I \ NW N WWI ‘ I I M STUDIES ON THE BACTERIOLOGECAL CHARACTERIS'S‘ECS AND OTHER PRO?ERTEES OF A FEW MICROORGANESMS CAUSENG ROPINESS 1N 51611.2( ‘Y‘i’wsi: for 4.110 flag”. of M. S. MECWGAN 3TA?E COLLEGE Eadamee Cfiaadiwifzomez $946 mass-ms This is to certify that the thesis entitled "Studies on the bacteriological characteristics and other prOperties of a few micro-organisms causing ropiness in milk." presented by Radamee Orlandi Gomez has been accepted towards fulfillment of the requirements for . m. 8. degree m_B@9.§£3_ETin0t‘§Y and Public Health Date AUEUSt z3821946 ”-796 p33». . . it»... :a. u If .... til 5 (I... ~2....tll11. ....e .. u .l s . . . t. . . .... .2 n. 71:53 .. :F:...:.. i... ...........,....._ 1 n . . a .. 2 . a. 3:53.121... 33:35.” ....33...22.5.3.2“:a:::..v:v..$ .33... ... . ~ . . . a. I. .. . .I . ”“15 Drier 9.!v3'5—2ui .139 ‘pl :0 . . a . . . STUDIES ON THE BACTERIOLOGICAL CHARACTERISTICS AND OTHER PROPERTIES OF A FEW MICRO- ORGANISMS CAUSING ROPINESS IN MILK by RADAMEE QRLANDI-GOMEZ A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 1946 THEmS ’ ACKNOWLEDGMEng The author wishes to express his most sincere thanks and appreciation to the following people: To Dr. A. L. Bortree for his encouragement and invaluable assistance in carrying out this work: To Dr. G. S. Bryan for his supervision in writing this thesis and the many kindnesses and courtesies extended by him: To Dr. Ward Giltner of Michigan State College, who as Head of the Department of Bacteriology, gave all the facilities and made possible this work. Table of Contents Introduction . . . . . . . . . Review Of Literature . . . . . Experimental . . . . . . . . . Description of First Organism . Cultural Characteristics Biochemical Properties Description of Second Organism Cultural Characteristics Biochemical Properties . Description of Third Organism Cultural Characteristics Biochemical Properties Other Observations . . . Resistance To Desiccation Viability of Organisms When Suspended in Water . Page 11 ll 12 15 18 18 22 24 24 28 50 SO 55 Resistance to Holding and Short-time High- Table of Contents (conte) Temperature Pasteurization . Resistance To Disinfectants Population Densities and Growth Rates Discussion . . . . Summary . . . .. Literature Cited . O Page 55 36 43 46 51 53 INTRODUCTION The present studies were undertaken for the purpose of identifying some organisms isolated in pure culture from rcpy milk samples brought to the dairy bacteriology laboratory of Michigan State College for microscopical examination. Ropiness in milk is one of the best known of the abnormal fermentations Which frequently appear in milk supplies. As the condition is so well defined, a detailed description of the term is quite unnecessary at this time. In addition to the identification of the cultures isolated, it also has been the purpose of this work to study the behavior and peculiarities of some of these micro- organisms causing ropiness of milk when submitted to various conditions of environment and habitat. We have tested their resistance to desiccation, pasteurization temperature, disinfectants, etc. and viability in water suspensions. Numerous organisms have been reported and described in the literature as the cause of ropiness in milk. Undoubtedly, we could also eXpect a lot of variations as to the way in which they would behave under varying conditions. A brief study of the relation of a 37° C. and a room temperature incubation to the generation rates and population densities of these organisms is also included in this work. Nine pure cultures were isolated in the laboratory. Eight of these cultures were identified as micrococci; the other one was found to be a rod-like organism. From the eight micrococci isolated, two were selected for detailed study, because of the peculiar way in which they produced ropiness in milk and on the basis of carbohydrate fermentation. They seemed to be of two distinct types. One of the micrococci has been labelled: 'M—1' and the other “183 H.F.' The rod-like organism was labelled: I'Bao." REVIEW OF LITERATURE Early History of Ropiness In the excellent monograph published by Buchanan and Hammer (5) in 1915, an extremely valuable and detailed study is presented on thirty-three microorganisms known up to that time as the cause of ropiness in milk. They, also, present an excellent summary of the work published by early bactericlogists on this kind of fermentation encountered frequently in milk. According to Buchanan and Hammer (5), the first one to observe specific organisms in slimy milk was Ehrenberg in 1840. The bacterium found by Ehrenberg 'as found to produce a oitron yellow color in milk. He named it Vibrio exaggnthug. Later,the organism was classified as ngillgg synxanthus. These same investigators, Buchanan and Hammer (5) found that Pasteur in 1857 was the first who "definitely proved the existence of slime-producing organisms, in this case in wines3' Lister in 1875, who 'associated bacteria with slimy milk formation;" and Schmidt-lfllhein, nine years later, in 1882, who found a coccus while examining microscopically some viscous milk. Schmidt-Mfilheim did not isolate the organism nor study it. A few years later, Adametz (1889) described and named Bacillus lactis viscosus as the cause of ropiness in milk. 4 Since that time, investigations have continued on this type of fermentation which is probably second in importance to that which causes the souring of milk. Many other organisms have been isolated and described as causative agents of ropiness. Organisms Associated with Ropinegg Hammer (7) states that “one of the more common of the ropy milk organisms is Alcaligenes viscosus. It has been isolated repeatedly in Europe and has been encountered in many sections of the United States.“ He also mentions organisms of the group Escherichigr Aerobccter as the cause of many epidemics of ropiness in the United States. Other organisms mentioned by him and associated with the rcpiness of milk are: Streptacoccus lactis var. hollandicus, Lactobacillus casei, and Lactobacillus bulgaricus. Hammer and cordes (8) reported an organism not previously encountered, a Gram—positive unpigmented coccus, which produced 'very little acid, if any, from the carbohydrates tested". They considered it to be an undescribed species and named it Staphylococcugpcremorigr viscosi. At the present time, this organism is known as MgcrococchLcremoris-viscosi. Macy (15) described an organism obtained through a groceryman, who received it from Finland, absorbed in ordinary blotting paper. Marked characteristics of the organism were “its failure to grow well on the ordinary routine media, its ability to produce marked persistent ropiness in milk and its resistence to desiccation and heat“. It was capable of producing ropiness even after having been dried on blotting paper for six months. The organism survived 60° C. for thirty minutes. It was found to resemble most closely Strep lactis var. hollandicus, and Strep tsette. But it differed in certain characteristics from these two. He proposed a new species: Streptococcus 2.1.1.95. Long and.Hammer (12) found organisms similar to Alcaligenes viscosus in every characteristic except for their failure to produce ropiness. These bacteria were regarded as non-ropy strains of Alc. viscosus. Some of the organisms, which in the beginning gave no ropiness, started doing so after a few transfers in milk. The name, Alc. viscosus. var. dissimilis was given to them. Bergey (3), reports five species of micrococci which may produce slimy or ropy milk: Micrococcus pituitoparus, M. mucofaciens, M. freudenreichii, M. cremoris-viscosi, and M. viscosus. The first two are classified under pigmented micrococci, M. pituitoparus as not liquefying gelatin, while M. muccfacienp_liquefies it. The other three micrococci are classified as non-pigmented ones. M, freudenreichii and M. cremoris-viscogilliquefy gelatin, while M. viscosup does not liquefy it. More recently, Prouty (15) has reported the isolation of a micro-organism which in some respects was related to Staph. cremoris-viscosi of Hammer and Cordes (8). Outstanding