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MASTER OF HOME ECONOMICS

BY
LYDIA ZAE NORTHRUP

i 1913

 

 

 

 

 
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THKH UTILITY OF LACTIC BACTERIA.

Thesis for the derree of
MASTER OF HOME EBFCOWOMICS .
by

Lydia zoe Northrup

=a on ww we 4 oe OR

1913

 
THESIS

 

 

 
THE UTILITY OF LACTIC BACTERIA.

Pernaps no other single group of micro6r-
ganisms has such a wide distribution in nature and
in addition, such a range of usefulness sas has the
group of lactic acid forming bacteria which sare re~
sponsible for the cheracteristic fermentative
change in milk known as curdling. These bacteris
are also responsible for the characteristic fer-
mentation in other types of food as well, ssauer~
kraut and dill pickles being well known examples:
ensilage also derives its peculiar odor and flavor
from the growth of these micro6rganisms.

The most common phenomenon produced by the
lactic bacteria, the curdling of milk, has been
recorded from time immemorisl. "Butter", the
translation of the Hebrew word “hemah" given in the
Fnglish authorized version of the Old Testament,
means "curds" or curdled milk (9). In Genesis 18:
8, we ere told that Abraham placed curds before
his guests (2250 = 2000 B.C.); in Deuteronomy 32:
14 (1750 B.C.), Moses told the Israelites that

curdled milk was one of the blessings which Jeho-

ve
103196
van had given to his chosen people (46). In Isaiah
7; 15 and 22, butter or curds is mentioned as one
of the few foods left to the Judeans during their
war with the Syrians and Ephraimites (735 B.C.)
(10.).

For centuries soured milk has been used as
a food by the peoples of Eastern Europe, Western and
Middle Asia and parts of Africa. Fermented milk,

a soured milk beverage, has also been used from
time immemorial by the wandering tribes of Arabia.

Those of the ancient fermented milk bever-~
ages best known today are koumiss, kefir, leben,
yoghurt and mazun, dadhi, gioddu, khilat, oxygala,
chiston, rayet, huslanka and taettamoelk being
less commonly known. Each of these milk drinks is
the result of the concerted action of one or more
species of lactic bacteria with other bacteria and
yeasts.

Koumiss (kumyss, kumiss, etc.) is a drink
originally prepared from mare's milk by the Kumanes
@ Russian trive which lived along the river Kuma.
The milk of, the mare was placed in leather bags
and a small amount of old koumiss added as a star-
ter. This beverage is strongly effervescent, due

to the evolution of gas (carbon dioxide) produced
Curing the alcoholic fermentation by the yeasts
present.

Kefir (kephir, kepfir, kefyr, kefr, etc.),
the fermented milk of goats, sheep or cows wes Oo-
riginelly made end used by the inhabitents of the
Caucasus Mountains and wos prepared by adding "“ke-
fir grains" to the milk. The origin of these
grains is unknown but the natives believe that
they were the gift of Mahomet and are carefully
preserved by them. The natives prepared this drink
by placing the dried kefir grains in milk in a goat
Bkin vag and vy shaking occasionally until fermen-
tation commenced, when the grains were removed and
dried, thus preserving them for future use. The
fermentation was also often sterted by adding some
of the freshly fermented kefir to a new lot of milk.
In kefir, as with koumiss, alcoholic fermentetion
with the accompanying effervescence is more desir-~
able than marked acid production. Four different
organisms were found in the Kefir grains, yeasts
and three different types of bacteria; the combined
action of all four seem to be necessary to the pro-
Guction of good kefir.

Leben or leben raib is the name given by

the Eeyptians to their fermented milk drink. This
 

 

 

 

oo,
~4~

beverage which they have used for centuries, is pre-
pared from the milk of cows, buffaloes and goats.
The fermentation process is very similar to that

of other fermented milk drinks.

Yoghurt (yoghourt, yogurt, etc.) is a sim-
ljlar fermented milk prepared by the Bulgarians.
This differs from the preceding beverages in that
the acid fermentation is allowed to predominate.
It is made in skin bags in much the same way that
the Russians prepare koumiss.

Mazun (matzoon, matzun, madzoon, etc.) is an
Armenian drink almost identical with yoghurt.

Dadhi and khilat are fermented milks used
in India, Calcutta and Bengal respectively, very
similar to yoghurt, leben, etc.(17).*

Gioddu is a Sardinian fermented milk and is
produced by micro@8rganisms similar to those found
in the more studied fermented-milk beverages.(31).

The oxygala and chiston of Rome and Greece,
the rayet of Algeria and the huslanka of the Ruth-
enians are other known varieties of curdied milk
drinks (17}( 44).

* Chatterjee notes that the type of fermented milk

"“khilat" used in Bengal alone, has no prototype to
his knowledge. "The production of the curd depends
on the action of the products of a bacillus and not
on the living bacillus itself****there are very few
Bengalee householcs in which food prepared from
this curd is not in daily use ond 2 large trade is
cerried on in curd" (17).

There are two types of Tactic bvacteris which
ore capable of producing the characteristic cos¢g-
ulation of milk; these are for convenience often
differentiated by the terms "short" and "long" lac-~
tic bacteria. The former is the type met with in
ordinary sour milk, comprising over ninety nine
ner cent of its microflora. These ordinary lactic
bacteria, Bacterium lactis acidi, sre very small,
being from 0.6 micron to 1 micron long and O.5
micron wide, are oval in form and from the fact
that some of the shorter cells appear nearly spher-
ical and of ten oceur in chains, hes led to the ap-
plication of the name Streptococcus lacticus to
the group.

The “long” lactic bacteria present in the
Eastern fermented milks, sare true lactic bacteria
also as they produce almost exclusively lactic
acid from the sugar fermented, only very small
quantities of formic, acteic and propionic acids
being formed. These differ sreatly from Bact.

lactis acidi in size, being large rod-shaped forms

~ microns to 3 microns long and 0.5 micron to 0.75
 

 

 

TT “~~? ,
=a6G=

micron wide. The bacteria belonging to this latter
group are known as Bacterium bulgaricum, socalled
because first isolated and studied from Bulgarian
soured milk.

Both types of organisms are alike in that
they do not form spores and consequently are easi-
ly killed by heat, have no organs of locomotion and
form lactic acid from certain sugars as a main
product of their metabolic activities. They dif-
fer physically however in their respective temper-
ature requirements, Bact. lactis acidi requiring
a temperature between 30° and 35°C. for its @pti-
mum growth while that for Bact. bulgaricum is from
40° to 45°C; they also differ physiologically, in
their acid production, Bact. lactis acidi producing
in normal milk a maximum acidity of from 0.9 to
1.25 percent only while with the long lactic bac-
teria the maximum acidity varies from 1.25 to 4.0
percent.

