ma DEVELOPMENT or CLOSTRIDIUM f BOTUUNUM TYPES 62 A AND 7 3 IN STERELE SKTMMILK»? - Thesis for 1110 chru 01 D11. D MICHIGAN STATE UNITERSTTY ,,,,, Andre R. BrnIIaud ” ' 1965 :'“ Willi/[WITHIN W T HM 1293 19756 68069 L I B R A R Y Michigan State This is to certify that the thesis entitled The Deve10pment of Clostridium Botulinum Types 62 A and B in Sterile Skimmilk presented by Andre R. Brillaud has been accepted towards fulfillment of the requirements for Ph .1). degree in Microbiology L/ flfl/(Z/fl/K/ 7770A Majfl/professor Date February 26, 1965 O~169 L1 University ROOM use ONLY ROOM USE ONLY 56??“ W” 6” goo A061. T ABSTRACT THE DEVELOPMENT OF CLOSTRIDIUM BOTULINUM TYPES 62 A AND B IN STERILE SKIMMILK by André R. Brillaud Culture development of Clostridium botulinum Types 62 A and B in sterile skimmilk, at initial levels of 10, 000, 1,000, 100 and 10 per ml, and stored at l8, 13, 10 and 7 C, was studied“ Growth of Type 62 A occurred at all levels of inoculation when sample was incubated at 18 and 13 C, Toxin was produced and visual and olfactory changes also occurred with culture development in the skimmilk medium. At 10 and 7 C no growth, toxin production, visual or olfactory changes were evident. Toxin appeared in all cases of culture development as the population attained a level of greater than 1. O x 105 per ml. Immediately prior to the appearance of toxin in the medium an amino odor was always detected. Lethal levels of toxin were present before visual and other olfactory changes became evident. Clostridium botulinum Type B grew in sterile skimmilk when incubated at 18 C at initial inocula levels of 1,000, and 100 per ml. The data obtained on the growth of Type B compared well with the data obtained with Type 62 A. The inhibitory effect of the following fatty acids: butyric, oleic, caproic, caprylic, capric, lauric, myristic, palmitic. on the culture development of Type 62 A was tested. The individual acids were tested at 03 01% in skimmilk and 0° 001% in a 4% Trypticase mediumo Caprylic, capric, myristic and stearic acids were found to have an André R u Brillaud effect in prolonging the lag phase of culture development, Mixtures of all the acids were more effective in prolonging the lag phaseo Monolayer cell tissue cultures were studied to learn if they were sensitive to the cytotoxic action of botulinus toxino Mouse C3H, Osgood J-lll, Mouse L Cell, Detroit—6-Parent, Detroit-6-Yale Clone and Embryo Monkey Kidney cell cultures were used. Purified Type A toxin and toxin produced in skimmilk were tested“ All cultures were found to have varying sensitivities to the toxin with the exception of Mouse L cell which was refractive to the toxina Heat inactivated toxin and specific antitoxin were used as controls, THE DEVELOPMENT OF CLOSTRIDIUM BOTULINUM TYPES 62 A AND B IN STERILE SKIMMILK BY André R. Brillaud A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1965 AC KNOW LED GMENTS The author wishes to express his appreciation to Dru 00 W. Kaufmann for his guidance during the course of this investigation. This study was supported in part by a grant from the National Institutes of Health° The successful completion of this work was made possible by the help and confidence given to me by my wife, Joan, to whom this work is dedicated. v TABLE OF CONTENTS INTRODUCTION . REVIEW OF THE LITERATURE MATERIALS AND METHODS 0 Preparation of Spore Suspension and Counting Procedure Toxicity Tests 0 O 0 Preparation of Skimmilk“ and Trypticase Media Tissue Cell Culture Study 0 RESULTS‘. 9 Culture Development of Co botulinum Type 62 A in Skimm 11k Studies at 18 and 13 C Studies at 10 and 7 C a Culture Development of Co 903142.133? Skimmilk o 9 Studies at 18 C Studies at 13, 10 and7C Type B in Effect of Fatty Acids in Skimmilk on Culture Development Culture development in agitated skimmilok Culture development in unagitated Skimmilk” Effect of Fatty Acids in Trypticase Medium on Culture Development Culture development in agitated Trypticase medium a Culture development in unagitated Trypticase medium Effect of Botulinus Toxin on Various Tissue Culture Monolayers 0 DISCUSSION SUMMARY BIBLIO GRAPHY 0 iii Page Nxomxl 16 16 16 22 26 26 28 33 33 36 38 38 38 41 49 59 6O TABLE 10. LIST OF TABLES Composition of Tissue Culture Growth Media a a Development of C. botulinum Type 62 A Spores In~ oculated Into Skimmilk at Different Concentrations and Incubated at 18 C 0 Development of (_3 botulinum Type 62 A Spores In~ oculated Into Skimmilk at Different Concentrations and Incubated at 13 C o 0 Development of C_., botulinum Type 62 A Spores In- oculated Into Skimmilk at Different Concentrations and Incubated at 10 C 0 Development of (_3 bgtulinum Type 62 A Spores In- oculated lnto Skimmilk at Different Concentrations and lncubated at 7 C o . Culture Development, Toxin Production, and Visual Changes in Skimmilk Inoculated with C botulinum Type 62 A Spores and Incubated at Various Temperatures 0 1 Development of C botul: num Type B Spores Inocua lated Into. Skimm: 1k at Different Concentrations and Incubated at 18 C .. a Development of C botlu inurn Type B Spores Inocu—a lated Into Ski mmilk at Different Concentrations and Incubate-d at 13 C o a Development of C_ botuli num Type B Spores Inocu— lated Into Skimmilk at Different Concentrations and Incubated at. 10 C 0 Development of Co botuli num Type B Spores Inocu- lated Into Skimmilk at Different Concentrations and Incubated at 7 C Page 14 17 21 23 24 25 27 29 3O 31 LIST OF TABLES - Continued TABLE 11. 12° 13° 14° 15. 16° 17° Comparison of the Culture Development of _C_. botulinum Types 62 A and B Spores Inoculated Into Skimmilk at Various Levels and Incubated at Various Temperatures 0 Effect of Various Fatty Acids (0° 01%) in Skimmilk on the Germination and Outgrowth of Spores of C_3_o botulinum Type 62 A~=Samples Shaken 0 Effect of Various Fatty Acids (0° 01%) in Skimmilk on the Germination and Outgrowth of Spores of go botulinum Type 62 A-=Samples Not Shaken Effect of Various Fatty Acids (0. 001%) in 4% Trypticase on the Germination and Outgrowth of Spores of g. botulinum Type 62 A~-Samples Shaken Effect of Various Fatty Acids (0. 001%) in 4% Trypticase on the Germination and Outgrowth of Spores of C__. botulinum Type 62 A--Samples Not Shakenoo.°o°°oo.oooo. Effect of Varying Levels of Botulinus Toxin on Tissue Culture Monolayers 0 Comparison of Lag Phases of Culture Development of C_ botulinum Type 62 A Under Varying Conditions 32 34 37 39 42 43 55 LIST OF FIGURES FIGURE Page 1. Botulinus Toxin in Mouse L Cell Tissue Culture . . ., 45 2. Effect of Botulinus Toxin on Detroit—6—Parent Tissue Culture......................... 47 INTRODUCTION The main objective in pasteurizing milk at 62 C for 30 min or at 72 C for 16 sec is to destroy the nonsporeforming disease— producing bacteria. Nonpathogenic thermoduric bacteria belonging to the genus Micrococcus and heat-resistant sporeformers belonging to the genus Clostridium or Bacillus will survive these pasteurization procedures if they are present in raw milk (Martin at a_t_1,, 1961)“ The presence of clostridial spores in pasteurized milk as a result of pre— 01" post-pasteurization contamination, however, has never presented a public health problem as no deaths from botulinus poisoning have ever been reported from milk The relative short storage period encountered in the dairy industry today, as well as the low storage temperature undoubtedly exert some controlling influences Studies involving prolonged holding periods at low temperatures are particularly important in View of the new advances in pasteurization which provide for a considerable increase in the shelfwlife of pasteurized milk, The advent of ultra—high temperature (UHT) pasteurization which utilizes temperatures from 88 to 121 C with “no intended holding period” has increased the storage life of milk but the milk is not sterile (Tobias e_t a_1u, 1955). This study was undertaken to determine the development of a culture of C kiofiilinum in sterile Skimmilk on pro- longed storage at low temperatures“ Particular attention was directed to the effect of fatty acids on culture development since it has been reported (Foster and Wynne, 1948) that oleic, linoleic, and linolenic acids inhibited the germination of spores of C_ botulinum but did not affect the growth of vegetative cells“ P An investigation was also undertaken to study the effect of botulinus toxin on tissue cell cultures. Tissue culture systems were selected as some information is available on detection of toxic material by this procedure (Rightsel e} all” 1958; Rope e_:_t a__1_lt, 1957):. REVIEW OF THE LITERATURE The genus Clostridium comprises a large group of organisms, some of which can be divided into two subgroups according to their physiological manisfestationsg The first is the food poisoning group; the most well-known member being Clostridium botulinum, The second group is comprised of bacteria which cause histotoxic infections in man and other animals“ The best known member of the latter group is Clostridium perfringens, also known as Clostridium welchii. The food poisoning group is significant since they cause toxicity without 111 xiv—o growth and multiplication, while the latter, the gas gangrene group, produces infections in vi_v9., The members of the genus Clostridium are sporeforming, anaerobic, rod—shaped bacteria which, under appropriate conditions produce neurotoxins that are among the most potent poisons known. Clostridium botulinum produces a neurotoxin which is released into the growth medium after the major increase in the bacterial population has occurred (Lamanna, 195910 The organism is generally unable to grow and produce toxin within the bodies of homeothermic animalsu They are generally not linked to an infectious process“ Botulism is, therefore, a matter of food poisoning“ Different strains of (_I botulinum produce antigenically distinguishable neurotoxins and the injection of graduated doses will stimulate the production of antitoxins in animals“ The several toxins of C botulinum are distinguished serologically (Oakley, 1954) and typed according to their antigenicity as A, B, C, D and E1 Types A, B, and E are most frequently detected in food poisonings in man" Bronfenbrenner e_t a_L (1921) have reported strain differentiation by immunological methods and Gunnison and Scheonholz (1927a, b) have shown serological classification by complement fixation and precipitin reactions“ The identification of botulinus toxin in canned foodstuffs by serological methods has been reported by Keiser (1923). A number of investigators have studied the development of spores of C botulinum in various foods. Halvorson (1949) studied botulism in canned bread, Segalove and Dack (1951) studied the growth of the culture in dehydrated meat, Wagenaar and Dack (1954, 