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2/05 p:/CIRC/DateDue.indd-p.1
A COMPARATIVE STUDY BEDVENY THD TORT&L Blot R2TAL FLORA

CF ULE UDDER AMD TUT CF TER GENITAL

OiGAiN OF VES COV.

THESIS

Submitted to the Faculty of the lichigan
scricultureél College in pertial fulfillment of the

reauirements for the de,ree of waster of Science.

Manuel Justo.

May, 1917.
THEDIS
it is with pleasure thet the writer
acknowledges his gratitude to ir. I. F.
Huddleson anc to Dr. Ward Giltner for the
helpful suggestions they have tendered to him

while in the course of these investigations,

40021
COLTE NTS.

Introduction.

neview of Literature.

Iluethods of Study.

liorphology of the bLectcria from the Udcer.
The streotococci.
ine uicrococci.
The Bacilli.

Comperison of the Lormal Flora of the Udder and tnet
of the Genital Orgens.

General Discussion @nd Conclusions.

neferences cited.
Introduction.

There are few agricultural products which have ate
tracted more interest in both science and every day life than
milk. This is by no means strange wien one considers its
widespread use, its high rank as one of the most complete foocs,
the comparative ease of production - cualities with which are
associated important inconveniences, such as the readiness with
whieh it lends itself to adulteration, and the dengers thet may
lurk in it in the form of pathogenic organisms.

The lest mentioned consideration has given to the
bacteriolo; ical stuay of milk an overwhelming importence which
hes been repeatedly enphasized through countless instances of
local epidemics of typhoid, sore tirost, infant morta’ity, end
other outbreaks, traced directly or indirectly to the milk
supply.

Iikturally, the first task to which science subjected
itself was the improvising of practical rethodcs to render im-
potent any possible contamination. This pertially accomplished,
it devolvec uron the scientific observer to investigete by means
of laborious experirents the most importent end at the same time
the most intricate source of contamineztion - the udder of the
COW.

I voes milk within the udder or the cow contain

TI How do these gain access to tie udder?
ow 2 an

I.

Robert Hell, in 1874, formuleted the theory that
milk, inmediately upon being Greéewn from tne udcer, is germ
freee In this assumption he wes suprorted by Crotenfelt (19),
wno asserted thet milk cGrawn from the ucacer of a heaithy cow
is germ free or sterile. The originel sterility of normal
milk, he believed, is due to the fact that bacteria cannot
gain access to the milk glaenas from without es long es the
ucder is not injured in any way.

Freudenreich (14) holcs a similar view: "In the
udder milk is germ free ez:cept wien tne milk gléenas cre dis-
eesed, aS with tuberculosis or mestitis. In such cases the
tuberale bacilli or those orgenisms causing the inflammation
are present in the milk when drawn." He added, however,
that immediately after bein; drawn it always contains bacteria
due to its contaminated surroundings.

These views were sheared by Lister (26) and es late
as 1895 by Conn (6), and others,

The downfall of the theory began as early es 1892,
when Schulz (37.) made the first quantitative study of becteria
in the udder. While he found that the first milk drawn con-
teined lerge numbers of germs, es the milking wrocess con-
tinued their numbers decreased. This determination was efter-
ward amplified by Lehman(24) who says: "The bacteria in the
milk cistern will be largely washed out by the first milk
drawn but not all will be removed until milkin; hes progressed

for some time."  Bolley and Hall (5) state that "germs nearly
always ,ain edmission to the interior of the teats and often,
perhaps, the milk cistern proper, where some types multiply
in greet numbers",

Moore (29) corroborated Schulz's work by fincin;
thet there is a widely ranging bacterial content comprising
several species in the freshly crawn fore milk, while the
last milk drawn at a reguler milkin;; contains very few ricro-
orgenisms, comparatively. Seldom, however, is a sarple ort
milk entirely free from germs, unless drewn from a single
quarter well neer the end of the milking processes This
work of Mioore succeeded thet of Werd (38) who concluded from
his first studies of the bacterial flora of the udder thet:
1st. Certain species of becteria normally persist in particu-
lar querters of the udder for considerable reriods of time,
end, 2nd, that it is possible for becteria to remain in the
normal udder and not be ejected with the milk,

The well founded results of these investi;zetors,
ccvpled with those of Herdin; and Wilson (20), Evans (13), and
others, have furnished us with entirely new viewpoints concern-
ing the initial content and subsequent véiriations of nmicro-
organisms in wilk and their significance upon the proper in-
terpretation of its sanitery bacteriolo;ical examinations,

The present peper is tre result of some studies by
the writer bearing upon the groundwork laid by these investi-
gators. It is based uron a number of rersonally conducted
studies on fifty-five samples of milk freshly drewn from the
normal, healthy udders of twenty-five cows. The organisms

isoleted from these Samples of milk were studied and compared
with a similer number isolated from the normal genital organs,
(especially the vezina), of the cow.

The results of those studies which ere here summar-
ized comprise two parts: first, the normal flora of the udder
end, second, the comperison of the normal flora of the udder

end thet of the genital orgens.

The lormal flora of the Udder,

By the normal flora of the udder is neant those
organisms which, under the apparently normel condition of the
animal, ere able to grow end rultiply in wnet is suprosed to
be their normal kabitat. They should be found in all individ-
uals under the same conditions of health, elthough their nun-
bers may verys they do not alter by their activities the nor-
mal functions of the orgens in which trey rey be found,

Deviation from tne normal concition of the udder
mey be due to physiolo; ical cisturbances, but more generally
to the incorporation of microorganisms from without whose
activities ere very often the ceuse cf serious ailments end

diseeses.
Methoas of Study.

