A STUDY OF THE THERMAL RESISY'ANCE 0F BACTERIAL SPORES T0 M035? AND DRY HEAT BY USE OF THE THERMORESESTOMETER Thosis for flu Dagme of M. S. MfiCHiGAN STATE UNIVERSITY Waiter Aian Sisier I961 IIIIIIIIIIIIIIIIIIIIIIIIIIII lllllllllllHHlllHIUITHIIII‘IIMUM]HUI‘IIHHIHlIW 312930140404 LIBRARY Michigan Stan University PLACE N RETURN BOX to remove this checkout 1mm your record. TO AVOID FINES Mum on or before date duo. DATE DUE DATE DUE DATE DUE .M F 399;“ I Tn l ] L II J ___J| T: LJC: fifim MSU Is An Affirmative Action/Equal Opponunlty lnotltwon t, Y 2: 4,. id“ :-. '1‘ “ r1 "1 fink-DJ iL-{J-LU J. : ("If "“‘T 1 =1 ‘“ 1 .‘1 ft“ ' ’, ,fl } fl"- .1 QLUUJ. C‘l‘ 4.-..111 .1.L-.L‘J.<....-.L.; “avian“. ‘ in ‘t‘ ’ nwi'vt'r" .' r~"r\'r-*";v m ".c-‘r: W1 '. hW'J "v“."90 m .4 ~ . > . Cad ~J-..U;.-LlLLL-l——3.H UL L‘.’;L.LJK) .LO ;.-\,!J.i..).l. .LLJ. AJLLJ. .L,-£Js_.l. ‘ r ‘ ~' -;1 ".“NT _ ~'- '._‘ 11. r r '3 .. -’ -. A." .. »_1.-- —-v V’TT'“ .43. an.) L); .--‘.- i.....14iL...bit_L4.D.LsJLU- 44-243. by galter ”lan_Sisler frelininary studies indiCated that the thermal destruc— tion rate of the spores of Continental Can Company strain 5230 was not logarithmic. This investigation was to determine the accuracy of this observation. Destruction rates (D values) were determined for three different spore concentrations in moist and dry heat (superheated steam) using a thermoresistometer. Signi- ficant differences were found to exist between the D values obtained with the different spore loads in some instances at the higher tenreratures used with both moist and dry heat. How- ever, since the data showed no consistent trend and a nunber of sources of error were not considered in the statistical analyses, these data are insufficient to establish a non-exponential de- struction rate. In fact, the data obtained at the lower temper- atures using both moist and dry heat would not likely have been obtained if the destruction rate was not exponential. Survivor curves were determined using three initial spore concentrations in moist heat at 235 F and 250 F and in dry heat at 320 F. with the exception of the lowest spore concentration galter Alan Sisler at 320 F, the slopes of all the curves appeared to be approxi- mately equal. Regression analyses established that none of the survivor curves varied significantly from a logarithmic order of death. Considering the experimental error involved, fairly good agreement was observed between the D values calcu- lated from LDSO determinations and the values taken directly from the survivor curves. from these results, it was concluded that the death rate was exponential. The thermal resistance curve (THC) for the average of all the D values deternined in moist heat revealed a z value of 12.8 and the TRC for dry heat (superheated steam) showed a 2 value of 37.1. Thus, the temperature of superheated steam must be raised above a reference temperature approximately three times higher than that of moist heat in order to reduce the destruction time tenfold. .-- 11 rfl‘:__‘""1 v *fifif‘v " “TV-"'3'“. fflfl'fl x” rfi f: ' ’1"‘fl""1"‘ r fi-j; A . I‘ 4.2.-..5 - 4;]; 1:1 RBJOJLU'J-Ai'.‘ v.11 UL‘ u:.vl .J_{Ix‘.L 0.“. Di v" * cm ”W inf T."'*"m *“H " Tb ink/Isl 1“,.) DJ»; ILJILl DY b.0311 CF ":1 m'II'1 .i '"-. ; «w. :* n "rfvn'f Ti “11 .LI;_.|--‘- -U...:. 401 :J -Oi..—-'J.L.J‘)B By Halter Alan Sisler A THESIS Submitted to the College of Science and Arts of Michigan State University of Agriculture and Applied bcience in partial fulfillment of the requirements for the degree of Department of Licrobiology and Public Health 1961 .~ '1“ T we ‘ w-n r1 A'vfxl .\.«‘ .a .L_...J—JG--.'L.. .L For definite and helpful criticism in the research and presentation of this material, the author is greatly indebted to Dr. Ralph I. Costilow of the Department of Microbiology and Public Health and to Dr. Irving J. Pflug of the Department of Agricultural Engineering. I am particularly appreciative of their much needed encouragement during the long gestation period of this research. Statistical assistance of the friendliest and most judicious kind has been made available by Dr. ailliam D. Eaten, Statistician, Agricultural Experiment station, Kichigan state University. T4333 CF CC-‘I‘wTZTTs T ”w ""17 "2" .Lo I&.I‘.L\.UL.\ala.Ll.oooooooooooooococoon-000000000000 "32* . ‘1 Tm 7, m 17-; II. Lb‘flqdkf .L+3-u;.L-_L..:Joooo00.000000000000000... Spore structure and iesistahce............. Causes of neath............................ Lethocs 3mplo;ed to study heath by neat.... Crder of Death............................. Factors .14. fectim Thermal iiesistance... Methods of Evaluating Destruction nata..... TI]: 1;" ”$1.9?" 1“ 71* ‘ :.‘1~1¢v*rn ' .L o .34." 4-..L..J..I1LJ .‘VJ-JL/x..LJOCOOOOOOO0.00.... 00000 Source and Ireoaration of Cultures......... Standardi zation of Susgensions............. lstance.000000000000000 yiJ (b 5 03' H (3)} ., _ in... If. -1.-.) L100.coco-cooooooooooooooooooocoooaooooooo Effect of Spore Topulation on Destruction RateSOOOOOCOOOOOOOOOOO000.00.00.00....00... do rison of Thermal Resistance Curves for E- t T180113 and QUEBPllbcteQ Qtecdliooooooooooo U? ’C} 1' Oi 53"! survivor Curves............................ Percentage of necovered Spores from Cups by .blaSI}.iIl&ISOooooooooooocoo-00000000000000.0000. V0 Dis.) LSDI-kl'xooooooooooooooooooOooOQoooooOooooooo r. v5» -- s A—l -.‘ , . . -‘— . '1‘ ’3' -" 7 ~-:- -.~ —, -» VI. QLlHQ-LL’L—‘b; .‘LJJ Vb... vLLQiL'l‘J o o o o o o o o o o o o o o o o o o o o o o o o V11. BI;3L:LK3L;LLL:;‘1VOOOO00......OOOOOOOOOOOOOOOOOOOOOO n “v ufi J1~~LL J l. ‘ ...- - :1". 5‘ 4. .c‘- \ ELL”: i. :30 u l (.I" D Values ftr Io Jig- LA-LcLJ-CIUAA‘L A—JiI L; : x .L ‘5 I“ 323" "s 3 1:4. bllb‘.’ U‘c+ T - 'f- I ‘ I - 1 'l' v.— f‘ I' l‘ ‘ ‘ ‘l‘. » 1“ P - ‘-—. '1" ." r “ J J glut: g; 1 vi .21 J 4-891. (J‘Ji‘dl‘lléd sea a UCQ$-A '_’ - '1- ’ .' . ' -52" ' , .;- -3 4.- 2| . ._.v . -- Gal-xi LCD-L IQI. .J-L.J.Lil; le-llb 41.1.41 SPCAlcebo o o O F‘ " - . ‘r‘r. ~‘ . ‘r‘ . T 'r q ." ‘ "‘ “ “~ r . r‘ ,- 1.. ,, - uL'..1.:-‘;,LJ. .LLkJii 9L .2 “J G,..L\A'CD (Jute l'..L 6:1 by! the ' . ~ : _ l- ;; , - -- -. H . - z... , ' r -A . .n achinliil. ...:: oigou N 11.1; in J 58 t. utali Qt. iron. the ourVivor Jurve...................... .[WIJiP of Spores lec ove re d after suc- CEENSive {a3;i.’i;SOC...OOOOOOOOOOOOOOOOOO. CA7 00 FIGLHE l. m [-G 'H '0) *3 IO M FLJ L4 Q) q ‘1 a) 0) Thermal resistance curve of the various D values determined in moist heat........ Thermal resistance curve of the various D values determined in superheated steam. Survivor curves of various spore concen- trations of strain 5230 in moist heat at 230— E‘OOOOOOOOOOOOOOOOOO0.00.00.00.00...O. Survivor curves of various sgore concen- trations of strain 5230 in moist heat at 250 FOOOOOOOOOO0.0.0.0...OOOOOOOOOOOOOOOC Survivor curves of various Spore concen- trations of strain 5230 in superheated steam at (320 FOOOOCOOOOOOOOOOOO00.0.00... 