defence, but I
wish to quote a well-expressed paragraph
from Professor Winslow’s Buffalo address
given last year before the Fourth Interna-
tional Congress of School Hygiene.

It is an obvious truism that education is meant
to prepare for living; it seems clear that the most
general and fundamental phases of the art of life
should receive proportionate representation in the
preparatory process. The average man uses his
history once a day perhaps, his arithmetic some-
what oftener. Even his English grammar is on
trial during a part of his working hours only, and
his whole mental equipment is put by for a third
of the twenty-four. He is living all the time,
however, and is either well or ill, happy or miser-
able, efficient or useless, largely as a result of the
conduct and management of the delicate physical
machine which is in his charge. He may be inno-
cent of historic fact, of the multiplication table
and of syntax and yet be a useful and a con-
tented citizen. He can not be either long without
observing the laws of hygiene and sanitation. I
fancy that any one with a child of his own will
have no doubt that knowledge of what to eat and
what not to eat and why, of the meaning and im-
portance of fresh air, of the claims of exercise
and rest, of the essential routine of body cleanli-
ness, of how germ diseases are spread and how
they may be controlled, of the methods of render-
ing water and milk safe and the reasons therefor,
of the dangers of insect-borne diseases and of the
essentials of public sanitation, these are of even
greater moment than those which prepare for the
intellectual and social life.

IV. Orientation, or a ‘‘knowledge of gen-
eral biological principles that underlie these
studies’’ is the fourth aim in teaching biol-
ogy in secondary schools. Only when the
pupil senses the larger values and has his

imagination correspondingly stimulated
will he enter upon detailed work with the
blessed enthusiasm of the true scholar. By
these larger values of orientation is meant
something more than appreciation of the
practical applications of biology to life.
They include an unshakable confidence in
underlying truth and in the orderly se-
quence of events, which unconsciously per-
meates and characterizes the scientific mind.
It is the lack of appreciating these larger
and underlying principles that renders any
subject petty, stupid and ‘‘dry’’ so that
aggressive interest gives way to neutral
acquiescence. It is the unoriented pupil
who asks the discouraged teacher that most
exasperating of all questions: ‘‘What do
you want me to do next?’’

High-school pupils are old enough to ap-
preciate being taken behind the scenes and
shown what the whole thing is about. As
soon as they once gain a panoramic view of
the great field of biological science their
study ceases to be a personally conducted
excursion and resolves itself into a real ad-
venture that is worth while.

V. Finally, the application of it all is
‘to show him his place in nature and his
share and responsibility for the present and
future of human society.’’ The moral of
the whole matter is to get rid of the rub-
bish in life so far as possible by rational
behavior and effective conduct. Proper
training in biology will do more than safe-
guard the individual against personal
blunders in the conduct of his own life, be-
cause it stands for more than the simple
acquisition of information and skill for
selfish ends alone. It has to do with the
discernment of truth and error, with the
unconscious habit of correct judgment of
cause and effect independent of any per-
sonal bias.

This unselfish, impersonal attitude of
mind which true biological study engenders

SCHOOL AND

is exactly what we want for making good
citizens in the world. In such a scientific
atmosphere prejudice, ignorance, greed and
passion do not flourish, and it is in the
development of this atmosphere rather than
in the more obvious, utilitarian benefits of
biological knowledge that the greatest ap-
plication of our science to human welfare is
to be found. So much for the aims and
ideals in teaching biology in the secondary
schools,
The report goes on to affirm:

We do not believe in rigidity of method in sci-
ence instruction. Quizzes, conferences, experi-
ments, individual reports, excursions, text-assign-
ments are all good. They offer a rich and varied
choice of pedagogical method, and each teacher
should be given freedom to develop the methods
best adapted to his own group of students and to
the environment of the school in which he is teach-
ing.

In laboratory work time should not be wasted
in detailed microscopical work, in complicated ex-
perimentation, in useless attention to drawing or
other ‘‘busy work.’’ All laboratory work should
be based on definite information ungrudgingly
and interestingly furnished, and should be in the
nature of a direct effort to acquire more knowl-
edge at first hand. Experiments, results and con-
clusions should be accurately recorded. Neatness
in notebook records is desirable but must not be
exalted above thinking and understanding.