characteristics of the organism were, its ability to cause rapid development of ropiness accompanied by the presence of an abnormal flavor and odor followed by very active digestion of the curd. Apparently, the organism differed mainly from M. cremorig-viscosi in that it fermented certain sugars. According to Hammer and Cordes (8), and Bergey (5), M. cremoriggvisccsi has no action at all on carbohydrates. Of considerable significance also, in Prouty's micrococcus, is the thermoduric nature of the organism which in certain instances survived an exposure period of thirty—five min. at 143° F. (61.60 0.). Saddler and Middlemass (16), describe in an outbreak studied by them an atypical E. neapolitana. This organism failed to produce indol and grew better at 21° C. than at 37° C. According to Bergey (4), Eppneapolitana forms indol and has an Optimum temperature for growmh at 37° C. Saddler and.Middlemass, therefore, considered their organism an atypical form of E. neapolitana. It should also be mentioned that their organism, on .:.n ll}. .. Kilns I... (T 32 13 primary isolation, on whey agar and in milk had capsules. The associates of L.A.Rogers (1) report among organisms described as the cause of ropiness: 'B. viscosuml B. lactis viscosus,Strep. lactis var. hollandicus, certain corynebacteria, and members of the Escherichia-Aerobdcter group, including the E. neapolitana, A. aero ones, and A. cloacae.' It is also stated that, 'ropy milk may result in some cases from strains of ordinary milk streptococci as Strep. lactis, Strep. cremoris, or Strep. thermochilus. Very few of the micro-organisms causing ropiness in milk are spore-formers. Buchanan and Hammer (5), report two aerobic or facultative anaerobic and two anaerobic spore-formers. The former two are represented by chterium peptogenes and Bgcillusp(mesentericus)vulgatus, the latter by chillus kleinii and Bacillus pruchii. SOURCES OF ROPY MILK ORGANISM There are various sources from which ropy milk organisms could be obtained. Commonly, the organisms are ‘traced to and found on utensils and other equipment, ‘surface waters, or milk cooling tanks, the stable, and the hair coat of animals. Many other related sources could be mentioned. Harding and Prucha (9) noted that samples collected on the farm ordinarily show the presence of ropy organisms in the utensils, and in many cases, in the water in the cooling tank. They, also, point out that the can-washing vat at the plant may be a distributing agency for all utensils washed in the same water where a can bringing milk contaminated with the ropy milk organism might have been previously washed._ Davis (6) points out several common sources: I'dirt from the cow or worker; contamination from utensils; contaminated drinking water or marshy land which may infect the udders or flanks; water used for washing utensils; straw, mouldy hay, and bedding; dusteladen air; certain plants, e.g. butterwort; and finally feeding stuffs“. Beck and Chase (2) determined the source of organisms causing ropy milk in one hundred and nine cases of ropy milk outbreaks. Milk submitted by farmers and dairy men constituted seventy-nine of the one hundred and nine cases, while thirty-three cases were related to some water source or supply, and seven cases were from the dairy cans returned to producers. The associates of L. A. Rogers (1) state that 'feed and improperly cleaned utensils are two chief sources of infection, and water from polluted stream may be a source”. Hammer (7) states that ”ordinarily ropy milk organisms are not found in the udder". However in an epidemic studied by Hammer and Cordes (8), Stihph. cremoris- visccpi was found in milk aseptically drawn from the producing cows. They state that in this case 'it was impossible to determine whether the udder invasion preceded or followed the outbreak". In a study concerning the wide distribution of rcpy milk organisms in city milk supplies, Ward (19) found organisms were present almost everywhere in the establishments under study. He makes the following statement: 'ropy milk organisms may be regarded as so common as to constitute an ever present source of a possibly serious outbreak". . . . . ' observations indicate that at least slight contamination occurs with a frequency not heretofore recognized" . 10 Control of Organisms Hammer (7) states that in trying to control an outbreak, the sterilization and disinfection of all utensils and equipment coming in contact with the milk should be done immediately. Stables should be cleaned and disinfected. The flanks and udders of the cows should be wiped with a cloth dampened with a disinfecting solution. While studying the resistence to heat of some micro- organisms causing ropy milk among others, Ale. viscosus and a few representatives from the Epcherichia-Aerobacter group, Harding and Prucha (9) found that none of these organisms would resist a temperature of 14o-145° r. for thirty minutes. According to these investigators, proper pasteurization, should destroy any ropy milk organisms which may be in the milk. They recommend, too, the use of chlorinated lime where steam or hot water are not at hand. They state that a twelve-ounce can of good bleaching powder added to one hundred gallons of water would give a powerful disinfecting solution. All instruments, utensils, and equipment should be put into this solution and allowed to remain in it for fifteen to twenty minutes. ll EXPERIMENTAL DESCRIPTION or FIRST ORGANISM The following is the information compiled from the first organism studied: Organism described: 'Bac" Form: Small, short, plump rod. Pleomorphic; some organisms look almost coccoidal while others presented a definitely elongated body. Size: Varies, on the average the short plump rods measure from 1 to 2 microns long by 0.5 to 0.8 microns wide. Arrgpgement: Very seldom appears in chains of two or three elements; mostly single. Motility: Nonrmotile. Staining reaction: Although some organisms remain Gram- positive, the majority of the organisms were Gram-negative. With methylene blue stain, a twenty—four hour culture shows very uneven staining, probably due to the presence of some sort of metachromatic granules. A few organisms showed bipolar staining. An old milk culture shows still more uneven staining, showing clear spaces inside the bacterial 12 sometimes resembling a true spore but in most cases Just a clear space or light staining area, a sort of vacuole. Spore formation: None. Qapsule formation: A large capsule could be shown surrounding the bacterial cells. Cultural Characteristics Agar slant: A distinct muccoid, tenacious, and viseid growth occurs after twenty-four hours of incubation. Growth along the lines of inoculation was more or less even,showing heavy growth. Growth was moist and white or a whitish gray. Agar plate: On Tryptose Glucose Extract medium growth was evident in twenty-four hours. Colonies on the surface grew luxuriantly with a size varying from 5 to 6 mm. in diameter. They were thick, viscous, whitish, raised, convex, very glistening, and smooth-edged. Subsurface colonies were smooth, smaller and lenticular. After forty-eight hours or prolonged incubation at room temperature, surface colonies grew to a size of about 8 to 10 mm. in diameter and were very sticky. Threads three feet long were sometimes pulled out of the colonies. Milk aggr plgte: Surface colonies, on incubation at 37° C. for twenty-four hours, showed very little, or no proteolysis 13 of the milk casein, while the subsurface colonies showed a more distinct although still faint proteolysis. Colony characteristics were the same as on T.G.E. agar plate. Gelatin stab.: Filiform growth along the stab. Surface growth appeared from undulate to lobate. No liquefaction occurred even after a month of incubation at 20° C. Potato agar: Moderate growth, viscous, mucoid, grayish white, semi-transparent. Dunham's solution: Heavy turbidity and no sediment after twenty-four hours of incubation either at room temperature or 57° C. After twenty-four hours of incubation, a pellicle about 2 mm. deep appeared on the surface. After forty-eight hours