‘ IWIIH W x M L | i WWI M 1 145 804 .THS EVALUATION OF CETREC AGED A3 A FOSSIBLE WEAfix/xENT FQR. STgfiPTQCSCCE-fi 34' “—‘TWS Thesis for flu Degree o§ M. S. MECEESAE“? STATE UNE‘5EE‘QSZTY Dineshwar Prasad Sinha 1964; THESIS LIBRARY Michigan State University ABSTRACT EVALUATION OF CITRIC ACID AS A POSSIBLE TREATMENT FOR STREPTOCOCCIC MASTITIS by Dineshwar Prasad Sinha Since it was shown by Huddleson that citric acid in blood prevented the growth of é;geptgggggggflggglggtigg, a study was undertaken to determine if citric acid would also exert a growth inhibitory action to‘étggpgggggggg W in milk. Initially a modified skim milk was used for in 11.29. work, but as the study advanced an attempt was made to ob- tain milk resembling as closely as practical the natural product. A technique was devised for collecting the milk aseptically and dispensing it into sterile test tubes with- out contamination. This gave a satisfactory quality of milk for in vigo work. The growth rate of Streptococcus ggglggtigg,was studied in skim milk, heat sterilized milk and nonsterilized milk in the presence of sodium citrate, citric acid, and a mixture of both. There was no significant growth inhibition of‘étggpg jggagzagg agalagtiae in milk when sodium citrate was added. When citric acid was added to the milk in a concentration of 0.8% or more, the pH of the milk dropped from 6.64 to Dineshwar Prasad Sinha 4.08 or less. Two experiments were set up to determine if the growth inhibition of §§ggpt9§2§ggg,§g§l§§t;§g in citrated milk (milk containing sodium citrate or citric acid) was due to the absence of magnesium ions, required for growth, or due to the low pH (acidity) of the media. For the first ex- periment excessive amounts of magnesium chloride were added to the citrated milk to provide sufficient magnesium ions for the growth of Streptoggggus agalaggi g. The addition of magnesium chloride to the citrated milk did not accelerate the growth rate of figggptgggggu§_agélggtigg, In the second experiment, mineral acid was added to the milk to lower the pH and it was found that the growth rate of §§£§p§g§2§§2§_ agalagtiae was similar to that in the milk which had its pH lowered by adding citric acid. It has been demonstrated that growth inhibition of Streptogoccus agalagtiag in milk containing citric acid was due to low pH of the media. Streptococggs agalactiae may grow in the absence of free magnesium ions in the milk or else it is impossible to chelate total magnesium ions in the milk by using citrate_as a chelating agent. EVALUATION OF CITRIC ACID AS A POSSIBLE TREATMENT FOR STREPTOCOCCIC MASTITIS by Dineshwar Prasad Sinha A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Surgery and Medicine 1964 f A 327723 ”LUVZ4 [“5 7‘ Mct/ ACKNOWLEDGMENTS I wish to extend my sincere appreciation to Dr. A. R. Drury for his encouragement, guidance and technical assistance. His valuable suggestions are framed in the form of this thesis. I express my appreciation to Dr. G. H. Conner for his wholehearted cooperation and advice. The many helpful suggestions of Dr. I. F. Huddleson, Department of Microbiology and Public Health, are acknowledged with much gratitude. His intense interest and devotion to the research will be always memorable. To Dr. w. 0. Brinker, Head of the Department of Sur- gery and Medicine, I express my gratitude for his keen in- terest in this project and the facilities provided. I would like to express my thanks to Mrs. Carolyn Easter who helped me in this work. Lastly I express my heartfelt thanks to all others who helped me during this work. 11 Respectfully dedicated to my mother Lalo iii Chapter I. II. III. TABLE OF CONTENTS INTRODUCTION . o . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . 1. 2. Streptococcic mastitis . . . . . Composition of milk. . . . . . . nonormallIIilkooooooooo be “aStitiC milk 0 o o o o o o o O O O O O 3. Magnesium required for growth of bacteria. 4. Influence of feed on mastitis. . . . . . . MATERIALS AND METHODS. . . . . . . . . . . . . 1. Isolation of W W. . . 2. Milk used as media . . . . . . . . . . . . 3. 5. 6. a. commerCialo o o o o o o o o o b. Fresh, hand milked, sterilized. . . . . c. Fresh, aseptically hand milked, non— Sterilizedo o o o o o o o o 0 Materials used for plate counting. . . . . a. Tryptose agar . . . . . . . . be DilUtion blank. 