With respect to how the work should be
planned the committee unanimously agrees that
two years of work in elementary science should be
the basis for more advanced courses in science.
Such work should deal with physical environment
(including a study of matter and forces), plants,
animals (including man), and the general appli-
cations of scientific principles to human welfare.
If, however, administrative conditions make a two-
year course out of the question, the committee
recommends that a first-year course be required of
all, and that a second year be offered as an elec-
tive.

There are added in the preliminary re-
port four appendices in which tentative
outlines of the content of each of the four
half years recommended are given.

In part these appendices are as follows:

SOCIETY 5

APPENDIX A. PHYSICAL ENVIRONMENT

This study should acquaint the pupil with the
nature of his environment and emphasize the ne-
eessity for his adjustment to it.

1. States of Matter.

2. Water—Changes under different tempera-
ture; evaporation; condensation; distribution;
the ocean as a modifier of climate; winds, eur-
rents, rainfall; solution, precipitation, filtration;
carrying power; water power; steam power; ac-
tion in modifying the earth’s surface; ice, snow
and hail; circulation of ground water; water in
living things; osmosis. ’

3. Air.—Physical features; elements, com-
pounds, chemical changes; oxidation; photosyn-
thesis; indestructibility of matter; the CO, cycle;
forces of heat, light and electricity.

4, Soil—Origin; nature; its constant changes,
soil bacteria; the materials for plant growth.

5. The Past History of the Earth—Forces pre-
paring an environment for mankind; geography of
to-day; man’s relation to it.

6. Environment Extended to the Limits of
Space.—The heavenly bodies and their effect on
the earth; fixed stars and the sources of our
knowledge of them; the solar system.

7. Summary of the Relation of the Physical
Environment and Life to Show the Responsibility
of Man.—The conservation of natural resources.
In this preliminary half year on the physical en-
vironment the stage is set for the biological stud-
ies proper that follow.

APPENDIX B. PLANTS

1. Introductory—The general relations of
plants to animals and man; a consideration of
the familiar parts of a typical green plant with
the functions of these parts.

2. Nutrition—The essentials obtained through
a study of roots, stem and leaves. Nutritive proc-
esses in plants that are not green.

3. Reproduction, the essentials obtained through
a study of flowers, fruits and seeds.

4. Applications of plant biology to human wel-
fare.

5. Survey of plant groups, and plant identifica-
tions.

APPENDIX ©. ANIMALS

1, Animals in General.—Types, structure, dis-
tribution and relationships.
2. Life Histories.—Reproduction, growth, meta-

morphosis.
3. Response to Physical Environment.—Geo-
graphie distribution, reactions.

6 SCHOOL AND

4, Response to Organic Environment.—Compe-
tition, parasitism, symbiosis.

5. The Continuity of Life—Genetic relation-
ship, the paleontological record.

6. Man’s Place in Natwre.—His rise and domi-
nance; his distribution and variety.

7. Man’s Opportunity to Use His Biological
Knowledge-—Medicine, sanitation, agriculture;
domestication and control of animal life.

APPENDIX D. MAN

1. Structure of the Human Body.—Similarity
and contrasts with the lower animals. Character-
istics of the most common tissues,

2. Physiology—wNutrition, food and diet; di-
gestion, absorption; distribution of food in the
body; removal of waste; the part played by oxy-
gen. Functions of the tissues.

3. Hygienic Care of the Body.—Healthful diets;
drugs and stimulants; prevention of constipation;
exercise and rest; clothing; cleanliness; first aid
to the injured; special hygiene of eyes, ears, nose
and throat.

4. Bacteria and Sanitation—General character-
istics of bacteria, distribution, form, size, repro-
duction and conditions of growth; food preserva-
tion, soil inoculation and disease prevention. Pub-
lic health; care of streets, water supply, sewage
disposal, milk and inspection of foods, quaran-

SOCIETY

tine, disinfection and methods of securing im-
munity.

It is hoped in the final report to include
various syllabi of courses in biology which °
have proved successful, but the committee
feels that at present it is more desirable to
gain greater uniformity and agreement in
the aims and purposes of biological study in
the secondary schools than in the content of
the courses themselves. Given a pupil of nor-
mal capacity, it is of greater moment to have
a teacher imbued with high ideals and aims,
with a reasonably good preliminary train-
ing and endowed with the indispensable
gift of gumption, than it is to have a defi-
nite program of topics outlined in the cur-
riculum. In fact it would be disastrous to
all future educational growth if we should
ever reach such a degree of standardized
efficiency in our plans and programs of
study that we could be content to rest upon
our laurels. H. E. WALTER

Brown UNIVERSITY

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Contributions from the Biological
(Anatomical) Laboratory of

Brown University

VOLUME I. ISSUED JUNE, 1898.