40 43 44 45 l. I“? TRCDUC TIC}? Bacterial spores have been of interest to the microbiolotist since the beginning of observations of bacteria. They have afforded grounds for much research pertaining to (a) their nature and biological role, (b) the reason for their formation, (c) changes during their Sporuletion and germination, and, eSpecially, (d) their resistance to adverse environments. The heat resistance of bacterial spores is of the utmost concern to the food canner. Canning processes must be designed to render a food product safe for human consumption and at the same time allow for only the minimwn loss in palatability, nutritive value, and quality of the finished product. A.glance at the literature will reveal the enormous “mount of work that has been carried out in studying the thermal resistance of bacterial spores. It is known that the biological properties of spores determine and control their thermal resistance. Therefore, the development and application of sterilization by heat must arise from and be based upon previous thermal resistance studies. Many contradictions and discrepancies appear from the records of the earliest workers to those of the present day. This lack of uniformity has been due, in part, to variations within the same species of organisms, faulty I methods of study, and factors of unknown nature. W ri oas methods have been employ to study the thermal resistance of bacterial srores. Amen» the new- saratus cells: the thermoresisto- 1' :— fp-w a a. ' A...“ - .. .p - ”Tie 91‘. ml“. .Lo (,0 llLLL: CJ. tile .chcf't Eht 0‘ l‘i’r'lcul- Tw- \ tural Engineerirg, fichipen State university, has de- velOped a thernoxesiStoweter cc.able of nein: operated at very high tee ;eratures for either moist heat or dry heat resistance studies. Irelirinary studies indicated that a non-logarithmic order of death occurred when lo er hes Qtin temgeratures were obtained with this apparatus. The pm rpose of this stud; was to investigate tre orru er of death obtained pith moist heat as com pared to the order of death obtained with dry heat. 3. REVISJ CF LITERATUfiE Spore Structure and assistance Bacterial Spores are recognized as the most resis- tent of all living organisms in their ability to with- stand destructive agents (Perkins, 1957). It was first believed that spore formation was stimulated by adverse conditions; however, it now apnears tna many Species of bacteria eporulate in favorable environments. Actually, the biological role of the endospore is un- known. Since one bacterial organism forms only one spore, which in turn yields one vegetative organism, the endospore cannot be said to be a device that multi- plies the number of individuals of a species as in the case of spores of higher plants. Cook (1932) concluded that some bacteria form spores because they form spores. Many ideas have been expressed as to why bacterial spores show so much resistance to destructive agents. Williams (1929) believed that the resistance was at- tributed to the low water content of the Spore, thus making protein coagulation more difficult. It has since been shown that little difference exists in the water content of vegetative cells and spores of a given species of bacteria, but the per cent of bound water in the cell compared to that in the spore is quite significant. 4. Henry and Friammu1(1937), and Friedman and Lenry (1935) demonstrated that Spores of the gagillgs group have a bound water content of the order of 60 to 70 per cent, as compared with 3 to 21 per cent in the vegetative cell. The bound water, of course, would be less reactive and more resistant to physical agents. This theory may be somewhat discredited by the recent work of haldham and Halvorson (1954), They prOpose the theory that the heat resistance is a result of ”bound protein" rather than "bound water”, and that the protein of the spore is immobilized by having its polar groups attached to some particle; thus, making the protein less susceptible to thermal agitation and denaturation. Additional evidence presented by Powell and Strange (1953) suggests that the Spore protein is stabilized by attachment to particles since the Spore contains very little "free water" to assist in the protein coagulation, and that in germination there is an exchange of solids for water from the suspending medium. Curran et_al, (1923) found that Spores were higher in calcium than the vegetative cells and, in general, high concentrations of calcium were associated vith in- creased heat tolerance and resistance. Powell and Strange (1956) report that.dipicolinic acid or a polymer thereof may confer thermal stability upon Spore protein molecules either by direct linkage or further chelate linkages between dipicolinic ac’d, protein, and calcium or heavy.metals. Sugiyama (1951) suggests that the resis- tance could be due to a stabilizing effect due to lipid protein combinations. Some investigators have found a correlation between the specific gravity of spores and their resistance, the Spores with greater density being the more resistant. Others have advanced the theory that the enzymes present in the bacterial Spore are in an inactive or resistant form, From the foregoing material, it is apparent that the basis for the resistance of the Spore is still obscure. However, increasing interest and study of spore problems have brouyht us nearer to an understanding of the re— sistance mechanism. Causes of Death Although the mechanisms of thermal destruction of bacterial sporeseuwanot clearly understood, it is held by some investigators (Isaacs, 1935; Virtanen, 1934) that death is associated with heat inactivation of cer- tain vital enzymes in the organism. Rahn (1929, 1043) concluded that.death by the heat inactivation of enzymes could not be correct because, mathematically, a logar- 6. ithmic order is possible only when death is due to the destruction of a.s‘nple molecule in the cell. williams (1929) supports the idea that the cause of death in cells exposed to a high temperature is the coagulation of bacterial protein. This idea is based upon the fact that conditions which render protein more difficult to coagulate result in an increase in heat resistance. aahn and Schroeder (l9al) reported that the inactivation of enzymes can not be the<3ause of death because the number of living cells shows an enormous de- crease when heated while the enzymes show only a slight decrease. Furthermore, Rahn.