u: v . a: r. .r. 1.1 ‘ THESUS This is to certify that the ,4 f thesis entitled , The Viability of Aerobic Turmophino x ,’ Bacteria in the Presence of Varying ._ ! Concentrations of Acids, Salt ? j and Sugars " 1 presented by ‘7 2‘32. Horace D. Graham y ' if: has been accepted towards fulfillment .. of the requirements for 1 2:? __M0_S_-_degree inML-P'logy & :5. Public Health f 4:5,: Major professor Sufi-1’5 Vii! J :1 THE VIABILITY OF AEROBIC TEERMOPHILIC BACTERIA IN THE PRESENCE OF VARYING CONCENTRATIONS OF ACIDS, SALT AND SUGARS BY HORACE IL ggEUUUd A,THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 1951 JHESiS / o a / s/ ~g"";, ( L51 I ACKNOWLEDGEMENTS Dr. F.W. Fabian, professor of Bacteriology and Public Health, gains the deepest appreciation of the author for his expert guidance and keen interest in this project throughout the period of inves- tigation. Thanks are also due to messrs. Ralph C. Zalkan, Hilliard Pivnick.and Sam.Rosen, graduate students in this laboratory, for ‘useful suggestions and guidance at various times. antral filth)‘; _ ’ar'K TABLE OF COHTEETS Page Ho. IntrOduCtiOngooooeococo... 1 Literature Review . . . . . . . . . . . 4 Procedure . . . . . . . . . . . . . . . 9 Results . . . . . . . . . . . . . . . . 13 Analysis of Resultsand Disdussion . . . .20 Summary and Conclusions . . . . . . . . .28 Bibliography.....o...oo.o:29 INTRODUCTION Despite the importance of thermOphilic bacteria in the food industry, especially in the spoilage of canned foods, a survey of the literature reveals a paucity of information on their classif- ' ication and physiological characteristics. Schmidtl significantly remarked "The group of organisms known as obligate thermophiles has been completely neglected". Thermophilic spoilage has for years been a constant and vexing problem of food canners, food technologists and food bac- teriologists. The ubiquitous nature of the thermophilic bacteria attested to by the great variety of sources from which they have been isolated, and the extraordinarihy high.heat resistance of their spores, greatly increase the probability of spoilage in industry. They may occur in food ingredients such as sugar and starch and the spoilage of sugar in storage and during shipment has been attrib— uted to their action." Several cases of wholesale loss of inad- equately processed canned foods due to thermOphilic spoilage have been reported. Tomatoes, beans, peas, corn, non-acid foods in general- are very susceptible. In all such cases the flat sour obligate thermophiles in the sense of the definition of Cameron andEsty2 are of particular importance. The retreat of the Allied armies in the North.African campaign during World war II was alleflgedly due largely to a shortage of rations incurred as a result of thermophilic spoilage of canned foods stored in the warm.African desert. Similar spoilage of canned foods occured in the Pacific. With.the Pacific theatre now being regarded as a stragetic area large shipments of edible materials to these areas may be susceptible to thermOphilic spoil- age. Further close study of the behavior of these organisms may therefore be profitable. tAcids, salt and sugar occuring either naturally or added in varying proportions play an important role in food preservation. This preservative effect was recognised even by primitive man. The hydrogenpion concentration of food influences its de- terioration by micro-organisms. Concentrated solutions of salt and sugars are used to preserve certain foods by repressing develOp- ment of micro-organisms; onions, peppers and cauliflowers being pre- served by salt and fruits by sirups. The influence of acids, salt and sugars on thermophilic bacteria has not been extensively studied. As a result, Opinions differ widely as to the kind and quantity of any of these ingredients to be used for effective but, at the same time, economical preservation. The phenomenal expansion of the food industry and the present emphasis on the microbiological aspects of foods s 2greet the necess- ity for further study. Objectives of this igvestigation The general objective of these studies was to ascertain