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A STUDY OF THE EFFECT OF MILK UPON THE
BACTERIAL FLORA OF THE INTESTINAL TRACT.
A STUDY OF THE EFFECT OF MILK UPON THE
BACTERIAL FLORA OF THE INTESTINAL TRACT.

THESIS

Submitted to the faculty of the Michigan Agricultural
College in partial fulfillment of the requirements
for the degree of Master of Science.

By

Robert L. Tweed

June 1920
Page

Discussion---------------~.~--------=~~-+--------.~-- Al
Summary---- wt ee ee ee ese eee eee ween 43
Conclusion---------------------------------- a 44
Acknowledgment---~---------~----- = 2-2 ee ee oo ee ene 46

References-------------~ =~ - ene ee ee ee ee ne 47
ee ce
INTRODUCTION

Problems arising in connection with the high
death rate among children in the United States have
long been a worthy field for investigational and social
welfare work.

It has been shown that of the 2,500,000 babies
born each year in the United States, something like four-
teen per cent or 350,000 die during the first year, This
death rate as has been pointed out (35) is over seven
times as high as that of the British soldiers engaged in
the world war.

This great number of deaths is mainly due to
two general causes: (1) neglect due to ignorance and
(2) intestinal disorders. The first of these can be un-
doubtedly largely overcome by the efforts of social
workers but the second must be controlled by the pedia-
trician who must have reliable data at hand in order to
develop a rational system of treatment.

While clinical experience has shown the de-
sirability of breast feeding over artificial feeding,
definite data is still lacking in respect of the pre-
cise effects of artificial milk foods upon the intes-
tinal flora of young children.
Tissier (1), Moro (2), Logan (3), Kendall
(4), and others found that the organism named by
Tissier as Bacillus bifidus communis was predominant
in the feces of breast-fed infants, while Bacillus
acidophilus was more predominant in the feces of some
bottle-fed infants. These two organisms, being able
to produce large amounts of acid in the presence of
carbohydrates, were considered as fermentative organ-
isms to distinguish them from putrefactive types.

It was thus thought that normal infants had
a predominance of fermentative organisms in their
feces and that these in turn produced sufficient acid
to inhibit the growth of toxin producing organisms,
which are believed to be the cause of many intestinal
disorders among infants.

Porter, Morris and Myers (5) have recently
show that the above assumption is not altogether
true. They found that the fecal flora of normal in-
fants consisted of both fermentative and putrefactive
organisms and that neither predominated, but that in
the case of infants suffering with intestinal dis-
orders that the putrefactive organisms predominated,

This project was thus undertaken primarily
with a view to determine the type of bacterial flora
existing in the feces, when various milk diets were
re
fed, and secondarily to determine whether the guinea
pig could be utilized to advantage in this line of

research.

HISTORICAL REVIEW OF LITERATURE

Escherich (6) was perhaps the first inves-~
tigator to study the fecal flora of both breast and
bottle-fed infants. He employed both aerobic and an-
aerobic culture methods in his investigation and in
his list of the commonly encountered bacteria of the
intestinal tract, we find some of the most well know
organisms completely discribed.

This investigator noticed that the majority
of the bacteria discernible in the freshly pessed
feces were gram-positive and that his pure cultures
derived from feces were for the most part gram-nega-
tive.

Schmidt (7), a student of Escherich, claim-
ed that he could bring about this change by growing
them in media containing fat. This, however, was
disproved by Lehman and Neumann (8) and finally by
Escherich (9) who published a paper in which he shows
the fallacy of Schmidt's observations.

This problem of these gram staining bacteria
remained unsolved until Tissier (10) and Moro (11)
took up the investigation. These investigators used’
special media and methods and as a result contribut-
ed two new organisms to the intestinal flora.

Tissier isolated an anaerobe to which he
gave the name Bacillus bifidus communis, Moro's or-
ganism was aerobic and was given the name of Bacillus
acidophilus because of its ability to grow in relative-
ly large amounts of free acid, Both of these organ-
isms were gram-positive bacilli, morphologically simi-
lar to those found in the stools of normal nurslings,

Finkelstein (12) working independently and
using different media isolated organisms similar to
and probably identical with the organisms isolated by
Tissier and Moro. |

A controversy then arose as to the relative
predominance of B. bifidus and B. acidophilus. Tissier
claimed that B. bifidus was the dominant bacillus in
normal breast-fed infants and that Moro's organism
occurred only in the feces of infants suffering from
intestinal disorders and in normal bottle-fed infants.

Moro re-investigated the subject a few years
later and found that B. bifidus was the dominant or-
ganieam of normal breast-fed infants, but that B.
acidophilus was also represented in the feces of such
infants though in fewer numbers.

Moro's view has now been substantiated by
the researches of Cahn (13), Rodel (14), Coppolina
(15), Jacobson (16), and others and it is definitely
established that this view is correct.

Johannesen of Christiania in 1897 was prob-
ably the first to use acid broth as a medium for
isolating bacteria which can grow in relative large
amounts of free acid. About a year later, Bruno
Heymann made use of dextrose broth to which acetic
acid was added. This medium which is know as
Heymann's medium is one of the best that has been
used for the isolation of aciduric bacteria (17).

Among the first investigators to study the
effect of diet on the types of intestinal flora were
Herter and Kendall (18) who fownd in their experi-
ments on kittens and monkeys that an abrupt change
in diet from a dominant protein to a milk and sugar
diet is followed by an alteration of physiological
conditions in three distinct ways: (1) in the nature
of the intestinal flora, (2) in the putrefactive
products of feces and urine, (3) in the olinical
conditions. These authors suggest that the change
in intestinal flora is due to the addition of car-
bohydrates rather than a diminution in protein.
About the same time that Herter and Kendall
did their work, MacNeal, Latzer and Kerr (19) made a
study of the fecal bacteria of healthy men.

Noguchi (20) published a paper "Pleomor-
phism and Pleobiosis of Bacillus Bifidus Communid' in
Which he draws the conclusion that B. bifidus has an
aerobic phase in which it closely resembles B. mesen-
tericus fusicus and that the one source of the organism
in the feces of breast-fed infants is the breast of
the mother where it occurs as an organism of the skin.

Kendall (17), in 1910 published a paper,
"Observation On Aciduric Bacteria" suggesting that
there are two types of aciduric organisms: "The true
or obligate aciduric bacteria of which B. acidophilus
is the best known member; and the facultative aciduric
bacteria which may include various organisms”.

One year later Kendall (21) published a
paper "Activity of Bacteria In Intestinal Tract", in
which he concluded that by the feeding of lactose and
dextrose the intestinal flora may be temporarily
changed from that of toxin producing bacteria to that
of fermentative bacteria. However, the after-feed-
ing will be the determining factor as to whether the
fermentative flora will remain,

In the researches of Logan (22) on "The In-
testinal Flora of Infents and Children", we find the
findings of Moro (2) confirmed; namely, that in the
flora of breast-fed infants the aciduric group is
predominant and is strictly of the type called B.
bifidus and that in the artificially fed infants the'
place of the B. bifidus is largely taken by the fac-
ultative aerobe, B. acidophilus. The cases on both
breast and bottle showed a halfway stage between the
two types of flora,

Cohendy (23) who fed four patients for an
extended period on milk curdled with B. bulgaricus,
concluded that this organism became readily estab-
lished in the intestine and that it persisted there
for a considerable time after the subject had ceased
taking the fermented milk, This was said to be es-
pecially true if a diet was adopted containing suit-
able carbohydrates for the ingested organism. It
is stated that the growth of these bacteria took
place in the upper two-thirds of the colon.

In a later paper the same writer shows that
intestinal putrefaction as indicated by the excre-
tion of ethereal sulphates in the urine was materially
reduced by the addition of sour milk to the diet, and
that this reduction may be reasonably attributed to
the disinfection of the large intestine, prolonged
 
- 8 =

after the ingestion of sour milk was discontinued,
This may be taken as an indication that the growth of
bacteria continued after introduction ceased.

Belonovsky (24) studied the effect of the
Bacillus bulgaricus on the intestinal flora of mice. -
In his experiment several lots of mice were fed on a
basic ration of sterilized grain and water and to the
ration of two of his lots were added milk cultures of
B. bulgaricus. Mice fed on this ration forty-five days
showed this organism in the feces fifteen days after
the last feeding. With animals fed the culture for
four months, it was present for four weeks after the
last feeding.

On the other hand, Herter (25) found that
in the digestive tract of a monkey killed after feed-
ing for two weeks on milk soured with B. bulgaricus,
this organism wes abundant in the upper part of the
small intestine only. In the lower part of the small
intestine and in the large intestine, B. bulgarious
was present in only moderate numbers as compared with
other bacteria.

Heinemann and Hefferan (26) found an organ-
ism answering to the description of B. bulgaricus in
ordinary milk in feces of cows, horses, and man, and

in soil, grains, and pickles. They consider that it
 

is normally present in small numbers in the digestive
tract and suggested that it may cause pathological con-
ditions under certain circumstances.

In 1915 Rettger (27) published a paper,
"The Influence of Milk Feeding on Mortality and Growth,
and On the Character of the Intestinal Flora", in
which he says "Practically the same results were ob-
tained whether sweet or sour milk was fed and no dif-
ference could be observed in the relative value of
ordinary sour milk and of the so-called bulgaricus
product, Henoe, the unique properties of this food
exists in the milk as such rather than in any milk
acids or milk bacteria that may be present". He also
concludes that milk and lactose diet exert a very im-
portant influence on the character of the intestinal
bacteria, especially in white rats and chickens.
Within a few days after the ingestion of milk on lac-
tose, a transformation of the flora takes place, the
usual mixed flora giving away to a more simplified
flora in which B. acidophilus and B. bifidus are
prominent,

According to Rettger's studies the inges-
tion of large numbers of foreign bacteria does not of
itself bring about an elimination or displacement of
the common intestinal microorganisms.
-10 -

i Sisson (28) found in his work with puppies ,
that the type of organisms occurring at the duodemun,
ileum, cecum and rectum are in all instances similar
and that one cannot speak of a characteristic local
flora occurring in these regions. He also disagrees
With previous investigators in that feeding puppies
with cow's milk mixed with high percentages of su-
crose and lactose does not cause characteristic
changes in the intestinal flora at any level.

In direct contrast to Sisson’s work is the
work of Torrey (29) who found that the intestinal
flora is changed by the feeding of carbohydrate diets
and that it is not only shown in the feces but may be
shown at the different levels of the intestines.
Torrey suggests as a reason for Sisson's failure to
observe a change in the flora, that the media he used
was not suited to the growth of the aciduric type
that is found whem a high carbohydrate diet is fed.

Porter, Morris and Myers (30) found in
their work with infants suffering with intestinal dis-
orders that these disorders were always accompanied
by a predominance of putrefactive organisms and that
by a change to a high carbohydrate diet that these pu-
trefactive bacteria could be replaced with more of the
fermentative bacteria, thus relieving a large amount

of the disorder. These investigators also found that
~l1-

in the feces of a norm&l infant neither putrefactive
nor fermentative bacteria predominated but that they.

occurred in about equal numbers.