(l945a) argued that the magnitude of te perature coefficients of death support the theory that organisms subjected to moist heat are killed by denaturation of protein. It is noW'generally accepted that the cause of death by moist heat is due to coagulation of some pro- tein in the cell. Even though much research remains to be completed with dry heat, it is believed that death is due primarily to an oxidation process (Hahn, 1945b). Eethods Employed to Study Death by Heat Much of the earlyxwork on death by heat, or heat resistance of microorganisms, was done by means of a "therna death point” determination. damples of a culture or adepension were exgosed for a fixed time period, usually 10 minutes, to various temperatures and survival or no survival determined. The "thermal 'efinefl as the lowest temperature re- 3 death point” is ( sulting in no survivors after a.given exposure time the cells. The first of the modern metho s was that used by sigelov and gsty (1829). Trey defined the thermal death point in relation to time as the time at dif- ferent temperatures necessary to completely destroy a definite COficen‘Tation of spores in a medium of known rydroven ion concentration. Their mettod employed the exposin; of sealed glass tubes containing a known num- ber of spores in a s ecifiei aeiium to heat treatment in an accuratelx controlled oil bath and then removing the tubes after Various exposure times and subculturing for survivors. since this method involved the death rip ive tern thermal (L (u (t - S C) point in relation to time, the death point was soon changed to thermal deutn time (inf). "1 The iQT was conceived to lie between the longest time of exposure showin; survivors and the shortest time of exposure showing no survivors. Esty and Jilliams (1924) noted the occurrence of "skips" when single tubes were removed as in the method of Bigelow and Bsty (1920). They suggested a multiple tube method in which as many as 25 tubes would be removed at each time interval, subcultured, and the resulting percentage of survivors plotted against time on semi-logarithmic paper. This resulted in a straight line. The principle of using multiple samples in order to avoid “skips” has since been used in almost all heat study work; however, it is highly doubtful that there is strict logarithmic relationship between eXposure time and percentage of survivors (Schmidt, 1957). Magoon (1926), and Stern and Proctor (1954) used capillary tubes to hold the standard suspension. This technique had the advantage of assuring uniform and instantaneous exposure of all spores to the heat, and of giving each Spore the Opportunity'to germinate. Townsend, Esty, and saselt (1988) described the use of steam as a heating medium and the use of thermal death time cans. This method consists of using small cans (208 x 006) filled with a sample containing the spore load, sealing the cans, heating them rapidly in an accurately controlled steam retort, and then incubating 8. them. Thi method has the advantages of being able to use viscous products wiicheare difficult to get 0 —‘ into tubes and of be a similar to conditions occur- .4 Tr. ring 3.; in the CQinerciel processing of products (Yesair and Jameron, 1912; stumbo, Gross, and Vinton 1845). Schmidt (1950) improved on this method when he made use of a sgecially constructed miniature retort in ad- dition to thermocouples and a recording potentiometer to \ five a more accurate come-u: time (the time required to ( reach retort temperature Within the can). Also, Schmidt (1850) explained a method for heating cotton plugged tubes in steam and cooling under pressure which permitted subculturing by adding the medium to the suspension in the tube after heating, rather than the transfer f the suspension to a subculture medium with possible loss of viable organisms. ochmidt, Sock, and hoberg (1855) de- scribed a retort system designed for the use of either thermal death cans or cotton plugged tubes. williams et a1, (1937) made use of the “tank" method to determine spore destruction rates at temp- [\3 eratures above 12 F. This consisted of heating a suspension in any liquid, or in a mixture of liquid and suspended solids, under pressure with constant stirring, mam u=h—__—~_-_ 7*.éH_ - 10. then removing a sample at any desired time with continued exposure of the remaining material. From the large amount of data presented by Reed giggl. (1951) on this method, one may conclude that it was not too successful. For studying thermal death times up to 270 F. an apparatus called the thermoresistometer, which was designed by Stumbo (1948), may be used. This ap- paratus is designed to heat to high temperatures and cool rapidly a small amount of suspension (0.01 ml) without any need for correction due to lag time. Pflug and Esselen (1953, 1954) described a thermoresistometer very similar to the one designed by Stumbo (1948). Both thermoresistometers were designed for use with moist heat. Pflug and Esselen (1953, 1954) have oper- ated their thermoresistometer in a heating range of 235 F to 305 F with an exposure time as small as 0.01 minutes. It is known that vegetative cells and Spores (are much more resistant to dry heat than to moist heat (Rahn, 1945a). According to Schmidt (1957) nearly all of the methods used to study resistance to dry heat in the temperature range above 212 F thus far have not experienced a high degree of precision. Eflug (1957) 11. D; esigned a thermoresistometer for the study of the resistance of spores to dry heat. This apparatus is capable of operating in the range of 212 F to 380 F with an average lag correction factor of 0.23 minutes for air and 0.20 minutes for superheated steam. Order of Death The microbiologist considers a microorganism to be dead if it fails to reproduce when suitable conditions for reproduction are maintained. The order of death has been found to be either of a logarithmic or non- logarithmic nature. The logarithmic order implies that the logarithms of the numbers of surviving cells, when plotted on semi-logarithmic paper against the units of time of heating, will give a straight line curve. The non-logarithmic order is said to occur when the plotted curve is statistically incompatible with a straight line. Jhether the order ofcieath is logarithmic or non-logarithmic, the curve is referred to as either a thermal death rate (TDR) or survivor curve. Chick (1908, 1910) concluded that the death rate of microorganisms showed a logarithmic order. Data pre- sented by Bigelow (1921) were sufficient to suggest that thermal death rate curves were logarithmic. Cohen (19 2) found that the mortality of bacteria whether by strong or mild disinfectants follows the laws of logarithmic decline and the disinfection pro- cess can be expressed by mathematical relations com- parable to those used in dealing with monomolecular chemical reactions. nahn (1982, 1943, 1945a, 1945b) reviewed much of the data upon the logarithmic order ofcieath of.miCroorganisms and was of the Opinion that a logarithmic order is only possible when.death is due to the destruction of a single molecule in the cell. Stumbo gp g1. (1950) reported thermal death time curves were essentiallyimraight lines, and nothing about the nature of the curves indicated that they should not be straight lines. Reynolds and Lichtenstein (1952) presented data that is inconsistent with the assumption of exponen- tial death. These researchers obtained survivor curves which were sigmoid in nature. Kaplan at al- (1953) found the initial deviation from linearity of the thermal death.rate curves described by Reynolds and Lichtenstein (1952) to be real and not an artifact of experimental methods. 