The samples of rilk studied were obtained end
handled in accord witn the following procedure:

Liost of the Samples vere collectcd from the after-
noon milkings. The uaders of the cows from wW-:ich the samples
were taken were first groomed, wished with a piece of cloth

moistened in water and toward the middle of tne milking streams
==

of milk were directed into separate sterile bottles or test
tubes correspondin: with the different aquerters of the udder,
The samples thus collected were immediately taken to the lab-
oratory where plating was done within an hour after being
dréwne .

In plating, plain meat infusion esar, containing 1%

lactose ang with a reaction 1.2 - 1.550 normal acid to pheno-
phthalein, was used. One cubic centimeter of al to 50 dilu-
tion of milk which hed been Sheken 20 times was pipetted in
each petri dish end to each e tube containing from 8 to 10
cubic centimeters of lectose ecer at a tencerature from 40 to
42° Centigrade wes added. the plates were incubated either
at 37.5 Centigrade tor 48 hours or at room temverature (20 to
25°C.) for five days, and et the end of this reriod of incube-
tion the sum of the colonies developin; on the plates were
teken as reprev-enting the germ content of the milk,
Occasionally, plates were found in which no colonies
developed, tnese Were texen €s coming from a sterile cuarter
of tne udder, or es having e number of orgenisms less tran 50
which wes tre dilution used for pleting.
Prom the colonies aprearin; on the plete sone were
isoleted and studied uncer tie followin,. headings;
le whorpholo;ical cherectecristics,
| The morpnology oi exch culture wes stuaied from
snecrs mece trom the .rowtn on the a cr streak. The
streaks 1sed Were never more tren Your cuys old end The
morrpnolo.y of the individual cells wes obscrvea citer
staining lightly with an aqueous solution of methylene

blue or fuchsin. Loo heevy staining is objectionable
-6=

Since it introcuces a serious error by erousin, the cells
into 1asseS Which rt.ake ditfiecult the observation orf tne
individual cells. By reans of an ocular nicroneter tre
Qimensiomof ti:e cells were recorded.

2. Steinin; cherecteristics.

These were observec by stainin, tne smear of one
ager <rowth, not over four ceys old, by the Gram method
End with aqueous solution of methylene blue end fuchsin.

4. Cultural cheracteristics.

In this stidy the general character of -;rowth on the
a-ar Slant wes recorced. In nutrient broth the cultures
were examined for the growth end the formation of pellicle,
turbidity and sediment in the licuid in the tube. In the
gelatin tubes the rate of liquefaction of all the cultures
has been recorded.

4, tiocheimical reactions.

@e Action upon milk. Both plein end litmus rilk wes
prepared according to the standard methods end tne actions
of the cultures observed efter three aeys of incubation.

be Action upon carbohydrates. The charecteristios
observed in the media were the gas procuction and acid
production.

The media for this fermentation reection wes mece by
acdin; to 1000 c.c. of the ordinary meet infusion broth
10 grams of the test substances. They were dextroce,
lectose, saccharose, maltose, raffinose, mennite «nd
glycerine. The reaction wes made neutral for these
fermentation tests in the various suger broths above

mentioned. a twenty-four hour culture of each organism
7.

in plain bouillon was used and to each tube three loop-
fulls of culture were added so that approximately the
Same amount of organisms were present in each tube of
media. The cultures were incubated for fourteen days

at room temperature and five c.c. of the culture titrated
in the cold eseinst twentieth-normal sodium hydrate with
phenophthalein as an indicator. From the results so ob-
tained is subtracted the titration of a blank and the re-
Sults are expressed as the rercentaz:e of normal acid.

The production of ljo norticl acid marks the line between
fermentation and no fermentation.

This method of recordin; the acid fermentations of
any organism which is the one more generally adopted meets
several inconveniences such as the use of a definite end
uniform cuentity of the culture growth in titration, the
use of well prepsered sodium nydrate used for the test,
the time consumed in the procedure of titration, etc.
While the study of the fermentations of the different
organisms wes being carried on es here outlined, the at-
tention of the writcr wes struck by the work of Hiss on
this matter.

Hiss aevised tne following media, in which the cleev-
ace of any carbohydrate is indicated by the production of
ean acid reaction end by the coegulation of the serum (A
Text-book of Bacteriology, by Hiss end Zinsser, 1914):
"Clear beef serum is obtained by pipettin; from clotted
blood. To the clear scrum is added three times its bulk
of distilled water. The mixture of serum and water is

heated in the trnold Stcrilizer for Tiftcen rinutes at
100°C. to cestroy the diastatic ferments in the serum.
After cooling the mixture, a concentrated aqueous solu-
tion of litmus is adced until a deep transperent blue is
obtained. The mixture is then divided into as many parts
as there are cerbohydrates to be used. To escr of these
parts of the mixture is added one vercent respectively of
the suger to be used.

For the preparation of inulin medium it is necessary
to sterilize the inulin dissolved in water in an autocleve
under 15 pounds pressure for fifteen minutes in order to
kill spores before adding it to the serum. The serum
water media is sterilized by the fractional method in the
Arnold Sterilizer at 100°C. for twenty minutes on three
successive days in order not to coagulate the serun."

By using the procedure of Hiss, fermenting power of
an organism on a certain carbohydrate is observed more
readily, more easily, and probably with the same degree
of accuracy.

