y
X

With the Compliments of

YALE UNIVERSITY LIBRARY

NEW HAVEN, CONN., U. S. A.

^^

A TREATISE ON THE TRANSFORMATION OF

THE INTESTINAL FLORA

WITH SPECIAL REFERENCE TO THE
IMPLANTATION OF BACILLUS ACIDOPHILUS

BY
LEO F. RETTGER

Professor of Bacteriology, Yale University
AND

HARRY A. CHEPLIN

Seessel Fellow in Bacteriology, Yale University

YALE

DISSERTATION.

, JROM THE >

r SHEFFIELD LABORATORY OF BACTERIOLOGY
YALE UNIVERSITY \

NEW HAVEN

YALE UNIVERSITY PRESS

LONDON • HUMPHREY MILFORD • OXFORD UNIVERSITY PRESS

MDCCCCXXI

COPYRIGHT, 1921, BY
YALE UNIVERSITY PRESS

QR
11/

The present volume is based on investigations conducted at the Sheffield
Scientific School of Yale University and presented in lectures delivered
during the autumn of 1920 by the senior author, on the Silliman
Foundation.

PREFACE

Fifty years have passed since Pasteur and Koch, the founders of
modern bacteriology, made their first contributions to this new field
of scientific study. No phase of the subject has been given more atten-
tion by investigators than the bacteriology of the digestive tract.
However, success in these endeavors has been limited, with few excep-
tions, to the discovery of microorganisms which are manifestly disease-
producing and which exert their baneful influence in comparatively short
periods of time, as for example the microbial agents causing typhoid
fever and epidemic dysentery. The more subtle bacterial processes
which take place in the intestine, and the laws which govern the pre-
ponderance of one type of bacteria over another, have as yet been but
little understood.

The authors have aimed to present here in as direct a manner as
possible the results of an extended series of observations on: (1) the
relation of diet to the character of the intestinal bacterial flora, and
(2) the possibility of implanting bacteria of known physiological prop-
erties in place of those which ordinarily hold sway after early infancy.

The demonstration that lactose, dextrin and milk, when taken in suf-
ficient amount, encourage a non-putrefactive flora, and that a non-
putrefactive organism like Bacterium acidophilus may be established
in the intestine by oral administration, should be of fundamentally
scientific interest, irrespective of any important bearing that it may
have on the many and complex problems related to intestinal disease
and minor alimentary disturbances.

The descriptions of methods employed in the examination of fecal
specimens are given in some detail for the use of other investigators
who are engaged in studies similar to those recorded in this treatise.
Particular attention is given also to the preparation and use of Bac-
terium acidophilus milk, since the experience of the last six months has
shown that the administration of this product is the most effective and
practical method of implanting the submissive aciduric organism in the
digestive tract, and one which is not only well-borne by the subject but
is as a rule welcomed by him because of its unique qualities as a beverage
and a real food.

The Authoes.

New Haven, Conn., October, 1920.

TABLE OF CONTENTS

PAGES

I. Historical review 1-10

II. Feeding and implantation experiments with white rats 11-64

Methods 11-13

Preliminary feeding experiments 18—16

Carbohydrate feeding 17-21

Administration of living suspensions of Bacillus acid-
ophilus 21-34

Bacillus acidophilus and carbohydrate feeding . . . 34—49

Bacillus acidophilus versus Bacillus bulgaricus feeding . 49-52
Slow carbohydrate absorption in the intestine and its

relation to Bacillus acidophilus implantation . . . 62-61
Relation of hydrogen ion concentration to the character

of the intestinal flora > 61—63

Conclusions 63-64

III. Feeding and implantation experiments with human

subjects 65-110

The basal daily diet 66-69

Carbohydrate feeding 70

Transforming influence of lactose 70

Transforming influence of dextrin 70-77

Implantation experiments with living cultures of Bacillus

acidophilus 78-79

Simultaneous use of the special carbohydrates and Bacil-
lus acidophilus 79-84

Transforming influence of milk cultures of Bacillus acid-
ophilus on the intestinal flora 85-94

The use of Bacillus acidophilus milk reinforced with lac-
tose or dextrin 94-99

Attempts to implant Bacillus bulgaricus in man . . . 99-106

Incomplete absorption of lactose from the intestine . . 106-107
Relation of hydrogen ion concentration to the character

of the intestinal flora 108-110

IV. A full account of the preparation of Bacillus acid-

ophilus milk for human consumption, and of its

known properties 111-113

V. Methods employed in the routine examination of feces 114-117

The use of whey agar plates 114-116

Veillon tubes 116-116

Direct microscopic examination of fecal suspensions . 117

VI. General discussion and summary 118-124

Explanation of plates 126-126

Bibliography 127-136

I. HISTORICAL REVIEW

Thk present studies mark a resumption of work begun in this labora-
tory eight years ago. The new data recorded here are the results of
investigation into phases of intestinal bacteriology which are as yet
comparatively new and the importance of which is attested by the
almost universal interest which is manifested in them by physicians and
laymen alike.

While it is a well-established fact that diet exercises a most profound
influence in determining the predominance of one or another type of
bacteria in the alimentary canal, the question of transforming and
simplifying the ordinary mixed intestinal flora through diet in con-
junction with the oral administration of bacteria, or by the latter
process alone, is still in its incipiency.

The first systematic study of intestinal bacteria appears to have been
carried out by Escherich (1886) who made an extensive investigation
of the microorganisms in infants' dejecta, both in health and disease.
He observed the great predominance of Gram-positive rods in the stools
of healthy nurselings, but failed to isolate two organisms to which con-
siderable interest is attached at the present time, namely B. bifidus and
B. acidophilus. Through the classical researches of Tissier (1900)
and Moro (1900a) these two organisms were added to the list of known
intestinal bacteria. The controversy as to whether Tissier's B. bifidus
or Moro's B. acidophilus is the predominating type in the normal
dejecta of breast-fed babies was ended by the admission by Moro of
Tissier's claim (1900b and 1905a), which was substantiated by Ro-
della (1901), Cahn (1901), Cippolina (1902), Passini (1903), Weiss
(1904), and finally by Jacobson (1908).

By means of a dextrose broth medium containing 0.5 per cent glacial
acetic acid, as first suggested by Heymann (1900), Finkelstein (1900),
working independently, isolated an organism identical with that of
Moro, which he called "Saureliebender Bacillus."

The meconium of the new-born infant is sterile. Billroth (1874) is
given the credit by Mannaberg (1898) of having first observed this
fact. Senator (1880) found the intestine to be free from bacteria, and
his statement was confirmed by Escherich (1885), Popoff (1892),
Schild (1895), Szego (1897), Tissier (1900) and Moro (1900a).
While Breslau (1866) has shown that the early infection of the meco-
nium may be entirely independent of feeding, the mouth and the anus

I

2 TRANSFORMATION OF THE INTESTINAL FLORA

serving as the chief portals of entry for bacteria, the establishment of
the characteristic flora of the infant does not take place prior to the
administration of food, as was pointed out by Escherich (1885),
Tissier (1900) and Moro (1900a).

According to the substantiated claims of Tissier, his B. bifidus is
the predominant organism in the stools of breast-fed infants. In the
change from breast- to bottle-feeding B. bifidus is in a large measure
replaced by B. acidophilus, as has been claimed by Logan (1914) and
others. While cow's milk brings about a less homogeneous flora than
mother's milk, there is a large proportionate increase of B. acidophilus,
not infrequently to the exclusion of all other organisms. As the child
grows older and the artificial feeding becomes more varied, there
appears a more and more complex flora which in the course of time
resembles that of an adult. Fischer (1903) was the first to call atten-
tion to this change. Sittler (1910) and others confirmed the obser-
vation.

The comparatively recent experiments of Cohendy (1912) and
Kiister (1913) have for the time at least answered the question first
propounded by Pasteur, namely whether intestinal bacteria are neces-
sary for the well-being of the host. On the other hand, the enormous
numbers of bacteria in the intestine of man and animal have been the
subject of numerous researches into methods of quantitative determina-
tion. Eberle (1896), Klein (1900) and Hehewerth (1900) employed
the direct microscopic count; Winterberg (1898) for the first time
made use of the Thoma-Zeiss blood-counting chamber in this connec-
tion, while Strasburger (1902) resorted to the gravimetric method,
which proved very efficient. Others have by means of the plate culture
and other methods estimated the total number of bacteria in feces.
Thus, MacNeal, Latzer and Kerr (1909) have estimated the average
number of fecal bacteria excreted daily by a normal adult subsisting
on an ordinary diet as thirty-three times ten to the twelfth power
(33x10^"), and that this number of bacteria is equivalent to 5.34<
grams of dried bacteria or 0.585 gram of bacterial nitrogen. Matill
and Hawk (1911) place the daily output at 8.27 grams of dried bac-
teria, and Sato (1910) at 8.54 grams. Berger and Tsuchiya's (1910)
estimate was much lower, namely 3.023. Strasburger calculated that
a healthy adult daily excretes about 8 grams of (dried) bacteria, or 128
trillions.

The influence of sterile food on the intestinal population was studied
by several investigators. Sucksdorff (1886) and Brotzu (1895)
claimed that sterilized food reduces the number of bacteria in the ali-
mentary tract. Similar results were obtained by Gilbert and Dominici
(1894). However, the researches of Stern (1892), Adrian (1895),
Eberle (1896) and Ballner (1904) failed to confirm this assertion.
Escherich had shown that the sterilization of the food had little or no
influence on the number of intestinal organisms. Hammerl (1897),

HISTORICAL REVIEW 3

Albu (1897), Wang (1899) and Belonowsky (1907a) also were unable
to establish any direct relationship.

Intestinal antisepsis has received considerable attention. Bouchard
(1887) may be regarded as the pioneer in this field. He administered
charcoal, naphthalene and iodoform internally, and observed a reduc-
tion of toxicity of the stools and urine. Wassilieff (1882) claimed that
calomel produced a diminution in the putrefactive products of the feces
of dogs. Baumann (1886) also noticed that calomel was effective in
reducing the ethereal sulphates in the urine. Sucksdorff (1886) con-
cluded that quinine and naphthalene reduced the number of intestinal
bacteria. Miiller (1887) and Bartoschewitch (1893) noticed a dis-
appearance of ethereal sulphates after the administration of calomel,
and Fiirbringer (1887) found that this agent materially reduced the
number of intestinal microorganisms. Kumawaga (1888) reported a
reduction of bacteria in the proportion of 37 to 1 through the use of
acetanilid in the dog. Salkowski (1889) obtained a considerable drop
in the number of bacteria in the intestine of a dog which had received
chloroform water. Sehrwald (1889) also obtained positive results with
naphthalene, and Griffith (1895) and Williams (1895) used chlorine in
the treatment of typhoid patients with apparently excellent results.
Rovighi (1892) employed turpentine, camphor, menthol and boric acid
with moderate success.

Morax (1886) could not confirm Baumann's conclusions, and main-
tained that the apparent diminution of ethereal sulphates was due to
rapid removal of the putrefactive products through active peristalsis.
Stieff (1889) and Biernacki (1892) arrived at the same conclusions
as Morax. Schuetz (1901) could demonstrate no favorable influence
through the use of antiseptics. On the contrary, he observed an in-
crease of intestinal bacteria after the administration of calomel. Von
Mieczkowski (1902) pronounced bismuth, silver nitrate, tannopin and
beta-naphthol as valueless, but did secure a reduction after the use of
menthol. Strasburger (1903) obtained negative results with naphtha-
lene, thymol, silver nitrate and beta-naphthol. Salicylic acid, however,
caused some reduction. Schonenborn (1903) found that naphthalene,
itrol and thymol increased the bacteria, while salicylic acid mixed with
the food diminished the number. Miiller (1898), after several years of
observation, is of the opinion that disinfection of the alimentary canal
with drugs is hardly possible, and that there are no useful intestinal
antiseptics.

Hoffman (1906) showed that iodoform when given by mouth is an
effective disinfectant and decreases the number of bacteria in the feces.
Herter (1907) noted that in certain instances salicylates, aspirin and
salol exerted some action in decreasing the output of indican, but ulti-
mately reached the conclusion that most of the so-called intestinal
antiseptics do very little good in effecting diminution of the putrefactive
organisms of the intestine. Feigen (1908) used calomel, magnesium

4 TRANSFORMATION OF THE INTESTINAL FLORA

dioxide and iodo-anisol and secured no reduction; and Harris (1912)
failed to obtain any reduction in the bacterial count by employing
salol, beta-naphthol and guaiacol carbonate as antiseptic agents. Oliver
(1907), on the other hand, used beta-naphthol with excellent results;
Ellis (1912) reports success with magnesium sulphate in chronic forms
of diarrhea, and Hand (1912) speaks of bismuth salicylate as an anti-
septic in acute diarrhea. On the whole, however, the use of intestinal
antiseptics has proven more or less disappointing.

As early as 1868 Senator declared that the decomposition of protein
within the alimentary canal under ordinary conditions results in the
formation of substances toxic to the host. Nearly twenty years later
Bouchard (1884) elaborated the theory of intestinal intoxication. He
claimed that the amount of putrefactive products eliminated in the
urine was a measure of the degree of intestinal putrefaction and called
his measurements "Urotoxic Coefficients." Jaffe (1877), Salkowski
(1878), Brieger (1878) and others introduced new methods which
played an important part in extending our knowledge of the processes
of intestinal putrefaction under different conditions.

Ortweiler (1886) and Miiller (1886) demonstrated that the admin-
istration of carbohydrates tends to lessen putrefaction in the digestive
tube. Krauss (1894) obtained similar results with dogs. Biernacki
(1892), Eisenstadt (1897) and Bachmann (1902), however, noted only
a weak inhibiting action of carbohydrates. Hirschler (1886) appears
to be the first to conclude that particular carbohydrates, sucrose,
lactose, dextrin and starch, as well as the alcohol glycerol, exercise
some inhibiting influence on intestinal putrefaction. Poehl (1887),
Biernacki (1892), Winternitz (1892) and others studied the influence
of milk and found that a milk diet tended to decrease the undesirable
products of protein decomposition by bacteria. These observations
were further confirmed by Herter (1897) and Leva (1908). Herter
and Kendall (1909) fed milk and dextrose to cats and monkeys and
observed a marked decrease of the ethereal sulphates in the urine.
Barker (1914) and Torrey (1915) reported favorable results from the
feeding of lactose to typhoid patients.

Solukha (1896) and Kopetski (1900) sought to establish a definite
relationship between specific constituents of milk and the diminution of
intestinal putrefaction, and since previous workers, Hirschler (1886),
Winternitz (1892) and Schmitz (1893) had demonstrated that lactose
could inhibit putrefaction, their efforts were naturally directed to this
sugar. They concluded that lactose will inhibit intestinal putrefaction
when it is fed by mouth. Wereschtschagen (1895) claimed that glu-
cose may exercise the same action.

Nencki and Sieber (1882) and Stadelmann (1883) administered
lactic acid to their patients with apparently good results. Grundsach
(1893), Schmitz (1894) and Singer (1901) also claimed that lactic
acid lessens intestinal putrefaction, as measured by the decrease of

HISTORICAL REVIEW 6

ethereal sulphates in the urine; encouraging results were obtained also
by Winficld (1912). On the other hand, Rovighi (1892) showed that
lactic acid exerted only a slight inhibiting action, and Wintemitz
(1892) asserted that it has no inhibitory influence, whatever.

Poehl (1887) noted that sour milk when ingested decreased intestinal
putrefaction. This observation was confirmed by Rovighi (1892),
Embden (1894), Brudsinski (1900) and Fischer (1903). Tissier and
Martelly (1902) stated that the chief agent in effecting inhibition of
putrefying bacteria is probably the lactic acid produced by the lactic
acid bacilli. Tissier and Gasching (1903) found that acid-producing
bacilli are able in a sugar-containing medium to arrest the growth of
putrefactive organisms, thus confirming the conclusions of Bienstock
(1899).

The feeding of buttermilk to infants has long been a custom among
the peasants of Holland. Within a short time after DeJager (1897)
and Teixeira de Mattos (1902) brought this method of infant feeding
before the medical profession, the administration of buttermilk to in-
fants, especially those afflicted with gastro-intestinal disturbances, be-
came quite popular, and most favorable results were reported by various
investigators.

From the earliest historic times the use of both sweet and sour milk
has been practiced by many Nomadic and Semi-Nomadic tribes. The
consumption of soured milk undoubtedly became popular on account of
its enhanced palatability and better keeping qualities. As the result
of long-continued practice in the preparation of the sour milk by
methods handed down from generation to generation the different
countries or tribes had their own established product, which differed as
a rule from those of other peoples. Among the best-known of these are
the kefir of the Caucasus, leben raib of Egypt, koumiss of Asiatic
Russia, matzoon of the Armenians, and yoghurt of the Balkan
Peninsula.

Numerous instances are on record where persons lived and retained
much of their early vigor to a very old age, particularly in Bulgaria,
and where from all appearances they owed their long life to sour milk
which was their staple, and in many cases practically the only diet.
Metchnikoff was a close observer of these conditions which were being
brought to his attention by his students and others, and as a result of
these observations and further extensive studies he propounded his
lactic acid bacillus and longevity theory and founded his sour milk
therapy.

Cohendy (1906a) performed experiments upon himself and thirty
patients, by feeding pure milk cultures oi B. hvlgaricus. He observed
a decrease in intestinal putrefaction which was noticeable for seven
weeks after the taking of the culture. In a second paper the same writer
(1906b) pointed out that B. hidgaricus becomes so thoroughly accli-
mated in the human intestine as to be found there several weeks after

6 TRANSFORMATION OF THE INTESTINAL FLORA

ingestion, and was led to conclude that the lactic acid bacilli themselves
were responsible for their implantation, without due regard to the milk
as a factor.

Belonowski (1907b) concluded from the results of his own experi-
ments with mice that B. bulgaricus becomes established in the intestine
about the tenth day after ingestion and persists for some time ; second,
that the putrefactive types of the flora are greatly reduced ; and, finally,
that B. bulgaricus owes its anti-putrescent power, not to the lactic acid
alone, but also to certain other inhibitory products formed by the
bacilli. Leva (1908) investigated the effect of B. bulgaricus when fed
in the form of "lactobacilline" on the excretion of putrefactive prod-
ucts, and reported a decrease in the amount of phenol, volatile fatty
acids and aromatic oxyacids excreted. He believed that B. bulgaricus
becomes acclimated in the intestine. Pochon (1907) drank milk soured
by lactic acid bacilli and after several weeks observed a marked reduc-
tion in the amounts of indol and phenol in the urine.

Harrington (1912) and Bogert (1918) reported favorable results
obtained from the administration of living cultures of B. bulgaricus.
Moro (1906) found it necessary to use large quantities of bouillon cul-
tures of lactic acid bacilli when fed per os, and showed that better re-
sults could be obtained when the bacteria are introduced per rectum.
Dunn (1907) used buttermilk containing large numbers of living lactic
acid organisms in the treatment of various intestinal disturbances in
infants, with apparently excellent results. Piffard (1908) directed
many of his patients to employ sour milk for both dietetic and directly
remedial purposes. He admits, however, that such use of sour milk has
been largely empirical. North (1909) reported encouraging results in
the treatment of disorders of the nose and throat with B. bulgaricus.
Brady (1910) used sour milk with apparently good success. La Fetra
(1909), while disappointed in the use of acidified milk, felt that there
was a distinct field for the employment of buttermilk, which had given
him very encouraging results in cases of intestinal disturbance. Pans-
ier (1917) claims to have obtained good results from the treatment of
wounds with living cultures of the bulgarian bacillus. Liefmann (1909)
suggested sour milk as a means of eliminating typhoid bacilli from car-
riers. Mayer (1910) held that the continued use of sweet milk by
typhoid carriers reduces the number of typhoid organisms.

Wegele (1908) reported favorably on the use of milk soured with
B. bulgaricus. He believed that the production of lactic acid in "statu
nascendi" in the digestive tube is of greater importance than the mere
presence of the acid already formed in the milk. Wejnert (1908)
demonstrated that the administration of milk soured with B. bulgaricus
reduces the number of bacteria in the feces. Litchfield (1914) fed fer-
mented milk reinforced with lactose to his typhoid patients, with excel-
lent results. Tollens (1908) reported favorably on the employment of
kefir in intestinal disturbances, while Klotz (1908) used yoghurt to

HISTORICAL REVIEW 7

the same advantage with his patients. Oehler (1911) conducted feeding
experiments with yoghurt on mice and monkeys and stated that B. hvl-
garicus could be demonstrated with ease in the feces during the entire
feeding period.

The claims of Metchnikoff and his followers could not be substan-
tiated, however, by the following investigators. Luerssen and Kiihn
(1908) failed to implant B. bulgaricus in the intestine of man by the
continued use of yoghurt. Herter and Kendall (1908) found that on
feeding a monkey for two weeks exclusively on milk fermented with
B. bulgaricus of Massol the organism in question did not establish itself
below the level of the ileo-caecal valve. Spiegel (1911) stated that the
therapeutic value of bulgarian milk or tablets is at least doubtful and
that there is insufficient evidence of beneficial action. Heinemann
(1912) claimed that B. bulgaricus is not a panacea for intestinal putre-
faction. Blodgett (1913) concluded that the administration of this
organism in cases of glycosuria is of no benefit. Distaso and Schiller
(1914) fed the organism to white rats and were forced to the conclu-
sion that it is impossible to bring about the acclimatization of B. bul-
garicus in the intestine.

Macfayden, Nencki and Sieber (1891), Lembke (1896) and Hammerl
(1897) failed to establish any definite relation between ingested food
and types of intestinal bacteria. Lembke (1897) continued his inves-
tigation and was ultimately able to demonstrate that by changing the
diet, namely by the substitution of bread for meat, a marked difference
could be brought about in the character of the intestinal flora. The
investigator did not, however, attribute any prominence to the aciduric
organisms.

Tissier's extensive study of the bacteriology of infants' stools marks
a big step in advance. He observed three distinct phases in bacterial
infection of the intestinal tract of infants, namely, the period of
sterility, the period of mixed or promiscuous infection, and the period
of transition resulting in the establishment of the characteristic
nurseling's flora. At birth the alimentary canal and its contents are
sterile, as had been shown by Senator, Escherich and others. The
first indications of bacterial contamination of the meconium are dis-
cernible several hours after birth. The early invaders are adventitious
microbes in every respect resembling those which are commonly met with
in the baby's environment, and probably gain entrance to the intestinal
canal through the mouth and anus.

As soon as the digestive tract becomes the recipient of mother's milk
and its digestion products a marked change is recognizable in the in-
testinal flora. The heterogeneous aggregation of microbes gives way
to a simplified flora dominated by B. bifidus, which persists as long as
the child's diet is confined to breast milk alone. When breast feeding
gives way, however, to artificial feeding and the diet becomes more
varied the simplified and essentially B. bifidus flora gradually shifts to

8 TRANSFORMATION OF THE INTESTINAL FLORA

one of a more complex character, which eventually resembles the mixed
flora of an ordinary adult.

Moro (1900a) claimed that his Bacillus acidophilus was the pre-
dominating organism in nurselings' stools, but later admitted Tissier's
claims. Passini (1903), Moro (1905), Tissier (1905), Rodella (1905),
Sittler (1908) and Bahrdt and Beifeld (1910) concluded that diet
plays an important role in determining the types of intestinal micro-
organisms. They corroborated Tissier's earlier observation, namely
that Bacillus bifidus is the predominant organism in the stools of breast-
fed infants and that there is a more varied and less constant flora in
the feces of bottle-fed babies. Spiegelberg (1899) reported a scarcity
or absence of liquefying bacteria in the stools of normal nurselings.

Rettger and Horton (1914) noted a marked simplification of the
intestinal flora of white rats soon after they were shifted from the
usual mixed diet of grains and vegetables to one containing starch, lard,
protein-free milk and purified proteins, the Gram-positive organisms
of the acidophilus and bifidus types constituting eighty-five to one hun-
dred per cent of the flora. Hull and Rettger (1914) observed that
mixed grain feed tends to transform the flora of the rat, but that no
profound change takes place until milk or lactose is administered in
addition to the regular diet. The preponderance of B. acidophilus
over other organisms was brought about within three days after a diet
of vegetables and bread was followed by one of mixed grain and milk.
When lactose was fed in appreciable amounts the transformation period
was very short, and the change in type of bacteria was practically 100
per cent. It was noted, however, that the B. acidophilus phase was
often more or less temporary, this organism giving way to B. bifidus.
In the milk-feeding experiments the acidophilus phase was, as a rule,
the more permanent, B. bifidus seldom gaining the ascendency. Of par-
ticular interest in this investigation was the failure of the authors to
implant B. bulgaricus by the ingestion of milk cultures and water
suspensions of this organism.

Rettger, Kirkpatrick, Jones and Card (1914 and 1915) employed
several thousand chicks in their studies on the influence of milk feeding
on growth and mortality, and found that the unique properties of this
food exist in the milk per se, and not in any milk acids or milk-souring
bacteria. The chicks received unpasteurized, sterilized and naturally
soured milk, as well as milk soured with pure cultures of B. btdgaricus,
and no diff'erences could be observed in the beneficial eff'ects of the
different preparations. These observations impressed upon the writers
the fundamental fallacies of Metchnikoff's interpretation of the sour-
milk therapy. Hull and Rettger (1917) confirmed the results of their
earlier work. They found, further, that two to three grams of lactose
are sufficient to establish an aciduric flora within three days. It re-
quired a longer time to transform the flora by milk feeding than by the
use of lactose, and the change was not so nearly complete. Meat or

HISTORICAL REVIEW 9

high protein diet caused an increase in the relative numbers of putre-
factive bacteria, which could be reduced again by the addition of lac-
tose to. the meat diet. Starch appeared to foster the amylolytic types.
They concluded that milk owes its beneficial action to the lactose, which
is absorbed slowly from the intestine. On several occasions it was
found in the feces of rats to which it had been fed. It was claimed
that the transforming influence of lactose could not be due to lactic
acid produced from the sugar, because it was impossible to demonstrate
increased acidity of the intestinal contents of the white rats.

Throughout the above series of investigations Rettger and his asso-
ciates observed that B. acidophilus was an almost constant inhabitant
of the intestines of chickens of all ages and of white rats, and that the
administration of milk and lactose, and to a limited extent raw grains,
stimulated proliferation of this organism, and that unless such favorable
dietary agents were fed the number of acidophilus bacilli was held at
a low level. It was quite apparent then why the ingestion of milk soured
with B. hvlgaricus has such a marked transforming influence on the
intestinal flora, and that what at first sight appears to be B. bulgaricus
is nothing more than an invigorated B. acidophilus (1915, page 27).

Weiss (1904) had demonstrated the presence of large numbers of
B. acidophilus in the intestine of man after the administration of milk.
Sittler (1908) called attention to the relation of intestinal organisms
to the diet. He attributed the predominance of B. bifidus in the feces
of breast-fed infants to the lactose in the mother's milk, basing his con-
clusions largely on the observation that cow's milk reinforced with lac-
tose causes the establishment of a similar flora. He noted also that
malt soup exerts the same influence, while sucrose eff'ected no trans-
formation. De Gasperi (1911) claimed that bread or mixed grain
caused the establishment of B. hifidus, whereas the substitution of meat
for bread or grain brought about a preponderance of B. coli and
Proteus vulgaris.

That a mixed diet of milk and other staple foods causes a more
marked change in the bacterial flora than an ordinary diet without the
milk was shown by Fischer (1903) and confirmed by Sittler (1910).
Steele (1908) came to the conclusion that the regulation of the amount
and character of food is the most efficient means of checking excessive
bacterial activities in the intestine, while Friedenwald and Leitz (1909)
held that regulation of diet and evacuation of the bowels constitute the
most effective method of reducing the excessively high bacterial content
of the bowel. Harris (1912) fully agreed with the conclusions of
Friedenwald and Leitz.

Herter and Kendal (1909) noted a definite correlation between
specific types of bacteria and the chemical composition of the ingested
food. They observed a gradual but rapid substitution of an acido-
philic, non-proteolytic type of flora for one that is strongly pro-
teolytic, when the diet was changed from meat and eggs to milk and

10 TRANSFORMATION OF THE INTESTINAL FLORA

dextrose. Distaso and Schiller (1914) reported that, of the various
carbohydrates used by them, lactose and dextrin alone exercised a
profound influence on the intestinal flora, bringing about a pre-
dominance of B. bifidus. No mention is made of B. acidophilus.
Wollstein (1912) studied the eff'ect upon infants of high carbohydrate
and protein diets, and found that the former encourage the development
of cocci, B. coli, B. acidophilus and B. bifidus, whereas the high protein
diets favored B. mesentericus, and brought about a very great reduc-
tion in the numbers of B. acidophilus and B. bifidus.

Torrey (1915) found that 250 to 300 grams of lactose were re-
quired to transform the fecal flora of typhoid patients from the usual
mixed type to one dominated by B. acidophilus. B. bifidus was occa-
sionally seen, but it was never present in sufficient numbers to be of very
much significance. In his very recent investigation on dogs Torrey
(1919) demonstrated that fifty grams of lactose or dextrin, when added
to a meat and rice diet, completely transformed the ordinary flora to
one strongly dominated by B. acidophilus. He showed further that
the feeding of a bread and milk diet favored the development of a flora
consisting almost enirely of B. acidophilus. Saccharose, maltose and
glucose exercised only a comparatively moderate, if indeed any, trans-
forming influence. Starchy foods in the form of white bread, potatoes
and beans tended to bring B. acidophilus into prominence, while rice
was less effective. Torrey claimed that while animal proteins en-
couraged a strongly proteolytic flora, vegetable proteins were without
this effect.

II. FEEDING AND IMPLANTATION EXPERI-
MENTS WITH WHITE RATS

The first investigation reported here is an attempt to implant B. acid-
ophilus within the alimentary canal of the white rat. The experiments
consisted essentially in the feeding, in connection with a basic diet made
up of bread and beef, of such special carbohydrates as lactose and
dextrin, and of uniform suspensions of B. acidophilus in saline solution.
In studying the transforming influence of these agents an effort was
made to determine the minimum amounts required to bring about an
appreciable change in the intestinal flora. Bacteriological examinations
of the feces were made on alternate days. White rats were used ex-
clusively. They offer many advantages, at least in this particular
instance. They were easily obtained in large numbers, and with little
cost to the laboratory; they occupy little room; they may be induced
to live on a monotonous and simple diet, and the feces may be obtained
fresh at the time they are needed. During the investigation 66 rats
were employed, and between 1200 and 1300 fecal specimens were
examined. The total number of complete rat experiments was 118, some
of the rats being used more than once.

METHODS

The rats were kept separately in wire cages having floors made of
half inch wire mesh and placed over paper-covered trays for collecting
the droppings. The basic diet consisted of bread and beef, ten and
three grams respectively daily. This was fed for at least five days
prior to the administration of the test substances. The bread and
meat diet was found to be sufficient to keep the rats in good physical
condition, and was as a rule consumed without waste. There was but
one feeding a day, usually in the morning.

The feces were collected easily according to the method of Hull and
Rettger (1917). The rats were held by the tails and gently rubbed on
the back. The samples may be collected directly into test tubes with
very little or no contamination. The specimens thus obtained were
moist and soft and were readily reduced to a more or less uniform
suspension by vigorous shaking with the aid of broken glass in test
tubes containing ten cubic centimeters of physiological saline solution.

12 TRANSFORMATION OF THE INTESTINAL FLORA

These suspensions were diluted to match tube eight of the McFarland
(1907) nephelometer scale. The methods of bacteriological examina-
tion of fecal specimens are given at some length in the special chapter
on methods (pages 114-117) which goes into the preparation and use
of whey agar plates, Veillon tubes and Gram-stained miscroscopic slides.
The amount of gas formation and the relative numbers of B. acidoph-
ilus colonies in the Veillon tubes are recorded by the following system :

Gas production

B. acidoph

ilus colonies

—

none

—

none

+-

very slight

+-

very few

+

slight

+

few

++

moderate

++

moderate

+++

marked

+++

marked

++++

very marked

++++

very marked

Special tests for B. welchii were at times made by the milk culture
and heat test, but these were regarded unnecessary as a routine pro-
cedure, since the Veillon tubes provide the right conditions for develop-
ment of this organism.

In selecting methods for the bacteriological examination of intestinal
contents and fecal specimens the aim has been to employ simple and yet
sufficiently reliable procedures to obtain a general insight into the num-
bers and biological activities of important groups of bacteria which are
capable of establishing themselves in the intestinal tract, and to make
it possible to correlate particular types of organisms with specific
dietary conditions. The methods chosen enable one to gain a clear view
of the relative predominance and activities of the fermentative and
so-called putrefactive bacteria, both aerobic and anaerobic.

During the investigation pure stock cultures have been grown in
either whey broth, whey agar, or in milk. The hydrogen ion concen-
tration of the broth and agar, unless otherwise noted, was adjusted
to pH 6.8. Whey agar has been found to be of particular value for
preserving the different strains of B. acidophilus and B. hulgaricu^,
transplants being made onto the entire slope of the agar with a liberal
amount of the inoculum.

Samples of intestinal contents were procured in the following manner.
After a fast of twenty-four hours the rats were killed with chloroform,
the abdominal cavity opened and the entire intestinal tract spread out
on a sheet of paper. The various sections of the intestine, namely the
duodenum, jejunum, ileum, caecum and the colon and rectum, were then
removed with shears. The contents of each section were forced out by
a process of stripping with forceps into test tubes containing sterile
physiological saline solution and broken glass. Standard emulsions
were prepared and subjected to precisely the same bacteriological
examinations as the samples of feces.

EXPERIMENTS WITH WHITE RATS 13

In order to conserve space, quite a number of charts, curves and
photograpliic plates originally prepared for publication have been left
out of this book. Those which do appear have been selected because of
their representative character. The curves were plotted to show
average figures.

Throughout the present treatise the generic term "Bacillus" has
been applied to the acidophilus and bulgaricus organisms. "Bacterium"
has the greater claim to recognition here, from the standpoint of both
motility and spore production. Furthermore, such usage would be in
harmony with the general recommendation of the Committee on Classifi-
cation of the Society of American Bacteriologists which, however,
recommends the use of the generic term "Lactobacillus" for the group
of milk-souring bacteria to which B. acidophilus and B. bulgaricus
belong. The term "Bacillus" has been retained because of its universal
use in connection with these two organisms by both bacteriologists and
men of the medical profession, and because of the lack of definite action
on the point at issue taken by organized societies.

PRELIMINARY FEEDING EXPERIMENTS

While chief emphasis was placed on the implantation of B. acidoph-
ilus in the intestine of the white rat, considerable attention was given
also to the influence of various carbohydrates on the intestinal flora,
both with and without the administration of suspensions of B. acidoph^
ilus, and to the possibility of establishing B. bulgaricus in the digestive
tract. In every experiment the animals were first given a basal diet
which was found to encourage the development of the usual complex
flora without completely eliminating B. acidophilus, a normal inhabi-
tant of the alimentary tract of man and animals. This diet, consisting
of bread (ten grams) and chopped lean beef (three grams), encourages
to a certain extent the "putrefactive" and gas-producing types of
intestinal bacteria. It was consumed freely bj' the rats, and appeared
in every way to serve, for the relatively short periods in which it was
fed, as a complete and wholesome food.

Tables 1 and 2 give the results of examinations of the intestinal
flora of rats receiving the basal or "normal balanced" diet alone. In
the first of these tables will be seen the relative numbers of B. acidoph-
ilus, gas production in the Veillon tubes, and the description of Gram-
stained films of the fecal suspensions, while the second table shows the
distribution of B. acidophilus and other types in the alimentary canal,
namely the duodenum, jejunum, ileum, caecum and the colon and rec-
tum. Few bacteria were found above the ileum. In the ileum, and to
a still greater extent in the large intestine, there were large numbers of
bacteria, with B. coli as the dominating organism. B. acidophilus was
found in the large intestine only, and even here it was comparatively
rare. (See Tables 1 and 2, and Charts 1 and 2.)