It is this physiological property, that of
acid formation, /which depends the importance of
these lactic bacteria in their many phases of use-
fulness. Milk does not lose any of its valuable

properties by undergoing acid fermentation, in

fact its benefits as a food are generally augmented
a7 =

by the presence of the acid produced by the activity
of the lactic bacteria. The longevity of the Bastern
peoples is attributed by Metchnikoff to the use of
acid milk as their staple article of diet. He offers
this explanation of its action: lactic bacteria when
introduced into the intestine transform its carbohye
drate contents into lactic acid; this acts as an in-
testinal antiseptic, destroying or inhibiting the putre-
factive organisms (which work in an alkaline medi-
um), thus preventing the formation and subsequent
absorption of toxic substances which have a degen-
erative influence on the body tissues.

To Metchnikoff is due the great credit for
having insisted on the importance of introducing
these lactic bacteria into the intestines for the
purpose of controlling the type of fermantation
therein. As a result of his teachings many lead-
ing physicians today are recommending acid milk Ber-
erages for use in intestinal therapy, and several
large laboratories have been organized solely for
the preparation and distribution of concentrated
forms of starter for these curdled milk drinks.

Two of such are the “Pasteur Laboratories of Amer-
ica" and the "Ferment Company" both located at New

York City. Other commercial firms put out start-
ers for fermented milk or artificial buttermilk for
therapeutic purposes. Such are the “Lactone" and
"Bacillus bulgaricus” tablets prepared by Parke
Davis and Company of Detroit. H. K. Mulford Com-
pany of Philadelphia also have a similar preparat-
ion on the market. Within the last few years a
number of the fermented milk beverages themselves
have also been placed on the market under such
trade names as zoolak, vitalac, yogurt, bacillac,
lactobvacilline, fermentlactyl, bulgarzoon, etc.
The flora of these seems to be practically the
same as that of the original yoghurt.

As has been previously mentioned, the lac-
tic bacteria of the Bact. bulgaricum type are pre-
sent in the Eastern fermented milks in which they
dominate the fermentation. This property can be
explained by the physical influence of temperature
upon the metabolic activity of this group of micro-
organisms (see p. 6), climatic conditions favoring
the optimum growth of the long lactic bacteria rath-
er than that of the Bact. lactis acidi type. This
is a partial explanation also of the greater thera-
peutic value of milk soured by the Bact. bulgari-
cum type of lactic bacteria, body temperature offer-

ing the optimum conditions for growth; the greater
=Q ua

amount of acid produced, adding materially to its
beneficial action in intestinal therapy by acting
as a strong antiseptic, completes the explanation.
After having hed the centenarians of Bul-~
garie and of other sour milk drinking countries
held up a8 a living example of the efficiency of
acid milk beverages as an intestinal sntiseptic,
many investigators were led to carry on experi-=
ments simulating natural conditions as far as pos-~
Bible for obtaining corroboration or refutation of
the theories advanced by Metchnikoff and his co-
workers. Quite widely vurying results were 8s0 ob»
tained. Helen Baldwin (2) studied the influence
of lactic acid ferments upon intestinal putrefact=~
ion in a healthy man forty three years of age,
feeding him upon lactobacilline malt, bacillac,
zoolak and plain milk and concluded that in a mixed
diet containing meat, the patient's condition was
better when lactic acid ferments were not added to
the diet. She found however that when the main
proteid portion of the diet was furnished by the
milk, intestinal putrefaction was seemingly checked
to quite an extent during the use of the fermented
milks but in only one experiment was the reoction

of the feces acid. Grekoff (30) to the contrary,
found an acid reaction in the feces after the fourth
or fifth day of treatment with fermented milk.

Boas and Oppler (11) and Kaufmann (42) found
a Bact. bulgericum-like organism constantly pre-~
sent in carcinoma of the stomach but Straus and
Bialacour (77) found this organism in normal gas-
tric fluid; Heinemann and Hefferan (35) also con-
clude that it is normally present in small numbers
in the digestive tract of many animals since it is
so widely distributed in nature, they having found
it in ordinary milk, feces of cows, horses and man,
in soil, grains and pickles. The inference may be
drawn from their work that since this organism is
so widely distributed, it must be taken into the
digestive system almost continuously, and if it
were adapted to growth in the intestines it would
become established there in the natural course of
events. Rogers (65) states that an acid condition
of the intestines is carefully guarded against by
the provision for the neutralization of the acid
juices of the stomach as they enter the intestine
and he presumes that this action is automatic, i.e.
en increasing acidity in the food coming into the
intestine would be followed by a corresponding in-

cre:sed flow of alkaline secretion. *** It seems
allie

improbable that the acid contained in any form of
fermented milk would be great enough to affect the
activities of intestinal bacteria even along with
the heavy inoculation of the acid forming organ-
isms. However, Cohemdy (18) wes able to establish
Bact. bulgaricum ‘in the intestinal tract by long
continued feeding.

Grtnbaum (32), in discussing lactic acid
therapy, describes the value of Bact. bulgaricum
as not being due to its section as a phagocyte, i.e.
ingesting harmful organisms, but rather that it
produces lactic acid and further, forms it where
it is of value, i.e., in the contents of the lower
intestine which otherwise are subject to continued
putrefaction. There are two advantages in the use
of Bact. bulgaricum, first that its purity is
readily determined by simple microscopic examin«t-
ion and second thet it is a more active acid pro-
ducer than the ordinary type of lactic bacteria.