Ge Action upon nitrates. litrate reduction was de-
termined in the ordinary peptone broth efter seven days
growth of the culture at 20 C.

de Pigment formation. Pigment formetion wes studied
macroscopically efter fourteen caeys growth of the cultures
on acer slopes at room temperature (20°C.).

e. Liquefaction of Gelatin. For determining the
emnount of liquefaction a crop of broth culture was spread
on the surface of a tube of intrusion gelatin, the surfece

of the gelatin is marked around the side cr the tube and
oD on

the cotton plug coverec with careffin to prevent evapora-
tion. If liguefaction texes place it »roceeds ina
stretiform feshion end its amount is read in rillimeters
of liquefaction after thirty cays incubation of the tubes
at 20°c.

Hemolysis. To a tube of ordinary infusion agar at
50°C., 10% of defibrinated cow's blood was added end
after carefully rolling the tube between the hands to
mix the blood uniformly with the esar they were poured
into each plate. when the coer hea solidified, streaks
of the cultures were meade on it end the pletes incubated
at 37°C. Arter twer:ty-four hours the first observetion
or the pletes was ..ace and the streaxs of the culture
Surroundec by a distinct, cleer sone of hemolysis were
designatcd as havin, a positive hemolytic power; those
not showing any white zone of hemolysis were designated
Gas having a negative hemolytic power, but since the rapid-
ity with which the blood is hemolysed by various organ-
isms is veriable it seemed more convenient to record the
observations on the plete for a longer period of time,

four or five devs being sufficient.

Morphology of the secteria from the Udder.

According to their morphology the bacteria of the
udder will be here considered separetely under three groups,-

the streptococci, the micrococei, and the becilli.
-10—

The Streptococcus Croup.

According to Lisgula's classification of bacteria,
to the genus Streptococcus belongs all those microorganisms in
which the cells are spherical, dividing in one plane, and hav-
ing no flagella. wilson describes some of the most selient
characteristics of this genus as follows:

"The genus Streptococcus has been defined by its
parasitic life, the occurrence of the cell in chains, positive
reaction to the Grem stain, faint effused growth on media,
feint fermentative power for most carbohydrates, but not for
inulin, end the general failure to liquefy gelatin or reduce
nitrates. They may appear in long chains of a hundred or
more elenents, or in peirs and short cheins of four to six
cells. Chains are best developed in broth cultures; but
some Streins display the tendency to chein formation much
more markedly than others".

The different streins of streptococci differ so
widely in morphology, cultural characteristics, and virulence
thet it hes been difficult to classify them satisfactorily.
Notwithstanding this fect, claessificatory exeninations of
numerous strains heve eventueted in the aiscovery of definite
groups which possess conspicuous physiologic end pathogenic
peculiarities; even emong the rembers of one oi these groups
there have been found types which cre differentiated by further
refinement of laboratory technic.

Significent physiological dissimilerities among
closely releted streptococei have been ceterziined by studying

their reactions on special laboratory media. Anong tucse
=} | =

laboratory media those which have been more greatly used are
the different sugar broths.

The question mey tnus be formuleted: ‘shat relation
does this group of microorganisms, which hss exerted so intense
an interest on the pert of many workers in all lines of bacter-
iological research, bear to the milk problem‘

maumerous investigators heave demonstrated the presence
of streptococei in milk coming from udcers affected by mastitis.
Streptococei have also been found in milk coming from apparent-
ly nealthy udders. Various local outbreaks of “septie sore
throat” due apparently to a streptococcus infection have been
traced occasionally to a certain milk supply. his milk sup-
ply.in question, which seems to have been the chief vehicle
of infection, may have become infected under the two possibili-
ties: kither that the pathorenic organisms were introduced
into the milk supply by some infected person who had to handle
the milk, or that they were derived from the cows which proe
duced the milk.

Von Lingelsheim (23) mentions the fect that strepto-
cocci are found in market milk, and susgests thet these bacteria
may be the cause of many infantile intestinel ciseases. whis
Suggestion by Von Lingelsheim wes supported and amplified by
retruscky (34) in 1904, who announced es the results of his in-
vestigations that the high infant mortality in summer was
caused by streptococedi which he found in milk in lorge numbers.

the presence of streptococci in milk has also been
reported by vonn end nsten (7), neinemann (21), Davis (11),

imller (31), #rost (15), xeed end Jara (35), and others, but

despite their labor a satisfectory interpretation of their
 

-]2-

Significance to the wholesomeness of the milk has not yet been
achieved.

Some of the cheracteristics of the orgenisms isoletcd
from this source ere es follows:

Strongly hemolytic power, ound in the same rropor-
tion in certified milk as in that which has been resteurized,
they ere more resistant to heat tan the human streins of
hemolytic streptococci. They possess little or no virulence
for rabbits, therefore in all probabilities not for man. They
rapidly acidify end coagulate milk and grow well at 20°C.

They mey form short or long chains, but as seen in milk they
often appesr in pairs or a chain of few elenents. These milk
strains are different from those found in cese of diseased
udders in cows in thst these latter resemble the strains of
hemolytic streptococci from humen sources énd are virulent for
rabbits. Methers (27) found thet hemolytie streptococei, vith
all the characteristics of the humen type, may be highly
virulent for cows when injected into the milk ducts, they pro-
duce a severe mestitis or mey grow end multiply in the milk
ducts without causing any visible changes in the udcer, but
the milk in this instence conteinet en increase of leukocytes
as well as streptococci.

liathers also 305°S thet hemolytic streptococci from
milk end the Streptococcus licticus wey vroduce en acute in-
flammation of the milk ducts, but ne observed it wes of a
transitory nature and the wemmary glands reseined their normel
functions very repidly. Contrary to the findings of these

_ , (2) ,
investigators are those of Laerr,who on examination of eighty-
One samples of wilk, j;ert from milk dealers end part from
individual cows, found Streptococcus pyogenes in only two of
The samples.