the trend of growth of thermOphilic bacteria in the specific medium which had been adjusted to different pH levels with different acids or to which increasing concentrations of salt or sugar had been added and, ultimately, to determine their possible use in the preservation of foods which are susceptible to thermophilic spoil- age. Specifically, the objectives were to obtain the following information: 1- The order of effectiveness, bacteriostatically and bactericid- ally, of the acids, salt and sugars against thermOphilic bac- teria. The acids were used to adjust the pH of the medium to the same pH level while the salt and sugars were added in in- creasing concentrations. 2- The minimum or probable time for which the medium so treated and inoculated with thermophilic cultures would have to be incub- ated in order to reduce the bacterial population. 3- The relative rates of death of thermOphilic bacteria isolated from various sources when incubated for varying periods in a medium so treated. LITERATURE REVIEW A.very lucid review of the literature on the nature of thermOphiles is given by Gaughran?. ThermoPhilic bacteria have been characterized by different investigators in a variety of ways. They may be simply defined as unicellular spore-forming micro-organisms depending for their existence on animal or plant tissues and which grow at relatively high temperatures. Prickettu after sifting the various definitions accepted the one given by Cameron and Estyz. It is as follows: M- Mate Thermgphileg Cultures growing at 55°C but not at 37°C or showing no growth at 37°C but growing at 45°C and higher. (Bj- IacultatingQQermgphiles Cultures growing both at 55°C and 37°C. Prickett further observed that, though about sixty(60) different species of aerobic thermo- philic bacteria are named and described in Bergey's Manual of Determinative Bacteriology, the data on the species of this group are so meager that it is not possible to offer a rational system of classification. Furthermore, many of the characteristics used for separating the various species of thermophilic bacteria are probably as variable as they are in mesOphiles. - 5 - Imsenecki and Solnzevas have divided them, on a temperature basis, into two main groups: 1.. 2” StenothermalrfhermOphiles These develop at 60°C but do now show any growth during several days at 28-30%. The amylolytic bacterium, racing: diastat— igug, a potent starch hydrolysed; which grows vigorously at 60°C on.potato decOction, but does not grow at 28-30°C on the same medium is an example of this class. Eurithermal Ihermthiles Representatives of this group also exhibit a growth Optimum at about 60°C but slight growth may also be evident at shch low temperatures as 28-3000. Though high.temperature is a primary requisite for the optimal growth of thermOphilic bacteria, several workers (5,6,7,8) have shown that thermOphiles are not necess- arily inactive at relatively low temperatures. Bergeyg divides thermophiles into two groups: True thermoghiles These show optimum growth at 60°C to 70°C and only feeble or no growth below 40°C or 45°C. Facultative ihermoghiles These develop at room temperature- about 20°C, have their Optimum temperature at about 50°C and their maximum at about 60°C. -5- Gaughran? points out that the multiple connotations of the terms used to describe thermophiles have led to much misinterpret- ation. He thinks that bacteria with any optimum growth temperature of between 50°C and 60°C may be called thermophiles and suggests that, if necessary, the terms of Imsenecki and Solnzevaj should be used for defining the magnitude of the temperature range for growth. The Effect of:Agidg: Erickson and Fabiango showed that thermOphiles are more sus- ceptible to increasing concentrations of acids than mesophiles. Anderson and co-workers11 demonstrated that lactic, acetic, and citric acids in increasing concentrations reduced the thermal des- truction rates of Bacillygzjhermoacidurans, the relative order of efficiency being lactic 7' citric :7 acetic. Pederson and Backer12 concluded that there seems to be a significant difference between the minimum