0 o o o o o o Precipitating point of normal milk with CitricaC1dooooooooooo Growth of Stggptggggggg a a a in milk in the presence of sodium citrate. . . . . Growth of Streptggggggg a a in the presence of citric acid. . . . . iv Page mmmmwwI-a 10 12 12 12 13 13 13 14 14 14 14 14 16 IV. VI. VII. 7. Study with another culture of gtggptggggggg agalaQELae................ RE SULT S O O O O O O O O O O O O O O O O O O O O 1. Effect of sodium citrate on growth of Wagalagggae. c o o o o o o o 2. Precipitation of milk with citric acid . . 3. Effect of citric acid on growth of §§£§p§2¢ goggus agalagtiae in milk. . . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . 1. Selection of milk as media . . . . . . . . 2. Effect of sodium citrate . . . . . . . . . 3. Effect of citric acid. . . . . . . . . . . SUMMARY AND CONCLUSIONS. . . . . . . . . . . . REFERENCES 0 O O O O O O O O O O O O O O O O O 17 18 18 18 18 36 36 37 37 44 45 Figure 1. 2. 3. 4. LIST OF FIGURES Method for dispensing milk aseptically into teattUbCSoooooooooooooocoo Growth of Stgeptoggcgus agalagtiae in hand milked, heat sterilized milk in presence of SOdiWCitrateooooooooooooooo Comparison of growth rate of'§§;§ptgggg§g§ agglaggiae between heat sterilized and non- sterilized milk in presence of sodium citrate Growth of Streptoggggus agalagtiae in milk in presence of citric acid . . . . . . . . . . vi Page 40 41 42 43 LIST OF TABLES Table Page 1. Effect of Sodium Citrate on Growth of §5£§p§2r ggggg§,§g§;§§;i§g in Milk (24 hrs. of incubation) 20 2. Effect of Sodium Citrate on Growth of §S£2232r ggggughggalggtigg in Milk (48 hrs. of incubation) 21 3,4,5,6. A Effect of Sodium Citrate on Growth of‘gtggptgr gogggguggaléggiag,in Milk. . . . . . . . . . . . 22-25 7. Effect of Citric Acid on Growth of fisggptgggg§3§_ ‘ggglggtigg.in Milk . . . . . . . . . . . . . . . 26 8. Precipitation of Milk with Citric Acid . . . . . 27 9. Effect of Citric Acid and Sodium Citrate on ‘ Growth of W W in Milk . . . 28-29 10. Effect of Citric Acid on Growth of,§§;gp§gggg§g§ .§g§;§§;1§§,in Milk . . . . . . . . . . . . . . . 30-31 11,12,13,14. Growth of We mass in Milk in Presence of Citric Acid, Hydrochloric Acid, Mag- nesium Chloride, and Sodium Citrate. . . . . . . 32-35 vii I. INTRODUCTION ~Mastitis is one of the most important diseases of the dairy cow. It may be mild and undetectable or it may be severe enough to cause a decrease in both the quality and the quantity of the milk, even terminating in the com- plete cessation of milk production or in death of the animal. Van Houweling (1957) pointed out that milk losses from mastitis in the United States during the year 1956 were estimated at about 5 billion pounds with a value of approx- imately 200 million dollars. This loss represents 4% of the total milk production. Total losses including cost of replacement of mastitic cows and losses due to death were more than 245 million dollars for 1956. The dairy cow appears to be becoming more and more susceptible to mastitis, due in part to man's continual ef- fort to develop her mammary glands far beyond the natural requirement of the newborn calf. In the age of antibiotics and sulfonamide drugs, it probably seems paradoxical that citric acid might be thought of for the treatment of mastitis. Nevertheless, this line of thinking is not without founda- tion since Huddleson (1959) has shown that citric acid is effective in preventing igwy;§£2_growth of Stgptogoggus agglagtiae, the organism most prevalent in cases of mastitis (Drury g; 91, 1961). Uebb (1953) stated that the absence of an adequate supply of ionic magnesium in the culture medium affects ad- versely certain aspects of the nitrogen metabolism of gram- positive bacteria. By removing the ionic magnesium from the udder milk, } it might be possible to inhibit the growth of the gram-pos- itive bacteria. Citric acid, which is one of the constitu- ents of normal milk (139-210 mg.