Harding and ‘Wilson who meade the first extensive
study on the normal flora of the udder in this country on an
examination of twelve hundred and thirty (1230) samples of
milk direct from the udders of seventy-eight cows (78) found
only two representatives of the genus Streptococeus end they
say further: "The absence of tnis genus was noted early in
the cualitative work and attention siven to the detection of
streptococci. Neither of the two forms here listec ere
typical members of the cenus, their tendency to Sorm chains
bein, rathcr weak. It will also be noted treat two strains
of Streptococcus lacticus ere amonc the named species with an
interrogation point. These streins were not streptococci but
acreed fairly closely with the aescriptions of that species in
other resyects"™,

Quite in opposition to tnese results are those of
Evens, described in her recent work. She begins by saying:
"™ most important nuatter in the study of the streptococci in
milk is the differentiation between the lons chained strepto-
cocei, which me; be of a virulent type, end the seprophytic
Streptococeus lactieus (Kruse). It is my opinion thet such
a differentiation cen be nade by the Simple method here de-
scribed, although there are undoubtedly variations which do
not conform to either of the two types",

"Long-chain streptococci usually curdle the milk,
and reduction of tie litmus may texe place efter curdling,

but the color is never completely reduced. with Streptococcus
-14-

lacticus cultures the reduction of litmus precedes the curde-
ling and is complete beneath the cherply cefineda pink surface
layer". "Streptococcus lecticus leexs the tendency to form
long chains ana sore or ell of tne cells ere elonvated with
teperin, encs."

From the one nunurea end ninety-two sarnples of milk
studied by her, Streptococcus lacticus (Kruse) was isoleted
only once and wes discerded «s contaninetion, but t’.ere were
found long-chain. d streptococci which fsiled to give the re-
duction of litmus, characteristic for Streptococcus lacticus,
in 15.1 percent of tre senples studied by ner, and in numbers
ranging: from a very few to 264,0C0O per cubie centimeter. All
the rccults of these investigetors, with the exception of
those of tierdin. ard Vilson, point to the evidcence thet strep-
tococei wey be found in milk in numbers veryin; from a few
hundreds to severel thousencs per cubic centineters. But by
a deteilea analysis or the studies ot these investi.stors, it
is secn thet their ovservetions i.ave been wece lergely on

wmerxet milk,

HH

becteriolozical exaninetions o

”~

The work of EerGgin and wilson end thet of Evens

Nw”

decls principally on ¢tidies of ireshly Grewa rilk, enc :et

ev

~

their results are strictly in opposition. unile ferding and
vilson found only two merbers of tne streptococcus -roup in
their observations on a lzerge number of samples of ireshly
drewn wilk, Evens claims to have isolated strestococeci in 15.1%
of the samples of milk studied by her.

without attemptinz to criticise the rcsults of these

investigators, the results of Evans' work reporting the find-
ings of streptococei in freshly crewn milk in mumbers rénging
from a very few to 264,0CC per cubic centimeters cannot re-
strein the writer from considerin; this high number of organ-
isms as rethner improbeble in fresrly dréewn milk supposed to
heve been crawn under the best of aseptic precautions. The
reliability of this assertion of Evans is accin questioned by
the results of the writer in fincin, thet the germ content of
freshly drewn nilk from apparently heelthy udders, celculcted

on the numbers of organisms cevelopinz on the eager plate,

Cee:

A)

.verages not nigher than rive hundred orgenisms per cubie cen-
timeters. snd her results are cvestioned avein when one con-
Siders thet it has been steted (by Committees on becteriological
studies of milk) thet it is possible to produce certified milk,
end one of the recuirements oi this milk is thet it shell not
contain more than 10,000 microor;eanisms per c.c. at any tine
efter it hes been drewn from the udder of the cow, and yet this
milk containing co few a number of microorgsenisms comes in con-
tect, eltnovegh for a very snort tine, with the eir of the sur-
roundin:s where it is produced or prepared to be carried to
the consumer.

in his present stuaies the writer hes failed to isolate
streptococei from the sarples of milk crewn uncer the best of
eseptie precautions. these failures (should these orgfenisms be

found to preveil generally in the treshnly crevwn milk) may be

“

aceribed: Sirst, to the smell number of sernlies ctudied;
second, to tne fect thet, cenerelly, tne streptococci srow
retner poorly in tne leboratory media commonly employed in
this work end they mey even feil to form chains of ceils,
their distinguishiny; cheracteristic; end third, to the

failure to sepsrete the colonies supnosed to rerresent the ae-
~1lb6—_

sired type from amonz ell the others ceveloping upon the egar
plate beceuse their Sirilserity is so striking es to require

both experience and judgement for its svrecesse
p

The Beaecilli Common in lormal Lilk.

The application of bacteriolosicel research to the
study of the sanitery conditions in milk hes revealed to us, nany
yeers back, thet the typnoid bacillus, the tubercle bécillus, end
a few other pathogenic bacilli are frecuently fouhd in milk.

Lilk in which these orgenisms heve been found hes been
considerec as unclean, unfit for humen consumption, and therefore
regerded as being contaminated, This contamination hes been
traced to the improper handlins to which milk is often subjected
before reaching the consumer, or to verious infections of the
udder.

But it is only during recent years, through the obser-
vations of iiohler end Treum (28), Schroeder and Cotton (36),
Fabyan (16), Evans (12), «nd other investijctors, that attention

hes been focused to the presence in milk coming rom a

~
r
'
a

parently
normal udcers, of an orgenism which a,rees in its cenerecteristics |
with e similer one isoletcd by Bang, ae Denish becteriologist, in
1895, from the uterine exudate and ebortca fetus of en infected
COW The orgenism today beers his name cnd is cenomineted, -
Becterium abortus (Beng).