METHOD OF INVESTIGATION

The literature reviewed has not revealed
any data or studies of the intestinal flora produced
by the feeding of modified and fermented milks (ex-
cept milk inoculated with B, bulgaricus) often fed
to infants.

This investigation was designed to cover
the following points: (1) a study of the intestinal
flora of infants on various diets, (2) a study of
the intestinal flora of guinea pigs fed on various
modified milks with particular attention to raw and
pasteurized milk.

The samples of feces studied were obtained
and handled in the following manner; (I) those from
infants were obtained through the courtesy of the
Sparrow Hospital, Lansing, Michigan and were collected
by means of a copper wire containing a loop in one
end; standardized with a No. 2 lath nail. This wire
was inserted into a test tube or small bottle through
the cotton plug and sterilized before being taken to
the hospital. The nurse in attendance at the time
~12 -

excreta was passed would fill the loop of the wire
from a representative portion of the feces and then
place the wire and plug in the bottle. The bottle
was then taken to the laboratory where proper dilu-
tions were made by addition of sterile saline solu-
tion to the bottle containing the specimen. (No ef-
fort was made to secure quantitative results).

From the above suspension the following
plates were poured:

(1) Endo plates that gave an insight into the
type of aerobic bacteria which develop rather char-
acteristically on this media such as B. coli, B. pro-
teus, B, mesentericus and Streptococci.

(2) Liver glucose agar plates + 4 to phenol-
phthalein, incubated aerobically (31). This media
offers a very good means of differentiating the
aciduric bacteria. tt also has the advantage of sup-
pressing Streptococci and to a large extent B. ooli.

(3) Liver glucose blood agar plates + 1 to
phenolphthalein, incubated anaerobically for the cul-
tivation of B. bifidus (31).

(4) Liver glucose agar plates + 1 to phenol-
phthalein, incubated anaerobically for cultivation
of such spore bearing bacteria that might not develop

on the other media (31).
etn iin. ee
~13-

(5) Litmus lactose agar plates, incubated
aerobically for the per cent of acid producers that
develop on this mediun.

(6) Gelatin plates, incubated aerobically for
the per cent of organisms prodycing liquefaction.
| (7) Fermentation tubes of dextrose, lactose,
saccharose and litmus milk were also inoculated and
the percentage of gas reoorded, likewise the physical
appearance of the litmus milk, such as reduction and
production of red and blue color.

(8) Acetic acid broth having acidities of N/20,
N/10 and N/5, respectively were inoculated and incu-
bated for the isolation of aciduric bacteria.

All cultures with the exception of the fer-
mentation tubes were incubated for 72 hours and all
but the gelatin plates at 37°C. Fermentation tubes
were incubated for 48 hours at 37°C. and gelatin
plates were incubated at 20°C. for 72 hours.

Novy's jars end the pyrogallic acid-caustic
soda methods were used in obtaining anaerobic condi-
tions.

(II) Fecal specimens from the guinea pigs
were collected in the morning at intervals of every
few days. Beaver-board, which had been scrubbed

with bichloride of mercury was placed in the cages
 

al
~14-

as a false floor for the deposit of the feces, The
pigs being closely watched until a deposit was made,
When a portion was immediately collected and placed
in a test tube containing about 10 oa. of physiologi-
cal salt solution. After a suspension of the feces
had been made, appropriate dilutions were prepared
from the suspensions and the same media used as was

used in the case of infants feces.

EXPERIMENTAL DATA .
(1) Results of Infant Study.
The study of the fecal flora of infants
was undertaken in order that the writer might test
the media to be used as well as familiarize himself
with the normal flora of healthy infants.
Table No. I gives the results of the studies

made on the fecal flora of infants.
——_——— =

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- 16 ~

From the table’ it will be noticed that Cases
I, II, V, and VI were fed from the breast. In these
four cases the writer found a strongly gram-positive
flora with B. bifidus predominating both in the stained
film and the cultures. Other gram-positive organisms
present were 8. acidophilus, Staphlococcus pyogenes, aml
B. proteus. In Case I, Staphlococous pyogenes aureus
and Bact. welchii were quite prominent. This case,
having a syphilitic history, was troubled to a great
extent with constipation, senna and oil being given in
order to produce bowel movement,

Case III which received Mellin's Food show-
ed a gram-positive flora composed of B. acidophilus,
B. bifidus and Streptococcus lacticus. This case al-
so showed a very high fermentative flora.

Case IV was fed both from the breast and
bottle. The latter (hospital formula) consisted of
l oz. Of milk sugar, 2 oz. of top milk, 11 oz. of
water, and 1 oz. of lime water dissolved by adding
the milk sugar to boiling weter after which the top
milk and lime water were added and stirred well.

This formula was fed at the rate of 1 to 2 oz. every
21/2 hours.
The flora in Case IV was very similar to

that of Case III except Streptococcus hacticus was
-17 -

not so much in evidence and the percentage of fermen-
tative organisms was lower.

It will be noticed from the table that the
early specimens collected after birth showed little
or no growth upon the culture media. This was in
every case the meconium and in most cases it was doubt-
ful whether the child had had the breast before motions

The writer also observed that the three
phases of bacterial infection of infants as mentioned
by Kendall (4) are quite distinct, that is, (1) a
sterile period in which the meconium is practically
sterile for about 24 hours, (2) "Period of mixed in-
fection" which occurrs about the third day, due prob-
ably to the presence of food in the alimentary canal.
The organisms found during this period are B. subtilis,
B. doli, B. proteus, B. mesentericus, etc. (3) "Period

 

of Transition” in which B. coli diminishes in number
but does not entirely disappear; 8B. bifidus becomes
predominant and B. acidophilus also appears.

The media used in identifying the organisms
found were as follows: plain agar, dextrose agar,
litmus lactose agar, blood agar, gelatin, plain milk,
litmus milk, lactose, dextrose saccharose, mannit, and
maltose broths in fermentation tubes as well as the

special media mentioned on page 12.
me
~ 18 -

(2) Morphological and Cultural

Characteristics of the Aciduric Group

Bacillus bifidus, the dominant organism
in the stools of breast-fed infants, morphologically
is a long thin rod, frequently slightly curved with —
tapering ends occurring singly in pairs or groups with
the long axis parallel, Typically gram-positive, but
under certain conditions may appear as gram-positive
granules in otherwise gram-negative rods. Culturally,
B. bifidus is an obligate anaerobe, fermentative in
character, producing lactic acid but no gas, Grows
best on glucose blood agar (Torrey 31) upon which its
aolonies are visible in 24 hours but more distinctive
after 48 hours incubation as globular opaque colonies
7 to 3 mm. in diameter, buff to reddish brow in color.

Bacillus acidophilus is described by Moro (32)
and by Finkelstein (33) as a somewhat pleiomorphic
bacillus of varying length which may oocur singly or
in pairs, chain formation not being uncommon in arti-
ficial media, The organism forms no spores or capsules
and is typical gram-positive, although in old cultures
it may appear gram-negetive.

B. acidophilus may be isolated direct from
suspected material in N/20, N/10 and N/5 acetic acid
dextrose broth by a series of transfers. Probably the
 
~-~ 19 =

best solid medium for the growth of this organism is
glucose liver agar + 4 to phenolphthalein and contain-
ing 0.2 per cent sodium oleate according to the pro-
cedure of Torrey (31). Most strains of B. acidophilus
form on this medium flat, dingy colonies with a serrated
edge, although a few produce a round convex smooth
edged colony. This orgenism like 3, bifidus is also
fermentative in character but produces no gas.
(3) Result of Experimental Work With Guinea Pigs

The experimental work which follows was
conducted with guinea pigs. Pigs were selected of
approximately the same age and size and were all fed
a basic diet of sterilized bran as a concentrate and
carrots which had been peeled and washed in a solution
of 1-1000 bichloride of mercury as a succulent food,
To this was added the experimental foods.

(4) The Effect of Feeding Raw Skim

Two pigs were fed fresh raw skim milk for
a period of 45 days. The fecal flora of these pigs
were studied at intervals of every 7 é@ays. The re-

sults of these studies are show in Table II.
Table II
Showing Results of Feeding Presh Raw Skim Milk Upon Fecal Flora of the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Guinea Pig

Bran ------ 2 enn wn een ew ewe 55 gr. ‘
Diet Carrots-------------~------ 100 er. ,
Raw Milk---------.-.-----.--- 100 coc.
J * Y T
Case ’ I ! II “?

Feeding period * YT r Yr fr ir T | a Y Yr Y v T YT T—
in days. —3_ 1° So 7 10 "13 eh WoL 450 Bl a 3 10 "17 BA 31 '45'
J ' ’ rT YY
B. acidophilyg * -"' ="4-"' +' +' +' +' +' +! ¥ -' a '4.! 7. +' 4!" 4!
y 1 Ty FF Fy tT 6D T Tt v qT | a Jv Ot t t t
B. bifidus ee ee ee ee ee ee ee ee ee ee ee
TT 1 v Tw ¥  Yy 1 Tt v v Tv tf qT T mm fi TT. T T ¥
B. bulgaricus ' -' -' 2’ a! ar oh ot oh ot et let et lt os
! v ? ’ v t TT ? ms oT v Tt TT v q v t Tv '
B. coli Tee ee Tepe t pl gp pet mw Mp pe eye lee gt et pt Wt Oe ht
T v ? yy ! q t Tr ’ T 7 a o T ! t T oF Tv ’
B. 2, g2rogenes' +! +1 +iHe!t ot wt wt wt +! + lee Hee ee! at wt ew! 8!
B. mesentericus’ a FF. T Fo OE ’ v Tv v T i 7 | ee qT q
Vulcatus Paeteetpe oh oh or lt oye ot lb rt br tot
B, proteus yF FF T qT ¥ , 8 6 t La qT T Tv v Tv v T T T
vulgaris Tee te) tt tte ge pe ee! tt eee ow et 8! et 8! Ce!
T 7 7 ' T T 7 wT t v qT Tv 1 T J J T v t
B pyocyhafigs a _t me ot gt gt ag tect oF Ctl ot oo!
T tT vo Tr ov v vv Ff v T ¥ FF ¥ ot Fo GUE Tv ?
B. subtilis Mg et tt ee pm Te Mp pe ee ge Tet gt gt at et wt 8!
Staphlococcus T T T TT ©6T YT Tv Y rs i Yr YT a | ? T Y T
pyogenes aureus '++' +! +) +" + 4o Hehe ee) tt tt et 4 A nt oe ee!
Staphlococcus TF FF v v v Tt v v v A Tv mf T VT v vt v J t
pyogenes albus ‘( -' -' -' =" -" =" =" 2" 2! 4)? He! =) -' =' =' -' =!
Streptococcus oF v ae Tt Tv wv oF T Tt Tv r T q q Tt v Tt oT !
lacticus ee a, Se RS Sa a a SS SA Se
S reptococcus Ff J Y t v qT v 7 a r , vt F Tt a To J J TT '
pyogenes ee Oe eee ee ee ee ee ee ee ee ee ee ee ee ee
v v rr re es | ’ Y YT TT | re ie ‘Spr ir:
Other vpacteria q -'s.! .? .? ~! a’! ~! .! +4 t .! = ‘sg. ! ~'y.! ~! .!
4% Gas in yee r 4 ro TTY Tl nd '
dextrose broth 750 35°30 '10°10" oe Oo” bt 525 20°20" 5! re O° Oo” 5” 5!