pH for the growth Of vegetative cells and the minimum pH for the germination of spores of Bacillus thermoacidurans:vegetative cells initiated growth in broth of pH as low as 4.30, while heat resistant spores were incap- able of germination at pH below 5.0. Nunheimer and Fabian;3 found that on a pH basis, the order Of effectiveness for the germicidal action of the acids against strains of food poisoning staphylococci was acetic :> citric lactic 7 maliC'v tartaric:> HCl. The orderffor growth inhibiting prOperties was the same except that citric and lactic acids changed places in the series. Levine and Fellers 14 noted that acetic acid was much more.I effective than either Hydrochloric or lactic acid against Saccharogyces cerevieiae~_or Salmonella aertrycke. wyeth;5 reported that on a pE'basis the order of effectiveness against coliform bacteria was acetic S lactic 7 hydrochloric. 16 found that in a cancentration of Shillinglaw and Levine 0.02N the order of effectiveness of the edible acids as germicides against Escherichia colL at 30°C was tartaric) lactic) acetic > citric. The Influence of Salt: The influence of salt on the viability and thermal resistance of micro-organisms has commanded the interest of various investig- ators who have reported both increased lethality and a definite protective effect. 7 found that concentrations of sodium chloride up to Viljoen1 3.5 percent in pea liquor gave a protective effect against heat to certain micro-organisms including thermophiles. The greatest protection was observed at 1.0- 2.5 percent while #.0 percent either gave no protective action or else had a killing effect. Anderson et al 11 showed that the addition of increasing amounts of salt to tomato juice resulted in a progressive lower- ing of the hydrogen ion concentration and a concomittant more rapid destruction rate for Bacillus thermoacidurans. The Influence of Sugars: Erickson andFabianlo demonstrated clearly that thermOphiles are much less resistant to the effects of sugars than.other micro- organisms such as Streptococcus lactis and Streptococcus ligpefaciepg. They found, for three cultures of thermophiles, a preservative action in the presence of as low as 17.5 percent fructose, 27.5 percent dextrose and 40 to 45 percent sucrose. Anderson et al11 found that increasing concentrations of sucrose and dextrose in tomato juice enhanced the thermal resistance of Bacillus thermoacidurans. Robertson;8 Observed an increasing protective action for several micro-organisms including Streptococcus thermo hilu., when heated in increasing concentrations Of sugars. Several workers including Baumgartner and WallaceIgand Fayzo found that the thermal resistance of some micro-organisms when heated in hypertonic solutions of sucrose, glucose, lactose and other sugars is increased. PROCEDURE The underlisted cultures of thermophilic bacteria, obtained from the National Canners Association, were used in this inves- tigation: : Type : N.C.A. Number : Source : : : : : : Obligate thermophile- 3 1373 : Hominy' : : flat sour : : : : ” ' : 26 3 Corn : 8 8 8 8 : " " : 1503 3 peas : : 3 8 8 : Facultative thermophile : 1&01 : pumpkin : : ' ' : 1518 : corn : 8 8 8 8 Culture NO. 1373 became contaminated during the experiment and was omitted from the later portions. The food ingredients whose effect on the viability of the above thermophilic bacteria was studied and their sources are as follows: (A)- Apgdg: Acetic- Glacial, C.P. grade Citric- Baker's analysed, C.P. grade. Lactic- Edible lactic acid, 50 percent. - 10 - Bl- §2l£. Sodium chloride, Merck. Cl- §ggars Dextrose, DIFCO, Bacto dextrose, anhydrous Sucrose, C.P. Fructose (Levulose) C.P. Preliminapyflexperimentation: Growth curves were run on cultures 26, 1401, 1503 and 1518 in order to determine their rate of growth and death in dextrose tryptone broth and the approximate time intervals at which platings were to be done throughout the experiment. procedure: 50 m1. portions of sterile dextrose tryptone broth were pre— heated to 55°C to afford optimal temperature from the very start. Each was inoculated with 1 m1. of a suspension of each organism, washed