In its use in therapy the patient should be given
a carbohydrate diet, limiting the proteids and fat,
at least four days before beginning treatment.*

* Cohendy(18) found that the disinfecting action

of the lactic acid culture was not appreciably in-«
fluenced by variations in the amount of sugar eaten,

 

 
wlpea

indicating that the ordinary diet contains sufficient |
suger to support the growth in the intestines of '
the lactic bacteria.
Grfnbaum in conclusion says that although
many patients have been benefited by the use of
these fermented milk drinks he is inclined to think
that the ailment in many cases was only "maladie
imaginaire“. This view is also held by Dr. Har-
ley (33) of the University College of London; Rob-
ert Hutchinson (41), Assistant Physician to the
London Hospital, is also skeptical of results.
However, most physicians who have given the
lactic bacteria a thorough trial in intestinal
therapy, heartily endorse their use. Herscnhnell
(37) sags, that with reasonable use, lactic bac-

teria heave excellent therapeutical value for abnor-

 

mal proteid putrefaction in the intestine or for
some forms of constipation. Fresh (24 hours old)
cultures of lactic bacteria, preferably Bact. bul-
garicum, should be used for colonization in the
intestines, about two and one half months being

necessary to obtain a complete transformation of

 

the intestinal flora. <A proof of the therapeutical
value of the lactic bacteria is shown by the pre-

ponderance of Gram-positive bacteria in the feces

 
-13

of patients dieting on sour milk preparations; in
health, when B. coli ( a common intestinal organ-
ism) predominates, the intestinal flora are Gram-
negative (37). Previous to abdominal operations
he advises rendering the sastro-intestinal canal
aseptic by feeding the patient fresh liquid cul-
tures of the "Bulgarian" bacterium (38).

Herter and Kendall (39) investigated the
fate of Bact. bulgaricum in the digestive tract
of a monkey and found that an acid reaction could
be maintained in all but the Lleoececal region or
large intestine.

A frozen milk preparation "Lacto" contain-
ing lactic bacteria of the Bact. lactis acidi type
has been concocted by Mortensen and Gordon of the
Iowa Agricultural Experiment Station (54). The
purpose of this product has been to supply the
lactic bacteria in an attractive form to people to
whom loppered milk or buttermilk is objectionable.
Lacto is made from loppered whole or skim milk
with the addition of eggs, sugar, lemons and flav-
oring material; it has ao more pleasing flavor than
sherbets and ices and contains considerably more
nutriment, its protein content equsling that of

ice cream but containing less fat and more acid;
~l4@

the main argument however, is that the lactic or-
ganisms in this frozen seur milk preparation remain
alive for some time after the milk is frozen thus
allowing these bacteria to be supplied in large
numbers to replace the putrefactive forms present
in the intestinal contents. The largest percentage
of persons who sampled lacto preferred it to vanil-
la ice cream or sherbet; 30 it seems that if this
type of lactic bacteria ss. as beheficial as the
long lactic type, this frozen |jsour milk product may
become a wide spread benefit, especially so in the
Summer months when gastrosintestinal diseases are
predominant.

Many physicians, Hutchinson, Harley, Grfinbauwm,
Klotz, etc., are skeptical as to the therapeutic
value of lactic acid preparations but the greater
number who have employed them in one form or anoth-
er are convinced of their utility in intestinal and
other forms of therapy.

Dr. J. W. H. Byre (25) successfully treated
pathdgenic conditions of the colon with his pure
culture of lactic bacteria.

Hertz (40) and Grtinbaum (32) treated colitis
sucessfully. Dunn (24), upon feeding children suf<

fering with intestinal catarrhn, upon pasteurized
 

 

2a.

 

 

 

 

aw

———-- a
buttermilk inoculated with a known species af Jac-
tic bacteria caused the flora and the character of
the stools also to change to normal, the toxic symp-
toms passed away and the children gained in weight;
practically the same effect as is produced by yog-
hurt. Allen (1) effected a cure of South African
typhus fever and dysentery by the use of lactic
acid bacteria, Cummins, Fawcus and Kennedy (21) on
the contrary, found no cessation of the excreting
of B. typhosus in the feces nor in the urine by
treating with Bact. bulgaricum.

Other pathological conditions have been
successfully treated with liquid cultures of lactic
bacteria. Dr. W. Langdon Brown (15) successfully
applied this form of therapy to indicanuria. Grfin-
baum (32) treated uremia and diabetes mellitus and
found that the lactic acid is excreted as a carbon-
ate which tends to increase the alkalinity of the
blood and to defer acidosis; the lactic bacteria by
changing the carbohydrates in the food into lactic
acid, tend to diminish gitgcemia. Sahli (72) has
had good results from the use of lactic acid pre-
parations in some cases in conditions of anacidosis
in diabetes also and in pernicious anemia but says

that the presence of Bact. bulgaricum is not essen-
~l6u

tial as the same benefits were derived in most cas-
es from using the acid alone.

Liquid cultures of lactic bacteria have
been used also as a spray or wash for the disinfect-
ion of the mucous membrane of the nasal, oral and
genito-urinary cavities. Dr. Charles BE. North (55)
(56) of New York City was the first in the United
States to use suspensions of living lactic bacteria
for the disinfection of bvody cavities. Many cases,
chronic rhinitis, ethmoiditis, frontal sinusitis,
otitis, antritis, tuberculous sinuses, gonorrhea,
cystitis, leucorrhea, diarrhoea, acute and chronic
suppurating wounds and sinuses, inflammation of the
maxillary bones and palate, gonorrheal ophthalmia
and conjunctivitis, numbering three hundred in all,
were treated with injections of dextrose broth cul-
tures of Bact. bulgaricum. Of these, sixty three
were permanently cured, one hundred sixty two showed
marked improvement while with only eighty six was
there no result from the treatment. Dr. North con-
cluded by stating that the use of these cultures as
a wash or spray on inflamed surfaces or cavitiés
often diminishes odor caused by putrefaction; that
both acute and chronic inflammations caused by in-

fections sometimes appear to be checked by inject-
-17~

ing these cultures at the seat of infection; and
that the use of the cultures seems to be accompanied
by no special danger and that they cause no irrita-
tion.

The extensive work and favorable conclusions
of North suggested to Giltner (29) the possibility
of the therapeutic use of lactic acid cultures in
treating the purulent metritis following abortion
in cattle. Whey cultures of Bact. bulgaricum were
injected into the uterus and vagina after abortion
with the result that the discharges soon ceased and
portions of the retained placenta passed off without
any decomposition. No evidence has been noted of
sterility having been produced by the use of this
method, in fact, it is being used to combat steril-
ity or failure to breed in cattle with encouraging
results. In three year's application, no instance
of failure to restore a case of utero-vaginal trouble
to an apparently normal condition has been recorded,
@ large number of cases indeed indicating the value
of this treatment in assisting toward the restor-
ation of fertility to sterile cattle.

Since Dr. North's work in 1908 - 1909, oth-
er investigators have taken up the lactic acid

treatment of mucous membrane infections, especially
in connection with the genito-urinary tract. Dr.
Persson (62) of Mount Clemens treated thirty four
cases of chronic specific urethritis with the fol-
lowing conclusions: secretions in the infected ur-
ethra inhibited common lactic acid organisms within
15 minutes but when the lactic organism was grown
on nucleic acid media the organisms could be recove
ered in an active state after 10 hours sojourn in
the urethral cavity. The disappearance of the gono-
coccus bore a constant relation to the length of time
that the lactic bacteria retained their “virulence"
in the urethra.

