THE {BROW‘I‘H {3‘9 STAPHYLGCOCCUS AUREUS 2N MILK AND {1‘5 DE’E‘EC‘i'EG'Q- 3‘1” 234' SEROLOGEN, ME‘E‘893 '2'th for 9h. {23¢ng a? M. 3. MICHECSAN S‘E‘A‘IE UNIVERSW! Chasm; 5. 2311933 €962 THE GROWTH OF STAPHYLOCQCCUS AUREUS IN MILK AND ITS DETECTION BY A SEROLOGICAL METHOD By Charles G. Pheil AN ABSTRACT Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1962 2 / :l‘. _ .-/.~) fl. Vs.-.“- Approved //7;b%‘g/gggaléé:aaixarr \\~ ABSTRACT Whole raw milk was treated with rennin and calcium chloride to obtain clear milk whey for serological studies. Following removal of the curd, the whey was used to support the growth of Staphylococcus aureus PL-l2 and 8-42. The influence of the rennin and calcium chloride on the culture develOpment of S. aureus was studied. The effect of the initial level of inoculum, pH of the whey, and the tempera- ture of incubation on the growth of the culture was also studied; mixed culture studies were undertaken to determine the effect of other organisms on the growth of S, aureus. A clear whey which supported the growth of S. aureus was obtained by treating raw whole milk with rennin and calcium chloride. The initial level of cells did not appreci- ably effect the maximum concentration of cells providing the whey was adjusted to pH 7.0 and incubation was undertaken at 35 C. The presence of other bacteria commonly found in raw milk did not appreciably influence the development of the Staphylococcus cultures. In whey, S. aureus PL-12 and 8-42 reached cell concentra- tions suitable for agglutination studies. The immune fluids were prepared from rabbit serum and ascitic fluid from mice. Agglutination was noted, in all cases, when each immune fluid was tested at a dilution of 1:5. THE GROWTH OF STAPHYLOCOCCUS AUREUS IN MILK AND ITS DETECTION BY A SEROLOGICAL METHOD by Charles G. Pheil A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1962 3.. A’ ' A ! ,1/ .1. l X n, /r; 3 ACKNOWLEDGMENTS The author wishes to express his sincere gratitude to Dr. 0. W. Kaufmann for his guidance and patience during the course of the study and during the preparation of this manuscript. TABLE OF CONTENTS 2253 INTRODUCTION ....................................... 1 MATERIAL AND METHODS Preparation of immune fluids ................... 3 Factors affecting the growth of S. aureus in whey - ...................... I--:::::: ....... 6 Agglutination procedure in whey as .............. 9 RESULTS Immune fluids ................................. 10 Growth of S. aureus in pure culture ----------- 12 Growth of S. aureus in mixed culture ---------- 23 DISCUSSION ......................................... 30 SUMMARY -------------------------------------------- 33 10 ll TABLES A comparison of the agglutinating antibody titers in blood sera and ascitic fluid. The effect of rennin on the growth of S. aureus PL-l2 in whey. The effect of calcium chloride on the growth of S. aureus PL-12 in milk. The effect of rennin, CaCl and rennin plus CaCl on the time required for clarification of w ole raw milk at 37 C. Development of S. aureus PL-12 in whey at various pH leveIs. The effect of temperature on the growth of S. aureus PL-l2 in whey. Comparison of growth of S. aureus PL-12 in whey and brain-heart infusion Broth at various initial cell levels. Development of S. aureus PL-12 in the presence of other bacterIa in wEey. Deve10pment of S. aureus PL-12 in the presence of other bacterIa In brain-heart infusion broth. Agglutination with S. aureus PL-l2 in whey in combination with varIous organisms. Agglutination with S. aureus S-AZ in whey in combination with various organisms. Page 11 13 1h 16 17 19 21 25 26 28 29 FIGURES The effect of pH on the growth of S. aureus PL-lZ in whey. The effect of temperature on the growth of ‘S. aureus PL-l2 in milk. The growth of S. aureus PL-l2 in whey and brain-heart inTusion broth at different levels of inoculum. Page 18 20 22 INTRODUCTION Staphylococci in milk taken directly from a bovine udder showing clinical symptoms of infectious mastitis can be readily detected by several plating procedures since the organisms are present in large numbers, (Chapman 1945; GilleSpie and Alder 1952; Slanetz and Bartley 1953; Clark and Nelson 1961 and Finegold and Sweeney 1961). Detection by plating is more difficult, however, if the organisms are present in small numbers as might occur at the onset of the disease or if the addition of other milk has reduced the level of cells per ml to the point that they cannot be readily detected. SerolOgical procedures have been used to aid in differ- entiating pathogenic from non-pathogenic