%) appears to be one of the effective agents available for the binding of ionic magnesium in udder milk. If a solution of citric acid can be used as an intramammary injection to bind magnesium ions, a cure might be effected. If citric acid is effective in prevent- ing the growth of Streptogggcgs agalagtiag, a logical follow- up study would be to determine its effect on udder tissue. This study was designed to determine the i_r_1_ ym effect of citric acid and sodium citrate on giggptggggggg, agalagtiag in milk. II. REVIEW OF LITERATURE Extensive work has been done on mastitis, but still there is a vast area remaining for mastitis research. A survey of the literature reveals nothing whereby citric acid has been evaluated for the treatment of mastitis (strepto- coccic). There were only a few supportive articles on which this project is based. In addition, the literature on the streptococcic mastitis, composition of cows' milk, magnesium required for growth of bacteria and the influence of feed on mastitis was reviewed. 1. Streptococgig mastitis Laing, gtugl (1956) stated that in western Scotland ‘the average incidence of mastitis in the year 1956 was 37% of all cows. In about 60% of the cases specific pathogens were present in the milk (specific mastitis) and in the re- maining 40% there were no organisms either in milk or tissue (nonspecific mastitis). Of the specific infections, about 75% were due to staphylococci (in small proportion mixed staphylococcal and streptococcal infection) the rest were chiefly due to streptococci. Little,‘g§.§l (1946) stated that up until approxi- mately 1940, all forms of bovine mastitis were considered to be an incurable disease. In 1939 a mastitis survey of the Lansing area was done by Bryan (reviewed by Drury, 1961) who found that 86% of the herds and 26% of the cows in these herds had mastitis. He also determined that 98% of the mastitic infections were associated with streptococci. In the Fall of 1959, a mastitis survey consisting of 25 herds involving 380 milking cows in the Lansing area was done by Drury, gtflgl (1961). He found that 59% of the cows showed infection in one or more quarters. 0f the in- fected quarters, 58.8% were streptococcus, 39.4% were staph- ylococcus and 1.8% were other organisms. Carpenter (1922) observed that age of the cow and the level of milk production had an influence on the effec- tiveness of mastitis producing organisms. He mentioned that nonlactating cows showed no ill effects from the intramammary injection of organisms but clinical symptoms of mastitis were observed in the same animals a few months after parturi— tion, when organisms were introduced by intramammary infusion. Young cows were more susceptible than older cows. Spencer,lg§”gl (1950) reported that roughening of the surface of the epithelium of the milk-duct and the occlusion of ducts by clots of fibrin are probably important in the pathogenesis of bovine mastitis. It has been mentioned in the book Diseases 2§_Cattle (2nd ed.) that, "gtg, agalactiae enters the gland through the teat opening and apparently resides mainly in the lumen of ducts and on the lining surface of ducts and alveoli." The action of the streptococci on the tissue is through an irritant which forms in the milk. Little (1937) reported that the intact duct and sphincter of the teat may act as a natural barrier to the passage of pathogens into the udder. 2. C m i n of Mi a. Normal milk: Field (1960) stated that magnesium is a constituent of milk with a concentration of 4, 12, 16, and 31 mg./100ml. in human, cow, goat and rat whole milk respectively. Only about 20% of the magnesium in cow's milk is in the ionic form. Robertson,,g§.gl (1960) have found that the concen- tration of magnesium in milk can remain constant over a wide range of serum magnesium levels. A Kon and Cowie (1961) reported that there is insig- nificant variation in magnesium content of milk of differ- ent breeds of cows. Stewart, gtugl (1956) have reported a large seasonal variation in the magnesium content of herbage with the low- est content being in April and May when magnesium tetany is most prevalent. He further suggested that higher mag- nesium content of the herbage in April and May produced by magnesium limestone dressing considerably reduced the degree -of hypomagnesemia in a herd. Vankreveld, §£_§l,(l955) mentioned that calcium and magnesium in milk occur in different forms. They are bound partly to the protein and partly to phosphate or citrate and thirdly there are free Ca and Mg ions. This last frac- tion is probably the rate determining factor in the various coagulation processes. Kemp,lgt.