Dr. F. S. Mason (47) in his article on the
‘Application of Lactic Ferments tn Genito-Urinary
Work' finds that the lactic ferments inhibit the
growth of the pathogenic organisms in the genito-
urinal passages.

Dr. Watson (78) treated gonorrheal and mixed
infections of the female genital tract with a fil-
trate of sour milk containing large numbers of lac-
tic bacteria. This liquid culture was introduced
into the vagina after a thorough disinfection and a
subsequent washing of the infected parts with sterile
water. This treatment caused a stimulation of the

secretions which gradually changed to normal in

stihl ete

 
~l19u

character, then the amount of secretion also dimin:
ished to normal. This type of infection seems to
respond encouragingly and with rapidity to this
form of treatment. The use of liquid cultures of |
lactic bacteria in gonorrheal infections seems to
have a most promising future.

Affections of the nose, ear and throat have
been successfully treated also to a large extent by
liquid cultures of these bacteria. North's work
stimulated investigations of the therapeutic value
of these cultures as applied to the respiratory
passages. Dr. H. Holbrook Curtis (22) of New York
City began experiments along this line with a cer-
tain amount of skepticism but obtained such markedly
good results in certain cases that he concluded
that a very valuable advance had been made in the
treatment of pathogenic conditions of the nose and
accessory cavities. He examined the secretions
from the nasal fossae before the lactic bacteria
were introduced and again after successive appli-
cations of the culture. In some cases after a limit-
ed number of injections, not only were the pyogenic
bacteria destroyed but there was a total absence of
all bacteria in the secretions. In an acute case of

frontal sinusitis of over a year's duration, the pro-

 
=20—
fuse discharge was caused to diminish ninety per cent
after the second dose of a cubic centimeter of the
culture, Other types of cases were cured or greatly
relieved by this treatment. Dr. Curtis says “Should
future experimentation substantiate the evidence of
results obtained thus far, it would seem that a new era
in the treatment of pathogenic bone cavities has
been instituted, and that the theories of Metchni-
koff admit of a anch wider application than he has
claimed.

Berthelot (7) used the lactic culture suc-
cessfully on three cases of “Nasenrachenraum" and
freed a nine year old child of adenoids and "Fieber-~
anfallen". In another publication (8) he tried the
action of Bact. bulgaricum on the meningococcus in
vitro and found that the destruction of the patho-
gen followed in a short time the strong acid pro-
duction of the former. This lactic culture was to

be used as a nose and throat spray for annihilating
the meningococcus from "carriers". Practical ex-
periments had not been carried out at this time.

Dr. Francis Bond (12) used a whey culture
of Bact. bulgaricum as a throat BpraAy with fair
success in the treatment for Bact. diphtheriae.

Gilpatrick (28) however says that although

the pure culture of Bact. bulgaricum is sometimes
ae)»

of benefit as a lecal antiseptic it must ve |used
with caution, especially in the oral cavity because
the action of the lactic acid on the enamel of the
teeth may give rise to caries, In Pyle's Bersonal
Hygiene (63) this statement has been fefuted by
investigations carried on by Black of Indiana who
subjected a large number of extracted teeth to a
great variety of tests including the action of
chemicals and the attacks of bacteria and found
that strong and feeble teeth were affected alike.

Miller (52) sides with Gilpatrick in believ~
ing that caries is the result of lactic acid proe
duced by certain bacteria; Hopkins (63) of Harvard
has shown that of the one fifth of the fifty varie
eties of bacteria in diseased teeth which produce
lactic acid, none formed over 0.5 per cent. He
then subjected good and poor teeth alike to the
action of 0.5 per cent lactic acid for four months
wbthout any carious action taking place , and
stated further that caries is often accompanied by
a free secretion of saliva, i.e., that the mouth
is alkaline.
Other miscellaneous conditions of widely

different nature have been benefited by treatment

with lactic bacteria. Dr. C. H. Miller (52) treat-

 
mooe

ed rheumatoid arthritis with but little benefit to
the arthritis, the patient's health was greatly im-
proved however.

McCarrison (48) in treating endemic goiter
stated that the administration of Bact. bulgaricum
as an intestinal antiseptic had a favorable healing |
action when used when goiter was just beginning.

McLaughlin used a powder prepared from s&ter~
ilized and dried milk solids inoculated with Bact.
bulgaricum for dusting into suppurative wounds such
as carcinoma, chancroid, purulent ulcers and septic
wounds with good results (49),

Giltner (29) had equally good results in
packing large abscesses in cattle with sterile cote.
ton soaked in a lactic culture; such abscesses were
healed completely in a few days, characterized by
the absence of discharge. [In all cases the healing
was rapid and complete,

Much work has been done upon the effects of
Bact. bulgaricum and Bact. lactis acidi upon differ-
ent organisms in vitro. As early as 19035, Bonska
(13) determined that there is a marked antagonism
between bacteria of the B. subtilis group (some of
which are pathogenic) and lactic bacteria, the lat-
ter showing their specific energy in media whether
—ee a
=

 

\
--. -. . yf” eee

 
sugar-containing or not. Tne proliferation of the
subtilis bacteria soon ceases and their number rap- |
idly diminishes. Bonska does not attribute this phen-
omenon to the accumulation of lactic acid alone as
this diminution in numbers occurs in eugar-free or
in strongly alkaline solutions. These results taken
generally are contrary to those obtained by other
investigators, who claim that the antiseptic power
of lactic cultures is due alone to the sacid pro-
duced.

