Till-V? ‘L. 'YHI A STUDY ON THE GROWTH OF AEROBIC THERMOPHILIC BACILLI OVER A WIDE TEMPERATURE RANGE BY John B. Otting A THESIS Submitted to the School of Science and Arts of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1957 ”7H?! ABSTRACT OF THESIS The probability of food spoilage in a hot food vending machine was determined from the growth of aerobic thermophilic bacilli over a wide temperature range of hS-BB C in an artificial medium. At 45 C growth proceeded at a very slow rate. Generation times ranged from 11 minutes to 30 minutes at optimum temperatures for growth. The optimum temperature range for growth extended from 55 to 71 C. At optimum temperatures, growth and death proceeded at a rapid rate. At temperatures above optimum, the number of viable organisms dropped immediately after inoculation. After continued incubation at elevated temperatures, the number of viable organisms again reached appreciable numbers. "er-m. TABLE OF CONTENTS Page INTRODUCTION........................ 1 LITERATURE REVIEW ................... 3 PROCEDURE........................... 11 RESULTS ............. ....... . ........ 22 DISCUSSION ............ .. ....... 50 SUMMARY :55 BIBLI'IJGAYI'XPETIoooooooooooooooooooooooo S7 TH!!! INTRODUCTION Despite the economic importance of the thermophilic spoilage in the food industry, a survey of the literature reveals a scarcity of information on the classification and physiological characteristics of thermophilic bacteria. Thermophilic spoilage has been associated most fre- quently with canned foods. The thermophilic bacteria have a wide distribution in Nature and are difficult to destroy in foods due to the high heat resistance of their spores. Todays modern methods of processing have largely eliminated the spoilage of canned foods, although the thermophilic bacteria still remain as a constant threat to the canned food industry. Thermophilic bacteria may also be a problem in other areas of food dispensing besides canned products. Recently, machines have been designed to vend hot foods that are held continuously at serving temperatures within the machines. In most instances the temperature of storage is approximately 66 C. This temperature exceeds the maximum growing temp- eratures for most thermophiles, however, some species may have maximums above 66 C or they may under some conditions adapt to this temperature. THpq The objectives of this study were: 1. To determine the generation time of aerobic thermophilic bacilli at optimum and near optimum temperatures for growth. To determine the growth of aerobic thermophilic bacilli in an artificial medium at vending machine temperatures of 66 C and up. "TH!!- LITERATURE REVIEW Since the discovery of the first thermophilic aerobic sporeforming bacterium by Miquel in the year, 1879, (1) these bacteria have been objects of scientific interest. A survey of the literature reveals, that the bacilli are the most commonly encountered types of thermophilic microorganisms associated with the spoilage of food; particularly processed foods. Gaughran (2) states, "Among the cocci, we find sarcinae, staphylococci, and streptococci described as thermophilic, although it is more likely that their capacity was one of resistance rather than active growth at elevated temperatures." The majority of bacilli isolated at high temperatures prove to be aerobic spore— forming bacteria, nonsporeformers are rare, and facultative forms are more common than obligate anaerobes. Thermophilic bacteria have a very wide distribution range. Thermophilic bacteria have been isolated from deep ocean bottom cores, (3) to sands of the Sahara Desert. (2) Climatic conditions bear little influence on their distribution. Thermophilic bacteria have been characterized by different investigators in a variety of ways. A few of the common characterizations are as follows: THPQV? Bergey (h) classifies the thermophiles into two main groups: True thermophiles - Microorganisms showing optimum growth at 60 C to 70 C and only feeble or no growth below no C or hb C. Facultative thermophiles - Microorganisms that develop at room temperature, but have their optimum temperature at above 50 C and their maximum at above so C. Cameron and Esty (5) classified as follows: A. Obligate thermophiles - Cultures showing no growth at 37 C but growing at hS C and higher. Facultative thermophiles - Cultures growing both at 55 C and 37 C. Imsenecki and Solnzeva (6) have classified them, on a temperature basis, into two main groups: A. Stenothermal thermophiles - These develop at 60 C but do not show any growth during several days incubation at 28-30 C. An example of this class is, Bacillus diastaticus, which grows vigorously at 60 C on potato decoction, but does not grow at 28-30 C on the same medium. TH?