The failure of the common laboretory techniogue as ap-
plied to milk to reveal the presexce of this orgenism hes made
it necesscry to employ speciel methods for its isolation, One

of these methocs consists in incubatin, the plate from which

4
-17-

observations of this organism ere to be made, uncer aneerobic
conditions es devised by towak, end the other by confirming
the presence of these orgenisms in milk by inoculating labora-
tory animals, especially guinea pigs, with the milk and waiting
for the development of lesions in these animels.

The pathogenicity and deleterious effect of this or-
gonism upon man hes been cuestioned by Larson and Sedgwick (25),
Mielvin (52), Iicoll end Pratt (25), Cooledge (9), Williams and
Kolmer (4C), and others, and tne significence of this orgenisn
(Bacterium abortus) in milk coming {rom apperently normal
udders is one of the most keenly discussed cuections that the
SGience of Deiry secteriolo;sy is called upon to solve,

In the light or the new researches on this problen,

it is premature to essune whether the Becterium abortus is to

 

be regarded es a type or the normal ucder, but the following
fects ere known:

first; ‘ne Becterium abortus is not found in all
udders or in the milk coming from them,

second; Organisms to be conci dered a type of the
normal udder shoula be found in ell ceses exposed to the sare
variability of conditions end environment,

Third; ‘The letest researches on this problem have
strengthened the conviction thet this is the only type of bacil-
lus that preveils in the apparently healthy udder, but it is
not found in all apparently healthy udders.

If we rest our assu:ption on these well known facts, it

is not unressoneble to conclude that Bectcrium ebortus cannot

 

be regerded as a type present in ell normal udders, but yet we
-lB—

cannot make this conclusion for we leck definite methods of

study to prove that this is the case.

the wierococel.

Amons the microorgenisms inhabiting the normal flora
of the udder, the micrococci are the oneS which occur there
more frequently and the accumulative evidences that are begin-
nines to be derived trom studies of the becterial flora of tne
udder may prove, finaily, thet perhaps this is the omly type
thet predominetes in tne normal udder of the cow.

the classification or the streptococci, closely re-
lated to the micrococei, carriec to &é very high degree of re-
finement end verfection in the hancs of cordon (17), Andrewes
and Horder il), Winslow (42), end other investisetors, has
laid the steps by which a systematic classification of the
micrococci will soon be achieved.

Upon examinetion or an aescr plete containin: several
colonies oi micrococci, one resully perceives the close sini-
lerity in shape, color, and appearance amons all the colonies
developfng upon the plete. The similarities are often so
striking as to lead the observer to cuestion whether all the
colonies do not represent the Same type of orgenism. whe n
exemined under the microscope the doubt is strerathened es to
whether they all possess a morpholo, ical identity. However,
by further study of some of these colonies it becomes certain
that there exist sharp limits of variability amon: then,

Morphological differences among the bacteria are so

Slicht, especielly in the members of the family Coccaceae,
en
that physiological properties heve to be relied upon for their
classification, end yet such properties ere so extremely
veriable - especielly the fermentation reactions, the patho-
genic power, etc. - that it has been necessery to find out
whether they exhibit eny decree of constency.

On the morphological besis the micrococeci have been
divided into two types -- the staphylococci end the micrococci
proper.

The staphylococci are cocci whose planes of division

 

are not at right angles but divide at cifferent intervels with
a consequent irregular grouping ef the cells ruch resembling
erapes in clusters. ane genus is frecuently included under
uiecrococecus. It contains some of the more cormon pyogenic
cocel. It mney be sepereted by its Gram positive stain and
the production of white or orenge pigment.

The pyogenie staphylococci are found very widely
distributed in nature and ere found in the air, soretimes in
yeter, in man on the Skin end tuccus membranes, in the aliment-
ery cenel, end in the mouth, also in suppurative conditions
occurring in the mammalia and birds. In humen paetholozical
lesions they cre frecuently found in pus, csrecially in ab-
scesses on verious verts of the body. Occesionally, the
stephylococei enter the blood stream givins rise to e& purulent
infection known es pyaenia.

Under the grcup of stazhylococei, the following
microorgenisms sre included: sStephylococcus aureus, citreus,
albus, rosaceus, flavus, end sriseus, the first three being
the ones Which ocevr more frecuently. In tneir biolocical

properties, those wicroorgcenisis ere very siniler, citfering
~20-

4

from one another principally in the color of their growths.

4.

Aside from their difference in pigisent production, etterpts
have been mede to vurther aiffcrenticte them on the besis of
gelatin liquefaction.

Gelatin is licuefied by staphylococcus eureus,
citreus, rosaceus, cnd elbus.

Gelatin is not liquefiec by stephylococecus flevus,
and griseus.

The luicrococci. This .enus is rrecuently defined
to include the genus Stesnylococeus just discussed. It moy
be differentiated from this cencrally by its Gram-negative
cneracter. They exnibit e gsrest vericbility of pigment pro-
duction, elthough the mejority of them produce the caifferent
Shades of yellow cigment.

Before ettemptin; to ceseribe the cheracteristics
of the Liierococei composin:z the flore of the normal udder, it
is importent to distinguish the different kinds of veriability
anong them which contribute to the comple::ity of their classi-
fication.
The Licrococci from the ucder exhibit these differ-
ent veriations:
Pirst: Those aue to concitions of enviromicnt, such
eas color. Thus, the intensity of color of the pigment
of a good zany of the :ucrococei from this sovree veries

mr @
~

with the secge of the culture. at Lirst. some evltures
appear to possess e Wnitish color which, ctter ceveral
weexs, cnanges to yellow, orencve, etc. The intensity
of the color is €l1lso devendent uvon the suvrly of o:yrcen,

bedoming more intence with the greeter suctly oF ony Gene
c)
= C, an

Second: Certein physiologicel cherectecristics
which ere influenced by the ece end the frecuency with

YTEesn meala.