§1 (1960) reported that 33% of the calcium, 33% of the phosphate, 75% of the magnesium, and 90% of the citrate of milk are present in the dissolved state. There is a gradual increase in the magnesium content of milk from the beginning of lactation to the end as reported by Vanschoubrock,,gt.g; (1957). This increase, however, was less in the second half than in the first half of lac- tation. Gueguen, gt,§;_(1960) observed variations in the P, Ca, K, Na, and Mg content of milk with 10 cows of 3 dif- ferent breeds which calved at the beginning of the winter and had the same diet. He found that in the course of lac- tation, the K content diminishes regularly whereas the Na content increases, especially at the end of lactation. Cal- cium and phosphorus content were at their peak at the begin- ning and end of lactation. Mineral composition of milk is not affected if the animals are put out to pasture. Rook and Storry (1962) published an article on the nutritional aspect of magnesium in farm animals. In their literature review it was found that there is a small varia- tion in magnesium content of milk with change in diet. There was no fall of magnesium level of milk even though the in- take of feed or magnesium was reduced. There was no signif- icant fall in milk magnesium in the case of a hypomagnesemic cow. Todd, gtngl (1962) injected parathyroid extract into parathyroidectomized lactating Jersey cows and observed that parathyroid treatment caused an increase in milk magnesium with a corresponding decrease in urine magnesium whereas the citric acid content of urine and plasma increased, but remained constant in the milk. Davidov,‘§t.g; (1960) studied variations in the cit- ric acid content of milk on the basis of bulk milk received in a Moscow dairy and milk produced at one farm. The average monthly level of citric acid over a 2-year period was 108 to 210 mg.% for bulk milk and 139 to 210 mg.% for the farm milk. In a feeding experiment he found the average citric acid content of milk decreased from 177 mg.% in December— March to 163 mg.% in May and rose to 185 mg.% during the first few days after the transfer of the cows to pasture. Anagama,,gt.§l (1961) determined the citric acid (by Marier and Boulet method) in 180 samples of bulk Holstein Friesian milk taken between May, 1960, and April, 1961. The average was 192 mg.%. The monthly average ranged from 137 to 134 mg.% in July and August to 221 mg.% in March. He suggested that the decrease of citric acid content was due to the high temperatures of summer. Nickerson (1960) pointed out that milk from various areas differed significantly in their content for all con- stituents except soluble organic phosphoros, citric acid and magnesium. b. Mastitic milk (streptococcic): Redaelli, g; a; (1957) have studied the behavior of inorganic substances in mastitic milk. In the course of streptococcic infections there was a remarkable increase of the chlorine and sodium values and a diminution of cal- cium and potassium content. There was no significant vari- ation for magnesium. Little, gtng; (1933) stated that the udder capillaries under severe inflammatory conditions are permeable to blood protein. Under these conditions the same structure may per- mit the passage of blood alkali. In many cases of bacterial invasion, reaction of milk is usually altered towards the alkaline side. 3. Magnesium Required for Growth of Bacteria Webb (1953) stated that an inadequate supply of ionic magnesium in the culture medium adversely affects certain aspects of the nitrogen metabolism of gram—positive bacteria. Webb(l951) found that the requirement of magnesium for growth of gram-positive and gram-negative bacteria depends upon the organic composition of the medium. In general, gram-positive bacteria failed to grow when the magnesium content was less than 0.6 ppm., whereas this concentration is almost sufficient to maintain maximum growth of gram-negative species. Webb (1949) reported that the concentration of magnesium neces- sary to support optimal growth of gram-positive bacteria, some 20 to 30 ppm., is about ten times greater than that required by the gram-negative organism under the same con- ditions. Huddleson (1959) reported that gtaph. augeus and §5_, agalagtiag failed to grow in blood in the presence of citric acid due to the binding of the particular metal ion Mg, which is essential for growth. He also observed that citrate even in low concentration in blood inhibited the growth of §££p ggalactiae. Higher concentrations inacti- vated an agent or agents of the anti-microbial system that suppressed growth of gram-negative bacteria. Abelson,,§§.