^

^1

8 ^

•ildopp y

a

SVQ

"Udopio Y
•qdopio y

a

SVQ

«

to ce

p O

,2 w'tS eg a5 '^i£
X T3 O O O e ~

4* £ ,*J lU i; o V

^•.-i.^

U <J

§ ^ »2 CO CO • M

CO o o o =Po

S '2 JJ (U 4j O 4,

o c s *< *- i ^-

^ CO to ce CO CO w

CLi'O 'a fQ fQ rrt 'O

•^o O o o o o

'o ^ ^ ^ '^

o ii sJD be be ti) Sib

{J 4) 4J 4) 4J 4J 1)

o c c c c c c

l-s s s s s a

fc w c6 03 eS cS cS

Xj t *- *« »- ►- *-

coOOOOOO

+ *J + 1:J +

i-H O O « O* O fH

PQ

'O to

O 'O

>H O

. f-> . to . .

to . . to fT5 to CO

O CO J2
. CL P

o S o o

UiiMl

« bfe ^ I ^ ^

•rer

•3 M ^ ^ ^ ^ 55

S 58 OS * ^ * *

£ a s s s g H

CO o o o o o o

_ -a re 'O 1< 'O 'O

c £ £ ^ t; *- ^^
c6 &,&,&< f^ft &i

to CO to to to CO to
1X3 "T) 'O '^ 'O "^ '^
O O O p g p p

ti ti) ti bb bb bb bb

4> U V V V V V

s c c c c s c

s s a s s a s

ct3 cd ^ cS 03 cS cS
»« M b b »^ ^ i4

oooooo o

I I i I I i I

+ + 4^ + + :|: +

1 1 1 1 1 1 I

O OT 1-1 O CC >-< G»

CN tj" «> 00 O G< •»ii

m

P3

O

to to •
P P^

"££'

to O I

2 " I

ft> O I
u ft,

I iai^ai

*in *n "^ CJ ^ aM CJ

±3 J3 O +j C O +J
ft ft-K ft OS +; ft

y y V i. 1^ ID u
« =8 S « w § «

iiiifii

ft ftftPn'S ^e-

to to CO "> to to J£

'O "B fCO -o -a '5
p o o p O o P

t' %^ u 1^ u a, >^

bb bb ib bb bb C^ bb

V V 4) V 4) u V

C C C C C s c

a a a a a a a

Co c3 03 CO c6 cd cS
u u ^t t. U u f-t

OOOOOOO

I I I I I I I
I I I i I I I

OS '-' o iH eo iH o

•ildopioy

a'

'i(dopto y

a'

a)'«

2^'

^ftn

qq:

n

«

o

M

3 ^
a »

CQ o

« S

Ph u

-§

o o

h <» Cfi

B>

c

e S

S S

« si

60 bO

C C
O O

S C

be*" be

S > S >
c8 tS

o o o o o

11

a ^

o

»

a "

c

■H to

^'^
Co o
PQ S

"a

2 £

o .
. (^

i CO
4, |i|

S 5

O 1^

^ « «

o
p<

a
o

2 2

P^

►^ *M »-'

^ b a; .-s

CO

CO CO

1^ s

'bb
c
o

^O, &, CO 3 CO 3

O 2

bc^

c c

a a

ee cS

a
o
»->
<u

a c
§a

^ 3

!o ^ CO ii

2|2a

«bsb

a > a >

ce 03

h ^ ^ ^ ;h

oo o o o

be Ji

^a

s
c

I I I I I

a

•*->
u

is

a

a
1

'5

Q^

H^

O

O

«

o

Q

a a

s a

c a a

4-> 3 3

2 ^ '=' ►^ ^

4J ."S C* « W

y 3 0* > >

CS ly »* • •

. ^ O W

■g O ee C8

CO * *«W

-apqW . .

P CO CO

P, *" CO CO

2 ^^a a
o a a 2 2

CO 2 2oo

goo ^ ^

O^ cPhPh

as oS

^2^^£

« 4; « ee e8

i s s a a

'5 T3 '5 T3 ro

p o o 5 o

^- ^H t. c g

til be bb bb bb

4^ 0^ 4^ OJ OJ
C C S C C

a a a a a

ee 03 ^ ce c3
>^ u t^ 1^ ^^

O OOoO

I I

O O cy» G< 00

a

3
■•->

a«

3 c

s J?^ ieo

16 TRANSFORMATION OF THE INTESTINAL FLORA

CHART 1

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a "Normal, balanced" diet

I-

Bread lo
Beef v3

Z5

s

no

0

15

X

10

s;

0

/vvmAer orpcfays aff^r gdmtnis/ra/'or} afa/'e^

CHART 2

Graph indicating average percentages of B. acidoph-
ilus in the various parts of the alimentary canal
of rats fed on a "Normal, balanced" diet

25

20
15
10
5
0

1^

Bread /O
Beef <3

Fhr/-3 07 aJ/menTar'^ canctf

g

•I

^

ft?

o

EXPERIMENTS WITH WHITE RATS 17

CARBOHYDRATE FEEDING

In all of the experiments with rats the test carbohydrate was added
in dry form directly to the regular diet, namely ground bread and meat.
The meals were completely consumed. By comparison of the results
of daily examinations with each other and with those obtained during
the preliminary period the progressive changes in the intestinal flora
could be discerned easily. Each of the carbohydrate feeding experi-
ments was carried on for two weeks.

Lactose. — This sugar was fed to sixteen rats. The ingestion of two
grams daily effected a radical transformation of the fecal flora from
the ordinary mixed type to one almost entirely dominated by B. acid-
ophilus. (See Tables 3 and 4.) A progressive change was clearly
evident two days after the first administration of lactose, but did not
reach its maximum until at least the fourth day. In some instances six
to eight days were required for this maximum. The acidophilus phase
persisted with very little or no fluctuation as long as the diet remained
unchanged.

With the actual increase in the numbers of B. acidophilus in the feces
there was practically complete suppression or elimination of the types
constituting the ordinary mixed flora. Large numbers of the char-
acteristic fluffy colonies of the Moro bacillus appeared in the Veillon
tubes and agar plates within a few days after the first administration
of the lactose, and there was gradual diminution in the amount of gas
production in the Veillon tubes until finally it was entirely absent.
B. bifidus could be observed only occasionally in these tubes by its more
or less typical smooth, disc-shaped colonies occurring in the deeper
layers of the whey agar. The direct microscopic examination of Gram-
stained films offered additional confirmation of the transition to the
acidophilus type of flora. After the first six or eight days the films
contained practically nothing but Gram-positive rods. (See Table 3
and Chart 3.)

Table 4 and Chart 4 show the relative numbers of B. acidophilus in
the different parts of the intestine. The prominance of the aciduric
type, particularly in the lower intestine, is again noteworthy.

Dextrin. — The sixteen rats employed in the lactose-feeding experi-
ments were again placed on the basal diet until the simplified flora had
completely given way to the usual mixed type. The rats were then
given two grams daily of ordinary commercial dextrin, and the feces
examined as before. A change in the character of the intestinal flora
became apparent within two to four days. The dextrin stimulated the
proliferation of B. acidophilus in the course of the first four to six days
to such an extent as to overshadow all other bacterial types. The
typical colonies of B. acidophilus became very abundant on the whey
agar plates and in the Veillon tubes, often to the exclusion of practically
all other forms, and gas was absent from the Veillon tubes. The Gram-

CO Q

be

"n 2 y o5 «

4* >-i O

re s

'e

09

.„• O « • '. J3 ^

o g o W 2 -S 'G

4) O -^ 4) Cl-

« ^< ca Ch

_ fe tc to

«oi .-SI-?

§ § ^'S « ^ s;

•^ ^ fe ^-x: .ti .ti

1) 4J 5 O £3 2 ~

J; li S ^H cS 2 2

'2 'O TS ^o :- s :=:

2«

•ydopto y

■qdopp y

a

•ydopio y

a

c8 cS

fcr\ ,J " • ►■^^ ^ t>>

QO CO CO CO ""^ '^ ^^

<o o o o '13 '^ "S

c a a, ft s I g

S S S S tj u tj

M to 83 c6 c8 rt c8

>- »- t« », tH ti fc,

«

»

t+ 1 I I I I

0> O <3< O «5 0> OD

CO V CO o> o> O o>

0< ^ «0 » © G< •«J'

ai CO

73 ns

O

O

(i4

u

ti) be

(U

lb

c

C

S S

C8

CB

CO f, 'O rQ

O Q. C C

^ 57 be * *

S^« 4^ .« .-H
■M C « W

C8 CO "^ Q C

S ^ a o o

'•-^ S ? O O

fe co-cJiirS:^

._: o " -: -J

w ^' o 4) 4j tr< jr
o be w «*S «t- o o

W 41 o 'O 'O

+:; ± ^- CO CO

. ^ CC B B o O

•^ 1^ ^ O, Ph+j +j

«

S S E
ego
'O « ^o

41 V V

be be ft ft
§ o g S

Gnftftco tO^ 1^
CO CO CO CO CO ^ ^^

'O 'O 'O 'O 'O -^ -^
o /-s n (^ r\ ^^ •"?

be CO CO o5 CO X*F—

4) o o o o r: 'S

C ftftftftg g

B H E S c u u

pS cd cS CO CO cO o3

i£ CO fn

P O »H

CO CO

T3 T5
O P

be bo

4J 41
B B

be 4)
« B

Jf P S S

CO CO
. u u

= > CO

s

. CO _ »

'r";; B « -

is

2 2

^O CO (^W

r_ B IV, t~^ ■ J^

5fe

. B . O I.

V. B

CO

■y W «J i," w

w ?:! 5^ o 2i o ?r
o o o o CO g-:g

■g^ftftO ^£<-
Sj « JJ -^4) Cfi J'

S B B j» O >.^
S CO CO P "O t- 4>
M 13 >^ V 4; 4) >

s s s Pt, ^> s
£ s s s -^ s I

o o O o **- "^

■B "B "B ITJ B

V 4) 4> £ O

^H tH t. C *-

ft tC^ft*^
i2 i2 iS «> =c -.

'O 'O 'O 1X3 TS 'H —
O O O O o o 'S

ti) CO to CO to CO —

4) o o o o o "3
B ft ft ftft ft S

s s s s s s ■■§

CO CO cO cO cO cO CO
u u u t^ u U U

OOOOOOdn

-I- 4- I I I I I

+ + 4. 1 I I I
It* I I I I

e» •«? «o OD o o< ^

en
H

<

O

<
s <

fi Q

►J u,

< -'
H d

'■ Q

CO

s

Oh
O
Q

O

<

[^
o

o

H

H

^

»

"qdopio y
•ff

•i{dopio Y

a

SVjr,

K):«

^fts

•2 1 1

ta

a

s

B

n

c

s

CO

■4-1

o

c

1

c

o
c

>H

t

•M

cd

«

c

«

S

in

P

o

CO

O

>1

O

B

ii)

si

05

c

C

d
u

S

a

b

Scfi

O

o ^

73

'O

B

c

c6

>»
b

u

t

CO

^ S

^

^-S

u

a

V

fo

fe

ii

>>

'O

"^

c

(U

4>

o

u

^

b

a.

p.

CO

02

CO

CO

'S

-OtS

o

o

o

u

b

^

CO

to

CO

O

o

O

ACP,

s s s

cS

(S

Oi

ooo

o o
6 S

C S

mm

^<j5

CO CO

o o

CO ID

O O

-ii

3 eS TO
O «- ^

goo

S CO cS

S(

s

u

a
O

m

m

o

o

3 „

2 O 3

. w li >-

Sm I s

m . ^ 2

CO « «

o ^ >

. tJ V *■'

bcc ts y

£ * /-^ • •

f5 w n »^ '^
cS

'mm

a, <u •R S •«
ST >- 2 w «

. ^W to CO

4) fc^ O O O
^ ^ ^H »-

. e S "5 SS
S S g O O

g O CLi O, &i

'Sl s S S
J^coOOO

g S * CS ts

CO 55 —3 3 3
o 2 cs Bj ce

pin Ph y y ^y

S E W « W
cS cS rt CS CS
^ >« Vi ^ ^

OOOhPhPh

en ix> <o a> Oi
00 CX) o> o> 9>

3

-S S £ H c
o 2L3 v^

m

m

o

c^

^o

V s

• a, «J

<<

fi| '^ c

CO tj CO

O 2 O -I

£ £ £

Oj 03 OJ
^ ^ ^

OOO

*l

I I

tj

t- (S« rH

CO A OS

-S g £

§.2.3

£(ti

ce o
OO

20 TRANSFORMATION OF THE INTESTINAL FLORA

CHART 3

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a lactose diet

/oo

^ /

LJ/er /n ^/T3nr>5 _— « — " "

fo

Br-eoc/ /O y^

80

5

Beef 3 X

^

Lac^as^y

10

i

/

60

1

/

50

/

43

0

/

30

0

/

W

V

10

/

0

/

/Viymier- ofc/a^s af/er- ad'TrMvs:^n3^/on <y*o'/<?/

/o

14

CHART 4

Graph indicating average percentages of B. acidoph-
ilus in the various parts of the alimentary canal
of rats fed on a lactose diet

Irs Oie-f /n grams.

-k

v$ Bread /O
%, Bs<^f «3

\

/oo
30

\ 1

60
JO

1

60

SO

Parts of alimentary canal

Si

o

8

05

EXPERIMENTS WITH WHITE RATS 21

stained films frequently appeared to be almost purely Gram-positive
rods of the B. acidophilus type. (See Table 5 and Chart 5.)

The rise in the acidophilus curve is slightly more marked even when
two grams of dextrin are fed than during the administration of the
same amounts of lactose, the maximum in the former instance being
reached on the eighth day. In other words, dextrin has shown itself
to be at least as effective in simplifying the intestinal flora as lactose.

Several of the dextrin-fed rats were killed for the purpose of making
bacteriological* examinations df different sections of the intestine.
B. acidophilus was present throughout the intestine, even as high as the
duodenum. With the exception of a few Gram-negative rods and strep-
tococci, it was practically the only organism found above the ileo-
caecal valve; whereas below this level it was strongly predominant.
(See Table 6 and Chart 6.) Gas-forming bacteria appeared to be
almost entirely eliminated.

The results obtained thus far demonstrate conclusively that both
lactose and dextrin, when fed in sufficient quantities to albino rats, have
a most pronounced influence on the character of the intestinal flora,
transforming it from the ordinary complex type to one in which the
dominant organism is B. acidophilus.

When smaller amounts of lactose or dextrin than two grams were fed
daily the transformations were less profound. Thus, one gram en-
couraged the development of B. acidophilus to only about 50 per cent
of the total fecal flora, as will be seen in subsequent pages (35-40).

Glucose, maltose and sucrose. — None of these sugars, when fed in the
same manner and in the same amounts as the lactose and dextrin,
exerted any transforming influence upon the intestinal flora. This was
clearly brought out in the agar plates, the Veillon tubes and the Gram-
stained slides. Throughout these feeding experiments the flora pre-
sented the same character as when the rats were kept on the "normal
balanced" diet of bread and meat. A total of six rats was employed,
two for each sugar. (See Table 7 and Charts 7, 9 and 11.)

Bacteriological examinations of the different levels of the intestine
gave negative results also. B. acidophilus was absent from the small
intestine, and was found below the ileo-caecal valve in numbers only
that are normally met with in rats subsisting on the basal diet. (See
Table 8 and Charts 8, 10 and 12.)

The above results are in harmony with those of Hull and Rettger
(1914b, 1917).

ADMINISTRATION OF LIVING SUSPENSIONS OF
B. ACIDOPHILUS

All of the strains of B. acidophilus that were employed in these
experiments were isolated from the feces of albino rats during periods of
lactose or dextrin feeding. Typical colonies of this organism were

<

H

;z;

8

H

H

l-H

K)

Q

W

^-;

►H

n

o

<

Oh

H

CO

.8

§

HO

I

s

m

o

O 'O to
V, p ^O

. '-' o

^ be

O 6C .

•qdoppy

a

* S c -S M -T T
"3 -r '* «^ > -c .a

-M o w fe >;«« o

_g 4; g,fe > WW
rn ■'-' t< . 03 to
>- ^ CO g S O O

5! ^2 « 2i 2^ > >

■^ S &H A o^Z! -+3

f; ;h t- -i-> i c3 03
»* & fc «= -S ^ ^

*- S »H >- S '* *
be t<5 to g to s's'

s s s s e "-g ■•§

w ^ CO ti ^ C0 Cd

*1 tl »-l fc, t< »H fc,

I to qj

*- S c

g I c ,

*JIJ

—■ '3 & V"^ -«= -c
o H « c p,a a

w 5 -M a; o o o
o oco^33 2

+J *? to to to

CO ^ e s '^ '^ ''3
^ E C O O O

. . ►. CO to S

'g'S § i s s s

C ^. i- i c8 C6 c6
to to oj to ^^ ^^ ^^

^O "TJ fQ 73 ~ — ~

P P O P CS CS eS
f^ >-, f^ U ^ ^ ^

bbo5 to to:^""^
- -^ - PTS-^-^

PQ

S S 2 S o « "3

^ c6 ^ cC c6 q3 03

(h ll »H hi ;h bl ^H

to . .

'S 'w be

o U V

^ o e
. «

CO O S

S£0

eS CO-d

'" ^ a

0 ^ «

<u .„ . . . .

r o JJ « sj (U
'Go I T T f

•3 £ "Sh^' -s -c -s

>1 U Cm p, CL, Oi

o aj Ai 'O -C -o "O
3 c "-^ '3 "3 « "3

a; ,t w to CO CO

CO U . 'O 'O 'tJ TJ
(^ ^ C p O O O
g> X g ^< >i ^- »<

D,S 5 S ."5

to *to 'to 'as

P c >»2 o o o

1 i'bc'='«^^»'
4j 'S o § S E S

*-i fc, t< S« 83 e8 e8

toJ^OOOO

g O o 03 eS C6 08

bO to to 1^3 S Z3 Zh
ti q3 03

S<o,S

a u u

B S S U U U U
C3 C0 03 c$ c$ c8 c8

»H *« >H fcl fc, V, »H

O O O Oh pLi (1, Oh

4- I I I I I I 4- I I I I I I

EXPERIMENTS WITH WHITE RATS

23

picked off whey agar plates, introduced into whey broth and incubated
at 37° C. for forty-eight to seventy- two hours. By this simple tech-
nique eighteen strains were procured from the rats, and three from
human subjects who had been taking 300 grams of lactose daily in
connection with their regular diets.

The bacterial suspensions were prepared by washing off with sterile
saline solution the surface growths of large whey agar tubes which had
been incubated for forty-eight hours at 37° C. These washings were
reduced by dilution to the degree of turbidity corresponding to tube five
of the McFarland nephelometer. This bacterial concentration was ad-
hered to in all of the feeding experiments in which living bacilli were
employed. The final suspensions were added to the basal diet, being
thoroughly incorporated in the ground bread. No fewer than three,
and as many as eighteen, mixed strains were administered in individual
experiments.

The bacterial suspensions were given in amounts varying from one
to five cubic centimeters, and the usual bacteriological examinations of
the feces made. After several preliminary trials, beginning with five
cubic centimeters, two cubic centimeters of the standardized suspension
were finally chosen, as less than this amount was found to be inadequate
for bringing about a profound change in the intestinal flora.

CHART 5

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a dextrin diet

Die^ in omms

Sreod

Beef

Dexf/-in

^Uftyher-ofcJa^ ofjefoe/mi'n/srm^tbn of d/er

/Z

/4

O

<3
:z;
<!
O

Pi

<

Eh
^ CO

H d

CO

t3

1-1
>— I

Ph

o

Q
I— I
O
<J

«'

o

iz;
o

H

I— I

H

?b

'tidopwy

a'

•i{dopp y

a'

8VO

^ftn

m

S 3 o o o

C C t- Jh >-

3 ^ 4> <U «

«2 a s a

+? £3 3 3 3
g^ C C G

+J IS (h fH «H

w aa « 4) 4)
cs*^ > > >

rrt .5 .2 2
^ 'g "E "E 'u

4^ '^ -4-1 .M •+-)

»- ^. o u w
^ gfQfQM

y CS (U 4) lU

• rrt "^ '^ '^

til 4) 'S '0 'G

,z £ o o o

U Ph ;, ^< t,
jj. . <0 4) (0

« ^ .^ .^ .S

Ei-i '-' ."ti ."tJ ."S
. ^ o o o

-§ c s s a

« g cS cS c3
^< J^ t^ ^, j<

O O <S cS cS
u u

. . >%>>>.

cc w ;2 ^ ;2
g O cS 03 c3

art +j '+3 'XS
B o y w
cd ^ cd c6 c(3
»H ti (h ;h )m
OOPiPmPl,

a
1.Mi

tf

fQ

Q

SCO -1-5

3 W

3 O tS

I 3 «

.2^2
u o

ii c bb

w . (U

CQ C8 rt

" 2 «

si) . 2

•s

a.

V
Ih
■M

S a;

4) .

■fc a

cc o

^ 4)
- - U

a,

i a be

Ji « 2

'O rQ '5

O o ®

^ 2 ^

u5 CO W

O o o

C^ Ph S2h

a a a

cd c6 cj

M t4 ^

+ 4:*
I I I

I I I

3

3 S

E,3

O 3

05 to

Q»

3 3

.5

O O

;-< b

'C

41 V

■M

a a

d

3 3

Q

>>>>

««

t- Eh

3 <U 41

«i-i

> >

tJ

.5 .5

CQ

"E *E

4) 41

o

-l-> -M

l-H

4) W

eS eS

TS

fQ«

oi

4>

U

^^

«

4^ 4)

1

^3

i

T* ^

g

< jiji

s

Oh a.

o

o o

'2 'S

s

'v 'S

:H

■fc^

V

CO CO

5

TSTS

O O

^ U

4) V

,>,>

IS iS

'cc 'co

O O

PhO,

. a a

2 CS c8

3 jh fc,

goo

4) ^ _^

1^^

c >>X

•*J c8 oj

•- y U

3 — H .-H

eS C8

t^ ;->

pLHpk

1 1

1 1

1 1

O) Oi

05 OJ

a

3

•M

w

a;

eti

rH

3 C

G«

«J^

•M

ei) "o

a

oo

tf

c a a £

13

2

r; 3 3

■S « c ti

bb

fl 4J «; V

4)

. O -w C

c

e8 • CT E

a

«-y^2

e8

-«l^

o

'B .«co

CO

£ CO 03

eS

.T) CO W

4)

V ^ oO

4)

o

c . >-

-• be . •

a <u bcfc

d,

S C 4> 41

41

£ C ^^

»H

°iE-

^

^ £ (S K.

^

^

41 5 c C?

b

4)

>

PhS 4) .

41 ■!->>-■ c

a

HJ^-'CO o

o

cc > ^ro

&|E2

ti CO

«fep^PH

0<3
p

t . >»

CO

>>ii

dom.
edom
edom,
rongl

3

o

4)

a

F— 1 4>

^a

*> S I- *^

^f^p,=«

CO CO "^

3
C

CO 4)

1111

4)
>

1^

*^.2
■ 'E

CO t" CO 52

CO IJ

g O O o
SH<^Ai^

■M

o -t^

C8

^s

a a a a

25

ao

oooo o

-^ + tl I
I I I I I
l + tl I

UN I

cc t- «fl OS

t- CD 00 OS

3 C *>

gi a tf

3 S'^ « O

EXPERIMENTS WITH WHITE RATS

25

CHART 6

Graph indicating average percentages of B. acidoph-
ilus appearing in the various parts of the intes-
tine of rats fed on a dextrin diet

joo

60
JO
60

•50

<0

T

o

Bread /O
Beef v5

Parts of alimentary canal

i

a

Of the many experiments conducted on the implantation of B. acid-
ophilus in the absence of any added carbohydrate the following are pre-
sented here as typical. Three rats were placed on the basal diet for
ten days ; after developing the usual mixed flora, two cubic centimeters
of the suspension of living organisms were added daily to the bread and
meat. Two days after the first feeding of the suspension B. acidophilus
was observed in appreciable numbers in the plates and Veillon tubes.
The maximum transformation was reached within three to eight days
and persisted throughout the experiment. B. acidophilus was found
to predominate to the same extent as in the experiments in which lactose
and dextrin were fed. (See Table 9 and Chart 13.)

Post-mortem examinations of the different sections of the digestive
tract revealed the presence of the above organism throughout the in-
testine. (See Table 10 and Chart 14.) There was a higher propor-
tional representation of B. acidophilus in the duodenum and jejunum
than in the lactose- and dextrin- feeding experiments ; in all other re-
spects the bacteriological findings were essentially the same.

en

Pi
<
o

en
en

o

I— I

fi
<
>

O

5zi

<

8

I— (
O

m

Oh
CO

Q

CO

H

H

CO

H
CO

»

M

O

o

CO ^

O 05

M o
o'S

o 'O
^ o

CO •

O cc

2 o S 'O o S a

c6 O

!=^ P^ * a, ^£ S

n

n

i2 'T3 CO CO 03 03

'2 O 'C 'O 'O 'O
P g O O O p

o 2

CO CO CO CO ►-

O p P O^

&I >=^ P, Oh P, Ph

1 1 s s s sE

o O !^ o o o .^
^, ^, ^ & & ^ «

^ .« • • • • P,

=* " '3 •« "y 'y 4)
« U « « _h

CJ<P

o p

SCO
o

g S^PhPhP^p-S

c6 ;5: 4-1 -4-> -4-) -i->
CO u3 CO CO CO -•

i(doppy

a'

P cs

o

pq

»

" * cS cs cS aJ 4$

M)S S S S S ^

C • . • • • fc^

SB S S 2 S Tji

_ o o o o o a"

£ 'O ^3 'U 'C 'O §

ij « (u (U 4, (U g

o ti ti) tJo sib ii ti)
y V V u 4; V V
5 C c C C c C

&S S S s e S

7^ c(3 oS c6 cd cS 03

o

^ CO

S • o

Q. -^ • ^ • •
►-H CO "O CO ^ CO CO

S'O O 'O CO 'O "O
O *« O O O p

»-i . CO . . .

^ p g,o i p o

^ p. P.£ P<P,

£r^ S CS £ S S
" es ^- e8 > 08 c6

•d o ^ o ^ o o

S ^ £ ^ b^&

p. -5

4) .„• cs .-: .J .-; .-

ti o 3 -. .— -.

CO o

^ o ~ w S « y
e8 Ph o P» p, Ph Pc

4^ W 4J i 4J -kJ

. CO .-c CO M '^ "^

'2 § g § 5 § C8

.. . « . . .
-^S -M S P S 6
fecgJo o § o o

jr *- c >- £ *- t
•g Pirt p,p,p,Pi

yj CO CO CO CO CO CO
rQ 1^ 'P 'P fp 'O 'P

w o^y

} «3 W3 CO CO

; Fp fp rO frt I
> O O P 5

, L, h (I L.

c^ U2 ti) ti) be be tb

4) 4; 4; 4; 4; 4> 4>

s c s c c c c
S B S a £ S g

A c($ c8 c8 ^ ^ ^

Ih »4 Vl ll hi l-> ^

ooooooo

I I I I I

+ -1 1 + + * +

I I i I I I I

I I I I I

i-< 1-1 OT O f-" 0< i-<

en •«}' to oD o e« *

H
<

o
<:
o

^ CO

X ^

CO

K

Cu,
O
Q

<
pq

o

:?;

o
•— I

H

2
S

H
CO

.8

*s

I

g

8 ^

'tldopto y
•ff

STOQ

•ildoptay

a'

'qdopto y

a'

Kill's-

U SB

» « B

o 8 5
*S * >?*

-%

a B

B

.2

a s

s

V

a a

S

c

tj

■4-< ■*->

3

>>«

o o
a c

C

CQ

.■S

>

-t-i -M

to

O O

*3

4J

TS

es es

D' «

0

nn

CB

>-,

■t-J

n

w

to

05 u5

cS

0

'O 'O PQ
o o

to

O,

(h »h

0

s

»3 to

to

(«

o o

a B

o

to

O

to
0

s

c6

0

Oi

b

to to

S

o

>>

OO

c

1^

ire

d

c

cS

d,

S

^ t«

V

4-J

•!-> 4->

h

CO

COCO

■M

>>

s

0

S S S S-^
o 5 5 o be
ts 'O -TJ '^ S

V OJ oj <u O •

t^ t-i iL l^ ti (D
CO to to to to *^

TS 'a -a T3 -a V

0 o 5 p o S

tc ^H M »H ^, 2

sb be si ti ti ^

4J W 4; 4) 1) t».
C CI C C G C*

a s s s s >

cB cQ cS 03 ^
(h b bi ;h »H

00000

1 I I I I

14- + + +
I I I I I
+ tttj
I I I I I

o o o •<}< e«

a

3

v

5 a«
■Mil

4) 1> C8 O

•-saoo

g

03

a r.- s s

3 i S § 3
C 3 I C C

t! C >>>>

O 4) J^ ««

•S u > >

^ • . ^ ^

y y CO ro
^««to»

" 2 ?

O '" tt

Cu • •

e o o

i ^ft^a a
r^ a a 2 2

S^O 5; ^

cj — ■ ■ 4> aj

to CO

2 o

OS cS

ft^f^

4j 4) 1) w C3

a a s a a

00 O O O
t; 'O T3 ft) rrj
4; U 4) U u
»H »H >- ^< fc,

CO to CO to CO

'O 'o 'O 'O 'O
00000
t-i ti ^^ )^ u

ti ti ti ti ti

4) V 4) 4) 4)

C C C fl C

§ s a a a

CO cS CO c8 cC

»H (h b (h »H

00000

I I I I I

I 4- + + +

I I I I I

+ + 1:4:J
I I I I I

O O O «5 (S

a
-Si

O 3
P'7?

atf
34;^

« C6 O

O

a a
3 3

a a

. a a

c 3 3

§ c c

CI V 4) u

• S > >

CO CS

3 . .
^ y y

s 4,- y c6 O

W PQ jj CO C8

opQpq

to to *

o

to to rrt
00^

a a 50

CO c8 O

^ »- »H

to CO

O O

a a

a ki (^

loo

00
^ & -

D 4. »- K^ ^

y y C

. . £^^ y y
Oh&h y i £

« «icoco
+e +e M i^ i^

iiJII

mil

CO to «" J"

g g t, »H ^H

ti (i ti bb ti

y y y y y
C C C C C

a a a a a

cij 03 cB 03 r3
1^ U ti t-i t^

00000

I I I I I

I I4- + +
I I I I I
-^ + + 4:J
I I I I I

o o ©« ^ to

a

if

O 3
^ I ^ '« -

a
3
4^

y

y

a«

CS O
00

28 TRANSFORMATION OF THE INTESTINAL FLORA

CHART 7

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a diet containing glucose

25
4o
/■s
/o

O

{

V

Br&jd /O
Beef J

z 4- e a /O /Z /4

CHART 8

Graph indicating average percentages of B. acidoph-
ilus in the various parts of the alimentary canal
of rats fed on a diet containing glucose

Bread /O

Beef Z)
G/ocose 2.

^

\

23

ZO
IS

(f

/O
S
O

f^r/s of a/imc<rA:>fy cono/-

- - ■ 1

1

a
Q

a

8

a

Q

O

EXPERIMENTS WITH WHITE RATS

29

CHART 9

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a diet containing maltose

25
20
/•5
lO
5
O

1

J2isti

/n oram^

Bread /O
0eef v5
Mo/fose a

2 4 (> 3 /o 12 /^

CHART 10

Graph indicating average percentages of B. acidoph-
ilus in the various parts of the alimentary canal
of rats fed on a diet containing maltose

25
Zo
16

JO

5
o

D/e^ /n arair>S

BreoJ /O

5

Beef J

^

^Q//ose 2

s:

t

d

0

"JJ

V

Hs

^

«

U

«^

!(

Fh/-/^ of oZ/mcn^ry ca/^o/

^ ■ ■

8
«.

CI

3

ft?

Si

o

O

C5

CO

X
o

Q

<
n
o

g

^

I— I
o

Q

CO

H

OS

I '■qdo'ppy

so

^ I a

I •^dop^oy

Pll

« be

be 2

« g s

'S B c M — — '.

2 * § ^ '. '. j=

• u •- 4) A a» o

^ o 5 ??o «^

o

o^K s

."JOT

o C g

l£lll.ll

o o S ^(^Oh^

'S'S'S I S S s
5; t; ^ £ cs cs £

05 03 M 02 ^ ^
'O 'O 'O fQ ^H ,-< •—

£ S 2 2'3'3 rt

(U O o o

C 9 S S U U "u
^ ^ c8 cd ej ed c8
»^ (^ ;h ;.< b b b

o» ■* w CD o e« •*

«

m

220

a flO
2 * •
o o 'S -^^^'^

C e O S S S S

* S "S '. '. '. '.

o u ■•-> o o o o
S O CZ5'0 'O ■« 'O
P o fe '43 *3 ■« 'S

STfe

CO

03 09 ce ce
J 'C 'O 'O ts

^ S O O O O

S ^ 'O 4) « 4; 4J
w 4^ H ? L. S S

<< Ph-*-* ■*->■*->■*->

, • . 'cC CO *M 09

B S ,C» o o o ®
§ o be (^ ^ P^ ^

'2'S § S 6 S S

g >- »5 c8 c3 cS e8

I^ 0000
09 M 05

'O 'O fQ — < ^ -^ —

2 £ 2"* «'3'^

..V ,- •>%>%>>>»

bC^ 5S US S S =3

*J 2 2 cS eS aj eS

C Mh Ph u o w w

Sa q tp tp ;C V>

C Q w w w w

^ ^ 03 03 cd c6 cd

>H ^ b <H >-l ^ (h

+ 1 I I I I

J + 4- i I I I

©« •* « CD O 0« •*

-o

0

^

ti)

u

B

CO

i

0

«

u

^

0

0

03

ft

^

0

a;

09

a.

>-

C8
0

aj'O

0

ft

CO

n) V V V

'^j

^
^

^

ji

'*='5'=

u

u

ft

ftft ft

0

bb'*-

0

°S 0

u

0

>>

2

22 2

4^

b

'3

'S '« "S

ft

1

1^^^

■«->

CO

i

03

0
»1

rods

; rods

rods

ce

M

(U

4>

V V V

S^

ft

•5

itlv
itiv
itiv

►>.

CO

CO O! CO

s

0

0

rt* 0 0 0 0

bCft ft ftft

'S'S2 § S S S

ft ft^ ,t ^ t *-
OC0OC5

CO CD 03 ^^ '»^ *-' ^^

'O 'O 'O S S S '"'

2 2 S"*"* '*'*

bti CO X ^- — "-< ^-«

*j 2 2 "3 13 "3 "3

B ft fti « w o y

Be 2 +j tjj 4-> ^

B B U U U U

cd cd cC q3 cd cd cQ

h ^ (« t« bi h h

-^ I I I I I I
t-l- 1 1 I I I
t+ I I I 1 1

G< Tj< <C CD O 0< •«*

EXPERIMENTS WITH WHITE RATS

31

CHART 11

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a diet containing sucrose

£5
zo
/J
/O

O

f

0

V

P/e/

//7 gram s

SreocJ /O
BsLftf 3

Sucrose £.

A/iy/rthcr afc/a^s crf^r oc/m/z^/s^^vr/von of c^/e/

IS

/4

CHART 12

Graph indicating average percentages of B. acidoph-
ilus in the various parts of the alimentary canal
of rats fed on diet containing sucrose

•0

Oie^ /n Qroms

BreaJ /O

1

Beef ^3

■Sucrose 2 .

U

<s

<0

V

-k

«i

Z5

%

zo

15

V

^

lO

,5

/^r/s of a/t/i'Tcn^ry ca/to/

O

II

s

o

8

e

03

o

o

<

a

f^
o

<

<5
O

>^

<

»J «>

^ Q
CO f^

do

K Q
Pk o
O *^

<
o

O

I— I

H

l-H

H

03

"tidoptoy

8VO

j§ I •%idopioy

I 8VO

^Rh

.2 8

cq:

«

fQ

PQ

EO CO Cfl 10 CO

3 3 3 3 0

o o o o o

b ;^ ^ ^ b

4> 4^ Q^ 4J 4)

a a s s s

3 3 3 3 3

a a fi c a

^ OS cS ^ 03
'2 'C 'E 'E 'u
r V v V V

*i +J 4J +J +J

•g o o o y
g* cO cd cd q3

(^pqpqpQfq

'<n- « « « «

^^ jM jg j«
'T f I* "V T

Qj </} CO CO CO

rrl 'O 'O 'O 'O
O O C O O
g ^ ;h ^ M

4) (U U <U (U

> .^ .^ .& .^

^ |m +3 43 +3

"S CO 'co 'to 'cQ

O O O O O

P^ Ph &4 D4 P<

a s s s s

oj ^ oj cd ^
q b ^ b ^

ooooo

ccS ^ cct cC cd

^^^^^

c0 ^ c(3 ^ c8
tj V V w _y

a tjj in in '-P

w y w o o

cS cd 03 o3 cd

^ b> M »-c »-c

PLl PL| PU( P^ (Ll

a

"^ 3

a

atf

, 3 V ^
' « b! q

«

SCO CO CO CO
-3333

2 o o o o

0 t^ ti U ti

u V <u a

o a a a a

C 3 3 3 3

. a c a a

* .ti tJ c^ i^

« 3 3 « ^

i3 cS a!.2.2

beg 5 c8 ca

^ D 41 4> 4J
± ^ ^ ^ -M

rn rt5 Ef3 '^ 13
. 'V T " '.