1955, and 1958) investi- gated the development of the culture in canned bread, cheese, and cheese spread, and Grecz e_t a_l° (1959a, b) studied the development of g, botulinum in cheeseo No data are available on the development of the culture in milk“ Other investigators have studied the factors influencing the inhibition of growth, toxin production, and spore germi— nation of C botulinum by various substances and/or compounds. Bonventre and Kempe (1959) and Tanner e_t a_1, (1939) studied the effect of pH and temperature on the culture development of spores of V 9,, botulinum in a medium consisting of casein and yeast extract, They found that the organism would not grow when the pH was 4, 5 or lower and the temperature was 5 C or lowera At 10 C Tanner e_t a_l, (1939) found that toxin production was negligible, The effect of canning of corn and mushrooms on the culture development of g botulinum was studied by Denny e_t a_lu (1961). Grecz e_t a_l. (1959c, 1961) reported that the growth limiting total fatty acid concentration for C_ botulinum in aged cheese was approximately 2%. They also reported the production of antimicrobial agents, presumably fatty acids, in surface ripened cheese" Humfield (1947) reported on the inhibitory action of fatty acid- like constituents of wheat on germination of Clostridium spores° ' Germination of spores of _C botulinum was shown to be inhibited by IP oxidative rancidity in unsaturated fatty acids (Roth and Halvorson, 1952). Tarr (1953) and Townsend e_t 341. (1954) studied the effect of amines and acidity on the germination and growth of (_3. botulinum in fish muscle and Trypticase medium. Foster and Wynne (1948) reported on the inhibition of germination of spores of C.botu1inum by unsaturated C18 fatty acids. Little or no information is available on culture development and toxin production of C. botulinum in milk stored for long periods of time at low temperatures. Similarly, no information is available on the visual and olfactory changes which occur when this organism grows in milk. Some research has been conducted, however, on the inhibition of germination of endospores of C. botulinum, C. pasteurianum, Bacillus subtilis, and B. stearothermophilus by oxidized unsaturated fatty acids (oleic, linoleic, and linolenic) at a concentration of 0° 1% (Roth and Halvorson, 1952). Krukovsky (1961) stated that there is an increase of lipolytic rancidity and acidity in unpasteurized milk which had been aged; the concentration of fatty acid is directly associated to the time of storage. Foster and Wynne (1948) reported the inhibition of germination of spores of C. botulinum in synthetic growth medium using 0. 01% oleic acid and higher concentrations of linoleic and lino- lenic acids. In bovine milk production environmental temperature and humidity affected the fatty acid composition of the product (Richardson e_t $11., 1961), hence the fatty acid composition of milk varied according to at least these two parameters. Richardson e_t a_1. (1961) found that low molecular weight fatty acids (C6 to C12) were depressed by approxi— mately 2% at 85 F and 70% relative humidity. Increases of 6% and 4%, respectively, in the production of palmitic and stearic acids were obtained under the conditions noted above. Studies by Harper e_t a_1_1. (1961) on the volatile fatty acids in milk showed increases in fatty acid concentrations in pasteurized milk during storage. There were tenfold increases in formic, acetic, butyric, valeric, caproic, and caprylic acids in pasteurized whole milk when it was stored for 21 days at 40 F. Propionic acid increased only twofold. The bacterial count of the milk after this storage period varied from 2. 6 x 106 to 6. 7 x 10? organisms per m1. Botulinus poisoning is manifested in humans as a paralysis of the efferent branches of the autonomic nervous system. The specific sites of this paralytic action are the efferent parasympathetic nerves and/or the somatic nerves. Impulses transmitted across. synapses are chemical in nature. The mechanism for synaptic transmission is composed of two subsystems one depending upon the adrenalin-like substance, sympathin, and the other on acetylcholine. Only the cholinergic system is affected by the botulinus toxin (Lamanna, 1959). The central nervous system is not involved. The effect upon the other tissues in the body is now known, although Lamanna (1959) speculated that tissue cultures of mammalian. cells are not affected by the botulinus toxin since an organized cholinergic system is lacking. The white mouse test is used almost exclusively for the detection of botulinus toxin; however, tissue culture monolayers have been employed to assay diphtheria and tetanus toxins (Rope e_t a_l. , 1957). Gabliks and Solotorovsky (1961) used tissue culture derived from susceptible and resistant animals to detect diphtheria toxin. General cytotoxic experiments have been described by Eagle and Foley (1956) in which tissue culture was used to study the toxicity of carcinolytic material. Tissue culture techniques have been used to assay other metabolically produced bacterial products (Rightsel e_t a_1., 1958) . MATERIALS AND METHODS Preparation of Spore Suspension and Counting Procedure C. botulinum Types B and 62 A were used in all studies. The medium used to grow the culture for spore production consisted of 1. 5% Trypticase (Difco), 1. 0% Peptone (Difco), 0. 5% sodium chloride, 0. 25% potassium phosphate-di-basic, 0. 3% glucose, 0. 2% sodium thioglycollate and 0. 0004% methylene blue chloride (Zoha and Sadoff, 1958). To obtain a spore crop, 200 ml of this medium were dispensed in a 1-liter Erlenmeyer flask fitted with a rubber stopper, equipped with a glass inlet tube projecting below the surface of the medium and with an outlet tube for flushing with natural gas. A magnetic stirring device was used to produce agitation. The outlet tube was connected to a trap containing a 1:1, 000 dilution of aqueous quaternary ammonium compound to minimize the contamination of the atmosphere. One ml of. a heat-shocked spore suspension of C. botulinum was added as inoculum. Heat-shocking of the spores to stimulate germination was accomplished by placing the spore suspension in a water bath at 95 C for 30 min (Evans and Curran, 1943). To remove air from the culture medium, filtered natural gas was bubbled through the apparatusfor approximately 30 min at which time a yellowish color became evident. Excess gas was burned off as it escaped from the outlet tube. The inlet tube and the outlet tube were clamped to maintain anaerobic con- ditions during incubation at 31 :l: 1 C; after 7 days approximately 90% sporulation was observed. In preparing some spore inocula a medium consisting of 4% Trypticase and 1 ppm thiamine hydrochloride was used (Day, 1960). Under these conditions sporulation was enhanced and approximately 95% sporulation occurred in 3 days. The spores were harvested by centrifugation in a chilled Servall centrifuge at approximately 10,000 x g for 15 min. The spores were washed with Sorensen‘s phosphate buffer at pH 7. 0 until the supernatant solution no longer showed a blue—green color due to the presence of residual methylene blue indicator. The stock spore suspension was stored in buffer (pH 7. 0) in screw-cap glass bottles containing a few glass beads to facilitate mixing. The spore count of the stock suspension was 4.4 x 10'I spores per m1. Little, if any, loss of viability in the spore suspension was observed on storage at 7 C for 17 months. In all instances the spore suspension was heat-shocked immediately before use. All counts were made by diluting the samples in Sorensen‘s buffer solution. One ml of each dilution was added to each of 3 Prickett tubes containing 0° 5 m1 of a sodium thioglycollate—buffer solution made up as follows: 0. 2% sodium thioglycollate, 0.2% potassium phosphate— di-basic and 0. 1% sodium bicarbonate. Approximately 20 m1 of a modified medium of Wynne e_t a_l. (1955), at 45 C, were added rapidly to each tube. Adequate mixing of the inoculated sodium thioglycollate- buffer solution and the medium was accomplished in this manner. This medium (YESB) consisted of 0.1% soluble starch, 1. 0% yeast extract, 1. 6% agar, and O. 0004% methylene blue chloride. After the medium solidified an additional 2 to 3 m1 of sterile medium were added to main-- tain anaerobic conditions. Inoculation was undertaken at 37 C for 36 hr at which time colonies of E. botulinum Were counted. Toxicity Tests Toxicity tests were performed to ascertain when toxin was present in, the growth medium. The standard adult white mouse toxicity test was used for the detection of botulinus toxin in the milk medium. When lethal levels of toxin were suspected a 0. 5 ml sample of milk was injected into the peritoneal cavity of a white mouse. All mice were observed for 4 days following injection and those which survived were considered to have been inoculated with a nontoxic product. In most cases the test mice died within 24 to 36 hr. Inoculated animals which died within an hour after injectionwere con— sidered to have died from traumatic injury and were eliminated from the experimental data. Mice which died in this manner were replaced with new animals so that at least 4 mice were used for each sample tested. Controls were inoculated with 0. 5 ml of sterile Skimmilk and Skimmilk which had been heated to 95 C for 30 min. 9. botulinum Type A antiserum was used in these tests to determine if the animals inoculated with the Skimmilk containing toxin died due to botulinus poisoning. Equal volumes of antiserum (1:100) and Skimmilk samples were incubated at room temperature for 10 min prior to injection. Preparation of Skimmilk and Trypticase Media Two hundred and eighty ml of raw, fresh Skimmilk, sterilized at 121 C for 10 min, were added aseptically to each of 16, 12-oz screw— cap prescription bottles. One-liter quantities of Skimmilk contained in 2-liter flasks were similarly sterilized and used as a reservoir to fill the bottles to within 5 ml of their maximum capacity. To determine if the Skimmilk samples were sterile, duplicate 0. 1 ml samples were plated on plate count agar and incubated at 37 C for 48 hr. Heat- shocked spores of C. botulinum Type 62 A and Type B were added to provide initial spore concentrations of approximately 10, 100, 1,000 and 10, 000 spores per ml of Skimmilk. Each sample was shaken vigorously for 1 min to assure mixing. One ml was removed for an initial or 0 hr 10 count and a sample of each spore concentration was incubated at 18, 13, 10 and 7 C. The temperature varied :1: 1C. At 48 hr intervals each container was inverted 3 times and 1 m1 of the Skimmilk was removed for counting. Culture development was followed until the total count was in excess of one million cells per m1. Visual and olfactory observations were made on each sample. To study the effect of oleic, butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, and arachidic acids on the development of C_. botulinum Type 62 A, 1% suspensions of the fatty acids were prepared in hot (90 to 95 C) buffer solution at pH 7. The mixtures were shaken until emulSions were formed. Each fatty acid emulsion was added to approximately 150 ml of fresh, raw Skimmilk in 12-oz screw-cap bottles. In studies using Skimmilk the fatty acid concentration was 0. 