Crithari (23) in studying the symbiosis of

Bact. bulgericum with B. butyricus (non-pathogenic)

 

determined that when the acidity of the medium was
maintained at a constyunt amount that the phenomenon
of fermentation was reduced to a minimum. Very
Similar are the results obtained With pathogenic
and lactic organisms in vitro, by other scientists.
T. von Kern (43) attributes the inhibitive
action of Bact. bulgaricum upon B. coli to the nas-
cent lactic.acid. In the intestine this is followed
by a diminution of undesirable intestinal products.
He advises the use of “lactobacilline" to prevent
the accumulation of these products.
G. Rosenthal (67)(68)(69)(70)(71) has car-

ried out extensive experiments on the associative
 

 

 

—— an +e
action of Bact. bulgaricum upon a number of organ-
isms, B. perfringens, the gas-bacillus found in
lung gangrene and ear-disease; B. pyocyaneus, the
bacillus of blue or green pus; "l'anhemobacille
enterocogene du rhumatisme"; B. diphtheriae and
bacteria of the B. typhosus group with almost uni-
form conclusions, that the destruction of the path-
ogen is soon effected by the introduction of Bact.
bulgaricum into the culture, even if the former
has oeen allowed to gain a start. The delay in
the destruction is inversely proportional to the
quantity of lactic culture superadded and if the
reaction of the medium is carefully kept neutral,
hoth organisms grow. In the case of B. typhosus
he concludes that the acid is essential to and per-
haps the only cause for the death of the pathogen.
In my own investigations (57) on the influ-
ence of the products of lactic organisms upon B.
typhosus, the results obtained led to the conclusion
that, of equivalent amounts of acid formed by Bact.

lactis acidi and Bact. bulgaricum respectively,

 

the acid produced by the former was twice as ef-
ficient in antiseptic properties as that of the
Kni¢ch sold producing organism. Whether these same

results and conclusions would be derived from a
 

» + wom
SS er 9 See
parallel set of experiments with other strains of
the different organisms is a matter of question.
The most marked results were obtained with another
high acid producing lactic bacterium. This par-
ticular organism resembles Bact. bulgaricum in
nearly all respects, but produces acid in larger
ymounts and more quickly at room temperature, has
a much greater vitality and resistance to its own
products and produces a color reaction in litmus
milk culture similar to that produced by the or-
dinary sour milk bacteria. This bacterium, at 60°
acid (Fuller's scale), caused the destruction of
the typhoid organism within 24 hours and at 72°
acid within 2 hours. The acid produced by this
lactic organism however was not as efficient in
small quantities as that of Bact. lactis acidi.
Rodenwaldt (64) in his paper given at the

German Tropical Medicine Convention in Hamburg,

 

April, 1912, said that in vitro, Bact. bulgaricum
inhibits B. typhosus and B. dysenteriae principal-
ly in the first twelve hours of their development,
the typhoid bacilli still growing in culture after
6 hours growth with Bact. bulgaricun.

rrom these many examples of the influence in

vitro and in vivo of the different types of lactic

 

 
 

 

Cae

aCe...

 

 
@26-

orsanisms, it is evident that these bacteria already
have an extensive use in therapy and from the prom-
ising results obtained thus far in the various
cases, it is not altogether presumptuous to predict
a much wider range in this phase of their useful-
ness.

From the type of references just cited, one
might be led perhaps to entertain the erroneous i-
dea that the lactic bacteria find their greatest
utility in therapy. A much older and more firmly
established application however is the use of these
organisms for controlling the type of fermentation
in the butter-making industry. Lactic bacteria of
the Bact. lactis acidi type are employed almost ex-
clusively for this purpose.

A quarter of a century ago the use of pure
culture starters for butter-making was unknown.
The buttermaker prior to this time possessed lit-
tle or no control over the growth of the organisms
that produce cream ripening. The cream that was
received from a wide territory by the creameries
necessarily contained a numerous flora both as to
species and number, and if it chanced that the
proper species predominated in such mixed crean,

the ripening would go on in a normal fashion and
 

 

a — —_—_—
227

the results obteines would be satisfactory. If it
chanced however that the cream brought to the crean-
ery contsined in great numbers, species of bacteris
which were unfsevorable to good flavor production,
the butter would be likely to be poor in quality

and no care which the buttermaker could take in the
subsequent preparation of the butter could remedy
this particular defect, and consequent financial
losses would ensue. (19).

The revolution which had been produced in
the brewing industry by the use of pure yesst cul-
tures led to the suggestion that a similar control
might be possible in cream ripening by the intro-
duction into the cream of the organisms responsible
for the ripening process. It was Professor Storch
(76) of Copenhagen who first (about 1890) conceived
tne idea of obtaining from cream those species of
bacteria which produce the best results and then
furnishing them to the buttermeker for the purpose
of ripening the cresm, somewhat as yeast is fure-
nished to the brewer.

This plan as suggested vy Professor Storch
was put into practical use in Denmark, and later
in other countries in northern Europe. In one

sense this plan was not new. For many years pre-
f= a

 

 

 
vious, buttermakers had added what they called a
"starter" to hasten the ripening of the cream in
winter weather. A: starter was simply a lot of
good-flavored already-soured milk or cream contain-
ing of course the lactic bacteria in large numbers
in an actively growing stute. The new feature of
the method suggested vy Storch was the artificial
preparation of such a starter by the use of pure
bacterial cultures obtained by bacteriological
methods. This method soon distributed itself more
or less in the various butter-making countries.
Nowhere however has this method been used to a very
great extent except in the countries of Northern
Europe and anly then at times when the butter of a
creamery shows an off flavor or aroma as a result
of improper ripening of the cream. The analogy may
be drawn between this off-fermentation in cream
and butter and disease, the lactic bacteria added
in pure culture acting as a preventive of disease
largely through the agency of the lactic acid pro-
duced. This illustration holds true for all phas-
es of the usefulness of these micro68rganisms.

In the use of Professor Storch's method of
cream ripening the pure cultures of lactic organ-

isms can be obtained only by bacteriological methods.
 
=O Gu

To obtain such a culture bacteriologists have nature
ally had to select the type of bacteria found most
often in ripened cream which produced a high grade
butter. There is no difficulty in obtaining from
such cream tie lactic bacteria that are present in
the greatest abundance as they form over ninety
nine per cent of the flora.

The problem of a proper culture for cream
ripening is quite different in different countries
Since the public taste for butter is so dissimilar;
no type of ripening which is satisfactory in one
country will meet the demands of another neccssar-
ily. The cemand in the United States fs for a highly
flavored butter while Denmark and the rest of North-
ern Europe prefer a very mild flavored product.

By a happy provision of nature, many quite differ-
ent flavors are produced by the different varieties
of lactic bacteria so that these varied demands

may be wet without difficulty.

The lactic bacteria as obtained either dir-
ectly by isolation or in a powdered or liquid form
as furnished by commercial firms are not added di-
rectly to the cream to be ripened, for although
the organisms in thése commercial or newly isolated

cultures amount to many billions, still the number is
—— jee on ee” Ce "OC 7 =r

 

 
=-30~

qguite insufficient to inoculate properly a large
quantity of cream. The culture must of necessity
be "built up", i.e., the number of lactic bacteria
must be greatly increased. This is done simply by
adding the small emount of culture to a quart or

so of cream or milk which has been heated previous-~
ly to the temperature of boiling, covering such
inoculated cream or milk to keep out the dust and
placing it where it is moderately wurm for a period
of 48 hours. During this time the bacterie increase
enormously in numbers, thoroughly impregnating the
railk or cream, This large amount of culture is
then added to several gallons of pasteurized milk
or creem, stirred thoroughly, covered and set aside
for the further development of the starter orgen-
isms. In the course of two days the "starter" is
ready for inoculeting the body of the cream which
is to be ripened.