13. Falk and hinslow (1926) concluded that even though the logarithmic curve best expresses the death rate, the correspondence is not and cannot be exPected to always be a close one. Rahn (1931) analyzed the work done up to 1930 and found that out of 154 experiments, 83 resulted in sagging survivor curves (concave upward), 39 resulted in bulging survivor curves (concave downward), and only 32 resulted in a straight line. El-Bisi and Ordal (1956b) presented four types of abnormal curves most frequently encountered and an eXplanation for each. Frank and Camp- bell (1957) observed a steady increase in D values with increased time of heating, indicating that the survivor curve could not be a straight line. The order of death is of most importance since all calculations for obtaining sterility are based on a logar- ithmic order. Schmidt (1957) felt that enough data had been presented to warrant the use of a logarithmic order. Factors Affecting Thermal Resistance Schmidt (lc54) proclaimed that it is very difficult to make any definite statements on the work involving factors affecting thermal resistance due to the many vari- ables introduced by each researcher. However, we can not disregard this topic since there has been much research concerning it. l4. Esty and Meyer (1922) performed experiments using young and old moist spores of Clostridium botulinum and found the young Spores to be more heat resistant. Magoon (1926) reported that length of time of storage, storage temperature, and humidity had an effect upon the resis- tance of spores of Bacillus mycoides. Sommer (1930) found the greatest resistance of spores of 91. botulinum to occur between 4 to 3 days. Curran (1934) found aging as long as one year would increase the resistance of acillus cereus. However, during the first three months the resistance did not show any appreciable change. Wil- liams (1936) could not find any correlation betweenzage of the Spores of several different organisms and their heat resistance. From the work done to date in this area it appears that no definite conclusions are really justifiable. The temperature at which the Spores are grown apparently affects their resistance. Jilliams (1929), Curran (1934), hilliams and dobertson (1954), and El-Bisi and Ordal (1956b) found that the resistance of spores increased with an in- crease in temperature of incubation, and Lamanna (19a2) noted that within the genus figgillgg a general relation- ship between maximum growth temperature and Spore resis- tance existed. Therefore, this factor must have a defin- ite influence on resistance. 15. Nutrient conditions have been found to produce various effects on the resistance of Spores. williams (1929) ob- served that the resistance of a strain of Bacillus sub- tilig could be changed by varying the nutrients in the sporulation medium. However, an increase in resistance could not be transferred from one generation of Spores to another. Curran (1935) found that Spores produced and aged on soil or oats were more resistant than Spores produced and aged on artificial media. Sugiyama (1951) noted the resistance of Spores of 9;. botulinum to be affected by the constituents in the medium. Lowering of iron or cal- cium concentrations decreased the resistance, while the ad- ditions of long chain unsaturated fatty acids tended to increase the resistance. A noticeable decrease in resis- tance of Spores of Bacillus coagulans (thermoacidurans) was observed by El—Bisi and Ordal (1956b) when a high phos- phate concentration (1% th P04) was present in the sporu- lation medium. weiss (1921), hahn (1928), Braun, Hays, and Benjamin (1941), Anderson §L_al, (1949), and Sugiyama (1951) found high concentrations (25 per cent to 50 per cent) of soluble carbohydrates generally increased the resistance of bacteri- al Spores. Amaha and Sakaguchi (1954) could find no effect upon the resistance of spores of PA3679 (Clostridium sporo- agggé) after adding 10 per cent to 50 per cent sucrose, glucose, or glycerol to the suSpending medium. 16. The addition of various salt concentrations to the Sporuleting medium ias given conflicting results. weiss (1921) observed a decrease in the resistance of 21. bgpg: linum spores upon the addition of 3% salt. however, Bsty and heyer (1922) noted an increase in resistance with con- centrations up to 95. Headlee (1991), and Anderson, Esselen, and Fellers (1949) found that low salt concentrations (1% to 9.53) would increase the resistance, while concentra- tions of 8% to 10% would decrease the resistance. Yesair and Cameron (1942), using a medium consisting of 3.5% salt in phOSphate buffer, observed a decrease in the resistance of Spores of £1. botulinum at temperatures below 230 F, but no appreciable change at 230 F to 235?. schmidt (1957) believed the effect of salt depends upon the type of medium being used and upon the organism under test. The pH of the suspending medium may have a great ef- fect upon the resistance of the crganism. Even though there are variations in resistance of different organisms, n general, resistance is at a maximum in the pH range of L- H- 6.0 to 8.0. Sognefest g; a1. (1948) concluded from his work on the resistance of Spores of 21.'botu1inum and P33579 that in the pH range of 4.5 to 9.0, the lower the pH, the less the resistance; and with pH 5.5 and below a rapid increase in the destruction rate occurred. Little 17. change in resistance was observed between pH 6.0 to 9.0. Reed, Bohrer, and Cameron (1951), and heynolds g3 g1. (1952) offered the general belief that for spores which are heated in food products below pH 5.5 a recovery medium near pH 7.0 will show longer survival times. This is an indication that pH not only has an effect upon the organ- ism but may alter the degree of dissociation of many sub- stances in solution in the product or shift the oxidation- reduction potential, which may have effects upon survival or recovery of those that have survived (Schmidt, 1954). The use of antibiotics to lower thermal resistance has increased the interest of the researcher since Anderson and hichener (1950) reported the successful use of subti- lin and mild heat to preserve food. however, many workers believe that the action is only sporostatic and not lethal (Adams g3 g1., 1951; Cameron and Bohrer, 1951; Williams and Campbell, 1951; Williams and Fleming, 1952). Le Blane gt a;. (1953), Lewis g3 Q1. (1954), and O'Brien and Titus (1955) found subtilin would either cause a reduction in destruction time of Spore of PA3679 or reduction in the outgrowth of those spores surviving drastic heat treatment. It now ap- pears that there is still much research to be performed in this area before conclusions may be drawn. 