01%. A mixture containing each fatty acid at a concentration of 0. 001% was added to a sample; the total concentration of mixed acids was 0. 01%. In addition the mixture of fatty acids was added to a Skimmilk sample so that the concentration of mixed acids was 0. 1%. A sample containing only Skimmilk was maintained as a control. A bottle containing uninoculated Skimmilk was maintained as a sterility control. All Skimmilk-fatty acid combinations were sterilized by autoclaving at 121 C for 10min. To completely fill these containers to approximately 375 ml, 225 ml of Skimmilk previously sterilized in l-liter volumes in 2-1iter flasks, were added to each bottle. The Skimmilk sterilized in the l~liter volumes used to fill the storage bottles, was cooled rapidly in a water bath, pooled in a 4——-liter flask and inoculated with heat-shocked spores of _C. botulinum Type 62 A. The concentration of spores was 2 x 103 per ml. An air space of approximately 5 ml re- mained in each bottle. Samples were taken for bacterial cell counts at 0 hr and at 24 hr intervals. The counting medium was YESB medium and counts were made in Prickett tubes. Each bottle was inverted 3 11 times before sampling to adequately mix the Skimmilk before sampling. Contaminants were tested for by transferring 1 ml of‘sample to 25 m1 of Brain-Heart Infusion broth in a 25 x 150 mm test tube. The tubes were incubated at 37 C for 48 hr. One ml was transferred from each tube to a similar tube and incubated for an additional 48 hr. This transfer was necessary since the opacity of the Skimmilk masked the turbidity produced by the growth of any contaminant which might have been present. To determine the effect of agitation on the development of heat- shocked spores of C. botulinum Type 62 A, one series of samples was not shaken. Five bottles of each Skimmilk-fatty acid mixture were prepared for this study. To provide some basis for comparison a simple peptone medium, containing 4% Trypticase, was used in a series of experiments on the effect of fatty acids on culture development of C. bowling Type 62 A. This peptone medium was selected as it represents a tryptic digest of casein. A 40% solution of Trypticase was prepared in distilled water and extracted 4 times with n-hexane (Skelly Solve B) to remove any fatty acids. Four hundred m1 of the 40% solution of Trypticase were extracted with an equal volume of n-hexane. Each extraction lasted 15 min and was carried out in 2-liter flasks on a Burrell wrist—action shaker. The solvent was removed, pooled and. fresh solvent added to the Trypticase solution. The solvent was evaporated and the residue resuspended in 10 ml of n-hexane. Following extraction the 40% Trypticase solution was heated to 95 C for 10 min to remove residual solvent and a 4% Trypticase medium was prepared, containing 2. 5% sodium thioglycollate-buffer solution. The buffer solution consisted of 4% sodium thioglycollate, 4% potassium phosphate—di—basic and 1% sodium bicarbonate. Culturing was under- taken in screw—cap test tubes rather than bottles to conserve medium; 12 since these samples were not agitated changing containers did not seriously affect the experimental design. Other studies have shown that the organism grew well under these conditions. A duplicate series of samples were set up in 12—oz bottles and were agitated. Each 12—oz screw-cap bottle received 375 ml of the Trypticase medium and each screw-cap tube received 28 ml of the medium. Fatty acids were tested at a concentration of 0. 001%. All 10 fatty acids were tested in the bottles and only 4 acids were tested in screw-cap tubes (caprylic, capric, myristic, and stearic acids). Eight tubes of each fatty acid- Trypticase mixture were prepared. The fatty acid—Trypticase mixtures were sterilized by autoclaving for 10 min at 121 C. Each bottle and tube received heat-shocked spores of _C. botulinum Type 62 A at a final spore concentration of 103 spores per ml. Incubation was at 18 C. Samples for bacterial counts were taken at 0 hr and at 24 hr intervals. Counting procedures were identical to the method previously described. Each bottle from the agitated series was inverted 3 times prior to sampling. The tubes were sampled by selecting a tube from each replicate set of 8, a 1 ml sample removed and the tube discarded. The remaining tubes within a replicate set were not disturbed. To determine the effect of the extracted material from the 160 g of Trypticase the material was added to 375 ml of the 4% Trypticase medium in a 12~oz bottle. This sample was incubated at 18 C and agitated at the time of sampling. The initial spore concentration was 103 per ml. Tissue Cell Culture Study The use of tissue cell cultures for the detection of other bacterial toxins (Rope e_t a_1., 1957; Gabliks and Solotorovsky, 1961) encouraged the use of tissue cell cultures for the assay of purified toxin and toxin 13 produced in Skimmilk. Lamanna (1959) proposed that tissue culture of mammalian cells would not be affected by botulinus toxin. In an effort to. show that other mammalian tissues are affected by botulinus toxin and to develOp' a better test for the detection of the toxin, various tissue cultures were studied using cytotoxicity as the index of toxic action of the toxin of C botulinum Type 62 A. Cell cultures of Mouse C3H, Osgood J-lll, Mouse L Cell, Embryo Monkey Kidney, Detroit-6-Parent and Detroit-6-Yale Clone were used in this study.1 Based on their reproductive capacities each culture was planted at various initial concentrations; Mouse C3H at 100, 000 cells per ml, Embryo Monkey Kidney at 75, 000 cells per ml, Detroit-6-Parent and Detroit-6-Yale Clone at 125, 000 cells per ml, and Osgood J—lll and Mouse L Cell at 150, 000 cells per ml. Cell stock suspensions were prepared by trypsinizing a monolayer cell sheet, washing the cells in cold buffer at pH 7, and replanting the cells in sterile glass bottles. These bottles were used as culture reservoirs. Cell concentrations were determined by microscopic examination of the cells stained with gentian violet in a Petroff—Hauser chamber. Cytoplasmic material was removed by adding citric acid to the staining preparation and the cell nuclei were counted. Tissue cell cultures were transferred from the reservoir bottles and planted at cell concentrations as listed above in 25 m1. plastic tissue culture flasks. Cultures were used when confluent monolayers were evident. The growth medium (Table 1) was removed and replaced with test medium. The test medium was composed of 10% horse serum (pretested for cytotoxicity) in Medium No. 199 (Cappell Laboratories), The medium contained phenol red as an acid—base indicator. The toxin 1Obtained through the courtesy of the Virus Research Division, Parke,Davis and Co. , Detroit, Michigan. 14 Table 1. Composition of Tissue Culture Growth Media Culture Type Media Composition Embryo Monkey Kidney Eagle's Tissue Culture Medium 15% Tryptose Phosphate Broth 10% Calf Serum Mouse L Cell Eagle's Tissue Culture Medium 20% Horse Serum Osgood J—lll Eagle's Tissue Culture Medium 20% Human Serum Detroit-6-Parent and Hank' 8 Solution Detroit-6—Yale Clone 40% Human Serum Mouse C3H Eagle's Tissue Culture Medium 10% Human Serum 15 produced in the Skimmilk was contained in the material harvested from the clear liquid portion of Skimmilk remaining after incubation in the presence of C. botulinum Type 62 A at 18 C for greater than 90 days. Purified Type A toxin was also used.1 The volume of the toxin produced in the skimmilk in each tissue culture flask was as follows: 1. 0, 0. 5, 0. 25 and 0.1 ml. The purified toxin was diluted in a 1%. saline solution so that the final concentration of toxin in the flasks was 1. 0 x 10"5 (13 1.1350), 1.0 x 10-6 (1.3 1.1150), 2.0 x10'6(0.65 LD50) and 5.0 x10'6 ml (0. 26 LDSO). The LD50 of the toxin produced in the Skimmilk was calculated to be 0. 000033 m1, and the LD50 of the purified toxin was calculated to be 0. 00000075 ml. The method of Reed and Muench (1938) was used in calculating the LDSO. The volume of liquid in each flask was adjusted to 6 ml with medium and the pH was adjusted to pH 6. 8 to 7. 0 with a 1% solution of sodium bicarbonate. Duplicate controls of heat inactivated purified toxin, and heated toxin, produced in the skim- milk, (90 C for 30 min) were maintained. Cell controls of test medium in the presence of cells were also employed. Antiserum against the toxin was also used as a control at a dilution of 1:500. The Skimmilk in which the toxin was produced was filtered through a bacterial retentive sintered glass filter to remove all cells. All flasks were incubated at 37 C for 72 hr and observed for toxic action at 24 hr intervals. The tissue cultures were recorded photographically. 1Obtained through the courtesy of the U. S. Army Biological Laboratory, Fort Detrick, Fredrick, Md. RESULTS Culture Development of C. botulinum Type 62 A in Skimmilk The results obtained on the growth of _C_. botulinum Type 62 A in Skimmilk are shown in Tables 2, 3, 4 and 5. Each value reported is an average of three replicate counts. A summary table showing the relationship of culture development of C. botulinum Type 62 A with toxin production, visual and olfactory changes in Skimmilk is presented in Table 6. Studies at 18 and 13 C. At 18 C the samples containing 10, 000 heat—shocked spores per ml remained in the lag phase of culture development for approximately 9 days (Table 2). It was interesting to note that the total viable count decreased 90% during this period. Since the Skimmilk was not heated prior to plating the total count ob— tained on each Skimmilk sample represented vegetative cells and/or spores which may have germinated on the plating medium without heat- shocking at the time of plating. The decrease may be due to the failure of the spores to grow without heat—shocking or the failure of the spores to remain viable in the Skimmilk after germination. After 8 days of storage reproduction was rapid and the cells attained a level of 4. 9 x 107 per ml on the 22nd day. A slight amino odor, but no visible deterioration of the Skimmilk, was detected after 10 days of storage. The amino odor became increasingly more pronounced on subsequent storage until a strong putrefactive odor was noted. This occurred after 26 days of storage and after the population attained a level of greater than 4. 