Altnough this process is simple, it is some-
what troublesome ond for this reason the wider ex-
tension of the use of pure cultures in butteremak~
ing has been prevented. Several commercisl firms
heve attempted to put on the market s form of pure
culture that could be added directly to the cream

without previous cultivation. The tests of such
—_— ae re 8 re C-

 

 
cultures however heve not been satisfactory; the
bacteria are not present in sufficient abundance to
accomplish the best results. This is true of all
comumercigl pure cultures of lactic bacteria of eith-
er type; the organisms succumb either to the dry-
ing as in preperations in powdered or in tablet
form, or to the antiseptic influence of their own
products, lactic acid mainly, in the liguid form.

At the present time there is no reason for
believing that such pure cultures for direct use are
of any practice#l value and the use of pure cultures
in buttermaking still depends upon the "building-
up" process.

The starter prepared by the building=up
process is added to the cream to be ripened in the
proportion of about 10 per cent. After the cresm
has ripened and is ready for churning, a few gal-
lons are removed to be used as a starter for the
next day's churning. In this way the starters sre
carried from day to day for several days. About
once a week however, it is commonly found desir-
able to build up 3 new starter from the commercial
pure culture,

The more common method of utilizing the

lactic bacteria for buttermaking is vy the'natural'
On
ow bun

starter prepared by the buttermaker himself without
the necessity of purchasing a commercial product.
This natural starter is nothing more than ae lot of
good-flavored, naturally-soured milk or cream and
was used for many yeers previous to Storch's methe
ocd.

By the use of this method the dairyman hes
no method of knowing what hacterila sre present or
of how to control them. Logically then and theo-«
retically too, the method of the use of natural
starters is incorrect end practical experience a-
mong dairymen goes to show that although natural
starters are as a rule, quite successful and pros«
duce a satisfactory ripening of the cream, they
are not uniform and occasionally are not reliable.
Even though produced under similar conditions they
do not always give rise to the tyre of ripening
that is desired. The fact however, that such ir-
regularities seldom occur, in contrast with the
trouble and attention necessary to prepare a pure
culture starter from commercial products has led
to the use of the natural starter almost exclusive-~
ly vy dairymen who wish to make o high grade of
butter.

Experimental tests of the value of butter
ee -
Sa os Oe — —

 
made with and without pure cultures have shown
that the best quality of butter made with pure
cultures is apparently not quite equal to the best
quality of butter meade by spontaneous ripening.
Thses exceptionally fine grades of butter however,
are not the ordinary yield but have been made in
an experiment station or unon an individual farm
where special cere has been taken to produce cream
in the best condition,

Under these circumstances 4 pure culture
moy be of no advantage, but in general dairying
and especially in cregmeries, 911 sorts of cream
both good and bad are accepted. Here, pure culture
starters should always be used, and while these
cultures cannot correct entirely the imperfections
resulting from the miscellaneous organisms in the
mixed cream, they act as an effective check upon the
development of a heterogeneous microflora and their
use is thus clearly of benefit.

The use of pure culture starters kas an ad-
vantage along three distinct lines: the buttermaker
can handle his cream more uniformly, being able to
regulate the ripening, the grade of ripeness and
the amount of starter to add to a certain amount

of cream; the product posses 3 greater uniformity,
-d4ua

Fluctuations in quality due to 6 miscellaneous flora
or to seasonal variations tend to dissppear. This
seems to be the most distinct edvantsege of the pure
culture method, being the one which gradually
brought about the introduction of this method of
buttermaking throughout northern Europe. Ag to the
third advantage, on the average, the flavor of
butter is somewhat improved by such cultures although
it is difficult to obtain any proof of this owing
to the uncertainty in greding flavors. Markets
that demand a clean, mild-acid-flavored butter
prefer that made from pure culture starters.

It is not essential that the butter fat be
present during the acid |fermentetion in order to
impart flavor to the butter. Sweet cream may be
mixed with sour cream or milk and churned at once,
the flevoring compounds being absorbed from the
soured milk by the fat, and the butter will have
much the same flavor both as to intensity and kind
a8 though the fat had been present during the fer-
mentation. This method is identical with those
employed by the manufacturers of oleomargarine and
renovated butter. The flavor of these butter sub.»
stitutes is identical with and derived from the same

source as the flavor of butter,
“35 —

The question may now be asked, what becomes
of the immense number of lactic bacteria necessarily
introduced into the butter in the process of churn-
ing? and do they play any further part in the bute
ter after it is resdy for consumption?

A large part of the bacteria are removed
from the butter with the buttermilk, also by the
ywober in thé processes of washing and working the
butter, but the completed product still contains
them in great numbers. Freshly made butter contains
millions of bacteria per gram, but es they do not
find favorable conditions for growth on account of
the compactness of the butter and the comparative-
ly small amount of moisture and the lack of oxygen
penetration, they begin to die qtickly. Conn gives
the figures for the decline of bacteria in a same
ple of butter, starting out with 50,000,000 bacteriz.
per gram the nunber is reduced 50 per cent in one
day, down to 4 per cent of the original number in
four days and 0.6 per cent inside of thirty days.
This may be looked upon as the normal history of tha
bacteria contained in butter.

The salting of butter has some influence
upon checking the growth of lactic bacteris, and

other microSrganisms contained therein and thus,
- <————" oe
— Eee ee A

 

 
-J36<

within certsin limits, hos a preservative action.

The lactic acid contained in the butter,
contrary to what might be supposed, is not present
in sufficient quantity to serve as an important fac-
tor in its keeping quality, the total acid in fresh
butter titrating from 1.5 per cent to 2.8 per cent
(73). In fact, Rogers, Thompson and Keithley (66)
state that butter of as much superior keeping qual«
ity was almost invariably produced by limiting the
acidity to the region of 0.3 per cent and below.
The presence of larger quantities seems to sid mater=
ially in the decomposition of the butter proteins
and fat, resulting in aff flevor and rancidity,
i.e€., butter made from overripened cream, which is
high in acidity, possesses much poorer keeping
qualities than butter from properly ripened cream,
However, Hastings (46) says that sweet cream butter
is morked by the rapidity with which it undergoes
decomposition chenges, especially when it is made
from raw creem, and atates that as a rule, sour-
cream butter has much better keeping qualities
than those of sweet cream butter.