18. Methods of Evaluating Destruction Data The graphical plot on semi—logarithmic paper of the logarithmic order of death results in a straight line. This being true, then the rate of the reaction, K, could be calculated from the formula: 1 K = - (log No - log N) (1) t W I ' a constant, depending upon the organism, temperature, subtrate, and assuming logarithms to base 10 t '-' time of exposure in minutes No 3 initial number of organisms at start of t N : final.number of organisms at end of t The calculated K value from the above is small and inconvenient to use. This led Baker and McClung (1939) to measure the time needed1x>reduce the original number of organisms to 99.99%. Katzin QEDQL3 (1943) pointed out that in applying the above equation (1) to the length of time it would take to reduce the initial number by 90%, the equation would thereby become: F4 A [\I) V K = - log NQ or, t (1011\‘13 l p : - t or, 1 t z " a K The time t was defined as the Jecimal neduCLion Time (DnT), or the time required for the survivor curve to transverse one 105 cycle when 103 N is plotted against time. Ball (1943}, and otumbo (1948, 1949) used 3 to represent this time. However, Gillespy (1946) suggested the use of 3 because of the chance for confusion between Z of the sur- vivor curve and z of the thermal death time (TUT) curve. According to Ball (l943) a plot of the D values for different temperatures on semi—logarithmic paper would re- sult in a “phantom thermal death time curve". The phantom curve would have direction but no position and the slope of'the curve would measure the number of degrees Fahrenheit required to transverse one log cycle in the same respect as 2 of the TQT curve. D values may be calculated from survivor curves or from multiple sample method determinations. For the mul- tiple sample method, Stumbo (1948) used the formula: 20. (3) D = U'or t - log A.- log B A = the total number of samples heated multiplied by the num- ber of spores per sample B 3 the calculated total number of survivors based on the assumption that there is one surviving Spore per container when less than the total num- ber of containers show survival U or t 3 the exposure time at a given temperature Stumbo gt_al, (1950) used the basic method of Stumbo (1948) but calculated the number of survivors, (B) in equation (3), by the "most probable number" procedure of Ialvorson and Ziegler (1933). Schmidt (1954) describes a method fbr obtaining the D value based on the procedure for bioassay suggested by Reed (1936). Schmidt (1954) made ‘the assumptions that any sample not showing survivors at a.given exposure'time, would not show them at a longer exposure time, and any sample showing survivors at a given exposure time would show them at a shorter exposure time. The probability of sterility may thus be calculated from the formula: n / 1 (4) m / n K 2 probability of sterility the cumulated number of samples showing no growth at each exyosure time the cumulated samples showing growth at each embmmetmm By plotting the probability of sterility, (P), against exposure time on arithmetic probability p'per and fitting the best straight line to the points, the L050 point may be found. This point represents the time at which 50% of the samples will be negative. ”ith this knowledge the 3 value may be calculated as foll ms: t) I U u 1-1 A 0.6 C) V or, log A - log 0&0 log A Z 0.16 the initial number or organisms per tube 9 3 the surviving organisms per tube at L050 point (halvorson and Liegler, 1933) omparisons have been made of the methon of Stumbo (1948), Stumbo gt g1. (1950), and Schmidt (1954) for cal- culating D values, and, in general, these values all agreed within approximately 10 per cent (Schmidt, 1957). Lewis (1956) criticized the Stumbo gt El. (1950) and Schmidt (1954) methods and suggested the use of the weighted probit method or the Spearman-Karber method. however, these methods are extremely laborious (Schmidt, 1857). mammarrgm PROCEDURE Source and Preparation of Cultures The test organism for use in thermal resistance deter- mination should be one which may be grown easily on ordin- ary culture medium and one which will produce an abundant yield of spores in a minimum time. In addition, the or- ganism should have a characteristic type of’growth or pos- sess some distinguishing characteristic which will serve to differentiate it from contaminants likely to be encountered (magoon, 1926; Williams, 1929). For this study, a Spore forming mes0philic aerobe known as strain 5230Lwas selected. This organism appears to be identical to Eggillus subtilig in.morphological and biochemical characteristics, except that strain 5230 will grow anerobically in the presence of fermentable carbohy- drates, whereas, a. subtilis will not. The original cul- ture was obtained from Dr. C. F. Schmidt, Continental Can Company, Chicago, Illinois. The sporulation medium used was nutrient agar (Difco) plus 1 ppm MnSO4. The initial slants were prepared in 20 ml screw cap tubes. However, it was noted that the culture would lyse within 2 or 3 days in the tightly closed tubes. Thereafter, the screw caps were tightened just enough to hold them in place and to prevent air contamination. The —--._.- ~——— .m— .— ~.—- .— - _.- . .--.-—.