4% Gas in

lactosé broth 125! 25' eS 7' 2 qe O- zr 518 bo 15° 5 Oo" OT Oo" T 2"
% Gas in Saccha- ¢

 

 

 

 

 

 

 

 

 

rose broth "10'10' 8' T' : OQ 0 0: 0'15'LO'10'T ' o1 o: o: Q' 0:
Action on "BAT AT AD T° TT TY
Litmus milk ' Pt PP! PAC 1AC TAG *AC tAC "AC"AP'AP'AC'AC'AC "AC 'AC ‘ACTAG
0 Gelatin YT T Tv tT YOU OY T vy Y Y T Y TO Te T Y ?
liquefiers 125 '21'16 WS1L49, 915" 31 6130126349 3' O' O' O12 '21!
+4 Acid Tt v , OCT | to OU a |
producers , 4 3' 2 11,616.63. 2'33 '30 138 21.625 195 7 (E22 20! 27'51'56"
7 Alkaline r~ Yr a
and Inert 173! 76'82 '741'72 "2,5'52'67 '62 ‘48.551 '47.2:90 "7.880'73 67,689
N/5 Acetic ’ J ’ 1 t ! ? ? ’ ’ ’ ' ! ! 1 1 1 I I
acid broth i eee ee ee ee ee ee ee ee ee ee ee eee ee
N 10 Acetic T TT T T v ) re a | FO GO yr Y¥ yy OF rT f
acid broth Pt et oe et a tt at nt ape te te gale 1 +! 4!
N/20 Acetic ’ TY Y Y T Yr vy v Yr vr is iv TY #xY &#i’Y* iY YY ’
acid broth a $i++ ++ + ++ te ee ee ee, ae Se, ee ee
Y T —r— T T T YT T_T T_T ~¥ TY TT," 1
Yeasts Pelt Lh Lp lrg atta tg tg et OF lt lr lr tpt Ct gt gy!

 

*
High percentage of liquefiers due to Staphlococcus aureus
"=

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fhe Symbols used in the tables are listed below.

++ = Very numerous
+ = Many

Few °

+
I
WW

None found

SA = Slightly acid

* « Peptonized
T = Trace

A = Acid

SC = Coagulation

B.ME = Be. Edematis malimmi

 

CT
be
it

Spore former

B = Before receiving milk

 

 

 

BW = Bact. welchil

MT = Micrococcus tetragenus
HD = Hemolytic diplococcus
Dip = Diplococes

G+B = Gram-positive bacilli

 
 

~ 21.

A brief study of Table II will first reveal
that the feeding of raw milk to these pigs resulted
in a change of flora within three days; second that
the new flora consisted largely of organisms which are
capable of decomposing carbohydrates rather than those
which attack and break down proteins. It will like-
Wise be observed that while bacteria which are able
to ferment carbohydrates were present in the greater
number, that they were not gas producers, and that the
gas producers originally present were greatly diminished

The number of organisms capable of liquefy-
ing gelatin decreased in Case I from 23% to 6%, while
those in Case II decreased from 30% to 2.1%. ‘Those
capable of fermenting carbohydrates during the same
period increased in Case I from 4% to 32%, and in Case
II from 21.5% to 36%, thus showing some individual
variations.

| The per cent of fermentative bacteria showm

in the table and in the tables which are to follow
were determined by adding together the percentages of
those which developed as acid colonies on litmus lac-
tose agar and those belonging to the aciduric group,

as shown on the special glucose liver agar used.
 

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(5) Effect of Feeding Pasteurized
Milk Upon the Intestinal Flora of the Guinea Pig

As was previously mentioned in this paper,
these pigs were fed raw skim milk for a period of 45
days. They were then transferred to milk which had ,
been pasteurized at 145°F. for 30 minutes and cooled
to approximately 50°F,

The results obtained from the study of the
fecal flora from pasteurized skim milk is show in

Table No. III.
Table III

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Showing Results of Feeding Pasteurized Milk Upon the Fecal Flora of the
___Guinea Pig

Bran << awe amwmww nen awn eww eww ne dD er. T
Diet Carrots-----------------=+ 100 er. ’
Past. Milk-----.~-------- 1Q0_cc. '

a Tr”
Case ! I ! II !
Feeding period 1 qT Tt Yr vr Tr v TT tr hUT T T vy T Ts) TT T TT
in_days V2' SLOT 24 B145'  ' 2) SION 24 t 321451

T 7 TT oe J vt
B. acidophidus ' +' +'+a'+—'+-'+e +e? | +! ¥ tlenteetentee oT!
t i ! 3 qt ' Tq oT ' T Yr fF t rT T TY
Be bifidus Teer ot org et CP Lt tg att or
_— To r.6~SCCMSC*SY T rT T Y TT TY YT TTT
B, bulgaricus ' -' -' -' -' o' 2" -' ' tot wt =! A! ee ee ee
TT t 1 7 ¥ Tr FO UF Y ' “Ty T T YT . TT "y T
B. coli eS SS SS SS
| ? | J Sr ee t qT ) a | tr F- T TT ) re tr ot
B, L, Aerogenes! +! + 4ateaten ge tent § Mee eee te tebe tee!
B. mesentericus’ © © © TUT TT TT TTT
vulgatus ! ~! a | ~! oe J te t —! a ww! -! a t !
B. proteus br a rs ee ee eS ee es ee a ee ee ee Se
vulgaris Dogg ete tpe tpt OTE LY ot petpetpe lpg et oT!
T v tT q T t qT ’ ’ Ty. a T 1 JT t q T qT T
B. pyocyaneus ' -' -'a'ean' wwe tae et et at et et
TT T T T > J T - a T v T T TT T Fr YF T J T
B. subtilis +! +' apogee!’ + 40! ' tlt o t=! t ftp te! ’ !
Staphlococcus * * " %% 5 FO SKN NS SS : ee cee ee ee soe
0 enes aureus ' +! + ' + "44 They They Thy ! ’ t +! +! + Thy Th Ty Thy ' ! '
Staphiococcus °" °° YY YY YC RENN
pyogenes albus ' -' =! ~' oot a! 4! ! rooyror oroor or orot ’ 1
Streptococcus ° °° © "FT YT Yr PY yy.) 0r6 SN
lacticus Tw "He a a! a" a! ' ! ee pe he 4, 5 .! .! ! 1
Streptococcus "©" " © ° ‘© TT TF Pr pr, ees
pyogenes a ee ee ae ee ee ee Paerygtyro4rioef
"SF'SF'SF'SF'SF'SF'SF’ yo ~ . "SH "SE '
Other bacteria t= Meee tt tt et tt 8 Mee te be pete! + Fe 1 of
0 Gag in Y T T Y rT T Y Y T Yr —y Yr YY. T tT t
dextose broth *20'20' 115115 °10'10015' '20'15'15'20'20'15'20'' |!
y Gas in Yr vr iy Yr Yr Y YY YY TY Yr ) a | 1
lactose broth ‘'10' 15°10" 107 8 5! 100 ' '20'15'10'10'16'10'15' '  '
% Gas in Sacch-" ° or
arose broth 100 '10"10! 5" n . 5'__' __110'10' 5'10'10'10'10"
Action on rrr 1
Litmus milk ‘AG "AQ "AG 'AP'AP'AP’ ap! ! TAC'AC'AC' AC AP! AP'AP! toy
w Gelatin TV Tv T T T Tv vv T rT  F¢ 7 tT TT TT T ’
zuquetiors ae A 59 "5°10 "0,9" rs!
Acid r 1
roducers (40718 6 6" 716.5 7.9 ' 150" "20 166n5"7.7' 9°67 ro!
% Alkaline . rT 1
and inert 59 7a.789" 87'83'9.8"6.68 68 1 6L4I79.F845'R 5'601826 '

N/5 T Ft oF oT pCO T Tt TT

neta toate tot ! ~~? '.! ! .! .! ? tet -! .! _! .! - .! | a
N/10 Acetic TY “TT Y T ) me | YT" TTY Y Tr "T Y T T !
acid broth a ae ee ae ee oe oe ' ry! eigen gang non iy! t ’
N/20 Acetic v T T 1 Y Y ¥ ¥ tT 6 YF T t T T T Y T T
acid broth ee a ee a a ee ' Pehigt gt gt gtyntyn! ! '
— T YY Ct lt TT T T Y T TY rT v3
Yeasts i ! Patagtelotyplry ot 8! ! 1

 
I
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~ 24 -

From Table No. III it will be observed that
when pasteurized milk was fed, the per cent of fer-
mentative organisms decidedly decreased and particu-
larly true was this of the Streptococcus lacticus group.
It will likewise be observed that the putrefactive
organisms increased slightly upon this diet. This
possibly may have been due to the fact that the
Streptococcus lacticus group was greatly decreased
during pasteurization while the putrefactive group,
which is more resistant, (many of them being spore
formers) was not decreased to such an extent, thus
making it easier for them to gain a foothold.

(6) Effect of Feeding Sterilized Milk
Inoculated With Streptococcus lacticus

As a direct contrast to feeding pasteurized
milk in which the Streptococous lacticus group is
greatly decreased, Sterilized skim milk which was
freshly inoculated with a pure culture of Streptococcus
lacticus was fed for a period of 45 days to another
group of pigs. The results of which are recorded in
Table IV.
--
Table IV a
Showing Results of Feeding Streptococcus lacticus in Milk Upon the Fecal
Flora of the Guinea Pig.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A Gas in

Lactose broth '30'30O' 5' 5' 7'20'10'

” was in sacch-' j f FS 886

arose broth "25'10' f' T' O'15' 7" ' ' 5
' T T Dé T qT T a

t
q tf v

 

f15'10'10'10' £T'20'15'
TT T a T T tr Ff

q

5' T' T' 0'10'10'
oF q Yr TF ' J

 

Action on
litmus milk "AP'AP'AC'AC'AC'AP'AP' ' 'AP'ACTAC'AC'AC'AC'AC'
7 J Tv 4g |  *§ yy. a qT T

 

% Gelatin rr or ror?

 

 

 

 

 

 

Bran-------------------=- oD gr. "

Diet Carrots----------------- 100 er.
Milk + Strept. lact,----100 co,

t ?

Case Lil IV
Feeding period ? ' ? 1 t ? TT #+T T YT nd
in days ' B' 3'20'17'24'31'45' "Bt 3'10'17'24'31'45' 8
YT T Y Y T Y YT T Y T rT T Y YT ?