Lectic bacteria of the Bact. bulgaricum type
have been used instead of the ordinary Bact. lactis

acidi as a starter for cream ripening and buttermak-
- ~<a re” _.

 

 
i

ing with good results es to flavor and aroma of the
finished product. The dairymen finds difficulty
however in maintaining this starter at a, suffici-~-
ently high temperature to insure the curdling of the
milk within 18 to 24 hours; es 2 consequence, this
type of lactic organism seems to hseve no prospect
of being utilized in this way on a large scale.

Cheese is another of the important cairy
products in whose manufacture the lactic bacteria
play an importeunt part. In the process of making
cheese it is necessary to curdle the milk, that
the cheese constituents may be separated from the
whey and soluble elements of the milk. Two methods
are employed to accomplish this purpose, and as a
result two types of cheeses are produced, tne “acid-
curd" and the "rennet-curd" cheeses, in both of
which the lactic acid forming organisms are neces-
sary for producing the principal flavor.

In the acid-curd cheeses, the curdling is
accomplished by allowing either spontaneous acid
formation through the agency of the normal flora
of the milk, or by the addition of a pure lactic
culture os a starter. These cheeses which are eat-
en fresh, owe their flavor to the products of the

acid fermentation, especially lactic acid.
 

 

— -- en 8 -
In the preparation of the second type of
cheese, rennet is made use of for curdling the milk.
Rennet-curd cheeses undergo a process of ripening,
in contrast to the sacid-curd cheeses and if harm-
ful forms of micro§rgeanisms are present in the milk
they will pass into the cheese and there produce
their harmful effects. If the milk is inoculated
with » pure culture of lactic bacteria before the
addition of rennet, the proportion of desirable
Organisms can be increased and a partiel control
of the fermentation thus secured.

The proper ripening of rennet-curd cheeses
can only be accomplished in the presence of a con-
siderable number of lactic bacteris. In the eorli-
er process of cheese-making the curd is cut into
small cubes which enclose 95 to 99 percent of the
bacteria in the milk. The development of the acid
in the curd is rapid, due to tke concentration of
large numbers of bacteria in a small volume and to
the favorable environment. This elaboration of «cid
in the curd through the agency of the lactic baec-
terise is an important step in the process of mak-
ing cheddar cheese as it brings about the condi-
tion known as “matting". The curd particles at

e

first show little tendency to cohere, but as the
_ we eee ———"— ae

 

 
acidity increases, the nature of the curd changes
and this “matting or fusion of the curd particles
takes place. The nature of this change though not
well understood, is probably due to a combination
between the peracasein and the lectic acid, the’
resulting compounds differing from the parsacasein
in physical properties and solubilities.

In regard to the further reletion of the
lactic bacteria to cheese, i.e., in the ripening
process, de Freudenreich, se Swiss microbiologist,
ascribed an important role to the acid-forming
bacteria in the ripening of Swiss cheese for the
reason that great numbers were present in fresh
cheese, and when cheese was made from milk drawn
under aseptic conditions, no ripening Occurred;
also, that lactic bacteria of the Bact. bulgaricum
type predominating in Swiss cheese have a solvent
effect on the casein of milk.

It is generally conceded that acid forming
bacteria are essential factors in the ripening of
cheddar and probably all kinds of rennet-curd chees-
es.

At this point it is pertinent to mention
the possibilities of dairy products as carriers of

infectious diseases. This would naturally raise
~40-

the question as to whether the lactic bacteria
which occupy such a pertinent place in therapy
vill not exercise the same functions in this con-
nection also. As naturally soured milk is not
commonly used in this country as a food or bever-
age, the chances for diseases like typhoid fever,
diphtheria, scarlet fever or tuberculosis being
transmitted by this means is very slight.

Sour milk and cream products however, such
as buttermilk, cottage cheese, milk and cream
cheeses and butter hold an important place in the
diet of most people in the United States and should
a type of pathogenic organism find its way into
one of these products, the question arises immediate-
ly ‘how long will this organism retain its vital-
ity and virulence under the conditions existing in
‘this product?!

Obermttller (58)(59) found that both the
tubercle and the typhoid bacillus are capable of
living for many days in butter and therefore may
possibly be a source of these two diseases in the
consumer. Various investigators have found the
limit of life of the tubercle bacilli in butter to
vary from 12 days to 8 months, and although both

certainly cannot be right as the difference in the
———— ee
—_ —~ - -

oA} «

respective limits of time is too great, one or the
other must approach accuracy. Most investigations
show that the organisms of tuberculosis are not
killed in ordinary salted butter in four to five
months even though the butter has become rancid
and moldy and unfit for human consumption.(74).

If we take the figures of European investigators
we may conclude that butter is more frequently in-
fected with tubvercle bacilli than milk, in fact,.
from the individual investigations of Herr (36),
Cornet (20) and Bro@érs (14) tubercle bacilli could
be found thirteen times in butter for every ten
times in milk.

It is a simplé matter to destroy tubercle
bacilli in milk and cream by pasteurization, but
just how to make infected butter safe is a more
difficult problem. As it is a well known fact that
these organisms are unaffected in milk or cream by
the presence of the acid produced by active lactic
acid producing bacteria, the only safe-guard left
is pasteurization. Butter apparently contains no-
thing except salt which has an inhibiting influ-
ence upon the vitality and virulence of the organ-
jams of tuberculosis, but the germicidal value of

salt in the proportion in which it is used in com«
mercial butter is very low (74).

As to the effects of the products of lactic
bacteria upon the organisms of typhoid fever when
contained in butter, Barthel (3) states that in but-
ter which is strongly acid, typhoid bacteria may be
found after ten days, the strong brine enclosed in
such butter being a good nourishing medium. On
the other hand, Bassenge (5) has attempted to show
that during the process of cream ripening, the lac-
tic acid produced by fermentation organisms serves
to diminish the number of typhoid bacteria present
in the cream, and after the cream is thoroughly
ripened and churned, the butter after being worked
will contain no living typhoid organisms.