of incubation (either at 57° C. or room temperature), the solution remained turbid but began to show some sediment. This sediment on shaking rises as a coherent viscous swirl. The solution had become ropy. gpprile gkim milk: Sterile skim milk quickly became ropy at room temperature and 57° C. It was observed that at 57° 6., 10°°' of sterile skim milk inoculated with a 4 mm. loOpful of organisms, became ropy in about four hours; while at room temperature, rcpiness developed in about five hours using the same amount of inoculum. Sterile skim milk became thick and ropy on twenty—four hours of incubation at 14 57° 0., then after forty-eight hours, coagulated with a soft curd. 0n the fourth day, the curd was very hard with very little or no proteolysis at all. At 57° C., ropiness lasted only six days. At room temperature, the milk became much more ropy after twenty-four hours of incubation. A pellicle of mucoid material was evident at the surface. In four or five days, the milk was coagulated in a compact mass, but looked very finely precipitated. The sample was very thick in consistency. A few days later, the whole sample coagulated in a compact mass, proteolysis became evident by the seventh or eighth day. The ropiness of the samples lasted fifteen days. Rough agitation of ropy samples reduced or destroyed ropiness. Litmus milk: Incubated at 37° 0., the changes were essentially the same as in sterile skim milk, except for the litmus which was reddened indicating acid production. After a few days of incubation, the acidity increased. The curd Showed reduction of the litmus and reddening of the whey or serum. Sterile cream: After twenty—four hours of incubation at room temperature, the cream was ropy only in the upper &' layer of the sample. On further incubation, ropiness was doubtful and on the fourth day, no ropiness was 15 was observed in the cream sample. Biochemical Properties Gas production: No evidence of gas production was observed in milk cultures or a fermentation base solution containing different carbohydrates, 0.5% sodium chloride, 1% tryptose, and Andrade's indicator. Nevertheless, later, it was observed repeatedly that the organism started producing small bubbles of gas after the ninth or tenth consecutive transfer in sterile milk, incubated at room temperature. A lOOpful of the milk samples showing this gas production, inoculated into a fermentation base containing lactose and Andrade's indicator showed acid and definite gas production at 57° C. and room temperature. Attached to the walls of the lactose fermentation tubes was noted a sort of flocular growth, something not observed in recently inoculated samples with the non—gas-produoing organism. An attempt to show any kind of variation from the original cultures used was unsuccessful when dilution pour plates or pour plates streaked from the lactose fermentation tubes, showing this flocular growth, was tried. The colonies developing from these tubes looked exactly the same in appearance and stickiness to the ones obtained from the original cultures. Due to the production of gas under the conditions 16 stated, an organism related to the Escherichia-Aerobacter group was suspected. The organism was submitted to the methyl red and Voges-Praskauer tests. Methyl Red Test: Positive Voges-Praskguer: Negative Acid production: Using the same fermentation base mentioned before, the organism showed a strong fermentation power for most of the sugars used. See Table 1. Acid and no gas was produced from rhammose, glucose, fructose, galactose, mannose, lactose, sucrose, maltose, trehalose, cellobiose, raffinose, starch, dextrin, glycerol, adonitol, mannitol, sorbitol, and salicin. Fermentation of arabinose and xylose, was slight after twenty-four hours of incubation although after six days there was a definite fermentation. Inulin, after twenty-four hours, turned toward the alkaline side. The organism did not ferment dulcitol. As previously pointed out, it was observed that the organism would start producing gas in milk after eight or ten daily transfers in sterile skim milk. If lactose fermentation base was then inoculated from these milk cultures producing gas, the organism would show acid and gas production. Indol: No indol was detected after various incubation periods at 57° C. and room temperature. 17 Ammonia: Was produced from peptone. Nitrites: Not produced from nitrates. 18 DESCRIPTION OF SECOND ORGANISM The second organism under consideration in our study of ropiness in milk presented the following characteristics: Organism described: “M-l“ Form: Spherical, occurring mostly single or in pairs. Tetrads were seen as well as irregular groups. 'Size: The organism was found to vary in size with some less than one micron while other cells were about 1.5 microns in diameter. Motility: Non-motile. Staining reaction: Gramrpositive. Spore formation: None. Capsule formation: Throughout the course of our work, we have been unable to show any capsular material surrounding the cells. Culturgl Characteristics Agar slant: Growth along the line of inoculation showed heavy, somewhat echinulate growth. Growth was opaque, raised, sticky and dirty white in color. 19 Agar plate: (Using T.G.E. agar). Colonies on the surface were dirty white, round, entirely smooth, and opaque having a pin-point central portion slightly raised over the rest of the colony surface. This feature was not observed in all of the colonies although it was present in most of them. The colonies touched with the end of an inoculating needle were found to be sticky. The average size of a surface colony (24 to 48 hours old) was around 2 mm. Subsurface colonies were discoid in shape and about '1 mm. in diameter. Milk ggar plpte: After twenty-four hours of incubation at 57° 0., surface colonies as well as subsurface ones, showed marked proteolysis of the milk casein. Colony characteristics were the same as on T.G.E. agar plate. Gelatin stgb: 0n twentybfour to forty-eight hours incubation, no noticeable liquefaction occurred. After four days of incubation, a small infundibuliform to orateriforl liquefaction was observed. After a month of incubation, liquefaction progressed a little more showing a napiform configuration. Gelatin was proved to be very very slowly liquefied. Potato agar: Growth was poor showing tiny, semi-transparent pinppoint colonies. rwll. E... 20 Dunham's solution: The medium showed turbidity and a viscous sediment on twenty-four hours of incubation at 5700,, but it was not turned ropy by the organism. 0n prolonged incubation, this turbidity of the medium was decreased. Sterile skim milk: Sterile skim milk quickly became ropy at 37° C. and room temperature. It was found that lO°°’ of sterile skim milk inoculated with a loopful of a twenty-four hour milk culture (using a standard 4 mm. loop) and 1DCUbated at 57° C. or at room temperature started developing ropiness in about four and one-half hours. By the end of the fifth hour, the samples were definitely ropy. Ten cc. amounts of milk inoculated with a lOOpful of organisms incubated it 37° C. for twenty-four hours became ropy but not coagulated. After fortyaeight hours of incubation, a soft curd and some peptonization were observed. Peptonization progressed for a few days while rcpiness lasted twelve days at 57° c. Tubes of inoculated milk incubated at room temperature were very ropy at the end of forty-eight hours. By this time, no curd was formed but peptonization was quite rapid. On the fourth day, a very soft curd was formed at the lower half of the sample where apparently peptonization was not progressing so rapidly. By the seventh day, this soft curd had developed into a hard one; proteolysis was still taking place. 