§; (1950) has demonstrated the influence of magnesium on the toxicity of Ni, Cd, Zn, and Mn, on the growth of g, 921;, Aerobacter aerogenes, Aspergillus ni r, and Torulgpsis utilis. The toxicity of these cations is lowered in the presence of much magnesium. If magnesium is not present in the media these elements are toxic at very low levels for all four organisms. Higher levels of mag- nesium diminish the amount of nickel and cobalt bound by the cell. Bienvenu,,gt.gl (1963) found that the brucellacidal mechanism in nonvaccinated unbred heifers was dependent upon the level of ionized magnesium in their blood. After injecting 10 100,000 I.U. of human chorionic gonadotrophin into unbred heifers, brucellacidal activity and serum magnesium was sharply decreased by lowering renal threshold for the min- eral. 4. I f e f f ma t Pounden (1952 & 1957) observed two groups of eight cows in the same herd. One group was fed a ration of a1- falfa hay, forage crops, silage and moderate quantities of grain. The other group received grass hay, mostly timothy of relatively poor quality, and a more liberal feeding of grain. Both groups were observed for 16 weeks. In the first group mastitis (clinical) was evidenced in one quar- ter of two cows and the second group had clinical mastitis in eight quarters in S cows. Streptggogggs ggglagtigg, or unidentified streptococci or staphylococci were the causa- tive organisms. Pounden (1952) stated that the resistance for Streptococggs agalactiae in the milk was lessened as lactation progressed. Pounden,,g§.a1 (1956) observed that the inhibitory action of milk to the growth of'§fi;35§35§§gagi agalagtiae was lowered for a few days when corn silage was added to the hay and grain ration which had been fed contin- uously for a year and a half. Inhibitory action was retained or improved slightly when cows which had been on pasture all summer were fed forage crop silage as a part of the ra- tion in the fall. Pounden,,g£.gl (1958) mentioned that the number of mastitis cases increased in a large herd each spring 11 when fresh green feed was added to the ration of hay and grain. Care (1960) reported that a daily supplement of 2 ounces of magnesium oxide in feed per head of cattle main- tains the level of plasma magnesium within the normal con- centration. He concluded that the daily supplementation of the diet of adult cattle up to the extent of 4 ounces of magnesium oxide per head for a period of six weeks is unlikely to be attended by a loss of condition or to pro- duce undesirable plasma levels of magnesium, calcium and inorganic phosphate. Rook and Belch (1958) reported that when dairy cows were changed from winter ration to cut young grass, there .was an immediate fall in urinary magnesium excretion and progressive development of hypomagnesemia was noticed. When more mature herbage was fed, less marked changes in urine and serum magnesium levels occurred for the first few days following the changes from the winter feed. Thereafter a gradual rise in serum magnesium, followed by urine magnesium, levels took place, which appeared to result from a slight increase in the intake of herbage magnesium in the first few days following the change of the diet. Burt and Thomas (1961) studied dietary citrate and hypomagnesemia in the ruminant and suggested that the high citrate content of spring grass may be the cause of hypo- magnesemia. III. MATERIALS AND METHODS l. Isglation of Streptgggggus agalagtiag Streptogoggus agalagtiae was the test organism used in this study. It was obtained from one of the Michigan State University (MSU) Holstein cows affected with a clin- ical case of mastitis. Milk was aseptically collected and incubated at 37°C for 24 hours and the following tests were performed to identify the organism. a. Newman's staining b. Streaking of blood agar plate (5% bovine blood) c. Inoculation of tryptose broth d. Hotis test e. Inoculation of sodium azide crystal violet agar f. Streaking on Edward's medium The isolated strain of Streptococgus agalactiae was maintained by monthly transfer on thick tryptose agar plate and stored at 5C. Actively growing 22-24 hours broth cul- ture that previously had been transferred for several con— secutive days were used for the citric acid testing procedure. Transfer of the organism tends to stimulate a constant uni- form, rapid and more vigorous growth. 