Most investigators agree that the lactic
fermentation in milk has a deleterious effect upon
the vitality and longevity of the typhoid bacillus
and when the acidity in milk or its products has
reached 0.4 per cent the typhoid bacilli are killed
within 24 hours (5)(6) although Fr&nkel and Kister
(27) found them living even after 48 hours. How-
ever if one wishes to insure the safety of butter
for human consumption, the efficient pasteurization
of mixed lots of cream before being manufactured

into butter, or of the butter after having been
~43-

made from such cream, should be accomplished.
Cheese made from raw unpasteurized milk

Should also be considered as a possible carrier of
infection, is the conclusion drawn from several
experiments cited by Mohler, Washburn and Doane (53).
Harrison (34) also recommends the pasteurization
of the milk in order to make cheese absolutely
safe. I believe that the conclusions in my pub-
lication on the "Influence of the Products of Lac-
tic Organisms upon Bacillus Typhosus® may be applied

to all forms of dairy products: the acid (and oth-
er products) formed by lactic bacteria is efficient
a8 an antiseptic in amounts of 0.7 per cent and a-
bove in pure culture, but if certain species of
micro8§rganisms are present in milk or its products
they may either by checking the production of acid
or perhaps by living in symbiosis with the patho -
gen allow of its existence and proliferation there-
in. While butter and cheese allow of comparatively
little multiplication of pathogenic and other bac-
teria on account of their physical nature, still,
conditions exist which permit of the continuance
of these organisms in a virulent state for surpris-
ingly long periods of time; the many factors enter-

ing under natural conditions prevent any definite
- —

OW E_Ce
~44-

conclusions being deducted wnen these natural condi-
tions exist.

Bacteria of the two lactic acid producing
types are responsible for the principal fermenta-
tion and flavor in other food substances besides
in milk and its products. The best known of these
finished food products are sauerkraut and dill
pickles. In general practice these are produced

Ay spontaneous fermentation. Although many differ-
ent species of bacteria of an indifferent or harm-
ful type are initially present in the food sub-
stance, when thé process is successful the lactic
bacteria multiply rapidly from the first and quick-
ly produce enough acid to restrain growth of the
micro8rganisms which would otherwise injure the
final product.

The Russians produce "“barszcz" composed of
red beets which have undergone a lactic fermentation.
Other vegetables such as beans, string beans, corn,
asparagus, etc., sourec 3pontaneously vy lactic
bacterla are popular articles of diet in some coun-
tries. Fresh meat is often submerged in sour or

buttermilk for keeping it from putrefactive changes

for a short time; its flavor is improved consicer-
y
a45 =

ably by this treatment.

Sour dough bread is a German product made
from rye flour, and although the fermantation is
not always the same, bacteria of both the Bact.
lactis acidi and the Bact. bulgaricum type are re-
sponsible for its high acid content.

A certain amount of lactic fermentation is
sought in the preparation of some beers. This is
true in the so-called ginger beer prepared from gin-
- ger, sugar and water inoculated with “ginger beer
plant" an impure culture of yeasts and lactic bac«
teria preserved in much the same way as are grains
of kefir for the fermentation of milk.

Formerly,a lactic acid fermentation consti-
tuted an early step in the preparation of tomatoes
for ketchup.

Ensilage, composed of shredded corn, alfal-
fa, sorghum, beets or other similar materials, un-
dergoes a lactic fermentation and owes its preserv-
ation largely to the presence of the acid.

Still another phase of the usefulness of

lactic bacteria has been determined by Barthel and

Rhodin (4). Their experiments have ascertained
@

the utility of pure cultures of lactic bacteria
for the conservation of the fertilizing efficiency
*
~A6<

of manure. They added from fifty to one hundred
liters of whey to one thousand kilograms of manure
and found that the loss of ammonia was greatly re-
duced thereby; the untreated manure possessed 3
fertilizing efficiency only 59 per cent as great

as the treated manure. This addition of whey fur-~
nished the culture medium for the lactic organisms
which quickly taking advantage of the favorable
conditions, began active acid production, thus ef-
fectively checking the over-growth of the organisms
of the putrefactive type which other-wise would
soon have broken up the complex proteins largely in-
to their volatile gaseous constituents, NHs, HS,
PH3, COg, Hg0, N, etc.

Thus it is evident that the lactic bacteria
of both types have not only a wide distribution in
nature but have a number of diverse and important
uses each of which covers a wide field in itself,
the action in each case seemingly dependent mainly
on the physiological property of acid production.

These different phases of the utility of
the lactic organisms show no signs of undergoing a
decline and bid fair to retain their plece in the
order of things although some are but fads at the

present time,
It is also not impossible that new phases —
will be discovered ere long. Nature has placed
everything here for a purpose; sometimes this is
very evident, many times however, the purpose and
application have to be attained either by a process
of reasoning or by accident. When man thoroughly
understands the intricate workings of the lowest
forms of life he will have made large strides to-

wards attaining the millenium,
 

ae

9.

10.

ll.

12.

13.

oA.

BIBLIOGRAPHY.

Allen, R. W. Observations on Lactic Bacilli.
British Medical Journal. Nov. 28, 1908.

Baldwin, Helen. Observations on the Influence
of Lactic Acid Ferments upon Intestinal
Putrefaction in a Healthy Individual.
Journal of Biological Chemistry, Vol. 7,
1909.

Barthel, C. Bakteriologie des Meiereiwesens,

Barthel, C. and Rhodin, S. A Biological Meth-
od for Preserving Manure.
Deut. Landw. Presse. Bd. 39, 1912.

Bassenge, R. Das Verhalten der Typhusbacillen
der Milch und deren Produkten.
Deutsche Med. Wochenschr. Bd. 29, 1903.

Behla, Handbuch d. Techn. Mykologie, Lafar,
Bd. Ze

Ber thelot,A. Neue Anwendung der Milchsaure-
bpakterien Theraple.
Revue de Medicine, T. 1910, No. 8.

Berthelot, A. Antagonisme du Bacille Bulgare
vis-a-vis du Méningocoque.
Compt. rend. Soc. de Bigii.T. 68, 1910.

/

Bible, Century. Vol. Judges and Ruth.

Bible, Holy. Genesis 18:8, Deuteronomy 32:14,
Isaiah 7:15 and 22.

Boas and Oppler. Deutsche Med. Wochenschr. Bd.
21, 1895.

Bond, Franci8. Eactic Acid as an Antiseptic.
British Medical Journal, Vol. 1, 1910.

Bonska, F. W. Antagonism between the B. gub-
tilis group and Lactic Bacteria.
Landw. Johrb. d. Schweiz. 1903.
 

a4Qu

14. Bro@rs, C. We. Onderzoekingen over den tyd, ge-
durende welken tuberkelbacillen hunne
virulentie behouden in melk, karnmelk
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