TOWER, R. W. THE EXTERNAL OPENING OF THE “BRICK-RED”
GLAND IN LIMULUS PoLyPpHEMUS. Zool. Anz., No. 491, 1895.

GORHAM, F. P. THe CLEAVAGE oF THE Ec@ oF VIRBIUS ZOS-
TERICOLA. J. Morph., Vol. XII, No.3, 1895.

FIELD, G. W. ON THE MORPHOLOGY AND PHYSIOLOGY OF THE
EcHINODERM SPERMATOZOON. J. Morph., Vol. XI, No. 2, 1895.

MEAD, A. D. THE ORIGIN OF THE EcG CENTROSOMES. J. Morph.,
Vole Xi Nok 2511897:

MEAD, A. D. THE Earty DEVELOPMENT OF MARINE ANNELIDS.
J. Morph., Vol XII, No. 2, 1897.

STONE, E. A. SomME OBSERVATIONS ON THE PHYSIOLOGICAL FUNC-
TION OF THE PyLORIC CAECA OF ASTERIAS VULGARIS. Amer.
Nat., Vol. XXI, Dec., 1897.

BUMPUS, H. C. THE VARIATIONS AND MUTATIONS OF THE IN-
TRODUCED SPARROW, PASSER Domesticus. Biol. Lec. M. B.
L., 1897.

BUMPUS, H. C. A CONTRIBUTION TO THE STUDY OF VARIATION.
J. Morph., Vol. XII, No. 2, 1897.

BUMPUS, H. C. THE VARIATIONS AND MUTATIONS OF THE IN-
TRODUCED LitroRINA. Zool. Bul., Vol. 1, No. 5, 1898.

VOLUME II. ISSUED OCTOBER, 1901.

BUMPUS, H. C. Tue ImporTANCE OF EXTENDED SCIENTIFIC
INVESTIGATION. U.S. Fish Com. Bul., 1897.

MEAD, A. D. THE ORIGIN AND BEHAVIOR OF THE CENTROSOMES
IN THE ANNELID Eae. J. Morph., Vol. XIV, No. 2, 1898.
MEAD, A. D. Tue RATE OF CELL-DIVISION AND THE FUNCTION

OF THE CENTROSOME. Biol. Lec. M. B. L., 1896-7.

MEAD, A. D. THE CELL ORIGIN OF THE ProroTrocH. Biol. Lec.,
M. B. L., 1898.

MEAD, A. D. Tue EFFECTS oF CHEMICAL AND PHYSICAL INFLU-
ENCES ON THE DEVELOPMENT OF THE Empryo. Trans. R. I.
Med. Soc., 1900.

COGHILL, G. E. Tue RAMI oF THE FirrH NERVE IN AMPHIBIA.
J. Comp. Neur., Vol. XI, No. 1, 1901.

BUMPUS, H. C. Tue ELIMINATION OF THE UNFIT AS ILLUS-
TRATED BY THE INTRODUCED SPARROW, PASSER DOMESTICUS.
Biol. Lec., M. B. L., 1898.

to
oo

36.

BUMPUS, H. C. Facrs anp Trrortrs or TreLegony. Amer.
Nat., Vol. XXXIII, No. 396, 1899.

BUMPUS, H. C. On tHE IDENTIFICATION oF FisuH ARTIFICIALLY
HATCHED. Amer. Nat., Vol. XXXII, No. 378, 1898.

WALTON, L. B. Tuer Basan Seaments or THE Hexapop LEG.
Amer. Nat., Vol. XXXIV, No. 400, 1900.

BUMPUS, H. C. Tur Work or THE BIoLoGicAL LABORATORY OF
THE U. 8. Fish Commission at Woops Houu. Sci. N. S.,
Vol. VIII, No. 186, 1898.

BUMPUS, H. C. THe BreepIna oF ANIMALS AT Woops Hort
DURING THE MONTH oF Marcu, 1898. Sci. N. S., Vol. VII,
No. 171, 1898.

MEAD, A. D. THE BrReepiIng oF ANIMALS AT Woops Hot purR-
ING THE MontTH oF ApRiIL, 1898. Sci. N. S., Vol. VII, No.
177, 1898.

