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 The Persistence of bacteria in the mill( 
 
 3 1924 003 235 698 
 
The Persistence of Bacteria in the Milk Ducts of 
 the Cow's Udder. 
 
 BY 
 
 ARCHIBALD R. WARD, B. S. A., 
 
 CORNELL UNIVERSITY, ITHACA, N. Y. 
 
Cornell University 
 Library 
 
 The original of this book is in 
 the Cornell University Library. 
 
 There are no known copyright restrictions in 
 the United States on the use of the text. 
 
 http://www.archive.org/details/cu31924003235698 
 
tEteprinted.ftom the JOURNAL QP APPWED MICROSCOPY, Vol. I, No. 12.) 
 
 The Persistence of Bacteria in the Milk Ducts of 
 the Cow's Udder. 
 
 Read at the twenty-first annual meeting df the American Microscopical Society, Syracuse, N. Y.' 
 
 The constant presence of bacteria in 
 freshly drawn milk is a matter of con- 
 siderable importance. The fact that milk 
 when drawn from the udder may contain 
 bacteria is of the greatest interest in 
 connection with the observance of meas- 
 ures designed to reduce the bacterial 
 content of milk to the minimum. Here, 
 if that fact be true, is one source of the 
 infection of milk which can not be elimi- 
 nated by the exercise of precautions dur- 
 ing the milking or the subsequent pro- 
 cesses to which it is subjected. 
 
 The earlier investigations undertaken 
 to throw light on the question of the 
 presence of bacteria within the healthy 
 udder consisted in counting the bacteria 
 in samples taken during different periods 
 of the milking. Schultz' found a decrease 
 in numbers as the milking progressed. 
 L'ehmann' obtained like results. It might 
 be concluded from the work of Schultz 
 and Lehmann that the teats, or at most 
 the lower portion of the cistern, only 
 contain bacteria. 
 
 Gernhardt' found a larger number in 
 samples from the middle of the milking 
 than at the beginning, although some of 
 the samples from the last milk drawn 
 were sterile. To e^^lain his results, 
 Gernhardt suggests that the bacteria 
 make their way up through the millf 
 ducts of the teats, through the cistern, 
 and into the smaller ramifications of the 
 ducts whicl) connect the cistern with the 
 ultimate follicles. Such an assumption 
 explains the wide variation in numbers 
 obtained by him. 
 
 1. Leopold Schultz. Archiv. f. ■ Hygiene, 
 B. S. XIV (1892). 
 
 2. Lehmann. 17te Versammlung d. deut. 
 Ver. f. offent. Gesundheltspflege. 
 
 8. Gernhardt. Quant. Spaltpilzunters. d. 
 Milch, Inaug. Dissert. Univ. Jurjew. 
 
 Von Fceudenreich' states that, when lo 
 the udder, milk is free from bacteria 
 except when the milk glands are in a 
 diseased condition. He mentions the 
 fact as having been demonstrated by 
 Pasteur, who drew samples directly from 
 the cistern by means of a sterile cannula. 
 On the other hand, BoUey and Hall' com- 
 pared the species of bacteria in the milk 
 of several cows, the samples taken 
 through a sterile milking tube Inserted 
 into the milk cistern. 
 
 Russell" found that bacteria are present 
 in the udder proper in case of mastitis. 
 In Russell's Dairy Bacteriology we find 
 the following: " How far these different 
 forms of germ life are able to penetrate 
 into the healthy udder is as yet un- 
 known. In all probability, tbei glandular 
 tissue of the udder is not affected, 
 although it Is possible that microbes 
 might work their way up the open chan- 
 nel of the teat into the udder proper." 
 
 Grotenfelt' says that " When the milk 
 is drawn- from the udder of a healthy cow 
 it is germ free, or sterile. The original 
 sterility of normal milk is due to the 
 fact that the bacteria can not gain access 
 to the milk glands from without as long 
 as the udder is not injured in any way." 
 P. W. Woll, the translator of Grotenfelt's 
 work, adds in a footnote: " The bacteria 
 in the milk cistern will be largely washed 
 
 4. Bd. von Freudenrelch. Dairy' Ba.oteriol- 
 ogy, translated by J. R. A. Davis'. Page 36. 
 
 5. Boiley and Hall. Cent, fur Bakt'. und 
 Parasit, II, Abt. I: 795, (1895); Association 
 Ag. Coll. and Bxp't Stations (1S96); Ab- 
 stract In Experiment Station Record, Vol. 
 VII, No. 11, p. 991. 
 