21 A slight rcpiness was observed on the twenty-first day after inoculation, and on the twenty-fifth, it had completely disappeared. By this same time, proteolysis evidently was diminshed or stopped. Moderate, momentary agitation destroyed the ropy character of samples no matter whether these were incubated at 37° C. or at room temperature. It was observed, also, that the organisms imparted a light buff color to sterile skim milk. Litmus milk: Incubated at 37° C., the changes were essentially the same as for sterile skim milk. Very slight acid production occurred in the samples as shown by the slight change in color of the litmus even after a few days of incubation. Sterile cream: After twenty—four hours of incubation at room temperature, rcpiness was observed on the surface of the cream samples. No ropiness was observed in layers lower than 5/16' below the surface. On further incubation, .ropiness did not progress downward. On the third day, ropiness was doubtful in the cream layer,whioh in the first twenty-four hours was definitely ropy. By the fourth day, the samples showed no ropiness. 22 Biochemical Properties Gasgproduction; No evidence of gas production was observed in milk cultures nor in the fermentation tubes containing the fermentation base which previously has been described. Acid_production: Using the fermentation base described under “Bac', the organism produced acid from a few sugars. See Table 1. Acid was produced from glucose, fructose, mannose, lactose, maltose, and trehalose. Fermentation of galactose after twenty-four hours, was doubtful although after six days, it was more definite. Fermentation of sucrose and starch were very doubtful even after six days of incubation. Cellobiose and dextrin were slightly fermented after twenty-four hours of incubation. Cellobiose remained slightly acid even after six days, but dextrin turned negative. Glycerol was negative after twenty-four hours of incubation and slightly fermented after six days. Arabinose was negative after twenty-four hours and doubtful after six days of incubation. Rhamnbss, xylose, raffinose, inulin, adonitol, mannitol, sorbitol, dulcitol, and salicin were not fermented even after six days of incubation. Indol: No indol was detected after several incubation 25 periods at 37° C. and room temperature. Ammonia: The production of ammonia from peptone was doubtful or very slight as detected by several trials at 37° C. and room temperature incubation. Nitrites: Not produced from nitrates. 24 DESCRIPTION OF THIRD ORGANISM The third organism, (185 H.F.), included in this study is an organism which on account of certain definite and consistent reactions and behavior, we believe is a different species or strain from the one Just previously described. It presented the following characteristics: Organism described: ”183 H.F.” Form: The organism is spherical, occurring mostly single or in pairs. Irregular groups and tetrads could, also, be found. Sige: Cells were found to vary greatly in size from 0.8 micron to 1.9 microns in diameter. Motility: Non-motile. Staining reaction: Gram-positive, but very few scattered cells occasionally Gram-negative. Spore formgtion: None. Capsule formation: No capsule was observed. Cultural Characteristicg . ;§gar slant: Growth along the line of inoculation showed 25 echinulate margins. Growth was grayish white or slightly dirty white, glistening, and sticky. Agar plate: Surface colonies were Opaque, flat, glistening, slightly dirty white or grayish white. The organism was observed to produce some colonies having unevenly-shaped margins, although most colonies were round. The round colonies appeared evenly-colored and finely granular under the microscope. The ones having unevenly-shaped margins presented tiny darl spots inside the colony when observed under the microscope. Both types of colonies were sticky when touched with an inoculating needle. Sterile milk inoculated with organisms from any of these colonies made the milk ropy,_and if plated again on agar, it again showed the two types of colonies. Several trials were made to detect any mixture of two micrococci. The colonies developing from previously well-isolated colonies always showed the two types of colonies. The size of the colonies after forty-eight hours of incubation at 57° C. was about 2 mm. in diameter. Subsurface colonies were discoid in shape and much smaller, about 1 mm. in diameter. Milk agar plate: On incubation at 57° C. for twenty-four hours, the colonies growing on the surface showed a much Fillet ul U1... H. . ..l. . kill. . 26 more marked proteolysis of the milk casein than those growing in the subsurface. The area of proteolysis produced by surface colonies was definite. Susurface colonies showed proteolysis rather faint or incomplete. Colony characteristics were similar to those grown on T.G.E. agar plates. Gelatin stab: After twenty-four hours of incubation, no noticeable liquefaction was observed. After four days of incubation, a small crateriform liquefaction started to occur. After a month's incubation, saccate liquefaction was observed. Gelatin liquefaction was poor and slow. Potato agar: Growth was poor with semi-transparent, tiny or pin-point colonies. Dunhgg's solution: Showed turbidity and a viscous sediment after twenty-four hours of incubation. On prolonged incubation, the turbidity decreased. The medium was not turned ropy by the organism. Sterile skim milk: This organism showed a distinct and peculiar way of producing ropiness when inoculated into sterile skim milk. It was observed that when incubated at 37° C., no ropiness at all was detectable, although the organism was reproducing very fast as is shown by plate counts, and growth curves presented in Table 4 and Figure 3 . Microscopic examination of milk samples 27 incubated at this temperature, also showed tremendous reproduction of the organism. This particular characteristic of the organism, partial or complete inability to produce ropiness when incubated at 37° C. was observed at different trials. Somehow, a 37° C. temperature proved to be inimical to the organism as far as production of ropiness is concerned. I The milk looked normal during the first days of incubation. By the fourth day, a hard curd was formed with some proteolysis starting at the bottom. Sterile skim milk inoculated with about the same amount of organisms (standard 4 mm. loop was used) and incubated at room temperature started showing definite ropiness along the surface rim touching the inside of the test tube in about twelve and a half to thirteen hours. In twenty-four hours, ropiness was definite on the entire surface of the sample. The ropiness produced at room temperature was observed to be more definite only at the surface regardless of length of time the samples were incubated. Deep layers in any sample were Just like normal milk. It was, also, noted that when some small amounts of cream were present in the milk sample, the ropiness was easier to detect. The ropiness produced by this organism ' was delicate, the threads pulled out of a twenty—four hour milkLculture being very much like fine threads of silk. 28 At room temperature, no coagulation of milk occurred until the fourth day. On the third day, it was observed that the milk samples appeared a little thick in consistency and presented a very fine percipitate. Ropiness at room temperature lasted nine days, but it was not marked from the sixth day on; the threads formed being rather short and breaking sharply when about one-half inches or less long. This ropiness was noticed in the serum which apparently was extracted from the hard curd formed. This serum could have been, too, a mixture of normal serum and part of the products of proteolysis. Litmus milk: The changes which occurred in litmus milk were essentially the same as in sterile skim milk. A very slight change in color occurred indicating a very slight production. Sterile cream: After twenty-four hours of incubation at room temperature, ropiness of the cream was doubtful. After forty-eight hours and up to seven days, the samples gave negative results for ropiness. Biochemical Propertieg Qas production: No evidence of gas production was observed intmilk cultures nor in fennentation tubes containing the fermentation base described under 'Bac' - Gas production. ‘1 lllu‘li‘ ‘\ Isl]., ..l. 1.5.. irur. ,4.) J . . .. 