BUMPUS, H. C. THE Breepinc or ANIMALS AT Woops HoLyt
DURING THE MONTH oF May, 1898. Sci. N. S., Vol. VIII, No.
185, 1898.

BUMPUS, H. C. Tur BreepIng oF ANIMALS AT Woops Hott
DURING THE MonTHS OF JUNE, JULY AND AveusT, 1898. Sci.
N. S., Vol. VIII, No. 207, 1898.

THOMPSON, M. T. Tue BreEepiInc oF ANIMALS AT Woops Hott
DURING THE MONTH oF SEPTEMBER, 1898. Sci. N. S., Vol. 1X,
No. 225, 1899.

MEAD, A. D. PERIDINIUM AND THE “RED WATER” IN NARRAGAN-
SEIT Bay. Sci. N. S., Vol. VIII, No. 203, 1898.

MEAD, A. D. OBSERVATIONS ON THE SOFT-SHELL CLAM. 380th
An. Rep. Com. Inl. Fish. R. I., 1900.

MEAD, A. D. ON THE CORRELATION BETWEEN THE GROWTH AND
Foop Supply IN STARFISH. Amer. Nat., Vol. XXXIV, No.
397, 1900.

MEAD, A. D. THE NAtTuRAL HISTORY OF THE STarRFisu. U. S.
Fish. Com. Bul., 1899.

BUMPUS, H. C. THE REAPPEARANCE OF THE TILEFISH. U. S.
Fish. Com. Bul. 1898.

BUMPUS, H. C. On THe MovEMENTS oF CERTAIN LOBSTERS LIB-
ERATED AT Woops Hoxtu. U. S. Fish. Com. Bul., 1899.

TOWER, R. W. IMPROVEMENTS IN PREPARING FISH FOR SHIP-
MENT. U.S. Fish. Com. Bul., 1899.

GREEN, E. H. Tue CHemMicaL COMPOSITION OF THE SUB-DERMAL
CoNNECTIVE TISSUE OF THE OCEAN SunrisH. U.S. Fish. Com.
Bul., 1899.

GORHAM, F. P. Tuer Gas-BURBLE DISEASE OF FISH AND ITS
Cause. U.S. Fish. Com. Bul., 1899.

VOLUME III. ISSUED OCTOBER, 1903.

MEAD, A. D. Tue StarFisH. 29th An. Rep. Com. Inl. Fish. R.
I., 1899.

MEAD, A. D. OBSERVATIONS ON THE SOFT-SHELL CLAM. (Second
paper.) 31st An. Rep. Com. Inl. Fish. R. I., 1901.

MEAD, A. D. OBSERVATIONS ON THE SOFT-SHELL CLAM. (Third
paper.) 32nd An. Rep. Com. Inl. Fish. R. I., 1902.

43.

44.

MEAD, A. D. and BARNES, E W. OBSERVATIONS ON THE SoFT-
SHELL CLAM. (Fourth paper.) 33rd An. Rep. Com. Inl. Fish.
R. I., 1903.

RISSER, J. Hasrrs anp Lire History or THE Scauiop. 3lst
An. Rep. Com. Inl. Fish. R. I., 1901.

MEAD, A. D. Hasits anp GrowTH oF Youna LOBSTERS AND
EXPERIMENTS IN LOBSTER CULTURE. 31st An. Rep. Com. Inl.
Rishe (Reel. TOOT:

MEAD, A. D. Hasits anp GrRowTH oF YOUNG LOBSTERS AND
EXPERIMENTS IN LOBSTER CULTURE. (Second paper.) 32nd
An. Rep. Com. Inl. Fish. R. I., 1902.

MEAD, A. D., and WILLIAMS, L. W. Hasirs AND GROWTH OF
THE LOBSTER AND EXPERIMENTS IN LOBSTER CULTURE. (Third
paper.) 33rd An. Rep. Com. Inl. Fish. R. I., 1903.

WILLIAMS, L. W. THE VASCULAR SYSTEM OF THE COMMON
Seurip, Lotico PEALIT. Amer. Nat., Vol. XXXVI, No. 430,
1902.

WALTON, L. B. Tuer MeETATHORACIC PTERYGODA OF THE HEXA-
PODA AND THEIR RELATION TO THE WINGS. Amer. Nat., Vol.
XXXV, No. 413, 1901.