B. acidophilus Poor lh gt gt gt gt 4! ’ Poor lh gt gt gt gt 4! t !
T Y Y T rT T T Y T T TTY T YT !

B. bifidus Poort oot ote tg oo! -! ! Poor ott ot ott ot !
T Y T Y TTY ee | Y T YT Y Y Y !

B, bulgaricus 1 ~* .~' oF SF oF ot Lo! ? Poor ot lt ot or ofr ot I
t i T qT TT T q t T 5 T qt v T T tT v ’

B. coli lop Tee tee tee! tt ett lea ee 4 tte +t!
T y TY Y T Y Y Y Y a Y Y YT !

B. l. aerogenes ' pigetgatge!? op ate e! ’ ee ee ~'polye! !
B. mesenvericus’ °° Y Yr YT Yr vr YY YT YT vr YT T TT 1
vulgatus Peotoltgor ot oot oot oa! t rooroor ot or ok ot ot 1
B. proveus “"T Y ee | Y Yr T TY T tT Tv Yr f y Y Y 1
vulgaris Pat gteltyec!? olypitee! ? Pat gy. tg ipatgi'ye! 1
Y re | Y Y rT i y ’ 1 1 1 TT" 3 1 J

B, pyocyaneus reotootyg er otp et oy! 1 Meote et ot L! 1 _' .!
Y T YT YT Y Y Y T rT YY Y T T T 1

Be subtilis ry! ee ? a 1
Staphlococcus Yr Y Yr T Y YT r Y rT Y T Y “Y TY Tr t
pyogenes aureus 44 Pep pen pnp egy! +! ' 8 pe peg thy! gy! +! +! !
Staphlococcus T Y Y YT YT T Y Y Y ¥ Tr Y Y Y Y Y T ’
orenes albus "4! =" «' a! oteatye! ! ! ’ 1 ! ' 1 ! 1 1
oe toocous Ty YT T Tr rn TY | Yr YT 1
lacticus ee Se ee ee Se ee
Streptococcus Y T Y Y Yr T_T T T T Y Yr 1 Yr vr Y !
pyogenes Peg high pteotgant gt 4g! ’ ' Tet gtecteaotgn! 4! ’
TBWSE’  ° SRTBW BW’ ° MIMT’SRT TOT

Other bacteria '" 4%4.!' a'eotge ty lye! ! Per ge yt hoor ot ot 1
% Gas in T T rT rT T TT ddan T a | 1
Dextrose broth '35'30'10'10' T'30'12' '  '25'25'10'10' 7'25'28'
YT TTT Y T Y Y TTT Y T Y Yr TY '

1 1

T !

1

!

?

?

!

t

1

'

’

’

'

’

1

t

!

8

’

-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-

liquefiers '24'2292810'39'20T5' ' #%'25'331289.7 'e7'ar' 4'
Rp Acid t q t ' vv T Yr rf T Y TT “Tr tT
producers '14'24'40 52763780 B7' ' 15622'40 '483'50'59 '67'
% Alkaline YY Y Y Y T T T T T T TT vv TY TT
and inert. "62'54 AT OTE 6081.6 ' DOA’ 45 WZ" 42 445378 29 '
°

acid broth Pt oT otpetpet ot ot tt ot tp tg ten lentee!
¥/10 Acetic YT "Tr YT rT TT T TT TT TY -T"""T T
acid broth Tt ee ae ee bette TT eet +t 4! tent +!
ee ic TT. Th TCO OF ) y J ) f Tv T a , a ) a 7 §

acid broth Tyla e! a ee leet alee ot Tee + lee lee ee! + lee!
o I Tv Tv La oF T a T v t T Tt q T T v

Yeast ? .! .! a! ~’ ~! .! .! q T a! =~! ~.! .! apm! .!

 
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Table V.
Showing Results of Feeding Streptococcus lacticus in Milk Upon the Fecal
Flora of the Guinea Pig

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Bran--~------~------------ 5D gr. aR

Diet Carrots----------------- 100 gr. :
Milk + Strept. lact.----100 cc. :

T T 1

Case ; VII ; VIII ;
Feeding period T T T T T rr f° ur oF T T T T 7 TY Y 1
in days Br 5 tO" Died 'of "pndumdnia’ B’ 3'10'17'24'31'45' '" _ '
T T T YT TT T YT YT 1

B acidophilus Pe teete: fo 1 1 1 I 1 M oie sees e' ty! 1 !
T T T 7 1 T T 7 T T T T T T T T T T 1

B, bifidus t oot oho 1 ' 1 ’ 1 Cb eit toa? cit 1 !
7 ye 7 T T T T T T Y T T T TT T T T T T ’
B bulgaricus 1 2 et ’ 1 1 ! 1 Pog oat eo ak ' ?
Me 1 T TT YY T T T T t= T T T T T 1

B. coli eh a ta? ! 1 1 ' 1 toy that pt Wate! ! 1
5 1 aerogenes T T T T T T rT T T T T TT Y T T !
© ! a 1 ' 1 ' ! rn oe Met ok tt ' 1
B, mesentericus T Tt T T T rT T T T ¥ T T T T T T TT 1
te I 1 1 1 ! 1 Tt ot oF oa 2 ' 1

ees us T ag T T T T ac T T T T T a T T T T 1
' 1 1 ’ ! ' t 1 1 1 1 1 1 ! 1 1 1 1 1

wulperis. 1 + t=, TT T tT TF T T + + + ms T +t =o oF
' 1 r 1 1 1 ! 1 ' ' ' 1 1 ' 1 | ' 1

Be procyaneus = ‘a = Y ~ T T TT T T T as hs — T —T T - T T 1
B. subtilis te Fie te 1 1 1 1 1 Bg tg Og a ae Poe ' 1
Staphlococcus T T T T T T TT Y YT Y TY TT T YC 1 1) 1
ogenes aureus’ +' +' +' ’ : : : ' Py th Mig tel ge te Tele : ,
Stap Tococcus ° r YT T T T 1 oY T T T aT T T T T '
! T 1 1 1 1 1 ! 1 1 tt! 1 ! 1 1 1 1 ! 1

| So r— r = T ee T T TT T a ers T T T 1
lacticus = 1 es ‘cn! ~ 1 1 1 ! ! ! 1 - La be 1 Th th + 1 1 1
Streptococcus T T a fee T 3: Ne ee ee T T aT be fe T 7 i 1
0 venes 1 1 + 1 + ! 1 1 1 ! 1 1 + 1 + ! + ! “a 1 + tas! ax 1 1 !
DYae' 'S "SF! 7 ie T ) T 'SF 'SF "SRT mT ‘SR gpl =o 1
omar baetenia |. 418 feat tag t et sty et eee! ae. ow gm he y 3
% Gas in = 90: maa T T T Tr 7 T T ¥ T 2 cor Tt ae T 1
Dextrose broth '30'20'10? ' ' 2' '' '20'50'25' '15' 6' 6" 8
b Gas in T =e T T T T T T 7 T T T Tei T T e T T
Lactose broth '25'20' 6' ' ' ' ' ' 'go'ao'1s' 0" mt! mo
% Gas in Sacch-" T “T T CW T T Y Y T T eke O T T T T 1
ayoue. broth. "10710? Or Yt ao ee 12" 2 * BPO Ort 2
iction on T T “ee T TT T T ea 7 "Ty T T T T T T 1
Litmus wide . ‘apise tags ft 7 tt ap ap AG" "AOAC tos t
% Gelatin T T TT ) ee T Tr T T T T YT TY T T YY 1
liquefiers 1467420800 Yt tS home's" "1 Oe 8
® Acid Te Tr T T YT Yt T rT. .-  . 7 x 7 Y T 1
roducers ta ted tb eos”. MAb bo ee.
Alkaline T T c= oy a: 7. T T Eh a T > oe 1
and inert we'5o4iy o' | 1353, 6588 '40'40 Rm
Getic ee T YT T T T T T 7. v2 T a. 1 T 1

acid broth Pet a toq@mw 1 1 ' 1 La oot 1 tee teat 1 1
710 Acetic io ee ae T T Tr T ho = oe i) a i (i ia TT Fr TT T T T
acid broth Me eheeteet 1 1 1 1 1 toe teu? yt ™ Th yy! 1 '
N/20 Acetic YT T T T T T “T T Lo foe, oe TY T T T T Y 1
acid broth Leet get et FS gt ete! See tee tet
Pr. ae SPO ae TY T T T T T T rt aC YT T YT YT T rT YT 1
1 1 1 1 ! ’ 1 1 ' 1 1 ! 1 1 1 1 ! ! ’

Yeast wt ae ws

 
-~ 27 .

Table No. IV shows a decided increase in
the Streptococcus lacticus group and also in the nun-
ber of B. acidophilus present. However, in Case III
it does not show the reduction of putrefactive organ-
iams that might be expected, while in Case IV, the |
results are decisive.

As Bact. welchii persisted quite consistent-
ly in the feces of Case III and as that case did not
respond to the feeding of Streptococcus lacticus as
did Case IV. It was decided to try the experiment on
two more pigs which had a very high percentage of
putrefactive organisms to commence with. The results
as shown in Table No. V correspond quite closely with
Case IV in Table No. IV. Therefore, it may be said

that the flora of the milk consumed has a temporary
effect on the fecal flora of the guinea pig.
(7) The Effect of Feeding Sterilized Skim
Milk Inoculated With Bacillus bulgaricus

As many pediatrists find that feeding milk
inoculated with Bacillus bulgaricus to infants and
children suffering with intestinal disorders is ben-
ificial and in that there have been many recent
attempts to establish this organism in the intestinal

tract without success, it was deemed advisable to feed
- 28 -

the organism to guinea pigs and study its effect on
the intestinal flora.

Sterilized milk which had been inoculated
with a pure culture of B. bulgaricus was fed for a
period of 90 days, after which the 3B. bulgaricus was
discontinued. Upon being discontinued, specimens
Were collected every day to determine how long after
feeding the organism could be isolated from the feces’
when suitable food, such as plain milk was fed to the
subject.

The flora resulting from feeding milk inocu-
lated with Bacillus bulgaricus is shown in fable No.
VI.
ee
 