29 Acid_production: Using the same fermentation base used for the organism labelled 'Bac', the organism showed acid and no gas from glucose, fructose, lactose, maltose, celloise, and dextrin. See Table l. The fermentation of sucrose, which after twenty-four hours of incubation, was slight turned completely positive after a few more days of incubation. The fermentation of galactose and mannose was doubtful after twenty—four hours of incubation, but after ‘ six days of incubation, it was more definite. Trehalose was very slightly fermented after twenty-four of incubation but after six days a definitely positive reaction was observed. The fermentation of starch and adonitol remained doubtful even after six days of incubation. ,Arabinose which after twenty-four hours was negative, turned doubtful after six days of incubation. Rhamnose, xylose, raffinose, inulin, glycerol, mannitol, sorbitol, dulcitol, salicin were not fermented even after six days of incubation. Indol: No indol was detected after several incubation periods ‘t 57° C. and room temperature. .Ammonig_: Several trials for the production of ammonia from peptone at 37° and room temperature gave positive results. Nitrites: Are not produced from nitrates. 50 Other Observations During the course of this work, other tests were developed and conducted to get as much information as possible on the behavior of these organisms under study. The three organisms: "Bac", "M-l”, and "183 H.F.", Just described in the previous pages, were used for the purpose of getting this additional information we wanted to obtain from ropy milk organism. As it will be remembered from the descriptions made, I ¥~ 'Bac' is a rod-like, Gram—negative, encapsulated organism; 'M-1' and ”185 H.F.' are Gram-positive micrococci. For the purpose of obtaining the desired information, the following tests were carried on: resistance to desiccation, viability of the organism when kept suspended in water, resistance to holding and short-time high temerature pasteurization, and resistance to disinfectants. We, also, include here a brief study of the relation of a 37° C. and room temperature incubation to the population densities and growth rates of the micro-organisms under study. The paragraphs, which follow, are a short relation and discussion of the results obtained in the experiments ‘we have Just mentioned. IResistance to desiccation: The organisms were tested on a 31 hard, smooth surface anda rough, porous surface. Small , pieces of cover glass were smeared and filter paper was impregnated, by means of a loop, with two different suspensions of each of the organisms under study. One suspension consisted of organisms washed off from a twenty- four hour agar slant culture, while the second was merely a twenty-four hour old milk culture thoroughly shaken so as to insure even suspension of all organism. The small pieces of glass and paper were allowed to dry in sterile petri-dishes which were held at room a" temperature. Periodically, pieces of glass and paper were taken out from each petri-dish and dropped separately into 5 cc. of sterile skim milk. After forty—eight hours of incubation at room temperature, these milk samples were tested for rcpiness. The results obtained are presented in Table 2. The organism named "Bac' suspended in water and smeared on glass, resisted desiccation for nine days while on filter paper it resisted for seventeen days. When suspended in milk and smeared on glass, they resisted seventeen days; while those on paper resisted fifteen days. The micrococcus "M—l' suspended in water and placed on glass resisted thirty-three days while those on filter paper resisted seventy-eight days. The organism suspended in milk and treatedthe same way resisted fifty days on glass. Those impregnated in paper are still living one 52 hundred and one days after the tests were started. The micrococcus I183 H.F.” was found to possess more or less the same resistance as 'M-l“. Yet, a great difference has been observed when the former is suspended in milk and soaked on paper. With "183 H.F.', it was found that when suspended in water and placed on glass, they resisted for thirty-three days, while on filter paper, they resisted for sixty-eight days. Suspended in milk and placed on glass, they were resistant for fifty-four days, while on filter paper, they were resistant for seventy-three days. It will be observed that up to the present time When this work is being reported, there is a difference of twenty-eight days between the resistance found for "183 F.H." and 'M—l' when both organisms are suspended in milk and allowed to dry on paper. We have been unable to find an explanation for this difference in resistance considering that both organisms had about the . same resistance to desiccation when suSpended in milk but placed on glass. From the results obtained, itis concluded that organisms causing ropiness of milk when suspended in this product or in the presence of it, no matter whether on a hard, smooth surface like glass or a soft, porous object like paper, the organisms have better chances to withstand desciccation than when they are suspended in plain water and allowed to dry. This might be explained by the fact 53 that milk, itself, affords that protection, or perhaps because the capsule, which surrounds the organism, or the gum or mucin-like substance produced by the organisms and present in the milk, gives that protection against drying. The water used to suspend the organisms could have dissolved the capsular material, the gum, or mucin—like substance, or both things together (capsule and gum material) to such an , extent as to render the organism unprotected and exposed to the direct effects of dryness. This, of course, would have as a result a much decreased resistance when suspended in water. It, also, has been shown that the two micrococci producing ropiness of milk are much more resistance to desiccation than the rod-like organism producing the same fermentation. Viability;of organisms when suspended in water: Most of the investigators, who have worked with organisms causing ropiness in milk, agree that the majority of the ropy milk outbreaks are caused either by contamination from unclean utensils or from washing or cooling water which in some way come in contact with the milk while it is being handled or processed. It, therefore, seemed to us desirable to test the viability of ropy milk organisms when suspended in water. In testing for the viability of the three organisms 54 under study, when suspended in water, we have tried to simulate the conditions which generally would be found in a dairy. One—tenth mi. of a twenty-four hour milk culture of each of the organisms under study was added to 100 cc. of sterile water in dilution bottles. There was,also, added to each bottle containing the different organisms, 0.5% of milk. Two sets of bottles were prepared: one to be kept at room temperature and another one in the ice box. The set kept at room temperature would represent the water used for the washing and rinsing of dairy equipment; while that kept in the ice box would represent the water used in the cooling tank or cooling system of a dairy. After waiting for a week, tests were started by, adding 1 cc. of water from each of the bottles in each set to different 5 cc. of sterile skim milk. After forty—eight- hours of incubation at room temperature, the 5 cc. samples: were tested for ropiness. It was found that at room temperature, the organism labelled “185 H.F.' survived only for sixteen days. The organism labelled 'M—l" survived for twenty-five days, while the one labelled 'Bac" still is living . . . fifty-four days after the experiments were started. Tests for the bottles kept in the ice box show the three organisms are still living (fifty-four days after starting the experiments). Furthermore, they resisted 35 low temperature. In one instance, the bottles in the refrigerator had their whole contents frozen; yet, when allowed to thaw, the water in each bottle gave positive results with each of the three organisms, when tested for living ropy milk organisms. It is rather interesting to note that the results Obtained in the experiments are the reverse of the ones obtained when the organisms were submitted to desiccation. One desiccation, the micrococci "M-1' and "185 H.F.' resisted better than the rod-like organism I'Bac". Suspended in water, "Bac" keeps better than ”M—1” and “183 H.F." It has been shown by this experiment that ropy milk organisms are kept viable a much longer time in water held at very low temperature than in water kept at room temperature. Cooling waters in a dairy plant could, therefore, be considered a greater menace in so far as any ropy milk outbreak is concerned, than water kept at room temperature. fiesistgnce to holding and short-time high-temperpture pasteurization: Each of the three micro-organisms under study was tested for resistance to holding and short-time hightemperature pasteurization. None of the three organisms resisted the long-time pasteurization, 143° F. for thirty ..L‘a..