2. M k sed as media The milk used to test the growth of gagggugggagayg agalaggiae organisms was obtained from the following sources: 12 13 a. Commercial-Modified skim milk‘ sold by the M.S.U. Dairy was heat sterilized (autoclaved) at 115C and 10 pounds pressure for 15 minutes. b. Fresh, hand milked, sterilized--A cow not receiv- ing any antibiotics and showing zero (negative) reading on the California Mastitis Test (CMT) for all four quarters was selected for taking milk samples. The udder was washed with disinfectant and the cow was hand milked into a flask and then the milk transferred to a separatory bottle and placed in the refrigerator (7 C) for 12 hours after which the fat separated milk was dispensed into test tubes and autoclaved at 115 C and 10 pounds pressure for 15 minutes. c. Fresh, aseptically hand milked, nonsterilized-- A cow not receiving any antibiotics and showing zero (nega- tive) reading on CMT was selected. The udder was washed 5 times with a warm aqueous solution of Novadine." Special care was taken to wash the orifices of the teats. The cow was milked directly into a sterile flask with utmost prac- ticable aseptic precaution. Then the milk was transferred to a sterile separatory bottle and placed in the refrigerator (7 C) for 12 hours. A special fitting burett3(Fig. 1) was attached to the bottle to dispense the milk into sterile test tubes without contamination. Before using, the milk *Skim milk to which 1% dried skim milk and 2000 USP units of vitamin A and 400 USP units of vitamin D were added. u1/2 oz. CLENESCO NOVADINE in 2 l/2 gallons of water, Cowles Chemical Company, Cleveland, Ohio. 14 was examined for sterility. 3. a 0 un a. Tryptose agar (Difco)'--Tryptose agar was recon- stituted and autoclaved at 121 C at 15 pounds pressure for 15 minutes, and stored at 10 C. Whenever required, agar was melted and poured into petri dishes (sterile and dis— posable). After solidification all petri dishes were in- cubated at 37 C for 24 hours and were only used if they were negative for bacterial growth. b. Dilution b1ank—-Composition: Peptone 10 mg. Sodium Chloride 500 mg. Distilled Water 100 ml. The dilution blank was dispensed in 99 m1. amounts into rub— ber stoppered bottles and autoclaved at 121 C at 15 pounds pressure for 30 minutes. 4. Precipitating point of normal milk with gitric agid Citric acid ranging in concentration from 0.2%-4.0% were added to milk. Degree of precipitation and pH at dif- ferent concentrations of citric acid in milk was determined. Beckman's pH meter and Beckman's standard buffer were used. 5. G owth of Stre ococcus a alacti i m h - gnge of sodium citrate Sterile solutions of sodium citrate" were added 'Difco Laboratories, Detroit 1, Michigan. "Sodium Citrate (Na C H 0 ' 2H20):F.W. 294.111, J. T. Baker Chemical Co., PRiTliprurg, New Jersey. 15 to the sterile test tubes containing 9 m1. of skimmed milk (obtained from M.S.U. dairy store). One milliliter of the appropriate concentration of sterile sodium citrate solution was added to 9 ml. of milk to make final concentrations vary- ing from 0.1% to 4%. Thus in each instance the milk was diluted 9 to 1.