Fly

wnieh the eultures cre treusferred to

it is observed thet scme Licrococeci, which et first

thus
do not ferment sore cerborydretes, efter they ere ,rown

for a considereble period cf tine seem to eeccuire the
ebility to ferment such substances.

Third: Certain leboretory media used es a meexs
of aifferentiation between other closely releted organ-
isms seem to be of no avail as an index of differentia-
tion when epplied to the physiclogical veriations of
the licrococci. For exemple -- nilk. ilk is a
feavoreble nutrient mecium for bacterial ¢rowth beceuse

of its »roperties, ond the chenges prodveed in it by

many organisms cre important es dicgnostie reens.
pilk hes no Sveviel Giecsnostie value for the Licrc-

cocci since éli the cheonges it urcaergocs cre correleted

C)

with those wnich occur in the verious suger brotns end
ere dependent upon the general activity of the organisns.
The chenges produced in milk by these Micrococci ere the
production of eacid or elkeli, coc;ulation, cnd decolor-
ization of the litmus.

Decolorization of the milk hes no significence ex-
cept «es an indicetion of the activity of growth of an
organism. when ean crgenisn is .rowing most ectively
it uces up onysen and so recuces the litmus which is
therefore decolorized, cnd the reverse is trve when the
activity is less. auycen is no loncer used by the

rowing orgenism end consecvently it diffuses down from
=22.

the surface, -iving back some of the color to the litmus.

The coazuletion of the milk is lergely brought about
by the amount of ecid produced end this is more easily
and accurately studied in a broth containing the different
cerbohydrates.

It is then understood et this point thet the reactions
of the iticrococei in different laboratory media used in the
differentiation of other organisms do not furnish us with a
means for their differentiation, but by studying their aetivi-
ties on broths containing the different cerbohydretes end
other releted substances the pnrosrecsive divergence from their
apperently fundamental resemblances is observed.

The following protocol exerplifies the rermentation
reactions of the wicrococci isoleted from the rilk arawn from
the udders of normal cows. For the denomination of the color
of the pigments in the followin: teble, Ridgwey's "Color

otenderds end omenclature"™ was used.

’
Characteristics of the bicrococei from the Udder,

Culture Coagulation Rtecuction of Reduction of Liquefaction cof Dextro
of Lilk. Litmus luilk. tditrates. Gelatin.

 

OM~NIA MP WY BH
re lil+ernee 1 + + +
tt+t+tti+ttt¢
++i+r++e++-tt +4

+
+

Pirrere
Lteeitit+et+eee eit

+iririteititit+tt+ett+te it tite it i+
1rrt)erttteti+f tet bri ttt

+!

oO
tLPritiriririegeeei ere irrrritei rr eeeeeeeere
+ierceteeeeiresrti

++etettetettettttretetreteteetetereteetreeteeetetett+t
waltose Raffinose iuannite Glycerin hemolysis

Chromorenesis

 

(_t++eeeeteeliteteetetet tt

Heese eeeetereetttesi

1+ tf + }

it | +

ie i+t+ti+et+i+si+ ++ |

(i+ i+r+itri¢-i

le++

i+ !

+ +

+

11+ tip-t tt e+]

+ |

pale orengse yellow
white yellow
yellow

pele yellow orenge
white yellow

pele orange yellow
White yellow
orenge buff

orange

light orange yellow
yellow

light orange yellow
creem

cadmium yellow
white yellow
orange

buff yellow

Slizht pele vellow
deep chrome

yellow

cadmium

cnrome

light cednium
cream

deep chrome

orange

yellow

yellow

chrome

echnrome

oranse buff

orenge

ccanium yellow
orange

pale orenge yellow
orenge

ehrome
In conclusion, the following is a brief summary of
the selient cherecteristics of the licrococci studcieu in this
paper.

The lergest proportion of the izicrococeci from th
udder ere aerobes, followin,; the f:cultative anaerobes. It is
possible that there mey be in the udder some anaerobic -icro-
cocci, but no special technique was emvloyed to Show whether
they were present.

The charecter of the facultative anaerobism wes ob-
served espzcially on the esar plates on which colonies grown
on the under side of the efer When transferred to the afer
Slant secied to grow equally cs well as those isolcte: from
the upper surfece of the solidified aj ar.

the reaction of litmus milk veries from complete
coazulation end reduction of the lituus to no appsrent change.

The nejority of the orgenisms included in the fore-
soins List liquefy gelatin, verying in number of millimeters
and repidity of liquefection. The lorger percente e of or-
Ganisms do not recuce nitzrctes.

Dentroce end Leetoze fomnentations were ~ositive for
all the cultures.

lio ges ic formed wnen tveztcc in tue Fermentation tuvc.
lio cttengpt wer uede to divide them into jrovps according to the

substances acted upon.
Comparison Between the Orgenisms Isolated from

nx

the Ucder and those from the Ceniteal Orgens.

Jeélising th:t the fector governing the fitness of

the lecteel fluid for cixzcet or indireet consviption lies lcrge-

tot

thre microorrseniscns in-

aos

Hy

ly in its germ content and thet some o
cluded in the letter, eiter the milk is aréewn or yet within the
udder, may lieve imperiled the wholesomenecs of the milk by con-
ing from the discnerges of en infectious genital ciseese, the
weiter thougnt thet a coisperison showing vwnether eny reletion
exists between the flora of the normal udder and thet of the
normal genital orgens, (especially the vagina}, wight throw some
lisht upon the cuestion or the contamination of the milk while
yet within the udder.