“ B. Eurithermal thermophiles - Representatives of this group also exhibit a growth optimum at about 50 C but slight growth may also be evident at such low temperatures as 28-30 C. The authors used a bacterium designated as Bacillus s . to illustrate this definition. Hansen (7) has also demonstrated that ther- mophiles are not necessarily inactive at relatively low temperatures. Gaughran (2) designates bacteria with an optimum temperature for growth between 50 C and 60 C as thermophiles. He points out, that the multiple connotations of terms employed to describe the ther- mophiles has led to much misinterpretation. According to Gaughran the terms of fmsenecki and Solnzeva should be used only to define the magnitude of temperature range for growth. The thermophilic bacteria have been inadequately de- scribed in literature. These inadequate descriptions have led to the synonymity of species names. Morrison and Tanner (8) and Prickett (9) have contested the identity of many species found in the literature. Species were sepa- rated upon insignificant differences as the relative time required to effect a particular biochemical change, the abundance of growth on a particular medium, etc.. ”W! 3-1: .1." An attempt to standardize the terminology of this group has been made by the Committee on Classification of the Society of American Bacteriologists under Bergey (10). At a later date, Chester (ll) developed a revision of the classification of the thermophilic organisms, and presented descriptions of 21 species of the thermophilic group, Family Bacillaceae. The present system of classification is still unsatisfactory, for it is based upon slight dif- ferences in spore size and location, pigments, shape of sporangium, etc.. The anaerobic sporeforming thermophiles are likewise inadequately classified. All obligate anaerobes are given the generic name Clostridium. McClung (l2) and Damon and Feirer (13) did considerable work to clear up this confusion among the species of genus Clostridium. The question is asked, "How are the thermophilic bacteria capable of growth at such high and seemingly un- favorable temperatures?" No single answer to the question can be given. Various theories are offered as explanations in making thermophily possible. The type of external environment is offered as an explanation for thermophily. It is well known that bacteria are capable of surviving dry heat at a temperature which is lethal when moist heat is used. Allen (1) explains the TH!“ importance of the external environment by saying, "Suitable media may increase heat resistance by providing favorable conditions for active metabolism so that the organism is better able to repair damage done by heat, or they may aid growth at high temperatures because they contain certain substances which protect proteins from thermal denaturation. Another seemingly important point of thermophily is the structure of the cell. The spores of the thermophilic bacilli are known to have a high heat resistance. Most all of the vegetative cells are long and slender. Assuming this shape they have a large amount of surface exposed to the external environment. The thermophilic actinomycetes are likewise small in diameter. Lamanna (1h) discovered that organisms with lower minimum and maximum growth tem- peratures are as a rule larger in cell size than those organismm which have a high minimum and maximum growth temperature. Edwards and Rettger (15) made a study on the relation of certain respiratory enzymes to the maximal growth tem- perature of bacteria. They found a relationship between the maximum temperature of growth of the bacterium and the maximum temperature of activity of some respiratory enzymes as catalase, and peroxidase. Allen (1) does not i THE-1 believe that the respiratory enzymes are the controlling factors of growth, because many thermophilic bacteria will not grow at ordinary temperatures while their respiratory enzymes still function. Therefore, literature reveals that the question, "What factors make thermophily possible?" is not fully answered and not wholly agreed upon. Much work needs to be done in the field of thermophily. The first study of growth curves of thermophilic bacteria was conducted by Tanner and Wallace (16). Their results showed that at 55 C a more rapid increase in cell numbers occurred than at 20 C and 37 C, and after the period of active growth, a rapid death was observed. Gaughran (2), Imsenecki and Solnzeva (6), and Hansen (7) noted that the growth of thermophilic bacteria began almost immediately after inoculation of the culture medium, the lag phase was either absent or very short. Growth was characterized by high reproduction and rapid death rates. The