- 6Y =

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table VI
Showing Results of Feeding B. bulgaricus in Milk Upon Fecal Flora of
Guinea Pig
Bran caw an ae we ew aw eweeae nee ee 3D 6
Diet Carr 0ts--~-------------=-- 100 gr.
Milk + B. bulgaricus----- 100 cc.
T T
Case ' V ' VI
Foeding period ° Yr 7 rr T TTT YT T Y TT rT
T t t ! J ? ! ! T T t 1 ! 1 1 ! ! ?
in days ~ BO 5 10 17 24 Si 45 - —+ 510 17 24 51 45 -
B. acidophilus ' -' +++ "ee ee! +) tt bt at at eee ee tee ee!
1 T Tt Ba qT T T ' qT T J qT T | qT TT T T
B. bifidus Pt ppp pe Hee FF heehee He!
7 T tr 4 qT 1 JT qT T q T T TT qT v J T v
B. bulgaricus ' —!' 2! +! +'+elee tee! FT nt ate tte tet tee!
' 1 ’ y T 1 Y Y rr 6 T T 7 a oT
B, coli Tep  agtgatgeth Othoot ot ob Myth ae tell lb lb loos
1 q T qT Fr 6A J qT J T J T ov qT J q q ’
B, l. aerogenes'+—'+-' = ' .? .! .? .! 1 ‘1! .? _! .! ._! .! .! ’
B, mesentericus qT T v qT Tv v TT TT qT t J qT qT a qT qT oT J
vulgatus ! .! q .! .! _! .! ~! t 1! .! .! .! .! .. .! 1
By proteus v 1 Tt T 1 T q T qT qT Tv T T v T TT tT T
vulgaris Pye t gteltectec to lt oo! ! a 1
T TT T T t T tT T TJ T T tT T T Tt v J TT
B. pyocyaneus '+ea'+—' alentten!? 21 ott Tee tee tte tte eT eT!
- J Ty v T TT oT TT a T 7 T 1 yr OCF q T T
B. subtilis a Sk
Staphlococcus ° ° " ‘YY FPP PN
orgenes aureus’ +!' +! +lenten!) ot ett Tg tte teeta lta tte!
ere hiocoocus T t J Tr T v V 7 T T vv T F Tv 7 7 tT Ff
pyogenes albus ' -' a a re ? roof or or lb or or ot !
Streptococcus UE T T Tt T J T TT J qT 5T.lUCMW&SF Tt T FF TT ma oF
lacticus t .! .' 'y t .! .! .! 1 ! .? .- . .! ~.! .! ~? !
Streptococcus 7 TT T T Y Y YY TT Y Y T Y T Y Y Tr
pyogenes Tee gh gt gt gtyctge! ! tee! t'gatgaloe tee tee! '
ae Rropt TT T Tt T v T "St "St T 7 Tt tT * 7,
Other bacteria '4+' 4+' -' -' of 2) af 1 Te leet ot at ot alee!
‘» Gas in Y T Y Y Y T Y Y Y Y T Y T 1 T rv
Dextrose broth 20 720 om 5 ciopt gts! e015 5. O0' 0o'o' o' !
% Gas in YT Tv TT T Y T T T T
Lactosé broth "18°15" re z Oo’ o' o' !' 20 10' T' 0' 0’ O' O°
% Gas in Sacch- JT qT T Tr FF Tq Tt Tt. | qT a q T T
arose broth ‘10! 10" 2 —T' 5'10'10' ' +'10' 5' 5' 5' 5* 5' &' !
Action on T 7 T a ov o YF Ff Tr a a q FUE TT T 1
Litmus milk "AP'AP'AS'AC'AC'AG'’AC' ' 'AP'AC'AC'AC'AO'AC'AC' !
k Gelatin > a is Tv T T ¥ v TT v TT J, TT Da TT Tt YF FF F¥
liqudiess 1"24'21' 6' 5%3' O' O' ' '442.51L2143' 8' O' O'_ !
% Acid Tv Y v r Y Y YT —T T_T rT Yr YY TT
producers '14'16'40'44'5el'55 eae ' il ' eo 27 ~41,3 49 '77,6'69.4' _'
%® Alkaline i ee i re ee | TTT Tr
and inert '62'63'54'51 49,6 45 462)  ' '45 tease 'HhE45 S2A4'SE '
W/6 Acetic TT TT rr Tr TY Tt
acid broth Pe ppp et tT Tt mt pe te tt et!
N/10 Acetic " “ . ‘ re rt tT TY TT TTT T
acid broth Pet tat gt ate tee TT et tt tt tt tle lee!
N/20 Acetic TF TT TT v of T v ¥ TT FF FF TT v Ty T v
acid broth Teale lee et et ee ee 1 tel tt ee te te ee ee!
UE FF Fo ULE FOUL TT v oe FTF UE TT - a ft t v OF
' ’ ' ! ! ’ ! t ' ? ! ? ! .- .! .' a !

Yeast ai wm lm ll Ut mt me! gy

 

ee ee ee ee, ee ee ee ee) ee ee ee ee ee ee ee ee ee ee ee ee ee, ee ee ee ee i ee | | ee ie
e
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~ 30 -

While the organism was being fed to the milk,
the writer was able to isolate them from the feces in
large numbers. The writer is aware that the above is
contrary to the results of Rettger, who fed the organism
to chickens.

In the past, there has been considerable con-
troversy as to the differentiation between B. bulgari-
cus and B. acidophilus, which may have resulted in B.
bulgaricus not being found in the feces of chickens.

The writer has found that while the growth
of B. bulgaricus is very similar to B. acidophilus on
liver glucose agar as used, the colonies can be readi-
ly picked because they are much thicker and have a
more thread-like edge. The writer further believes
that B. bulgaricus can be readily distinguished from
B. acidophilus by its lack of ability to ferment mal-
tose (34).

The presence of B. bulgaricus in the feces
of Case V continued for a period of 17 days after in-
gestion of the organism. In Gase VI, it was continued
in the feces for a period of only 135 days, thus show-
ing that it could not be permanently established in
the intestinal tract of the guinea pig which corres-
ponds with the investigations of Herter, Kendall,
Rahe, Rettger, Torrey and others.
" = $1 -

Table No. VI further shows that B. bulgari-
cus milk increased the fermentative flora to a greater
extent than did the milk alone as shown in Table No. II.
This might seem to suggest that the organism itself |
exerted an influence upon the flora. To determine
Whether this was or was not the case, the following
experiment was conducted.

(8) Effect of Feeding Sterile Water Inoculated With
B. bulgaricus on the Intestinal Flora of Guinea Pig.

Bacillus bulgaricus was cultured on liver
glucose agar, washed off in sterile water and fed to
Cases XI and XII for a period of 45 days. The flora
produced by this diet did not show any appreciable
difference or change from the normal flora of the pigs.
In just one instance was the writer able to isolate
B. bulgaricus from the feces which suggests that the
organism dies off in the intestinal tract when no
suitable carbohydrate is fed. Table No. VII shows

the results of the feeding of B. bulgaricus in water.
~ ae
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Table VII

—_—w as ee ee _ -

Showing Result of Feeding B. bulgaricus in HzO Uvon the Fecal Flora
of the Guinea Pig

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Bran--------------------- 35 er. '

Diet Carrots--------.----..-- 100 er.
Water + B. bulgaricus---100 cc. '

q Y '

Case XI XII
Feeding period t T v - T T v Tv v T , tT T ' tf Ot Tv , t
in days ' B' 510717 24 31457 Y_' B’ 3'10'17'24'31'45" '* '
v vr TY T Y ov Tv T Y ¥ ee a Y Y Ff Y t

B., acidophilus ' -' -' -' 2" 2" 2) 2) 8 Tet at at ott at tt!
Tt v yY OCOFF TT T TT ot tr OF T TV J T r T 5 Ft ?

B. bifidus eS a a
T ' T T Y Y rT. EMT a | ¥ YY YF is Y 7 Y Y !

B. bulgaricus ' =" =" 2) w=) ate? oF TP et lr ot lt lt tt
v T vv TF FO T T Tv TO Te COM r !

Be coli ae ee a SS Se
t Y T 1 1 1 1 rT vv Y 1 T Yr rr | T '

B. 1, aerogenes '+—-'+a +e!) 2) meee tee Bet eet eet!
B. mesentericus’ °" ° © © TT Ty vt Ty tT tv?
vulgatus a
B,. proteus . / i. | 7 - re . r~—T re a 1
re ’ J ' t ! ’ q v 1
vat Eig ' = — — = T ~ Yr Y Yr tito tote + tata YT !
Be pyocyaneus |! =a! asa! pt wh 2) 8 Ft eet nt pet tT!
? r oF ! ' ? ? ro ' ’ ’ ? 1 y t Y T !

B. subtilis Tapeh eto et et at tT gg et et et et tT!
Staphlococcus ' °° © © oT FUT TT TT Tyr
pyogenes aureus’ +' 414+!’ + rete? § Teta tent apt a teet eo! ot!
Staphyococcus  ~ 5 Vr
orpenes albus ' w'te! 2! alee lent ot TT a eee eet et eT!
reptococcus * °° Y Fv

= ipe Seieh Poor ot or of lt or os ! a a es i ’ 1
Streptococcus *° %" TF
pyogenes inte Pog) gp lg ’ a ' 4! ’ t + en's Th. hye' geet ? ?
SF 'SFSF SF SF SSF - TSFTSF'SF'SF SF re re ae

Other bacteria ' +' +’ + ham! Por Pyar ygr gr yr gr gr yr or ot
‘R Gas in ' t ’ y Y — ~ Cerner ee eld et !
Dextrose broth 20° 20°20" 20 '20'20'10' ' 35 '30'20'20' 10. T'10' ' !
%® Gas in Y TT rr dr re a ee | 1
Lactose broth 25 20" 10'10'10'10' 5' ' '20'20' 107 TT’ 5' O' fT |
” Gas in Sacch-* re re es ee ee ee es ee ee Yt
arose broth "15'10' 4' @' T' 07 oa eno Lon 10' T' T' Oo" rH ro!
Action on rrr T TT rT
litmus milk AP AP Ae ‘ACTAGTAG TAG" ' AL AP AP. AC VAC AC. AC' '!  !
4 Gelatin TY Yr
liquefiers mo, 4 21 n7g1L913 '™.7'8.9' ' ~35.87.3'23 nay 9° 9 "128 rf
% Acid CTT tT YT NT tT ris J T ?
producers 11.99,9'122%6'64149147 = ' '16711,297'17'15.72119.6 ' '
% Alkaline Y “Ty YT T T Y YT "T rT se ir ir iy ir Yi YY OF
and inert "60.36R270F81,570675'78' _' _ '67.871, 56137096 966.2781
N/5 Acetic Lr re ee ee a ae ae eS '
acid broth a moorior or ot ! i ee es ee ee ' '
N/10 Acetic a ii
acid proth ? .! .! .! .! .! _! .! ’ t .! .! .! a er a ? !
N/20 Acetic TTP
acid broth Tepe! pig lg tg he! ’ Poteet gt gt gt 4! ' !
TT tT Tv T Tv TT , TF FF qT EO UE EG a Tt t

Yeast roor or lr lot lt lon ler ’ ’ ! t rs ’ '

 
It having been definitely determined by
the investigations of Kendall (17), Torrey (31),
Rettger (27), and Porter, Morris, and Meyers (5)
that the addition of lactose to any diet would change
the flora from a putrefactive to a fermentative type.
This experiment was added as a control or check on
the other experiments conducted. The results are
Shown in Table No. VIII and correspond with the find-
ings of the investigators mentioned above. The
aciduric group consisting of B. bifidus and B. acidophil-—
ys predominating and almost completely crowding out all

other forms.
—_— ew he

Table VIII
Showing Results of Feeding Milk Plus 54 Lactose, Upon the Fecal Flora
of the Guinea Pig

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Bran------- <= oe ee em Om om SE Se oe on oe we om 45D Zl. !

Diet Carrots---------------- 100 gr.
Milk + 54 Lactose------ 100 «co.

"Y Tr"

Case , TX ' X '
Feeding perioa ~~ °" © © * vy !
in_ days ' B' 3'10'17'24'S1'45'  ' ' B' 3'10'17'24'31' 45"
1 ? ? Y YT 7 7 Y Y T T | YT Y T T Y Yr t

B, acidophilus Potgor pt gt pet pei get Fo oT tee et et et ati et UT!
tT J ' a t , 7 ! 1 1 ' q T q Tt | re | v !

B, bifidus ee ee ee 2
SF qv vv T tT T v T T T q ' t Tv q y of t Y

s t ! T ! ! ! t t 1 ? ’ ’ 1 t v t ! t !