-’ ' 5.. I». “~- 36 minutes nor the short-time pasteurization, 1600 F. for fifteen seconds. It was decided then to measure the time required to kill the organisms at 143° F. Table 3 presents the results obtained on the different trials made. The two micrococci 'M-1' and "185 H.F." were killed at this temperature somewhere between ten to fifteen minutes. The organism labelled 'Bac' was found to be extremely sensitive to heat. It was killed in less than three minutes. Taking into consideration the two most resistant I- micro-organisms studied, it is considered that a holding time of thirty minutes offers a safe range of about fifteen minutes for these organisms to be killed. Resistance to disinfectantp: Due to the great importance of controlling organisms causing ropy milk outbreaks, it seemed desirable to learn something about the resistance of these organisms to dairy disinfectants. The resistance to disinfection was measured by means of‘the use-dilution method. Glass rods, one inch long by one-eighth of an inch in diameter with a hook at one end were dipped into a 10 cc. saline suspension. of the organisms to be tested. The saline suspension was prepared from a twenty-four hour agar slant. The glass rods impregnated with this coating of organisms were allowed to dry for half an 37 hour before the trials were started. Then, they were dipped into the disinfecting solution for a given time-interval, after which time, the rods were rinsed in 10 cc. of plain sterile water for one minute. The rods, taken from the rinsing water, were then dropped into tubes containing 5 cc. of sterile skim milk. The tubes, which after forty-eight hours of incubation at 37° C. showed ropiness of the milk sample were considered positive. Only two organisms were tested: the one labelled 'Bac', as a representative of the rod-like organisms, and 'M—1' '*w as a representative for micrococci. ”M—l” was chosen for the tests rather than the "185 H.F.“ because as it has been shown, 'M—l' possesses a little more resistance to desiccation or pasteurization temperature than "183 H.F.". Besides, the organism "M-l", as explained on preceding pages, produces a more definite and longer-lasting ropiness in milk than micrococcus "183 H.F.' Three common and frequently recommended disinfectants for use in the dairy industry were used for the trials. The three disinfectants used were: commercial sodium Jhydroxide (lye), chlorine (HTH—ls), and roccal. Most of the trials were made in quadruplicate. All the tests were run at a 25° C. _ 0.50 temperature. Tables 5,6 and 7 present the results obtained during these trials. 38 Ly_: Was tested in three concentrations: 1%, 0.5%, and 0.25% solutions with 2% milk added. The addition of a small amount of milk was considered advisable since it seems most probable under dairy conditions that in washing and sanitarizing dairy utensils, there would always be present in the disinfecting solution small amounts of milk. It also i has been proven by McCulloch (l4) and Tilley and Schaffer (17) that the effeciency of sodium hydroxide is but very slightly changed by the addition of moderate amounts of '51-'11 t. .w 13-. organic matter. Micrococcus 'M-l" with a 0.5% solution resisted an exposure of five minutes. Since we were interested in getting disinfection in rather a short time, a 0.25% was not tested for this organism. Even a 0.5% solution seems undesirable if time is to be saved. A 1% solution killed the organism in four minutes but not in three minutes. For the control of micrococci causing ropy milk outbreaks, lye does not appear to recommendable since too strong a solution would have to be used if time is to be saved. A 1% solution is considered too strong a solution to handle conveniently. The organism labelled 'Bac' proved to be very sensitive to lye. See Table 5. Solutions of 1%, 0.5%, and 0.25% lye killed the organisms in less than thirty seconds. 39 Since a 0.25% solution was very effective against this organism in a very short period of time, there would be little difficulty in controlling outbreaks caused by this organism if lye is used as disinfectant. A 0.25% solution of lye is considered very easy to handle and very economical. CHLORINE: Was tested in three dilutions: 200 p.p.m., 100 p.p.m., and 50 p.p.m. of available chlorine. The available chlorine in each of these dilutions was tested in each and every case Just before starting the experiments. The compound used in these chlorine tests was a freshly-opened can of HTH-l5. Again, 2% milk was added to the different dilutions for the same reasons mentioned above and while working with lye. 'M-1' and ”Bac" were not killed in nine minutes exposure to any of these dilutions. See Table 6. Due to the results obtained when 2% milk was added, it was decided to try the compound without adding any milk. The results obtained by doing this were highly satisfactory. 'See Table 6. Using a dilution of 200 p.p.m., ”M—l” was killed in two minutes but not in one minute. With the dilartion of 100 p.p.m. available chlorine, one series of inrbes showed the killing of the organism in one minute, but in the same series of tubes, one test tube showed a positive 4O reaction at three minutes exposure. In a second series of test tubes, the organism was killed in two minutes but not in one minute. In a third and fourth series of test tubes, the organisms were killed in three minutes but not in two minutes. It would be safe to state that the organism would be killed in three minutes but not in two minutes, although in the first series of tubes, there was a positive case at three minutes exposure. .5] ‘.‘I“— .. With a dilution of 50 p.p.m. available chlorine, three series of tubes shows killing of the organisms in five minutes but not in three minutes. A fourth series of tubes showed a positive tube at five minutes although the tube at three minutes was negative. It would be safe to state that 50 p.p.m. available chlorine would kill the organisms in about five minutes but not in three minutes. The organism labelled ”Bac" was again found to be less resistant than the micrococcus "M—l" when submitted to the action of chlorine since 200 p.p.m. available chlorine killed "Bac" in thirty seconds but usually not in fifteen seconds. In one instance; it was killed in fifteen seconds. See Table 6. 