strains of staphy- lococci, (Kolle and Otto 1902; Hucker 1926; Kourilsky and Mercier 1942; Blair and Hallman 1936 and Oeding 1952). In most instances the serological reaction was carried out in saline. Serological procedures have also been used to detect _SgaphleCOccus antitoxins in blood, milk and colostrum, (Minett 1937 and Lasmanis and Spencer l95h). These workers treated milk with rennet and tested the clear suspension for antitoxins; the concentration of rennet was not reported. Serological procedures have been employed to aid in the detection of infections based on the fact that antibodies resulting from eXposure to microorganisms can be detected. I The antigen in this case is prepared by culturing the organism.under laboratory conditions. Since antibodies may remain for long periods, some difficulty may be encountered in determining whether the infection is one that is active or is one that has subsided. This technique may also be unsuit- able for detecting early infections since few if any anti- bodies are present. Gruber and Durham (1896) were among the first to use an agglutination test to detect the presence of specific bacteria in liquid preparations by reacting these samples with specific antisera. The current study was undertaken to determine some of the factors affecting the growth of Staphylococcus aureus in raw milk using a modified agglutination procedure. MATERIAL AND METHODS Preparation of immune fluids: Staphylococcus aureus PL-l2 associated with bovine mastitis and 8-42 an entertoxin producing culture wemeused to stimulate antibody production in rabbits and mice. The antigen used to prepare the immune fluids was prepared by streaking each culture onto 20 plates of brain-heart infusion agar and incubating at 35 C. After L8 hrs, the growth was harvested in 10 ml of sterile phOSphate buffered saline at pH 7.0 and centrifuged at 800 g for 10 min to remove clumps of agar. The supernatent fluid containing the organisms was decanted and centrifuged at 15,000 g for 10 min. The cells were washed three times in phosphate buffered saline. The con- centration of the bacterial cells used for injection was estimated using a standard curve made from turbidmetric measurements at 640 mu. The bacterial cells were heated at 60 C for 20 min on two successive days. The viable count after heating was determined by plating two ml of the sample; in all cases the estimated count was less than one organism per two m1. In preparing antiserum, three New Zeeland White rabbits weighing approximately 1.7 kg were injected intravenously with a heat killed culture containing 1 x 109 cells per ml as follows: first day—0.5 ml, third day—0.75 ml, sixth day-1.0 ml, ninth day-1.25 ml, twelfth day-1.5 ml and fifteenth day—2.0 ml. Five days following the last injection, the animals were bled by cardiac puncture, and the blood was pooled. Antiserum was also prepared by subcutaneoumy’ inoculating three Dutch Belt rabbits weighing 0.8-1.3 kg as follows: 0.1 ml on the first, third, sixth, ninth and twelfth days and 0.5 ml on the fifteenth and eighteenth days. Seven days following the last injection, the animals were bled by cardiac puncture, and the blood was pooled. All whole blood was held at 37 C for 30 min to hasten separation. The serum was decanted and centrifuged at approximately 15,000 g for 30 min at 3 C; Merthiolate (1:10,000) was added as a preservative. Antibodies were also prepared from ascitic fluid (Lieberman 22 g; 1960) using white Swiss mice weighing approximately 18 gms. To stimulate antibody production, five animals were first injected subcutaneously with 0.1 ml of adjuvantoantigen mixture at the site of the axillary and inguinal lymph nodes. This mixture consisted of equal parts of the cell suspension, containing 1 x 109 heat killed cells per m1, and the complete Freund adjuvant (Freund and McDermatt 19h2). The adjuvant was prepared by mixing one part of Mannide Monooleate1 with 5.5 parts of Bayol F2. Following the subcutaneous injection, mice were inoculated intra- peritoneally with 0.L ml of the adjuvant-antigen mixture on the 15th, 18th, 2lst and 23rd days. To obtain higher titers a booster injection was made intraperitoneally on the 28th day. Mice having distended abdomens, typical of ascites, were tapped three days after the last injection with sterile 17 gauge needles and the fluid collected in sterile tubes. To separate the antibody fraction from the fatty materials and leukocytes, the samples were frozen and thawed twice. The clear portion containing the antibody was decanted into sterile tubes and stored at 3 C; Merthiolate (l;10,000) was added as a preservative. The titer of each immune fluid was determined using a tube agglutination procedure. A washed cell suspension of each S. aureus culture, grown on brain-heart infusion agar,was suspended in 0.005 M phosphate buffered saline at pH 7.0 and used at the standard antigen. The Optical density of the cell suSpension was 0.1 at 6h0 mu; this yielded a concentration of approximately 108 cells per ml. Sterile phOSphate buffered saline was used to prepare a two-fold 1Obtained from the Atlas Powder 00., New Castle, Delaware. 