B, bulgarioug "=" =" =" =" -7 a) 8 nt nt tt
B, coli lee ee pepe ge geen! Ot Nee gt ape lee tee tee UT!
? v ! 1 T ! a ! 7 ! tT FO TT t ) a J T ,

B. 1. aerogenes'’+—-'+-' -' =" 2) 2) att Meet wt et ne eT
Bo mesentericus" °° °° © © © © RO
vulgatus Petestootlotooroor lh ot yl tiget ot lt lt tt!
BL proteus rt , f ' rT Y Y Y Y TT T nT Te
vulgaris 1 +h. ! ~! .! .! .! .! t "h.! ~! . ! .! .! .! .! ? T
T t wT v t TT T 1 t Of 1 1 T qT ? Tv T a 1

! .! .! .! .! ~! ~! .! 1 1 .* .! .! .! .! .! ._! ’ }

B. pyocyaneus on Teotpotee’ at 28
Be subtilis hetyet or orot ot 2! 1 my' w' 2! ' ' ? 1
Staphlococcus *~ ° *° *° vy 560 TTT
omenes aureus’ +' + e—-'te eet a! ot Tg tee ene tee
Staphlococcus *~ * * * ' © © © 6" 2 ee ee ee eee eee eee eee eee
pyogenes albus '-' -' -' «!' -«' =" =! ' a ! !
Streptococcus ° ° © "YT FF NR RN '
lacticus ! ~! .! ~ .! ~.! .! .! , ¥ .! .! _! .! .! .! .? ’ t
Streptococcus °" ° ° ° TF FRB rT 1
pyogenes Peet gta tgp pete FT gt gt a lpg et oe OT!
Toy'spr4or Tt rT T T—Tonropr 7 T T77 Tt rT '

Other bacteria '+' +’ brat at oh ah Tt tee wt wt et et tT
% Gas in tT T TTT corn eel T '
Dextrose broth '75'15' t' 5' 7’ pm o' ' 'go'20'10' 5' T' T' Tt
% Gas in Ty TY Y iY , ee a re ae ae ee ee ae ae t
Lactose broth '50'10' @' T' Oo' O' T' ' '20' 5' T' T' O' O' O' _ *
% Gas in Sacch-" * *" ' © ' F TTT rT rT rr ror rer
arose broth ''30' T' o' o' o' O' O' ' = '22' DT pt gr’ OF O' OF FF
Action on * rr ne ee ae as ae Se i, i i aS i: ie i en !
Litmus milk TAP'AP'ACTAG‘AG'AG'AG' ' '‘aptactactactac'actac' 1!
% Gelatin TT
liquefiers '50' 4' 1' 4'42' O' O' ' O35" 5'5,67.3' 1'.4"7 0"
* Acid Oo rT oT oT t TT FF ot TT F FO qT J TT Tt TT t
roducers ' 5'20'30 '365'468447'51L9 ' 2.250'53'46 '618'646e33' 1 !
4 Alkaline “TTF r vy vY YY YY YY vY VY vv 1
and inert 145'76'69 '695949.256,348,1 = ' — '57.5'45 "AL ABL 2! 33 "BOT r o4
N/d Acetic c Tv TT T Tv tv Tt oF tt FF TT Tt TT 7 TT ¥ v 1
acid broth ' w ! mpm tp at gn pe! +! s row’ .' .! wt’ ran +! ’ 1
Acetic oF a T T r v oF vt FT UE TT TT ot a Tt FOU ?

acid broth Up ee et ee ee FT te tt tee tee ee oT!
NjzO Acetic Trt
acid broth Vee gee ee ee ee eet OT Meet ee ee tee ee tee OT
I zg 1 v 1? v q t I t J f ! f t J , ? T

t ! q t ' ! t v 1 ’ y t q J . q ! ’ !

Yeast

 
- 35 -

(10) Effect of Feeding Borden's Eagle
Brand, Condensed Milk, Horlick's Mdalted Milk and Mel-
lin's Food on the Intestinal Flora of the Guinea Pig.

These kinds of milk mentioned above being
used in 4 great many instances as food for infants and
especially so among the pediatrists of Lansing; it was
considered of sufficient importance to determine the
typical fecal flora resulting from their feeding.

The guinea pigs used in these experiments
were first fed upon formulas recommended by the respec-
tive manufacturers for infants of me month of age and
gradually increased until they were receiving the
formulas recommended for infants of twelve months of
aZe.

The type of flora and the relative numbers
of the different organisms are shown in Tables No.

Ix, X, and XI respectively.
7 - 56 =

Table IX

Showing Results of Feeding Borden's Eagle Brand Condensed Milk Upon
the Fecal Flora of the Guinea Pig

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

—_ BYVAaN aww new ewww n meen gr. '
Diet Carrots---------~--=------ 100 gr.
Borden's Condensed Milk---100 cc. '

T T T

Case ' XIII ' XIV ’
Feeding period — rT Yr" Tr YY YY VY YY VY YY © rr rr Yr TY
‘ q 2 ? Y 122 129 v ’ t v ! ! ? 122 12 t ’ ’ 1

in_days TT 5 Jv 2 7b v i” a 7 T Tt rr Be ae 15 Ft 9 T t
B, acidophilus ‘t-<'t='+' +? +74" 1 1 Thee! eiag tgp i gt boro
TT TT T | TT T 1 T T T qT oT tT J 7 4 tg qT !

B. bifidus Pororoomylr oh ot PF UP hy
Y 1 T Yr TY Y TTT Y Y T TT Yr TY Yr Y ?

B. bulgaricus ' =! -' o' w=) oh ett or tt ot et et et et tt!
f r Tv ' ! q yy 6 TT v ee | T t r T w ’ !

B. coli oe eS ! t lop T ple hp he! ' ' '
1 ' T T J ee | ot T T i qT , T t oT T T 1

B. 1. aerogenes'"+-'+-' -' -' =) = ee ta
B. B. mesentericus | a a a ns a OS a ee
Vulgatus ret wt te! ON! U8! ro Mobrorotmet ot ot ! 1 7
B. proteus a
vulgaris a 1 ’ Peg tent otgotyen! .! 1 1 1
Ff Tt J TT 7 oF TT Tr oT nm a Ds v TT qT FF FD Ty yf

B pyocyaneus q .! .! .? .! ~! .! t q ',..! .! .? .! .! .? t ! 7
= qT T r J qT q TT oy ft v J T T t T T Tt TT t
B. subtilis her gtorooboor et or or byt yt oh ot ot ot ttt
Staphiococcus " *" '" " © ' ' " TT YY Pr ry,
ogenes aureus’ +' +'¢al+— lente ' TT OT gt te 4 otha ent ne
a@phiococcus " * " * ' 1 1 TF © TOY YT CT
ogenes albus ' -' ~' o' o' «' .! ! 1 Pe f of t
pyonense oceus "° * *F F FF FO FRR ees aon Sen Sones Seen See aoe See
lacticus ae ae Se eS ee + +4 +4! ‘t+ t+ moog
Streptococcus ' ' " ' © ' t YT TrrT 4s T TT
bYOReEneS Pet ten te tn bette tT tte beteetee! ae! root!
Mr ‘sp’. 1 ' ' 16pfspr TTT rw y To's

Other bacteria ' @' #1 =" #' aw! =F TT Fatiant ot ot et wt TT!
as in T qT T T v T gt T Do T oF t T qT ¥ T Y y ?
Dextrose broth '40' 20) B72 10° . rs! 20°10! B' 8'10'10' ' ' !
% Gas in rf f TO YF YT" T T_T TT !
Lactose broth ‘'30' 10! m' §' T! 2 rot 10" g' of pt pt 5 rrr ot
ya Gas in Sacch-' YT Y TT T TT” TY Y' v vy vv. TF fF T T !
arose broth "20" . riot Tt O' ' ' "10' f' O'o' m FT I 1 !
Action on T v SF a T Tt T T OO - TT Tr iT Ft T -_ T T
litmus milk Tactactac'ac'ac'ac' '  ! "AC" AC ‘AC "Ac "AC TAG! 1 of 4
® G Gelatin TY TY tT TO Y tO UY T Tr Y "Ty T !
liquefiers 11.2'7,3'3,74.1' 6'2.7' ! 1 88! "5 tL. Q! 370,963" ' 1 t
% Acid . rT oF ' Gr YT T ’ YY YY Vv Y T T !
roducers '9, 2'16.4'266 60'41'33' ! ! '33'5Q0'55'73'44,'51' ! ! ’

4 al ne Jy J T TD 7 TT oT T Tv yy FD TT JT t
and inert 719.6 T2559. PDT 53'449 ' ! 'Q22.542 1) 24" 1A L427 en
N/5 Acetic TTT TY
acid broth a ae +- oe i t The "4. leetge! »'y.! ' t '
H/10 Acetic r ree a re ce re a i a a. ee i i Se ee, Fe t
acid broth Parag t ath gt gh gt boot bgt gt ateatoet 4" TT!
N/20 Acetic ¥ YF YF F vy UF yy, FF FO Y TT Yr FF FF YC t
acid broth Tee tap page ge ae!’ 8 Ot Neelam teeteetagteg' TOUT!
v T Tv TT v T FE oF T TT TT yr, OFF Tv Ty TY y 1

! ? ’ .! t ’ ’ t ! ? ’ .! ’ t t ’ ’ ’ !

Yeasts

 
Table

x

Showing Results of Feeding Horlick's Malted Milk Upon Fecal Flora
Guinea Pigs

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

BYan- ---------- --- eee SO ZT. '

Diet Carrots------------------ 100 gr. '
Horlick's Malted Milk----100 ac.

T T t

Case ' VII 1 VIII '
Feeding period eo oy eimzvemimnat 14
T 1 t ' T t

in days : Be e216 £5 50 ——r BT er 16' eS 00 ——,
B. acidophilus ' -'e=" +" + ' 4% 4% 1 1 Meet et tt tt tee
t T J T Tf ! T Tt T Tt T T qT T T 7 7 T !