100 p.p.m. available chlorine killed the organism in thirty seconds but not in fifteen seconds. In one instance, the organism survived thirty seconds, but was killed in one minute. It was found that 50 p.p.m. available 41 chlorine killed the organisms in three minutes but not in one minute. In one instance, it survived three minutes but was killed in five minutes. It has been found that chlorine is a very satisfactory disinfectant to be used intme control of ropy milk outbreaks. It is economical and rapidin its killing action. "32:5“ 0‘, D. The efficiency of chlorine is tremendously reduced by small amounts of milk. Therefore, it is suggested that in disinfecting against organisms causing ropiness and which might be found on the surface of the dairy equipment and a utensils; the latter be washed and rinsed in water before applying the chlorine solution. According to the results obtained, and when dealing with organisms causing ropiness, a dilution of 200 p.p.m. available chlorine would be one offering a good killing action in a reasonably short time i.e., two to three minutes. It should be remembered that chlorine solutions are unstable, therefore, the solutions should be freshly prepared before use so as to provide for the proper amount of available chlorine in the disinfecting solution. JROCCAL: A quaternary ammonium product was also chosen for the tests since these compounds are being used and recommended as good disinfectants for all kinds of utensils axui glassware. Roccal in the presence of a small amount of milk was also studied. As with chlorine, the results were 42 not satisfactory. Dilutions of 1:4000 and 1:5000 with 2% milk added did not give favorable results. See Table 7. It will be observed that the results are ”spotty” and variable. In this respect, the data are in agreement with those obtained by Miss Leavitt (11) in her studies about these products (quaternary ammonium compounds) tested by the use-dilution method with Staphylococcus aureus as the t test organism. Better results were obtained when the compound was i tested without adding any milk. See Table 7. But, still the L results present much variation in the killing time. A stronger solution (1:2000) was tested with the most resistant of the two micro-organisms, the micrococcus 'M—l", but again variation in the killing time was obtained. Due to these variations, no definite recommendations can be made on the use of roccal. From the results obtained during these experiments with commercial sodium hydroxide (lye), chlorine (HTH—l5), and roccal, it is concluded that chlorine (HTH-l5) is a highly desirable chemical for disinfection against organisms causing ropy milk. I. It has been found that milk coming into contact or xnixed with a solution of chlorine or roccal will reduce the activity of the solutions to such a degree as to render 'themtundependable. Therefore, the presence of small amounts (yr milk (2%) should be avoided when disinfecting for the 43 control of ropy milk organisms. The results obtained, also, indicate that in the control of rcpy milk outbreaks as well as in the disinfection of utensils, the detection of the type of organism causing the trouble might be helpful since there seems to exist differences in the viability and resistance among the various types of organisms causing ropiness in milk. Among the micro-organisms causing ropy milk, micrococci appear to be more resistant to disinfection than the rod-like, non—spore forming organisms. Population densities and_growth rates: Reference made at. throughout theliterature, concerning the most favorable temperature for reproduction and the growth of the micro- organisms causing ropiness in milk. A study was made on the pOpulations densities and growth rates for the three organisms when grown at 37° C. and room temperature. Table 4 presents the population densities of the three organisms, 'Bac“, “M-l', and ”183 H.F.' at different intervals of time when incubated at 37° C. and room temperature (27°- 50° 0.). From the plate counts shown in this Table as well as the growth curves in Figures 1, 2, and 3, it will be noticed that in all three cases the growth rate is more rapid at 57° C. than at room temperature (27° - 30°C.). In fact, the generation time calculated for the organism 'Bac" during the most active period of production (two to twelve hours) fluctuated between seventeen to thirty—two minutes when 44 incubated at 37° C. while at room temperature (27° - 50° 0.), during the same period of time, it fluctuated between twenty-one to thirty-three minutes. For “M-l',the generation time during the most active period of reproduction (two to eight hours) fluctuated between twelve to twenty-seven minutes at 37° C. and for the same period at room temperature fluctuated between twenty to twenty-five minutes. For "183 H.F.' the same observations were made. In the two to eight- hour period, the fluctuation in generation time was between fifteen to thirty-one minutes at 37° C. For the same a period of time at room temperature, the fluctuation in the generation time was between twenty-four to twenty-seven minutes. It is interesting to note that, while the three organisms spent so much energy reproducing faster at 37° C. than at room temperature, the population densities at 37° 0. never reached the peak which they reached at room temperature, where reproduction was slower. It will, also, be observed Lnthe growth curves of Figures 1, 2, and 3 that with the organisms labelled 'Bac', the highest population densities, whether at 37° C. or room temperature (27° — 30° 0.), were found to occur a few hours before a twenty- four hour period of incubation. With the two micrococci 'M-l' and '183 H.F." the population densities at 37° c. and room temperatures continued to increase until a maximum was reached at twenty-four hours of incubation. 45 From the observations made, it is concluded that as far as pOpulation densities are concerned, room temperatures (270 - 30° 0.) should be considered a highly favorable temperature for micro-organisms causing ropiness in milk, while considering speed of reproduction or multiplication, a 37° 6. should be considered better for the micro-organisms. rkx'ufi‘ 46 DISCUSSION Throughout the course of this work, the identification of the three micro-organisms isolated in pure culture has been difficult due to certain variations and differences from the cultural characteristics and biochemical reactions of the organisms already reported and to which we think these I are related. The organism labelled "Bac' was found to start i producing gas after several daily transfers in milk at E room temperature (270 - 30° 0.). Although when freshly isolated, it produced only acid and no gas, the fact that it did produce gas after several transfers, made us suspicious of a possible relation to the Escherichia-Aerobacter group. We have found in the literature that atypical or intermediate form of this group (Escherichia-Aerobacter) might in some occasions produce ropiness of milk. Topley and Wilson (18) state that besides a 29;; group on the one hand and an aerogenes—cloacae group on the other, "further work revealed the occurrence of a third group of strains possessing properties intermediate between < those of the two main groups. This group is,as yet, not completely defined, and is therefore most conveniently referred to as the 'intermediate' group. The 'intermediate' group is differentiated (1) from B. coli mainly in being citrate positive, in generally failing to form indol and by 47 their inability to produce gas in MacConkey's medium incubated at 44° 0.; (2) from B. aerogenes in being methyl red positive, Voges—Proskauer negative. . . . . . . . . ." Jordan and Burrows (10) in discussing Escherichia ppli, state that "atypical strains are not infrequently found". They, also, state that strains of Bact. coli might be isolated, which do not immediately ferment lactose . . ." Discussing the ”Colon-Aerggenpp intermediates" they explain: "these bacteria present extremes which are connected by a variety of intergrading forms. 0n the basis of the IM VIC tests, sixteen combinations are possible . . . . . . . . . . The allocation of these intermediate forms to pplg_or aerogenes is, obviously, a difficult matter and probably not advisable". In the review of literature, reports were presented indicating the possibility that members of the Escherichia- Aerbacter group could produce rcpiness of milk. Esch. coli var. neapolitagg is one of those intermediate strains that have been reported as producing rcpiness of milk. Saddler and Middlemass (16) have reported an atypical Esch. coli var. negpolitana producing ropiness of milk. What is stated about Each. coli could, also, be said about Aerob. aerogenes. Strains of latter organism could produce ropiness of milk. We have brought into discussion the words and works of Topley and Wilson, Jordan and Burrows, and other investigator 19 have 1801( atypical or group which The 0 wwapsulated methyl red ; test. After gas, Undoub characterie being a ”I therefore, and labell type of or 3WD Whic 'The found to 1: fr w ("5'1" and W me mannose, J ce110biose 1M1: sugars. investigators to support our conclusion that the organism we have isolated and labelled "Bac” is most probably, and atypical or intermediate form of the Escherichia-Aerobacter group which produces ropiness in milk. The organism we have studied is a Gram-negative, encapsulated organism which did not produce indol, gave a methyl red positive test, and a Voges-Proskauer negative test. After a few transfers in milk, it started producing gas, Undoubtedly, these variations in properties and characteristics eliminate the possibility of the organism being a typical Esch. coli or Aerob. aerogengp. It is, therefore, concluded that the organism we have isolated and labelled “Bac' is most probably an atypical or intermediate type of organism belonging to the Escherichia-Aerobacter group which produces ropiness in milk. The organisms labelled "M-1" and "183 H.F.