2 J3 j3 -c -5
5^ &,&,&' f^
o 00 o o

'-' -3 w w «

+j CO CO i5 J2

ceo TJ 'S 'S
^ o o P 0
^ ^H ^^ »- »-

« 4, <u 4) 4)
>■ > > >

fH .« .« •- .rt

4; CO CO CO CO

0022
a,p,a,ft

>

'd a a a a

y cS 03 03 cS

f-l t< ^1 »H ^.

50 ^^^^

5 ^ cfi ed 03
&c_« y _o _y

alp +3 43 '^j
y y y y
cS c(3 cS cd 03

tc ti ^4 Vi (4

art

a

3 g

1 .a 3 § O
QhsSOO

n

fQ

CO CO CO CO

3 3 3 3

• 0000

2 C 2 g S

3 V y V y

s a a a a

y 3 3 3 3

a a a a c

s S y u

* «8 .2 .2
'E "E *^ *-
y y « «

08 cS " *

PQPQ WfQ

'-^ y IJ" y y
fro o 00

'O o o o o

g S 2 ^ ^^

4j y y « g»

•- 43 V *i -g

M w w 2 2
o o o 2 2

a a s a a
2 s 2 2 2
ooooo

03 03 s) e8 c«
q3 c8 c8 c8 c8

.y .H .a .a M

^ ;j3 tl3 IC Ifl
y y y y y

cd c8 c8 83 cd

^ »4 ll tH kc

I I I I

■s§i

a«

EXPERIMENTS WITH WHITE RATS

33

CHART 13

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a diet containing B. acidophilus

100

90
60

70

60
60

s30

ao

10

£^/e///7 oronpyS

Bread /O

3.aa'dopn//us2. CC.

M"^ocr of ofays afrer aclifiin/sTnrrion of c/As^

2 4 6 8 /O /a /4

CHART 14

Graph indicating average percentages of B. acidoph-
ilus appearing in the various parts of the
intestine of rats fed on a diet con-
taining B. acidophilus

loo

90
QO

JO
60

v50

o

V

Bread lO

Beef O

3.ocic/ophI/u^£.c.C.

(■SuSPer'SiorJ in J"/i«.//veJ

Parts of alimentary canal S

8
o

a

0?

34 TRANSFORMATION OF THE INTESTINAL FLORA

These experiments indicate that B. acidophilus is able to establish
itself in the intestine of the white rat when administered per os without
any accompanying carbohydrates. In every instance where two cubic
centimeters of the suspension were given it became the predominating
form throughout the enteric tract, almost completely eliminating or
suppressing all of the other bacterial types. The results warrant the
assumption that this organism is capable of adapting itself to intestinal
conditions and that positive implantation can be brought about.

As will be seen in the following pages, the administration of only
one cubic centimeter of the standard B. acidophilus suspension did not
suffice to maintain for more than six to eight days the characteristic
acidophilus flora established through combined B. acidophilus and lac-
tose feeding. From the experiments thus far conducted it appears as
if amounts of the suspension much smaller than two cubic centimeters
do not possess any marked transforming influence.

B. ACIDOPHILUS AND CARBOHYDRATE FEEDING

The purpose of this series of experiments was to determine the com-
bined influence of relatively small amounts of either lactose or dextrin
and suspensions of B. acidophilus which by themselves had very little or
no transforming influence on the intestinal flora. Accordingly, a num-
ber of white rats were put on the basal diet until the flora was found to
be of the usual mixed type, when they received the different test sub-
stances according to a plan involving eighteen individual rat experi-
ments.

Three of the rats were given two grams of lactose daily along with
the bread and meat ; three received two grams of dextrin each ; three, one
gram of lactose; three, one gram of dextrin; three, one gram of lactose
and one cubic centimeter of B. acidophilus suspension, and three one
gram of dextrin and one cubic centimeter of the bacterial suspension.

In all of the rats which were fed two grams of the lactose or dextrin
there was a marked simplification of the intestinal flora.* This was
demonstrated by the examinations of the fecal suspensions and of ma-
terials from the different levels of the enteric tract. In striking con-
trast to these findings, however, were the results obtained in the feeding
of one gram of each of these sugars. The small amount of lactose or
dextrin stimulated only a partial transformation, and B. acidophilus
at the best attained a maximum of only about 50 per cent of the total
bacterial population. There was no radical suppression of other types
of ordinary intestinal microorganisms, and gas formation in the Veillon
tubes, while considerably reduced, persisted throughout the experiment.
(See Tables 11 to 14, and Charts 15 to 18.)

*The tables are omitted to conserve space. The results were essentially the same
as in Tables 3 to 6.

CQ

^

8 ^

I '^dopioy

* 8

n- .'ii 8
>» 8 8

«

Etib

CO CO

"^ 'O • • • 'tl

*« >- 'O 'O ^ 5
. . o p p ,^ s

CO CO ^- •* *" «J O
O O . . . W -t
^^^ S O 8 S"

a s f^^^5i3

v< v> C S C v

C C C ,«^ CO

cS C8 C8 flH 'O

,„• .„• _ _ .„ O

W W « W W -J *"
W W W W O cS .

P p o o o a 55
o O o o o c o,

^^ *^ 4J ■*-> •♦-»

« « 4) « « 3 5

cfi en cfl cfi cfi -§ o

^ >i> u> t>» >» to >»

C C c C Cn3 a
S3 w cS cS cS o OS

s a B B 6 aB

o o c o o ^ o
-a -a T3 'O TS S T3

t> 1) OJ « « S 4)

^- J- u fci >-. £ »H

Oi a- i2, cih a.j5 f^

CO CO CO to to to
•B 'O 'C tS 'O 'C 'O

p p o o o a o
f-> u >^ t^ u ^ u

ii ti sb ib bb tib tib

0) 0^ ^ li i; a; 4)

a a a a a a a
a S S S S S S

A cO ^ c6 ^ c6 c6

t^ U ^^ t-, U U >-i

ooooooo

I U + + -1- +

+ 1 I I I I I

I U + + + +

4:t + + + i I

I I + 4- + + +

0« H* CO X O 0» ■*

«

O O O .J

So.

CO CO „ „ „

® . . .

Oh &I uj to to

_ ^ O O O « g

* 2 s a a g-^

(^ Vi b ■•-> V

fc fe, 'O 'O 'O ^ .
^ **^ a a a « "

. . c6 ce cS Ph ^
no +J +J +J "^

« « « a S

S 5 a a a-o S
• • ... be .

i § s a a a a

0 o o o o '^ o

« « u 4; 4, H 4)
^; J; »H ^< »- 2 ^H

A p- o, a, (i^ Ph

i£ i£ to CO CO CO
'C 'O 1^3 -O "B "O 'O

g g o o o a p

be si) sb ib tb CO bb
u 9j V v <u o V
a a a a a P.C

a a a a a a a

A CQ cd C(J qS 02 Co

^f-r y^ y^ ^ y^ y-,

0000000

1 U+ 1 + I

I I I 1 I u

I I I + + + +

:{:4:t + + + +

I U + + + +

cj« •* to xi o o» •*

«

O

Q

to to

O O

to to "
O O .

'O . to to

O CO fQ rg

^- -a p o

. o ^ ^
a vr s* tlO

be tl (D

" c a

a a

00

a c
^ a

a a

33 ca

Bo

g 2 E o' o o 2

^ •-I >H 4) ^ y »-

• . o

be be CO -y cr -^ to

« 4) O S 6 &i O

C a 0,0 p, « A

a a a * B^ a

cS ce cd ^ 5J 03

Sh tri t-< 13 t^ ^ '^

O O O 2 o c o

- - ^ S ^'S a

a a a

ce cs

SSS

a*^ i'*'
a a e|a| ^

o c o O o t o
'U 'C 'O 'O Ph'O

4) <U 4> -^ 4) U

M ^. >- c »- i2 ^H
a. p, o, 5 a^ a,

'o 'S *S "S *S *^ *3

u y w y u ,• y

0 o o o o S o
w o « V « g y

0 o o 5 o P« o

+J +J +J ^j 4-> _ +J

o. ci, &, d, &, S P<

V a V lu u ^ ij
t- »- ^- >H «- 2 ti

cn cK 1X1 CO cfi O cc

1 Ul 1 + I

+ 1 I I I I I

I I + + 4- + +

4:t + i + + +

1 I + + + 4- +

0» («■*■*■«}■ «5

©> t}< CO 00 o ©« -.fi

r-i

CO

H
<

O

<

o

>^

<

So

"< '^

CO

h- 1

o

Q

B

<

n

p^
o

!zi
O

H

n

l-H

P3
H
en

•■qdoptofr
•■qdopio y

8^1

m

^

%

a a

i-i

a* . a a

(3

. 3 13

0)

3 S !3 3
fi . § C S

+5

1

«

S ., v V

U t* i>

hJ

B C « V a>
e3 "^ . w o

.

§ '3 +: *J

CO

CO

« tj W W

^

"5 SPQP5

s

e8 ^

pq

4) "^ &, Ph

p<

o

CO 03 4J +J

U3 to « +J +J

i

'O'ocnco
2 g ^ ^

1

-5 J- COM

0

CO

m

O . > V V

d fl CO CO

a

. e . CO CO

CO C CO 'O 1^

^

2 goo

^ *-l L- t-

o,^; o »i fc.

&

CO CO

. CO CO

oT

rCO O O

a

cofd ss o o

£

C3 S O^Oi

•fH

o c g a,&,

a.

ce S

•M

Cl(Cd Ph

CO

..as

« « 2 2

.S3
Q

a • fi a a

cj V n b (h

>

^ ^ § g

mil

i

a ^ a a

o o o o

iiiii
Hill

'S

v ^ u v

^

^ £: ^ »4

Pi

Oc&iPiPh

u,u,%^^^

CO

CO CO CO CO

<n vi ^ xn m

'o

^tttt

'u 'O prt 'O 'O
O 5 o o o

^

3 ^ f, ^ *-

»-.»-£ »H ^1

bC

h bb si) be bb

si ti) bb ti) ti

(U

Q 4J «; 4) w
3 fl C C C

c a a a a

1) 4> flU O 4>

c

c c c c c

s

iiiii

(d

Oj C3 03 Oj

^

»-l ^ Vl »-c

^4 ^ ^ »4 >4

O

oooo

ooooo

1

+ + + +

1 + + + +

1

1 u +

1 14- + +

1

+ + + +

1 + + + +

4-

J- + + 4:

4-4- + 1:t

«o

CO fH U5 W

O CD Q 00 t-

eo

.* «5 «5 «5

s

g

a

s 1

i

1 stf

o

ii i^

0

§i3§

hsSOO

C8

3 5^ « cS O

. a a a a

a 3 3 3 3
g C S C B

C « >>>»>.

.T^ ^ ^ ^
4J g 4) (U 4)

c .

. ^ 4-> *J *J

+j y W W «
<^ ^ vt <& ^

««mmpQ

. CO CO CO o5

CO 'O "v 'O "O

'O O P O O

. OT bobChO
CQ O u u u

o PhC c a

fl i s a a

g cS e8 (3 !8
M *; »- ti Vi

2 O O O O
^ ^ ^ ^ ^

rQ « « 4, 4;

Co

bb'tSiSiSiS
Sg.P^^

•^ y L- L^ L^

g

4->

3

CO CO CO

O O O

o

PhPhP,

03

a a a

&

CO

cd s: c3

V

'C

>H k, ^

fo

E

a
p

^c c c

V

CS 03 (S

c

SSS

p<

i

a a a

•FN

O

OOO

u

'ti Ti '^

w

-o

ii V <u

O

h, u u

g

cj

PhP^Ph

4^

p^aap^a,

u

V

4) 4) 4)

h

>«

U U U

•4-1

■4->

*J ^J +J

COCOCOMCO

I I I u

4- + + + +

art

3 V ^
« C8 p

=!oci3

EXPERIMENTS WITH WHITE RATS

87

CHART 15

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a diet containing lactose
100

30
ao
10

60
50

JO

20

10

0

DieT in qram:5

AJumher ofohys offer aeimimsfr-cff^ion of- di^T-

SL 4 6 e to IZ J^

CHART 16

Graph indicating average percentages of B. acidoph-
ilus appearing in the different parts of the intes-
tine of rats fed on a diet containing lactose

too

9'0

ao

60

50
40
30
20
lO
o

V)

Oie^ in Oranns

0

Breach lO
LaCTose 1

^

I

V

^

1 1

Parts of alimentary canal U

s

Si

o

ft?

o

o
o

H
CO *-;

< ?

t^ CO

Q
H

'a

so
I «»iO

"§ I -tidoptoy
I 'tidoptay

^O,

5>58

»

CO «3

T5n3

P P tB 03 tC to 05

'O TS 'O 'O ts
• • o o o o o
g g b ^ ^ ^ ^1

&I P< 03 05 o5 o5 o5

_, _, o o o o o

2 2 s g g g g

O O 0! eS C8 cS eU
u u u u u

rr. rv. 'd 'O 'O 'O 'd

'*^ ^ c c c c c

. . cd q3 cd cd oj

?j y "O O "S ■« 'S

o o w w « y cj

§ S o o o o o

o o « w y w o

^ ^ o o o o o

Q^ Q^-*-" 4-1 4-' ■!-> *J

57 57 &( &I Oh P, eii
i; 1^ v v <u V V

rT^ rj; -u 4J +J -t-i +J

■" ^ 02 cn CO cfi CO
c C c c c c c

* * * * =8 * =8

i i s a B a s

o o o o o o o
'S 'S ■« -a -B ^3 T3

« W Hj 4) 4) 4; 4)

^ ^ h ^ h kl M

A &H o, Ph Oh a, Ch

i2 i2 to 03 V5 oj aj
'U 'C rO T3 -a ^3 'O
P g o o o o o

»-l ;h IL, )h ^H ^ ^H

ti) !ab bb !« !:£ bb t<)

4J 4J 4; 4J 4; 4J 4^

c c c c c c c

s a s s s s s

cd CO cd cd ^ cd cc

Vi b b ;i ti b »a

ooooooo

I I 4- + + + +

4- I I I I I I

I I + + + + +

t1: + + i + -l-

I I 4- + + + +

to CD to ■* CD «5 0«
1-1 rH ^ Tj< ■t}< t}" U5

0» ■* 50 CD O (S« •»fi

P3

'B O

|2

S 2

to to

o o

-d 'O

S cd

* t r-

O «

^ So

^1 '^ 'O

. c - -

03 2.-1

'2 ^yc-o

O p O^Oh

to 03

O O

&P.

e s

s s

cd ed

u u

'OO

iso

^-OScd

si) 3 o

. . O O
Q. 4) 4) &< Oh

cd 57 cd cd
§£ cSS g g

o c S o o »- »-
'O cd Q,!^ 'O Ph 0*

4) 4) y

>H to 05 ^. ^, to to

^Q 5 >-^ i-^o o
'S ^ ^H -^ -3 *H ^.

0 li) !i o o ^^

y V 4J o S 4^ lU
O B C O O C C

■M 4^-1-'

Ong g PH&Hg g

«§!««§§

coOOmmOO

1 I+ + 4- 14-

I I I I I U

I 4- + + + + +

4: + + + 4- + +

I 4- + + + + +

©» H* O CD ©< to OD
0< CO '^ ^ ^ ^ ^

0< ^ « CD O 0» •*

PQ

O

CO to

'O'O
o o

05 X „ „

&.&Htn g

SSa,2
" « -O

btfrt

OO « >.cd ^ £
^, S-O cdOtJ^*^

« 4) _

5 . c -S 'O

o o

■&£-||is|

2; fc. «-2 lU ed g

4J 4J f-J cd t. I. ►<

COW:

'C03'

S S g £ g g g

0 o gco 5 o o

'O "u «T5 _ »n 'H 'O

t J; *- c S «^ t;

to to (c CO cc »3 CO

'D 'C ^o 'O frt "B '3

P P O O o O P

»- *< C t< i; >« *H
ti) bo tx) "^ 03 bcih

V V 4; O O V V

c c c OiPhS a

s s a s a s a

cd cd cd cd cd cd cd

(h (a b b> b t-> bi

ooooooo

1 I4- + + 4- +

-^11114-1
I I + + + + +

4: + ^+ I+ +
I I + -t- + + +
Jj4: 4:^4:4:

'J' ^ C« O C5* CD .-I
C* M «5 «5 U5 ■* «>

m 'J' to CD O Ol •*

1X1

H

<

Pi

O

<

<

Pi
<

H

Sq

H d

O CL,

o
o

p— <

H

S

H

U7

•t(dopia y

a

'qdopto y

a

5 «

qq:

A

CQ

O

a a a s

S 3 3 S

. C C C C
tn

U Cl ^ i) ^

^ ■ u<> >

I o ^- ^

C ,5 w w w

^fQ Jg c3 eS

.'? . 05 CO

o o

CO CO

O O

e8 . O
4) .

;0 g

sgs

J^^

S-l .

cS

CS cS

O 2 cii£,(i
e V S -t-" -M

a c8

d ei ^

I £ s s s

O . o o o

. CO fQ fQ 'O
^ O S V V
4) &. »- fe fc

&^ a ^^'^

g CO ''J to
TS 'CO

o o o

so

be be be

<U V <u

ace

JJ ?J cS eS C6

I I+ +

+ + + + +

I I + + t

g 3

s e

m

o

s s e e

C 3 3 3 3
§ C C C C

C 4J >.>,>.

+i ^- fci >-
■M '3 lu aj «
o g,> > >

. • -M J-J -W

y y rt cs cs
;§««««

. • &l CO CO

-c^i O O
g t-iXl ^ "^

S S

s s
5o£^^

'O 'o 55 c c
c S 2 =* «

cS cS Ph _ _

_• ^ C f^ Ph

Qh O4 3 V i;

(U t-i nS t-i ti

J- »- fcl *J +->
D 4; jg eS S

s a g s s

o o 5 o o
'O 7, ts 'O Ti

41 W 5 4; 4)

CO CO a; CO CD

'O TS rrt 13 T3
O O o O O
t-i U f^ ;^ U

t^bb ££i be bb

4^ 4^ 4; 4^ V

e s c c c

a a a a a

03 CO cij ^ cS
V> h b h b

00000

+ + + + +

I U + +

4- + + + +

4-l- + 1:1:

a

11

SPi

-S s a s c

3 3? « C8 o

©

.3

3

'E

4->

4-i

X

W

V

Ctf

Q

P3

fQ

(?

O

fM

a a a
333

C 3 C

a 9 4J «

1=! ^ ca c3
^j 02 P3 P3

CS . .

CO Q^ O- Oh

2 ^ ^ ^
. £ 4J 4J

^^^^
G . . .

to to CO

a'o 'S'S
222-

'^s ae
^000

CO >»>>>.
fa. 13 c c

t> CQ CO 03
^ ^ S S

s a a a
§000

to CO CO CO
■ Frt 'O "O 'O

1 V S ^ /-s

CO bb be be

O 41 4) v

CuG a a

a a a a

^ cS cS c6 cd

^ 0000

o-a

. 3

OhC

I + + + +

1

atf

c

g

S c

3

3

1^5

83 0

>-i

-^

uo

40 . TRANSFORMATION OF THE INTESTINAL FLORA

CHART 17
Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a dextrin diet

Z^/g/ //•? groz-ri-s

BreacJ /O
Beef 3

C'e>drirt I

S. 4 6 5/0/2/4

CHART 18

Graph indicating average percentages of B. acidoph-
ilus appearing in the different parts of the intes-
tine of rats fed on a diet containing dextrin

100
30
60

70
60

SO
40
30

20

10
0

Diisrf- in qroms

<3

Brcod JO,
Beef J.

•^

■

>

1

^

1 1 1

Parts of alimentary canal

s

SI

o

o
O

EXPERIMENTS WITH WHITE RATS

41

When, however, the diets furnishing only one gram of lactose or dex-
trin were reinforced with one cubic centimeter of the standard suspen-
sion of B. acidophilus, a very marked transformation of the intestinal
flora from the mixed type to one completely dominated by B. acidoph-
ilus was effected. Within six to eight days after the administration of
these diets this organism constituted 90 to 99 per cent of the total flora.
The Veillon tubes contained numerous colonies of B. acidophilus, and
no gas, while the stained films revealed a picture of practically all Gram-
positive acidophilus-like bacilli. (See Tables 15 to 18, and Charts
19 to 22.)

The following experiments which extended over a period of thirty
days were undertaken to determine whether the ingestion of one cubic
centimeter of B. acidophilus suspension alone would maintain the sim-
plified character of a flora already established by the feeding of two
grams of lactose or dextrin, or the above-mentioned combination of
sugar and B. acidophilus suspension. Nine rats which had been on the
basic diet for five days and had developed the typical mixed flora were
divided into three lots of three each. The first lot was given two grams
of lactose daily ; the second, one gram of lactose and one cubic centi-
meter of the bacterial suspension ; and the third, two cubic centimeters

CHART 19

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a diet containing lactose and
B. acidophilus

C>/c/- m grams
/OO Bread lo . OGeiS, Laci-o-sG J
S-acidojohi/c/s 1 c.

So

70
60

s50

40

20

ire.MS'O'4 Ift S^

z A e e /o /s 14

I-H

CO

o

Q

o

<

m

Q
;?;

en
O
H
O
<

o

8 ^J

6

o

l-H

^

<u

c

«

CO

.s

2

c

s

.2

_Ci

'3

1

e

0)

«

a.

a.

03

1

9
to

QQ

N-^

K.a

to

e

^

u

la

o

g

s

o

'S

•t.

<1

J

n

s

S

c8

'«

u

so

be

8

I-H

V

°Q

CO

O

g

■»->

1-1

^

00

«H

u

V

n

o'

I— 1

13

C8

V

»H

1

1

■4->

•xidopioy
•ff*

5

svo

'Udoppy

a'

svo

'Udopp f>

a'

svo

-i e

?!,§

cq-§^V

•s* *-

to ®

^fi.

fc e-S

« i» •»

Days aft

Initial

dminist

ion of D

■5

- T!3«

tf

Tj

c/>

O TS

2

CO

O

fir

l-l<

V

F1

a

O

B
a

O

^-V/-N

/-v^^/'^v

73

v u

V V V

t!

^ J<! Jd J<-i<!

et

C

1 1

1 1 1

J3 J= J3^X

(•>

•^K

p,a,(i,a<a,

O

O O

o o o

u

S

TS 'O'C'C'C

t;

u u

www

a

O

<<^<;«ji<j

^

a.

^^^

^v.^^-^

4->

f.

ce 09

CO w cc

•i-i

-CB 'O'O'a

^

T

o o

o o o

^ f-l

ert

;s

V o

www

S

1^

> >

> > >

4J+J

-M -M -M

CO 03 CO

S p

o o

O O O

o

o

&0,

o-aciH

'S

S S S S 6

Ih

ce c8

ce ce ce

a,&

f- p

»H tl t,

r/1

ooooo

TS'O

1 ^^

,—fr^^^

£

O

cS cS

ce ce ce

>>>.>-.>^>>

s

S

c8 c6

ce ce es

W W

www

6

•4-> •*->

■M 4-> -M
WWW

C8

?

ce ce

>-<

u u

^ u u

oopHa<Pk^pH

CD ©» OS «0 b- 0> «5
CO 03 0> OS 0> 0> OS

o* •* to cc o 0< -*

m

P5

CO

O .
ti CO

03 O

o ^

Oh .

rJ^ ^ W W U W 4)
(^ rC J«! Jr! J«! J« J«!

a*3 &I Oi Ch Ch &,
g^y O O O O O
^^ O "O 'O 'C 'O '3
•S y "w 'w "w *W 'w

1 3 <: <j <3 <i <!

>" CO 03 09 to 05
•n rS 'O "W "O 'O tJ

w cn o c o o o

g ^, >« ^H >H >,

u r V V V w w

£ fa .>; •>; > > >

&I .-S .ti .ti .ti .ti

W . to CO CO CO 03

J- g O O O O O

^ § aH&c-G,&,

ns'^BSBBB
§^22222

„„ooooo

T3 TS -3 -H j-j —. j2

2 g'ee'rt'ee eele

ti) Oi jb^jb;^;^

« 2 "3 "ce "S "3 13
^.w _w _w _w ^w

Se ^ ^ '^ +3 *j
c w w w w w
ce ce ce ce ce ce ce

tH U IM I-, u t^ t^

O O Pi Ph Ph Oh PU

o 50 » OS ^ oe •*

GO CO OS OS OS OS OS

0» ^ <0 CD O ©* 'J'

pq

be

P3

T)

09

2

1

en

0

?w

A

g

fl

ce

Hi

m

(*)

u

, a

, ,

0

^^^•■V

,^v

^^''■N

w w

w

u w

c8 C

Jd^^ Jd J^

j: js^jsx

r>

a,&&.&,a.

n

f-

0 0 '

0

0 0

w

0

TS-CCt) 'O

i!

w
0

w w

w

w'w

a

<<3<j<j*sj

u<

w

*^

b

09 03

CO

CO 00

*^

•^ r^ r^ r^ r^

.

Jj

0 0

0

0 0

1*

1

b

h

h b

W V

w

u w

S

> >

>

> >

•M ■<->

-M

•4-1 ••->

to

0; M

g B

0 0

0

0 0

0

n

a.&&.&a4

w a>

S E

C

s s

u

h

a ce

ce

ce ce

pL,CL

k. (<

u

ti u

03

00000

'^'S

S S

^

;=a

2

C

ce ce

le

ce ce

>.>.

>>>.>»

0

c

ce ce

e«

at ce

w w

W

w w

a

w w

w

w w

ce

es

0^ «

ce

ce ce

b

0 0 Ph Pk Ph pL, Pk

I I I I I I I

i 1 1 1 1 1 1
+ 1 1 1 1 1 1

b- O OD CD OS OS OS
00 OS OS OS OS OS OS

G< •* to 00 O 0< 'J'

to

H
«U

O

<
o

>^
(Si
<

& CO

"^

^ us

H d

X ^

CO ^

do

K Q

Oh O

Q fl.

<

n

o

^2;

o

t—t

H

pq

I— <

Pi

H

CO

•ildopp y

d

SVQ

S I 'qdoptoy

•ff

8VO

°5S

PQ

ce CO CO 03

3 3 3 S

O O O O

»M ». tl h

U V V V

s s s a

3 3 3 3

a a a a

C

bbb

o

■|

>

>

>

c

cd

.5

.s

a

."

w

eg

'E

'E

*E

*E

V

V

V

t3

1

fQ

(Q P3 n

.— s «j (U « «

=! ^ 7 1 1

&10 o O o

•2 "S "w "S «

(O CO CO CO
O ^H Vl >H >H

4, t) « (U

> > > > >
■-3 i*' is 'm is

•S 'So 'co to 'co

g O O O O

a s a s s

2 2 2 2 2

ooooo

cO c8 cd cd ^

h U bi be b

Oh Ph Qh PHP^

tfJJf
I I I I I

tJJJJ
I I I M

m

CO <0 CO CO

3 3 3 3

■ O O O O

CO u b b< (^

3 V V V V

s a a s a

« 3 3 3 3

3 e C S C

.•S.-Sbb

« « « «

"^ J: Jj J3 js

f^o o ° o

O fQ rQ ^ 'O

'S 'S '5 *w '"

^^^<:^

ooooo

OS

c3 eS cij 03

.XX

^ ^ CO c6 cd

O V V (J V

+j +j '+j ^ +3

u u u w u

cd cC CO c6 cd

^H tH U ^ kl

I I

I I

a

g 3

'i 3 S

O 3 —

CI

atf

CO O

«

P5

be

P5

PQ

CO CO <o CO

3 3 3 3

,/ O O O O

^ ^ IL, (^ bi

3 i; u V 4>

2 a a a a

« 3 3 3 3
S C C 3 3

c.^bbb

* T" 't< 'E *E

V V V

4j

V

o-S o

^ y « w o

O rt * c8 C8

*Qgp5 wpg

^ "Y T 1 T

&I o o o o

,2 'O IS !2 2
3 '3 '3 '0 'w

ui CO CO CO

i2 'O 'O 'O "O
'5 o o o o
^ u u u u

., V V V u

> > .£; .i .t

"2 '+3 |*j 4-" *j

•^ *co to CO m

5S o o o o

a a a a a

2 2 2 2 2
ooooo

cO cQ ^ CO cd

xxxxx

tH ^ tH tH >-<

P^P^PhPhPui

O M 0> 05 0>
Oi 9) 0> O) 0>

3 3 1)

gi art
'5 s a s =!

S 0? « c8 O

44 TRANSFORMATION OF THE INTESTINAL FLORA

CHART 20

Graph indicating average percentages of B. acidoph-
ilus appearing in the different parts of the intes-
tine of rats fed on a diet containing dextrin
and B. acidophilus

1

-<>

1

0

^

li^

loo

V

90

>,

^

t)

eo

v^

1o

«

60

Dteti.

/n qrarns

'-^

Bread /o

J3. acidotJ7i/vs J C C

Parts of alimentary canal

8

05

of the standard suspension for the first two weeks, followed by one
cubic centimeter of the same suspension during the remainder of the
experiment.

The results obtained with the different rats during the first fourteen
days were very consistent and essentially the same as in previous ex-
periments with the special flora-simplifying diets, B. acidophilus becom-
ing the dominant organism and remaining so to the end of the fourteen
days' period. At the close of this period one cubic centimeter was sub-
stituted for the two cubic centimeters of B. acidophilus suspension ad-
ministered to the third set of rats, and this plan followed for the remain-
ing sixteen days of the experiment. The diet of the other rats remained
unchanged.

The daily administration of one cubic centimeter of B. acidophilus
tended to preserve the simple character of the intestinal population for
six to eight days, after which a somewhat sudden reversion to the usual
mixed flora took place. (See Chart 23.) On the other hand, the flora
of the other six rats remained simplified and completely dominated by B.
acidophilus. These results lead to the conclusion that in the white rat

m

o

52;

<!
H

iz;
o
o

H

i

iH

^

^■^

V

J§

Ct3

(0

_n

g

13

0

•S

CO

1

g

fi

9

9

at

QQ

t">4

03

Q

0

«)

IS

g

0
2

0

4:.

<5

j

n

S

s

c3

'S

u

«

be

rH

e

-M

•S

QQ

^

^

CO

n

0

I— 1

I

'ildoTpioy
•ff'

SVQ

•ildopti

»F

a'

svo

•qdopti

»F

s'

SVQ

rfs e

?i,«

«-• ® c5

s~.

CQ'« 7

0

•e» V.

u «

1

HO

h s

i»

^ »>

•«*

c^

Days a

Initi

A dmini

0
8

••a

1s

■W

Pi

O ^ 5 "^ *3 &>^

« ^ .^ « 4> «
CS >-i kT O

•s «.-

5 o S '3 £ «« o

>»>> CO go S
>§ S r^ £ ?^ ^ £

s s B a-^ul

« V « r, o li 5

05

'^ S P p p g *

si) M 03 o3 05 03 —3

« 2 2 P P 2 *
o a.P<acaH&io

a a a s s a "-g

0} cj ti cS c6 03 q3

>4 »H ^1 >^ ^ >4 |L|

OOOOOOP4

+ 1 I I I I

«© G« CD »C ■* to 0>
CO t- 00 00 O) 0> OS

0* •* «0 CD O G> ■*

m

«

£

■?

o

a«

>H CO 03 ^ S

g H flO ti

>. 2 g -d . .

. ;S a Oh J. I

-^ - " « te J- _e

p g' p,f^ >■ >-'C'

CS ■<->

.-,c«

§a

'O fO

Pom

li « a '-->J^ P p

iaj^^ao,
'««|2gia

to 03i2i5i5^^
rrt rrt 'O '2 ^a ^ ^

ti) 03 03 w 03 :^:§*
« 2 2 S 219 "rt

a a a a a t; tj

ct3 c(3 cd ce3 c9 cS cS

M ILi ^ ^ ^ fH M

OOOOOPMpLi

*+ 1 1 I I I

•* 05 to eo OS t- 05

Tfi to CD 0> OS 03 OS

CS* •* to 00 o cyi •<?

fQ

"tj 03
CO
»-. O

be

a a

bp2

So

_, «5

^^'

g a

4) fQ O W S i^ Si

p o -^ -Si ^ra ,2

S o cc 'O Is ^3

+J ■4-> 03 to to

Ji222

• • .. h^ w to '3

53 fl >» >» p P p
i § M-bi) ft ft ft

(u m p P g g H
^^ f^ u fl ei cs as

« to aj M^'-'^

'O "O r^ ra -H ^ j2

2 2 g S «3 « «»

bo to M 03 ~ S S
S Q.2 2 « « «

fl fii &< Ph « y w

a a a a « « «

03 ^ c9 Co cd cd q8
u ti 1^ u u u ^^

OOOOPnPkPk

*+l

©» O ff> OS CD Oi CO

00 t* OS OS OS OS OT

01 ■* .r 00 O ©« ^

46 TRANSFORMATION OF THE INTESTINAL FLORA

at least the ingestion of one cubic centimeter of B. acidophilus suspen-
sion is insufficient to stabilize and maintain a simplified flora of the
purely aciduric type, although two cubic centimeters of the bacterial
suspension when fed daily are able, not only to bring about a complete
transformation, but also to preserve the acidophilus picture indefinitely,
that is as long as the suspension is administered. However, one cubic
centimeter of the B. acidophilus suspension, which in itself exerts little
or no transforming influence, becomes very effective in the course of but
a few days after it is reinforced with one gram of lactose or dextrin.
The same results are obtained, of course, when one cubic centimeter of
the acidophilus suspension is added daily to the diet containing one
gram of lactose or dextrin which in itself brings about only partial
transformation.

The conclusions arrived at thus far regarding the transforming in-
fluence of lactose, dextrin and B. acidophilus, when given in sufficient
amounts, alone or in combination, were further corroborated by the
following experiments. Nine rats harboring the usual mixed flora were
divided into three groups of three each. The first group received two
grams of dextrin; the second, two cubic centimeters of B. acidophilus
suspension ; and the third, one gram of dextrin and one cubic centimeter
of the bacterial suspension. The results were quite uniform throughout

CHART 21

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a diet containing dextrin and
B. acidophilus

90

GO
70

60

60
40

20

10

Diei in gram's

Bread /O

Beef J

Dexirm I

3. Qcjdophi/tis Ice.

(SusretsioM It Jaunty

/\lumbcrofdqys qf^rocfmmis^mr/on 0/ c//e^.

to

JZ

/4

en

H
<

o

<

<
o

>^
<

< ^

Q

»— (

o

Q
I— (

o

«

o

o
»— (

H

PQ

Pi

H

CO

•■qdopto y

a

gr>o

§ I •tidopioy
* ff'

^7

*ȣ)

^ft.

n

n

CO CO CO

3 S fl .

O O O CO

^ ^ ^ ^

V V V o
B 9 5 a;

3 3 S a

a c c g

V V V >>

> > > Jt

«

be U OJ « ^

^ ^^ J^w

t O O O Oh

O "B 'O 'C o
'S 'D "O S

CO CO CO

. '2 'C 'O M
S p o o 'O

'O 4; 0* 4)

■« "- "- •*->

CO CO CO •»

c o o ss

a s s s

2222
OOOo

g j3 s ce « cj

• 3 >>>.>.>.

g C 3 S S 3
(-1 . > cj o3 rt rt

CO o

^ w y « «
03 ^ ^ 03 cd

O PhPliPhPu,

0> O) A Cj*D

00 a> 9) o>

s c
o

4J « 03 O
HsSOO

W

bbb

cd cs

CQ

OO PlfLi^l

II III

a

3

So

bbb

<«

looo

^+^ s « s

^ Vi b

I I

1 1 i

^ Q ^^

SUCJ

48 TRANSFORMATION OF THE INTESTINAL FLORA

CHART 22

Graph indicating average percentages of B. acidoph-
ilus appearing in the different parts of the intes-
tine of rats fed on a diet containing lactose
and B. acidophilus

JOO
90

3d

JO
60
30

f

0

Breaol /O

Beef O

Dextrin 1

B. oc/dop/}//u<s Ice

Parts of alimentary canal

s

8
O

s

e
O

a
o

O

CHART 23

Curve indicating average percentages of B. acidoph-
ilus appearing in fecal specimens from rats
fed on a diet containing B. acidophilus

I
/oo

Dief- momnrs

Brood /O

See/ v3

Bread /o
Beef i

\Bacidomlos Ic.c.

/dumber ofdoeis offer odmi/^/:s/n:/^ort afeJis.-^

S. 4

to IZ 14 lb la So S2 Z4 Zb £8 JO

EXPERIMENTS WITH WHITE RATS 49

the experiment. The transformation of the flora was practically com-
plete in each of the three groups of rats, and B. acidophilus was present
in the fecal specimens to such an extent as to almost completely exclude
all other types. The examination of the different sections of the intes-
tine showed the presence of B. acidophilus throughout the length of the
digestive tube, and in relatively large numbers.*

B. ACIDOPHILUS VERSUS B. BULGARICUS FEEDING

The methods of preparing and administering the suspensions of living
organisms in these experiments were identical with those employed in
the preceding work. The five strains of B. bulgaricus which were used
were isolated from commercial preparations placed on the market by
five different laboratories. The strains of B. acidophilus were the same
as those employed in the previous experiments.