 6. H. L. Russell. Dairy Bacteriology, pp. 
 42, 43. 
 
 7. Gosta Grotenfelt. The Principles of 
 Modern Dairy Practice, translated by F, 
 W. Woll, p. 23. 
 
LSeprlnted from the JOIJENaL OF APPLIED MICROSCOPY, Vol. I, Ko. 12.] 
 
 Page 206 out .by the first milk drawn, but not all 
 removed until milking has progressed 
 some time." 
 - Rotch" concludes that the few cases in 
 which contaminated samples were 
 obtained from the strippings, were due 
 to faults in technique and not to bacteria 
 from the Interior of the udder. 
 
 Moore' reviews the conclusions of 
 Schultz, Gernhart, and Rotcb and gives 
 the results of his own investigation. In 
 every examination made he found the 
 last milk from at least one quarter of the 
 udder to conta:ln 1^act;^^a. In concluding 
 his paper Mi^re suggests that a bacteri- 
 ologic exan^inatlon of the larger milk 
 ducts and of the acini themselves might 
 throw some 4ight upon the assumption of 
 Oernhart. Such an investigation was 
 rendered impossible at the time on 
 account of / his inability to procure the 
 udder of a freshly killed milch cow. 
 
 That sterile samples may frequently be 
 obtained directly from the teat is a fact 
 that has been demonstrated by many 
 investigators. But the frequency with 
 which these same workers have failed 
 leads to the conclusion that the last 
 milk contains otily a. few bacteria and 
 which may,' or may not be contained in a 
 given small sample. Schultz, Gernhart, 
 fiussell, Rptch, and Moore have all been 
 unable to ^et sterile milk in every case. 
 Informatl(Ai is not at hand concerning 
 the amount of milk taken for a sample, 
 except that Moore took 50 cc. of the last 
 milk. Conp" suggests that the reason 
 the earlier workers obtained sterile milk 
 so readily was because they did not col- 
 lect large samples. He says: " Essen- 
 tially the same facts have been demon- 
 strated iaj..iegard to human milk. • * * 
 Honigmann", Knochenstiern", Ringel", 
 and Falleske" have all independently 
 found that it Is impossible to get human 
 milk from the mammary gland in such a 
 way as to be sterile." 
 
 Von Freudenreich" states that he 
 failed to obtain sterile milk in large 
 quantities although the udder was 
 
 8. Dr. T. M. Rotch. Transactions of the 
 Association of American Physicians. 18M. 
 
 9. V. A. Moore, preliminary Investiga- 
 tions Concerning the Number and Nature of 
 Bacteria in Freshly Drawn Milk. Twelfth 
 and Thirteenth Annual Report of the 
 Bureau of Animal Industry, U. S. Dep't of 
 Agr., p. 261. 
 
 10. W. H. Conn. Bull. No. 25, U. S. Depart- 
 ment of Agriculture, Office of Experiment 
 Stations, p. 9. 
 
 11. Honlgmann. Ztschr. Hyg. 14 (1893), p. 
 207. 
 
 12. Knochenstiern. Inaug. Diss. Dorpat. 
 (1898).. 
 
 13. Ringel. Munch. Med. Wochenschr. 
 (1893), p. 613. 
 
 14. Falleske. Vlroh. Arch., ISO (1892), p.- 185. 
 16. Ed. von Freudenreich. Landwirt- 
 
 schaftllches Jahrbuch der Schwelz, (1891) 
 n, p. 18. 
 
 washed and smeared with lard to pre- 
 vent contamination. In an attempt to 
 collect ten liters of sterile milk for an 
 experiment in cheese making, he was 
 unable to reduce the nuniber below 212 
 bacteria per cubic centimeter. He calls 
 attention to the ease with which a few 
 cubic centimeters are collected, using the 
 same precautions, but he does not recog- 
 nize the presence of bacteria from with- 
 in the udder. 
 
 Those who believe the last milk to be 
 absolutely sterile when drawn from the 
 teat must necessarily explain the con- 
 stant presence of bacteria in the fore- 
 milk. The explanation is substantially 
 as follows: Bacteria in the air or in 
 stable filth accidentally gain a foothold 
 in the milk remaining on the end of the 
 teat after milking. The favorable condi- 
 tions for bacterial growth offered by the 
 ducts favor the multiplication of the In- 
 vading bacteria, which increase so rapid- 
 ly as to account for the presence of the 
 multitudes always found in the fore milk. 
 Experiments by the writer have shown 
 that it Is possible for this to occur under 
 certain conditions, but the more probable 
 explanation is embodied in the results of 
 the investigations about to be described. 
 These will be treated under three separ- 
 ate heads, as they have in common only 
 the fact that they lead to the same con- 
 clusion. 
 