‘ All tubes having different concentrations of sodium citrate as well as control tubes were inoculated with the same number of Streptogogcus agalagtigg. In the control tube 1 ml. of 0.5% sodium chloride solution was added. All tubes were incubated at 37 C and dilution plates were made at the end of 24 hours and 48 hours of incubation. Each tube was mixed on a Vortex‘ mixer for one minute to obtain uniformity of the mixture before sampling. Dilution plates were made by plating out different dilutions on 24 hour incubated tryptose agar plates. Tube contents were plated in original form as well as in different dilutions for which dilution blanks were used. Care was taken to place the plating materials on the center of agar plate and petri dishes were slowly rotated to obtain a uniform spread all over the surface. The petri dishes were set on a level sur- face until the plating materials were absorbed into the agar (approximately 2 hours). After 24 hours of incubation col- onies were counted. The same procedure was repeated with the fresh *Vortex Jr. Mixer, Scientific Industries, Inc., Queens Village, New York. 16 sterilized and nonsterilized milk. The pH was measured after 48 hours of incubation by using Beckman's pH meter. 6. Growth 0: Streptogpppup agalactiae in mil; in the prgs- pnpg pf pitric acid The same procedure was adopted as in #5, the only difference being that citric acid was used as a source of ++ in the milk. A mixed solution of citrate for binding Mg sodium citrate and citric acid was also tried which gave a higher pH than that of citric acid, and lower than sodium citrate when used alone. The pH was measured by Beckman's pH meter in the beginning and end of 48 hours of incubation. To determine whether the growth inhibitory action of citric acid in milk on Streptococcus agalactiae was due to binding of Mg++ or to acidity of the medium, two methods were used: a. Addition of magnesium chloride b. Addition of hydrochloric acid to lower the pH of milk A sufficient amount of magnesium chloride was added to the citrated milk to provide enough magnesium ions to satisfy the growth requirements of Streptocopcus agalaptiae. The growth of Streptococcus agalaptiae in this milk was com- pared with its growth in citrated milk without the additional magnesium chloride. 1 The amount of hydrochloric acid required for a de- sired pH of 9 m1. of milk was determined. Then by adding 17 hydrochloric acid, the pH of milk was made to correspond to the pH of the citrated milk. 7. S .o . anoth-_ u _ume o_ S ‘9 o_- -- aoa a 'a‘ Another culture of Stpeptopopgup agglaptiap was ob- tained from Dr. Huddleson' on which he worked in 1959. The dilution and cultural procedures were the same as previously described (Part 5). *Department of Microbiology and Public Health, Mich- igan State University. IV . RESULTS 1. E ect of odium t at on ro h of Stre oco s agalactiae in milk' Results obtained from the experiments have been pre- sented in Tables 1 through 6. No growth inhibitory effects on Streptopoppus agagaptiae in commercial skim milk occurred when sodium citrate concentrations varying from 0.1% to 4% were present in the milk. Furthermore, there were no significant inhibitory effects on fresh milk (see Tables 4 to 6) when concentrations of 1% to 4% sodium citrate were present regardless of whether the milk had been sterilized or not. Growth of the organism, however, was more vigorous in the sterilized milk than it was in the nonsterilized milk (see Tables 5 & 6). 2. Prepipitation of milk with citric agid A small amount of precipitation occurred when the citric acid concentration in the milk was 0.2%. This low- ered the pH of the milk from 6.65 to 5.64. With a citric acid concentration less than 0.2%, there was no gross pre- cipitation of milk. Moreover, concentrations of citric acid higher than 0.3% in the milk produced a heavy precipitation (see Table 8). 3. ‘ 0 i ' - '0 01 9 o. o - Str‘o 0-0 s a-a a - tiae in milk Growth of Streptococcus agalactiap was inhibited 18 19 when concentrations of 1%, 2%, and 4% citric acid were added to the milk. The minimum level capable of inhibiting the growth of Sttgptppppppp,ppplppttpg,was found to be 0.8% (milk pH 4.08-4.12). However, at this concentration, results were variable, there being total inhibition on some trials and some growth on other trials. When the citric acid concen- tration was increased to 0.9%, there was complete inhibition of organism growth on all trials (Tables 10 to 14). When, by the addition of hydrochloric acid, the pH of the milk was lowered to the same pH as that produced by 0.9% citric acid (3.90 to 4.10), inhibition of §tpgptppppppp_ ,pgptppttpg likewise occurred (see Tables 11 to 14). 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