2Obtained from Esso, Standard Oil Co., New Jersey. serial dilution of each fluid starting at 1:10; one ml of antigen was added to each tube. The tubes were shaken and incubated for 12-18 hrs in a water bath at 10 C. The highest dilution showing agglutination was considered the end-point; the end-point was recorded as the reciprocal of the dilution of the immune fluid before the addition of the antigen. Factors affecting the growth of S. aureus in whgy: To obtain a solution of sufficient clarity for this study, the milk was treated with U.S.P. grade C rennin. Since Tewes and SchlosSberger (1952) noted that rennin exerted a bacteriostatic effect in milk, the effect of rennin on the growth of S. aureus in milk was studied. Raw whole milk was sterilized at 121 C for 10 min and rennin .added to provide a concentration of 10.0, 1.0 and 0.1 mg of rennin per m1 of milk; controls without rennin were also included. Duplicate tubes at each concentration were inoculated with S. aureus to provide an initial concentration of approx- imately 10 cells per ml. Incubation was at 35 C; the develop- ment of the culture was followed by plating periodically on tellurite-glycine agar until a maximum cell level was obtained. The effect of calcium chloride on the growth of S. aureus was also studied. Milk samples containing 11.1, 6.66, h.hh and 2.22 mg of calcium chloride per ml of milk were inoculated with S. aureus to contain approximately 10 cells per ml; calcium chloride was omitted in the control samples. Culture development at 35 C was followed as above. The growth of S. aureus in whole raw milk containing 10 mg of rennin per m1 and 2.22 mg of calcium chloride per ml was also followed as above. The effect of various concentrations of rennin and calcium chloride on the time required to obtain a clear whey was studied. The influence of pH and temperature on the growth of S. aureus was investigated to determine the minimum time required to obtain a cell population suitable for use as antigen in the modified agglutination test. In these trials 100 ml of milk, inoculated to contain 50-100 organisms per ml, were clarified by adding one mg of rennin per m1 of milk andZLZEmg of calcium chloride per ml of milk. Following clarification of the milk, the whey was adjusted to pH 6.5, 7.0 and 8.0 with 0.5 N sodium hydroxide and the growth of S. aureus was followed using plate count agar. Temperatures of 36 and 45 C have been reported as optimum temperatures for the growth of S. aureus by Breed 22‘31 (1957) and Raj and Liston (1961), respectively. In view of this, the effect of temperature on the growth of S. aureus PL—l2 was studied in milk inoculated with ~8- approximately 100 cells per m1. Rennin and calcium chloride were added and the whey adjusted to pH 7.0 after clarifi- cation. Culture development was studied at 25, 35, L0 and #5 C to determine the time required to obtain a maximum number of cells. The effect of initial inoculum was determined by inoculating milk so as to contain 10,000 and 10 cells of S. aureus per ml. Rennin and calcium chloride were added at a level of one mg per m1 and 2.22 mg per ml, respectively. Culture development at pH 7.0 and 35 C was determined by plating in triplicate using plate count agar. For compara- tive purposes, growth studies were made with S. aureus in brain-heart infusion broth. To determine the effect of other bacteria on growth, ‘S. aureus PL-l2 and S—h2 was grown in the presence of Strepto- coccus lactis, Bacillus globigii, Pseudomonas fluorescens, Micrococcus §E°: Closridium perfringens, Escherichia coli, Streptococcus agalactiae, Lactobacillus casei, and Aerobacter aerogenes. One hundred m1 of milk were inoculated to contain approximately 10 cells per m1 of each organism; S. lactis was also added at an initial level of 10,000 cells per ml. The concentration of S. aureus was adjusted to contain approximately 10 cells per m1. Immediately following inocula- tion, rennin and calcium chloride were added so as to provide a concentrationcxeremg