B. bifidus ee ee ~'49' ! 1 i a 1 ! 1
_ T Tr vr YT iY TY Y Y Yr Y YY T Y T 1
B,. bulgaricus ' a! .’ .! .! _! .! 1 ? ? .! .! .! .? .! .! Y ? 1
T T T T Tr Y , v Y ,  v T r Y Yr Y T r 1

B. coli Toy ln en .! ? q q a ae elg eg! 7 ' ?
SJ a Y T ’ T oT a FU J T TT yF FF FF ot qv 7

Be. 1. aerogenes b+ - rete ot ot ot TT Mp tee Bet eT eT
B. mesentericus pd
vulzgatus _! . .! .' ~! .! t t 1 ! ~! .!? .! .! ~! 3 q t
B, proteus I r I 1 1 T T iY r tT TT To 1
vulzaris Pater oh ot oot os 1 ! Meet pet oF ot tL! 1 ’ 1
| 1 T Y Y rw oY T Y Y Y T Y vr T Y T T ?
py Cy. T ! Y 1 t ! 1 ! t ! ! t ! q q q t ’ f

B. c aneus nas —y — —r = — r tT r—r —r —r - + — YT Y !
Be subtilis ry! .! .! .! .! .! ! ! I +! ~~ To! ~.! .! ._' 1 t ’
Staphlococcus J, v qT 7 T TT T T qv T T TO v to y, oF J TT '
ogenes aureus!’ + 'ee-'te tet e ee TT llee! ateaten Pome or ont
Siachlocoscas , . ' 7 TT Y Y TT T rT Y Y T r 1
pyogenes albus 1. ! .! .! .! .! .! v t T . .' .! .! .? .! t J t
Streptococcus T Tv v J T q T mn oi T vr J q q JT T Vv } FT t
lacticus Te eee te pe te TUT le! + ttt [+t tt lot ror}
Streptococcus tC T Tv qv tT qv TT qT Tn JT T T F v T vy '
pyorenes ! + po Tw ! talent! ! ? t 4! aw tet ptt lg! 1 1 J
TSR? TT Y T Y Y T TSP SH THD" Tr Y 7 ) re | 7

Other bacteria ' +! —~' .' 2? ot 2! 1 ! a ee ee ’ ' oof 1 ’ 1
A Gas in tT qT 1 T T Tv 7 tT 7 T J qT T 7 v ) Tv 7 '
Dextrose broth 120! 8'10'65%' v' p' ' 10710 T'10' 10° 5’ FUT
% Gas in 5 ee ee ee Se a 7 —y" ——rT '
Lactose broth ‘'10' T' 5' T' Oo” roof of! 5. I 0" 5' p' or ot fs!
P Gas in Sacch-' TT Ty tr Off , J TT OF Tv J Tv qT t
arose broth "10' T' T' QO' 91 o' ' ' 3 51 7 0" p'op' or or fs!
Action on ee come memes memes meee epee ems meen emus mene cas eee eae Se ee SO RO
litmus milk WAP TAC TAC TAS AC TACT rf VAP IACTAC TAC AC ACT roof of
%® Gelatin , Ff ' ry !
liquefiers 5.25.6 18 | "3.1 29 Bs or '! 1191, 4 4'3. '1,5'2. 25 roto
yy Acié oT v tT oT TT TT J T '
roducers 18. 525 400 80 ‘622 61 rt RITA BS 45' as 30' ' ' !
Alkaline rrr CrP
and inert 78,872. $507 169540'96.7 rf “"SRS'56A5AIOEE 54 69. 95 ' | I
N/5 Acetic v T T T T | Tt T Tt qv v 1
acid broth Y .! ~! .! .! am'ea! t , t a w! a += 4. ! ? J 1
N/10 Acetic oF v oF t J FO ULE J oF a a ot a oy J oT T J Tv !
acid broth ! aly! +! +! »! 4+! 1 ? t ~! ~! +! »! >! >! ? , t
O Acetic a T 7 ' Tv FF T TT v qt Jy. oF y 7 Do T T i !
acid broth. Mee a ee te te ee! TT Meee leet ee tet tee TT!
J. qT | a T Ty. T ma T T T FT a J T OF v y 2 q

Yeasts q .! .! ~ .! .! .? ! ! y .? .' ~! . ! 1 Y ’ y ’

 
 

cos —_— - ST —_— —— —_-—

wwe lhl

Table XI

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lactose broth '10'20" 10" 7 9! Q' ' 5' o' oT F' O' O'

 

 

 

 

 

 

 

 

 

 

Showing Results of Feeding Mellin's Food Upon the Fecal Flora of the
Guinea Pig —_—
Bran----9------------- 65 gr.
Diet Carrots-------------- 100 gr. '
Mellin's Food-------- 100 cc.
T T
Case ’ TX t xX 1
Feeding per ion Y T Y Y T Y rT TTT a | a | rr a a |
in days t B! 3'10 "17 124 21 J ! 1 B! 3'10 '17 124 131 ' ! '! t
a oT T T T oT T T T J T T T 7 TT vt T Y
B, acidophilus 't-$"¢' +' #tate! 7 ' Tat et + + +! ~' ro roo
1 T T J T T T qT TT T Tv qT vu r q T TV Tt !
B. bifidus Po pe ee pee dT pe te be be te tet FT!
? Tv , 7 y 7 ! J T T T T Tv Fo Ff T Bi qJ 1
B. bulgaricus rw? «!? ’ 1 ro! ! ! roeoproruot ? 1 1 ? ! !
Tv r q T qT Tv 7 a fi 7 ¥ L i t t qT q T Ty 1
B,. coli Tree th gt gp Tp lp FE Tg pe pepe pe pee TUT
7 T T T qT qT T T of t v T T t 1 Tr T V !
B, 1. aerogenes += Momeoror oh ot oF FP ht
B. mesentericus' "©" ° ° %§" %Y YT b RR Ne
vul gatus ee ' 1 rorotrorororaet ! ! 1
B. proteus Y Y Y Tr Tr T T T 7 Y T T YT T Y T Y '
vulgaris Te Why! ~~!) wt ow! «!? ! 'h! .' .! troofr ot 1 1 ’
. T 7 J tT T t ty J q ’ v T 7 qT T T TV }
B, pyocyaneus ' -' -' -' -' =" =" ee
7 T 7 Of v J Tt T T | TY q OT v T Vv f
B, subtilis | ryt wt! ot ow! eC! ! ryt .! ! rooroof ! ! !
Staphlococcus - i rr a ae ae, rr a T_T t
ogenes aureus’ +' +'eo't+e'4¢—'4~)  ' en
aphiococcus mm T v T y “T Y “TT Yr rf rT TT Fr FC ’
pyogenes albus ' -' -' -' =" =" ~~" ! a es es en
pris toeoccus ! ot ! T 7 —y tr ’ q ’ Y ’ T Y Y Y T t
lacticus ae ee a 2 2 Ve! pt te ete ee te TUT!
Streptococcus . ' . ' J ' ! ' J ! tr TT Y rT Y 1
pyogenes ts Vp pe pepe '¢—e! Patel tg tpotpoant 2) FT FT
Toyr 1 ‘LipDipDip T GeB! T YT Tf Y Y Y T 1
Other bacteria '+-' -' -"' #+' 4!' +! ' a
"b Gas in rT 7 Y YY Y Y Yr Tv YY Y Y YT Y T t
Dextrose broth 120 130 120° os ro Tr ' "10' 5' pt 6' pt gt ot fs!
% Gas in ' 7 tT Y T T 1 7 T ?
1 z ’ 1
of oT 1
? ! y
as Tv '
! 1 t
T a tT
! ! 1
t “Ty ?
’ , 9
' T T
! ' '
ae T I
? ! ?
it oT !
g q q
Tt Tt
t q I
' Tt
' ’

PEEVE yaa:

rose broth LO 5 re re Oo” os 7 om! On Oo” Q' Ov o- '
Action on ‘ (rT r “"T
litmus milk "AP 'AG'AG*AG AG "AS" AP ae "AC" AG "AB "AO" '

! 1 ? t 1 ’ v ’ Y ! T
es “T4117 25! Oo" Oo. eel 7g + +) +
producers 96.7 30' p00 oS ' Ot ' We 50 "37 A68580. 63— rf
7 Alkaline Tv
and inert 4 4mn2e. 30552! 133 187.9 63 ‘el! A444 371
N/o Acetic For rr yf
acia broth poor ohooh ot ota Poort Cry lt ity! '
N/10 Acetic . tt iv ¥ T Y “TOT rT TT" T T
acid broth Mpelg et pe! ol 4! +! Pett gt gt gt gy!
N/20 Acetic TF FD 3 q TT oT OF qt TT — v J 7 Ft J.
acid broth Tin) gg pe lee tte oe! Mgt tee te te lte!

TT T T yF OF T TT 1 qT T qT TT. T T
Yeasts 1! 4! &lpa tye! ~! 1 a! .! .! . a ~! ?

 

~~ ~e
- 39 «

From the foregoing tables it will be ob-
served that the flora produced by the various milk
diets are very similar, all of them coming under the
fermentative type and, to a large extent, consisting
of B. acidophilns and Streptococcus lacticus.

Since these kinds of milk all contain a
large percentage of carbohydrates particularly maltose,
the results obtained with Horlick's Malted Milk and
Mellin's Food correlate with the previous work done
by various investigators with this particular sugar.

(11) The Effect of Feeding Sterilized
Milk Inoculated With Bacillus acidophilus
Upon the Intestinal Flora of the Guinea Pig

The intestinal tract being the natural
habitat of B. acidophilus, the writer considered it
of interest to determine if it could be established
in the intestinal tract of the guinea pig, which animal
was found to contain few if any of this organism in
the intestinal tract. Therefore, milk which had been
sterilized was inoculated with a pure culture of B.
acidophilis and fed to Cases XI and XII for a period
of 45 days, during which time B. acidophilus became

the predominant organism of the feces.
 

raae

 

 

 
 

- 40 -

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table XII
Showing Results of Feeding B. acidophilus Upon the Fecal Flora of
Guinea Pig |

bran Se 3D gr. !
Diet Carrots------------------ 100 gr. '
Milk + B, acidovhilus----100 cc, '
qy “\" '
Case XI ' XII
Feeding period Tv T v T J T t ThA Tl ¥ #F| qT TT J T Fr hE T
a T Y 1 0! 1p ! t t q J ! ! ? ? , 1 1 ? !
in days oF 5 ae ye! $51 TT yr CU B 7 S10 17 24 51 Tv TT) !
B. acidophilus ' m'+# 44 "He lee ee) tt OT mee te te lee Tee TUT!
og FT T T TT tT T T J a a TT a Fo FOU Bi Ft FF 1
B, bifidus Bt pepe tbe tot TTT et at tee et nt TT!
ees | rr T Yr r rT TT Y Yr vr fr iy, i. 1
B, bulgaricus ' -' =" -' =-' 2=' 2) T PF tat ot at ot ot ot tt!
- Fo = TT wT T qT FO g Tv TT) ? FT Fo TT qT Tv !
B, coli "++! +! + ipalee! -! ' ' Th Tey Tete tgp ty! 1 1 ?
Be le aerogenes” TT TT Y Yr Yr 1 “YT YF YY YT YT" T 1
Tew lye! -! .! .! -? ' v a ~! .! . .! ? ! !
B, mesentericus er ee ee a cee ee eee eee eee ee ee eee ee
vul atus q a ~! .! .! ~? .! 1 ! a a! +! .! ~! .! t ’ 1
e proteus I
1 ! 1 1 ’ ' ? 1 1 ' ' ’ 1 1 1 ’ ! ’ 1
vulgaris ~ ans T T T T T Tt yt ~ , a _ + v J , a FTF FF !
B oc eus ' m' -' of oF oto! t 1 Poor ltr ot lot ltoos ’ ’ !
T J Fr FD . T v 7 Tt TT J TT qv FO OU Tt v T t
B, subtilis Poor grok Lh or or or ror utr 1
Staphlococcus *° "° Fv
pyogenes aureus’ +' + 4n'en'e ete TTT gt tye tee tet eet
Staphlococcus *° *F YF FO
pyogenes albus '+-' -' -' -' -' 2) ' fo tee tt et ttt
Streptococcus tT Df TT 7 T T T T JT ne TT TT J TT J T 7 a !
lacticus ee ee ae ee ee a a ee
Streptococcus 7" 7 7 7 7 7 7 7 7 7 7 Tf Tt —r
! t ' ’ ? ’ ! Y ? 1 q v ' z t q ’ ,
byoRene 8 of * J = I r i a + — FE q t + t= ~ a vv T vv TT t
Other bacteria '+' +' +' «' .' 2! ! ’ PMarygto oroor on ot ! ’ 1
% Gas in T TOT ene lal dl Y Y ?
Dextrose broth '20'10'10' T' mt f' # § '2575'10' Bt pt Bt tt!
b Gas in T T T Yr Tv TY Tr Y Tr yr iy iv #v #7 rT YT T ’
Lactose broth ‘'10' T' T' O' O' O' ' ' ' 5'10' 5' O' Of O' tr +r of
ra Gas in Sacch-' TT T T T Tt TT qt Ft UL Fo UE TF 6 J FF Tt tT T
arose broth "1I0' Tro'oro'o' ' ' 'tmprqyo' protroror ' ' 3
£c tion on rf Fee re CTT Y v TY rT}
litmus milk 'aprac'ac'ac'ac'ac' ' ' ‘'aptactap'ac'ac'tac' ' '  !
“0 Gelatin T Ff T tT T a T TF. T Di Ty Tr Tt T Tq T t T g
liquefiers '99.378.0'9.' O' O' O' ' ' =+4,9%3.9'10' O' O'O' ' ' 3
» Acid T T TT v Tv Tv Tv yy OCU 7 TT qv ot qT v qT v v !
roducers 127448 -' -~' =" -' ' I "7.239, '20'27.913' «.' t ' '
SS Alkaline T qT v J TT J qT TT oF oT v gv J 7 vv tT —F TT T
and inert '78 482 «! a ~.? =m! ? ’ "74,957,1' 70 "72.5" a" ~! ! t 1
N/5 Acetic Y—"Y"T Y T T T Y T Y Y TT Y Y T oT 3
acid broth a OO
N/10 Acetic T Y Tr Y Yr v T TT YY iY Yv Yr YT Y “T r Y Tr 1
cid broth ee ee ee ae A, Se
[20 Acetic T Ty T Tt tT T T T TT T T T vv J Tt TT v Tt q
acid broth Tepe pp pe pe pee ee TTF a gee ee oe toe TUT!
7 Ft T T oy T TT T FF T Fo ot vt 6 T oF ?
t 7 Y T t t ¥ ! ! ? T ' 1 ~ .! .' t t !