from a 48 hour dextrose tryptone agar lepe and incubated in a water bath maintained at 55 t 1°C. Plate counts were made at 0,2,4,6,8,10,12,14,16,18,20,24,30,36,42,48, 72 and 96 hours. Preparation of Test solutionsi (3)- Acid solutionpi .A sterility test was run on each batch of acid as follows. A drop of the acid was added to a test tube containing 10 ml. of dextrose-tryptone broth. The tube was then incubated at 55°};100 for 48 hours. In no instance, however, did thermOphiles appear in any of the acids used. B- - 11 - Using aseptic precautions throughout all manipulations, sterile dextrose tryptone broth was adjusted with the aid of a Beckman pH meter, to the required pH values diown in Tables 2, 3 and h. Fifteen m1 portions were then diapensed into sterile test tubes. Salt solutions: Sodium.chloride in concentrations of 0, S, 1.5, 2.5, 5.0, 10, 15, 20 and 25 percent by volume was added to dextrose tryptone broth and the mixture heated in.the steamer to dissolve all the ingredients. The broth was then.dispensed in 15 ml. quantities into test tubes and the tubes autoclaved at 121° 0 (250° F) for fifteen minutes. Sugar solutions: In the case of dextrose amounts equivalent to 5, 10, 20, 30, ho and 50 per cent by vdlume were added to dextrose tryptone broth and.the procedure repeated exactly as in.the case Of the salt solutions. For fructose and sucrose 100 per cent weight by volume solutions were made up and autoclaved at ten pounds pressure for ten minutes. Enough of these stock solutions were then aseptically pipetted into sterile flasks to make up 100 ml. of each of the concentrations required. From.these flasks 15 ml. portions were then aseptically pipetted into sterile test tubes. Preparation of stock cultures: All cultures were grown on dextrose tryptone agar slants forsh8 hours. This allowed the use of cultures of a standard age of #8 hours throughout the experiment. Inoculation andpplate counts: All the tubes of broth for the acid, salt or sugar test were pre-heated at 55°C for two hours. The 48 hour cells were washed off the dextrose tryptone agar slepes using 10 ml. of sterile distilled water. Into each of the tubes, 1. m1 of the well—shaken cell suspension was inoculated. kept in.a water bath maintained at 55 f 1°C during this process, and tubes then incubated at 55 £ 1°C. Plating on dextrose tryptone agar by the pour plate method was done from each tube after 0,6,12, 24, 98 and 96 hours of incubation and plates incubated at 55 f 1°C for 36 hours . All inoculations of tubes and platings from tubes were done in duplicate. _ 13 - RESULTS Table 1 shows the number of viable cells for cultures 26, 1401, 1503 and 1518 after increasing periods of incubation. Growth curves based on the above numbers are shown on ézééh 1. Table 2 shows the effect of acids on the viability of the organisms. Table 3 shows the effect of increasing concentrations of sodium chloride while the effect of increasing concentrations of dextrose,fructose and sucrose are shown in Table h. Explanation of Tables: Tables 2-8 inclusive, } :_That percentage increase over original inoculum after period of incubation indicated. e.g. ,L 300 :. an increase of 300 per cent :_a threefold multiplication. - =.That percentage decrease or destruction based on the original inoculum e.g. - 59 :_59 per cent of the original inoculum died. 0 :,Bacteriostasis - no increase or decrease relative to the initial 11100111111110 T elem. H 2035”... Homeowners? media @543 on «and .3853 uh mangoes gone dudes me uuo o. m 0 O C a a m. V ‘1”, F O 7. r.\\ Inl/ # N W. \ / y . a w . l a Ow 3.»me H... E 9.933 ogoesodwtwwo deceased. 4536 095+. 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".2 -__-‘"':_‘ g ‘(m‘o .n 0 e :2 24 no «‘90 96 TIME IN HOURS ] a... w u z 4 3 /~\\ I @4000 3‘ —._——_~~ a ”"‘ > ¢ u. o 20- I .- E \‘ and». 