 THE PERSISTENCE OF CERTAIN SPECIES OF 
 BACTERIA IN THE FORE MILK. 
 
 The Work of Bolley and Hall is the 
 only investigation on the subject that 
 has come to notice. Samples of 
 milk were taken by means of a sterile 
 milking tube inserted through the duct 
 into the milk cistern. Some species were 
 found common to both the first and the 
 last milk drawn. Only one organism 
 was found common to the milk of all the 
 animals examined, that one having no 
 effect upon the milk. The writers con- 
 clude that a given form, once present, 
 may be quite constant in its occupancy 
 of the udder in an IndividuaL 
 
 In the Investigations which I have 
 made to determine the nature of the milk 
 duct flora, the following methods were 
 adopted. Before collecting samples, the 
 udder and flanks of the cow were thor- 
 oughly moistened to prevent the dlslodg- 
 ment of dust by the movements of milk- 
 ing. In addition, the teats were moist- 
 ened with a solution of mercuric chloride. 
 Samples were drawn directly from the 
 teat into sterile test-tubes, which were 
 provided with cotton plugs. In this 
 respect the work of the writer differs 
 from that of Bolley and Hall. Cultures 
 were made immediately after collecting 
 the samples. Five two hundred and fif- 
 tieths (5-250) of a cubic centimeter of 
 
[Reprinted from the JOtJRNAt OF APPLIED MICROSCOPY, Vol. J, No. 12.1 
 
 .ge 207 milk was found. In general, to Introduce 
 a sufficient number of bacteria for con- 
 venient study upon a plate culture made 
 in 15 cc. of media. For a time both gela- 
 ' tin and agar plate cultures were made, 
 but the use of the former was discon- 
 tinued, as agar was found to be more 
 
 The milk of each of the four teats of a 
 cow was examined on two successive 
 days, and after a lapse of two weeks, 
 some of them were examined upon four 
 more day^. Four or possibly five species 
 were observed, only one being common 
 to the four teats. Although the bacterial 
 
 A pbotogiaph of a section through the teat and one quarter of the udder of a cow. The parts rep- 
 resented by the letters A, B, C indicate the three arbitrary divisions into which 
 the gland was divided for purposes of examination. 
 
 satisfactory. The total number of colo- 
 nies did not appear until after several 
 days in the Incubator at a temperature of 
 37.6 degrees C. The plates were then 
 examined and sub-cultures were 
 made from the colonies of the apparently 
 different species. The various forms of 
 colonies were carefully described and the 
 number of each recorded. 
 
 flora of each of the teats differed from 
 that of its neighbors, the same species 
 were found to persist in the same teat 
 from day to day. They were not present 
 in the same relative numbers on each 
 occasion. 
 
 The milk of another cow was examined 
 on five occasions, covering a period of 
 eight months. In the milk of this animal 
 
[Reprlutedfroin tlie JOUKNALQF APELIED MlUfiOSCOfY, Vol.li"Na. 12J 
 
 Page 208 but three species of bacteria were found. 
 On the first' day that the milk was 
 e3eS,Sii'ned,' streptococcus ■ was found to 
 ■prfedominate in numbers in all four of the 
 teats. The' cither two species occurred 
 only occasionally, but in the later exami- 
 nations.^bey .were found to exceed the 
 streptococcus in number. 
 
 The presence of streptococci in millt 
 fi:om "a normal udder is, in the experience 
 of the writer, unusual- -None, have been 
 fourid»"in the milk of eight cows in the 
 saine stable, or for. that matter, in any 
 examination of fore milk from cows else- 
 where. ' The persistence of the strepto- 
 coccus in the milk of tHe one cow is 
 therefore of special significance. 
 
 The mathematical probability that the 
 same organism will invade the same 
 sterile milk duct, even twice in succes- 
 sion, is infinitely slight. It is therefore 
 necessary to seek other explanation for 
 the constant presence of bacteria in tfie 
 fore milk, when we consider the persist- 
 ence of species In the milk of certain cows 
 or in particular quarters of the udder of 
 the same cow. 
 
 AN EXPERIMENT IN COLONIZING THE 
 CISTERN WITH BACTERIA. 
 