9 x 107 cells per ml. Although the identification of the amino odor was not attempted it might signify early utilization of the Skimmilk l6 17 Table 2. Development of _C_. botulinum Type 62 A Spores Inoculated Into Skimmilk at Different Concentrations and Incubated at 18 C Days of Approximate Initial Inoculum per m1 Storage 1.0 100 l, 000 m, 000 01 7 64 800 5, 500 z 42 212 100*2 1, .2002 4 3 8 100 800 6 l 3 43 530 8 1 2 40 730 10 l 1 33 2,200 12 1 1 50 5,100 14 1 2 37 1,500,000 16 1 1 5 2,600,000 18 1 1 9 6,600,000 20 1 1 5 2,600,000 22 1 1 14 49,000,000 24 13 32 38 -- 26 430 800 1,000 -- 28 530,000 1,000,000 1,600,000 -- 30 4,300,000 7,300,000 8,500,000 -- lZerohrcounts representspores. 2Represents the number of vegetative cells per ml in Skimmilk. 18 substrate, since the odor was noted only in those cases when subsequent microbial growth occurred. Sterile Skimmilk controls and inoculated Skimmilk stored at inhibitory temperatures of 4 and 7 C did not contain any unusual odors. The presence of the amino odor was used, there— fore, as a definite indication of metabolism of the Skimmilk substrate by the microorganism. In all cases, deterioration of the Skimmilk eventually occurred within 4 to 6 days after the amino odor was first noted (Table 6). Toxicity tests performed after 14 days of incubation indicated possible toxin production since one of the 4 mice injected with this material died within 24 hr. On the 16th day of storage at 18 C the count was approximately 2. 6 x 106 per ml, and all animals injected with this Skimmilk died within 36 hr. Control mice injected with sterile skim~ milk and mice receiving the inoculated l6-day—old Skimmilk which had been heated, survived. On the 26th day of storage a strong putrefactive odor became evident. The first visual changes were observed after 28 days (Table 6). Based on the exponential phase of culture development the gener~ ation time (g) was 13 hr, calculated by the method of Monod (1949). This figure is based on one trial using replicate tubes. There is a dif- ference in the calculated generation time of 6 hr between the organisms inoculated at the 10, 000 level and those inoculated at 1, 000, 100 and 10 levels (Table 6). The limitation of the method of calculating the gener— ation times was i: 2 hr. If a comparison is made between storage temperatures at an initial level some differences in the generation times can be seen. There is no significant difference between the generation. time of the organisms at the 10, 000 level at 18 C and the organisms at 10, 000 level at 13 C (Table 6). Comparing the initial levels of l, 000, 100 and 10 at both temperatures, 18 C resulted in generation times of 19 7, 7 and 7 hr respectively, while 11, 11 and 8 hr were obtained, respectively, at 13 C. The greatest difference in the generation times was 4 hr. A shorter time can be expected at 18 C since it approximates more closely the optimum temperature for the organism (30‘to 37 C), than does 13 C. At 18 C with an initial concentration of approximately 1., 000 spores per ml the duration of the lag phase was about 22 days (Table 2). This represents an increase of 13 days in the lag phase when compared to the lag obtained at an initial level of 10, 000 cells per ml. There was a decrease of 99% in cell count during the lag phase when the initial inoculum was 1, 000 cells per ml. A test for toxin was undertaken after 26 days of storage. All animals survived which indicated the absence of toxin in the Skimmilk. The cell count was approximately 1, 000 per ml and a slight amino odor was noted. After 28 days of incubation the viable cell count was approximately 1. 6 x 106 per ml, and all test animals injected with this Skimmilk died within 24 hr. Putrefactive changes were observed after 32 days of incubation. The generation time under these conditions was 7 hr. At 18 C with initial inocula of 100 and 10 spores per ml the lag phases were 22 days in both cases (Table 2). In the former the count decreased more than 98 %, and in the latter the decrease was approxi— mately 86% during the lagphase. Rapid growth was initiated after 24 days of storage. All tests for toxin at both 24 and 26 days of incubation were negative. On the 28th day the cell counts were 1. 0 x 106 and 5. 3 x 105 per ml, respectively. All animals injected with this material died within 24 hr. No deaths were obtained in the. animals injected with control Skimmilk. This control material consisted of sterile and heated samples of stored Skimmilk. Visual changes, exemplified by precipi- tation of the casein, and olfactory changes, recognized by a putrefactive odor characteristic of C. botulinum, were noted after 32 days of incu- bation. The generation time was 7 hr in. both cases. 20 At the incubation temperature of 13 C the Skimmilk inoculated with an initial inoculum of 10, 000 spores per ml remained in the lag phase of culture development for 38 days (Table 3). A decrease of 97% in the bacterial count occurred during this period. Rapid growth of the organism occurred after 40 days of storage. An amino odor was noted after 42 days of incubation. The count was approximately 1. 0 x 106 cells per m1 on the 44th day. No toxin was present at this time. When the count reached 2. 0 x 106 per ml on the 46th day of incubation, however, all animals injected with the Skimmilk died within 24 hr. The character- istic odor of E. botulinum was noted after 48 days of storage, and the visual changes occurred 2 days later. The generation time based on the exponential phase of culture development was 14 hr. Although this generation time was slightly longer than the generation times calcu- lated for the lower initial inocula at this storage temperature the dif— ference was not appreciable when the degree of accuracy in calculating the generation times (i 2 hr) is considered. When the initial inoculum of spores was 1, 000 per ml and the incubation temperature was 13 C the lag phase was 44 days. A decrease of approximately 99% in the cell count was noted throughout this period. After 48 days of incubation rapid growth was observed and the population attained a level of 1. 0 x 106 per ml. An amino odor was noted but toxicity tests proved negative at this time. After 50 days of storage however, all test animals injected with Skimmilk died within 24 hr. No deaths were incurred in the control animals. Olfactory and visual changes, identical to those previously described, occurred on the 52nd and 54th day of incubation, respectively. The generation time was 11 hr. At 13 C the organisms in the Skimmilk at an initial level of 100 heat-shocked spores per ml exhibited a lag phase of 48 days (Table 3). A 96% decrease in cell count was noted during this period. Rapid growth 21 Table 3. Development of C. botulinum Type 62 A Spores Inoculated Into Skimmilk at Different Concentrations and Incubated at 13 C Days of Approximate Initial Inoculum per ml Storage 10 100 l, 000 10, 000 O1 6 67 900 5,300 z 72 462 5702 3,4002 4 5 24 240 1,700 6 2 20 160 1,200 8 3 23 250 1,200 10 2 18 270 1,300 12 1 9 230 670 14 1 9 67 470 16 1 9 80 300 18 1 5 47 730 20 1 3 84 200 22 1 3 29 370 24 l 10 17 300 26 1 26 24 300 28 1 14 18 330 30 1 34 11 230 32 1 41 11 170 34 1 34 19 270 36 1 33 22 230 38 1 27 24 300 40 1 23 20 600 42 1 33 62 ~— 44 1 42 63 1,000,000 46 1 38 1,000 2,000,000 48 1 63 1,000,000 8,000,000 50 1 1,000 2,200,000 -- 52 70 950,000 8,200,000 -- 54 1,000 2,800,000 -- -- 56 1,400,000 6,800,000 -- -- 58 6,300,000 -— —- -- 1 Zerolnrcounhsrepresentspores. zRepresents number of vegetative cells per ml in Skimmilk. 22 was initiated on the 48th day of incubation, and the cell count was approximately 63 per ml. Toxicity tests performed on a sample after 48 days proved negative. No amino odor was noted. After 50 days at 13 C the characteristic odor was evident. When the viable count reached 2. 8 x 106 per ml, on the 54th day, all animals injected with Skimmilk died" within 24 hr. Animals injected with this Skimmilk which had been heated survived. The putrefactive odor due to C. bOtulinum was noted on the 56th day of incubation with visual and olfactory changes occurring 2 days later. The generation time was 11 hr. The lag phase of the culture, initially inoculated into the Skimmilk at approximately 10 spores per ml and incubated at 13 C, was 50 days (Table 3). The decrease in count of the culture was approximately 83% during this prolonged lag phase. After 54 days of incubation the count attained a level of l. 0 x 103 cells per ml, and toxin was not present. A very slight amino odor was noted in the sample at this time. On the 56th day of storage 3 of 4 animals injected with the Skimmilk died within 36 hr. After 58 days all test animals died within 24 hr. Olfactory and visual changes occurred on the 60th day of incubation. The generation time was 8 hr. Studies at 10 and 7 C. The culture did not develop in the Skimmilk inoculated with spores of C. botulinum at approximate initial levels of 10, 000, 1, 000, 100 and 10 per ml and stored at 10 and 7 C. A gradual decrease in cell count over the test period of 56 and 76 days, respectively, was noted (Tables 4 and 5). At 10 C the toxin assays carried out on the 7th,. 14th, 28th and 56th day of incubation indicated that toxin was not present in the Skimmilk. No visual or olfactory changes were noted at any time during the 56 days of the experiment (Table 6). At 7 C tests for toxin performed on the 14th, 26th, 40th and 76th day of incubation were negative. No visual or olfactory changes were noted at any time during the 76 day incubation period at 7 C (Table 6). 23 Table 4. Development of C. botulinum Type 62 A Spores'Inoculated Into Skimmilk at Different Concentrations and Incubated at 10 C Days of Approximate Initial Inoculum per ml Storage 10 100 ' 1, 000 10, 000 01 5 56 600 5, 400 2. 63 4.32 4302 4,41002 4 7 42 400 3,300 6 4 35 270 3,200 8 3 31 500 4,300 10 2 24 130 2,600 12 2 23 100 2,700 14 Z 24 100 1,000 16 2 18 230 1,700 18 2 19 200 1,300 20 2 12 130 6,000 22 3 13 200 4,000 24 1 17 100 4,600 25 1 19 100 4,000 26 2 19 100 1,300 28 1 18 100 1,300 30 1 12 100 1,000 32 1 14 83 1,300 34 2 13 90 1,000 36 1 5 83 1,000 38 1 5 70 1,000 40 1 4 67 1,000 56 1 10 100 1,000 1Zero hr counts represent spores. 2Represents number of vegetative cells per ml in Skimmilk. 24 Table 5. Development of C. botulinum Type 62 A Spores Inoculated Into Skimmilk at Different Concentrations and Incubated at 7 C Days of Approximate Initial Inoculum per ml Storage 10 100 1, 000 10, 000 01 10 73 800 5,200 2 82 ' 512 5002 5,7002 4 6 36 300 4, 500 6 5 43 190 2,300 8 5 21 280 5,100 10 4 40 380 5, 700 14 4 36 330 2, 700 16 4 27 500 2,600 18 5 29 330 3, 700 24 3 30 150 1, 000 26 4 24 130 730 28 2 21 60 1,900 34 3 16 100 l, 500 38 3 15 130 2,000 40 2 15 110 1, 300 42 4 16 120 1,500 44 4 16 120 1, 700 46 6 16 100 1, 500 74 3 8 60 790 76 4 7 60 600 1Zero hr counts represent spores. 