THOMPSON, M. T. A RARE THALASSINID AND ITs LARVA. Proc.
Bos. Soe. Nat. Hist., Vol. XX XI, No. 1, 1903.

THOMPSON, M. T. A NEw Isopop PARASITIC ON THE HERMIT
Cras. U.S. Fish. Com. Bul., 1901.

GREEN, E. H., and TOWER, R. W. THE ORGANIC CONSTITU-
ENTS OF THE SCALES oF Fisu. U. S. Fish. Com. Bul., 1901.
TOWER, R. W. TuE GAS IN THE SWIM BLADDER OF FISHES. U.

8S. Fish. Com. Bul., 1901.

TOWER, R. W. BritaRy CALCULI IN THE SQUETEAQUE. U.S. Fish.
Com. Bul., 1901.

GORHAM, F. P. MorpnontocicaL VARIETIES OF BACILLUS DIPH-
THERIAE. J. Med. Res., Vol. VI, No. 1, 1901.

GORHAM, F. P., and TOWER, R. W. Dors Potassium CYAN-
IDE PROLONG THE LIFE OF THE UNFERTILIZED EaG@ OF THE SEA-
Uncuin? Amer. J. Physiol., Vol. VIII, No. 3, 1902.

COGHILL, G. E. Tae Crantau Nerves or AmBiystomMa TIGRI-
NUM. J. Comp. Neur., Vol. XII, No. 3, 1902.

VOLUME IV. ISSUED DECEMBER, 1905.

GORHAM, F. P. Recenr Dests to Brontogy. Prov. Med. J.,
Vol. VI, No. 2, 1905.

THOMPSON, M. T. THE METAMORPHOSES OF THE HERMIT CRAB.
Proce. Bos. Soe. Nat. Hist., Vol. XXXI, No. 4, 1903.

MEAD, A. D. THE PrRopLemM oF LOBSTER CULTURE. Proc. Amer.
Fish. Soe., 1905.

MEAD, A. D. EXPERIMENTS IN LOBSTER CULTURE AT THE WICK-
FORD STATION OF THE RHODE ISLAND COMMISSION OF INLAND
Fisuertes, 1904. 35th An. Rep. Com. Inl. Fish. R. I., 1905.

HADLEY, P. B. Pretimrinary REPORT ON THE CHANGES IN ForRM
AND CoLOR IN THE SUCCESSIVE STAGES OF THE AMERICAN
LossTeR. 35th An. Rep. Com. Inl. Fish., 1905

64.

“J
bo

s~J
or

HADLEY, P. B. PuxHororropism In THE LARVAL AND BARLY
ADOLESCENT STaaes oF Homarus AMERICANUS. Sci. N. §&.,
XXII, No. 569, 1905.

EMMEL, V. E. THe REGENERATION oF Lost PARTS IN THE LoB-
STER. 35th An. Rep. Com. Inl. Fish., 1905.

MEAD, A. D., and BARNES, E. W. OxsseRvVATIONS ON THE SOFT-
SHELL CLAM (Fifth paper). 34th An. Rep. Com. Inl. Fish.,
1904.

FULLER, C. A. THE DISTRIBUTION oF SEWAGE IN THE WATERS
oF NARRAGANSETT Bay, wWitH ESPECIAL REFERENCE TO THE
CONTAMINATION OF THE OystTeR BeEps. Appendix to Rep.
U. 8. Com. of Fish, to Sec. of Com. and Labor, 1904.

GORHAM, F. P. Tue BacrerioLoay or DIPHTHERIA. Prov.
Med. J., Vol VI, No. 3, 1905.

MARSH, M. C., and GORHAM, F. P. THe Gas DISEASE IN
Fisues. App. to Rep. U. S. Com. of Fish. to Sec. Com. and
Labor, 1904.

SULLIVAN, M. X. SyntHetTic CuLturE MEDIA AND THE BIo-
CHEMISTRY OF BACTERIAL PIGMENTS. J. Med. Res., Vol. XIV,
No. 1 (N. S., Vol. IX, No. 1), 1905.

SULLIVAN, M. X. THE PuHysioLogy oF THE DIGESTIVE TRACT
IN THE ELASMOBRANCHS. Amer. J. Physiol., Vol. XV, No. 1,
1905.

VOLUME V. ISSUED JULY, 1907.