- ._.. —- .4..._. -O- --- _-——---——-— letained from Dr. C. F. Schmidt, Continental Can Company, Chicago, Illinois 24. culture was transferred on each of two successive days before inoculating the slants for Sporulation from a 24 hour culture. After incubating for one week at 98 F, the spores were ready to harvest. Ten m1 of chilled 35 F sterile distilled water were added to each tube and the growth freed with a sterile inoculating loop._ The spore suSpension'was then filtered through sterile cotton con- tained between 2 pieces of cheese cloth. The filtrate was washed by centrifugation in sterile h/15 phosphate buffer at pk 7.0, resus;ended in the buffer, and then stored in a sterile 16 oz glass bottle containing glass beads. This was used as the stock spore suSpension. Standardization of SuSpensions The stock spore suspension was shaken by hand for 5 minutes in an effort to break up any clumps that might be present. «Two m1 of the Spore suSpension were heat shocked at 212 F for 15 minutes to enhance germination. A series of dilutions was made, 5 plates were poured for each -ilution using dextrose-tryptone agar (Difco) containing 04 ("J .004 per cent brom cresol purple modified by the addition of 0.5 per cent soluble starch. Since this organism will produce large Spreadin~ colonies, which cause difficulty (‘1 as well as inaccuracy in plate counting, it was necessary .1. to pour the agar in three layers. The first la er served 5 as a base to eliminate moisture on the bettom of the cul- ture in the petri dish, the second layer contained the spore dilution, and the third layer was a thin overlay which eliminated surface moisture on the second layer as reduced the abundance of oxygen available thereby U) P. e 11 Ca. reducing the size of the colonies. The plates were incubated at 98 F for 48 hours. Counts were made to finfi the :rorer dilutions, and then 25 repli- - $ 1 ure 1‘ (D t A cate plates were poured per dilution. This oroce sulted in a standardized stock suspension containing 3.30 x ltd sp res per ml. 3 ietroff-Lausser count indicated 1.0 x 10 spores per ml. Three dilutions of the stock suspension containing 3 . 3.80 x 10 SfOPeS per ml were made with h/io phosphate buffer (pH 7.0) in order to obtain three spore concen- r . \ r ‘ r‘ ‘r H. r - A 6 7 p ,':: 8 ~ f' '1 trations of approximately 10 , 10 , anu 10 per m1. These suSpensions were placed in 16 oz glass bottles, containing glass beads, each suspension was then standardized accord- .1 i. (D at 3 Lb ing to the above counting procedure, and store . A microsc0pic examination of eacn suSpension revea’ed approximately 100 per cent spores, no excess of foreign materials, and mall clumps occurring in about 10 per cent of the fields viewed. 26. of Thermal Resistance An imgroved model of the thermoresistoneter designed t‘f‘ C—4 by lfluy end Buselen (loco was used in this research. This thertoresistoneter is cafable of being used in stuoies of the resistance of microorganisms to wet heat, surerheste: steea, or hot dry vses. er heat studies go have been conducted u; through 850 F; however, the apparatus will operate as high as 350 E. The dry hea system is accurate to f_O.l minute of exposure times. The lea correction factor is 0.20 minute for suferneeted steam -‘ 0L1 and 0.23 minute for air. r0 lag f ctor is involved during 9:. wet heat operation. In wet heat studies, five tin—plated cups containing the test sanples were transgorted into and out of the ex3osure chamber by five gistons. The exposure chamber is connected to a pressure controlled steam reser- voir containing approximately 6 g cubic feet of saturated steam. The temperature in the r rvoir is measured with {D (D «D an industrial thermometer. In C sing superheated steam, four replicate cups are tested at each EXPOsufe time, the wiston) cortains a thermocouple fifth sample cup (150. l g used to measure the ten erature at the cu; position. The top, bottom, and front plates of the exposure chamber are heated electrically by manually operated variable trans- formers. Saturated steam leaves the pressure controlled steam reservoir, passes through a pneumatic needle valve, 3 then to an elec rically heated exposure chamber. The de- 27. sired exposure time is set on an automatic cycle timerl. A Precision timer2 is used to record the actual time of exposure since there may be a slight difference between actual and desired eXposure time. The subculture tubes are placed in the receiver and at the end of the desired eXposure time the cups are automatically withdrawn:from the exposure chamber and dropped into the tubes. The sample cups used were punched from 0.008 inch thick tinplate. These cups have an outside diameter of ll mm and a depth of 8 mm. In order to remove the machine oil from the cups, the cups were washed in boiling soapy water, rinsed in‘distilled water, rinsed in ethyl alcohol, rinsed in acetone, and then air dried. They were steril- ized by placing 25 to a petri dish and heating at 320 F for 2 hours. A.0.0l ml sample of the Spore suspension.was dis- pensed into each cup with a calibrated a 179E micrometer screw3 combined with a 2 ml syringe. The inoculated cups were dried in an electrically heated vacuum oven‘nhich contained a drying agent (Ca504) and was under 20 inches of vacuum. To facilitate drying the suspensions, the oven was heated to 104 F and then the heater was shut off. T". 1Hanufactured by maéle Signal Corp., holine, Illinois. 2manufactured by The Standard Electric Time 00., Spring- field, Kass. q ' V I ' r‘ n Omanufactured by x. M. welch hanufacturing 00., lolo sedg- wick St., Chicago 10, Illinois The dried snore suspensions were stored in dessicators which contained the drying asent. Thermal death time studies were made for each Spore concentration using wet heat and superheated steam. The dried spore suSpensions were heated and subcultured in tubes of dextrose-tryptone-starch brOth plus brom cresol r‘ for a days at which time "1 purple. Incubation was at 85 r the results were recorded. a second reading was made at ‘14 days and the tubes discarded. Acid formation and a typical pellicle are characteristics of a positive tube; no growth indicates a negative tube. Any growth different from that which characterized a positive tube was con- sidered to be due to a contaminant. D values were calcu- lated according to the method described by Schmidt (1954) and presented in the above review. A statistical analyses was performed to determine if any significant difference existed between the D values for each Spore concentration at each Specific temperature. The analyses were conducted as follows: (a) The standard deviation error of each line drawn to best fit the points plotted on the probability paper was calculated: (6) $.32 n - 2 65 (Te standard deviation error fez = total sum of the square of each distance from the plotted points on the probability gaper to the line which best fits these points. This diStance is measured on the vertical axis (time scale) of the probability paper. n = total number of points to which line has been fitted. (b) The standard deviation of D at LDéQ was found: as 9:. K (7 ) 05 - standard deviation of D at LD5Q standard deviation error a K = log a - log 6.9 (constant for.a given spore concentration for a given set of heating data times number of replications, lO replications used thus log 0.69 becomes log 6.9) (c) The difference between standard deviations of D values at LD50 was calculated: 071:2 - D1) {02132 “.231 (8) “(DZ - D1) = difference between standard deviations of D values at LD5o 0230 = standard deviation of D at LDso squared. (d) The t test was conducted to investigate whether any significant difference existed between D values: (9) «(D2 - Dl) calculated value of t t : D2 - D1 3 difference between calculated D values 30. 