2Represents number of vegetative cells per ml in Skimmilk. 25 owcmflo OZ». Exop OZN Spa/ohm OZH OZ OZ .HZ OZ 1.. 1- on OH DZ 02 HZ OZ .1 I: on OOH oz oz , ez oz -- -- os ooo .3 oz oz ez oz -- -- os ooo .3 1W N. oedomHoQEoH ommHon OZ OZ HZ OZ u- .1 pm 0H DZ 02 HZ OZ .. .. om OOH oz oz ez oz, -- -- om ooo; oz moz N.ez Hoz .. -- om ooo .3 O OH manoeuomEmH omeOPm co co om ooH xv; mm m om OH mm om em co.” x mfm co 2 we on: em 2 om .3 x N .N 3. S E. ooo .3 om 3. 3. .3 x 3N so 3 mm ooo .3 O Ma oudfimuomgoefl. ommHOom NM mm mm mod xm.m ow e um OH mm mm mm ooH x o .H mm N. MN 2: Nm mm 3 .23 x o .3 3. s S ooo .3 ow ow 3 .3 x 3N oo 3 o ooo .3 0 ma mhflumummaom. owMMOHm Amanda 3sz SE\.O.GV Gov Ayes 3sz :5\.ocv Hmdmt/ .uommHO gamma/w cowuodpohnm Qua/0H0 65TH omMQnH wmq EsfidoocH mowflmzfiu 3x08 33on one oH0mmm :w: mo SOSGHSQ 133: Mo 08TH mo 08TH. ..odoU Sou ommoeooQ monsomhomEoH msofium> Hm pmHmQSoQH paw mohomm 4 me osz gcflduon ..w £33 3335005 MSEEfiMm a“ mowcmflO Hmsmf? pad .Qofiodponnm cinch. JGoEQ0$>oQ 6.39350 .0 63mm. 26 Culture Development of C. botulinum Type B in Skimmilk The data showing the effect of inoculating spores of C. botulinum Type B in Skimmilk are presented in Tables 7, 8, 9 and 10. A summary table showing a comparison of Type B with Type 62 A is presented in Table 11. Studies at 18 C. Heat-shocked spores of C. botulinum Type B inoculated into sterile Skimmilk at an initial concentration of 1, 000 spores per ml and stored at 18 C had a lag phase of 18 days (Table 7). The lag phase with Type B was 4 days shorter than the lag phase obtained with Type 62 A culture under similar conditions (Table 11). The maxi- mum decrease of Type B culture during the lag phase was 70%. Tests for toxin production at the end of 18 days of incubation were negative and the bacterial population was 8. 6 x 102 cells per ml. Growth was rapid on the 20th day of incubation and the bacterial count was 1. 3 x 104 cells per ml. No toxin was present. On the 22nd day of incubation the population was 1. 6 x 106 cells per ml and all animals injected with a sample of Skimmilk died within 36 hr. No deaths were obtained in the control animals. Coagulation of the milk protein and the appearance of the putrefactive odor of Clostridium were evident on the 22nd day. A slight amino odor was noted one day prior to the appearance of the putrefactive odor. The generation time was 7 hr. The data on both types of cultures, inoculated into Skimmilk at comparable levels and stored at 18 C, were generally in agreement. Type B culture produced a more rapid destruction of the substrate than did Type 62 A culture as visual and olfactory changes occurred 10 days earlier (Table 11). Type B culture produced toxin 6 days earlier than Type 62 A and had a shorter lag period. At 18 C using a concentration of 100 spores per ml the culture remained in the lag phase for 30 days (Table 7). There was a difference 27 Table 7. Development of C. botulinum Type B Spores Inoculated Into Skimmilk at Different Concentrations and Incubated at18 C Days of Approximate Initial Inoculum per ml Storage 10 100 1, 000 01 21 170 2, 200 2 192 1802 1, 7002 4 15 180 1,700 6 14 200 1,500 8 15 130 1,400 10 11 130 1,400 12 11 120 1,300 14 9 110 830 16 7 77 660 18 8 87 860 20 4 40 13,000 22 4 53 1,600,000 23 6 71 -~ 24 6 84 ~- 26 6 120 -— 28 6 120 —— 30 3 140 -— 32 4 110,000 —~ 34 4 3,000,000 ~- 36 5 6,600,000 —— 38 4 -~ -— 40 5 ~- -- 56 5 —= -- 1 Zero hr counts represent spores. 2Represents the number of vegetative cells per ml in Skimmilk. 28 of 8 days in the lag phases of Type B and Type 62 A at this level at 18 C. Type B culture had a longer lag phase. The decrease in total count during this lag phase (Type B) was 69%. Tests for toxin at the end of the lag phase were negative. - After 32 days of storage toxin was present as 3 of 4 ahimals injected with Skimmilk died within 48 hr. The count at this time was 1. 1 x 105 cells per ml. On the 34th day of incubation the count was 3. 0 x 106 cells per ml and all mice died in 24 hr. ‘No deaths were obtained in the control animals. A slight amino odor was noted after 32 days of incubation, and the strong putrefactive odor of Clostridium was present after 34 days of storage. Visual changes were noted on the same day. The generation time was 10 hr. The data obtained with both types of cultures, inoculated at 100 spores per ml and stored at 18 C, agreed fairly well. Type 62 A culture had a gener— ation time of 7 hr, and Type B had a generation time of 10 hr. The lag phase was 8 days longer with Type B culture. The generation time was not appreciably longer with Type B culture. At the 100 per ml initial level the production of visual and olfactory changes became evident at the same time (Table 11) . When the initial level of spores of C. botulinum Type B was 10 per ml and the incubation was at 18 C a decrease of 85% in cell count was noted over the test period of 56 days (Table 7). No growth, toxin production, visual or olfactory changes were noted at any time. Type 62 A culture at comparable levels and incubation temperature brought about visual and olfactory changes of the Skimmilk in 32 days (Table ll)o Studies at 13, 10 and 7 C. With initial inocula of 10, 000, l, 000, 100 and 10 per ml in sterile Skimmilk a gradual decrease in cell popu- lation was observed over the test period of 56 days (Tables 8, 9 and 10). The storage temperatures of 13, 10 and 7 C did not permit the growth of C. botulinum Type B. No toxin was produced nor was there any evidence of visual or olfactory changes during this prolonged 29 Table 8. Development of C. botulinum Type B Spores Inoculated Into Skimmilk at Different Concentrations and Incubated atl3 C Days of Approximate Initial Inoculum per m1 Storage 10 100 1,000 10,000 01 20 183 2,200 16,000 2 163 2002 1, 9302 21, 0002 4 15 170 1,800 12,000 6 16 190 1,760 12,000 8 15 170 1,430 16,000 10 11 180 1,760 13,000 12 11 170 1,470 11,000 14 10 190 1,330 12,000 16 9 200 660 14,000 18 11 180 630 12,000 20 7 77 570 6,600 22 8 70 630 5,300 23 4 76 430 9,300 24 4 84 430 9,000 26 6 88 460 6,300 28 4 80 530 6,300 30 3 82 330 2,300 32 3 93 260 ‘2,300 34 3 83 360 2,700 36 2 42 160 2,000 .38 2 51 230 1, 700 40 Z 37 200 2,000 56 1 28 100 2,300 1Zero hr counts represent spores. zRepresenhstheInunber ofvegeiafive ceUs perrnlin.skunnnflkr 30 Table 9. Development of C. botulinum Type B Spores Inoculated Into Skimmilk atmDifferent Concentrations and Incubated at 10 C Days of Approximate Initial. Inoculum per ml Storage 10 100 1, 000 10, 000 01 24 160 3,000 16, 000 2 162' 190‘2 1, 8002 18, 000‘2 4 19 150 1, 900 17, 000 6 18 160 1, 500 20, 000 8 16 150 2, 000 19,000 10 16 160 l, 500 20, 000 12 15 160 1, 500 20, 000 14 17 150 1, 300 18,000 16 15 120 1,300 15,000 18 17 140 1,100 16,000 20 13 130 1, 000 16, 000 22 13 120 1, 000 16, 000 23 11 94 1,200 13,000 24 11 9.3 1,300 11,000 26 13 120 1, 000 14, 000 28 13 120 900 11, 000 30 9 120 1, 400 9, 300 32 10 110 1,400 9, 300 34 8 1.10 1,500 8,300 36 10 69 900 12, 000 38 10 72 1,100 10,000 40 8 78 1, 100 9, 000 .56 5 34 500 9, 300 1 Zero hr counts represent spores. 2Represents the number of vegetative. cells per ml in Skimmilk. 31 Table 10. Development of C. botulinum Type B Spores Inoculated Into Skimmilk. at Different Concentrations and Incubated at7 C Days of Approximate Initial Inoculum per m1 Storgge 10 100 1, 000 10, 000 01 20 180 1, 960 15, 000 2 172 1902 1, 8003 16, 000'2 4 19 150 1,600 15,000 6 17 120 1,800 15,000 8 15 140 1,700 15,000 10 15 170 1,500 17,000 12 16 180 1,500 17,000 14 15 130 1,900 18,000 16 13 140 1,100 14,000 18 14 120 1,000 14,000 20 13 93 1,300 14,000 22 13 97 1,300 12,000 23 11 100 1,700 11,000 24 12 100 1,200 10,000 26 9 120 1,200 10,000 28 10 120 1, 200 11, 000 30 6 110 870 9,000 32 7 98 800 8,600 34 8 120 860 6,000 36 8 100 960 6,000 38 6 91 830 4,300 40 7 94 1,000 6,300 56 8 50 430 5,300 1 Zero hr counts represent spores. 2Represents the number of vegetative cells per ml in Skimmilk. 32 owcmgo 023” 3.338“ oZN Lox/Ohm 023 oz oz oz oz ez ez oz oz o? o? 3 oz oz oz oz ez ez oz oz o7 o7. o3 oz oz oz oz ez .3.z oz oz o? o? ooo .3 oz oz oz oz ez ez oz oz o7. o7 ooo .3 o s oz oz oz oz ez ez oz oz omA omA 3 oz oz oz oz ez ez oz oz of omA o3 oz oz oz oz ez .3.z oz oz of omA ooo .3 oz oz oz oz ez ez oz oz om». omA ooo .3 o3 oz oo oz oo ez om oz o omA om 3 oz 3 oz om ez em oz 33 fix 3. o3 oz 3 oz Nm ez om oz 33 of N3. ooo .3 oz om oz 3. ,3z 3. oz 3.3 omA om ooo .3 o 3 oz N... moz Nm Nez wN 3oz e omA NN 3 3..., Nm 3 Nm Nm oN 3 s om NN o3 _ NN N... NN Nm NN wN s s 3 NN ooo .3 o 3 $3.3 $3.33 to A9393 :8\ .65 m .UOHnm c388 0839 :m: ommfinfi wmiH 133:3: omdnoum moWCMQO mo mEMH mo BETH. mo GOSMHSQ mmusudHoQEoH msozum> um poomQSofl pcm £984 mdozhm> 3.3m vfizgflfixm SCH ovumHSoocH monomm m pad 4 No momzH. 8353:1500— ..U mo “CoEQoHoN/ofl 93333.50 3: mo GOmCmQEoO .: @389 33 incubation period. At 13 C the cell populations decreased 89, 95, 86 and 95% when the initial spore levels were 10, 000, 1, 000, 100 and 10, respectively. At incubation temperature of 10 C the decreases in cell counts of all the initial levels were approximately 82%. Similarly, at 7 C the decreases in cell counts were approximately 75%. In summary, at 18 C C. botuliniin Type B grew, produced toxin, visual and olfactory changes characteristic of Clostridium when inocu— lated into sterile Skimmilk at levels of 1, 000 and 100 per ml (Tables 7 and 11).. At this storage temperature an initial inoculum of 10 spores per ml did not deve10p into an active vegetative culture within 56 days. Regardless of the initial inoculum, incubation at 13, 10 and 7 C did not permit the growth of the organism. When the increase of bacterial population became evident a slight amino odor was present in all cases immediately before the appearance of toxin. Toxin could not be found in the Skimmilk at the time of the appearance of the amino odor. The appearance of the amino odor immediately beforetoxin production corresponds with the reaction of Type 62 A culture in Skimmilk. Effect of Fatty Acids in Skimmilk on Culture (Development The data showing the effect of various fatty acids, at a concen- tration of 0. 01%, in Skimmilk, on the culture development of spores of C. botulinum Type 62 A are presented in Tables 12 and 13. Culture development in agitated Skimmilk. Spores of C. botulinum developed in Skimmilk and in 22 days the cells attained a level of 9. 3 x 105 per ml (Table 12). The lag phase of the culture in this skim- milk sample, which did not contain any fatty acids, was 20 days. Butyric, caproic, arachidic, and oleic acids exerted little if any effect on the lag phase of culture development. In the presence of caprylic, lauric, and palmitic acids the lag phases were 24 days as compared 34 Table 12. Effect of Various Fatty Acids (0. 