generation time was short and the loga- rithmic growth phase was of short duration. Due to the rapid death rate of thermophilic bacteria, their generation time can not be calculated from viable counts, taken during the logarithmic period of growth. A generation time, based only on a viable count, will prove "mm to be several hours longer than the generation time obtained, under the same conditions, when both viable and total cell counts are used in the calculation. The method, for the determination of generation time, using a viable and total count is explained by Wilson and Miles (17). If the viable and total counts are known both at the start and at the end of x generations, then the gen- eration index can be ascertained at any time during the logarithmic period of growth. Knowing the generation index, the number of generations can be calculated, and the gen- eration time obtained. Slater (lb) devised another method for the determina- tion of generation time. He used a nephelometric method and in this way measured mass, or both the viable and dead cells. The organism used was Lactobacillus delbruchii and the medium employed was a malt wort broth. The experiment was conducted at MS C. A standard number of tubes was prepared and from these tubes the number of bacilli in the broth was determined. The growth rate constant was calcu- lated and the generation time obtained. Ybumans and Youmans (19) presented a method for the determination of generation time similar in principle to Slator's. Their method was used to determine the growth 'I‘HF‘ -10- rate and generation time of tubercle bacilli. They used inocula containing different quantities of tubercle bacilli. The time was recorded at which growth with each inoculum first became visible. The time of first appearance of growth of each inoculum was plotted against the logarithm of the amount of inoculum employed and a linear relationship was obtained. The slope of the straight line, so obtained, represented the rate of growth of the tubercle bacilli and from this line the growth rate constant and generation time was determined. 1‘. THVQ PROCEDURE The following cultures of thermophilic bacteria, with their source of isolation, were used in this investigation. Organism Source Bacillus Sp, # 1 A Compost Bacillus sp. fip2 Soil Bacillus sp. 3 Q Manure Bacillus sp. ¢_5 Flower pot soil Bacillus sp. i 2 Soil Bacillus sp. ill Compost Isolation_procedure: All of the above thermophilic bacteria were isolated by the pour-plate method using Dextrose Agar, Difco. Incubation was at 55 C for 2h hours. Isolated colonies were picked from.the pour-plate and restreaked upon the surface of Dextrose Agar to obtain a pure culture. Stock cultures were prepared from the pure cultures. Only thermophilic bacteria which showed growth on Dextrose Agar at 65 C were maintained as stock cultures. Preparation of stock cultures: Pure cultures were streaked on Dextrose Agar slants and incubated at 55 C for 2h hours. The slant cultures, -11.. ’fHP' -12- after incubation, were sealed with para-film and refrigerated at S C. The stock cultures were transferred every four weeks. Proof of method employed to determineggeneration times: In a preliminary experiment a comparison of methods for generation time was done on Escherichia coli and Aerobacter aerogenes. This comparison was made to show justification for the method used in later experiments to determine the generation time of thermophilic organisms. E; coli and A; aerogenes were selected as test organisms because of their rapid growth and relatively slow death rate during this logarithmic period of growth. The generation.tine of §L_22li and A; aerogenes was determined by two methods. The first method employed viable counts during the logarithmic period of growth. The second method employed the principles of spectrophotometry, during the logarithmic period of growth. A comparison was made between the generation times obtained by each method. The inoculum used for each method consisted of a 12 hour, Dextrose Broth culture of §;_ggli or A; aerogenes. These organisms were grown at 37 C and transferred daily for 3 days prior to use. Viable count method_procedure: Five hundred ml flasks containing 250 ml of sterile Dextrose Broth, per flask, were preheated I‘M 'THP- -13- to 37 i.l C in a water bath. The preheated flasks of Dextrose Broth were inoculated with 1 ml of a l-lOOO dilution of the broth culture tested. Saline was used as a diluent. After inoculation, incubation was continued in the water bath. Viable counts were de- termined after increasing periods of incubation. Platings were made at O, 2, h, 6, 3, and 10 hours. Bacto-Tryptone Glucose Extract Agar was used for plating. Plates were incubated at 37 C