4 *‘F'#‘ _ 3 CD or.” - o .1 0 o :2 «'90 9e 24 30 "ME IN HOURS Fig. 2 logarithmic growth curves of five thermophiles whengrownat 55°C indem- trose tryptone broth with the pH adjusted to 5.5 with acetic, citric and lactic acids respectively. -21- 3~ The rapid formation.and accumulation of acids through.the de- composition of carbon compounds in the medium if such are not neutralized, since the fermentation capacity of thermophiles has been calculated to be perhaps thirty times as great as that of Streptococcus lactis at 200 C. h- Cytolysis induced by autolysis with the resultant accumulation of toxic products in the medium have been suggested for observed decreases in fine total cell count. Evidence of antolysis has been obtained by way of the detection of an accumulation of enzymes in media inoculated with thermOphilesS. The effect of acids,gsalt and sugars: The effect of increasing concentrations of acids, salts and sugars was determined in terms of per cent increase or decrease of call numbers relative to the initial inoculum. Results are recorded in Tables 2, 3 and h. Ap The effect of Acids: “M3 9” .7» From Table 2A it may be noted that at pH 5.5 and incubation time of 6 or 12 hours the germicidal powers of the acids used follow the order citric 7»acetic.7 lactic. In the case of citric acid an increase in acidity beyond pH 6.0 down to pH 5.5 resulted in an increase in cell destruction. Beyond pH 5.5, however, an increase in acidity resulted in no significant decrease in the number of cells. For acetic and lactic acids, multiplication occured at pH 5.5 and germicidal action started at pH 5.0. A decrease in pH below pH 5.0 germicidal action afforded no significant decrease in cell numbers. as» “I \(UXM Q \ z \ ( I \ o x “x M v- :s. \ 7 I i ui \ > < ENC—1.53“ ‘ \ u t o Q \ . \ ‘t 2 0» \ I .- ‘\ C \ ( \. ‘3 O “ J O 3 :2 2‘4 is «”90 96 mm: m nouns ‘MI ’5: ”O“ * KL \m'szsf' ~. oxfiq Leeannnm or was cums: OW b / o/./” / I I I I I I XI I a I I’ {F 3 , t I ’J I» I’l” s f2 2‘4 is «"90 96 mm: m nouns ‘U. ‘s---‘- +..g__v o , LOGARITHM OF AVE. 1 CHANGE O'——¢— 6 l2 24 86 48 90 96 TIME IN HOURS 0' § 9 LOGARITHM OF AVE! CHANGE TIME IN HOURS fl“! “' a... 06.3-: g A” d I 0 :1 u! 2 ‘~ is fit! 0 «a 2 0- 1 s. - U-ltfl“ : -4‘fi_--- s9 0 J CDII———$r ' 3 :2 24 as mu: m nouns 4O 90 96 Fig. 3 logarithmic youth ms of five thermophiles when grown at 55° C in dem- trose tryptone broth to which various percentages of Neel had been added - 22 _ Explanations for the mode of action of_9rganic aciggz Egg; are the reasons for the order of the germicidal;powers of the acids tested? Several investigators have attributed the effect of added acids in suppressing the growth of bacteria to the change in the hydrogen 11 and Levine and ion concentration. However, Anderson et al Fellers 1“have pointed out that the toxicity of certain organic acids is due in part to the undissociated molecule and the anion in add- ition to the effect of the hydrogen ion. Cohen and Clark 21 suggested that the action of acetic acid may be due to the free acetate radical exerting a synergistic effect upon the disinfecting power of the hydrogen ion. Reid 22believes that the anion and undissociated molecule may act independently in exerting their disinfectant action or as positive catalysers increasing the bactericidal power of the hydrogen ions. The influence of acids on the surface tension of the medium and the size of the molecule have been mentioned by Each 23 as possible reasons for observed germicidal action. He also stated that the hydrogen ions control the antiseptic effect but the unp dissociated part of lactic acid is the germicidal factor when the pH value is important. ‘lraube's rule 2“ states that in a homologous series of fatty acids the lowering of surface tension is parallel to the increase in molecular weight of the fatty acid. This may explain why citric u? -23.. acid is more germicidal than either acetic 0r lactic since of the three acids citric has the largest molecular weight Acetic acid :. m.w. 2'. 60.0 Lactic acid m.w. :-_ 90.6 Eitric acid m.w. :192.0 The dissociation of the acids may also account for the order of germicidal action observed: Dissociation conjsjants oLthe acids usegd_ Acid Dissodiation constantsfli'.) _5 1.. Citric 8.7 x 104mg :.-. 