01%) in Skimmilk on the Germination and Outgrowth of Spores of C. botulinum Type 62 A-— Samples Shaken _— Days of Fatty Acids Incubation Control Butyric Arachidic Oleic Caproic Caprylic at 18 C (no./ml x 100) 03 18.0 18,0 18.0 18.0 18.0 18.0 3 18.0 12.0 16.0 11.0 14.0 16.0 6 1.3 1.8 1.0 1.1 1.1 1.2 9 .55 7 .7 .8 5 5 12 .35 .4 .3 .4 2 2 15 .30 .3 .3 .3 .2 .3 18 .20 1 .2 .1 .2 <.1 19 .15 1 .1 <.1 1 <.1 20 .15 1 .1 .1 .1 1 21 6.0 .4 .7 .6 <.1 .1 22 9300.0 10.0 40.0 120.0 <.1 <.1 23 -- 25000.0 32000.0 61000.0 .1 .1 24 -- -- -- -» 1.5 .1 25 —- —— -— ~~ 7000.0 6.5 26 -— -- -- -- 31000.0 9000.0 2.7 -- -_ -- -- -_ _- 28 -— -- -- —- -- —- 32 -- —- -- -- —— -- 1Mixture of all acids, each acid at 0. 001% (total acid concentration 0. 01%). 2Mixture of all acids, each acid at 0.01% (total acid concentration 0.1%). 3Zero hr counts represent spores. 35 Laurie Myristic Capric Stearic A.A.M.1 A.A. E.Z Palmitic 18.0 11. 18. 18. 18.0 l4. 18. 18.0 18. 14. 12.0 18.0 <. <. <. 1200 . 0 40000 . 0 4100.0 60000,0 >10000.0 >10000.0 >10000. 0 36 with the 20 days lag period obtained in the control. Rapid growth of the culture in the presence of these acids was initiated after 24 days of incu- bation at 18 C. Myristic, capric, and stearic acids extended the lag phase of culture development of C. botulinum Type 62 A in Skimmilk 6 days. This extension of the lag phase for 6 days is an appreciable increase over the 20 day lag phase of the culture in the control Skimmilk medium. A mixture of all 10 fatty acids in Skimmilk, each at a concentration of 0. 001%, completely inhibited culture development over the test period of 32 days. When each fatty acid was added to Skimmilk at a concen- tration of 0. 01% (total concentration of acid was 0. 1%) growth was also completely inhibited during the test period. It is apparent that certain individual fatty acids were inhibitory to the development of the culture, but the mixtures of all acids were more effective. Althouth each of the 3 most inhibitory acids at a concentration of 0. 01% extended the lag phase 6 days, the mixtures of acids consisting of individual acids at a concen- tration of 0. 001%, increased the lag phase for periods of time greater than 12 days (Table 12). A mixture of myristic, capric, and stearic acids each at 0. 001% may have been as effective in prolonging the lag phase of culture development. Although sampling was terminated after 32 days no physical and/or olfactory changes were evident in the Skimmilk after 45 days at 18 C. This indicated that the mixtures of all 10 acids, each at one-tenth the concentration at which the individual acids were tested, gave greater inhibitory action. Culture development in unagitated Skimmilk. The study on unagitated medium was devised to determine the effect of introducing air into the con— tainer at the time of sampling. Results showing the effect of fatty acids on culture development in unagitated Skimmilk are presented in Table 13. In Skimmilk the lag phase of culture development, determined from the growth curve, of C. botulinum Type 62 A was approximately 15 days. Of the four acids tested, caprylic acid had the least effect and the lag phase of the culture in the presence of this acid at 0. 01% concentration .mwhomm “ammonaoa mozdoo Ha oHon .AooH .o doHHHmHH—Goozoo HoHom H3303 oeHo .0 3m p.336 «Howe .mHUHom. HHe mo oeouxSN/HN .AoeHo .o GowpmHuCoocoo pHom H3303 oeHoo .0 pm pHom CHomo .mpHom HHm mo whoexHHZH 37 H.v H.v .. i i a- i ow H.V H.v c.0000 o.oooH© 06002.. .i I- pN H.V H.v on o.ooom 06on odoooHN .... mN N. H. N. H.HN 0;. o.ooHHu a- HVN N. H. N. N. H. H.H .. mN N. N. m. N. H. m. u... HN u- u... ..i In an .2. o.oooow wH u- u- -1 a- ..... .:. 0.00No N.H m. N. m. m. HN. HN. 0.0 0H H.H N.H o.H N.H mo. H.H m.H oH 0.x o.HNH o.HNH o.NH o.oH o.mH o.vH m o.wH o.wH o.wH o.wH o.wH o.wH o.wH mo 83 x 38\.o£ N.m.<.< e.H>H.<.< 3.33am ,oHpmHeze/H 3.250 033.230 HonucoU U wH pm. mp3o< zuumh coflworoozH . mo TAMQ .96vasz ooZ moHnHEwman< No 2:9. 83582553 ..0 Mao monomm mo 3.3393950 Hose 330346339300 33 so .VHHHEEHva a...“ AoeHo .3 33.3% 33.3mm mdoHHm> W30 poowwm .mH 033mm. 38 was 22 days. This represents an increase of 7 days in the lag phase beyond the control. Capric and myristic acids inhibited culture development for 23 days, and stearic acid extended the lag phase to 24 days (Table 13). The mixture of acids, at both concentrations, inhibited culture development for the entire test period. No growth was present in the unagitated Skimmilk after 40 days of incubation at 18 C (Table 13). Effect of Fatty Acids in Trypticase Medium on Culture Development The effect of various fatty acids, in a 4% Trypticase medium on the culture development of C. botulinum Type 62 A is presented in Tables 14 and 15. The fatty acids were tested at a concentration of 0. 001% in contrast to the previous study in which the concentration of fatty acids was 0. 01%. Culture development in agitated Trypticase medium. Butyric, caproic, lauric, arachidic, palmitic and oleic acids did not inhibit the growth of the culture. The lag phase of culture development in the ex- tracted Trypticase medium, the unextracted medium, and the presence of these 5 fatty acids was 3 days. The lag period of the culture was 5 days in the presence of caprylic and capric acids, and 6 days in the presence of myristic and stearic acids. The mixture of all 10 fatty acids, at a concentration of 0. 001%, extended the lag phase for 3 days, and the acid mixture with each acid at 0. 001% (total concentration of acid was 0. 01%) extended the lag phase for 4 days. The lag phases were 6 and 7 days, respectively." The material which was extracted from the 40% Trypticase solution and tested in the 4% Trypticase medium, extended the lag period for 3 days. The lag phase was 6 days. Culture development in unagitated Trypticase. Caprylic, capric, myristic, and stearic acids were tested in this study. In the control 39 Table 14. Effect of Various Farry Acids (0. 001%) in 4% Trypticase on the Germination and Outgrowth of Spores of C. botulinum Type 62 A--Samp1es Shaken Days of Incu- Fatty Acids bation Control1 Control2 Control3 Butyric Caproic Lauric at 18 c (nO./ml x 100) 06 9.8 8.7 9.5 11.0 10.0 12.0 1 9.0 6.9 7.0 9.0 8.2 10.0' 2 8.1 7.4 6.2 7.7 8.0 7.5 3 6.8 5.2 4.4 7.1 6.8 6.8 4 6,500.0 1,200.0 4.1 110.0 9.7 480.0 5 >100, 000. 0 >100, 000. 0 5. 0 >100, 000. 0 9, 300. 0 64, 000 0 6 -- -— 6.6 -- -- -~ 7 -- -— 2,400.0 -- ~~ -~— 8 ..- __ _. .._ M. _ _ 9 -_ _- __ __ _ - __ lHexanew extracted Trypticase ZUne’—,xtracted T rYPticase 3Trypticase extracted with hexane plus soluble material from Trypticase. 4Mixture of all acids each at 0. 0001% (total acid concentration 0. 001%). SMixture of all acids each at 0. 001% (total acid concentration 0. 01%). £’Zero hr counts represent spores. Oleic Arachidic Palmitic Stea 9.7 9.6 9.9 9.1 9.5 9.3 8.4 8.1 8.1 6.1 4.9 8.0 970.0 9.1 1,000.0 >100, 000.0 16, 000. 0 >100, 000.0 —- -— -— 26, 41 sample of extracted 4% Trypticase medium the culture developed in 3 days (Table 15). The lag phase of the culture in the unextracted Trypticase medium was also 3 days. The lag phase was 5 days in the presence of capric and myristic acids. Caprylic and stearic acids prolonged the period of culture development; the lag period was 6 days. The mixtures of acids, at concentration of 0. 001% and 0. 01%, affected the development of the culture; the lag phases were 6 and 7 days, respectively. Effect of Botulinus Toxin on Various Tissue Culture Monolayers The results of this study are found in Table 16 and in Figures 1, 2, 3 and 4. With the exception of Mouse L Cell culture all cultures tested were found to be adversely affected by botulinus toxin (Figure 1, Table l6)° The most sensitive cultures were Detroite6‘Parent and Detroit-6-Yale Clone. The cytotoxicity in the sensitive cultures was essentially the same in all cases. The confluent monolayer of cells was disrupted with the appearance of intracellular spaces. In all. cases of sensitivity early cytotoxicity of the monolayer occurred after 24 hr of incubation at 37 C. Further degeneration of the cells occurred, exhibited by granulation and shrinking of the cytoplasmic mass and of the nucleus. Long slender streams of cytoplasmic material connected masses of condensed cells (Figure 2F). In the final stages of degener— ation the cells were reduced to tightly packed masses of cells. At the end of the test period of 72 hr a few cells remained unaffected. Generally, the cytopathology exhibited by the cells was that of old, naturally deteriorating cells in culture. The 72 hr test period was selected since cells maintained in culture without a change of medium begin to show some slight deteriora- ation after storage for extended periods of time. After 72 hr the pH of 42 .mohomm anemonmop messoo H: OHon .3020 .0 CofiwnucoodoU pwom Hanoi 00H00 .0 p.80. Ludo mpflod 2.0 mo end—flange .Aobfioo .0 cofimficoocoo Bum $303 80500 .0 pm Ludo mpflon Ed .05 ensuing m .ommoflhcflpk Umpcdngxocbu .ommUSm>HH popomnuxo 16:88me 8.888.888 -- -1 -- -- -- E. -- 8 8.83 8.88.83 -- -- .. i 3 i w 2.8 8.2: 4 8.83.8 8.88 .2 8.888.83A 8.888 .887. .. E. 8 8.8 2.8 8.8 8.8, 8.83 8 8.83 .N -- -- 8 06 040 0.m 18. 0.N 06 a- u- m 8.8 18 8.8 8.8 8:8. 8.8 8.88.3. 8.88 .3 8 NJ» 0.0 N.m 0.0 0.m 1% 0.8 0.8 m NJ. 0.8 0.0 m.m 06 N6 mg 0.0 N 0.0 v.0 0.8 0.0 T0 0.0 8.8 10 H 8.8 N8 8.3 8.8 8.8 8.8 8.: 8.8 ..8 80H x HE\.o£ U 0H on 8 .mm 3% .< n .2 .< .< oflcnnmmu oflhmopm 0.38332 oHHQmU NHOLSQOU Lonpcou £0,333. mpflo/V Knox—mm 1505 mo 33mm Goxmflm poZ moamgmmuudfl N0 0858.0 my? .10. mo menomm wo £8.30pr50 pcw Cofimcfieth 93 Co ommoflmcwnH 00¢ CH. $1.00 .3 mpflnxw 5.0mm mnownm> Mo Hoowflm .mH oHQmH. .Qonu HoonHHdnHv .00mnH EdwomHu—Qm HE H 0858-2:wa HuonHHSnH .ESHHoocQ Luz/Ohm HoodHflcoo Hofidoo Edhomficdfln .conp VHHHE 0088,3993 “do: Ho HE H Hocm EDHHooE QHBOHw Hoochucoo Hofidoo conHN .ESHHooE QHBOHm >30 pofimuqou Hofiaoo 200H ..1 mcnfionoHHOZO QBEHXNE n ++++ EwHHonoHofno on H 0 H powwou pom 43 ++++ +++ +++ + ++++ 8 i. 8 N8 ++++ ++ ++ 8 ++++ 8 + 8 88 t++ ++ + 8 +8 8 8 8 8N ocoHo 8 8 8 8 8 8 8 8 NH 8898-8.8flo8880H ++++ ++ 8 8 +++i 8 8 8 N8 ++ + 8 8 ++++ 8 8 8 88 -8. 8 8 8 -u. 8 8 8 8N 8888.2 8 8 8 8 8 8 8 8 N8 888882 8.8.385 -w+++ +++M M+ - -8 1 M1 -uu-l- +- I- - 8 ..... N8 ....... ++++ +++ + 8 -- -- 8 8 88 +++ ++ 8 8 -- a- 8 8 8N 0 0 0 0 1: 1.. 