07D2 - D1) = difference between standard devi- ations of D values at LD5O Statistical t - value obtained from t table at the 95 per cent confidence in- terval. The D values were used in the procedure for ob- taining the survivor curves. The length of time for exposure at each temperature and for each different spore concentration was based upon 5 D value, 1 D value, 1 g D values, 2 D values, etc. Each cup was drOpped into 10 ml of sterile saline, shaken mechani- cally for 10 minutes, shaken by hand for 1 minute just before plating, and five replicate plates per dilution were poured with dextrose-tryptone-starch agar plus brom cresol purple. The zero times were obtained by drOpping the cups into 10 ml of sterile saline and heat shocking at 212 F‘for 15 minutes, and then the above shaking and plating procedure was repeated. Incubation was at 98 F for 48 hours after which counts were made. The survivor curves were plotted statistically by the method for the standard error of the estimate. Regression analysis were performed to see if the data would best fit a straight line which would be an indication of logarithmic order of death. The standard error of the estimate was conducted as follows: 81. a % bx = basic equation for a straight (10) line predicted be the standard d error of the estimate predicted number of survivors predicted initial number of organisms: found by (ll) a 3 § - b X ..here: - = average of the log number of survivors b - same as below x - arithmetic average exposure time for complete run x = the lenith of time of eXposure for a given sample b= ixy - (Ex) (ty) (12) n txg - (tx)2 I1 where: X I]. - same as above 3 log number of survivors at each exposure time number of samples taken‘during the run The retreesion analysis was carried out as follows: :::=,Statistical Fg¢,l per cent and 5 per cent H V 32. T3 Zy2 - £2312 (15) n E' It 2y2- aZy - bey (16) where: T = total sum of squares of the log of survivors E' 3 sum of squares of the log of survivors representing the regression line ' mean square of the deviations from the regression line Ed I same as above (12) n Statistical F = values obtained from Goul- den's tables y, a, and b are the same as above (11, 12) Since it was believed that the number of spores remaining dried to the sample cup could have an effect upon the resulting survivor curve, randomly selected sample cups representing the three spore concentrations were drOpped into 10 ml of sterile saline and heat shocked at 212 F for 15 minutes. Each tube was shaken mechanically for 10 minutes and by hand for 1 minute prior to plating with<1extrose-tryptone-starch agar plus brom cresol purple. The cups were removed aseptically from the first tubes and drOpped into a second set of tubes of saline. The cups were passed through four tubes (A) (n for washing and plating in this manner. A series of 5 washings in 8 ml of dextrose-tryptone-starch broth plus brom cresol purple followed. Plates and broth tubes were incubated at 98 F for 48 hours and observed. 34. RESLLTS Effect of Spore POpulation on Destruction Rates The Schmidt method was employed in calculating the destruction rates for three different spore concentrations. Values were obtained for four temperatures with moist heat and for two temperatures with dry heat (superheated steam). t tests at the 95 per cent confidence level were performed to investigate any significant differences that might exist between the values for each temperature. Significant differences were found to exist between destruction rates observed with.different initial spore populations in some instances (Table 1). Specifically, differences were found between D1 and D2, D2 and D3, D4 and D5, D4 and D6, and D7 and D9. No test for significant differences between D7 and D8 and D8 and D9 (Table l) could be computed, since the plot of the probability points used in the Schmidt method fell on a straight line. Hence, no standard deviation error could be obtained for use in the statistical analysis. However, a significant difference was found to exist between D7 and D9 with an arithmetic dif- ference less than that between D7 and D8 (0.597 minute compares to 0.991 minute). Therefore, it is safe to con- clude that a significant difference exists between D7 and D8' The arithmetic difference between D8 and D9 (0.394 minute) is of doubtful significance. Table 2 presents the D values obtained for superheated steam. Signifi- cant differences existed between D1 and D2, D1 and D3, and D2 and D3. No significant differences were observed between destruction rates at 320 F. A clumped or nonhomogenous Spore suspension could vary the destruction rates to show significant differences. However, a microscopic examination of each spore suspen- sion revealed approximately 100 per cent Sporulation, no excess of foreign material, and only small clumps occur- ring in about 10 per cent of the fields viewed. Although each suspension was shaken by hand for 5 minutes prior to being used, there is the possibility that some clumping occurred in the 2 ml syringe during the dispensing of the 0.01 ml aliquots into the thermoresistometer cups. The suspension containing 1.27 x 108 spores per ml was extremely heavy and trouble was encountered in passing it through the orifice of the needle of the micrometer syringe. This could have resulted in a somewhat low initial pOpulation. However, should either of these events have occurred, it does not seem probable that the cups containing the clumped Spores or low popula- tions would be selected for testing only at the higher temperatures. Furthermore, no significant differences 36. in the destruction rates were observed among those values obtained at 235 F‘with moist heat or among those calcu- lated at 320 F with superheated steam. 37. .mwmaamcw Hmoflpmwpmpm may aw mm: mom omqflmpno on UHsOO “chum GOHgmfi>mb 09m0qmpm on .moCmn «mafia pzmflmnpm 6 do Hamm mpcfiom hpflawbmnoem 0mg mo poam map mmomomn mm paw mg no mm 0am 0Q Cmmspmn pmuosoqoo pod who; mocmoflmfldmflm how mumma + .mmmpsdq pump 0pm0flmcw mpmfipomndm e ammo pom no.0 pm .cmam no I m0 mqoa mm I an psoo Mom mcoz on I mm #:00 pom 00.0 as .qun 00 I 00 p200 9mg no.0 as .amam no I an mam.o omm.0 n 00 som.o u on osm.o u so 00m #dmo pom o0.0 as .qum .+00 I s0 mmm.m 0mm.m u 00 was.m u m0 mms.a u so owm mac: man I Ham 0:02 mam I OH: mqoa Ham I 0am 000.3 0H0.