1.8mm ) 2- Acetic 1.752 x 10"5 3- Lactic 1.37 X 104 The above order indicates that if dissociation was the prime factor in the germicidal action then the order should be citric 7 lactic 7 acetic. This again may explain why citric acid was more toxic than either acetic or lactic acid, Since acetic acid was found to be more germicidal than lactic acid the undies- ociated molecule may be toxic as suggested by Anderson and co- workersll and Levine and Fellers 11+. The effect of Salt“? 3 From Table 3Ait may be seen that up to 1.5 per cent sodium chloride was non—germicidal except in the case of organism #1503. For I' 1503 germicidal action took place at 1.5 per cent but not at 0.5 per cent. Concentrations of 2.5 per cent or greater proved germididal in all cases. With culture 17' 26 total destruction took nus ° sum w W .5 d: z z \. flirt." < “ V x , ff, ‘r \ 0 " \\ “M U- - E __ __ ._.——‘ I. \\/I/4 :1 Y, --&-—— - \\ \ “J \ \ {mo . was“ \ > 2_ “Nap—ML __ . g ”I ‘ x .\ < \ u ‘\ u \ o \ O \ 5 0P \ 2 . I \ I I- ‘\ I- C ‘\ E auras: ‘3‘2 , f—O——-*—( I V O V V V ‘— O O .I .I o s I2 24 as is o 96 0 s :2 24 as « ‘60' J9s 'I’IME IN HOURS TIME IN HOURS m 6 “NI. 3 ‘\ g z \\ Z ( \ ‘ new I \ “new 5 h--~‘* o x ’ ‘ ““““ a! .l we: 3 ‘_ ~~~~~~~ ‘ / \ the \ ~—..____4I . u IL 0 V’mvm o 20L s. 2 ‘\ 1 I I- \ t s g N— ? ' - : \"-— ‘5‘?“ \ ‘5-2- HT; 0 O J J o s. I2 24 as so 0 9s 0 s :2 24 as «‘60 9s TIME IN HOURS TIME IN HOURS Fig. 4 Logarithmic youth curves of five thermOphiles when grown at 55° C in tryptone broth to which different percentages of dextrose, fructose and sucrose respectively had been added. -214... place at 5.0 per cent and greater. The reason for the action of salt in inhibiting the growth of bacteria or destroying the cells is not readily apparent. Anderson er al 11 observed that there is obviously no toxic entity in salt. Moreover, as pre-mentioned, certain investigators in! cluding VilJoen;7 have attributed a protective effect from heat on some micro-organisms by certain concentrations of salt. Anderson and his colleagues think that, in as much as the addition of increasing amounts of salt to media is known to result in increased hydrogen ion con- centrations, the lethal effect of salt may be due at least in part to the lowered pH of the substrate. The effect of salt on bacteria is thought by many investigators to be mainly and simply plasmolytic. Plasmolysis, however, is believed to last for only a few hours and not all bacterial cells are affected. Rockwell and Ebertz25 suggested that at least five factors are involved in the detrimental action of salt on bacteria, namely; 1- The direct effect of the chloride ion. 2- Dehydration. 3- The removal of oxygen. 4- Sensitization against carbon dioxide. 5- Interference with the rapid action of proteolytic enzymes. In.addition to the above postulates it may be that, due to the dis- sociation of sodium chloride in solution, the sodium and chloride ions, though necessary for the growth of some organisms, and acceleratory in small amounts, _ 25 - become toxic in large quantities 1.8. as the concentration of salt is increased. This is an established fact in bacterial physiology. The law of diminishing returns seems evident as the concentration of salt gets beyond 2.5 per cent since even a concentration of 25.0 percent gave no significant increase in germicidal action. The gffect of spgars From the average percentages of increase or decrease over the initial inocula in the presence of varyinga concentrations of dextrose, fructose and sucrose shown in Table 0, it h:;¥ be seen that 5.0 per- cent dextrose did not suppress growth over the first 6—12 hours of incubation in the case of cultures 26, 1373 and 1518. .A decrease of 66 per cent occured after 12 hours in culture 1503. For culture lhOl a decrease of 72 per cent took place after 6 hours. For all organisms a germicidal action ensued after 6 hours in the presence of 10.0 per cent or greater. During incubation periods of 6-12 hours multiplication took place in the presence of 10.0 per cent fructose. Concentrations of 20 per cent or greater proved germicidal in all cases. Except for organisms #1A01 sucrose in equivalent concentrations gave results in the same trend as fructose. Sucrose however, was less destructive beyond 20.0 per cent. The reverse of this