0 0 NH HConnHuoupHouqu 8 8 8 8 -- -- 8 8 N8 8 8 8 8 -- -- 8 8 88 8 8 8 8 -- -- 8 8 8N 8 8 8 8 -- -- 8 8 NH HH80_A 888888 +++ ++ + + -- -- 8 8 N8 ++ ++ + 8 -- -- 8 8 88 + + 8 o -- -- o o 8N 8 8 8 8 -- -- 8 8 N8 :78 88880 -++++ ++++ M+++ - +++ -- -- - ++ + N8 - - 8 8 8 8 -- -1 8 8 88 8 8 8 8 -- -- 8 8 8N 8 8 8 8 -- -- 8 8 N; 3.0 88.822 8.4 8.8 8N8 H8 8.8.0 8.0.4 8.0.8 2.0.0 89:28.: 83:80 £088 VHHHH>H mo oEDHo> mo mHSOTH HHoU Ho 2:8 woowmm 0088.0wa mHo>wHoqu>H oHSuHSD 3638 CO ConH mDGHHsuom mo mHo>oiH wGH>Hm> wo pooEmH .0H 03.0% 44 the test medium became acid, as indicated by the change in color of the phenol red indicator from red to yellow. Since the production of. acid by metabolizing cells in culture is normally expected no significance was attributed to this change. A complete or partial change of the test medium would have prevented any change in the cells. The medium was not changed since the flasks which contained the toxin did not receive a change of medium. When the medium was harvested from the culture flasks after the test period, and injected into white mice, the toxin titer was not decreased. All mice injected with the unheated toxin produced in skim- milk died within 36 hr, while those mice receiving the same material which was heated lived. The toxin and antiserum mixtures from the test flasks were not toxic to the mice. 45 A- Heat inactivated B‘Cell control botulinus toxin (1. 0 ml) 72 hr 72 hr D-Botulinus toxin (0.1 ml) 72 hr (0. 25 ml) 72 hr Figure l. Botulinus toxin in Mouse L Cell tissue culture. Figure l — continued E—Botulinus toxin (0. 5 ml) 72 hr 46 F-Botulinus toxin (1. 0 ml) 72 hr 47 A- Heat inactivated toxin B-C e11 control (1.0 ml) 72 hr 72 hr c‘i" I, ’4. l . J.“ ‘ r I' . 8. C-Botulinus toxin D-Botulinus toxin (0.1 ml) 72 hr (0.25 ml) 72 hr Figure 2. Effect of botulinus toxin on Detroit-6-Parent tissue culture. Figure 2 — continued E—Botulinus toxin (0. 5 ml) 72 hr 48 F“ Botulinus toxin (1. 0 ml) 72 hr "P DISCUSSION Recent developments in ultra-high temperature pasteurization (UHT) have increased the storage life of milk but have not resulted in sterility (Tobias e_t a_1., 1955; Speck, 1961). Although sterility is not assured there is evidence of a state of ”practical sterility" being established. This term describes a state where not all bacterial spores are killed but the survivors are unable to grow and cause spoilage due to a number of factors: 1) degree of anaerobiosis in the container, 2) storage temperature, and 3) the formation during sterili- zation of various compounds inhibitory to spore germination. The increased storage life of this nonsterile food might present a public health problem if the heat resistant spores of C. botulinum survived the UHT pasteurization or if the spores present as post—pasteurization contaminants overcame these inhibitory factors and germinated. This study has shown that heat-shocked spores of C. botulinum Types 62 A and B were able to germinate and grow in sterile Skimmilk when stored at relatively low temperatures. Conditions for germination and growth varied slightly between the two types. The Type 62 A culture was able to grow and produce toxin in sterile Skimmilk at all inoculum levels when the temperature of incubation was 13 or 18 C. Type B grew in sterile Skimmilk only at 18 C, and only when the initial inoculum level of heat=shocked spores was 100 per ml or greater. Lower inocu- lum levels of Type B at temperatures below 18 C resulted in no growth, no toxin production, and no physical or olfactory changes. Invariably a slight, but distinctly noticeable, amino odor im- mediately preceded the appearance of botulinus toxin in the Skimmilk medium. No toxin was evident when the amino odor first became 49 Ilr 50 noticeable. Some speculation may be made at this time as to the origin and eventual fate of the compound causing this odor. Since the amino odor occurred in the Skimmilk medium at the beginning of active multiplication, it can be assumed that the odor indicated the early proteolytic activities of the culture. No such odor occurred in control samples of Skimmilk in which the organism failed to grow or in sterile Skimmilk stored for long periods of time. The amines present in the medium due to the early proteolytic activities might constitute a variety of structures depending upon the amino acid source. Since Skimmilk proteins are composed of a variety of amino acids the identification of the source amino acid would be very difficult. The presence of the amines could be attributed to a number of factors. Among these could be included the early degradation of casein into shorter units and the subsequent decarboxylation of the amino acids into amines. The amines could be detected by their characteristic odor in more advanced stages of putrefaction as part of the putrefactive odor of Clostridium. Typical reactions can be illustrated as follows: 1. H3N(CHZ)4CHNH2COOH HZN(CHZ)5NHZ Lysine C02 Cadaverine H H 2. th-ée CHZCHNHZCOOI-I C02 HZCHZNHZ Tyrosine Tyramine Both cadaverine and tyramine have very distinctive amino odors. All members of the genus Clostridium display an active amino acid metabolism. The nutritional requirements of organisms vary considerably and it has been shown (Nisman, 1954) that the principal chemical reaction by which C. botulinum obtains energy is coupled 51 deamination. This occurs when the culture is grown on amino acids as the sole source of carbon and nitrogen. The reaction, called the Stickland Reaction, is described as a coupled deamination between two amino acids Where one is the donor and the other the acceptor of protons. The reaction can be illustrated as follows: Alanine Glycine HZC HCOOH + NH3 + C02 \ N H I Alanine Proline 1 Milk constituents, which are water, protein, fat, carbohydrate, inorganic salts, and vitamins, can be placed into 3 groups on the basis of their solubilities: 1) some are in true solution, 2) some are partly in solution and partly in suspension or colloidal suspension, and 3) some are present entirely in the colloidal state. Casein, which is the principal protein of milk, is present in milk entirely in the colloidal state and can be utilized for growth and reproduction by C. botulinum. The utilization of the protein is accomplished through hydrolysis followed by the Stickland Reaction, under anaerobic conditions and in the presence of cofactors. Milk contains not only casein, which can be hydrolyzed into its component amino acids, but also some free amino acids which can be readily utilized by the organism in the Stickland Reaction. Although this study has shown that C. botulinum Types 62 A and B can grow and produce toxin in Skimmilk it must be pointed out that this study, carried out in experimentally inoculated sterile Skimmilk, investigated the natural development of the pure culture of C. botulinum. 52 The observation that toxin was produced in certain instances under these experimental conditions does not necessarily imply that the presence of this organism in pasteurized Skimmilk poses a health hazard. The assumption that C. botulinum spores would constitute the only microbial contaminant in milk is possible but unlikely. Undoubtedly other sporeforming or nonsporeforming bacteria would be associated with the contaminants responsible for the presence of go—stridiurn. The development of Clostridium in association with other bacteria, a condition more truly representative of nature, may considerably alter culture development and/or toxin production. The development of Staphylococcus aureus (Oberhofer and Frazier, 1961) is affected when grown in the presence of other organisms. Likewise, toxin production by C. botulinum (Grecz e_t a_1., 1959a) is reduced in the presence of other organisms. With respect to the possibility of C. botulinum becoming a health hazard in milk, the incubation temperature of milk today and its storage life have not approached the experimental con= ditions of this investigation. But as was previously stated, with the advent of new processes of pasteurization (UHT), longer storage periods at higher temperatures can be expected. As these factors approach the experimental conditions of this investigation, a health hazard, due to growth of C. botulinum in Skimmilk or whole milk, might become a reality. Literature reports on the inhibition of culture development of C. botulinum have emphasized complete inhibition of growth by certain fatty acids. Foster and Wynne (1948) reported that linoleic and lino— lenic acids at 0.1% concentration inhibited growth of C. botulinum. Roth and Halvorson (1952) have inhibited spore germination of C. botulinum using 0.1% rancid fatty acids. Grecz e_t a_l. (1959a) have found that in cheese a 2% concentration of mixed fatty acids became 53 the inhibitory level for C. botulinum. In the present study on the effect of fatty acids on the culture development of heat-shocked spores of C. botulinum Type 62 A individual fatty acids had a slight effect in partially inhibiting culture development, but a composite mixture of the 10 fatty acids appreciably extended the lag phase of culture develop- ment. In Skimmilk the composite mixtures of fatty acids at 0.1% and 0. 01% completely inhibited culture development of C. botulinum while in a 4% Trypticase medium the fatty acid mixture at 0. 01% and 0. 001% merely extended the lag‘phase by approximately 4 and 3 days, respectively. In all cases of partial inhibition of culture development the mixtures of fatty acids were more effective than any individual fatty acid at comparable concentrations. Mixtures of fatty acids more closely represent their natural distribution in milk. The concentration and type of fatty acid would be important in controlling the metabolic activities of C. botulinum in milk. Since the inhibitory effect on the culture is enhanced by increasing the spectra of acids it becomes evident that fatty acids are at least one of the constituents of milk which can partly inhibit the culture development of heat—shocked spores of C. botulinum Type 62 A. i Krukovsky (1961) has shown a high lipase activity in milk before I pasteurization. Hilditch and Longenecker (1937) made some attempts to characterize the component fatty acids in milk but not until recently i have Harper et al. (1961) made some critical analyses on the concen— tration of free fatty acids in milk. Their investigation was on the free volatile fatty acids in milk and they have found that butyric, caproic and caprylic acids, among others, were present. These three fatty acids were included in the present investigation. Although additional evidence of the presence of higher molecular weight fatty acid constituents of milk was not found in the literature it is not too unrealistic to expect the presence of these higher molecular species of acids in milk. 54 A comparison was made of the lag phases of the culture in agitated (sample inverted three times prior to sampling) Skimmilk versus un~ agitated Skimmilk (Table 17). The lag period of the culture in unagitated skirnmilk was 5 days shorter than the lag phase in the agitated Skimmilk. The introduction of air into the storage container during sampling appreciably extended the lag phase of culture development of C. botulinum. A difference of at least 2 days between