for 2h hours. Inoculations and plate counts were done in duplicate. Results were recorded, and a generation time based on a viable count during the logarithmic period of growth was obtained. Spectrophotometric methodprocedure: The spectrophotometric method is similar in principle to the methods employed by Slater (18) and Youmans and Youmans (19). The principle of the method is as follows: If a series of broth tubes are inoc- ulated with various amounts of inocula, and the time is determined at which each inoculum grows up to a certain standard mass; then a linear relationship should be obtained when the time at which each inoculum grew up to a certain standard mass, is plotted against the logarithm of the amount of inoculum employed. ‘A‘_ TH! -1u- The slope of the straight line so obtained will represent the rate of growth of the organism and can be used for the calculation of the growth rate and generation time. In this experiment a spectrophotometer was used to measure mass. The certain standard mass or "deciding point" in this case, was when each inoculum grew to give a decrease of 5 per cent transmission upon the spectrophotometer. Various dilutions of the organisms were made to give inocula containing a different number of organisms. Spectrophotometer readings were made at half-hour intervals, and the time at which a certain standard mass was reached, was interpolated to 15 minutes. The equations used for determining the generation times were as follows: The growth rate constant K was determined in the following manner (20): K = 2.§O? log t B” Bt o where Bt = the log of the largest inoculum used. and B0 = the log of the smallest inoculum used. and t = the difference in time required for the smallest inoculum to reach a certain mass and the time required for the largest inoculum to reach this same certain mass. "TH! After the growth rate constant K is determined the generation time can be calculated from the follow- ing equation (20): .692 K C? where g = the generation time and .692 = the natural log of two. A series of new, 16 by 150 mm, pyrex test tubes containing Dextrose Broth were standardized with a Bausch and Lomb Spectronic 20 Colorimeter and were used in this experiment and again in later experiments. A wave length of 530 millimicrons was used through- out the entire experiment. A set of nine, standardized, sterile, test tubes was used for §;_ggl$ and a similar set was used for g; aerogenes. One control tube was used for both sets. Each sterile test tube, including the control tube, was filled with exactly 9 ml of sterile Dextrose Broth. Each set of nine test tubes was divided into three series, with three tubes per series. Each of the three tubes in a series was inoculated with a different number of organisms. Three series of tubes were used to obtain results in triplicate. The inocula used for determining the generation time TH! -16- of §;_ggli and g; aerogenes were 10'“, 10-5, and 10-6 dilutions of the original number of organisms present in 1 ml of a 12 hour broth culture. Sterile saline was used as a diluent for the preparation of inocula. Each set of tubes containing exactly 9 ml of sterile Dextrose Broth was preheated to 37 1’1 C in a water bath, then each series of tubes was inoculated with exactly 1 ml of the above dilutions. For example: One set of tubes consisted of series A, B, and C. Series A had three test tubes, numbered 1, 2, and 3 respectively. Number 1 tube was inoculated with 1 ml of a lO"'LL dilution of a 12 hour broth culture. Number 2 tube was inoculated with 1 ml of a 10'5 dilution, and number 3 tube was inoculated with 1 ml of a 10-6 dilution of a 12 hour broth culture. Series B and C were arranged in a similar manner. The time of inoculation was recorded and the tubes were again incubated in a 37 :_1 C water bath. Each set of inoculated test tubes was shaken, and read in the spectrophotometer at zero time. The per cent transmission was recorded. This procedure was repeated every 30 minutes after inoculation, and the per cent transmission was recorded each time. All readings were made using the same control tube, which contained 9 ml of sterile Dextrose Broth plus 1 m1 of sterile saline. The control tube was incu- bated with the other tubes in a water bath at 37 i 1 C. All spectrophotometer readings were made by placing the control tube in the spectrophotomete and setting the scale at 90 per cent transmission. In this manner, an initial reading could be recorded above 90 per cent transmiSsion as well as below 90 per cent transmission. If the control tube were set at 100 per cent transmisSion, a reading of 103 per cent transmission, due to a slight defect in the tube, could not be accurately determined. A decrease of 5 per cent