The result of these stucies, Showin. thet there exists
some relation between the organisms found in the narméel udder
end those found in the normal genital orgens, was necessary in
order to neesure successfully the degree to which the cischerges
of the genital orgens of the cow prove to be & factor in the con-
tanination of the milk.

In a study of this nature, the first question thet
commends our ccnsideretion is: How do the organisms gain access
to the udder?

Scientifie opinion concerning: the answer to this cues-
tion is divided between two factions of becteriolosists; on the
one hand, those believing the theory thet the organisms make
their way into the ucder through the circulatory system, end on

the other hand, those supporting the assumption thet they cain
--6~—

entrance to the udder through the cischerges from the genitel
organs.

Before stetin, the erguments by which each theory is
upneld, let us turn our attention for a moment to the anatomy
of the cow's udder to find what features in its enetomicel
structure will favor the incorporetion into the udcer of orgen-

isms from the outsidee

The Cow's Udder.

The vrerticular glands in which the milk orizinates,-
the milk glends - form the most importent rortion of the milk
secretins udcer. The cow's udder is divided by a strong
fibrous vertition, running lon itudinaelly. Eech of the helves
contains @ lirge silk cglend of & revcisn-grey color, or more
correctly Ssveexin,, en accviuletion of ,lanculer structures
cellea the gland basket. The milk glancs very creatly in sive
in different inaividuels end contein embecded in them a white
connective tissue, the delicate glend-lobules in which occur
numerous round cevities, the microscopic gleand-lobules, or
elveoli, which ere terrinel oz léterel ciletetions of nwm.erouvs
End extre:.ely tine canals in which the xcteriel for the forme-
tion of milk circuletes through numerous ljwph tracts, unitin;
into ever widening cuets - milk ducts - end eventuelly into
lerge, nollow cavities - the so-ceiled rilk cisterns or milk
reservoirs. Four or these ere ¢~resent in eéech udder and cor-
resvonding to thece ere four teats.

The teat is @ céenal strrounded by a museuler wall and
closed at the extremity by an involuntery sphincter ruscle

which varies wren in riziadity in different ecnimels. Often, the
oT

pressure of &@ small emount of nilk in the cenal is sufficient
to open it and permit the milk to escepe. In other individuals
it reauires strong eifort ot the nana of the milker to draw the

milk. This canal with a terrerature of the animel body and

v

on

contsining always, even erter the most complete wilking, a small
amount of milk offers ideal conditions for becterial srowth.
when the animal lies down, be it in the stable or elsewhere,
the udder and teats come in contsct with dust end dirt teeming
With bacteria. The pressure exerted by the animel upon the
ucder, added to the pressure of the milk in the udder, opens
the canal of the teat and thus provides an avenue for the en-
trence of microorgenisms into tne udder.

By studying; the anatomical structure of the udder
it is observed that its different verts ere not closely con-
nected. This fect has been confirmed by observing certain
infections to which the udder of the cow is often subject;
for example, mastitis, which has been ¢roveu to be a local in-
fection which does not trevel from one cuarter to another,

we are in @& position now to return to the theortes
of our discussion. Do tie Orgenisms gein access to the ucder
through the blood stream?

The shiftin; of the balance of opinion on this ques-

tion may =ossibly be treced to the statement in res:rd to

Co

1

Bacterium cbortus in the udder, -iven py Cotton (10). He seys:
"The bacillus of infectious abortion rey end in sore ceses does
persist in the udders of cows thet heve eborted for yeors end

possibly for tne balance of their lives end, Gurin, this time,

is elimineteu more or less continually With their wilk. It
=~ B=

mekes its appearence in the milk months before abortion occurs,
even before a conception that is terminected by en ebortion. It
mey be eliminated for yeers from the udders of cows thet never
aborted."

This periodicel eppeerance of these ».rticuler kinds
of organisms in the udder he: to have some source of supply.
That the genital trect is not a source of suzply is rroved by
the conclusions of the same writer thet Secterium abortus persist:
in the genital tract for only 46 deys (or even a shorter tine).
Thet it does not come from the outside is shown by the fact thet

Becterium abortus persists, when exposed to weether conditions,

 

for a very short time, even as short es ten dzeys.

Tnen it is left for us to assume thet it gains access
to tne udder threugh the blood strea:.. The assumption thet
becteria gain access to the udder through the blood stream is
imncsrobably for veriovs reasons.

first; If the bacteria of infectious abortion gein
eccess to the udder through the blood stream, we would expect
to find the abortion germ equally distributed in each of the
cuerters of the udder which is fear from bein: the cese; this
is elso true of other becteria apvezrin, in the udder.

second; Cooledge, by means of the acglutinsetion
tests, found thet antibodies for this orgcenism could be demon-
strated to exist in the milk coming from some cuerters of the
udder, but they were not found to be present in the milk coming
from all of the quarters. snd further in the ciscussion of
this point, Huddleson (22) joins Cooledze when he says: "the

entibody content of the milk anda thet of the blood in the seme
me =

cows is not in a direct proportion." This shows a:'ein thet
the infection does not cozre through the blood stream.

It is e well xnown fect that some drugs, especially
bitters and arometics, sre very lergely excreted in the milk
end guided by this fect there is reeson to believe thet bac-
terial toxic vroducts rey cvecome excreted through this channel,
but yet this does not appesr to be the cese with microorganisus.