MEAD, A. D. SPONTANEOUS GENERATION AND EVOLUTION. Ad-
dress delivered before the A’soc. Alumni of Middlebury Col-
lege, June 26, 1906.

BARNES, E. W. METHODS OF PROTECTING AND PROPAGATING THE
LOBSTER, WITH A BRIEF OUTLINE OF ITS NATURAL HISTORY.
36th An. Rep. Com. Inl. Fish., 1906.

HADLEY, P. B. RecARDING THE RATE OF GROWTH OF THE AMER-
IcAN LosstreR. 36th An. Rep. Com. Inl. Fish., 1906.

HADLEY, P. B. REGARDING THE RATE OF GROWTH OF THE AMER-
IcAN Lopster. Biol. Bul., Vol. X, 1906.

HADLEY, P. B. Osservations ON Some INFLUENCES or LIGHT
UPON THE LARVAE AND EARLY ADOLESCENT STAGES OF THE
AMERICAN LOBSTER. 36th An. Rep. Com. Inl. Fish., 1906.

HADLEY, P. B. Tue RELATION OF OPTICAL STIMULI TO RHEO-
TAXIS IN THE AMERICAN LoBpstTeR, HoMARUS AMERICANUS.
Amer. J. Physiol., Vol. XVII, 1906.

HADLEY, P. B. GaAtvanoTaxis IN LARVAE OF THE AMERICAN
Losster, (Homarus AMERICANUS). Amer. J. Physiol., Vol.
XIX, 1907.

EMMEL, V. E. Tue RELATION oF REGENERATION TO THE MOLT-
ING PROCESS IN THE LopsTEeR. 36th An. Rep. Com. Inl. Fish.,
1906.

EMMEL, V. E. Torsion AND OTHER TRANSITIONAL PHENOMENA
IN THE REGENERATION OF THE CHELIPED OF THE LOBSTER (Ho-
MARUS AMERICANUS). J. Exp. Zool., Vol. III, 1906.

EMMEL, V. E. Tre RecGEeneration or Two ‘‘CRUSHFR-CLAWS’’
FOLLOWING THE AMPUTATION OF THE NORMAL ASYMMETRICAL
CHELAE OF THE LossteR (HomMARUS AMERICANUS). Arch.
Entwich. Mech., Vol. XXII, 1906.

76.
rie
78.

79.

80.

81.

82.

83.

84,

85.

86.

87.

88.
89.

90.
91.

92.

93.

94.

WILCOX, A. W. Locomorion 1n Youn@ COLONIES OF PECTINA-
TELLA Maenirica. Biol. Bul., Vol. XI, 1906.

WILLIAMS, L. W. Notes oN Marine CopePpopA OF RHODE
IstaNnp. Amer. Nat., Vol. XL, 1906.

TRACY, H. C. A List oF THE FisHEs OF RHODE ISLAND. 36th
An. Rep. Com. Inl. Fish., 1906.

HADLEY, P. B. Rapres—ItTs OriGin, Cause, Symptoms, D1aq-
NOSIS AND TREATMENT. Proy. Med. J., Vol. VIII, 1907.

VOLUME VI. ISSUED OCTOBER, 1909.

WILLIAMS, L. W. List or THE RHODE ISLAND CoPEPODA, PHYL-
LOPODA, AND OSTRACODA, WITH New SPECIES oF CoPEPoDA.
37th An. Rep. Com. Inl. Fish. R. I., 1907. Special Paper No.
30.

EMMEL, V. E. REGENERATED AND ABNORMAL APPENDAGES IN THE
LozszveR. 37th An. Rep. Com. Inl. Fish. R. I., 1907. Special
Paper No. 31.

WILLIAMS, L. W. THe StToMacH oF THE LOBSTER AND THE
Foop or LarRvAL Logsters. 37th An. Rep. Com. Inl. Fish. R.
I., 1907. Special Paper No. 32.

TRACY, H. C. Tue Fisues or RuopE ISLAND, V.—THE FLatT-
FISHES. 38th An. Rep. Com. Inl. Fish. R. I., 1907. Special
Paper No. 36.

TRACY, H. C. Tue Fisues or RHopE Isuanp. VI.—A DEscrIP-
TION OF Two YouNG SPECIMENS OF SQUETEAGUE (CYNOSCION
REGALIS), WITH NOTES ON THE RATE OF THEIR GROWTH. 38th
An. Rep. Com. Inl. Fish. R. I., 1907. Special Paper No. 37.