transmission from the original per cent transmission at the time of inocu- lation was recorded as the "deciding point". The time of this "deciding point" was recorded for each of the three dilutions employed and from this infor- mation the generation time was calculated. Generation times of thermophilic bacteria: Preliminary experiment: Each of the six thermophilic organisms was incubated in 250 ml of Dextrose Broth in a 35 1.1 C water bath. Platings were made with Dextrose Agar after increasing periods of incubation. All plates -m- were incubated at 55 :_l C for 2h hours. In this manner the approximate length of the logarithmic phase of each bacterium could be determined. Generation time procedure: From preliminary work it was determined that 9 hour cultures could be satisfactorily used for inoculation purposes. 'Most of the thermophilic organisms were still in their logarithmic period of growth after 9 hours of incubation. Cultures q :or inoculation were grown in 15 ml of sterile 3 used Dextrose Broth, for 9 hours at 55 i l C. Each culture was transferred every 12 hours for 3 days prior to Use. For each temperature at which a generation time was determined, 3 series of sterile test tubes with 3 tubes per series were used. Each of the 3 tubes_ in a series represented a different dilution, hence, a different number of organisms per original inoculum. The procedure used for determining the "decidin; (1 point" was the same as the procedure explained earlier for determining the "deciding point" in the .1 experiments conducted on g; coli and A; aerqgenes. The dilutions used for determining the gener— ation time of thermophilic organisms were different -19- than the dilutions used in determining the gener— ation time of E; coli and g; aerogenes. The di- lutions used for thermophilic organisms were lO'l, 10'2, and 10'3, respectively, of the original number of organisms present in 1 ml of the 9 hour broth culture. Sterile saline was used as a diluent and was preheated in a water bath to SS i’l C before the dilutions were made. Sterile test tubes, (in a series identical to that used with E; coli and g; aerogenes), were filled aseptically with exactly 9 ml of sterile Dextrose Broth and preheated in a water bath to the temper- ature at which the generation time was determined. The tubes were then inoculated with 1 ml of the above mentioned dilutions. The time of inoculation and the per cent transmission at zero time was re- corded. All tubes were incubated in water baths at the desired temperature. The per cent transmission was read on the spectrophotometer every 30 minutes after inoculation until the "deciding point" was reached, or until a tube recorded a decrease of 5 per cent transmission from the original per cent transmission at the time of inoculation. -20- Calculations based on data of time required to reach the "deciding points" and the dilutions used gave the various generation times. Growth studies at above optimum temperatures whereggeneration times were not obtained. At temperatures of 67 C and above, growth was too slow, with most thermophilic organisms, to obtain a generation time by the spectrophotometric methoa. Therefore, viable count studies were made at above optimum temperatures, where the spectrophotometric method could not be employed. As in previous work, 9 hour Dextrose Broth cultures were used for inoculation. These cultures were grownin the same manner as described for studies on generation times. Ninety ml portions of sterile Dextrose Broth contained in 250 ml flasks were preheated to the temperature desired. Each 90 ml of Dextrose Broth was inoculated with 10 ml of a l-lOO dilution of a 9 hour broth culture. Therefore, the number of organisms per ml was closely related to the number of organisms per ml in the 10"2 dilution used in determining generation times. Saline was used as the diluent, and was preheated in a 55 1'1 C water bath. Flask cultures were incubated in a water bath at various temperatures from 67-83 C for 72 hours. Platings were made from the flask cultures with Dextrose Agar, after -21- O, 6, 12, 2h, h8, and 72 hours incubation. Plates were in- cubated at 55 i l C for 2h hours. Plate counts were made at the end of 2h hours incubation. Inoculation of flasks and platings from flasks were done in duplicate. Growth curves at various thermophilic temperatures: Two of the 6 thermophilic organisms studied were se- lected, and growth curves were run on these organisms at MS, 55, 59, 63, 67, 71, 75, 79, and 83 C. Nine hour Dextrose Broth cultures were used for in- oculation as in previous experiments. Portions of sterile Dextrose Broth contained in 500 ml flasks were preheated to the temperature desired. Bach 250 ml of Dextrose Broth was inoculated