Since two cubic centimeters of the standard suspension of B. acidoph-
ilus, as well as one cubic centimeter of the same suspension reinforced
with one gram of lactose, were found to effect profound transformation
in the flora of the albino rat, parallel sets of experiments were set up in
which the administration of B. bulgaricus and attempts to recover this
organism from the feces constituted part of the plan.

As it is now a well-known fact that colony formation, morphology,
staining properties, etc., are unsatisfactory bases for distinguishing
between B. acidophilus and B. bulgaricus, and that even the milk acidity
test is not always dependable, the fermentation test of Rahe (1914)
was employed. The pure strains of these two organisms were inoculated
into maltose broth adjusted to pH 6.8, and incubated at 37° C. for
forty-eight to ninety-six hours. The maltose solution and the broth
were sterilized separately. None of the five laboratory strains of
B. bulgaricus were able to attack the maltose with acid production,
while all of the many strains on hand of B. acidophilus fermented the
sugar with sufficient production of acid to be readily demonstrable.
This method of distinguishing between the two types of aciduric bac-
teria has been of very material assistance in the carrying on of this
phase of the investigation.

In all of the experiments in which B. bulgaricus was administered its
ingestion had absolutely no influence on the character of the intestinal
flora. At no time during the entire investigation was B. bulgaricus
recovered from the feces, regardless of whether the suspension was fed
alone or in the presence of lactose as an energy-supplying pabulum. In
all of the animals receiving the bulgaricus suspension alone the usual
mixed flora prevailed to the end of the feeding experiment. Upon the

*For a complete record of results the reader is referred to the doctorate thesis of
H. A. Cheplin in the Yale University Library.

50 TRANSFORMATION OF THE INTESTINAL FLORA

ingestion of one gram of lactose in connection with one cubic centimeter
of the bacterial suspension there was an increase in the number of
aciduric bacteria in the same way and to the same extent as in the
experiments in which one gram of lactose or dextrin alone was fed.
The aciduric organisms were found to be B. acidophilus, and not
B. hvlgaricus. (See Charts 24 and 25.) The characteristic fluffy
colonies were picked off the whey agar plates and inoculated into the
maltose broth.

The stimulation of the proliferation of B. acidophilus, and not of
B. hulgaricus, as the result of the ingestion of one gram of lactose and
one cubic centimeter of the standard bulgaricus bacillus suspension, is
a clear indication that B. hulgaricus is incapable of adapting itself to
the intestinal environment of the white rat. The intestinal flora of the
control rats receiving two cubic centimeters of B. acidophilus suspen-
sion or one cubic centimeter of the suspension plus one gram of lactose
was completely transformed from the complex to the simple aciduric

In order to determine whether any of the ingested bulgaricus bacilli
survive for any length of time in the intestine, the test animals were
killed twenty-four hours after the last feeding of B. hvlgaricus material,
and bacteriological examinations of the different sections of the intes-
tine made. It became quite apparent that B. hulgaricus did not sur-
vive in any portion of the enteric tract, and the results were the same as
when one gram of lactose or dextrin alone was fed.

CHART 24

Curves indicating average percentages of B. hul-
garicus and B. acidophilus appearing in fecal
specimens from rats fed on a diet con-
taining lactose and B. bulgaricus

6o

40
JO
SO
lO

Dief

\ ^roms.

BreQ<i /O

c LacTos^ /

fn e.bulooncus ICC

2 4 e O /O 12 14

EXPERIMENTS WITH WHITE RATS

61

CHART 25

Graphs indicating average percentages of B. bul-
garicus and B. acidophilus appearing in the
different portions of the intestine of rats
fed on a diet containing lac-
tose and B. bulgaricus

too
so

eo
70
60

50
40
JO
20

/o

D,-ef

1 qmr.

Bread lo.

Beef J.

Locrose I.

B- acic/otJii/as I
3- bvlooricos u

— H-" — tt^ H-" H-

Parts of alimentary canal

i

s

s

05

In the following experiments larger amounts of B. bulgaricus sus-
pensions were employed than heretofore, namely two to five cubic centi-
meters. When these amounts were administered to rats daily without
any accompanying carbohydrates no change whatever could be ob-
served in the character of the intestinal flora, and B. bulgaricus failed
to appear in the feces.

Nine rats which had been on the basal diet for seven days were divided
into three groups of three each. One of these groups received two cubic
centimeters of the standard suspension of B. bulgaricus ; another five
cubic centimeters of the same material, while the remaining three rats
were given two cubic centimeters of the living suspension together with
two grams of lactose. The results were similar to those obtained in the
preceding experiments. The fecal flora of the first two groups of rats
remained apparently unchanged. The ingestion of even five cubic centi-
meters of B. bulgaricus suspension exercised no transforming influence

52 TRANSFORMATION OF THE INTESTINAL FLORA

whatever. (See Tables 19 and 20, and Charts 26 and 27.) In both the
plates and the Veillon tubes essentially the same colony picture was
obtained as in the preliminary tests when the rats were reciving the
basal diet alone. Gas formation in the Veillon tubes was very pro-
nounced at all times. There was some increase in the relative number
of Gram-positive rods in the films prepared from the fecal suspensions
directly, due in all probability to the passage through the alimentary
canal of dead organisms of the B. bulgaricus type.

It was impossible, also, to recover B. bulgaricus from the feces of the
rats that were fed two cubic centimeters of the living bulgaricus sus-
pension together with two grams of the lactose. However, the intestinal
flora of these animals was thoroughly simplified, the transformation
effect being indistinguishable from that obtained with the feeding of
two grams of lactose alone.

Post-mortem examinations, made twenty-four hours after the last
feeding, failed to reveal the presence of B. bulgaricus in the different
parts of the digestive tube. (See Table 20.) Not one of the nine rats
receiving the suspensions of B. bulgaricus showed any evidence of this
organism in any portion of the intestine. On the other hand, all of
the animals which received two grams of lactose in connection with two
cubic centimeters of the bulgaricus suspension harbored a flora in the
feces and at the various levels of the intestinal canal which was wholly
dominated by B. acidophilus. (See Tables 21 and 22, and Charts 28
and 29.)

The foregoing results corroborate the earlier findings of Hull and
Rettger and in themselves most conclusively demonstrate that B. bul-
garicus cannot be implanted in the intestine of the white rat even when
administered in enormous numbers. The decisive results obtained with
B. acidophilus proved beyond a doubt its adaptability to intestinal
conditions and its capability of implantation within the intestinal tract
of the albino rat.

SLOW CARBOHYDRATE ABSORPTION IN THE INTESTINE

AND ITS RELATION TO B. ACIDOPHILUS

IMPLANTATION

Of the various carbohydrates fed to white rats lactose and dextrin
alone exerted a simplifying action on the intestinal flora, bringing B.
acidophilus into great prominence. Since this organism is a normal
inhabitant of the lower intestine, it was thought that perhaps both lac-
tose and dextrin, in virtue of their slow absorption, reach the colon
where they provide optimum cultural and environmental conditions by
serving as readily utilizable sources of energy for B. acidophilus and
the closely related B. bifidus of Tissier. According to this theory, the

^1

u

w

*o

•s

V

a

a

ea

«>

fl

1

a

.2

i

s

Si4

Q*

CO

QQ

CO

»^

«3

CO

u

^

»

U

Bs

c3

g

^

9

•c.

«

a.

.

J

«

s;

1

ts

§

«

u

.e

bC

§

CO

CQ

fi

V

(J

PQ

i.

Cb

o

1— (
T3

PQ

i

Q

•i(dopii

V

•a

8VO

•tldoppy

S

svo

■tldopiz

V

a

svo

^■§

Si<»

• o t>

«*^

05 :§^

o

u »

^Oh

•id

h "3

«

<» s.

•••

Days aft

Initial

A dminist

•2

5

CO CO CO ^ ,

'O 'O '^ CO CO CO , •

O O O rrt t3 TJ ij

^, M >- o O O^

. . . ^H M M O

CO CO to "

g g 2 w "5 CO ^

g 2 s a a a a

m « iuts -d ^3 rrt
fefe&ii fl g S a

eS w w ej
- o 3

O o

g w

« 0 -

O o

o o o
. • +-> +J -<->. ^

CL, A Pm &, p< A a,
« « « ST <u lu 4)
^ b M £ >-i ;-i ^H

+-> +J -t-> ^ 4J •)-> ^

CD cn M M CO cc cn

>>> t>^ >> X **> >» >-.

c e c c*c c fl

Cd cd cd ert Co ^ ^

a a a a a a a

o o o o O o o
'O ^O 'B 'o tj xi 'O

" &, p, o^ &< &H a, a*

CO CO CO CO CO CO CO

T3 TJ 'T3 "^ "d 'd 'd
O O O P O O O
fn ^H Vi ^< tn ^4 M

bb sb Sab si bb bb tJD

4) 4J qj 1> 0^ U 1)

c a a a a a a

a a a a a a a

^ cS ^ 03 ^ 03 cj

M |L| ^ ^ tH (-1 M

ooooooo

M I I I I I

I I I I I i I
I I I I I I I

o o o o o o o

G* 'Ji W 00 O G^ >*

W

«

CO

. . ro

JS i2 O CO CO CO CO

2 g . o p o p

" " CO M tH >H ;<

§ § ftco c/S g CO

^^^aaa^E,

a a g a a a a
2 2o § § 2 g

cS c8 » eS

^\^

-• § y y «

u y a* ;
o o y
y y

o '

55-51.1

g "

^ w o o

y y g y

- o o

y y i 2J 2i 2i 2i

^ Fh Cd ^^ ^J ^^ .^

s. >>^y >>>>>>>>
c c S fl s S o

a a -^ a a a a

-S 'S — 'O 'O 7, TS

S ajT3 y y y y
Ji M c t^ »- t *-

P, P, CS Dh Ch Ph Oh

CO CO CO CO CO CO CO
rrt rrt 1^ ifl 'O 'O 'a

5 O O O O ? O
C M >H M ^^ »- I-.

bb tb bb bb bb bb fcb

4) gj Qj 1) 4> 13 D

c c s d a c p

a a a a a a a

^ ^ o3 o3 ^ o3 rt
^ M ;li »4 ^ >H 1^

OOOOOOO

tt + tt + l:

o o o o o o o

0» •* to QD O 0» -H*

CO CO CO CO

'O 'O 'O tJ

CO

o p p O
^< ^< »-< ^

2^1

CO CO CO CO

CO 2 ^

O O O O

ftco g

Pi Pi Ph P,

a a a a

i Gram
Gram po
Gram p

^ cQ ^ cd
>-i ;h b b

0C500

^ ^ ^ ^

y y y y

it%

y y y y o -ir! y

y y y y o S^ y

o o o o w g 5

y y y y o 2 y

o o o o +5 g o

PhPiPhP,

y

-i->

Pi

y a) y y

tq

Pi

y

U U t-i u

■4->

y

^

+J 4-> -l-> 4J

CO

c3

u

^.j

fl c a c

W
^

CO

cS c8 c3 cS

S

03

SS^S

03

S

§931

a

o

a a

o o o o

o

o

T3 t3 ^ 'O

'O

tS'O

y (u y y

y

y

;-l ^ (H P

u

u

P< P, Pi p<

&I

ft A

CO CO CO CO

CO

CO

CO

T3 "TU "^ 'd

T!

rCO

O O O O

O

O

O

^1 t< ^H f-i

P-i

;h

^

bb bb bb bb sij bb ti

y v y y

OJ

y

y

pace

a

a

a

a a a a a a a

^ c8 cj c^ o3 c0 cd

tH ^4 M ^ (q »4 (H

+ 11 + 1:1: + +

o o o o O o o

CJ» •* 50 00 O G* 'i«

o
pq

Qi
o

o

><
<

Kg

1^

H
S

fe

CO

P

fe

o

O

l-H

tf Q

<!

O

O

1— I

CM

«

Em

O

a
a

8VO

85 2

i S S E S
0 0 s s s

0
0

•o

S3fe«fi«

«5

c

3 3 41 4) «
C G > > >

.s

«

•4-> V • • •

«

to

g .-S -5 -S -5

CO

c

•^ 3 es es 03

c

""

rt O'CQ CQ PQ

c

'*» . ;

C

.2

D -4-; . . .

43 y CO CO to

.2

'cO

0 S 'O 'O 'O

'co

c

eS CO 0 0 0

C

fq^ u u u

I

CO

. M CO CO CO

^

to 1^3 0 0 0

3

w

rQ 0 &< &l &

2 . s s a

CO

to

CO

=1

. 5S c8 c8 c8

3

_«

CO 0 (4 (h h

«

'E

0 OhooO

•E

U)
3

rt B 'O 'O 'O

S * c C C

3

P3

C8 »- c6 c6 c8

«

n

73 (U « «
'y C ti fci >H

n

g=««^5?

CO

s

to

i

ti)tf >.>>>»

(h

g^« c c c

u

W)

j; R 03 rt *

bO

Vi

cs S* . . .

CO

<«H

<IH

4J

55111

pq

CQ

^ ^'^'2'S

•>

•^

o

£ (U ;h >-, M

0

I— 1

Pt^fe OhO^A

I-H

TS

_ CO CO CO

ts

etf

e B 0 0 0

c6

1

0 0 >- ^ ^H

'^'S • • •
^ « be be be

«

4L

15

S

2: 2i cs c3 cs
aicnOOO

Mill

+ + 1:JJ
II 1 1 1

00000

S

a 1

5

(W

U s«

•*

'B.iiy

td

3 ^4) o5 0

09

P5

QhsSUO

Pi

s a a

C d 3 3 3
g g C C C

"= *= bbb

4-> V V V a>
2 ."t! > > >
"53...
. cr'-*-' ■<-' -H
+j « u o
y jj eS 03 eg

j|«npQP3

CQ . . .

. CO to to

CO , TIS T3 'C
'O CO O O O
O rrt t-c »- »-l
'^ O . . .

. ^< CO CO to
CO .000
O CO &c &, C
p4 o

- f^a a a
i E 2 2 2
± gooo

'^^ S c S

C '2 c8 03 oj
d, • 4) a> «

2 4) -t-" 4J 4->

«2 >» >. >.
dj ^ ^ cQ 03

^ s s a s
05000

■13-0 ^3 'O 73
« 5 « (U v
u, jr u u ^

CO CO "^ <" "3

^O rQ 'O 73 "O
O o O O O

ii) bbi^b£ bb

y ij d^ d> 4^

c c c s c

a a a a a

c6 eg ^ rt 03

»M l> ^ »H (^

I I

00000

Stf

3

(U'JU OS p

lOO

PQ

be

PQ

«

. ^. a a a

I I c c c

•M *J t> 4) 4>

*» 4J « « y
W « C8 e8 cfi

, . CO CO aJ

CO m t3 'O -3
'B'O O O o

0 0

^ (-.

u u

CO CO

CO to

0 0

i

0 0

&&I

a a

a a

a
2

cs eS

b h

00

00

0

73 'O

tS'C

C c

c c

ce c3

cS as

. .

dp.

4)

&<&i

4, <u

41 V

h u

^

u u

■^ -t;

cocc

03

^ ^

B C

^

V V

ce c6

ce

fefmiss

s

a a a a a

0 0

0 0

0

'O TJ "O 'O "B

V V

V 4)

V

b ^

b ^

;^

Ph &! CI. C^ &l

to CO

to to

to

'O 'O 'O 'O 'O

0 0

0 0

0

M ;h

u u

^

be be be ti) 60

v 41

4) V

V

c c

c c

G

a E a a

e

cS OS

ce cS

3

hi u

^ u

b

00000

I II II

1 1 1 1 1

00000

« 2
- 0.2.

Eti

P 3 e
3^0

4J aj 9
S 00

EXPERIMENTS WITH WHITE RATS

66

CHART 26

Curve indicating average percentages of B. bul-

garicus appearing in fecal specimens from rats

fed on a diet containing B. bulgaricus

D/CT in grams

30

20

10

0

0

4

Brvod lO.

Beef J.

3-6tj/^ricus Sec

Alumhcr of- days of^ cxsfrrnnis^a'/fon e^ o/iis^.

2 4 6 8 /O /2 /4

CHART 27

Graph indicating average percentages of B. bul-
garicus appearing in the different parts of the
intestine of rats fed on a diet contain-
ing B. bulgaricus

30
SO
/O

0

.0

V

Bread JO.

Be^f J.

Bholparicus So.C.

-H-

-H-

M-

Parts of alimentary canal

a
a

<3

05
o

CO

o

I— (

Pi

n
n

Q

O

o

o

CO

Q

m

en
H

SI.

09

a

1

e

5 I 'iidoptoy
' J '^

i I «"o

"■ I "udopioy

SVQ

cqts

20'

^fts

n

t3

CO

2

O

U

u3

o

bb

s

S

2

g

o

2

^

o

i "^ 'Q f^ "^

^ (U V V V V

,„. « JM ^ jid jg jg

+j o o o o o o
P^qJ^ 'O 'O 'O 'O

JJ 4) ■« *3 *S '0 '0

>» !^ 03 CO 03 03 05

'2^ a a s s s

C J- eS cS c8 e8 ee
p: CL Ih »4 ;h ;h ;-(

^ ^O o o o o

O g cS CS cS cS c8

jf g a a :=) ;=! a

« S^ ee c6 as cS cS
C Ph O o CJ o CJ

CO CO CO CO CO

O O O O O
&4 Ph Ph (^ &i

y y « y w

a a ^ ^ _ .

cs 5* 5 5 5 03 rt

(h M ^4 »4 ;H »4 »H

O O (X, Oh (li d, Cu

o o o o o o o

-I. I I I M I

+ 4. I I I I I

f+l I I I I

©t tP «0 CD O CS< ^

n

PQ

03

O 03

. y o

OT y )h

2 o .
fty fcb

S9 y

" ?i S

•^ fc! '2 ^-^-A^

Ph C y y y y

03 ^ ^ ^ jd

y p-j .„ ;3 :q ;q ;:?

y c3 y I. I. I. I,

o s y ^ ^ ^ ^

^ *^ X o o o o
fl,4-> +j 'O 'O 'O 'O

tJ O y*'^ '^ '^ '^

55 -2 £ ^ *^ *^ "^J

>» CO CO CO CO CO

C '5 fe fQ "d •« 'a

2 2 ^ ? 9 ? 9

"^ ?* qj v-* \-^ w w

S' S e o o o o

-S i'S a a a a

F g S:^ c8 es 03 C8

»o f^o o o o

CO _ CO

g g g es cS c8 c8

... .>>>>>>>,
fee fcC oj jH j^ j-j j-j

Ji JJ 9 CS 03 C3 03
C C P. y u y y

a a a y y y y
c(3 cd ^ ctf 03 03 cd
ti ^^ ^^ ^^ u u u

OOO&HO-iaHaH

o © o © © o o

I I I I I I I

t-tl I M I
Jt-I--!- I I I

«o «o ®( O I

CO ^ 03 05 I

cs* ■* <o CO © m ><}<

ti

P5

»

'O y be

o o '.^ y

^- y y C

. o y

c c/3 y

CO >-• >

r^ 03 * y ^^^

'O «5 f> 4) u 1)

O fQ

' .„• 3 3 a

y '. •

^ c p , o P o

Ph S "^ +j 'O "O 'O
g « a g^ y y 'B

>> £> . 03 M 03

03 * y y o o o

s ;^ fe fo ^ »^ ^_

. . . CO 03 03

a a a a ^p^p,
ggggiig

Ol 03 w M ^ ^ ^

rQ rQ rQ rQ ^ j_j j^

O O o O es ca eS

^K *H ^H I- ** " "

bbtico B3 3*f:2*a'

« « O ° «! 03 C8
S C Pi Pi y y y

a a a a y y y

^ ^ cQ 03 03 c8 Gd

Ih h b »H h «H »H

OOOOPiCI^Ph

o o © © © o ©

I I I I I I I
t+ I I I I I

■* CS< CO O «0 OJ ■«J'
rt «0 CD CJ 03 OS o>

G» -* «0 CD © C* ■>*

/O 1U90 J,»d

•i(dopp y

a

800

i(dopto y

^"^

a

1* ^ o

III

«

n

'%

o

M in vi

3 3 3

O O O

t^ ^^ t^

V V V

ass

3 3 3

a a a
b> h ki

u .:^ .1:^ .s.

O O

&" fQ W PQ

s

o

(U

M .

3
CO O

O 4)

"^ s

I ^ ^ T3

rS U3 CQ CO

[^^ a; 03 u3 o5

^- o o o

c S

Ph Ph &

s s a

^ (U oS cci ^
■g +j tM t-i t^

^•3000

cd ^ ^ ^

5 O ^ C<3 Bj

=* .y .Si .Si

SCO +j *.P '.C
"^ C! t) «
CD ^ ^ Cd 03

U t-i U t^ Ui

S c

t t Hi

I I I I I

t t Hi

I I Ui

JJ _»J

S

3

w

<u

Stf

00

0

4J

4->

0

s

C3

cS

^J

«

«

u

c8

o5

CO

0

0

M

(H

CO

0

bb

o o

s s

3 3

02

^ o -n -3

c8 c3

O O

<<

S S >:

>- 3 M

o « o

&s
s§

c8

S§

c8

CO CO

O O

. 2 g I §

s -

3

c

g cS C8

CO '—!'-<

>> cS c6

3 > -t^ -t^

000 CLiOh

- t J fj
I I I I I

* t I JJ

Stf

CS O
00

fQ W

-?

a
o

c

03

m

«

CO to to

3 3 3

poo

^ M u*

V V a>

ass
333

C C 3

.t: »H ;..
a V «
&> >

"C 'C 'C
<u u V
4:: 4-> 4->

y W O
03 ti cd

«««

v u u

I I

I I

I I I

Hi

tf

a

3
•t->

stf

ii B»^

58 TRANSFORMATION OF THE INTESTINAL FLORA

other carbohydrates used would disappear from the enteric tract before
the colon is reached and hence would not offer the favorable pabulum to
the aciduric organisms.

To establish the validity of the above assumption, at least in so far
as the availability of the carbohydrates in the lower intestine is con-
cerned, the following experiments were conducted in addition to the
examination of fecal specimens for reducing substances. A number of
rats which had been receiving one or another of the carbohydrates, lac-
tose, dextrin, maltose, sucrose and glucose, as well as control rats sub-
sisting on the basic diet alone, were killed and careful examinations made
of the contents of the caecum and colon for the presence of reducing
substances and the corresponding predominant bacterial types.

The contents of both the caecum and colon of each rat, in amounts
ranging from one half to one gram, were placed in large test tubes
containing twenty cubic centimeters of water and broken glass and
shaken until a fairly homogeneous suspension was obtained. The tubes
were then heated in a boiling water bath for ten minutes to hasten the
extraction of the carbohydrate from the suspended particles. After
thorough centrifugation the supernatant fluid was filtered twice. In

CHART 28

Curves indicating average percentages of B. bul-
garicus and B. acidophilus appearing in fecal
specimens from rats fed on a diet con-
taining lactose and B. hulgaricus

too

Alumher of days a/risr adrni/i/s^rcf/ron of c/itf^.

2 4 e a lo iz 1^

EXPERIMENTS WITH WHITE RATS

59

CHART 29

Graphs indicating average percentages of B. hul-
garicus and B. acidophilus appearing in dif-
ferent parts of the intestine of rats
fed on a diet containing lactose
and B. bulgaricus

Die-f-

-H-

-If

-tt-

Parts of alimentary canal

g

e

05
a

executing the tests for reducing substances two cubic centimeters of the
filtrate were added to five cubic centimeters of Benedict's solution, the
mixture boiled for one minute, and allowed to remain in the refrigerator
for ten to twelte hours before taking the final readings.* The bac-
teriological tests were carried out in the usual manner.

The feces and the fecal material from the caecum and colon of the
lactose- and dextrin-fed rats gave positive reduction tests with the
Benedict solution, and revealed a completely transformed flora domi-
nated almost entirely by B. acidopMlus. On the other hand, the
samples from the rats which received maltose, sucrose or glucose gave
the same results as those of the control animals which received the basal

* In the testing for lactose heating with dilute acid was resorted to for inversion
of the sugar.

60 TRANSFORMATION OF THE INTESTINAL FLORA

diet only. There was no reduction of Benedict's solution and the intes-
tinal flora was of the usual mixed type. (See Table 23.)

According to the above results neither lactose nor dextrin is com-
pletely absorbed from the intestine of albino rats when two grams are
fed daily. In other experiments, reduction was obtained with the fecal
specimens of rats which received one gram of lactose or dextrin daily
together with one cubic centimeter of B. acidophilus suspension. The
reduction tests were not as pronounced, however, as in the experiments
described here.

The demonstration of incomplete disappearance of lactose and
dextrin before they reach the colon offers some foundation to the avowed
assumption that these carbohydrates favor the proliferation of B.
acidophilus in the lower intestine because they serve as pabulum which
is readily available to this organism for energy requirement and thus
establish for it an optimum environment. Maltose, sucrose and glucose
disappear from the intestine before they reach the ileo-caecal valve, and
therefore do not establish a favorable environment for the aciduric
group of organisms.

TABLE 23

THE RELATION OF DIET TO REDUCING CARBOHYDRATES AND TO

THE BACTERIAL FLORA IN FECAL MATERIAL FROM THE

CAECUM AND COLON OF RATS

7?A7-

or^jtAOtatcTi

PtACenT
or-

J5

C0J\lTKDL

IS Days

4

Bread 10
Beef J

J6

/

57

Breod 10
Beef (5
Lac^e £

+

98

33

3G

^f

Bread 10
Beef J
Maffose 2

"

—

a

60

J

6/

Sreod 10
3eef O

"

+

97

6a

99

63

Bread /O
Beef J
Sucrose 2

"

—

a.

64

6

65

Bread /O
Beef J

"

—

3

66

4

EXPERIMENTS WITH WHITE RATS 61

There appears to be, then, a definite correlation between the rate of
absorption in the alimentary canal of a utilizable carbohydrate and its
tendency to effect, when administered per os, a transformation of the
intestinal flora from the ordinary complex type to one in which the
dominating organism is B. acidophilus. This conclusion is further
strengthened by the results of the following observations.

RELATION OF HYDROGEN ION CONCENTRATION TO THE
CHARACTER OF THE INTESTINAL FLORA

As has been stated in the historical resume of this paper, the possible
relation of acidity of the intestinal tract to the prevailing flora has
been a subject of much debate. Metchnikoff's theory underlying the
alleged implantation of B. bulgaricus rests largely on the assumption
that this organism exerts its remedial influence on the host because of
the acid production which it causes within the enteric tract, particu-
larly the large intestine.

In the present study the hydrogen ion concentrations of the feces
and of the contents of the intestine at different levels were determined
partly by the Sorensen indicator method as modified by Henderson and
Palmer (1912-1913) and partly with the aid of the colorimetric stand-
ards prepared according to the description of Clark and Lubs (1917).
In preparing the fecal extracts for the determinations the general plan
first suggested by Howe and Hawk (1912) and later employed by
Nelson and Williams (1916-1917) was followed, with the exception that
500 milligrams of the fresh feces in place of two grams, and that twenty
cubic centimeters of practically neutral water instead of fifty cubic
centimeters of N/2 sodium sulphate, were employed.

The filtered fecal extracts were usually colored light yellow to brown,
and gave very little if any sediment upon standing. Contrary to the
experiences of Howe and Hawk, comparatively sharp readings could be
made. Four color indicators were used, namely brom thymol blue, brom
cresol purple, methyl red and sodium alizarine sulphonate, the first two
being chosen because they offer more distinguishable shades of color
within the same range of pH than does para-iiitrophenol which was used
by Nelson and Williams (1916-1917). Owing to the wide color range
of sodium alizarine sulphonate (4.7 to 8.0), preliminary tests were
made with this agent to determine the narrow ranges within which the
reactions fell, and thus to facilitate the more exact determinations made
with the other indicators. Brom thymol blue and brom cresol purple
often serve as excellent checks for each other.

Altogether 146 hydrogen ion concentration determinations were
made. The results of some of the individual tests are given in Table 24.
The regular bacteriological examinations and reduction tests with

<

H
O

<1
fQ

Q

<

<

Pi
Q

K
O

PQ

S^

rtf

o

H

Pi

o

o
o

•^ i-i

o
o

o

Ph

O

o

»— I

H
<J

h-;
Pi

W)

i-H

c

C8

C--

s^

o'O

V

;i^

s

-5 lU
O V

1

Ofe

<H1

o

rO >-

f— t

c <«

c

.2

§ii

o

*E

_. c8

1
&

4; CQ

o

CO

1— 1

be

V3

C

V

«n3 1

t>

V

Pm

1^

«w

O

c

'*1

^o

a

*w

c

CO

d)

>,

o^

es

Q

en

03

c

V

a

^

V

u

fe

Pm

««

o

»4

c

-M

CM

.2

a

'm

c

CO

u

t>>

p.

ns

3

Q

CD

o

1-H

CO

bC

(U

_fl

«

wa

^

<u

<«H

fo

O

b

c

4->

_0<M 1

'tn

CS

C

CO

v

PhCS" I

CO

3

Q

CO

CO

sn^mdoppv "9
JO ^U30 jgj

uoi^n^os

sjoipauag

JO uoipnpay^

Hd

snijqdoppv "9
JO :ju90 aaj

uoi^nps

s^:jDip3U3a

JO uonDnpay^

sn|iqdoppv '9
JO ^U90 ja J

uoT:^nyos

Sjpipauag

JO uoT^anpa^

Hd
sn^mdoppv '9

JO ^U3^ J9 J

uoi^npg

s,:i3ip3U39

JO uoi:}3np3^

Hd

SUIBJ£) UI

]BUI^S3^UI JO \[\2U3^ Sh 2 2 r-H

O* OT «5 ■*

O) O^ O)

o> o o>

1 1 1 1

+ + +

CO ot o t^

O OD t-;

»C « «0 «5

to wj «>

O .-H l-< O

05 Ol <*

Oi Oi Oi

1 1 1 1

+ + +

CD ■* ■«* »

CD O CD

«5 U5 »0 «5

«5 to* "J

0» CO CO «5

ifl Ol ^
0 0)0)

1 1 1 1

+ + +

t- iC to to

CD CD •*,

«0 AC «5 to'

to to' to

CO O i-H -.Ji

o> O CO

1 1 1 1

+ + +

«5 »C O CD

CD to O

«5 to to "J^

«5 "J to

O CO
I— (

O CO CS*

i-l

Control
Bread
Beef

Bread

Beef

Lactose

CD CD O
Jfl ui to

SUiBjr)
ui ^qSp^

^BH

CO CD O O
i-H O 00 «5
G< G< CS< rl

+ + +

«* CD to
lei ui ui

»C t-; »q

«5 to to

to CD Oi

ui ui iti

CD o> to

ui to ui

CD O CD
•O to "5

q •«*! O)
to' to ui

O^ to CD

<d ui ui

+ + +

OD CD O)

ui ui ui

+ + +

CD t-_ O

ui ui to

+ + +

«5 to CD

uj to to

+ + +

■,f_ q «5
«j to to

.S

(U 4> ><
»H V 4J

•«i< «5 0*

l-H l-H O)

©« o< en

CO

S.S

P-I^
« o fS

OJ W « CJ 111
u U ei •

mpQH-m

CS» «5 CD
•* CO C5<

i-l p-1 r-t

■>* CD »0
to OS r-(
©» rl ©t

IS e

fl O r^

4) « X CO C~,

>- w « .
mCQQPQ

O t- b-

05 b- i-H
I-l r-( C5<

EXPERIMENTS WITH WHITE RATS 63

Benedict's solution were made in connection with the hydrogen ion
determinations .

The results show clearly that, while the hydrogen ion concentration
varies normally within certain limits, the figures for the rats harboring
a simplified flora strongly dominated by B. acidophilus run almost
parallel with those of the animals having the usual mixed flora. The
hydrogen ion concentration limits remain practically the same.

It was found also, in association with the above tests, that the feces
of the lactose- and dextrin-fed rats contained reducing carbohydrates
and a corresponding flora consisting almost entirely of B. acidophilus,
thus further emphasizing the observed relationship existing between in-
complete absorption of the carbohydrates and simplification of the flora
through dextrin and lactose feeding.

CONCLUSIONS

Diet is a controlling factor in the regulation of bacterial activities in
the intestinal canal of albino rats.

B. acidophilus of Moro is a common inhabitant of the alimentary
tract of the white rat.

Lactose and dextrin, when given in sufficient amounts, bring about a
marked transformation of the intestinal flora in which B. acidophilus
assumes particular prominence and may even completely supplant all
other types of bacteria. Maltose, glucose and sucrose, on the other
hand, have very little or no tendency to induce such a change.

The same results are obtained when one gram of lactose or dextrin
is reinforced with one cubic centimeter of the standard B. acidophilus
suspension as by the daily feeding of two grams of either of these carbo-
hydrates alone in conjunction with the basal diet of bread and meat.
In both instances the bacterial transformation to the simple aciduric
type is practically complete.

The daily administration of two cubic centimeters of the B. acidoph-
ilus suspension (nephelometer five) without the addition of the carbo-
hydrates also leads to a profound change in the character of the flora
of the white rat, and an implantation of B. acidophilus to the extent of
90 to 99 per cent of the total flora is effected.

The simple character of the new flora persists so long as one or
another of the above diets or preparations is continued, but reverts
gradually to the normal or usual mixed type within five or six days
after a return to the basal diet.

B. bulgaricus, which is not an intestinal organism, is unable under
any circumstance to establish itself in the alimentary canal of the
albino rat. Even the daily administration of five cubic centimeters of
the B. bulgaricus suspension exerts no influence on the bacterial popu-
lation of the intestine.

64 TRANSFORMATION OF THE INTESTINAL FLORA

There appears to be a definite relation between the rate of absorption
or digestion in the alimentary canal of a utilizable carbohydrate and its
tendency to effect a transformation. Only those carbohydrates which
reach the caecum and colon unchanged have a transforming influence.

The process of elimination of the ordinary mixed flora and the
establishment of B. acidophilus apparently does not depend on any
changes in the hydrogen ion concentration within the intestine.

III. FEEDING AND IMPLANTATION EXPERI-
MENTS WITH HUMAN SUBJECTS

The following experiments followed closely upon those which were con-
ducted on white rats and which form the subject matter of the pre-
ceding chapter. Very little difficulty .was experienced in procuring
volunteers, most of whom were graduate students and assistants in the
laboratory. Thus far in the investigation seventeen human subjects
have been employed of which fifteen were apparently normal and two
had a long history of intestinal disturbances. Altogether 600 stools
were examined. Most of the subjects served in more than one experi-
ment, bringing the total number of individuals up to forty-six.

The attempts to duplicate the results obtained with white rats were
in a very large measure successful. The investigation resolved itself
into numerous phases in almost all of which the chief aim was to
establish a Bacillus acidophilus flora in the intestinal tract, either by
the administration, in conjunction with the ordinary daily diet, of
special carbohydrates, namely lactose and dextrin, or of living and
viable cultures of B. acidophilus in whey broth or in milk, or of a com-
bination of either of these carbohydrates with the living cultures.

Following a brief period of preliminary observation, and after several
bacteriological examinations of the stools had been made, the subjects
entered upon a period of daily administration of the above-mentioned
test materials, which usually extended over ten days, and in some
instances twenty and thirty days. The feces were collected in paraffined
paper containers, and immediately brought to the laboratory where
they were placed in the refrigerator, and examined on the same day.
Daily examinations were made, the technique being essentially the same
as that employed in connection with the feeding experiments on rats
except that the fecal suspensions for the inoculation of whey agar for
plating and of the Veillon tubes were made up to match tubes ten to
twelve of the nephelometer scale, instead of eight, and that the bacteria
on the Gram-stained slides were counted and the numbers of the different
types given in terms of percentage. The greater turbidity was found
desirable owing to the decidedly smaller number of viable bacteria
present in human as compared with rat feces.

The various tables indicate a fairly close correlation between the
results obtained through cultural procedures and- the direct counts of
the Gram-stained films prepared from the fecal suspensions. The

66 TRANSFORMATION OF THE INTESTINAL FLORA

cultural tests furnish the more reliable information as to the actual
bacterial activities within the alimentary canal, owing partly to the
uncertainty of identification of the more important organisms by
morphology alone, and partly to the fact that an examination of the
Gram-stained film can offer no clue regarding the viability of the bac-
teria. The relative number of viable bacteria in human feces, as com-
pared with the total bacterial content, is extremely small, as has been
shown by various investigators. McNeal, Latzer and Kerr (1909)
have estimated it at about one to three thousand. The absence in some
instances of complete correlation between the results of the direct and
indirect methods of determination of intestinal flora is in all probability
due to an unusually large proportion of dead bacteria.