 With reference to determining the pos- 
 sibility for an organism to persist in the 
 cistern for a considerable period it was 
 determined to introduce, into one quar- 
 ter of the udder, a culture of an easily 
 distinguished bacillus. For this purpose 
 Bacillus prodigiosus was selected because 
 the red color of its growth on agar would 
 render its presence in milk easily recog- 
 nized when cultures were made. Four 
 cubic centimeters of a bouillon culture 
 were introduced into the cistern by 
 means of a hyperdermlc syringe length- 
 ened with a milking tube. Both the milk- 
 ing tube and the syringe were scalded to 
 guard against introducing any other 
 micro-organisms along with prodigiosus. 
 It was known from work ^.Iready done 
 that the organism in question was not a 
 natural Inhabitant of the udder with 
 which the experiment was being made. 
 
 The use of the milking tube, as is 
 nearly always the case, occasioned an in- 
 flammation of one side of the udder. The 
 Inflammation is attributed to the use of 
 the milking tube rather than to Bacillus 
 prodigiosus. The threatened obstruction 
 ^ of the teat by the accumulation of irreg- 
 
 ularly shaped masses of casein, rendered 
 it necessary to frequently draw out the 
 purulent liquid from the diseased quar- 
 ter of the udder during the two following 
 days after which the inflammatory con- 
 dition subsided. Plate cultures were 
 made each day. On the day following r.he 
 inoculation, ten colonies of Bacillus pro- 
 digiosus appeared on the plates. 
 Although the same amount of milk was 
 
 used in making the cultures on the days 
 following, the number of colonies was 
 observed to decrease in number^ On the 
 sixth day, the colonies of that bacillus 
 ceased to appear. During the whole 
 period, with the exception of thei ' fitst 
 two days, colonies jot the native bacterial 
 flora were observed in each plate culture. 
 The fact that an organism selected at 
 random, without considering its fitness, 
 for. inhabiting the udder,, should' succeed 
 in persisting there for six days' is signifi- 
 cant. The experiment denioris'trates the 
 fact that frequent and thof&ugh'milking 
 may not remove all bacteria from the 
 udder. That other species of bacteria, 
 better fitted for that; eltivironment, are 
 able to persist in the udder for longer 
 periods seems highly probable. 
 
 A BACTERIOLOGIC EXAMINATION OF THE 
 GLANDULAR TISSUE OF THE UDDER. 
 
 The writer is indebted to Dr. Moore 
 for the suggestion of this line of work 
 and for the privilege of associating with 
 him in an investigation based upon it. A 
 partial report of the results obtained has 
 been published elsewhere"- In attempt- 
 ing to draw conclusions from the facts 
 which have already been presented, the 
 writer finds himself unavoidably influ- 
 enced by the facts brought to light in 
 the work to which reference has been 
 made. That his conclusions may not ap- 
 pear to be based upon a less firm foun- 
 dation of fact than Is the case, he feels 
 justified in here referring to the joint 
 labor. 
 
 The fundamental method underlying 
 the investigation consisted in making a 
 large number of cultures directly from 
 freshly exposed glandular tissue. Sterile 
 tubes, tubes containing about 15 cc. each 
 of gelatin, and some containing slanted 
 agar were taken to the place of slaughter. 
 
 The purpose was to compare the bac- 
 teria found in the fore milk with those 
 which might be found in the udder. 
 Samples of the fore milk and in one case 
 of the strippings, were taken Immediately 
 before the slaughter. In order to obtain 
 more definite results, each quarter of the^ 
 udder was arbitrarily divided Into three 
 divisions. The first (A) Included the teat 
 and milk cistern. The second and third di- 
 visions (B,C) Included horizontal zones of 
 equal thickness constituting the r^jnain- 
 Ing portion of the udder. (See illiistra- 
 tion). 
 
 Each cow was milked before killing. 
 Immediately" after slaughtering ^he cow, 
 the udder was carefully removed, Th^ 
 skin was reflected and a flamed knife 
 was used to make a dorso-ventral Inctsloii 
 several inches in depth in one quarter of 
 
 16. ■y. A. Moore and A. R. 'Ward. Bull, 
 No. 1S8, Cornell University A^lculturkl 
 Experiment Station. January,' 1S99'. ' 
 
[Reprinted from the JOURNAL OF APPLIED MICROSCOPY, Vol. I, No. 12.] 
 