with 5 ml of a l-lOO dilution of a 9 hour broth culture of the organism. Saline was used as a diluent and was preheated in a 55 i l C water bath. Flask cultures were incubated in a water bath for 72 hours. Dextrose Agar platings were made from the flask cul- tures at O, 2, h, e, d, 10, 12, 2h, hd, and 72 hours. Plates were incubated at 55 i l C for 2h hours. Plate counts were made at the end of 2h hours incubation. Inocula- tion of flasks and platings from flasks were done in duplicate. RESULTS Table l-a shows the viable cells of E; ggli and fl; agg- ogenes during their legarithmic period of growth. Table l-b shows the generation time of E; ggli, and A; aerggenes cal- culated from viable cells. Table 2-a shows the time in hours for growth of E; ggli and g, aerogenes to show a decrease of 5 per cent transmission on the spectrOphotometer. Table 2—b shows the growth rate constant and generation time of Ehwfiflll and A; aerqgenes cal- culated from spectrophotometer readings. Table 3-a, through table 8-a show the time in hours re- quired for the different thermophilic organisms to show a decrease of 5 per cent transmission on the spectrophotometer at various temperatures. 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In this experiment, the laborious process of obtaining an accurate total and viable count was eliminated, and a generation time was determined by mass measurement with the aid of a spectrophotometer. The preliminary experiments conducted on g; ggli_and A. aerogents justify the use of the spectrophotometric method for determining generation time. The viable count method gave a generation time of 19 minutes for E; coli and 20 minutes for §;_aerogenes. Under the same conditions, the spectrophotometric method gave a generation time of 22 ndmurtes for E; coli and 20 minutes for §;_aerogenes. The rwmnilts arc in close agreement and warrant use of the spec- trophotometrhznmthod. Hansen (7) reported a generation time of 16 minutes at 55 L:_for'a thermophilic organism. The generation times ob- tairmxi in this study ranged from a minimum of 11 minutes at an optimum temperature of 71 C for Bacillus sp. #2" to a zuininnnn of 30 minutes at an optimum temperature of 59 C for Bacillug 8p. #11. The optimum temperature range for some -50- -51- organisms is very broad, while the optimum temperature range for other organisms is very narrow. For example: the optimum temperature for Bacillus s . w ranged from 59-63 C, while the optimum temperature for Bacillus §p.g§ll is 59 C. An increase or decrease of h C from the optimum temperature of 59 C for Bacillus sp. 111 shows a considerable increase in generation time. At A; C, the lowest temperature studied, growth was found to be very slow, and did not follow the regular ther- ’1 mophilic pattern of growth. Figures number 1 and a show a long logarithmic growth phase with a slower eie away phase at AB C. The total number of viable organisms obtained at AS C was no larger than the total number of viable organisms obtained at optimum temperatures for growth, but the number <1f organisms surviving after hU hours of incubation at MS C *was larger than the number surviving after he hours of in- CLflxation at optimum temperatures for growth. Hansen (7) also rxyted a.longer growth phase at lower temperatures, and at- trijnited the length of the growth phase to no acid being _forn£fli, because the fermentation processes are also slower at lxfider temperatures. Some thermophilic bacteria will grow verfir slowly at 37 C and even at 20 C. Gaughran (27) believes the ranimum temperature for growth to be rixed by the consistency of fats elaborated by stenothernal thermophiles, nnny approach solidity as the minimum temperature for growth is reached. Figures number 1 and 2 show that at optimum, or near optimum tenperatures for growth the lag phase was either very short or absent, followed by a short logarithmic phase and then by a rapid die away. Due to the rapid growth and death rate of thermophilic bacteria the maximum viable count is never very great in comparison with other bacteria. Although the maximum crop of thermophiles in a culture is of low mag- nitude, the chemical changes are considerable in a short period of time because of their high 'ermentation capacity. Various explanations are found for the rapid growth and death of thermophilic organisms. 