holds true for organism lflOl. For all three sugars increasing the concentration beyond that needed for germicidal action endowed no increased lethality. The - 26 - effectiveness of the sugars in reducing the bacterial count may therefore be written as dextrose > fructose 7 sucrose. Reasonsigpr the action of the sugars and the order ofggermicidal action found The reasons for the germicidal action of sugars against micro- organisms are not well established. Concentrations of 10950 per cent of sucrose and dextrose are known to cause no appreciable change in the pH of culture media, hence no significant portion of the germic- idal action of the sugars can be attributed to pH changes. Fayzo thinks that in both the protective and germicidal actions the influence of sugars on osmotic pressure determines their effective- ness. In his opinion the permeability of the individual cell app- arently plays an important part in regulating the rate of death in water and in sugar suspensions. He hypothesises that resistance to the effects of sugars is limited to those cells which are highly sensitive to water.at slightly increased temperatures. Plasmolysis of the microbial cell may explain why dextrose exerted a germicidal effect at a lower concentration than either fructose or sucrose and why fructose exerted a greater germicidal action at the same concentration as sucrose. Osmotic pressure and hence plas- molysis depends on the number of particles in solution. Dextrose and fructose each with a molecular weight of 180 would therefore contain more molecules per unit of weight than would sucrose. The activity should therefore tend to decrease as the molecular weight increases. -27.. On a chemical basis, dextrose, an aldehyde sugar) should be less reactive than fructose, a keto sugar, as evidenced by the fact that many chemical tests can be carried out on the latter in the cold while the former must be heated. This order of activity apparently does not hold biologically since dextrose was found to be more germ- icidal than fructose. The reason for this is not apparent. SUT‘MARY AND CONC LUSIOIIS The growth of thermophilic bacteria was found to be very rapid reaching a maximum.within 8-1h hours followed by a.rapid decline in cell numbers. The lag phase of the growth cycle is not very pro- nounced and after 96 hours there were less cells present than in the initial inocula. The order of germicidal action of the acids studies is citric> acetic ;7 lactic. An increase in acidity beyond pH 5.0 gave no increased gennicidal action. Concentrations of’sodium chloride up to 1.5 percent were not germicidal except for organism # lhOl in which case a lethality took place at this concentration. Concentrations of 2.5 per cent and greater were germicidal in all cases. An increase in salt concentration beyond 2.5 per cent gave no added germicidal effect. The order of effectiveness of the sugars as germicides was found to be dextrose ;. fructose 7' sucrose. In all cases concentrations beyond 20.0 per cent gave no significant increase in germicidal action. From the growth curves and tables 1, 2, 3 and h it may be COD! cluded that the deleterious action of various food ingredients on thermophilic bacteria occurs during the first 6 to 12 hours of incuba- tion. This interval corresponds roughly to the phase of physiological growth and the early logarithmic phase of the bacterial growth curve. 2. 3. 4. 5. 9. 10. -29.. BIBLIOGRAPHY 5chmidt, Clarence F. 1950 Spore Formation by ThermoPhilic flat sour organisms. The effect of nutrient concentration and the presence of salts. Hourg of Bacteriology. 359:2, Aug.; 205. Cameron, E. J. and Esty, J.R. 1926 The examination of spoiled canned foods. Classification of flat-sour spoilage organisms from non-acid foods. Journal of Infectious DiseaseL, 19: 89-105. Craughran, Eugene, R.L. 191+? The Thermophilic Microorganisms. Bacteriological Reviews 1_1 3 189-225 0 Pricket, as. 1928 ThermOphilic and Thermodunc microorganisms with special reference to species isolated from milk. 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(1‘ A AMU/ _ , ‘* \ ., lx \ x *"i I“ I“); ‘x‘. l L