“ have been found to be most closely associated to Micrococcus freudeureichii and Micrococcus cremoris-viscosi since both ("M-l" and ”183 H.F.“) are unpigmented, gelatin-liquefying micrococci. Yet, the organism 'M-l' differs from !.cremoris- viscosi mainly in its ability to ferment glucose, fructose, mannose, lactose, maltose, and trehalose. Galactose, cellobiose and glycerol were slightly fermented. M. cremoris- viscosi is reported by Bergey (3) as having no action on sugars. 4! “'55. 49 The organism ”M-l“ differs mainly in two properties from M. freudeureichii. First, 'M-l" gave a doubtful result as to the production of ammonia from peptone and according to Bergey (3) M. freudenreichii produces ammonia from peptone. Second, according to Buchanan and Hammer (5), this latter organism ferments lactose and sucrose and in this respect the organism ("M—1') also differs from M. freudeureichii, since it fermented dextrose and lactrose but gave doubtful results with sucrose. ‘ It should be mentioned that this organism resembles in some respects the one reported and described more recently by Prouty (15). The organism described by this investigator differs from ours in that his was Gramfvariable (staining) produced rapid liquefaction of gelatin, ammonia from peptone and had thermoduric properties. Organism ”183 H.F.' also resembles in some respects M. cremgris—viscosi. Yet, we think the organism is more closely related to M. freudeurichii than to M. cremoris-viscgpi. The production of ammonia from peptone; fermentation of dextrose, lactose, and sucrose; and the fact that "183 H.F.” produces ropiness mostly in the surface layers of milk in test tubes, while the milk below, is normal in consistency and still liquid, relates it more to M. freudenreichii than to M. cremoris—viscosi. This last property mentioned is one ascribed by Buchanan and Hammer (5) to M. freudeureichii. 50 Due to these differences in properties and reactions, a definite identification of the two micrococci 'M-1' and "183 H.F." is not established. A discussion on other observations made with the three organisms studied is presented with the results obtained under the item: OTHER OBSERVATIONS. 51 SUMMARY 1. The three micro-organisms isolated and studied in the laboratory are shown to produce ropiness of milk. 2. The organism labelled "Bac" is most probably related to the Eschericgiijerobacter group. It is a Gram-negative encapsulated organism which does not produce indol, gives a Methyl red positive test, and Voges-Proskauer negative test. After a few transfers in milk, the organism produced acid and gas from lactose. The organism is probably an atypical or intermediate form of the EggherichggsAerobgpter group. 3. Micrococci ”M-1" and "183 H.F." were found to be related to Micrococcus freudenreichii and Micrococcup cremoris-viscosi. Yet, due to certain properties and differences from the latter two mentioned, a definite identification is not established. 4. The two micrococci studied were found to be, in general, more resistant to desiccation, pasteurization temperature, and disinfectants than the red-like encapsulated organism "Bac." 5. Suspended in water, the organism "Bac'I was more resistant than the two micrococci "M-1" and “183 H.F.”, when the waters in which they were suspended were kept at room temperature 52 (27° - 30° C.). When suspended in water and kept at low temperatures (ice box), the three organisms were viable for a much longer time than at room temperature. 6. Commercial lye, chlorine, and roccal might be good disinfectants against ropy milk organisms if used under certain conditions and dilutions. Yet, chlorine seems to be ~ a more desirable chemical againstfgalk organisms due to its If rapid action and low price. The solution of chlorine should be freshly prepared and any contact or mixture with milk should be avoided. As far as population densities were concerned, room temperature (27° to 30° C.) was highly favorable for rcpy milk organisms, but insofar as the speed of reproduction was concerned, the 37° 0. temperature was preferable. (1) (2) (3) (4) (5) (6) ('7) (8) LITERATURE CITED Associates of L.A.Rogers. Fundamentals of Dairy Science - 2nd. Edition pp. 357-360. Reinhold Publishing Corp. New York, N.Y. 1935 Beck, E.C. and Chase, F.E. Some Experiments Dealingiwith Ropy Milk - Scientific Agriculture 19 (I): 48-54. 1938 Bergey, David H. Manual of Determinative Bacteriology - 5th. Edition pp. 236-261. Williams and Wilkins, Baltimore, Md. 1939. Bergey, David H. Manual of_§eterminative Bacteriology - 1st. Edition pp. 204. Williams and Wilkins, Baltimore, Md. 1923. Buchanan R.E. and Hammer, B.W. glimy and Ropy Milk - Iowa Agr. Exp. Sta. Research Bulletin No. 22. 1915. Davis, J.G. Ro Milk — J. Ministry of Agriculture (Great Britain) 42 (7): 658-662. 1935. Hammer, B.W. Dairy Bacteriology - 2nd. Edition pp. 54—63. John Wiley and Sons, Inc. New York, N.Y. 1938 Hammer, B.W. and Cordes, W.A. AniUnusual Outbreak of Ropy Milk - J. Dairy Science 3 (4): 291-299. 1920. (9) (10) (11) (12) (13) (14). (15) (16) (17) 54 Harding, H. A. and Prucha, M. J. Is Ropy Milk A More Serious Dairy Trouble - J. Dairy Science 3(6): 502-521. 1920 Jordan, E. 0. and Burrows, W. Textbook of Bacteriology - 14th. Edition revised pp. 373-376. W. B. Saunders Co., Philadelphia and London. 1945. Leavitt, Anita Harriet Comparative Study of the Use-Dilution Method and the F. D. A. Phenol Coefficient Method of Testing Vetering:y Disinfectants - p. 15. Thesis presented for M. S. Degree at Michigan State College. 1946. Long, H. F. and Hammer, B. W. Studies on Alcaligenes viscosus - Iowa State College J. Science 10 (3): 261-265. 1936. f Macy, Harold A Ropy Milk Organism Isolated from the Finnish Piima or Fiili - J. Dairy Science 6 (2):127-l30. 1923. McCulloch, Ernest C. The Germicidal Efficienc of Sodium H droxide - J. Bacteriology 25 (5): 469-393. 1933. Prouty, C. C. A ROpy Milk Outbreak Caused by a Thermoduric Micrococcus - J. Milk Technology 6 (5): 263-265. I943. Saddler, Wilfred and Middlemass, J. D. 0011 Types and Ropy Milk - A case of rcpy milk caused by an a-typical Escherichia neapolita_g - Scientific Agriculture 6 (5): 297- 302. 1925- 26. Tilley, F. W. and Schaffer, J. H. Germicidal Efficiency of Sodium Hydroxide Sodium Carbonate nd Trisodium Phos hate - J. Agric. Res. 42 (2): 93-106. 1931. 55 (18) Topley, W. W. C. and Wilson, G. S. The Principles of Bacteriology and Immunity_- 2nd. Edition pp. 523-26, 570-71. The Williams and Wilkins 00., Baltimore, Md. 1936 (19) Ward, Archibald R. The Wide Distribution of Ropy Milk Organismsjig CityMilk Supplies - J. Dairy Science 6 (6): 616-525. 1925. obwpfimom cowpwunmanmu obflpom I. .pom I + noflpaunmsnuo pamfiaa II .Hm aseppsoe I I coauwvcmanom aanHm hum» I. .Hm.> oPprMoq a I "ovoz I. I I. I II It. on I. H. II 4 Iv I+ on I? I? .H@ I? 4. II I H onoaH N.$ l I II II I u" .Hm I H I. I... .+ I+ + IT .Hm + + I. I H. OH I. I I II I. If Hm I Hm I + I? + m ... If «Hm ... If I I H ohm-1H NH I I I I II Ha I I I. I A +I + I." 4 5. 1mm + + I I H ..m.m mm I I. I I I + .3 I I I E + I. «I I I. Am I I I I a mg I I I I I I... am I am I + 4. + .Hm I ... “fin. I If I I « OHI.Q.IH L I. I I H II .Hm I. H. I + ... If I? ... I? I 4. 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