BASAL DAILY DIET

In order to determine the influence of the ordinary daily diet on the
types of bacteria commonly developing within the alimentary canal the
stools of subjects C and I were examined for ten and fifteen days
respectively. Throughout the observation period these individuals
were found to harbor the ordinary complex flora. (See Tables 25 and
26.) B, acidophilus was present in extremely small numbers. Subjects
A and D, who had been consuming as part of their regular daily diet one
half to one quart of milk daily for weeks and perhaps months prior to
the experiments, were instructed to continue the use of a quart of milk
daily for ten days. These subjects carried a relatively high proportion
of B. acidophilus, and the amount of gas production in the Veillon tubes
was considerably less than in the tubes of C and I. Upon the removal
of the milk from the daily regimen the percentage of B. acidophilus
dropped gradually until there was a complete reversion of the fecal
flora to the ordinary mixed type as seen in subjects C and I. (See
Tables 27 and 28, and Chart 30.)

The examinations of the stools of subjects A and D supported the
claims of Hull and Rettger and others that B. acidophilus is a normal
inhabitant of the intestinal tract of man. We have had no difficulty
in later experiments to demonstrate again and again this important and
fundamental fact. Under ordinary conditions of diet, however, the
organism is present in very small numbers, and requires a dietary
stimulus or bacterial reinforcement to incite it to increased activity.

The ordinary daily diets of the various subjects employed in this
investigation have been well suited for studying the simplifying influence
of the various test agents, inasmuch as they encourage the development
of the so-called putrefactive and gas-producing organisms, and only in
a very slight degree or not at all B. acidophilus. With such diets as the
starting points, the administration of the various test materials which
have a transforming influence in the direction of simplification of flora

EXPERIMENTS WITH HUMAN SUBJECTS 67

should make possible an easy recognition of change in the bacterial
types.

No efforts have been made in any phase of the present investigation
on man to prescribe a definite diet. It was assumed that if changes in
the character of the intestinal flora can be brought about without fol-
lowing any definite or limited dietary scheme, it should be relatively
much easier to control the bacterial types by limiting the diet, as for
example by reducing the amount of protein food consumed.

TABLE 25— SUBJECT C

Ordinaey Daily Diet

1-

O ram-Stained Films from

Fecal

Suspensions

:s'=^

:!

Total

Gram-Positive

1"^

^

1-

Veillon Tubes

Organisms

Org,

anisms

cp

"^

•i

«

11

s S.

;.2
5^8

§.

t

?^

^2

«*2
^1

o

«s

^

«»

S

^i

.u,

.o

^

ft.

^

Cq

e

oq

^

Ct5

1

0

++-H-

++++

15

85

5

10

2

2

—

++-f-

—

14

86

5

9

3

3

—

—

21

79

10

11

4

0

+++

—

—

24

76

10

14

5

1

—

—

30

70

12

18

6

4

—

—

28

62

12

16

7

6

+++

—

—

25

75

8

17

8

12

+++

—

—

32

68

14

18

9

6

—

-H-f

—

32

68

12

20

10

4

—

4-ff

—

38

62

16

22

1

2

2

1

3

0

4

6

5

1

6

3

7

0

8

4

9

0

10

2

11

2

12

0

13

0

14

4

15

0

TABLE 26— SUBJECT I

Ohdinaey Daily Diet

— +++ — 23 77

— -H-+ — 26 74

— •+++— 41 59

— -H4- — 37 63

— +++ — 30 70

— +++ — 31 69

— +-H- — 28 72

— +++ — 34 66

— +-H- — 27 73

— ++ — 36 64

— -f+f — 29 71

— +++ — 35 65

— 4-H- — 24 76

— +++ — 33 67

— +++ — 38 62

10

13

12

14

14

27

12

25

10

20

12

19

8

20

12

22

9

18

14

22

11

18

10

25

'8

16

12

21

14

24

68 TRANSFORMATION OF THE INTESTINAL FLORA

TABLE 27— SUBJECT A

Oedinary Daily Diet, Including Sweet Milk 1000 cc.

A.

Gram-Stained Films from

Fecal

Suspensions

:i'^

•g

Total

Gram-Positive

^1

^

1

Veillon Tubes

at

Organisms

Orgi

%ntsms

-5-

^

-«

el

8

^1

1

1

^O^
♦-"V

^i

'^ i

1^

^

a.

%
^

^

oi

?5

^

^

1

12

++++

—

+++

—

32

68

12

20

2

30

-H-H-

-f-

++

+-

38

62

20

18

3

78

++

++

+

++

60

40

40

20

4

65

++

+

+

+

52

48

34

18

5

80

+

++

+

-H

65

35

50

15

6

85

+

+++

—

+++

62

38

48

14

7

75

4-

++

—

++

64

36

46

18

8

86

-H

++f

—

4-H

55

45

30

25

9

60

++

+

+-

4-

58

42

32

26

10

72

++

++

-h-

44-

54

46

35

19

OaDiKARY Daily Diet Without Milk

11

70

++

+

+

4-

46

54

28

18

12

22

44+

—

-H-

—

40

60

16

24

13

12

-m-

—

++

—

38

62

18

20

14

3

+++

—

■H-t-

—

36

64

16

20

15

2

+++

—

-1-H-

—

35

65

16

19

TABLE 28— SUBJECT D

Ordinary Daily Diet, Including Sweet Milk 1000 cc.

1

10

44-f

444

21

79

8

13

2

5

4+H-

—

444

—

30

70

18

12

3

3

++++

—

444

—

32

68

15

17

4

6

4+1-

—

44

—

27

73

12

15

5

12

444-

—

444

—

34

66

16

18

6

18

4-H-

—

44

—

38

62

17

21

7

42

444-

—

44

—

48

52

29

19

8

62

44

4

4

4

54

46

32

22

9

69

44

4

44

4

51

49

31

20

10

78

4

44

4-

44

58

42

36

22

Ordinary Daily Diet Without Milk

11

8

444

44

35

65

12

23

19

10

444

—

44

—

34

66

15

19

IS

18

444

—

44

—

31

69

10

21

14

12

4444

—

444

—

30

70

10

20

15

2

4444

—

444

—

28

72

12

16

EXPERIMENTS WITH HUMAN SUBJECTS

69

|.^^<^^'^'^'{i'5^«^

©

o

CO

|*.«'R,«g>^'^.{J.5^<5

li^^l'^^S'Jj-I.H'^^'*

©

IS.

l^^^^>^^5!<5^«?-^<i

70 TRANSFORMATION OF THE INTESTINAL FLORA

CARBOHYDRATE FEEDING

In all of the carbohydrate ingestion experiments on man either lac-
tose or dextrin was employed. These substances were administered
three times daily in 1 00 gram quantities, except when the total amount
per day was only 150 grams, in which case they were consumed in one
dose. The lactose was taken in the form of a heavy aqueous suspension,
while the dextrin was used in 50 per cent solution prepared by boiling
the dextrin in water. Owing to its unpleasant taste the dextrin was
flavored with a little orange juice. These carbohydrates were taken be-
tween meals.

TRANSFORMING INFLUENCE OF LACTOSE

This sugar was administered to six persons, and the results were
fairly uniform. The ingestion of 300 grams of lactose, in addition to
the ordinary daily diet, by subjects A, C, D and L effected a radical
transformation of the fecal flora within four to eight days, from the
usual mixed type to one dominated almost entirely by B. acidophilus.
The essentially aciduric flora persisted as long as the use of lactose was
continued. (See Tables 29 to 32, and Chart 31.) It will be seen,
however (Table 33 and Chart 31), that the flora of subject B, a sturdy
individual weighing almost 200 pounds, failed to respond to the influ-
ence of 300 grams of lactose taken daily for ten days, whereas the inges-
tion of 400 grams of the sugar brought about a complete simplification
of the intestinal flora within four days.

The use of 150 grams of lactose daily by subject I (Table 34 and
Chart 32) resulted in an appreciable multiplication of B. acidophilus,
but there was no radical suppression of the other types of intestinal
bacteria.

The simplification of the intestinal flora was characterized by an
enormous relative and actual increase in the numbers of B. acidophilus
appearing in the stools, and the almost complete suppression and elim-
ination of the other bacterial types ordinarily present. After the
expiration of the first four days following the initial administration
of the carbohydrate, the characteristic colonies of B. acidophilus were
found to dominate in the whey agar plates and at times were practically
the only colonies present. The Veillon tubes very strikingly indicated
a gradual diminution in the numbers of gas-producing organisms.
These observations were further confirmed by the increase in the rela-
tive numbers of Gram-positive rods in the Gram-stained slides.

TRANSFORMING INFLUENCE OF DEXTRIN

The ingestion of 300 grams of dextrin exerted a marked transform-
ing action on the intestinal flora, stimulating the proliferation of B.

EXPERIMENTS WITH HUMAN SUBJECTS

71

TABLE 29— SUBJECT A

Ordinary Daily Diet + Lactose 300 gms.

^

Gram-Stained Films from

Fecal

Susp*

ensions

•2

a.

-ef^)

Total

Oram-Positive

1 *

^

1-

Veillon Tubes

Organisms

Organism

s

cq

t

"S-

•«

•s

11

8
® as

8

*.2

8

1

1

?5

•^ i

0 g

T3

V.

90

^

^

^

c

^

Oh

CQ

Cs

cq

Cb

C3

1

6

-H4+

-K+

38

62

16

22

2

62

-l-H-

++

++

+

46

54

30

16

3

60

-l-H-

++

++

+

48

52

36

12

4

92

+

4-

68

32

51

17

5

99

+-

—

78

22

62

16

6

94

4-

—

80

20

68

12

7

92

-f-

—

84

16

70

14

8

98

—

—

88

12

76

12

9

99

—

—

85

15

72

13

10

97

—

+-H-

—

86

14

76

10

TABLE 30— SUBJECT C

Ohdixary Daily Diet + Lactose 300 cms.

1

0

++++

+++

25

75

10

15

2

0

++++

—

-H+

—

28

72

12

16

3

2

++-H-

—

+++

—

34

66

14

20

4

21

+++

—

•H-t-

—

40

60

18

28

5

30

+++

4-

++

-h-

42

58

29

13

6

28

+f+

-f-

++

+-

41

59

27

14

7

62

+++

+

++

+

43

57

30

13

8

95

++

+++

+

-f-H-

62

38

50

12

9

89

+

+++

-h-

+++

66

34

52

14

10

91

+

+++

—

+++

65

35

51

14

TABLE 31— SUBJECT D

Ordinary Daily Diet + Lactose 300 cms.

1

0

+44+

■H+

30

70

12

18

2

32

44-1-

4-

4-i-

■4-

52

48

40

12

3

84

4

4-

444-

64

36

48

16

4

92

—

—

444-

72

28

58

14

5

90

—

—

444

70

30

52

18

6

99

—

—

444-

87

13

72

15

7

94

4-

—

444

88

13

78

10

8

99

—

—

444

90

10

78

12

9

94

—

—

444-

86

14

72

14

10

99

—

—

444

89

11

73

16

72 TRANSFORMATION OF THE INTESTINAL FLORA

CO

<
o

^*^>SiS^'^^^^

8; <^ <^ vS «^ ^ .5^ .^ ^ 0

|§-'«^'SJ^^^<5-^'i

EXPERIMENTS WITH HUMAN SUBJECTS

73

acidophilus within three days to such an extent as to considerably
overshadow all of the other bacterial types. (See Table 35.) Similar
to the results of lactose feeding, 90 to 99 per cent of the colonies on the
whey agar plates were those of B. acidophilus. Acidophilus colonies
were very abundant also in the Veillon tubes in which there was no gas
production. Direct examinations of the Gram-stained slides offered

TABLE 32— SUBJECT L

Ordinary Daily Diet + Lactose 300 gms.

-s

Gram-Stained Films from

Fecal

Susp

ensions

•S

o-

:^^

-§

Total

Oram-Positive

•1-^

^

1-

Veillon Tubes

la

Organisms

Organisms

^ 8

v. o

cq

«

•^

«

.«

ll

8 2.

8

8

«o .2

•u

&.

^

t> o

o^

O-^

o g

|.8

8

-§

^

»-'V

»-^

*-f§

*-^®

o

"S

6

<^i

(^^

.u,

.o

^"^
^

1

cq

(.

^

^

1

0

•H-f

26

74

10

16

2

0

—

+++

—

38

62

14

24

3

0

—

4++

—

35

65

16

19

4

2

—

-f4+

—

36

64

15

21

5

0

—

+++

—

38

62

18

20

6

15

—

+++

—

40

60

22

18

7

37

4-

+++

-t-

44

56

24

20

8

70

+++

+

++

+

62

38

48

14

9

89

•H-f

+f

++

+

68

32

50

18

10

87

++

•H-

-t-

++

69

31

52

17

TABLE 33— SUBJECT B

Ordinary Daily Diet + Lactose 300 gms.

1

0

4-H-

34

66

12

22

2

0

—

4-H-

—

30

70

10

20

3

6

—

1 1 '

—

42

58

16

26

4

1

—

44-H

—

32

68

15

17

5

18

—

4-H-

—

30

70

14

16

6

3

—

4-1-1-

—

38

62

18

20

7

26

—

4-H-

—

44

56

20

24

8

12

—

444-

—

48

52

26

22

9

10

—

4-14

—

40

60

22

18

10

8

-H-H-

—

444

—

46

54

28

18

Ordinary Daily Diet + I^actose

400 GMS.

11

16

4-H+

—

444

45

55

25

20

12

48

+4+

4-

44

4

68

32

40

28

13

60

4++

+

44-

4

64

36

42

22

14

86

44-

444-

4

444

70

30

50

20

15

89

4-

4-H-

—

444

72

28

54

18

74 TRANSFORMATION OF THE INTESTINAL FLORA

TABLE 34— SUBJECT I

Ordinary Daily Diet + Lactose 150 cms.

^1

Gram-Stained Films from Fecal Suspensions

05

Veillon Tubes

-1 2-

Total
Organisms

^

8 2

^ ««

^i

5*5

CHART 32

Gram-Positive
Organisms

1

0

+44+

-l-H-

27

73

12

15

2

18

++++

—

+++

—

34

66

16

18

3

46

+++

+

++

4-

30

70

15

15

4

38

4-H-

+

-m-

-1-

42

68

24

18

5

40

+++

+

+

+

40

GO

25

15

6

49

++

+

++

+

42

58

28

14

7

66

++

+

+

+

49

51

38

11

8

58

+

+

-f-

+

54

46

36

18

9'

60

++

+

+

+

45

55

27

18

10

68

++

-H-

4-

++

50

50

36

14

/ 2 3 4^ -5 e y 0 f /a // /2 A3 A^ AS

Curve indicating percentage of B.
acidophilus appearing in fecal
specimens from human subjects.
Diet
Ordinary daily diet
Lactose 150 gms.
Ordinate — Per cent of B. acidoph-
ilus.
Abscissa — Number of days after
administration of diet.

EXPERIMENTS WITH HUMAN SUBJECTS

75

additional evidence of transformation of the bacterial tjpes. Gram-
positive acidophilus-like rods held prominence and at times their num-
ber reached as high as 83 per cent of the total number of organisms
counted. The simple character of the flora tended to remain permanent
as long as the ingestion of the dextrin was continued. However, within
five days after the last dextrin intake the fecal flora gradually reverted
to the usual complex type. (See Chart 33.)

TABLE 35— SUBJECT C

Ordinary Daily Diet + Dextrin 300 gms.

-«

Gram-Stained Films from

Fecal

Suspensions

SO

^

:s^

3

Total

Oram,-Positive

••pv

^

Veillon Tubes

Organisms

Organisms

^ e

1-

2'

-a

cq

<s

•S

"^

'd

-«

li

8

» 99

8

*.2

8

Si.
o

?i.

1^

^-e

V.

<n

^

05

^

e

e

^

Oh

cq

=q

^

^

1

10

-H-H-

+++

20

80

8

12

2

30

+-f-t-

++

++

+

32

68

15

17

3

96

+

+++

—

+++

78

22

60

18

4

98

-f-

++++

—

81

19

64

17

5

98

—

++++

—

80

20

70

10

6

99

—

++++

—

85

15

73

12

7

99

—

+-r4-f

—

88

12

80

8

8

98

-^

4+++

—

82

18

62

20

9

99

—

++++

—

92

8

81

11

10

99

—

++++

—

90

10

83

7

Ordinary Daily Diet

11

70

4-

+++

—

++

60

40

48

12

12

56

+++

-H-

+

+

42

58

32

10

13

40

+++

+

++

4-

46

54

28

18

14

8

+44+

—

+++

—

30

70

10

20

15

3

++++

—

+++

—

22

78

10

12

TABLE 36~SUBJECT F

Ordinary Daily Diet +- Dextrin 200 gms.

26
20
18
21
30
25
20
10
15

^^-H-

+4++

-H+

++

— -\-h\- —

++ —

28

72

16

12

36

64

20

16

30

70

18

12

44

56

28

16

40

60

25

15

38

62

20

18

45

55

22

23

33

67

15

18

34

66

12

22

76 TRANSFORMATION OF THE INTESTINAL FLORA

CHART 33

The administration of 200 grams of dextrin to subject F exercised
a slight transforming effect (see Table 36), whereas 150 grams of the
same carbohydrate to subject I resulted in much greater stimulation
of the development of B. acidophilus (Table 37). Idiosyncrasies of
this sort on the part of the human subject were observed in the lactose
feeding also. While the ingestion of either 150 or 200 grams of dextrin
encouraged the proliferation of B. acidophilus, there was no marked
suppression of other types of bacteria vegetating in the intestinal canal.

The rise in the B. acidophilus curve as the result of the feeding of
300 grams of dextrin daily is at least as sharp as in the administra-
tion of the same amount of lactose, reaching its maximum on the third
or the fourth day. Dextrin has been found as effective a transform-
ing agent as lactose (Chart 33). The decidedly objectionable odor and
taste of the dextrin solution will of necessity stand in the way of its
practical use, however, even though other substances like orange juice
may be employed to partly obscure the taste and odor.

(yd/nar/da/fy diet

/oo

/oo

0O

A /"^

, ®

do

0

7"

6o

1 V 1\

7"

6o

SO

i\ 1 \

5o

4o

/ 1

4o

3o

/ \

Jo

>►•*.

2o

/ \

20

A»^ ^^•"'^S.

/o

y I v^—^iw

lo

/ ^^

0

-^

^ %

0

/ z 3 4 £ 6 y a f /c // /z /3 /4 /s

/ 2 3 4 S a J 8 f /o // /Z /3 /4 /S

/oo

(h^inary dai//d/et

\ ©

Cur ve^ md/cat/n^ percentage of

■5pec/men.5 from humarj subject J.

Ordinary da/7y diet

\ /50grrj3. Curve J

Dextrin \Z00 •• •• ^

[JOO " " C

fa
8o

7°

6o
So
40
50

(

Zo

1 \ \

-

/o

J \

Ordjnates -fkr oenfafD. ac/dooh.

fjb^c/ssae —/dumber of days after
admini-sfrafion ofd/et.

/ Z 3 4 -5 6 J 3 <f /o n /Z /3 /4 /S

_

EXPERIMENTS WITH HUMAN SUBJECTS 77

TABLE 37— SUBJECT I

Ordinary Daily Diet + Dextrin 150 cms.

rfS

Gram-Stained Films from

Fecal

Suspensions

«

§-

:^'=^

Total

Oram-Positive

8 "=>

^

1-

Veillon Tubes

Organisms

Organisms

cq

~

«>

"^p

"S-

8.-S

8 e

8

8

'ti

>«

» S

» Oj

* 89

« •«

4^

Si-

Si-

O o

o «^

O-e

O §

8

1

Oh S

^-e

V

»9

^

00

'^

8

Q

^

ft.

cq

C5

K5

Cb

55

1

4

+4++

44+

30

70

12

18

2

10

+++

—

4+

—

35

65

18

17

3

28

++f

—

4+

—

34

66

20

14

4

60

++

+

+

+

52

48

38

14

5

42

++

+

+

+

50

50

32

18

6

88

—

44+

—

44

68

32

54

14

7

62

++

+

+

+

60

40

45

15

8

84

—

+4+

—

+4+

64

36

51

13

9

86

—

4+

—

4+

62

38

50

12

10

80

+

4+

4-

44

59

41

42

17

Ordinary Daily Diet

11

46

+

44

4

4+

42

58

30

12

12

10

+++

—

4+

—

31

69

20

11

13

8

4+4+

—

44+

—

37

63

18

19

14

10

+44+

—

44+

—

33

67

16

17

15

1

444+

—

++

—

32

68

14

18

TABLE 38— SUBJECT A

Ordinary Daily Diet + B. Acidophilus 300 cc.
(Whey Broth Culture, Neph. 5)

1 0 444+ — 44+ — 38 62 12 38

2 80 +4 + + +

3 85 — 44+ — ++f

4 80 4- 444 — 44+
6 90 — 444 — 4++

6 90 — +++ — +44.

7 90 — 44+ — 4++

8 90 — +44 — 444

9 92 — 44+ — +44
10 94 — 44+ — 44+

The experiments have demonstrated conclusively that both, lactose
and dextrin, when administered in 300 gram quantities or more daily,
exert a most profound influence on the character of the intestinal flora,
transforming the latter from the ordinary mixed type to one dominated
by B. acidophilus.

52

48

40

12

56

44

42

14

50

50

40

10

75

25

68

7

80

20

72

8

84

16

78

6

88

12

80

8

86

14

78

8

85

15

76

9

78 TRANSFORMATION OF THE INTESTINAL FLORA

IMPLANTATION EXPERIMENTS WITH LIVING CULTURES
OF BACILLUS ACIDOPHILUS

B. acidophilus was administered in the form of whey broth cultures
which were incubated for forty-eight hours at 37° C, and reduced, when
necessary, to five of the McFarland turbidity scale. Four different
strains of the organism were employed; they were isolated originally
from human stools. In some instances the subjects were given their
own strains in conjunction with others.

TABLE

39-

-SUBJECT

' B

Ordinary Daii

.Y

Diet + B. Acidophilus 300 cc.

(Whey

Broth Culture, Neph

:. 5)

^

^

Oram-Stained Films from

Fecal

Suspensions

il^*

Total

Oram-Positive

^

Veil Ion Tubes

-f

<j>

Organisms

Organisms

BQ

"^g

'^

,

e.-s

8 e

8

8

■^ i;

>«

•«

^ «

» «95

SB .«

1

a,

Si.
o

^1
ft. S

*- o

V.

ag

^ *

'^

e

e

^

Ah

B5

^

^

1

0

-H4+

— +++

—

26

74

8

18

2

52

+4+

+ +

+

40

60

27

13

3

60

+

++ —

+

44

56

25

19

4

90

—

+++ —

+++

72

28

61

11

5

90

—

+-H- —

1 1 1

70

30

62

8

6

90

i-

+++ —

76

24

67

9

7

90

—

+++ —

81

19

74

7

8

90

i-

4-H- —

80

20

72

8

9

90

+-

+4-1- —

82

18

74

8

10

92

—

-H-t- —

88

12

70

18

TABLE 40— SUBJECT C

Ordinary Daily Diet + B. Acidophilus 300 cc.
(Whey Broth Culture, Neph. 5)

1

0

+44+

+++

42

58

25

17

2

0

+++

—

■i-+

—

51

49

38

13

3

50

+++

+

44-

+

55

45

40

15

4

70

++

++

+

++

60

40

48

12

5

70

++

++

4-

++

68

32

52

16

6

85

+

++

—

++

82

18

66

16

7

80

+

++

—

4+

80

20

68

12

8

90

. 4-

4+

—

++

88

12

79

9

9

92

—

+44-

—

+++

87

13

80

7

10

90

+-

+++

—

+44-

86

14

72

14

EXPERIMENTS WITH HUMAN SUBJECTS 79

In the attempts to implant B. acidophilus in the absence of special
carbohydrates five subjects received 300 cubic centimeters each of the
whey broth cultures, in addition to their usual daily diets, for a period
of ten days. The bacterial culture was taken in one dose, usually
before the noon luncheon.

Within two to four days after the first administration of the viable
living bacteria, B. acidophilus colonies were observed in both the whey
agar plates and in the Veillon tubes in appreciable numbers. The
Gram-stained slides gave additional evidence of the simplification of
the intestinal flora. Gram-positive rods resembling B. acidophilus con-
stituted 75 to 80 per cent of the total bacterial count. This trans-
formation reached its maximum on the fourth to sixth day, and the
simple flora persisted as long as the B. acidophilus cultures were ad-
ministered. (See Tables 38 to 42, and Chart 34.) B. acidophilus
dominated the fecal flora to practically the same extent as when 300
grams of lactose or dextrin were fed. (See Chart 34.)

The conclusion may be drawn from the above experiments that B.
acidophilus is capable of establishing itself in the intestinal tract of
man when administered per os without accompanying special carbo-
hydrates. In each of the subjects it became the predominating organ-
ism in the enteric tract, almost wholly suppressing or eliminating all
of the other viable bacterial types. The results warrant the assumption
that the organism in question is able to accommodate itself to the
environmental conditions of the human intestine, and that positive
implantation can be eifected. This assumption has been strengthened
further by the results of subsequent experiments.

SIMULTANEOUS USE OF THE SPECIAL CARBOHYDRATES
AND BACILLUS ACIDOPHILUS

The purpose of these experiments was to determine whether half
or even less of the transforming doses of lactose and the whey broth
cultures of B. acidophilus can effect a marked simplification of the in-
testinal flora in man when these agents are administered together.
Accordingly, five human subjects whose flora was known to be of the
usual complex type were given, in addition to their regular daily diet,
150 grams of lactose and 150 cubic centimeters of the acidophilus
cultures.

The results are summarized in Tables 43 to 47. They show a pro-
nounced change in the character of the intestinal flora, with a strong
predominance of B. acidophilus as the outstanding feature. A com-
parison of these results with those obtained with the subjects who had
received only 150 grams of lactose reveals striking differences. Fol-
lowing the combined feeding the development of B. acidophilus was so
pronounced that, barring one exception, within four to six days after

80 TRANSFORMATION OF THE INTESTINAL FLORA

CO

©

^ ^ ^ ^

1 ll

/

0^

1

■k

^1

v^

•0

1
1

1

II

1

|,^<^«^-Sv^^<55<§-^^

15

.is

®

5^

®

!5

5?

^
^

1

^

^

/

K

\_

N

^^^

«>

" X

«^

^

M

N

*^

^

N

!,*.« »^<§ '^ ^-^ ^ ^ <»

-l^*.'^ -^.^ ^ ^<<5<^

^ Q

i^

!5

®

3^
K

®

5^

>

^

N

^^ — .

N

l,^« ^j ^ -^ JJ ^ ^ ^

•i'^'^ iR^vS >5^ -51 ''^ <?

^

<s

EXPERIMENTS WITH HUMAN SUBJECTS 81

the beginning of the experiment it constituted 90 to 99 per cent of the
viable intestinal organisms. Subject H failed to respond very readily,
but after the sixth day the transformation was practically complete, as
indicated by the agar plates and Veillon tubes.

In every instance when simplification of bacterial types was effected
there was a sharp diminution of gas production in the Veillon tubes and
eventually complete or almost complete absence of gas. Furthermore,
the Gram-stained films revealed a picture in which 60 to 80 per cent of
the organisms were acidophilus-like rods.

TABLE 41— SUBJECT D

Ordinary Daily Diet + B. Acidophilus 300 cc.
(Whey Broth Culture, Neph. 5)

•«

O

ram-Stamed

Films

from Fecal bus

pensions

•S

»<

:§'=>

T

Total

Gram-Positive

^

1-

Veillon Tubes

ta

Organisms

Organisms

^1

8

•w

8

'^^

'ti

-JS

« S

« Oj

« 09

«•-

1^

SI-
o

o

ft. S

^1
1^

ft^

V.

as

"^

»»

^

e

e

^

a.

CQ

Pq

^

^

1

50

+++

+

+

+

64

36

42

22

2

60

++

+

+

+

62

38

44

18

3

90

4-

-H+

—

-H+

72

28

64

8

4

90

—

+++

—

-f+f

85

15

72

13

5

98

—

+-I-H-

—

++++

92

8

86

6

6

93

—

++++

—

+-I-H-

90

10

82

8

7

90

4-

+++

—

4-H-

88

12

78

10

8

95

—

+++

—

+++

94

6

88

6

9

95

—

+++

—

-H-h

90

10

80

10

10

92

—

4-H-

—

+++

93

7

85

8

1

TABLE

42-

-SUBJECT

E

Ordinary Daily Diet + B.

Acidophilus 300 cc.

(

[Whey Broth Culture,

Neph.

5)

1

0

+++

—

+++

—

15

85

6

9

2

0

+++

—

-m-

—

22

78

12

10

3

0

-m-

—

+++

—

30

70

18

12

4

60

++

+

+

+

45

55

35

10

5

70

+

++

4-

++

52

48

40

12

6

84

+

—

-H-l-

68

32

57

11

7

99

—

—

1 1 1

83

17

78

5

8

95

—

—

-H+

88

12

72

16

9

98

—

—

444-

84

16

75

9

10

96

—

—

44-1-

82

18

70

12

82 TRANSFORMATION OF THE INTESTINAL FLORA

These results are in perfect harmony with those obtained in the
feeding of albino rats, and may be summarized in a few words. In the
combined feeding of lactose and whey broth cultures of B. acidophilus
the same degree of transformation of intestinal flora may be obtained
with half of the amounts of the lactose and cultures as when either of
these agents is administered separately. Thus, in man the ingestion
of 150 grams of lactose and 150 cubic centimeters of the whey broth
daily results within the course of a few days in a profound transforma-
tion of the intestinal flora in which B. acidophilus becomes the domi-
nating organism, with no reversion to the usual mixed type until
several days after the use of the test materials has been discontinued
(Chart 35).

TABLE 43— SUBJECT F

Ordinary Daily Diet + Lactose 150 oms., B. Acidophilus 150 cc.
(Whey Broth Culture, Neph. 5)

54.

Oram-Stained Films from

Fecal

Suspensions

If^

Total

Gram-Positive

•l"^

'^

1-

Veillon Tubes

Organisms

Organisms

. 8

fiq

«

•♦^ '♦^

■» -s

"*-. 8

"^

,

e."§

8 e

s

e

'^ i

.«

!«

5" *

» &j

'^ <K

« •«•

« *

**

Si,

su

O o

o «

O-S

^ g

s

^0,

^-e

!.

»5

^

<K

'^

c

e

^

cq

oq

^

03

1

25

+++

—

+++

35

65

15

20

2

68

++

+

+

+

38

62

25.

13

3

92

-t-

—

+++

74

26

62

12

4

90

4-

—

+-H-

72

28

65

7

5

90

i-

—

+++

75

25

66

9

6

98

—

—

+H-

81

19

70

11

7

95

-f-

—

4-H-

78

22

68

10

8

95

-t-

—

+++

76

24

62

14

9

90

+

4-

\^

80

20

68

12

10

60

++

++

+

++

73

27

60

13

TABLE 44— SUBJECT G

Ordinary Daily Diet + Lactose 150 cms., B. Acidophilus 150 cc.
(Whey Broth Culture, Neph. 5)

1

90

+

4-H-

2

80

+

4-H-

3

95

—

4-H-

4

85

4-

444-

5

85

4-

444

6

98

—

4-M-

7

90

4-

4-H-

8

98

4-

44+

9

80

4-

+44

10

94

—

4-l-K

— 444-

64

36

45

19

68

32

50

18

74

26

62

12

70

30

56

14

72

28

60

12

82

18

74

8

78

22

69

9

80

20

70

ID

76

24

68

8

83

17

72

11

EXPERIMENTS WITH HUMAN SUBJECTS

83

S^^R.'S'S^-I.^^^'s

CO

H

^ "5$ N ^ <»

®

lSs*'^5>^*<?i'^^

ft^vS >? ^ .? .5 5^ <i

B^«S<1,4>^^^^^^

84 TRANSFORMATION OF THE INTESTINAL FLORA
TABLE 45— SUBJECT H

Ohdinary Daily Diet + Lactose 150 gms., B. Acidophilus^ 160 cc.
(Whey Broth Culture, Neph. 5)

S4<

Gram-Stained Films from

Fecal

Suspensions

:§'='

Total

Oram-Positive

•l"^

^

Veillon Tubes

Organisms

Orgi

<xnisms

^ ff

1-

3-

PQ

'^^

^

<

rf!

I'S

II*

8

e

as •»

•»

&.

54.

O §

O S'

O-e

^ S

1

•s

Oh g

^ i

^^3

i.

»»

^

09

^

g

^

05

^

Ki

■;b

^

1

0

-H-H-

30

70

12

18

2

0

-H-H-

—

—

32

68

10

22

3

0

-H-f-

—

—

36

64

15

21

4

0

+++

—

—

35

65

18

17

5

3

-K+

—

—

40

60

21

19

6

70

44+

+

++

+

52

48

42

10

7

90

+

+++

-H+

66

34

42

24

8

95

+-

+++

++f

70

30

62

8

9

80

+-

+++

+++

73

27

60

13

10

92

—

+++

+H

77

23

66

11

TABLE 46— SUBJECT I

Ordixary Daily Diet + Lactose 150 gms., B. Acidophilus 150 cc.
(Whey Broth Culture, Neph. 5)

1

2

2

20

3

34

4

90

5

90

6

93

7

95

8

95

9

90

10

93

++ —

+++

31

69

10

21

32

68

15

17

35

65

20

15

64

36

48

16

60

40

48

12

71

29

62

9

70

30

60

10

86

14

68

18

81

19

70

11

85

15

73

12

TABLE 47— SUBJECT K

Ordinary Daily Diet + Lactose 150 gms., B. Acidophilus 150 cc.
(Whey Broth Culture, Neph. 5)

1

0

2

6

3

18

4

98

5

99

6

99

7

99

8

99

9

99

LO

99

36

64

12

24

34

66

14

20

38

62

18

20

68

32

54

14

85

15

76

9

77

23

70

7

79

21

73

6

85

15

75

10

88

12

76

12

86

14

80

6

EXPERIMENTS WITH HUMAN SUBJECTS 85

TRANSFORMING INFLUENCE OF MILK CULTURES OF
BACILLUS ACIDOPHILUS ON THE INTES-
TINAL FLORA

In the more recent experiments on intestinal implantation B. acidoph-
ilus was administered in the form of pure milk cultures. The strains
which were employed were the same as in the preparation of the whey
agar cultures previously described. Since B. acidophilus is primarily
of intestinal origin, and not a milk-borne organism, many difficulties
were encountered in the preparation of the sour milk. However, within
three weeks the strains employed were by frequent and liberal transfers
from milk to milk adapted to the new environment, so that ten cubic
centimeters of inoculum added to one liter of sterile milk effected dis-
tinct acidification and creamy coagulation within twelve to fifteen hours
at 35 to 37° C. A full description of the method of acclimating B.
acidophilus, and of preparing and utilizing the sour milk culture will
be found in one of the following chapters.

The B. acidophilus milk was given to quite a number of subjects, of
whom some received 500 cubic centimeters and others one liter daily, in
addition to the regular diets. The milk cultures were taken as a rule
in 500 cubic centimeter quantities, and between meals. Occasionally the
daily amount was divided and the portions taken at definite intervals.

Subject A whose intestinal flora was easily simplified by any and all
of the transforming agents employed throughout the investigation,
responded readily to the consumption of 500 cubic centimeters of the
acidophilus milk per day (Table 48). B. acidophilus soon made its
appearance and in a very short time became the dominant and practi-
cally the sole viable organism occurring in the stools. After the first
two days of sour milk feeding no gas was produced in the Veillon tubes,
and the stained slide showed a sharp increase in the Gram-positive rods
of the aciduric type, which soon reached 75 to 80 per cent of the total
number of fecal organisms counted. After the discontinuance of the
sour milk feeding the flora gradually reverted to the ordinary mixed
type. This return required about five days.

Again, the ingestion of the same amount of the milk culture (500
cubic centimeters) daily by subjects F and G exerted only a slight
transforming influence on the fecal flora. (See Tables 49 and 50, and
Chart 36.) The whey agar plates and Veillon tubes indicated a moder-
ate multiplication of B. acidophilus, and the stained films showed the
Gram-positive rods to be outnumbered by the other types of intestinal
organisms. However, when one liter of the acidophilus milk was taken
daily by these same subjects the complex intestinal flora was practically
completely reduced in a few days to the aciduric type. (See Tables 51
and 52, and Chart 37.) Abundant proliferation of B. acidophilus was
clearly evident after four to six days, and its complete predominance

86 TRANSFORMATION OF THE INTESTINAL FLORA

over the other intestinal organisms was maintained as long as the sour
milk consumption was continued.