AS @ conclusion to tnis side of the cuestion, Williexs
(441, writes: "We ere bezinning to believe thet the tubercle
becillus coes not recs from the narmmaery cepilleries into the
milk ecini while the secretory epithelium is intect, end that
the danger of tubercular infection from the milk is chiefly
or wholly dependent uzon tyberculer agisecce of the  ,land or to
conter.ination of the milk from becilli eclcewrere elininated,
especially to becilli vcided with the feces," agein he says:
"The diseeses of the genitel orgens of cows nay be related to
dairy inspection from three different viewpoints:

sirst; Certein types of genital ciseeses mey induce

sepraenita, pyaemia or serticeeuia, and through such avenves
imperil the wholesomeness of the milk by the ;roducts of
disease being excreted in the rilk.

recond; Disec:es or the genital orgsns tenu to cause

& discherge through the vulva which, soilin: tre teil,
thishs, end udder, threatens finelly to crop in pert into
the milk while bein: drewn.

Third; Genital aischarges pessing downward trom the

vulva alon, the tsil, thighs. end udder, come finally in

contact with the teut orifice and the infection 1.e) pass

alon; the teat canel, ¢gcin the secretory surféeces or the
glend, rultiply tnerc, estebdlisnin;,, a new disec:ice
end conteninete the rilk aireectly before it is drewn,"

Dut if it is true that this occurs in a diseased con-
dition it is Within the seme ground of possibility thet it
should heppen during the normal condition of the animal, al-
thovgh to a lesser degree,

finally, in sup ort of this letter view it hes been
pointed out by some investigators thet the fact thet organisms
are generally found in lesser numbers in the recr cuarters
than in the front cverters of tne udder wey explain the rela-

3

tion thet these orgenisms ray heve with the genital cischarges.
Althou;;n the writer's personal observetions do not werrant him
in méxins a definite stetenent et this tire, however, he is
inclined to believe thet the fact thet microorganisns are
found in lersger numbers in the reer querters then in the front
ones lies in the essumption thet wren the cow lies down the
reer teats ere mere ezsosed to the conterinaetion from the
floor tnan ere the front ones. une writer is thus encouregec
to believe thet neither of tne theories here ststed is supported
by their correspondin; evidences, end the ettention of the ob-
Server needs to be directed to other outloovzs in order to elicit
proofs that will be sufficient to settle this cuestion of con-
veyance of the contsnminating orgenisms to the inside of the
udder.

The organisms from the genitel orsens of the cow were
isoleted and studied by lr. Keck’ and Mr. Bolton end the folloy-
ing is taxen from their observations:

¥

Nia, 7. CO. Keck end ir. 2. 3. Bolton of this leboratory were
working on the flora of the genital orgens.
—Sl=

Cows from which studiesS were made:

lumber of cows from Which swebs were made 350
Iunaber of swabs rede from the ve;inas 30
iuriber of ewabs made from the uteri 20

Iorber of cows from which swebs were tiade of
both vazines and uteri st the sare tine 4,

Orscnisms isoleted:

Ibmber of orgenisms isolated from the vaginas 34
umber of organisms isolseted from the uteri £4
Total nuxber of different orgenisms isoleted D2

Iumber of organisms occurring in both uterus
end vagina 5
In going over their results they found a very wide
range of organisms. There were no two cows which showed an
identical flora in either the uterus or the vesina, end, be-
Sides, they found ver; little sSirilerity between the orcenisns
isolated from any two cows. In «ceord with their morphology,

they divided the organisms isoleted es follows:

umber of Bacilli isoleted Lk
" "Wicrococci " 11
" " Streptococci " 4

Due to the fect thet the complete study of the orsan-
isms isoleted by them hes not termineted, the cescriptions of
their rorphology, cultural end physiologicel cherecteristics,

neiiolytic power, etec., ere here omitted.
aon

General Discussion and Conclusions.

The results set forth in this vaper have been besed
upon the study of a number of orgenisus isoleted from 55 sarples
of rilk drawn from the normal ucders of 25 aprerently nealthy
cows and comperead with those similerly isoleted from the normal
senital organs of 50 dirferent cows.

The samples of milk were taken from cows which did not
Show any sign of genital discharges. In the majority of the
ceases, both samples of milk for study were drawn and svabs
from the genital organs were mede from the sare cows, elthough
in a good many instences only one of these was taxen from each
individuel.

In comperin, the orpenisms isoleted from these two
sources, the writer hes found thet e@ wide renge of verious
orgenisms prevailed in the genital orgens of the cow. A lerge
number were bacilli while none were isolsted from the normal
udder. streptococci were found in the fsenitel orgens, but
none were isoleted from the samples of milk taken from the
normal udders. The majority if not the entire number of
orsenisms found in the udders were liicrococci, end in the
genital organs only 11 iicrococei were isolated and were found
in less numbers then the bacilli of wnich 22 representatives
were studied. Furthermore, the licrococci isoleted trom the
genital organs did not seem to possess a Similarity to those
isoleted from the udders.

The result of the writer's present observetions,

while not determinative in cheracter, lead him to assume the

following brief conglusions:
i
0)
C
i

First; Thet the normal flore of the udcer comprises
largely of microorganisms which belons to two tzpes, the
wicrococci and tre Staphylococci, varying in pigment pro-
duction, fermentation reactions, hemolytic power, and the
other tests to which they have been subjected,

Second: That although in the few samples of milk
here studied these two t;pes have been considered as
representin;, the bacteria of the normel vdder, this does
not yreclude the possibility tioat had a large number of
serples been studied and more colonies subjected to ex-
aiiiretion, other types might heve been encountered.

Third; That a comperison between the organisms
isoleted from the ucder and those isolated from the genital

orgens shows that there is not a close similerity vetween

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