¥easts - - - - - - = +> on

 
~ 4] -

After the 45 day period sterilized milk
was fed without being inoculated with B. acidophilus.
However, B. acidophilus remained the predominating
organism during the next 45 days that the milk was
fed. Subsequent to this, milk was also discontinued
as a part of the diet and B. acidophilus almost in-
mediately commenced to diminish until the 3Slst day was
reached, at which time the writer failed to find them

in the feces of the guinea pigs.
DISCUSSION

From the experimental work done, the writer
has shown that the fecal flora of the guinea pig may
be influenced by diet. However, he does not believe
that they meet the requirements of an experimental
animal for this kind of work, because (1) their stools
do not resemble closely enough those of the human, (2)
they do not consume their food quickly nor cleanly,
thereby making it difficult to keep sterile food from
becoming contaminated, (3) it is difficult to obtain
uncontaminated stools, (4) they are hard to maintain
on a monotonous diet.

The data presented also suggest that it
might be well to inoculate pasteurized milk with a
pure culture of lactic acid bacteria before being
rf

 
- 42 -

consumed as has been proposed in a recent patent (36).

If for any reason it should not prove ée-
sirable to inoculate pasteurized milk with some lactic
acid bacteria, the writer would suggest that cows milk,
particularly in the summer and in case of intestinal
disorders among children, be modified by the addition
of lactose. Human milk contains almost four times as
much lactose as protein. Cow's milk contains not
quite twice as much lactose as protein and produces a
putrefactive flora more commonly than does human milk.
It has been repeatedly shown that when lactose is fed
a fermentative type of flora results, which closely
resembles that of breast-fed infants.

By the addition of lactose to cow's milk as
mentioned above, such milk would be made to resemble
more closely that of the human, thereby reducing the
chances of successful invasion in children of putre-
factive bacteria which tend to be inhibited in fer-
mentative surroundings.

Aside from the possible therapeutic value
of fermented milks there seems to be no question but
that they are nutritious and refreshing and that their

use should be encouraged among adults because of their

food value.
- 43 -

SUMMARY

The writer's experiments in feeding different
diets to guinea pigs may be briefly summarized as
follows:

(1) The feeding of fresh raw skim milk alters
the fecal flora from a putrefactive to a fermamtative
type.

(2) The feeding of pasteurized milk, however,
does not produce this change to as great an extent
as does raw milk.

(3) Milk inoculated with a pure culture of
Bacillus bulgaricus results in a highly fermentative
flora and shows B. bulgaricus in large numbers during
the period in which fed but immediately disappears
When suitable carbohydrate food such as milk is with-
drawn from the diet.

(4) The addition of 5% lactose to the milk
fed, produces a flora very similar to that of breast-
fed infants, B. bifidus and B. acidophilus predomina-
ting.

(5) Feeding pure cultures of Bacillus bul-
garicus in water produces no change.

(6) Feeding of Borden's Eagle Brand Conden-
sed Milk, Horlick's Malted Milk md Mellin's Food all

produce fermentative flora,
 

(7) B. acidophilus predominated in large
numbers in the feces of the pigs fed milk containing
B, acidophilus and it remained dominant as long as
suitable food, such as milk, was furnished in the
diet. However, upon discontinuing food suitable
for the growth of B. acidophilus, it was supplanted

by putrefactive organisms.
CONCLUSIONS

The work presented in this paper has neces-
sarily been limited but from the data obtained the
following conclusions may be drawn:-

(1) The normal intestinal flora of the
guinea pig, which is composed of a relatively high
percentage of putrefactive organisms, may be changed
temporarily to a more fermentative type of flora by
the feeding of milk inoculated with lactic acid
bacteria or by the addition of lactose to the diet.
The writer also found that the feeding of Borden's
Condensed Milk, Horlick's Malted Milk and Mellin's
Food brought about a similar result,

(2) The length of time in which the fer-
mentative flora, brought about by the feeding of milk
inoculated with lactic acid bacteria, will persist
after ingestion of the bacteria depends upon the type
of food consumed thereafter.
(3) Guinea pigs do not meet the requirements

of an experimental animal for this kind of work.
ACKNOWLEDGMEN T

The writer wishes to acknowledge
his indebtedness to Mr. L. H. Cooledge,
Dr. Ward Giltner, and Mr. G. L. A. Ruehle

for suggestions and assistance received

during this investigation.
(1)
(2)
(3)
(4)

(5)

(6)

(7)
(8)

(9 )
(10)

(11)
(12)

(13)

~ 47 w~

REFERENCES

Tissier, H.*
1900. La Flore intestinale des Nourrissons,
Paris.

Moro, C.*
1900. Jahrb. f, Kinderheilk., 11, 1909,
p. 47; Wien klin. Woch; Heft 5.

Logan, W. R.
1914. Jour. Pathology and Bacteriology,
Vol. XVIII, p. 527.

Kendall, A. I.
1917. Text-book, Bacteriology, General
Pathological, Intestinal, pp 581-600.

Porter, Morris and Myer.
1919, Jour. of Inf. Dis., Nov. p. 349-377.

Escherich, T.*
1886. Die Darmbakterien des Sanglings,
Stuttgart.

Schmidt, a.*
1897. Wien klin. Woch., p. 643.

Lehmann and Neumann,
1897. Hyg. Rund., p. 849.

Escherich, Tf.
1899, Jahrb. f. Kinderheilk, Vol. xLIX,
1357 et seq.

Tissier, H,.
1900. La Flore intestinale des Nourrissons,

Paris.
Moro, E.
1905. Jahrb. f. Kinderheilk, Vol. LXI.
Finkelstein, Z.*

1900. Deut. Med. Woch., pp 22 and 263.

Cahn. A.*
#oOl. Cent. £. Bakt, pp. 30 and 731.
(14)
(15)
(16)
(17)

(18)

(19)
(20)
(21)

(22)

(23)

(24)

- = 48 -

—

Rodella, A.*
L903. Ibid., pp 29 and 717.

Cippolina, A.*

1903. Ibid., pp 22 and 576.
Jacobson.
1908. Ann. Inst. Past.

Kendall, A. I.
1910. Jour. Med. Res. pp 22, 153-166.
Herter and Kendall.
1909-10. Jour. of Biological Chemistry,
Vol. VII pp 203-237.

MacNeal, Latzer and Kerr.
1909, Jour. of Inf. Dis., Vol. VI, p. 123.

Noguchi » He

1910. Jour. of Exp. Med., Vol. XII, p. 182.

Kendall, A. I.

1911. Jour, of Med. Res., Vol. XXV, p. 117.
Logan, We. R,.
1915-14. Jour. of Pathology and Bacteriology,

Vol. XVIII, pp 527-551.

Cohendy, M.*

1906. Description d'um ferment lactique
puissant capable de s'acclimator
dans l'’intestin de l'homme. Comptes
Rendus de la Societe de Biologie,
Vol. LIX (Anne 58, tome 1), No. 11,
p. 558-560. Paris, March 30.

Belonovsky, Jd.

1908. Influence du ferment lactique sur la
flore des excrements des souris.
Annales de l'Institut Pasteur, Vol.
XXI, No. 12, pp 991-1004. Paris,
December 25, 1907. Abstract in
Centralblatt fur Bakteriologie.
Parasitenkunde und Infektion-skrank-
heiten. Abt., 2, Vol. XXI, No. 13/14,
p. 451, Jena, July 25.

me
(25)

(26)

(27)

(28)
(29)

(30)

(31)

(32)
(33)
(34)
(35)

(36)

Herter, C. A.
1909, On the therapeutic action of fermented.
Popular Science Monthly, Vol. 74, No. l,
pp 31-42, New ‘ork, January.

Heinemann and Hefferan. ,
1909. A Study of Bacillus bulgaricus. Jour.
of Inf. Dis., Vol. VI, No. 3, pp
504-318, Chicago, June 12.

Rettger, L. F.
1915. "The Influence of Milk Feeding on
Mortality and Growth, and on the
Character of the Intestinal Flora"
Jour. of Exp. Med., Vol. XXI, p. 365.

Sisson, W. R.
1917, American Jour. of Diseases of Children,
Vol. XIII, p. 117.

Torrey, J. C.
1919. Jour. of Med. Res., Vol. XXXIX, pp
415-447,

Porter, Morris and Myers.
1919. Jour. of Inf. Dis., Nov., pp 349-377.

Torrey, J. OC.

1917. Jour. of Bacteriology, Vol. II, No. 4,
p. 435.
Moro, &.
1900. Wien klin Wehnschr., Vol. V, p. 114.

Finkelstein, J.*

1900. Deutsch. Med. Wehnschr., Vol. XXII, p 263.
Rahe, A.
1915. Jour. of Inf. Dis., Vol. 16, p. 210,

Earnshaw, G. KE.
1918. The Literary Digest, Jan. 5, p. 22.

Buell, Hillhouse.
1920. Creamery and Milk Plant Monthly, April,
Vol. IX, p. 46.

* Not verified by the writer.
 

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