Similar results were obtained with subjects B, H, M, O and P, who
received one liter of the milk culture daily in addition to the ordinary
diet. The change in the intestinal flora of these individuals was quite
uniform, the ingestion of the acidophilus milk causing in each case such
a marked development of the B. acidophilus type as to overshadow
completely all of the other viable organisms ordinarily present. (See
Tables 53 to 57, and Charts 37 and 38.) It is of particular interest to
note that the dominating B. acidophilus flora of subjects H and O, who
received the milk cultures for twenty and thirty days respectively,
remained unchanged during the entire milk-feeding period, without any
apparent tendency whatever to revert to the original mixed flora. Fol-
lowing the discontinuance of the milk consumption the homogeneous

CHART 36

i^-»»i^t ■ •

Ordmaryda/ly
c//et

/ Z 3 4 £ a 7 8 J /o // /z /3 M /S

/oo

So

7>

6e
So
4c
3o
2o

A>

©

/ Z 3 4- S 6 y 3 <J /o // /Z /3 /4 /s

Curves /nd/caf/rx^ percentage of
B. ac/c/oph//u5 appear/ng m feca/
spec/ mens from ha/nan subjects.

Ordinary da//yd/et
fj.ac/dooML/s mi/k SOO cc.

Ordinates -Fercerrt of3ac/dooh.

ffhsr/ssae -Number of days after
adn?mfstraf/or? ofd/ef.

EXPERIMENTS WITH HUMAN SUBJECTS

87

character of the simplified flora gave way to the usual heterogeneous
mixture.

The application of pure milk cultures of B. acidophilus as compared
with the use of special carbohydrates like lactose and dextrin, and of
whey agar cultures, either with or without the carbohydrates, has very

TABLE 48— SUBJECT A

Ordinary Daily Diet + B. Acidophilus Milk 500 cc.

-«

Gram-Stained Films from

Fecal

Suspensions

«

a^

is'^'

'S

's

Total

Oram-Positive

•l-s-

^

Veillon Tubes

Organisms

Org,

anisms

^ e

1

^-

Kl

.g

-*-, CS

"^

,

8.»

8 e

8

8

"^ 4s

-«

iJS

«> S

«> Oj

»* «»

».*

*.2

^^

a.

a.

^«®

o «

o-S

^ g

1

ft. S

^•^

».

Oi

^

95

^

s

^

a.

CQ

05

C5

^

1

80

+

+++

42

58

28

14

3

99

—

+++

—

68

32

50

18

3

99

—

+++

—

70

30

54

16

4

99

—

+++

—

84

16

72

12

5

99

-f-

-H-f

—

82

18

78

4

6

99

—

-H4+

—

90

10

80

10

7

98

—

-H-++

—

86

14

78

8

8

99

—

-H+f

—

4+H-

85

15

74

11

9

96

—

+++4-

—

87

13

75

12

10

98

— ■

++++

—

82

18

71

11

Ordinary Daily Diet

11

80

+

-H-f

—

++

72

28

60

12

12

64

++

++

+

4-f

61

39

50

11

13

40

44+

+

++

-t-

40

60

23

17

14

15

-H-f

—

+++

—

42

58

20

22

15

6

+++

—

-H-f-

— •

38

62

14

24

TABLE 49— SUBJECT F

Ordinary Daily Diet + B. Acidophilus Milk 500 cc.

1

4

2

2

3

5

4

18

5

10

6

15

7

8

8

10

9

14

10

2

-H+ —

30

70

5

25

35

65

15

20

38

62

16

22

36

64

15

21

32

68

10

22

30

70

10

20

40

60

15

25

37

63

14

23

36

64

16

20

30

70

12

18

88 TRANSFORMATION OF THE INTESTINAL FLORA

great advantages, which will be dwelt on at greater length in another
chapter. Of special significance is the very marked and almost imme-
diate transforming power on the intestinal bacteria which such milk
culture possesses. Furthermore, the milk can be prepared by trained
specialists and technicians with comparatively little expense and with

TABLE 50— SUBJECT G

Ordinary Daily Diet + B. Acidophilus Milk 500 cc.

Oram-Stained Films from

Fecal

Suspensions

:i'=^

Total

Oram^Positive

•1"^

^

1-

Veillon Tubes

Organisms

Organisms

eq

<»

■" ■"

•»•§

4<&

^

,

,

e.'S

8 S

8

8

51h

C^-^

^1

8

1

*^ i

^(1

^TS

^

Qq

^

»»

^

8

^

^

cq

cq

?l!

^

1

68

+++

+

++

+

40

60

22

18

3

10

-H+f

—

+H-

—

30

70

13

17

3

8

+++

—

+++

—

33

67

15

18

4

6

-H-H-

—

+-H-

—

35

65

15

20

5

12

+++

—

++

—

38

62

16

22

6

10

44+

—

+++

—

35

65

14

21

7

6

■H-H-

—

+++

—

30

70

12

18

8

15

+++

—

-H-l-

—

42

58

15

27

9

30

+++

-t-

-H-

—

44

56

18

26

10

20

+++

—

-H-

— .

36

64

15

21

TABLE 51— SUBJECT F

Ordinary Daily Diet + B. Acidophilus Milk 1000 cc.

1

4

+4-H-

+4+

37

63

18

19

2

10

++4+

—

4++

—

35

65

20

15

3

20

44+

—

4+

—

38

62

22

16

4

8

+44

—

++4

—

41

59

23

18

5

65

4

H+

4-

44

58

42

38

20

6

78

4

44

4-

44

64

36

42

22

7

90

4-

444

—

444

80

20

60

20

8

94

—

4+4

—

444

78

22

65

13

9

88

4-

444

—

44+

76

24

64

12

10

92

—

44+

—

+++

75

25

60

15

Ordinary Daily Diet

11

60

44

4+

4

+

68

32

49

19

12

28

444

—

4+

—

62

38

40

22

13

4

4444

—

4++

—

44

56

26

18

14

10

4444

—

444

—

40

60

16

24

15

2

4444

—

44+

—

33

67

12

21

EXPERIMENTS WITH HUMAN SUBJECTS

89

|^^^v8<!$^<^<?^^

|.^<8'^>Sv§'|'S<^-^"^

l.^^^^^"?^.!^^-^^

®

§<R^8>^vS«5^'^'?~^"^

#v« >^^ '^^''J ^ ■^^

90 TRANSFORMATION OF THE INTESTINAL FLORA

a high degree of certainty of obtaining a uniform product which is
decidedly palatable and wholesome, to say the least.

It may be of interest here to state that subjects O and P were persons
who presented a history of intestinal disturbances. O had been subject
to most obstinate constipation requiring regular use of drugs to relieve

TABLE 52— SUBJECT G

Ordikary Daily Diet + B. Acidophilus Milk 1000 cc.

><

Oram-Stained Films from

Fecal

Suspensions

:i'^

'j;

Total

Gram-Positive

•IV

^

Veillon Tubes

Organisms

Orgi

inism^

^ a

1-

2

%.2

cq

<»

•S

'^ -is

V

<

^

§-1

§§.

8

8
» .«

OB .2

Hia

g-

S^

C) o

o J^

O-e

^ §

1

Oh g

^-e

1.

as

^

«, ^

e

e

^

a,

cq

^

C!

1

92

++

+++

+ +++

61

39

40

21

2

75

-H-

4+

+ ++

66

34

42

24

3

52

++

++

+ ++

60

40

44

16

4

64

-f-f

++

+ ++

58

42

42

16

5

78

++

4++

+ ++

64

36

48

16

6

95

4-

+4+

— +++

72

28

60

12

7

92

—

+4+

— 4++

80

20

65

15

8

96

—

+++

— +++

85

15

72

13

9

94

—

+H

— +4+

82

18

70

12

10

90

—

+++

— 4++

88

12

76

12

Ordinary Daily Diet

11

60

+

++

4- ++

70

30

57

13

12

32

■H4-

+

++ +

62

38

40

22

13

46

+4+

+

++ +

57

43

38

19

14

4

+++

—

4-4+ —

41

59

20

21

15

6

44++

—

+++ —

35

65

16

19

TABLE 53— SUBJECT B

Ordinary Daily Diet + B. Acidophilus Milk 1000 cc.

1

78

44+

+

++

+

44

56

28

16

2

88

++

+

53

47

38

15

3

94

+

4-

69

31

52

17

4

98

—

—

78

22

62

16

5

90

—

—

75

25

60

15

6

96

4-

—

82

18

70

12

7

98

—

—

86

14

75

11

8

97

—

—

80

20

70

10

9

98

—

+4++

—

4+++

87

13

78

9

10

99

—

+44+

—

+4+

84

16

76

8

EXPERIMENTS WITH HUMAN SUBJECTS

91

CO

<

i

|<5L^ rti. vj >§ ^ -^ .5 * <4

|<R>8'«t.vl'?'$<S^-5o

05
s

«9

«0 ^

Si. ««

« 5

Q ?»i

BQ

<0 -M

§H.S
Bq J.

8 GO

§^

s

Bq

1. (.

1 ^

8 S

92 TRANSFORMATION OF THE INTESTINAL FLORA
TABLE 54— SUBJECT H

Ohdinaet Daily Diet + B. Acidophilus Milk 1000 cc.

s-d

^

►si

Oram-Stained Films from

Fecal

Suspensions

:i'^

Total

Oramr-Positive

•1"^

^

/

Veillon Tubes

Organisms

Organisms

eq

-S-

A

•«

■s'-s

8

4«

h

e

«>

■ O

1

Oh i

^

an

§
^

^

oq

1

^

1

0

++++

444

28

72

10

18

2

4

++++

—

444

—

35

65

12

23

3

1

++4+

—

444

—

37

63

15

22

4

82

4-f

++

4

44

54

46

35

19

5

72

+++

4+

44

44

60

40

40

20

6

78

+

4+

4-

44

55

45

38

17

7

88

4-

4-H-

—

64

36

48

16

8

92

—

—

75

25

60

15

9

98

—

—

78

22

68

10

10

98

—

—

76

24

65

11

11

92

—

444-

—

70

30

58

12

12

94

4-

—

80

20

65

15

13

98

-f-

—

82

18

66

16

14

95

—

—

84

16

68

16

15

96

—

—

85

15

66

19

16

98

—

—

84

16

70

14

17

98

—

—

86

14

75

11

18

96

—

—

80

20

70

10

19

96

—

444+

—

83

17

74

9

20

92

—

44-1-

—

78

22

65

13

Ordinabt Daily Diet

21

84

+

4+f

—

444

66

34

52

14

22

70

+

44

4-

44

68

32

48

20

23

72

++

44

4

44

65

35

44

21

24

48

+ff

4

44

4

52

48

28

24

25

30

+++

4

44

4-

45

55

25

20

TABLE 55— SUBJECT M

Obdikary Daily Diet 4 B. Acidophilus Milk 1000 cc.

1

62

444

44

+4

44

64

36

38

26

2

96

44

444

4

4H

84

16

66

18

3

80

44

444

4

44f

80

20

55

25

4

88

—

+44

—

444

82

18

60

22

5

94

—

—

88

12

70

18

6

9Q

—

—

94

6

78

16

7

95

—

—

85

15

73

13

8

94

4-

—

90

10

75

15

9

98

—

—

92

8

84

8

10

96

—

—

90

10

86

4

EXPERIMENTS WITH HUMAN SUBJECTS 93

the condition for the time being. P, on the other hand, suffered from
chronic diarrhea acquired while he was residing in the tropics. The
stools were of the most offensive odor.

Both of these subjects responded readily to the acidophilus milk ad-
ministration, in so far as implantation of B. acidophilus is concerned,
as is seen in the accompanying tables (56 and 57). What is of further
interest is that subject O presented a specimen of stool for examination
every day but one during the thirty-five or more days that he took the
acidophilus milk. These specimens were from all appearances normal.

TABLE 56— SUBJECT O

Ordinary Daily Diet + B. Acidophilus Milk 1000 cc.

r«

Oram-Stained Films from

Fecal

Suspensions

«

&l

1"^

Total

Oram-Positive

•1'^

u
^

Veillon Tubes

Organisms

Organisms

^ 8

1-

2-

fe.S

Pq

<i>

p>

■id

i

1-1

^•8

^1

1

1

1

'^ 1

1^

|a

^

?5

s

^

s

^

1

86

+++

++

++

■H

39

61

22

17

2

92

•H+

■H-

-H-

++

65

35

41

24

3

96

+

4-

68

32

50

18

4

98

-f-

—

72

28

58

14

5

98

—

—

75

25

56

19

6

96

—

—

70

30

54

16

7

96

—

—

74

26

60

14

8

98

—

—

80

20

70

10

9

94

■h-

—

78

22

66

12

10

96

—

—

72

28

64

8

11

98

—

4-H-

—

76

24

65

11

12

98

-h-

—

82

18

70

12

13

99

—

—

85

15

78

7

14

90

-t-

—

84

16

74

10

15

96

—

—

83

17

75

8

16

95

—

4-H-

—

78

22

65

13

17

98

-f-

—

+++

80

20

72

8

18

95

-f-

—

82

18

70

12

19

96

+-

—

85

15

75

10

20

90

—

—

74

26

68

6

21

90

—

—

+H-

76

24

66

10

22

98

—

—

78

22

70

8

23

93

—

—

80

20

68

12

24

96

—

—

88

12

81

7

25

94

—

—

84

16

74

10

26

98

—

■H+

—

85

15

78

7

27

92

—

—

83

17

74

9

28

98

—

—

88

12

80

8

29

99

—

-H+

—

90

10

80

10

30

95

—

+++

—

85

15

76

9

94 TRANSFORMATION OF THE INTESTINAL FLORA

The subject claimed to have been improved in his general condition.
Within three or four days after presenting himself, a change was noted
in the stool of subject P from the diarrheal to what appeared to be a
normal specimen, and a complete disappearance of the offensive odor.
During the entire period of observation this individual expressed himself
strongly as having been very much benefited by the acidophilus milk.
These two cases are too few, however, to be of much significance.

TABLE 57— SUBJECT P

Ordikart Daily Diet + B. Acidophilus Milk 1000 cc.

^ ,c Gram-Stained Films from Fecal Suspensions

••• o

3 (^ ?§ Total Oram-Positive

'2 "o" 'w Veillon Tubes Organisms Organisms

^ e . 1 ^

^1 ^ f I l| l| o| ll

N I I ^ j^^ ^1 i'^ 1^

03 cq ?rs eq

C3

1

12

-H-H-

-fH- —

22

78

8

14

2

80

++

++

+ ++

38

62

28

10

3

92

+

— -I-H-

64

36

50

14

4

94

-h-

— +4+

79

21

64

15

5

98

—

— +++

75

25

65

10

6

96

—

— +++

74

26

66

8

7

93

—

— +-H-

76

24

66

10

8

90

-+-

— -f++

72

28

60

12

9

96

—

— +++

78

22

68

10

10

98

—

— -H-l-

84

16

75

9

Ordinary Daily Diet

11

82

■1-

+++

— -H4

80

20

68

12

12

60

-H-

+-f

-f- -H-

67

33

50

17

13

52

++

■H-

+ -H

48

52

36

12

14

50

++

++

+ -H-

45

55

30

15

15

30

++

++

+ ++

42

58

22

20

16

22

+++

-f-

4+ —

34

66

18

16

17

12

+-H-

—

-H- —

35

65

15

20

USE OF BACILLUS ACIDOPHILUS MILK REINFORCED
WITH LACTOSE OR DEXTRIN

The influence of the special carbohydrates as supplement to the milk
cultures of B. acidophilus was studied by adding 100 grams of either
lactose or dextrin to 500 cubic centimeters of the milk product, mixing
the two well by shaking in a large flask and administering them in one

EXPERIMENTS WITH HUMAN SUBJECTS

95

or two feedings (usually one) between meals to subjects who had failed
to respond very appreciably to the 500 cubic centimeters of B. acidoph-
ilus milk alone. In the single dextrin ingestion experiment the dextrin
was added to the milk in the form of a 50 per cent aqueous solution.

The results are given in the following tables (58 to 64). In every
instance the combination exerted a marked transforming influence on the
intestinal flora with a resultant simplification of bacterial types which
was comparable with the changes effected by 300 grams of the lactose
or dextrin or by 1000 cubic centimeters of the acidophilus milk alone.
(See Charts 39 and 40.) There was an almost complete absence of gas-
producing organisms in the Veillon tubes, and the Gram-stained slides
presented a bacterial picture in which B. acidophilus-like rods were
strikingly prominent. These observations were well supported by the
colonies on the whey agar plates. The discontinuance of the use of
lactose or dextrin and the milk cultures by subjects F and G after
feeding periods of twenty and ten days respectively resulted in the
return of the original mixed flora within five to ten days.

These observations are of particular interest in that they emphasize
the necessity of accompanying B. acidophilus administration with suit-
able carbohydrate and hence the value of the dextrin and lactose in a

CHART 40

\)/zf/mryCi7//y/P/e^

/ z 3 4- £ 6 7 a 9 /o /I /2 /3 /4- /SfeiJ

Curve indicating percentage of B.
acidophilus appearing in fecal
specimens from human subjects.
Diet
Ordinary daily diet
B. acidophilus milk 500 c.c.
Dextrin 100 gms.
Ordinate — Per cent of B. acidoph-
ilus.
Abscissa — Number of days after
initial administration of diet.

96 TRANSFORMATION OF THE INTESTINAL FLORA
TABLE 58— SUBJECT F

Ordinary Daily Diet + B. Acidophilus Milk 500 cc, Lactose 100 oms.

i

Oram-Stained Films from,

Fecal

Suspensions

:i'=^

^

Total

Oram-Positive

1:

1

Veillon Tubes

Organisms

Organisms

■S'-S

eq

H

8

09

1

•s

as

1

8

^

ftn

K5

8

BQ

05

^

1

30

44+

-H-

31

69

16

15

2

45

4-H-

+

++

+

37

63

12

15

3

42

■H+

+

++

■f-

35

65

23

12

4

78

-H-

++

+

++

42

58

28

14

5

86

++

-f-

++f

48

52

35

13

6

90

•t-

—

■H-f-

50

50

38

12

7

94

—

—

4-H-

64

36

50

14

8

88

-t-

+++

—

-m-

65

35

48

17

9

96

—

—

++f

60

40

48

12

10

92

-H

—

-H4-

70

30

56

14

11

90

—

—

+++

68

32

55

13

12

84

-1-

—

-H-f-

55

45

40

15

13

89

■f-

—

+++

63

37

56

7

14

95

-f-

—

4-H-

72

28

58

14

15

15

4-

—

4+f

69

31

53

16

16

76

+

++

-t-

-H

66

34

52

14

17

92

■t-

—

+4+

67

33

67

10

18

85

+

-H

+++

58

42

46

13

19

82

•f-

—

+++

56

44

42

14

20

87

+-

—

+++

53

47

40

13

Ordinary Daily Diet

21

62

+

-H

+

++

38

62

26

13

22

48

-H-

++

+-

++

40

60

25

15

23

32

4-H-

•f-

++

+-

34

66

20

14

24

16

-f+f

—

++

—

46

54

26

SO

25

6

+++

—

++

—

39

61

18

21

TABLE 59— SUBJECT G

Ordinary Daily Diet + B. Acidophilus Milk 500 cc. Lactose 100 oms.

1

42

2

84

3

92

4

96

5

90

6

95

7

98

8

99

37

63

18

19

55

45

42

13

60

40

48

13

76

24

63

14

70

30

60

10

68

32

56

13

72

28

64

8

82

18

72

10

EXPERIMENTS WITH HUMAN SUBJECTS 97

TABLE 60— SUBJECT G

OaoiKABY Daily Diet + B. Acidophilus Milk 500 cc, Dextrin 100 oms.

«

*

Oram-Stained Films from

Fecal Susp

ensions

:i'^

s

Total

Oram-Positive

•?'^

^

Veillon Tubes

Organisms

Orffanisms

^ f

1-

2-

CQ

«

•§

8

OS

a.

as

e

1

1

i

1

82

■f++

++

++

++

54

46

38

16

2

90

+

+++

-f-

68

32

54

14

3

90

-t-

+++

—

70

30

52

18

4

98

-f-

+++

—

88

12

78

10

5

98

—

++++

—

4-H-f

86

14

74

12

6

96

—

+++

—

89

11

75

14

7

99

—

+H

—

90

10

82

8

8

99

—

+++

—

85

15

76

9

9

99

—

-H-f

—

84

16

72

12

10

99

—

+++

—

88

12

81

7

Ordinaey Daily Diet

11

96

■H+

+++

80

20

68

12

12

84

+

-H+

■t-

+++

78

22

60

18

13

80

++

++

+

++

71

29

50

21

14

82

++

++

+

+

70

30

52

18

15

72

■H-

-H-

+

+

62

38

40

22

16

70

+++

+

•H-

+

60

40

44

16

17

62

+++

+

++

+

45

55

30

15

18

48

■H+

+

■H+

+

38

62

18

20

19

30

■fff

—

+4-f

—

31

69

15

16

20

6

+4+

—

-H+

—

33

67

12

21

TABLE 61— SUBJECT H

Ordinary Daily Diet + B. Acidophilus Milk 500 cc. Lactose 100 oms.

1

28

+4+

+

45

55

27

18

2

46

++

+

+

+

42

58

28

14

3

92

+

4-

65

35

50

15

4

96

—

—

73

27

61

12

5

94

—

—

75

25

65

10

6

92

4-

—

78

22

66

12

7

93

—

—

70

30

60

10

8

98

—

—

84

16

76

8

9

95

—

—

80

20

68

12

10

96

—

—

82

18

66

16

98 TRANSFORMATION OF THE INTESTINAL FLORA

flora-transforming dietary. There can be no doubt that the most
successful implantation of B. acidophilus is accomplished through the
combined use of the lactose or dextrin and B. acidophilus cultures,
which condition may be met by the use of sufficient acidophilus milk
alone.

TABLE 62— SUBJECT N

Ordinaby Daily Diet + B, Acidophilus Milk 500 cc.^ Lactose 100 oms.

►si

Qram-Stained Films from

Fecal

Suspensions

1*^

'S

Total

Oram-Positive

^

1

Veillon Tubes

a

Organisms

Org,

<xnisms

fe.^

Ol

'^

«

>

el

•M

"e

■^ ^

>*s

-«

^ S

« Oj

O-S

« •»

«B.2

<«>A

^

g.

O o

O S*

^ S

<s

1

Kftn

Ah g

ft.^

».

•9

^

«»

'^

Q

^

ft.

cq

i?

cq

5^

^

1

7

++4+

+++

33

67

12

21

2

28

++++

—

-H-f-

—

35

65

16

19

3

90

+

+++

4-

+++

63

37

50

13

4

98

-f-

H-H-

—

+++

76

24

62

14

5

96

—

+++

—

+++

82

18

70

12

6

98

-i-

•t-H-

—

■H+

80

20

72

8

7

99

—

+++f

—

-H-f+

88

12

76

12

8

99

—

++4+

—

-H-H-

85

15

74

11

9

96

—

-f-H

—

+++

86

14

76

10

10

98

—

+4-H-

—

+++

83

17

75

8

TABLE 63— SUBJECT Q

Ordinary Daily Diet + B. Acidophilus Milk 500 cc. Lactose 100 oms.

1

4

2

35

3

68

4

78

5

96

6

92

7

98

8

98

9

95

10

96

11

92

12

98

13

96

14

90

15

95

■H-

31

69

10

21

38

62

15

23

52

48

38

14

57

43

44

13

64

36

53

12

71

29

62

9

75

25

65

10

73

iW

66

7

70

30

60

10

72

28

58

14

69

31

58

11

78

22

68

10

74

26

65

9

70

30

58

12

80

20

72

8

EXPERIMENTS WITH HUMAN SUBJECTS 99

TABLE 64— SUBJECT R

Ordinary Daily Diet + B. Acidophilus Milk 500 cc. Lactose 100 oms.

rf*

Oram-Stained Films from

Fecal

Suspensions

«

1-

.1^

•••

Total

Oram-Positive

1-

Veillon Tubes

a

Organisms

Organisms

05

^2

-^

4

.«<

■svs

IS.

«> eg

s

s

^ i

Oh

fis

55

cq

^

oi

C5

05

1

0

++++

—

-H+

24

76

8

16

2

2

-H+f

—

+++

—

32

68

12

20

3

72

++

++

+

++

48

52

29

19

4

85

+

■f-

53

47

36

17

5

83

4-

—

62

38

48

14

6

87

-f-

—

65

35

50

15

7

96

—

—

72

28

62

10

8

94

—

—

70

30

62

8

9

98

■ —

—

78

22

68

10

10

96

-t-

—

84

16

72

12

11

90

+

■h-

70

30

63

7

12

94

-f-

—

73

27

62

11

13

97

—

—

82

18

74

8

14

88

+

—

75

25

66

9

15

96

—

—

76

24

68

8

ATTEMPTS TO IMPLANT BACILLUS BULGARICUS IN MAN

In the two following experiments B. bulgaricus was administered in
the form of milk cultures which were prepared with the same strains
that were employed in previous experiments on white rats. The method
of preparing these sour milk cultures of B. bidgaricus was identical with
the procedure followed for the B. acidophilus milk.

Two subjects were chosen who had been particularly susceptible to
the administration of B. acidophilus milk, 500 cubic centimeters of this
product being sufficient to bring about a very profound change in the
intestinal flora. The B. bulgaricus feeding was continued for ten days,
and the daily amount consumed was 1000 cubic centimeters, taken twice
a day between meals in 500 cubic centimeter portions. This was imme-
diately followed by a five day period in which the subjects were given
neither B. bulgaricus, B. acidophilus nor special carbohydrates. Fol-
lowing this control period, milk cultures of B. acidophilus were admin-
istered to the same two individuals, but instead of 1000 cubic centi-
meters being used, only 500 cubic centimeters were consumed daily.

100 TRANSFORMATION OF THE INTESTINAL FLORA

The ingestion of the large amounts of B. bulgaricus milk offered no
encouragement for the development of B. bulgaricus in the intestine.
(See Tables 65 and 66.) At no time could this organism be recovered
from the feces. On the other hand, an appreciable increase took place in
the numbers of B. acidophilus, and a partial transformation of the intes-
tinal flora was effected. The character and extent of the transforma-
tion were the same as when these same subjects (A and D) consumed
1000 cubic centimeters of ordinary daily milk along with the ordinary
diet. (See Tables 27 and 28.) The milk as such stimulated the growth
of B. acidophilus. In fact these two individuals showed some response
to even as little as 500 cubic centimeters of plain milk.

Within five days after the discontinuance of the B. bulgaricus milk
B. acidophilus gradually disappeared and the usual complex flora
again asserted itself. The results obtained in this series of experiments
with human subjects are in full accord with those of Hull and Rettger
(1917) and of our own on white rats (pages 11-64), and lead us to
the conclusion that B. bulgaricus is unable to adjust itself to the con-
ditions prevailing in the intestine of both man and the albino rat, and
that so-called implantation of this organism in the intestine is impos-
sible, at least under the conditions of the experiments thus far con-
ducted. (See Chart 41.)

After having demonstrated the inability of B. bulgaricus to implant

CHART 41

Bo

®

/oo
3o

@

SO
4c

A .^

Or(fmr/dff///c//e?

6o
So

4o

A 0rcf/nar/d7///6

3o

A A

"^*^***NJ

3o

t ^^» * ' 1 Y

Zo

l\ f

^V

2o

/o

/ V

^■"*>v

/o

\

0

/, T,. _

.

V>

o

£. — —

^ , a ^

/ z a 4- S 6

f e f /o // /z /d /4 /s

/ 2 3 4- 5 6 y a f /o n /z /S /4 /S

Curves indicating percentage of B. acidophilus and B. bulgaricus appearing i
fecal specimens from human subjects.

Diet — Ordinary daily diet, B. bulgaricus milk 1000 c.c.

B. acidophilus

B. bulgaricus ■

Ordinates — Per cent of organisms.
Abscissae — Number of days after administration of diet.

EXPERIMENTS WITH HUMAN SUBJECTS 101

TABLE 65— SUBJECT A

Ordinary Daily Diet + B. Bulgaricus Milk 1000 cc.

i

Oram-Stained Films from

Fecal

Suspensions

:i^

Total

Oram-Positive

•|v

'^

1-

Veillon Tubes

m

Organisms

Orgt

inisms

eq

t: fe

cq

«

<»
»

CQ

«.s

•i^

«*^

«*-, ts

"^

d.'ts

6 9

s

^

o

'^ i

>«

tt

« s

5> 5)i

J> *

«.j.

to °»

■♦.*

a

a.

O o

o ■»

O'e

O g

e

1

*.&.

(S:^

»« ®

.0^

^

1

09

B5

C3

1

fin

1

18

++++

—

-H4-

32

68

15

17

0

2

40

+

++

+

55

45

35

20

0

3

15

—

++

—

58

42

36

22

0

4

5

4+H-

—

+++

—

50

50

38

12

0

5

29

—

-H-

—

52

48

33

19

0

6

38

+

-H-

+

60

40

39

21

0

7

36

+

+++

■t-

65

35

48

17

0

8

30

+

+++

4-

62

48

40

22

0

9

32

+

++

+

64

36

44

20

0

10

26

+

++

-h-

66

34

45

21

0

Ordinary Daily Diet

11

16

—

—

46

54

30

16

0

12

15

—

—

42

58

28

14

0

13

10

—

—

39

61

21

10

0

14

0

—

—

32

68

10

22

0

15

4

—

—

30

70

12

18

0

TABLE 66— SUBJECT D

Ordinary Daily Diet + B. Bulgaricus Milk 1000 cc.

1

12

++++

+4-

40

60

21

19

0

2

32

+

44-

+

42

58

28

14

0

3

46

+

++■

+

38

62

26

12

0

4

30

+

+44-

+

55

45

39

16

0

5

28

+

++

+

48

52

30

18

0

6

36

+

++

+

66

34

38

28

0

7

35

+

4-+

+

62

38

40

22

0

8

32

+

++

+-

64

36

42

22

0

9

30

4-

++

4-

68

32

45

23

0

10

37

+

++

4-

60

40

40

20

0

Ordinary Daily Diet

11

22

++++

—

—

41

59

26

15

0

12

8

+44+

—

—

46

54

24

22

0

13

0

+4++

—

—

44

56

25

19

0

14

1

++++

—

—

45

55

25

20

0

15

3

++++

—

—

43

57

29

14

0

102 TRANSFORMATION OF THE INTESTINAL FLORA

itself within the intestinal tract of these human subjects (A and D),
the same individuals received 500 cubic centimeters of B. acidophilus
milk for twenty and ten days respectively in place of the 1000 cubic
centimeters of B. hvlgaricus milk. Within one to three days an almost
complete simplification of intestinal flora took place in both subjects,
as was shown by the whey agar plates, Veillon tubes and Gram-stained
films of the fecal suspensions. (See Tables 67 and 68, and Chart 42.)

TABLE 67— SUBJECT A

Ordinary Daily Diet + B. Acidophilus Milk 500 cc.

,^ -<! Orwm-Stained Films from Fecal Suspensioru

^ •<• o

.«^ ^ Total Oram^Positive

Total Oram^Positv,

Veillon Tubes Organisms Organisms

^1 ^ * * l-i l| I. I

8 "^

»3 8

§* §« O^ Oj* o-* o|

:2 I ^'^ fel fec^ t^

1

80

■ff

4

+

62

38

45

17

2

92

+-

444

—

69

31

54

15

3

99

—

444-

—

73

28

60

12

4

99

—

444

—

80

20

70

10

5

99

—

—

78

22

62

16

6

99

—

—

85

15

74

11

7

94

—

—

88

12

78

10

8

98

—

—

82

18

76

6

9

98

—

—

90

10

78

12

10

92

-f-

—

84

16

76

8

11

99

—

—

80

20

71

9

12

95

4-

—

83

17

72

11

13

90

—

—

88

12

76

12

14

98

—

—

80

20

66

14

15

92

—

—

85

15

78

7

16

96

—

—

87

13

75

12

17

99

—

—

90

10

82

8

18

99

—

—

80

50

72

8

19

96

—

—

86

14

75

11

20

98

—

—

88

12

78

10

Ordikaht Daily Diet

21

90

444

—

444

82

18

66

16

22

82

-f-

444

—

44+

77

23

58

19

23

70

+

44

4-

4+

68

32

50

18

Qi

44

++

44

4

4+

60

40

34

26

25

40

-H-

4

4+

+

39

61

19

20

26

45

++

4

4+

4

42

58

24

18

27

27

44+

—

4+

—

30

70

14

16

28

30

44+

—

4++

—

34

66

18

16

29

12

44+

—

444

—

35

65

15

20

30

8

4++

—

4++

—

36

64

12

24

EXPERIMENTS WITH HUMAN SUBJECTS

103

■k

^ ^

^

I® (^

^

^

1 J

^

^

^ y

^

^

1 r

«5
5{

y

i

t

^

y

>^

\

v»

\.

14

yi

>

^

•>

^^»..,,..._____^

N

^""•■^^.^^

V

l^^^s >5.<S .§ >$ <^ ^ ^ *>

t <

\

V J

'^

% ^.^■-^'^

«»

^ y'^

N

V

?l

5^

\

I

^

4

l^

\

^

1

^

i

^

\

«5

f

«J

i

is

\

^

/ .

^N

4

•O

\

K

/

«

•

•^

4

^^

V

n

\.

N

^»«s.^

•s

1 tS^«*<^vS'^ >$^ t? ^ ^

*&•

8

C3»

nS <3

8

c.c.

doph

inistr

A,

•M

o ^ S

1^

,

.*^

O "S -«

Co

'S ^ ^- «

r5

Oh

8

.S^'^

1

8

Q ^^ e S»

53
8

8

^

(>

S^Rh 'ir.

PQ

i-8

►13 7 O

o

'S i h

«+~.

« S ^

o

^ "O

8

B.

Ordinal
— Num

u

«>

^.

C3

^

2

a,

•B

Os

5

8

•o

'^

e

^o

^

8

•«>*

«0

^

bi

t.

8

O

104 TRANSFORMATION OF THE INTESTINAL FLORA

The typical mixed flora again appeared within ten days after discon-
tinuance of the acidophilus milk.

Note. — In the search for B. hulgaricus in the whey agar plates of the fecal sus-
pensions from the subjects receiving milk cultures of B. hulgaricus, several fluffy
colonies resembling B. hulgaricus and B. acidophilus were fished from each set of
plates. For identification the maltose test advocated by Rahe (1914) and studied at
some length by us was utilized. None of the transplants from the plates failed to
utilize maltose with acid production, and in every other respect there was lack of
evidence of the presence of B. hulgaricus.

TABLE 68— SUBJECT D

Ordinary Daily Diet + B. Acidophilus Milk 500 cc.

•ilb

•«

Orarrv-Stained Films from

Fecal

Suspensions

SD

a.

1^

Total

Oram-Positive

*>• .2

1-

Veillon

Tuhes

1

Organisms

Org,

anisms

a

"^

4

i«

el

« as

1-8

ll

e
*

a.

§

^

^

i23

1

44

+f+

+

++

+

41

59

22

19

2

88

+

-m-

-t-

63

37

51

12

3

96

—

■H+

—

60

40

48

12

4

90

—

-H+

—

67

33

56

11

5

98

—

+4-H-

—

82

18

68

14

6

92

—

+++

—

84

16

75

9

7

96

—

+++

—

85

15

72

13

8

98

—

H-H-

—

88

12

76

12

9

99

—

•H-H-

—

++++

86

14

78

8

10

99

—

+4+

—

■H+

90

10

76

14

Ordinary Daily Diet

11

90

-f-

+++

—

+J-f

78

22

66

12

12

85

+

4-H-

—

4+f

70

30

56

14

13

80

+

++f

4-

+4+

62

38

40

22

14

42

•H-

+

+

+

46

54

30

16

15

45

++

+

+

+

34

66

16

18

16

28

—

+

—

40

60

23

17

17

20

—

+

—

42

58

20

22

18

14

—

++

—

35

65

15

20

19

15

—

+4+

—

36

64

12

24

20

5

—

4-H-

—

31

69

14

17

The following two confirmatory experiments were carried on with
pure cultures of B. hulgaricus. Subjects F and H each received 300
cubic centimeters of a whey broth culture of the given organism daily
for ten days, in addition to the usual diet.

EXPERIMENTS WITH HUMAN SUBJECTS

105

The ingestion of the very large numbers of B. biUgaricus brought
about no apparent change in the intestinal flora. It was impossible to
recover B. hulgaricus from the plates, which were free from colonies of
this general type except an occasional B. acidophilus colony. There
was no reduction whatever in the amount of gas production in the
Veillon tubes, and the Gram-stained slides were typical of the usual
flora of so-called "normal" individuals. The results are shown in
Tables 68a and 68b, and Chart 42a. These results are in accord with
those obtained with white rats, and fully corroborate the findings in the
B. hulgaricus milk feeding experiments on man.