the lag phases was considered to be significant, whereas i 1 day was considered to be within the limits of error in determining the lag phases. The lag periods of the culture in Skimmilk containing capric, caprylic, myristic, and stearic acids were 3, 2, 3, and 2 days shorter than the respective lag periods of the culture in the agitated skirnmilk. When the limits of error in determin- ing the lag phases are considered these differences were insignificant. Skimmilk without sodium thioglycollate, which was agitated at 2—day intervals as previously described, required a prolonged period (22 days) in order to establish favorable conditions for culture development (Table 2). In this Skimmilk, not containing sodium thioglycollate, the culture eventually developed. It is theorized that milk contained com— ponents which acted much in the same manner as sodium thioglycollate since the Skimmilk was eventually reduced to a level consistent with growth (Table 2). The agitation introduced air into the medium which temporarily exhausted the reducing capacity of this naturally occurring oxidation-reduction system. Important factors which affect the auto- reductivity of the Skimmilk medium are the time periods between agita- tion, the amount of air introduced during agitation, and the effectiveness of the method of dispersing the air throughout the medium. It is obvious that the 2-day period between the agitations employed in the initial studies (Table 2) delayed but did not inhibit the culture development of C. botulinum. Somewhat longer periods between agitations in the control sample of Skimmilk (Table 12) reduced the lag phase of culture 55 Table 17. Comparison of Lag Phases of Culture Development of C. botulinum Type 62 A Under Varying Conditions Length of Lag Phase Fatty Acids Skimmilk T rypticase Agitated Unagitated Agitated Unagitated (days) Control 20 15 3 3 Capric 26 23 5 5 Caprylic 24 22 5 6 Myristic 26 23 6 5 Stearic 26 24 6 6 Hp 56 development by approximately 2 days; this difference was considered to be a slight decrease when the lag phase in this control sample is compared to the lag phase determined from the data in Table 2. However, when the Skimmilk was not agitated (Table 13) the lag phase was decreased by 7 days. Trypticase medium containing sodium thioglycollate, as previously described, provided favorable conditions for culture development. Early trials indicated that the culture would not develop in 4% Trypticase solution which did not contain sodium thioglycollate. In the control 4% Trypticase medium containing sodium thioglycollate the lag phases of culture development in the agitated and unagitated samples were the same, 3 days (Table 17). No appreciable differences were observed in the lag phases of the agitated and unagitated samples containing capric, caprylic, myristic, and stearic acids. Marshall (1963) reported that in 22 days of incubation at 18 C, 0. 2% sodium thioglycollate in agitated Skimmilk sterilized at 121 C for 12 min and which contained approxi- mately 1, 000 spores per ml, did not reduce the lag phase of culture development of C. botulinum. In a similar series, Skimmilk samples which were not agitated Marshall (1963) showed that sodium thioglycol— late reduced the lag phase of development by approximately 14 days. The ability of the culture to develop more rapidly in Trypticase, under the same conditions of incubation and agitation as the Skimmilk medium, can possibly be attributed to the fact that Trypticase, an enzymatic digest of casein, may present a more favorable substrate for microbial attack than does the larger and less soluble protein of milk, casein. Although the oxidation of the sulfhydryl groups of the thio- glycollate ions probably occurred during agitation, the Trypticase medium readily became reduced and permitted the rapid development of the culture. The introduction of oxygen into the unagitated samples was limited to that which could diffuse into the Trypticase medium from 57 the surface. In these samples the sulfhydryl groups were not inacti— vated by the oxygen as in the agitated samples, and Trypticase medium was readily reduced when sodium thioglycollate was present. The agitated Trypticase medium, containing sodium thioglycollate, was also rapidly reduced and permitted the culture to develop in the same period as the unagitated medium. The prolonged lag phase of culture development of C. botulinum (Table 2) can be attributed to a number of factors. The inoculum level directly affected the length of the lag phase of culture development. When the inoculum level was low the lag phase was always longer than the lag phase obtained with a larger inoculum. This relationship was true at all incubation temperatures which allowed for culture develop— ment of _C. botulinum. Another factor which influenced the development of the culture was the presence of fatty acids in the medium. Some fatty acids were instrumental in prolonging the lag phase, although the concentration of acids tested exceeded (lO-fold increases in some cases) the concentration of these acids reported in fresh milk (Harper e_t a_1., 1961). It is important to consider that the mixtures of the acids were effective in completely inhibiting the development of the culture. In a 4% Trypticase medium, which was more suitable for culture develop— ment than Skimmilk, a fatty acid mixture containing individual fatty acids at a concentration of 0. 0001%, extended the lag phase for 3 days. This concentration of each acid was consistent with the concentration of the acids reported in milk by Harper e} a_._l. (1961). They reported that caprylic acid was present in fresh milk at a concentration of 0. 0023%. Undoubtedly many more fatty acids naturally occur in milk and it would not'be inconsistent to ascribe to them some effect on culture develop- ment of C. botulinum. The last factor responsible for the prolonged lag phase of culture development was the presence of air introduced into the Skimmilk during sampling. When the periods between samplings 58 were increased the lag phase became somewhat shorter and when air was not introduced by agitation during sampling the lag phase of cul— ture development became appreciably shorter. Results of the present investigation on the effect of botulinus toxin on tissue culture indicate that the toxin exerted a toxic effect on some tissue cultures. Not all mammalian cells show sensitivity to the toxin. Some of the cells showed a high degree of sensitivity to the toxin, whereas one tissue culture was completely refractive to the toxic action of the bacterial metabolite. In all cases of toxic reaction, dis— played by the tissue cultures, the action could be prevented by the addition of specific antitoxin or by heating the toxin before use. Botulinus toxin is highly specific in its action on the nervous system. The peripheral nerves are most highly affected, but as indi- cated by this investigation other cells in culture can likewise be affected by the toxin. Since in xix/_o, the action of the toxin on the peripheral nerves is extremely rapid, the effect on other tissues in the body are masked by the more pronounced effect on the peripheral nerves. Also, Lamanna (1959) stated that tissue cultures of mammalian cells would be unaffected by the toxin. The action on the nervous system is acute and the peripheral nerves would constitute the major site of attack but other tissues may also be affected. Under the conditions of the present investigation it was found that even though an organized cholinergic system is lacking in tissue cultures toxicity of the com- ponent cells was evident. Cytotoxicity of non-nervous tissue can occur. SUMMARY Clostridium botulinum Type 62 A grew and produced toxin in sterile Skimmilk at 13 and 18 C. At 10 C no growth took place in 56 days. _C. botulinum Type B grew and produced toxin in Skimmilk at 18 C but did not grow at 13, 10 and 7 C after incubation for 56 days. An amino odor was always detected at least 2 days prior to the pro- duction of lethal levels of toxin in the kimmilk. Lethal levels of toxin were detected before a visual change occurred. Caprylic, capric, myristic, and stearic acids, at a concentration of 0. 01% in agitated Skimmilk extended the lag phase of culture develop- ment for approximately 5 days over that obtained in the control. In unagitated Skimmilk these acids extended the lag phase of culture development for approximately 8 days. At a total fatty acid concen— tration of 0. 1% or 0. 01%, a mixture of the 10 fatty acids inhibited culture development; the culture failed to develop in 40 days. Under agitated and unagitated conditions caprylic, capric, myristic, and stearic acids, at a concentration of 0. 001%, in 4% Trypticase medium, did not markedly prolong the lag phase. A mixture of the 10 fatty acids at a concentration of 0. 01% or 0. 001%, in the agitated and unagitated Trypticase medium, extended the lag phase for approximately 3 days. Tissue cell cultures were affected by Type A botulinus toxin. Detroit—6-Parent and Detroit—6—Yale Clone cultures showed cytopatho- logical changes in the presence of the toxin. Mouse L Culture cells were not affected by the toxin. 59 BIBLIOGRAPHY Bonventre, P. F., and L. L. Kempe. 1959. Physiology and toxin production by Clostridium botulinum Types A and B. III. Effects of the pH and temperature during incubation on growth, autolysis and toxin production. Appl. Microbiol. 11374—377. Bronfenbrenner, J., M. J. Schlesinger, and S. C. Calazans. 1921. Typing of different strains of B. botulinus by immunologic methods. Proc. Soc. Exper. Biol. Med. fill-22. Day, L. E. 1960. Studies on the sporulation of Clostridium botulinum Type A. M. S. Thesis, Michigan State University. Denney, C. B., J. M. Reed, and C. W. Bohrer. 1961. Effect of Tylosin and heat on spoilage bacteria in canned corn and canned 1 mushrooms. Food Tech. 338-340 (July). Eagle, H., and G. E. Foley. 1956. The cytotoxic action of carcinolytic agents in tissue culture. Amer. J. Med. 2_1_:739-749. Evans, F. R., and H. R. Curran. 1943. The activating effect of sublethal heat on spore germination in mesophilic aerobes. J. Bacteriol. fiz513-523. Foster, J. W., and E. S. Wynne. 1948. Physiological studies on spore germination with special reference to Clostridium botulinum. IV. Inhibition of germination by unsaturated C18 fatty acids. J. Bacteriol. 5_5:495-501. Gabliks, J., and M. Solotorovsky. 1961 . Effect of diphtheria toxin upon tissue culture derived from susceptible and resistant species. Bacteriol. Proc. M1422137. Grecz, N., R. O. Wagenaar, and G. M. Dack. 1959a. Inhibition of Clostridium botulinum by culture filtrates of Brevibacterium linens. J. Bacteriol. 31506-510. 60 61 Grecz, N., R. O. Wagenaar, and G. M. Dack. 1959b. 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