and 2.22 mg per m1 of milk, reSpectively. After incubation at 37 C for 30 min, the curd was removed by centrifugation at 800 g for 10 min. The clear whey was decanted and adjusted to pH 7.0. Follow- ing incubation at 35 C, a total bacterial count was made in each instance using plate count agar; the S. aureus count was determined using modified tellurite-glycine agar as described by Clark and Nelson (1961). Agglutinationgprocedure in whey: The antigen generally used in an agglutination test is obtained by harvesting bacterial growth from solid or liquid media and suspending it in saline to provide the desired concentration. The experimental bacterial antigen lused in this study was grown in whey and the agglutination test was undertaken using the whey directly. To remove leukocytes, casein flocs and other non-bacterial particulate material, the whey was clarified using a sintered glass filter of medium porosity. Two-fold serial dilutions of immune rabbit serum or ascitic fluid were made in 0.005 M phosphate buffered saline (0.85% NaCl) at pH 7.0; one ml of the diluted immune fluid was mixed with one ml of antigen. Incubation was undertaken in a water bath at 10 0, since preliminary trials indicated slightly better agglutination at this temperature, for at least 24 hrs. .10- RESULTS Immune fluids: The titers of the immune fluids are given in table 1. Since the titer of the various fluids varied, all fluids were standardized to give agglutination in the 1:320 dilution as the end~point. Non-specific interference due to antigenic substances in the whey was determined using sterile whey and immune fluids. With undiluted unheated immune blood serum, a precipitate was noted; after heating the serum at 56 C for 30 min and diluting 1:5 in phosphate buffered saline, no precipitate was noted. To minimize this interference, all serum was heated and diluted 1:5 or greater. Control serum collected from each animal prior to immunization showed no agglutination at the 1:5 dilution. Since ascitic fluid does not contain S. aureus antibodies as a result of a previous exposure, this fluid was also used as a source of Specific antibody for Staphylococcus in per- forming the agglutination test. Ascitic fluid was not heated since no precipitation was detected with undiluted fluid. The failure to observe agglutination with sterile whey and ascitic fluid diluted 1:5 indicated the absence of substances in the whey capable of reacting with antibody for S. aureus. .11 .- TABLE 1. A comparison of the agglutinating antibody titers in blood sera and ascitic fluid. Antibody Route of Source Animal Inoculation New Zeeland Intravenous Blood Serum Rabbits Intravenous Dutch Belt Subcutanteous Rabbits Subcutanteous Ascitic Fluid White Mice Intraperiton- eal Antigen Titer PL-12 320 S-h2 320 PL-12 6&0 S—42 320 PL-12 1280 S-h2 640 (a) These values represent the reciprocal of the highest immune fluid dilution causing agglutination. -12- Growth of S. aureus in pure culture: The effect of the composition and pH of the medium, the temperature of incubation and the initial level of cells on the development of S, aureus PL—12 is shown in tables 2, 3, 5, 6 and 7 and figures 1, 2 and 3. To study the effect of composition on the growth of S. aureus, sterile whole raw milk was clarified with rennin and calcium chloride. The data in table 2 indicate no significant difference in the cell levels obtained in whey after the milk was treated with 10.0, 1.0 or 0.1 mg of rennin per ml. The effect of calcium chloride on the growth of S, aureus in the milk medium is given in table 3. A slight decrease in total count was noted as the concentration of calcium chloride increased. In milk containing 11.1 mg of calcium chloride per ml and one mg of rennin per ml of milk, a population of approximately 47 x 107 cells per ml was obtained after 24 hrs incubation at 35 C. In milk containing 11.1 mg of calcium.chloride per m1, a level of 88 x 106 cells per ml was obtained. The increase observed in the former may indicate that the inhibitory activity of calcium chloride was lessened by the removal of the free calcium chloride in the formation of a rennin-calcium chloride complex. If the complexing action of rennin removes free calcium -13- .mcofimeHHmoa 03p so comma HE pom mHHoo mo nomads mmmao>4 .m ooo.ooo.oom.a ooo.ooo.oom ooo.ooo.omo ooo.ooo.ooo.H em ooo.omm.H ooo.OOm.m ooo.oos ooo.osm.a NH oom.a com 00H own A 0mg Am mm was 0 I): J as: causes 02 H.o o.H o.oH asap Nas\wev cofipmhpsoocoo cacsom cofipmpsodH I .smas as NH-qm muonsw .m mo negonm amp :0 madden 90 pomwwo was am mqm