TABLE 68A— SUBJECT F

Ordinary Daily Diet + B, Bulgaricus 300 cc.
(Whey Broth Culture^ Neph. 5)

Oram^Stained Films from Fecal Suspensions

1(=^

1.

Total

Oram-Positive

•?'^

s

Veillon Tubes

Organisms

Organisms

^ 8

CQ

2

a

1

cq ^

cq

•S

8

« as

e

99

2

^

03 i^s

CQ

^

^

1

0

++++

+++

—

16

84

6

10

2

0

44-H-

— +++

—

18

82

5

13

3

0

-H-H-

— +++

—

24

76

8

16

4

0

44++

— +++

—

22

78

7

15

5

0

++++

— +++

—

25

75

8

17

6

0

++++

— +++

—

28

72

10

18

7

No

Specimen

8

0

++++

— +++

—

35

65

12

23

9

No

Specimen

10

0

++++

— ++++

—

38

62

10

28

TABLE 68B— SUBJECT H

Ordinary Daily Diet + B. Bulgaricus 300 cc.
(Whey Broth Culture, Neph. 5)

1

0

2

0

3

0

4

0

5

0

6

0

7

0

8

0

9

0

10

0

— -fH- —

15

85

3

12

12

88

4

8

21

79

7

14

32

68

15

17

23

77

10

13

30

70

12

18

25

75

9

16

33

67

13

20

20

80

6

14

36

64

17

19

106 TRANSFORMATION OF THE INTESTINAL FLORA

CHART 42A

/oo

A>o

a,

0

6o

®

7"

^o

^

4o

4&

3o,

3o

Zo

2o

o

•

o

/ zayf-Se-zaf/o/r/z/a m/£

/ Z 3 -^ S 6 / S

T /o // /z a A^AS

Curves indicating percentage of B. bulgaricus appearing in fecal specimens from

human subjects.

Diet

Ordinary daily diet

B. bulgaricus 300 c.c.

(Whey broth culture Neph. 5.)

Ordinates — Per cent of B. bidgaricus.

Abscissae — Number of days after administration of diet.

INCOMPLETE ABSORPTION OF LACTOSE FROM THE

INTESTINE

In the feeding experiments with rats a direct correlation was estab-
lished between the transforming influence of lactose and dextrin on
the intestinal flora and the presence of a reducing substance in the
large intestine and in the feces. It was assumed, therefore, that these
carbohydrates owed their Bacillus acidop}iilus-?,imm[3iimg influence to
their incomplete absorption from the enteric tract. This view was
strengthened by our failure to determine the presence of reducing sub-
stances after the feeding of the other carbohydrates, glucose, maltose
and sucrose, which lacked the power of in any way effecting a change
in the flora.

Since lactose and dextrin so readily effect a simplification of the
intestinal flora in man also, attempts were made to demonstrate their
presence in the stools of subjects receiving them. The experiments on
lactose alone have been carried to completion, and are presented here.

The stools of four subjects (A, C, D and L) who received 300
grams, and of a fifth who took 400 grams, of lactose daily for ten days,
in addition to the daily diet, were studied at some length. Besides the
usual bacteriological examinations for the determination of type dis-

EXPERIMENTS WITH HUMAN SUBJECTS 107

tribution, tests were made for the presence of reducing substances, the
technique being the same as that employed in the experiments with white
rats (page 58), with the exception that three grams of the human feces
were employed and added to fifty cubic centimeters of dilution water.
The results are shown in the following table (69). In this table are
included also the results obtained with two control subjects, that is
persons who were not receiving the special carbohydrate. The stools
from the subjects taking lactose gave an unmistakable reduction with
Benedict's solution, and harbored a flora almost completely dominated
by B. acidophilus, while those of the controls possessed no reducing
properties and contained the usual mixed flora. The conclusion is
drawn, therefore, that in man, as well as in rats, the lactose, when in-
gested in sufficient amounts to transform the flora, is not completely
absorbed before it reaches the large intestine, and that it serves as
particularly utilizable pabulum for the development of B. acidophilus
in the large intestine, where the activities of intestinal organisms are
greatest. Hence, an optimum environment is created in the lower
intestine for B. acidophilus. This correlation between the simplifying
property of lactose and its incomplete absorption from the enteric tube
must be viewed as of considerable significance.

TABLE 69

The Relation of Diet to Reducing Carbohydrates and to the Bacterial Flora in
Fecal Specimens from Human Subjects

Reduction of Per Cent of

Subject Diet Period of Feeding Benedict's Solution B. Acidophilus

Control
F >. 10 days - 1

.s ^ .Si

I %^^ 10 days - 2

A -3* i 10 days + 97

*3 bo

C ^*.§ 10 days + 91

D 1^1 10 days + 99

L O ^ 10 days + 87

cd be

^•.a^ 10 days + 89

108 TRANSFORMATION OF THE INTESTINAL FLORA

RELATION OF HYDROGEN ION CONCENTRATION TO THE
CHARACTER OF THE INTESTINAL FLORA

In the feeding experiments with albino rats no definite relation could
be established between the hydrogen ion concentration and implantation
of B. acidophilus in the intestine. Similar attempts have been made
more recently to determine whether the rapid development of aciduric
organisms following lactose feeding is the result of increased acidity
of the intestinal contents. The same methods were employed as in the
work on the rats (page 61) except that for the hydrogen ion deter-
mination tests of human stools three grams of fecal material were sus-
pended in fifty cubic centimeters of neutral water. The subjects in-
cluded five individuals who received either 300 or 400 grams of lactose
daily, five who took 150 grams of lactose and 150 cubic centimeters of
whey broth culture of B. acidophilus, five who received 300 cubic centi-
meters of the broth culture, two that were given 1000 cubic centimeters
of B. acidophilus milk daily, and two controls subsisting on the ordinary
daily diet. A total of thirty-eight hydrogen ion concentration deter-
minations was made. The bacteriological findings are presented here
along with the pH figures (Table 70).

TABLE 70

The Relation of Diet to Hydhooen Ion Concentration and Bacterial Flora in
Fecal Specimens from Human Subjects

Suspension of Feces

Suspension of Feces

Diet '

8 Days Aft

er First Feeding

10 Days Aft

■er First Feeding

Subject

Per Cent of

Per Cent of

Ph

B. Acidophilus

Ph

B. Acidophilus

Control

F

dinary

Daily

Diet

6.4

0

6.1

1

I

O

6.0

3

6.2

2

A

ally
gms.

5.6

98

5.5

97

C

6.2

95

6.4

91

D

dina

D

;tose

5.0

99

5.1

99

L

o 3

6.4

70

6.3

87

EXPERIMENTS WITH HUMAN SUBJECTS 109

TABLE 70 {Continued)

Suspension of Feces

Susp

ension of Feces

Diet "

SDaysAfti

er First Feeding

10 Days

After First Feeding

Subject

Per Cent of

Per Cent of

Ph

B. Acidophilus

Ph

B. Acidophilus

aily
gms.

Q o

B

Ordinary

Diet

Lactose 40

6.0

86

6j2

89

^-v

*o

w s:

F

Diet

50 c
Nep

6.2

95

6.0

60

G

>,'=^'^ j:

6.0

98

6.1

94

H

y Dail
se 150
philus
th Cull

6.4

95

6J

92

I

inar

ICtO!

cido
Bro

6.5

95

6.S

93

K

t

6.0

99

6.4

99

^-s.

«5

A

Diet
00 cc.
Neph

5.9

90

6.0

94

B

aily
us 3
ult.,

6.5

90

6.4

93

C

6.0

90

6.6

90

D

inar
cido
Bro

5.8

95

5.6

9S

E

Ord

B.A

(Whey

6.0

95

6.8

96

4J J^

V 7S

Ss

F

Daily
3hilus
0 cc.

6.4

94

6.3

92

G

Ordinary

B. Acido

IOC

6.2

96

6.0

90

No correlation exists betwen acidity and the prevalence of B. acidoph-
ilus in the stools, according to the results of these experiments. While
there is an appreciable range within which the hydrogen ion con-
centrations vary, the differences and the extremes are no greater than

110 TRANSFORMATION OF THE INTESTINAL FLORA

for the combined list of controls and subjects receiving the whey
broth culture of B. acidophilus, except subject D and perhaps A whose
figures were as low at 5.0 and .5.5 respectively. These two subjects were
very susceptible to lactose feeding, requiring much less than 300 grams
to effect a simplification of the flora. When they received as much as
300 grams daily the implantation of B. acidophilus was practically
complete, and there was a tendency toward a diarrheal condition.
There was a large remnant of lactose in the feces, and the conclusion
is to be drawn that the excess of lactose in itself caused some intestinal
irritation either directly or through the production of more acid than
could be neutralized or absorbed as quickly as it is formed. However,
these figures are well within the range of normal stools, as will be seen
by comparison with the results of Howe and Hawk (1912) and Nelson
and Williams (1916-1917). Certainly there can be no question of in-
creased acidity in the stools of subjects who received 150 grams of
lactose together with the 150 cubic centimeters of B. acidophilus cul-
ture, as compared with those who took no lactose at all.

IV. A FULL ACCOUNT OF THE PREPARATION

OF BACILLUS ACIDOPHILUS MILK FOR

HUMAN CONSUMPTION, AND OF

ITS KNOWN PROPERTIES

Much time and effort have been given in recent years to the pro-
duction of sour milk and sour milk products. Milk soured with B.
bulgaricus powders or tablets and special preparations sold as koumiss
have gained wide usage, especially in this country. Irrespective of the
alleged therapeutic properties from the standpoint of bacterial im-
plantation in the intestine, these preparations are of undoubted merit.
They are a valuable food and serve as a substitute for ordinary milk
for which many persons have little or no tolerance and which to many
others is objectionable as such. Furthermore, these sour milk products,
when they have been prepared successfully, are beverages which have
attained considerable popularity.

Bacillus acidophilus milk bears some resemblances to sour milk pre-
pared with pure cultures of B. bulgaricus or with bulgaricus powders
or tablets. Coagulation of the casein takes place in both, due to acid
production, and no gas is produced. There is an absence also of other
products of the ordinary putrefactive type. Both are distinctly acid
to the taste, and unless the B. bulgaricus milk has been incubated too
long, they are palatable.

The two products differ in the following respects. The acidophilus
milk never attains the degree of acidity in old culture as does the bul-
garicus milk ; in fact there is little or no danger of the former becoming
too sour if the time of incubation is within reasonable range, which
cannot be said of the other product. The acidophilus milk acquires a
creamy, but never sticky or stringy, consistency, while the bulgaricus
milk has a thicker and at times a more or less slimy character, depend-
ing largely, of course, on the particular strains of B. bulgaricus
employed in the preparation. The acidophilus milk has a decidedly
perceptible aroma and taste, which add very materially to its palata-
bility and which are more or less absent from the bulgaricus milk.

The following method is employed in the preparation of the Bacillus
acidophilus milk.

Sweet skimmed milk is sterilized by autoclaving at an extra pressure

112 TRANSFORMATION OF THE INTESTINAL FLORA

of fifteen pounds for twenty to thirty minutes, or longer when more
than 1000 cubic centimeters are sterilized in a container. For this
purpose Pyrex Florence flasks have been employed in the present in-
vestigation. The cooled milk is inoculated with a pure culture, prefer-
ably mixed strains, of B. acidopJtilus, and incubated at 35 to 37° C.
for twelve to twenty-four hours. The inoculum should be a milk culture
not more than seventy-two hours old.

It is at this point that special effort is required to obtain a product
that is well soured and viable. Recently isolated strains of B. acidoph-
ilus are slow and ineffective. Therefore, it is necessary to employ
strains which have been grown in milk for at least two or three weeks
and which have been transplanted from milk to milk at frequent inter-
vals, preferably every day. The amount of inoculum for each trans-
plantation should be large, as compared with the usual procedure.
For every 1000 cubic centimeters five to ten cubic centimeters of the
inoculum are required to obtain maximum development within twenty-
four hours' time. The transfers are made with sterile pipettes made
from sections of glass tubing. These pipettes are drawn out at one end,
but the opening must be large enough to admit coagulated milk. Plati-
num loops are useless for making the transfers.

As soon as the milk has undergone coagulation it is removed and
placed in the refrigerator. It is important that the refrigerator com-
partment in which the milk is kept be clean and free from odors which
may be absorbed. When kept under these conditions the milk changes
very little in the course of a few days, but should be used very soon.

If it is desired to reenforce the milk with lactose, the sugar may be
added at the time the milk is taken. It will be expedient to shake the
lactose and milk well and allow the mixture to stand for thirty minutes
or more in a cool place. This will allow a goodly portion at least of
the sugar to dissolve and to reduce the gritty character of the lactose-
enriched milk. For those who crave sugar the addition of the lactose
greatly adds to the richness of the milk.

In the present investigation four strains of B. acidophilus have been
employed for the production of acidophilus milk. These strains were
isolated from human feces in October, 1919, and were grown on whey
agar for five months, transfers being made three times a month. For the
inoculation of the first milk tubes the four strains were combined.
Transfers were made almost daily. At first it required five or six
days, and at times even more than a week for the milk to show indica-
tions of acidity and coagulation. The coagulation time was gradually
shortened tintil three weeks after the first milk culture was prepared,
when coagulation was obtained within twenty- four hours. At the end
of the fourth week of almost daily transfers the coagulation time was
reduced to twelve to fifteen hours, where it stands at the present time,
many months after the first milk inoculation.

PREPARATION OF BACILLUS ACIDOPHILUS MILK 113

Experiments are in progress to determine the influence of temperature
on coagulation time. Most rapid growth and acid production take
place at 35 to 37° C. Very satisfactory results are obtained also at
30°, though in a somewhat shorter period. At 20° slow coagulation
occurs, while below this temperature little or no change takes place in
the appearance of the milk during the first three or four days. It
appears from these experiments as if incubation at 30° may be substi-
tuted for the 37° temperature, providing the organisms are quite viable
in milk, as in the present instance.

Experiments are under way also to determine whether sterilization of
the milk, prior to the inoculation, is absolutely necessary. Instead of
heating the milk in the autoclave it is heated in a double boiler or in a
flask deeply submerged in boiling water for forty-five minutes to one
hour. This treatment does not destroy spores and hence the milk is
not rendered sterile. Inoculation with B. acidophilus was followed by
incubation of duplicate samples at different temperatures, 20°, 30°
and 37° C. Control flasks, that is flasks which were heated in the same
way as the others, were incubated without being inoculated with B.
acidophilus, and incubated beside the others. All of the flasks were
examined at twelve hour intervals when whey agar plates were poured
and Veillon tubes were inoculated from the different flasks.

Within twenty-four hours the milk flasks that had received the B.
acidophilus inoculum and were incubated at 30 and 37° underwent the
characteristic acidophilus coagulation, and there was no indication by
odor or physical appearance that any putrefactive action was in prog-
ress. These observations were fully corroborated by the plates and
Veillon tubes, which contained pure or practically pure cultures of
B. acidophilus. The control flasks, on the other hand, gave marked
evidence of putrefaction within twenty-four hours. There was partial
coagulation and later a very extensive liquefaction of the casein, the
liquid appearing watery, accompanied by offensive odors. Similar
changes took place in the corresponding flasks incubated at 20° C,
but they were very much slower.

These experiments indicate that, while B. acidophilus does not pro-
duce as much acid in milk as do B. bulgaricus and Streptococcus lacti-
cus, it nevertheless protects the milk against undesirable decomposition
by so-called putrefactive organisms, and if viable and present at the
outset in considerable numbers, it should render milk safe against de-
composition harmful to the consumer. Whether boiled milk may be
substituted for the sterilized and the same satisfactory product can be
obtained has not yet been determined. Boiled or incompletely sterilized
milk must be inoculated immediately, however, before any bacterial
change has taken place. Hence, from the practical standpoint, sterili-
zation will be necessary to obtain constant and reliable results.

V. METHODS EMPLOYED IN THE ROUTINE
EXAMINATION OF FECES

No phase of bacteriology has been more seriously handicapped by
the lack of adequate methods than the study of intestinal micro-
organisms. The most recent contribution is the work of Morris, Porter
and Meyer (1919), who advocate the technique employed by them in
their bacteriological examinations of children's stools. While these
methods may be of considerable value in the hands of trained technicians,
and for certain purposes, they have not appeared practical and suffi-
ciently direct to be of material help in the present investigation. It has
been our aim to employ the simplest technique that is consistent with our
purpose, namely to determine at frequent intervals the relative numbers
of B. acidophilus-like bacteria as compared with the total of all other
organisms, and to follow the progress of gas-producing organisms as
they increase or decrease in numbers in the fecal material examined.
The following methods were adopted early in this work and have con-
stituted a large part of the daily routine examinations. They are pre-
sented at some length here because they have proven themselves prac-
tical and in a high degree satisfactory.

THE USE OF WHEY AGAR PLATES

At the outset 1 per cent glucose agar was employed for plating, etc.
This was soon discarded in favor of whey agar which by a series of
comparative tests was found to be superior in every way to the other
media under consideration. Many difficulties were at first encountered,
however, in the preparation of clear whey from skimmed milk. These
difficulties were eliminated as soon as the following method was
perfected.

The skimmed milk is heated to 80 to 90° C, and five cubic centimeters
removed to a test tube; while the test sample is still hot it is treated
with 10 per cent solution of hydrochloric acid drop by drop until com-
plete coagulation takes place. This is repeated two or three times.
The calculated amount of acid required to coagulate all of the milk i^
thoroughly mixed with the milk, the temperature of which is still 80
to 90°, and the casein allowed to settle. This is followed by filtration.

ROUTINE EXAMINATION OF FECES 115

at first through absorbent cotton and then paper, and neutralization
of the filtrate with sodium hydroxide to pH 6.8-7.0. Before further
filtering, 0.5 per cent peptone is added and the medium autoclaved for
fifteen minutes at fifteen pounds extra pressure. The albuminous
material is filtered off through paper, and the clear filtrate employed as
such for whey broth or for the preparation of whey agar. To convert
into whey agar add 1.2 per cent of standard shredded or powdered agar
to the whey broth.

The agar plates were prepared by the usual dilution process. The
first tube of liquefied whey agar was inoculated with a four millimeter
platinum loop, a bi-convex loopful of the standardized fecal suspension
(see pages 11-12) being employed. For further dilutions, three and
five loopfuls of tubes one and two were transferred. The plates were
incubated for forty-eight hours at 37° C. under aerobic conditions.

B. acidophilus colonies were recognized with the aid of the micro-
scope, and their relative number as compared with all other colonies
determined. The counting was facilitated by the use of an ocular
which is marked off in small squares. There are two types of agar
colonies of B. acidophilus designated by Horton and Rettger (1914)
as X and Y. The former have a decidedly fuzzy appearance and re-
semble small agar colonies oi B. tetani; these are indistinguishable from
the colonies of B. bulgaricus. (See Plate II.) The Y type is small
and round to spindle-shaped and is only partly fringed, at times appear-
ing almost perfectly smooth.

When in some of the experiments there was any doubt as to whether
the observed colonies were those of B. acidophilus or B. bulgaricus
transfers were made into maltose (1 per cent) broth and the tubes in-
cubated at 37° for at least forty-eight hours. B. acidophilus attacks
the maltose and causes a decided acidity which is easily recognized by
any of the ordinary tests. B. bulgaricus produces no such change.
Morphological studies and milk coagulation tests were made also in some
instances.

The study of whey agar plates gave the most valuable information
in so far as the relative numbers of viable B. acidophilus are concerned,
and in a most satisfactory way showed the changes that were taking
place in the flora from the complex to the simple acidophilus phase, and
vice versa. The results are given in the tables in terms of percentage.

VEILLON TUBES

The Veillon tube as described by Veillon and Zuber (1898) was em-
ployed, with the following modification. Instead of one end being
permanently sealed, both ends were left open, and the tube was nothing
more than a nine-inch section of one centimeter glass tubing. Previous

116 TRANSFORMATION OF THE INTESTINAL FLORA

to filling, one end of the tube is tightly plugged with a rubber stopper.
After filling, the other end is plugged with non-absorbent cotton.

The Veillon tubes are filled about twelve centimeters deep with the
1.2 per cent whey agar described above, and sterilized in the autoclave.
The inoculation of the tubes was accomplished in the same way as the
agar tubes that are used for immediate plating. It is of much impor-
tance to mix the agar and inoculum thoroughly, and this is done best
by tilting the tubes back and forth somewhat vigorously, even though
the cotton plug becomes soiled. The formation of gas bubbles must be
avoided, however. The tubes are incubated for at least forty-eight
hours at 37° C. The colonies are examined with the aid of a hand lens
which magnifies at least four or five times.

These tubes are well adapted for the study of both aerobic and
anaerobic organisms as well as the intermediate forms, as for example
B. bifidus. Furthermore, they present an excellent index of the presence
of gas-producing organisms found in the intestine, namely B. coli and
B. welchii, and of their relative numbers. For this purpose alone they
have been of inestimable value not only for the study of rat, but of
human feces as well. Gas-producing bacteria in rat feces are usually
relatively few, but in the stools of man they are as a rule very abun-
dant and it was feared at first that the agar would be so thoroughly
broken up as to make it impossible to study the individual colonies
with any degree of satisfaction. This obstacle is encountered in the
examination of ordinary (often termed "normal") feces of man, but in
direct proportion to the transformation that is brought about in the
intestinal flora by the agents which have been successfully employed in
this work, the number of gas-producing bacteria correspondingly
diminishes and often the tubes remain entirely free from gas in the
course of the experiments.

Bacillus acidophilus is recognized in the Veillon tubes by the rather
small fuzzy or sea-urchin-like colonies which develop particularly well
in the upper and middle layers of the agar. Colonies of B. bifidus are
small, disc-like and smooth, and are most abundant in the three to six
centimeter layer which begins about one centimeter below the surface.
The colonies of the strict anaerobes make their appearance in the depth
of the agar. The different colonies may be reached for subculture study
and microscopic examination by removing the rubber stopper, drawing
or forcing the agar column out of the lower end of the tube, and fishing
with a platinum wire or a very fine-pointed pipette, or by filing and
breaking the tube in the immediate vicinity of the chosen colony. In
the present work almost the entire emphasis was placed on B. acidoph-
ilus colonies and gas production. These are indicated in the various
tables by the following signs: — , -+- — , +, H — h> H — I — h and -\ — | — | — h>
as has already been explained on page 12. (See Plate V.)

ROUTINE EXAMINATION OF FECES 117

DIRECT MICROSCOPIC EXAMINATION OF FECAL
SUSPENSIONS

Films were prepared from the standardized suspensions of fecal
specimens and stained by the Gram-method. The slides were counter-
stained with dilute carbol fuchsin. Such slides are of considerable value
in that they show the relative predominance of the different types of
bacteria, and emphasize in particular the conspicuous large Bacillus
acidophilus-like rods when they are present in any numbers. This
method has serious limitations, however, in that it does not give a true
picture of the viable organisms which are present in the intestinal tract.
Many of the rods and cocci that are seen in these preparations are dead
at the time of leaving the animal host, although they take one or the
other of the stains. Furthermore, such dead bacteria may continue
for considerable periods to be eliminated, owing to their being held back
in the folds and on the inner walls of the intestine.

The results of the examination of stained films were recorded ver-
bally in the tables summarizing the data from the rat-feeding experi-
ments, while at the time the human subjects were employed they were
recorded in terms of percentage of Gram-positive and Gram-negative
organisms and Gram-positive rods and cocci. As will be seen in the
different tables these results correlated definitely with those obtained
by the use of whey agar plates and the Veillon tubes. The relative
numbers of aciduric organisms as compared with all other organisms
present in human feces were as a rule smaller by the direct than the
culture methods, and in many instances very considerably smaller.
In the examination of the rat feces, on the other hand, the direct count
was in perfect agreement quantitatively with the results of the other
two methods. In spite of the recognized limitations of this method, it
has proven itself to be indispensable in the present investigation.

VI. GENERAL DISCUSSION AND SUMMARY

Under conditions of normal functioning the intestinal flora is funda-
mentally a physiological unit rather than a heterogeneous collection of
adventitious microorganisms, and bears a definite relation to the diet
of the host.

The present study has shown that the types of bacteria developing
in the alimentary tract may be influenced in a noteworthy manner, not
only through special alterations in the chemical composition of the diet,
but also through the ingestion of living cultures of B. acidophilus with
or without accompanying carbohydrates. Only two of the carbo-
hydrates here employed, however, have the property of stimulating the
aciduric bacterial type, namely lactose and dextrin.

Lactose and dextrin, when added in given amounts to the basal diet
(bread and meat) of the white rats or when taken by men in addition
to the usual daily diet, brought about a marked transformation of the
intestinal flora within four to six days, and stimulated the development
of B. acidophilus to such an extent that it largely or entirely dominated
the flora, eff'ecting an almost complete suppression of all other bacterial
types commonly found in the enteric tract. The simple flora persisted
as long as the feeding of these carbohydrates was continued. B. bifidus
occasionally increased in numbers under these dietary conditions, but
usually remained relatively obscure. It never became the dominant
type.

These results are in harmony with those of certain other observers.
Barker (1914) and Torrey (1915) claimed to have secured favorable
results with the feeding of lactose and milk to typhoid fever patients.
Torrey demonstrated that the administration of a high carbohydrate
(lactose) diet tended to reduce the putrefying types of bacteria, and to
favor the growth of organisms of the B. acidophilus type. Our results
are in accord also with those of earlier investigations reported by Hull
and Rettger (1917) and the more recent announcements of Torrey
(1919), but they are diametrically opposed to those obtained by
Sisson (1917) in similar feeding experiments with puppies. Sisson
reported that a milk diet containing 10 to 15 per cent of lactose did
not cause an intestinal flora essentially difi'erent from that following a
diet of whole pasteurized milk. In fact, he failed to find any organisms
of the B. acidophilus type in any portion of the alimentary canal.

In a short paper by Distaso and Schiller (1914) some feeding

GENERAL DISCUSSION AND SUMMARY 119

experiments with white rats were reported which indicated that lactose
and dextrin exercise a marked transforming power on the intestinal
flora, bringing about a predominance of B. bifidus. These results are
in agreement with our own in principle, but differ in that they placed
chief emphasis upon the Tissier organism in their findings, whereas in
our experiments B. acidophilus played the role of primary importance.

Hull and Rettger (1917), as the result of limited observations on
this particular carbohydrate, reported that the administration of dex-
trin to albino rats did not cause any noticeable increase in the numbers
of B. acidophilus. These findings are in disagreement with the results
of dextrin feeding in the present investigation, and may have been due
to the idiosyncrasies of the few rats that were used by Hull and Rettger
in the dextrin feeding experiments.

Kendall's suggestion (1911) that the feeding of liberal amounts of
lactose to infants may cause an abnormal or excessive development of
B. welchii, and similar contentions of other observers, receive no support
whatever from the present studies. While there can be no doubt that
B. welchii ordinarily finds favorable conditions within the intestinal
tract of man and of the albino rat, the feeding of lactose has at no time
caused an increase in the numbers of this organism. On the contrary,
there was always a marked reduction, and at times complete elimina-
tion of not only B. welchii, but of B. coli as well.

Following the administration of two grams of lactose or dextrin in
association with the basic diet, B. acidophilus made its appearance in
all parts of the intestine of white rats, even the duodenum. Whether
the presence of this organism in appreciable numbers in the small intes-
tine was due to the proliferation of bacilli already occurring there, or
to an upward movement of bacilli from the large intestine must of
course still remain undetermined.

Maltose, saccharose and glucose appeared to exercise no transform-
ing influence on the types of bacteria present in the intestinal tract of
white rats. These results are in perfect agreement with those of Dis-
taso and Schiller (1914) and Hull and Rettger (1917). Torrey
(1919), however, claims to have obtained a moderate increase in the
numbers of B. acidophilus as a result of sucrose feeding in dogs. The
findings in the present investigation, which run almost parallel with
those encountered in the bacteriological examinations of the fecal
specimens of rats subsisting on the basal diet alone, emphasize the im-
portance of the selection of special carbohydrates (lactose and dextrin)
for favorable transformation of the intestinal flora, and not merely
a carbohydrate, as is so often recommended.

The most plausible explanation of the favorable influence of lactose
and dextrin feeding on the implantation of B. acidophilus is one which
to a large extent rests upon the fact that they are not completely
destroyed before reaching the large intestine. There they establish an
optimum environment by serving as a readily available source of energy

120 TRANSFORMATION OF THE INTESTINAL FLORA

for B. acidophilus. As this organism is normally present in the intes-
tine, though usually in small numbers, and merely requires a stimulus
to further development, which is furnished by the dextrin and lactose,
the conditions for its development upon the administration of these
carbohydrates become even more favorable than for any of the other
intestinal organisms. This theory was advanced by Hull and Rettger
(1917) who also reported the presence of a reducing substance in the
feces of rats fed on lactose. The other carbohydrates used in this
investigation, on the other hand, do not reach the large intestine, where
bacterial activities are at their greatest, and hence are unable to exert
any influence on the putrefactive processes going on here.

That the stimulating influence of lactose and dextrin is not due to
acids produced from these substances is strongly indicated by the
absence of increased hydrogen ion concentration in the fecal materials
from the caecum and colon of rats and from the feces of men receiving
these carbohydrates. This observation is in harmony with those of
other investigators. Hirschler (1886) concluded that the presence of
acid in the intestine is not in itself sufficient to prevent putrefaction.
Winternitz (1892) claimed that lactose when fed to animals or man
exerted an inhibitory influence on putrefaction, but that this action
was due to the sugar itself, and not the lactic acid formed from it.
Rovighi (1892) pointed out that the ingestion of lactic acid had only
a very slight influence on intestinal putrefaction, and Fischer (1915)
observed that acid played no part in inhibiting indol formation.

While the multiplication of B. acidophilus is stimulated to a greater
or less extent, the transformation brought about by the feeding of one
gram of lactose or dextrin to white rats or 150 grams to man seldom
amounted to more than 50 per cent, and with a few exceptions in man,
there was no radical suppression of other types of bacteria developing
in the intestinal tract. In order to obtain complete transformation
two grams of lactose are required for rats, and as a rule 300 grams
for man. This is in accord with findings of Hull and Rettger (1917),
who showed further that with increases of lactose beyond two grams
daily B. bifidus tended more and more to acquire prominence in the
feces of white rats, and that when as much as three grams was fed
daily the Tissier organism assumed the chief role, in place of B.
acidophilus.

The successful transformation brought about in rats by the com-
bined action of one gram of lactose or dextrin and one cubic centimeter
of B. acidophilus suspension, and of 150 grams of lactose and 150 cubic
centimeters of culture in man offers a new avenue of approach to the
intricate field of bacterial implantation within the intestinal tract.

What appears to us to be of no less significance is the demonstration
in the present investigation that the administration of the acidophilus
suspension alone, that is, without added carbohydrates, results in the
implantation of B. acidophilus and suppression of the other bacterial

PLATE I

Figure 1

.i:^

^

-fl

Figure 2

[See exnlanation of Plates on d. 1 25

PLATE II

Figure 3

■■

¥

■■

'^f*^

^H

W-^^

^ ^ I^^^H

i«|

M*V '•'

"a:^H

'*^^w

^'^

'i^^^^H

'#...

1

Figure 4

PLATE III

Figure 5

PLATE IV

Figure 6

^^*k^

4

Figure 7

PLATE V

Figure 8

PLATE VI

t»\

• • , ^ •• • ' • . . • •• - •^

I ^ - I •. I.

>

4".! ^ .-

* • '.

Figure 9

PLATE VII

/

K r ' • <

if *^ - ^

^4 •

Figure 10

PLATE VIII

\

•A.

J/:

^

I •

Figure 11

GENERAL DISCUSSION AND SUMMARY 121

types ordinarily present in the alimentary canal. It is of interest, too,
to know that two cubic centimeters and 300 cubic centimeters respec-
tively of the suspension, when fed daily to rats and to man, are suffi-
cient to effect the transformation, and the maintenance of the simple
flora.

Since the bacteria within the digestive tract procure their pabulum
directly or indirectly from the diet consumed by the host, it is logical
to assume that there is a definite relation between the chemical nature of
the ingested food and the metabolic activities of the intestinal organisms.
Many investigators have noted that marked changes in the intestinal
flora follow sharp alterations in the diet ; namely, that a proteolytic or
putrefactive flora results from high protein feeding, and that a carbo-
hydrolytic or fermentative flora develops when the protein intake is low
and the diet consists largely of carbohydrates. However, the ordinary
high-calory diets accomplish reformation only, and not a simplification
of the intestinal flora, that is, a change from the putrefactive to the
fermentative state of metabolism. As soon as the proper carbohydrate-
protein ratio is disturbed the reformed intestinal tenants begin to sin
and putrefactive metabolism again holds sway.

The harmful effects caused by, or associated with, pathogenic organ-
isms of exogenous origin, such as B. typhosus, B. dysenteriae, Ms.
cholerae, need no further comment. But it should be a matter of much
concern that the so-called "normal" organisms of the intestinal canal
may occasionally bring about decidedly abnormal conditions. Booker
(1897) claimed that Proteus vulgaris and certain streptococci may
elicit diarrheas in infancy. Bertrand (1914) found Proteus vulgaris
in each of his fifty-five patients suffering from diarrhea. Schumburg
(1902) reported the same organism as the cause of poisoning following
the consumption of sausages. Klein (1896, 1898) concluded that
B. welchii is an important factor in the causation of infantile diarrhea,
and Tissier (1905) described cases of acute diarrheas caused by this
organism. More recently Kendall and his associates (1911, 1913) and
Smith (1913) gave expression to similar observations and views.

Senator (1868) declared that the decomposition of proteins within
the intestinal canal under ordinary conditions results in the formation
of substances toxic to the host. Bouchard (1884) elaborated the
theory of auto-intoxication. Combe (1907) stated that food too rich
in nitrogen will cause intestinal auto-intoxication. Metchnikoff pub-
lished his views on the same subject, calling attention to a definite
alleged relation between premature senility and intestinal putrefaction.
He claimed that the proteolytic intestinal organisms are constantly
producing substances which are absorbed by the host and which act
as accumulative poisons. Metchnikoff proposed to fight these proteolytic
bacteria on their own battlefield by the introduction of an antagonistic
microbic army, the lactic acid bacilli, and thus ushered in a new and

122 TRANSFORMATION OF THE INTESTINAL FLORA

interesting field of study, namely bacterial implantation within the
digestive tract for therapeutic purposes.

According to MetchnikofF, the so-called lactic acid therapy consists
essentially in administering per os living cultures of lactic acid bacilli,
either in milk soured by them or as tabloids, or in pure culture together
with some fermentable carbohydrate. The lactic acid bacilli ferment
the carbohydrate in the alimentary canal, producing lactic acid and
thus exerting a direct influence on the bacterial processes, particularly
in the large intestine. Soured milk was considered to be of special merit
in this method of treatment, particularly milk which was rendered acid
with B. hulgaricus.

So general has the use of soured milk and of B. bulgaricus products
become that the market is flooded with various commercial preparations,
in the form of powders, tablets or liquid cultures or suspensions which
contain as their active principle the organism which has been shown to
be the most important souring agent in the sour milk products of the
Orient, namely B. bulgaricus. Not only has it been claimed that
beneficial results are obtained by the ingestion of milk which has been
soured by B. hulgaricus, but that this organism itself, without the
addition of milk or lactose exerts the same favorable influence by
destroying or eliminating harmful bacteria from the intestinal tract,
and thus preventing auto-intoxication.

Repeated attempts in the present investigation to establish B. bul-
garicus in the alimentary canal of albino rats and of man through the
administration per os of extremely large numbers of this organism
were entirely unsuccessful. At no time in the study was B. bulgaricus
recovered from the feces of man or the white rats or from any portion
of the digestive tract of the rats, irrespective of whether the organism
was fed alone or together with a utilizable carbohydrate. However, in
all of the subjects which received the suspension of B. bulgaricus in
connection with lactose or dextrin there followed a multiplication of
B. acidophilus, and to the same extent as when similar amounts of the
carbohydrates were fed without any addition of bacterial suspensions.

These results are in harmony with those of the following observers.
Kulka (1914) demonstrated in his experiments upon man that the
"B. metchnikovi," when introduced per os, cannot be recovered from the
feces. Rahe (1915) failed to procure any evidence by feeding experi-
ments that B. bulgaricus is capable of surviving in the lower intestine of
man. Morse and Bowditch (1906) found that practically the same,
and in some instances better, results were obtained with pasteurized as
with raw acidified milk or buttermilk, and concluded that the action of
the lactic acid bacteria is unimportant.