HADLEY, P. B. Tue Growrn and _ Toxin PRODUCTION OF
BACILLUS DIPHTHERIAE UPON Protrip-FrREE Mepia. Jour. Inf.
Dis. Suppl., No. 3, May, 1907, p. 95.

EMMEL, V. E. REGENERATION AND THE QUESTION OF “SyM-
METRY IN THE BiG CLAWS OF THE LOBSTER.” Science, N. S.,
XXVI, 1907, p. 83.

SULLIVAN, M. X. Tue Puysiotocy oF THE DiGEsTIVE TRACT OF
ELASMOBRANCHS. Bul. U. S. Bureau of Fish., XXVII, 1907,
ps

WALTER H. E. Tse Reactions oF PLANARIANS TO LIGHT.
Jour. Exp. Zool., V, 1907, p. 35

HADLEY, P. B. Tue Reaction or BLINDED LOBSTERS To LIGHT.
Amer. Jour. Phys., XXI, 1908, p. 180.

WALTER, H. E. Tueortes oF Birp MicraTion. School Science
and Math., April-May, 1908.

HADLEY, P. B. JOHANNES MULLER. Pop. Sci. Mo., LXXII,
1908, p. 513.

HADLEY, P. B. Tuer BEHAVIOR oF THE LARVAL AND ADOLESCENT
STAGES OF THE AMERICAN LOBSTER (HOMARUS AMERICANUS).
Jour. Comp. Neur. and Physiol., XVIII, 1908, p. 199.

KEYES, F. G. A Vacuum Storcock, Science. N. S., XXVIII,
1908, p. 735.

DOLT, M. L. Smuverte SyntTHeTIc MepIA FoR THE GROWTH OF B.
Corl AND For Irs IsoLATION FROM WATER. Jour. Infec. Dis.,

~ V, 1908, p. 616.

95.

99:

100.

101.

102.

103.

104.

105.

VOLUME VII. ISSUED OCTOBER, 1911.

KEYES, FREDERICK G. THe Gas Propucrion or BacriLius
Cour. The Journal of Medical Research, Vol. XXI, No. 1.
New Series, Vol. X VI, No. 1, pp 69-82, July, 1909.

WALTER, HERBERT E. An Iprat Coursrt 1x BioLoGy For
tHe Hian Scnoor. School Science and Mathematics, Vol. IX,
1909, pp 717-724 and 840-847.

KEYES, FREDERICK G. AN Improvep Mernop or CoLLecrina
GASES FROM THE Mercury Pump. The Journal of the American
Chemical Society, Vol. XX XI, No. 12, December, 1909.

MEAD, A. D. A Mernop or Logster Cutture. Bulletin of the
Bureau of Fisheries, Vol. XX VIII, 1908. Issued February, 1910,
pp 221-240.

MEAD, A. D. A New PrincrpLe oF AGRICULTURE AND TRANS-
PORTATION OF LivE FisHes. Bulletin of the Bureau of Fisheries,
Vol. XXVIII, 1908. Issued April 1910, pp 761-780.

CLARK, PAUL F. Tue RELATION OF THE PSEUDODIPHTHERIA
AND THE DiepHTHERIA BaciuLus. The Journal of Infectious Dis-
eases, Vol. VII, No. 3, May 20, 1910, pp 335-367. _

WALTER, HERBERT E. Variations In Urosatpinx. The
American Naturalist, Vol. XLIV, October, 1910, pp 577-594.

BUNKER, JOHN W. M. Tue Hycrene or THE SwimMina POOL,
American Journal of Public Hygiene, Vol. XX, No. 4, Novem-
ber, 1910, pp 810-812.

HADLEY, PHILIP B. Anppirionat Nores upon THE DEVELOP-
MENT OF THE LopstER. Fortieth Annual Report of the Com-
missioners of Inland Fisheries of Rhode Island, 1909.

TRACY, HENRY C. Awnwnoratep List oF FisHes Known To In-
HABIT THE WATERS OF RHODE IsLAND. Fortieth Annual Report
of the Commissioner of Inland Fisheries of Rhode Island, 1909.

GORHAM, FREDERIC P. Pretimrary REPORT UPON THE SANI-
TARY CONDITION OF RHODE IsLAND OystTER Beps. Report of the
Commissioner of Shell Fisheries of Rhode Island for 1910.

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