302‘ views are as follows: Allen (1) claims the rapia 'rowth and death of the'mophilic organisms is due to their extremely high biochemical activity. Gaughran (2) accounts for the rapid growth and death on the inability to sporulate readily due to low 02 tension in a liquid medium at ele— vated temperatures. Imsenecki and Solnzeva (6) showed that an increase in aeration greatly in- creased the total number of cells. Hansen (7) claims that the rapid growth and death of thermophilic organisms are due to the I -53... rapid form tion and accumulation of acids through the decomposition of carbon compounds in the medium, Since he estimated that the fermentation capacity of thermophiles was 30 times as large as that of Streptococcus lactis at 20 C. Imsenecki anC Solnzeva (6) suggest that rapid growth and death of thermophiles maybe due to cy- tolysis induced by autolysis with resultant accu- mulation of toxic products in the medium. They detected autolysis by an accumulation oI enzyme in media inoculated with thermophiles. My explanation for the rapid growth and death of thermophilic organisms is the increased susceptibility of young cells to many lethal agents. The high metabolic and biochemical ac- tivity of thermophi es and the temperature at which they are grown contribute to the number of lethal agents present, causing a rapid death. At temperatures above optimum, growth was very slow. Fiffllres number 1 and 2 and the data in Table 10, Show that the ViefiDle count took a sharp drop immediately after inoculation, anaerl incubated at a near maximum temperature for growth. .AJLL organisms followed this same pattern in their upper limits of' growth“ After continued incubation at these elevated v" I“ -Su- temperatures, growth again became apparent in appreciable numbers. A flask containing 3-15 thousand viable organisms per ml, dropped to less than 100 viable organisms per ml, after two hours of incubation. After Z-QU hours of continued incubation at an elevated temperature, the flask which dropped to less than 100 organisms per m1 again had a viable count in the thousands or millions. This action maybe due to the survival of resistant organisms. These organisms maybe few in number at first, but after continued incubation for 43-72 hours, or more, multiply to an appreciable population. figures number 1 and 2 show that after hU-72 hours of incu- bation at a near maximum temperature for growth, the viable count is higher than the viable count obtained after 43-72 hours of incubation at optimum temperature for growth. Near maximum temperature for growth, the fermentation rate is probably greatly reduced and the organisms may continue to live and reproduce for a longer period of time. SUMMARY The results of this study show the wide temperature range for growth of aerobic thermophilic bacilli. The temperature range may extend from AB C to 83 C, and even higher, with the optimum temperature range for growth extending from 55 C to 71 C. The generation time obtained for thermophilic organisms ranged from ll minutes to 30 minutes at optimum temperatures for growth. At up C, a below optimum temperature, growth proceed at a very slow rate, but, the total number of organisms sur- viving a?ter an hours of incubation is greater than the total number of organisms surviving after hd hours of incubation at optimum temperatu:e for nrowth. At optimum temperatures for growth the generation time is relatively short. The lag phase is very short or absent and the logarithmic phase is short, followed by a rapid die away. Growth and death proceeded at a rapid rate. The total number of organisms surviving after ad hours of incubation at an optimum temperature for growth is slightly above or equal to the total number of organisms present in the original i 1100 ulum . At temperatures above optimum, growth was very slow but proceeded for a long period of time. The number of viable organisms took a sudden drop immediately after inoculation. -55- -56— Afdoer'continted incubation at elevated temperatures, the 1 IDJUHDGP of viable organisms again reached appreciable numbers. Steurting with the same number of viable organisms per ml aftxer 72 hours of incubation, the number of viable organisms L) per‘ rd at elevated temoeratures was larger than the number { “ OL . Fabian, F. W. and Graham, H. T.—1953 - Viability of thermophilic bacteria in presence of varying concentrations of acids, sodium chloride, and sugars. Food Tech. 7 (S) 212—217. Smith, N. R. and Gordon, Ruth B. - 1955 - Questionable adaptations of cultures to higher tempera- tures. J. Bact. b9, 603-60h. Allen, M. B. - 1950 - The dynamic nature of thermophily. J. Gen. Physiol. 33, 205-214. Gaughran, E. R. L. - 19u9 - Temperature activation of certain respiratory enzymes of thermophilic bacteria. J. Gen. Physiol. 32, 313-327. Gasman, E. P. and Rettger, L. F. - 1933 - Limitation of bacterial growth at higher temperatures. J. Bact. 26, 77-123. Gaughran, E. R. L. - 19h? - Saturation of bacterial lipids as a function of temperature. J. Bact. S3, 506. b 1' , 4.1“ “I,“ l . L r V Demco~293 {‘7‘ MICHIGAN STATE UNIVERSITY LIBRARIES 3 1293 13103 7119