Although considerable doubt has been cast upon Metchnikoff's ex-
planation of the merits of sour milk therapy, the fundamental principle
of his arguments must in the main be accepted. That is, bacterial
implantation with the concomitant transformation of the intestinal

GENERAL DISCUSSION AND SUMMARY 123

flora is both desirable and possible. But it is possible only when living
cultures of properly qualified organisms are employed, or when such
specific diets are administered which stimulate and favor such a trans-
formation. B. bidgaricus is incapable of accommodating itself to intes-
tinal conditions. B. acidophilus, on the other hand, being of intestinal
origin lends itself readily to implantation in the intestinal canal.

That the value of sour milk feeding does not lie in the lactic-acid-
producing bacteria in the milk, nor in the acids which they produce, has
been conclusively demonstrated by Rettger (1915) and his associates
in their extensive experiments with several thousand chicks. Practically
the same results were obtained whether sweet or sour milk was fed, and
no difference could be observed in the growth- and health-stimulating
properties of ordinary sour milk and of milk soured with B. bulgaricus.

The feeding of B. bulgaricus, without due regard to the use of milk,
can have little or no importance attached to it. The beneficial results
which have been attributed to yoghurt and other Oriental sour milk
products have in all probability been due to the milk as such, rather
than to the acid-producing bacteria which they contain.

Milk by virtue of its milk sugar stimulates proliferation of B.
acidophilus in the intestine. This important fact was apparently over-
looked by Metchnikoff and his pupils. What they observed as B. bul-
garicus in the feces after the feeding of milk soured with B. bulgaricus
was in reality not this organism, but B. acidophilus. That such an
error of interpretation could have been made should not be at all sur-
prising in view of the fact that these two organisms resemble each other
so closely and are in fact indistinguishable in practically all of their
various aspects. The marked resemblance of B. bulgaricus and B.
acidophilus in cell morphology and orientation and in colony formation
on lactose or whey agar plates is well shown in the accompanying
photographs (Plates 1 and 2). It should be borne in mind that in
many of the experiments conducted by Metchnikoff and his enthusias-
tic followers large quantities of milk were consumed, and hence the con-
ditions for the development of B. acidophilus were more favorable, so
that instead of the acclimatization oi B. bulgaricus, as maintained by
them, B. acidophilus gained prominence and was the organism seen by
them in the feces. This assumption is strengthened by the announced
observations of Belonowsky (1907b) that the intestinal flora of nurs-
ing mice was of the same character as that of the older mice receiving
the B. bulgaricus milk culture.

It was demonstrated in the present investigation that the amount
of lactose or dextrin required to cause practically complete simplifica-
tion of the fecal flora could be reduced by one half, providing living
cultures or suspensions of B. acidophilus were given along with the
carbohydrate.

B. bifidus is also an inhabitant of the large intestine, and may be
looked upon as a logical candidate for simplifying the flora, but this

124 TRANSFORMATION OF THE INTESTINAL FLORA

organism is not so easily cultivated as B. acidophilus, and it has a much
higher energy requirement, so that much larger amounts would be
needed to establish it as the dominating flora than when the Moro
bacillus is employed. However, both B. bifidus and B. acidophilus ad-
ministration with a view to implantation has a thoroughly sound and
logical basis, which cannot be said of B. bulgaricus.

Milk cultures of B. acidophilus have proven themselves to be par-
ticularly effective in transforming the intestinal flora of man. In some
instances 500 cubic centimeters of a twelve to twenty-four hour culture
were sufficient to bring about simplification with the desired elimination
of the non-aciduric forms. A total of 1000 cubic centimeters taken
daily in two applications in every instance brought about the trans-
formation in the course of a very few days. Practically the same
results were obtained when a daily amount of 500 cubic centimeters was
reinforced with 100 grams of lactose.

B. acidophilus sour milk may be prepared readily, providing strains
of the organism are employed which have been grown for some time in
milk and which are adapted to this medium, and if transfers are made
to the sterile milk with liberal amounts of the inoculum, preferably a
young milk culture. When correctly prepared and cooled the milk is
of a creamy consistence. It should be shaken, however, for some time,
before using, to make it perfectly homogeneous and creamy in appear-
ance. Furthermore, the product has a pleasing odor and is decidedly
palatable. Aside from any therapeutic or medicinal properties which
acidophilus milk may possess, it should gain wide use as a beverage
alone, or as a food which has the nutritional properties of milk as such.

No definite claims are made at this time that whey broth and milk
cultures of B. acidophilus have distinct therapeutic properties, al-
though two subjects who presented a long history of serious intestinal
disturbance were among the 17 subjects employed in the 45 milk- feed-
ing experiments conducted on man, and appeared to respond favorably
to the administration of 1000 cubic centimeters daily. These clinical
cases were not sought at this time, but requested to be admitted to the
experiments. We hesitate to discuss further the very satisfactory re-
sults that have been obtained, from every standpoint, with these two
subjects until a large number of clinical cases have been under observa-
tion. Our real aim thus far has been to determine whether intestinal
implantation of B. acidophilus in the white rat and in man can be ac-
complished, and under what conditions such implantation, whether
temporary or relatively permanent, can be brought about. The experi-
ments here recounted furnish the answers.

EXPLANATION OF PLATES

Plate I

Fig. 1. B. acidophilus. Stained preparation from culture in glucose broth
incubated forty-eight hours at 37° C. Note the pleomorphism of the organ-
ism. X 1000.

Fig. 2. B. bulgaricus. Stained preparation from pure culture in whey
broth incubated forty-eight hours at 37" C. Note close similarity of the
individual cells and chains of this and the preceding figure. X 1 000.

Plate II

Fig. 3. Forty-eight-hour colony of B. acidophilus on glucose agar incu-
bated at 37° C. X 100.

Fig. 4. Forty-eight-hour colony of B. bulgaricus on whey agar incubated
at 37° C. X 100. Note the similarity of the two colonies.

Plate III

Fig. 5. Surface growth of B. acidophilus in a tube of whey agar grown
aerobically at 37° C. for forty-eight hours. Note small discrete colonies.
X4.

Plate IV

Fig. 6. Colonies on whey agar plates prepared from feces of human
subject immediately preceding a period during which 1000 cc. of B. acidoph-
ilus milk was taken daily. Note absence of small irregular and fluffy
colonies. X 40.

Fig. 7. Colonies on whey agar plates prepared from feces during the
period in which 1000 cc. of B. acidophilus milk were taken daily. Note the
dominating colonies of B. acidophilus which are characterized by their small
size and very irregular border. X 40.

Plate V

Fig. 8. Veillon tubes used in the present investigation, demonstrating
their particular value in the determination of gas-producing organisms in
the intestine of the white rat and of man. Note the gradual diminution of
gas production from left to right in the series of six tubes. The gradations
correspond to the different stages of transformation of the usual complex
flora to the simple flora dominated by B. acidophilus. Natural size.

126 TRANSFORMATION OF THE INTESTINAL FLORA

CAMERA LUCIDA DRAWINGS OF GRAM-STAINED FILMS OF

STANDARDIZED FECAL SUSPENSIONS FROM A

REPRESENTATIVE HUMAN SUBJECT

Plate VI
Fig. 9. Film prepared immediately preceding the administration of B.
acidophilus milk. Note relatively large number of Gram-negative organ-
isms. X 1000.

Plate VII

Fig. 10. Film prepared four days after the beginning of B. acidophilus
milk feeding (1000 ec. daily). Note increase in the number of Gram-
positive acidophilus-like rods. X 1000.

Plate VIII

Fig. 11. Film prepared eight days after the initial feeding of B.
acidophilus milk (1000 cc. daily). Note great preponderance of Gram-
positive acidophilus-like rods. X 1000.

BIBLIOGRAPHY

Adrian, C. 1895. Ueber die Abhangigkeit der Ausscheidung aromatischer

Korper im Ham, insbesondere der Aetherschwefelsauren und ihre Be-

deutung im tierischen Stoffwechsel. Arch. f. Verdauungskrankh., 1,

179-197.
Albu, a. 1897. Ueber den Einfluss verschiedener Ernahrungsweisen auf

die Darmfaulniss. Deut. med. Woeh., 23, 609-511.
Bachmann, W. 1902. Ein Beitrag zur Kentniss der Darmfaulniss bei ver-

schiedenen Diatformen unter physiologischen Verhaltnissen. Zeit. f.

klin. Med., U, 458-480.
Bahrdt, a. and Beifeld, H. 1010. Ueber die Wirkung der Nahrungs-

komponenten der Frauenmilch auf die Darmflora des Sauglings. Jahrb.

f. Kinderh., 72, Erganzungsheft, 71-93.
Ballner, F. 1904. Experimentelle Studien iiber die physiologische Bak-

terienflora des Darmkanals. Zeit. f. Biol., J^6, 380-419.
Barker, L. F. 1914. The diet in typhoid fever. Jour. Am. Med. Assoc,

63, 929-931.
Bartoschewitsch, S. T. 1893. Zur Frage ueber das quantitative Ver-

halten der Schwefelsaure und der Aetherschwefelsauren im Harn bei

Diarrhoen. Zeit. f. physiol. Chem., 17, 35-62.
Baumann, E. 1886. Die aromatischen Verbindungen im Harn und die

Darmfaulniss. Zeit. f, physiol. Chem., 10, 123-133.
Belonowsky, J. 1907a. Zur Frage der Wirkung steriler Nahrung auf die

Darmflora. Centralbl. f. Bakt., Iste Abt., U, 322-324.
Belonowsky, J. 1907b. Influence du ferment lactique sur la flore des

excrements des souris. Ann. de I'lnst. Pasteur, 21, 991-1004.
Berger, F. and Tsuchiya, I. 1910. Untersuchungen ueber die Bakterien-

menge der Faces unter normalen und pathologischen Verhaltnissen und

ihre Beeinflussung durch Calomel und Wasserstoff-Superoxyd. Zeit. f.

exper. Path. u. Therap., 7, 437-454.
Bertrand, D. M. 1914. Recherches sur la flore intestinale dans la diarrhee

des nourrissons. Ann. de I'lnst. Pasteur, 28, 121-131.
Bienstock, E. 1899. Recherches sur la putrefaction. Idem, 13, 854-864.
BiERNACKi, E. 1892. Ueber die Darmfaulniss bei NierenentzUndung und

Icterus. Deut. Arch. f. klin. Med., ^.9, 87-122.
Billroth, C. T. 1874. Untersuchungen iiber die Vegetations formen von

Coccobacteria septica; etc., Versuch einer wissenschaftlichen Kritik der

verschiedenen Methoden antiseptischer Wundbehandlung. Berlin, 244

pages.
Blodgett, S. H. 1913. Glycosuria and the Bulgarian bacillus. Med. Rec,

83, 1071-1075.

128 TRANSFORMATION OF THE INTESTINAL FLORA

BoGERTj F. V. 1918. Lactic acid cultures in the treatment of difficult feed-
ing cases. N. Y. State Jour, Med., 18, 13-15.
Booker, W. D. 1897. A bacteriological and anatomical study of the

summer diarrheas of infants. Johns Hopkins Hosp. Report, 6, 159-258.
Bouchard, C. J. 1884. Recherches experimentales sur la toxicite des urines

normales. Compt. rend. Soc. Biol., 1, 665-668.
Bouchard, C. J. 1887. Lecons sur les autointoxicationes dans les maladies.

Paris, 348 pages.
Brady, J. M. 1910. Acidified milk in pediatric practice. Arch. Pediat.,

27, 426-432.
Breslau. 1866. Ueber Entstehung und Bedeutung der Darmgase beim

neugeborenen Kinde. Monatschr. f. Geburtskr., 28, 1-23.
Brieger, L. 1878. Ueber Phenolausscheidung bei Krankheiten und nach

Tyrosingebrauch. Zeit. f. physiol. Chem., 2, 241-258.
Brotzu, L. 1895. Sulla desinfezione del canale intestinale. Centralbl. f.

Bakt., 17, 726-727.
Brudzinski, J. 1900. Ueber das Auftreten von Proteus vulgaris in Saug-

lingsstuhlen nebst einem Versuch der Therapie mittelst Darreichung von

Bakterienculturen, Jahrb. f. Kinderh., 52, 469-484.
Cahn, a. 1901. Ueber die nach Gram farbbaren Bacillen des Sauglings-

stuhles. Centralbl. f. Bakt., orig., SO, 721-726.
CiPPOLiNA, A. 1902. Ueber das Vorhandensein der sogenannten Saurelieb-

enden Bakterien im Stuhle des erwachsenen Menschen. Idem, 32, 576-

580.
Clark, W. M. and Lubs, H. A. 1917. The colorimetric determination of

hydrogen ion concentration and its applications in bacteriology. Jour.

Bact., 2, 1-34.
Cohendy, M. 1906a. De la desinfection intestinale obtenue, sans regime

special, par I'acclimatation d'un ferment lactique dans le gros intestin.

Compt. rend. Soc. Biol., 60, 602-604.
Cohendy, M. 1906b. Essais d'acclimatation microbienne persistante dans

la cavite intestinale. Delimitation du siege probable de cette acclimata-

tion. Idem, 364-366.
Cohendy, M. 1912. Experiences sur la vie sans microbes. Ann. de

I'Inst. Pasteur, ^6, 106-137.
Combe, A. 1907. L'auto-intoxication intestinale. Paris. Abstr. Brit.

Med. Jour., 1907, 2, 1718.
De Gasperi, F. 1911. Flore intestinale des rats blancs sur regime ordi-
naire et au regime carne. Centralbl. f. Bakt., Iste Abt., orig., 57, 519-522.
De Jager, L. 1897. Buttermilch als Nahrungsmittel fur Sauglinge.

Jahresber. u. d. Fortschr. der Thierchemie, 27, 271-272.
Distaso, a. and Schiller, J. 1914. Sur I'acclimatation de microbes

etrangers a la flore intestinale. Compt. rend. Soc. Biol., 76, 243-244.
Dunn, C. H. 1907. On the use of living lactic acid bacilli to combat in-
testinal fermentation in infancy. Arch. Pediat., 21/., 241-246.
Eberle, R. 1896. Zahlung der Bakterien im normalen Sauglingskoth.

Centralbl. f. Bakt., Abt. 1, orig., 19, 2-5.
Eisenstadt, H. L. 1897. Ueber die Moglichkeit, die Darmfaulniss zu

beeinflussen. Arch. f. Verdauungskr., S, 155-176.

BIBLIOGRAPHY 129

Ellis, H, A. 1912. Infantile diarrhea and its treatment. Australasian

Med. Gaz., SI, 30-33.
Embden, H. 1894. Beitrage zur Kentniss der Alkaptonurie. Zeit. f.

physiol. Chem., 18, 305-334.
EscHERicH, T. 1886. Die Darmbakterien des Neugeborenen und Sauglings.

Fortschr. d. Med., 3, 515-522.
EscHERiCH, T. 1886. Die Darmbakterien des Sauglings und ihre Beziehun-

gen zur Physiologic der Verdauung. Stuttgart, F. Enke, 180 pages.
Fansler, W. a. 1917. The use of B. lactis bulgaricus in the treatment of

infected wounds. Journal-Lancet, 37, 679-680.
Feigen, H. 1908. Bacterien-menge des Diinndarms und ihre Beeinflussung

durch Antiseptica. Inaug. Diss., Bonn.
Finkelstein, H. 1900. Ueber saureliebende Bacillen im Sauglingsstuhl.

Deut. med. Woch., 26, 263.
Fischer, A. 1903. Vorlesungen ueber Bakterien. Zweite Auflage, 347

pages.
Fischer, A. 1915. Hemmung der Indolbildung bei Bakterium coli in Kul-

turen mit Zuckerzusatz. Biochem. Zeit., 70, 105-118.
Friedenwald, J. and Leitz, T. F. 1909. Experiments relating to the

bacterial content of the feces, with some researches on the value of certain

intestinal antiseptics. Am. Jour. Med. Sci., 138, 653-661.
FiJRBRiNER, F. 1887. Zur Wiirdigung der Naphthalin und Calomel-

therapie des Unterleibstyphus und der Abortivbehandlung dieser Krank-

heit iiberhaupt. Deut. med. Woch., 13, 209-212; 235-238.
Gilbert, A. and Dominici, S. A. 1894. Action du regime lacte sur le

microbisme du tube digestif. Compt. rend. Soc. Biol., 117, 277-279.
Griffith, A. L. 1895. The therapeutic value of intestinal asepsis. Lancet,

2, 1136.
Grundzach, J. 1893. Ueber die Asche des normalen Kothes. Zeit. f.

klin. Med., 23, 70-79.
Hammerl, H. 1897. Die Bakterien der menschlichen Faces nach Auf-

nahme von vegetabilischer und gemischter Nahrung. Zeit. f. Biol., 85,

355-376.
Hand, A. 1912. The use of intestinal antiseptics in childhood. Arch.

Pediat., 29, 84-87.
Harrington, E. I. 1912. Lactic acid cultures; some clinical observations.

N. Y. State Jour. Med., 12, 73-76.
Harris, N. M. 1912, Intestinal antisepsis. Jour. Am. Med. Assoc, 59,

1344-1350.
Hehewerth, F. H. 1900. Die mikroskopische Zahlungsmethode der Bak-
terien von Alex. Klein und einige Anwendungen derselbei^. Arch. f.

Hyg., 39, 321-389.
Heinemann, p. G. 1912. The dietetic and therapeutic value of fermented

milks prepared from commercial ferments. Jour. Am. Med. Assoc, 58,

1262-1254.
Henderson, L. J. and Palmer, W. W. 1912-1913. On the intensity of

urinary acidity in normal and pathological conditions. Jour. Biol. Chem.,

13, 393-405.
Heymann, B. 1900. Cited by Finkelstein, 1900. Deut. med. Woch., 26,

263.

130 TRANSFORMATION OF THE INTESTINAL FLORA

Herter, C. A. 1897. On certain relations between bacterial activity in the

intestine and the indican of the urine. Brit. Med. Jour., 2, 1847-1849.
Herter, C. A. 1907. Common bacterial infections of the digestive tract.

New York, 314-346.
Herter, C. a. and Kendall, A. I. 1908. Fate of B. bulgaricus (in bacil-

lac) in the digestive tract of a monkey. Jour. Biol. Chem., 7, 203-236.
Herter, C. a. and Kendall, A. I. 1909. The influence of dietary altera-
tions on the types of intestinal flora. Jour. Biol. Chem., 7, 203-236.
Hirschler, a. 1886. Ueber den Einfluss der Kohlehydrate und einiger

anderen Korper der Fettsaurereihe und die Eiweissfaulniss. Zeit. f.

physiol. Chem., 10, 306-317.
Hoffmann, A. 1906. Beitrag zur Frage der Darmdesinfektion. Mittheil.

a. d. Grenzgeb. d. Med. u. Chir., 15, 696-606.
Howe, P. E. and Hawk, P. B. 1912. Hydrogen ion concentration of feces.

Jour. Biol. Chem., 11, 129-140.
Hull, T. G. and Rettger, L. F. 1914. The influence of milk and carbo-
hydrate feeding on the intestinal flora of white rats. Centralbl. f. Bakt.,

Abt. 1, orig., 75, 219-229.
Hull, T. G. and Rettger, L. F. 1917. The influence of milk and carbo-
hydrate feeding on the character of the intestinal flora. Jour. Bact., 2,

47-71.
Jacobson, G. 1908. Contribution a I'etude de la flore normale des selles

du nourrisson. Ann. de I'lnst. Pasteur, 22, 300-322.
Jaffe, M. 1877. Ueber die Ausscheidung des Indicans unter physiolo-

gischen und pathologischen Verhaltnissen. Virchow's Arch., 70, 72-111.
Kendall, A. I. 1911. Certain fundamental principles relating to the

activity of bacteria in the intestinal tract. Their relation to therapeutics.

Jour. Med. Res., 25, 117-187.
Kendall, A. I. 1913. Observations on the etiology of severe summer

diarrheas of bacterial causation. Boston Med. and Surg. Jour,, 169,

764-766.
Klein. 1896. Report on an inquiry into the relations of Asiatic cholera

and Cholera nostras, or English cholera. Report of Med. Officer to Loc.

Gov. Bd., 25, supplement, 173-204.
Klein. 1898. Report on the morphology and biology of Bacillus enteritidis

sporogenes; etc. Idem, 27, 210-250.
Klein, A. 1900. Eine neue mikroskopische Zahlungsmethode der Bak-

terien. Centralbl. f. Bakt., 27, 834-836.
Klotz, M. 1908. Ueber Yogurtmilch als Sauglingsnahrung. Jahrb. f.

Kinderh., 67, Erganzungsheft, 1-56.
Kopetski, I. A. 1900. Beitrag zur Frage der Wirkung des Milchzuckers

und der Milchsaure auf die Harnsecretion und die Darmfaulniss bei

gesunden Menschen. Diss. St. Petersburg, 68 pages.
Krauss, E. 1894. Ueber die Ausnutzung der Eiweissstoffe in der Nahrung

in ihrer Abhangigkeit, von der Zusammensetzung der Nahrungsmittel.

Zeit. f. physiol. Chem., 18, 167-179.
KuLKA, W. 1914. Studien zur Frage der fakalen Ausscheidung darmfrem-

der Bakterien. Arch. f. Hyg., 82, 337-350.

BIBLIOGRAPHY 131

KuMAWAGA, M. 1888. Ueber die Wirkung einiger antipyretischer Mittel

auf den Eiweissumsatz im Organismus. Virchow's Arch. f. path. Anat.,

lis, 134-202.
KiJsTER, E. 1913. Die Gewinnung und Ziichtung keimfreier Saugetiere.

Deut. med. Woeh., 39, 1686-1688.
La Fetra, L. E. 1909. Observations on the use of lactic acid bacilli. Am.

Jour. Obst., 59, 926-927.
Lembke, W. 1897. Beitrag zur Bakterienflora des Darms. Arch. f. Hyg.,

26, 293-328.
Lembke, W. 1897. Weiterer Beitrag zur Bakterienflora des Darms. Idem,

29, 304-363.
Leva, J. 1908. Zur Beurteilung der Wirkung des Lactobacillins und der

Yogurtmilch. Berlin, klin. Woch., ^5, 922-924.
Liefmann, H. 1909. Beitrag zur Behandlung der Typhusbacillentrager.

Munch, med. Woch., 66, 509-611.
Litchfield, L. 1914. Abstract of discussion. Jour. Am. Med. Assoc, 63,

935-936.
Logan, W. R. 1914. The intestinal flora of infants and young children.

Jour. Path, and Bact, 18, 627-551.
LuERSSEN, A. and Kuhn, M. 1908. Yogurt, die bulgarische Sauermilch.

Centralbl. f. Bakt., Abt. 2, 20, 234-248.
Macfayden, a. M., Nencki, M. and Sieber, N. 1891. Untersuchungen

ueber die chemischen Vorgange im menschlichen Diinndarm. Arch. f.

Exp. Path. u. Pharm., 28, 311-350.
MacNeal, W. J., Latzer, L. L. and Kerr, J. E. 1909. The fecal bacteria

of healthy men. Jour. Inf. Dis., 6, 123-169; 571-609.
Mannaberg, J. 1898. Die Bakterien des Darms. Nothnagel's Spec.

Path. u. Therap., 17, 17-40.
Mattill, H. a. and Hawk, P. B., 1911. A method for the quantitative

determination of fecal bacteria. Jour. Exp. Med., H, 433-443.
Mayer, G. 1910. Ueber Typhus, Paratyphus und deren Bekampfung.

Centralbl. f. Bakt., Abt. 1, orig., 53, 234-279.
McFarland, J. 1907. The Nephelometer. Jour. Am. Med. Assoc, J^9,

1176-1178.
Metchnikoff, E. 1907. The prolongation of life. New York, 161-183.
Morax, V. 1886. Bestimmung der Darmfaulniss durch die Aetherschwefel-

sauren im Ham. Zeit. f. physiol. Chem., 10, 318-325.
Moro, E. 1900a. Ueber den Bacillus acidophilus. Jahrb. f. Kinderh., 52,

38-65.
Moro, E. 1900b. Ueber die nach Gram farbbaren Bacillen des Sauglings-

stuhles. Wien. klin. Woch., 13, 114-115.
Moro, E. 1905. Morphologische und biologische Untersuchungen ueber

die Darmbakterien des Sauglings. Jahrb. f. Kinderh., 61, 687-734; 870-

899.
Moro, E. 1906. Natiirliche Darmdesinfektion. Miinch. med. Woch., 53,

2001-2002.
Morris, G. B., Porter, R. L. and Meyer K. F. 1919. The bacteriologic

analyses of the fecal flora of children. Jour. Inf. Dis., 25, 349-377.
Morse, J. L. and BowDiTCH, H. L. 1906. Acidified milk in infant feeding.

Arch. Pediat., 23, 889-908.

132 TRANSFORMATION OF THE INTESTINAL FLORA

MiJLLER, F. 1886. Ueber Indican-ausscheidung dutch den Harn bei

Inanition. Mittheil. a. d. med, Klin, zu Wiirzburg, 2, 341.
MiJLLER, F. 1887. Ueber Schwefelwasserstoff im Harn. Berlin, kiln.

Woeh., 23, 406-408.
MiJLLER, F. 1898. Autointoxieationen intestinalen Ursprunges. Verhand.

d. Kongress f. Innere Med., 16, 149-175.
Nelson, C. F. and Williams, J. L. 1916-1917. The urinary and fecal

output of calcium in normal men together with observations on the hydro-
gen ion concentration of urine and feces. Jour. Biol. Chem., 28, 231-236.
Nencki, M. and Sieber, N. 1882. Ueber das Vorkommen von Milchsaure

im Harn bei Krankheiten und die Oxydation in den Geweben der Leuka-

mischen. Jour. f. praktische Chem., 26, 41-47.
North, C. E. 1909. Reports of 300 cases treated with a culture of lactic

acid bacteria. Med. Rec, 75, 505-514.
Oehler, R. 1911. Ueber Yogurtkontrolle. Centralbl. f. Bakt., Abt. 2,

30, 149-154.
Oliver, T. 1907. Lectures on autointoxication in disease. C. J. Bouchard,

1907. Preface by Oliver, 3-13.
Ortweiler, L. 1886. Ueber die physiologische und pathologische Bedeu-

tung des Harnindicans. Mittheil. a. d. med. Klin, zu Wiirzburg, 2,

163-188.
Passini, F. 1903. Ueber das regelmassige Vorkommen der verschiedenen

Typen der streng anaerobischen Buttersaurebakterien im normalen Stuhle.

Jahrb. f. Kinderh., 57, 87-92.
PiFFARD, H. G. 1908. A study of sour milks. N. Y. Med. Jour., 87, 1-9.
PocHON. 1907. Cited by Metchnikoff in The prolongation of life. New

York, 1907, 161-183.
PoEHL, A. W. 1887. Bestimmung der Darmfaulniss durch Untersuchung

des Hams. Jahresber., u. d. Fortschr. der Thier-Chem., 17, 277.
PopoFF, D. 1892. Die Zeit der Erscheinung und die allmahliche Ver-

breitung der Mikroorganismen im Verdauungstraktus der Thiere.

Wratsch, 1891, No. 39. Abstr., Centralbl. f. Bakt., 11, 214-215.
Rahe, a. H. 1914. An investigation into the fermentative activities of the

aciduric bacteria. Jour. Inf. Dis., 15, 141-150.
Rahe, A. H. 1916. A study of the so-called implantation of Bacillus bul-

garicus. Idem, 16, 210-220.
Rettger, L. F. and Horton, G. D. 1914. A comparative study of the

intestinal flora of white rats kept on experimental and on ordinary mixed

diets. Centralbl. f. Bakt., Abt. 1, orig., 73, 362-372.
Rettger, L. F., Kirkpatrick, W. F. and Jones, R. E. 1914. Sour-milk

feeding and its influence on growth and mortality. Storrs Agric. Exper.

Sta., Bulletin 77.
Rettger, L. F,, Kirkpatrick W. F. and Card, L. E. 1915. Comparative

study of the value of sweet and sour milk. Storrs Agric. Exper. Sta.,

Bulletin 80.
Rettger, L. F. 1915. The influence of milk feeding on mortality and

growth, and on the character of the intestinal flora. Jour. Exp. Med., 21,

865-388.
RoDELLA, A. 1901. Ueber die sogenannten saureliebenden Bacillen im

Sauglingsstuhle. Centralbl. f. Bakt., 29, 717-724.

BIBLIOGRAPHY 133

RoDELLA, A. 1905. Reparition des microbes I'intestin du nourrison. Ann.

de rinst. Pasteur, 19, 404-410.
RoviGHi, A. 1892. Die Aetherschwefelsauren im Ham und die Darmdes-

infection. Zeit. f. physiol. Chem., 16, 20-46.
Salkowski, E. 1878. Ueber den Einfluss der Verschliessung des Darm-

kanals auf die Bildung der Carbolsaure im Korper. Virchow's Arch.,

73, 409-443.
Salkowski, E. 1889. Zur Kentniss der Wirkungen des Chloroforms.

Virchow's Arch., 116, 339-345.
Sato, T. 1910. Ueber die Bestimmungen der Bakterienmenge in den

Faces des Menschen. Zeit. f. Exp. Path. u. Therap., 7, 427-436.
ScHiLD, W. 1895. Das Auftreten von Bakterien im Darminhalte Neuge-

borener vor der ersten Nahrungsaufnahme. Zeit. f. Hyg., 19, 113-129.
ScHMiTz, K. 1894. Die Beziehung der Salzsaure des Magensaftes zur

Darmfaulniss. Zeit. f. Physiol. Chem., 19, 401-410.
ScHONENBORN, G. 1903. Vcrhaltcn der Bakterienmenge im Stuhl bei

Eingabe von Antisepticis. Inaug. Diss., Bonn, 40 pages.
ScHUETZ, R. 1901. Kritischer und experimenteller Beitrag zur Frage

gastro-intestinaler Desinfection. Arch. f. Verdauungskr., 7, 43-66.
ScHUMBERG. 1902. Wurstvcrgiftung. Zeit. f. Hyg., ^i, 183-184.
Sehrwald, E. 1889. Naphthalin und Typhus. Berlin, klin. Woch., 26,

447-450.
Senator, H. 1868. Ueber einen Fall von Hydrothionamie und iiber

Selbstinfection durch abnorme Verdauungsvorgange. Idem, 5, 254-256.
Senator, H. 1880. Ueber das Vorkommen von Produkten der Darm-
faulniss bei Neugeborenen. Zeit. f. Physiol. Chem., Jf, 1-8.
Singer, H. 1901. Die medicamentose Behandlung der Darmfaulniss.

Therap. Monatsh., 16, 441-445.
Sisson, W. R. 1917. Experimental studies of the intestinal flora. Am.

Jour. Dis. Children, 13, 117-127.
SiTTLER, P. 1908. Beitrage zur Bakteriologie des Sauglingsdarmes. Cen-

tralbl. f. Bakt., Abt. 1, orig., ^7, 14-30; 145-169.
SiTTLER, P. 1910. Die wichtigen Bakterientypen der Darmflora beim

Saugling, ihre gegenseitigen Beziehungen und ihre Abhangigkeit von

ausseren Einfliissen. Idem, Ref., Jf.6, 138-140.
Smith, R. M. 1913. Two types of infectious diarrhea in infants. Boston

Med. and Surg. Jour., 169, 756-758.
SoLUKHA, I. P. 1896. Beitrag zur Frage des Einflusses des milchzuckers

auf den Eiweisstoffwechsel und die Darmfaulniss bei gesunden Menschen.

Diss., St. Petersburg, 90 pages.
Spiegel, L. 1911. Natur und Bedeutung des Yogurt. Reichs-Medizinal-

Anzeiger, Leipzig, 36, 663.
Spiegelberg, H. 1899. Ueber das Auftreten von "proteolytischen" Bak-
terien in Sauglingsstuhlen und ihre Bedeutung in der Pathologic der

Darmerkrankungen. Jahrb. f. Kinderh., Jfi, 194-223.
Stadelmann, E. 1883. Ueber die Ursachen der pathologischen Ammoniak-

ausscheidung beim Diabetes Mellitus und des Coma-diabiticum. Arch. f.

Exp. Path., 17, 419-444.
Steele, J. D. 1908. Experimental observations upon the value of intes-
tinal antiseptics. Jour. Med. Res., 18, 93-105.

134 TRANSFORMATION OF THE INTESTINAL FLORA

Stern, R. 1892. Ueber Desinfection des Darmkanals. Zeit. f. Hyg., 12,

88-136.
Stieff, R. 1889. Ueber die beeinflussung der Darmfaulniss durch

Arzneimittel. Zeit. f. Klin. Med., 16, 311-324.
Strasburger, J. 1902. Untersuchungen ueber die Bakterienmenge im

menschlichen Faces. Idem, 4^, 413-444.
Strasburger, J. 1903. Ueber Bakterienmenge im Darm bei Anwendung

antiseptischer Mittel. Idem, 4^8, 491-505.
SucKSDORFF, W. 1886. Das quantitative Vorkommen von Spaltpilzen im

menschlichen Darmkanale. Arch. f. Hyg., 4, 355-396.
SzEGo, K. 1897. Die Darmmikroben der Sauglinge und Kinder. Arch. f.

Kinderh., 2£, 26-46.
Teixeira de Mattos, F. 1902. Die Buttermilch als Sauglingsnahrung.

Jahrb. f. Kinderh., 65, 1-61.
Tissier, H. 1900. Recherches sur la flore intestinale normale et patholo-

gique du nourrison. These, Paris, 85-96.
Tissier, H. 1905. fitude d'une variete d'infection intestinale chez le

nourrisson. Ann. de I'lnst. Pasteur, 19, 273-297.
Tissier, H. et Gasching, P. 1903. Recherches sur la fermentation du lait.

Idem, 17, 540-563.
Tissier, H. et Martelly. 1902. Recherches sur la putrefaction de la

viande de boucherie. Idem, 16, 865-873.
ToLLENS. 1908. Kefir als Sauglingsnahrung bei chronischen Verdau-

ungsstorungen. Miinch. Med. Woch., 55, 2331-2336.
ToRREY, J. C. 1915. The fecal flora of typhoid fever and its reaction to

various diets. Jour. Inf. Dis., 16, 72-108.
ToRREY, J. C. 1919. The regulation of the intestinal flora of dogs through

diet. Jour. Med. Res., 39, 415-447.
Veillon et ZuBER. 1898. Recherches sur quelques microbes strictement

anaerobies et leur role en pathologic. Arch, de Med. Experim., 10,

517-545.
VoN MiEczKowsKi, L. 1902. Desinfectionsversuche am menschlichen

Diinndarme. Mittheil. a. d. Grenzgeb. d. Med. u. Chir., 9, 405-414.
Wang, E. 1899. Fiitterungsversuche mit Indol. Zeit. f. Phvsiol. Chem.,

27, 557-574.
Wassilieff, N. p. 1882. Ueber die Wirkung des Calomel auf Gahrungs-

prozesse und das Leben von Microorganismen. Idem, 6, 112-134.
Wegele, C. 1908. Ueber die Wirkungsweise von Yogurt-Kuren und ihre

Indikationen bei Magen-Darmerkrankungen. Deut. Med. Woch., 34,

11-18.
Weiss H. 1904. Zur Kentniss der Darmflora. Centralbl. f. Bakt., Abt. 1,

orig., 36, 18-28.
Wejnert, B. 1908. Ueber den Einfluss der per Os und per Rectum dar-

gereichten gewohnlichen und nach Metschnikoff zubereiteten sauren Milch

auf die Bakterienflora im Kote. Wien. Med. Woch., 58, 754-763.
Wereschtschagin, W. 1895. Beitrag zur Frage des Einflusses des Trau-

benzuckers auf die Darmfaulniss bei gesunden Menschen. Diss., St.

Petersburg.
Williams, L. 1895. Two cases of infective fever showing the therapeutic

value of intestinal antisepsis. Lancet, 2, 1036-1087.

BIBLIOGRAPHY 186

WiNFiELD, J. M. 1912. Lactic acid and colonic irrigation in the treatment

of psoriasis. Jour. Am. Med. Assoc, 59, 416-418.
WiNTERBERG, H. 1898. Zur Mcthodik dcr Baktcricnzahlung. Zeit. f . Hyg.,

29, 75-93.
WiNTERNiTZ, H. 1892. Ueber das Verhalten der Milch und ihrer wich-

tigsten Bestandtheile bei der Faulniss. Zeit. f. Physiol. Chem., 16, 460-

487.
WoLLSTEiN, M. 1912. The influence of high protein feeding on the general

metabolism, on the intestinal flora and on the body temperature of infants.

Am. Jour. Dis. Children, ^, 265-300.

PRINTED IN THE UNITED STATES OF AMERICA

QR Rettger, Leo Frederick
171 A treatise on the trans-
R47 formation

Biological
fit Medical

PLEASE DO NOT REMOVE
CARDS OR SLIPS FROM THIS POCKET

UNIVERSITY OF TORONTO LIBRARY