 Pa^e 209 the udder. Samples of milk were col- 
 lected In sterile test-tubes as it welled 
 out of the cistern and its smaller rami- 
 ilcations. In making cultures from the 
 glandular tissue, care was taken to pre- 
 vent milk of the ventral region from 
 Coming in contact with the freshly ex- 
 posed surfaces. Bits of tissue were 
 detached with flamed scissors, and trans- 
 ferred to culture media by the use of a 
 flamed platinum loop. Tubes of gelatin 
 and of agar were inoculated in this man- 
 ner from ea,ch of the three arbitrarily 
 designated divisions of the quarter. The 
 same procedure was repeated with each 
 of the other three quarters of the udder. 
 Cultures were made from the udders of 
 six cows in the manner described. 
 
 Upon returning to the laboratory the 
 gelatin was liquefied at a temperature 
 not exceedli^ 37 degrees C and poured 
 into sterile ^etri- dishes, where it again 
 became soli^. Agar plate cultures were 
 made from the milk samples, and, to- 
 gether with those slanted agar cultures 
 already inoculated, were placed in the 
 incubator., ^he agar plate cultures were 
 designed to lae used as a check upon the 
 reliability of the conclusions reached 
 from an examination of the other cul- 
 tures. For instance: it might be possible 
 that organisms appearing to have been 
 obtained from the interior of the udder 
 may have lodged upon the bits of tissue 
 during the transfer. The identity in cul- 
 tural and morphologic characters of bac- 
 teria found in cultures made from the 
 fore milk and the glandular tissue of the 
 udder would eliminate a source for false 
 conclusions. 
 
 The tube,s of slanted agar, after stand- 
 ing in the incubator for several days 
 were exar]|lined particularly with refer- 
 ence to the presence or absence of 
 growth. Nearly all of the media which 
 had been in contact with material from 
 the udder . showed growth. Note was 
 taken of the color and character of the 
 growth of the colonies and sub-cultures 
 were made. 
 
 The gelatin plate cultures were in like 
 manner examined, furnishing a more sat- 
 isfactory method for obtaining pure cul- 
 tures. With these, a direct comparison 
 made it possible to trace the presence of 
 the same organism in the three localities. 
 In order to prove that these identities 
 existed, sub-cultures were made for a 
 more detailed comparison later. The 
 plate cultures made from the milk were 
 examined and sub-cultures were made 
 from all of the ap^ar«ntly different colo- 
 nies. 
 
 By comparing cultures from the various 
 sources, it was found that the same or- 
 ganism frequently occurred In the fore 
 milk and in each of the three parts of the 
 udder. (See illustration.) Most of the 
 bacteria obtained in pure cultures were 
 
 found to belong to one of three species 
 of micrococci. Cultures of the three 
 species were obtained from a sufficient 
 variety of sources to demonstrate their 
 general distribution throughout the 
 udder. 
 
 The apparently healthy udders of six 
 milch cows were in that manner found to 
 contain bacteria in the depths of the milk 
 secreting tissue. By the methods 
 employed, it was impossible to detect any 
 difference in the relative numbers of bac- 
 teria present in the three regions of the 
 udder. 
 
 The evidence at hand indicates that the 
 teats and the greater portion of the udder 
 may normally contain bacteria. It also 
 seems highly probable that a few at least 
 of the organisms found in the udder 
 remain there after each milking, becom- 
 ing the progenitors of the organisms 
 found to be present, in the milk when 
 drawn. This conclusion seems to be sup- 
 ported by the following facts: 
 
 1. Certain species of bacteria have 
 been found to persist In particular quar- 
 ters of the udder for considei'able periods 
 of time. This controverts the statement 
 that the milk ducts are sterile at the 
 close of the milking, becoming tenanted 
 from the outside by any organisms "which 
 by chance come In contact with the end 
 of the duct. 
 
 2. It is possible for bacteria to remain 
 in the udder and not be ejected along 
 with the milk. This has been proven 
 possible in the case of one organism. A 
 culture of Bacillus prodigiosus has been 
 introduced Into the milk cistern and has 
 succeeded in persisting there for six days, 
 as was shown by Its presence for that 
 period in the milk of that quarter of the 
 udder. 
 
 3. Cultures of bacteria have been 
 obtained by Dr. Moore and the writer 
 from the glandular tissue of the udders 
 of freshly killed milch cows. Identical 
 species of micrococci were obtained from 
 the milk and from the glandular tissue 
 of the udder. 
 
 4. It has not been shown by the investi- 
 gations published up to this time that the 
 last milk drawn is always sterile. 
 
 Archibald B. Ward. 
 From the laboratory of Comparative 
 Pathology and Bacteriology, New Tork 
 State Veterinary College, Cornell Uni- 
 versity, Ithaca, N. T. 
 
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