¢ u mam oma.m u Ham mmo.m u {can 0mm moqsOHMHcmam cmmapmm cams 0.5ma o.ma mm.H .ao ..mEmB 1 mmothmwmfifi onoHMHGmHn mom mpwme f HSNQIOH x COfipmAQC00QOO macaw on {14' mmommpmmmwfl pdmoflmfimnflm pom pmme tam pmmm pmwoa pom mosam>_m H 1.! rl...’ mdB 38. .mAmQajc pmmp mpMoaUflw mpafihompom * #Qmo pom no.0 pm .GMwa mg I NO #200 pom mp.o pm .Cnfim mm I an pamo pom no.0 as .cmam No I H0 mm.a 00.0 mm mH.H n m0 mo.H u H0 mflofl am I Ln 0:04 mg I mm moon on I we mm.w No.0 u on mo.w u mm mm.« I .00 mossOHMHQMam somepmn mama 0. ma m.ma mm.a go ..mEmB mmodmhmmufld pmsowwwumflh how mpmma HE\©IOH x Coflpmhpcmoaom whomn mmoamhummfid pumoflmflfinfln pom pmmh Mao.dfi3m9h bopsmmamMSnv pmmm Nun mom mmjam>.m N HdflfiH (L) (O 0 Comparison of Thermal Resistance Curves for hoist Heat and Superheated Steam The thermal death time (TDT) curve is a measure of the change in rate of destruction with change in temper- ature. In recent years it has been found more desirable to plot thermal resistance curves (TRC) than TDT curves since such resistanCe curves relate time to give a definite amount of destruction (90 per cent) at various temperatures. Therefore, any point on the T30 may be extrapolated to determine the correSponding time and temperature necessary to reduce the number of organisms by 90 per cent. The slope of the T33 is symbolized by z and is defined as the number of degrees Fahrenheit required for the curve to traverse one log cycle. The zvalue is the number of degrees the temperature must be raised or lowered from a given reference temperature to produce a tenfold decrease or increase in destruction time. The D values listed in tables 1 and 2 were used in plotting the T30 (figures 1 and 2). It should be noted that the z of 37.1 for superheated steam is approxi— mately three times as large as the 2 obtained for moist heat. Since it is known that heat in the absence of moisture is much less effective in killing than when moisture is present, it would be expected that the z 40. “mp ° D Values 0 Means Z=.|2.8 '0 U .0 7 Log of D Values (Minutes) 00' 255 230 255 230 255 260* Temperature (°F) Piqurs 1. Thermal resistance curve fitted to the mean of four different D values determined in moist heat. ’37-”1£)' .23 3 .E E V |.Or U) Q) 2 (U > o ,. . “5 OJ DValues m 0 Means O _l 00' sic 3&0 330 3510 350 360 Temperature (°F) Firure g. Thermal resistance curVe fitted to the mean of two different D values determined in superheated steam. 41. for superheated steam would be the larger of the two. However, figure 1 should be considered the most re- liable since it has been fitted to four different mean D values at four different temperatures, and yet, the means are found to lie approximately in a straight line. Figure 2 is fitted to only two means and, of course, these would have to lie in a straight line. Hence, any error involved in determining the mean D values would have a pronounced effect on the 2 value. 42. Survivor Curves The process of obtaining the survivor curves used the D values (tables 1 and 2) as a basis for length of time of exposure to the moist heat and superheated steam. The number of survivors was determined after each heating interval by a direct plate count. In all cases, the survivor curves were plotted statistically by the standard error of the estimate. This resulted in the best straight line possible for each set of data. The survivor curves obtained after subjecting the spores to moist heat are presented in figure 3 and 4. Figure 5 shows the survivor curves obtained when superheated steam was used. If the arithmetic mean of the number of survivors at each exposure time were joined together by a line, the resulting curves, particularly those presented in figure 5, might easily be considered as representing non-logarithmic order of death. Schmidt (1957) stated that the non-logarithmic order of death should be ac- cepted only after it has been shown statistically that the order is not logarithmic. A regression analysis of each curve revealed that the individual points did not vary significantly from a straight line. Therefore, it is most logical to represent the death rate as a constant. 43. 5-0 —— |.28 x IOG/ml ---l.26 xl07 ml ‘~\ ---f"l.27.x l0 / ml ~\ 0 arlthmehc ,mean \ ' of survwors Log of Survivors 2'00 é is 6 Ii)- [2 minutes Fivure a. Survivor curves of various spore concen- trations of strain 5230 in moist heat at 250 F. - .04 ‘ . 6.0+ —— I.28 x log/ml 0 "'"“'"" i 26 X '07 ml \ ----- -l. 27 x l0 /ml \ o arithmetic mean \ of survivors Log of Survivors I) ‘LCD 9’ o 2.0 1 1 L I J 1 O 0.! 0.2 0.3 0.4 0.5 0.6 0.7 minutes FL ure 4. Survivor curves of various s ore concen- tratio: s of strain 52 30 in moist heat at 250 F. 6-01 —— L28 x IOG/ml --— L26 x Io7 ml ----;-- |.27.x l0 /ml \ o arIthmejIc mean ‘. of survwors I I 5.0 Log of Survivors l 2.0 24.0 4:0 6:0, afo l0:0 liO I4.0 mInutes tn: .rrure 5. Survivor curves of various spore concen- trations of strain 5230 in superheated steam at 320 F. 46. It will be noted in figures 3 and 4 that the slopes of the survivor curves are very similar. Theoretically, if the destruction rates are logarithmic, the curves hown on each of these figures should be parallel. When one considers the error involved in plate counts and the possible fluctuation in temperature of the thermoresistometer chamber from.day to day, the rates indicated for the three Spore concentrations in figures 3 and 4 must be considered as reasonably parallel. In figure 5 two of the curves are reasonably parallel, but the survivor curve obtained for the 1.28 x 106 per ml spore concentration is quite flat in comparison. The regression analysis revealed that this curve was ap- proaching a non-logarithmic order of'death, whereas, the other curves were found to be highly logarithmic. The low death rate indicated in this instance may be due to the several breakdowns which were encountered with the thermoresistometer involving the heating chamber when the data at this temperature were being collected. The arithmetic means of the survivors shown on figure 5 seem to indicate that the temperature of the heating chamber may have fluctuated somewhat during the exposure time, causing these means to be located quite some dis- tance on either side of the statistically plotted sur- 47. vivor curves as compared to the variations observed in the arithmetic means for the curves presented in figures 3 and 4. It will be noted that on all survivor curves the arithmetic mean values for the initial numbers of spores were consistently higher than the intercepts. This may be due to the difference in the method of ob- taining the initial number of survivors. Table 3 shows a comparison of the mean D values ob- tained from the end-point destruction data and calcu- lated by the Schmidt (1954) method and the D values ob- tained directly from the survivor curves. Although no statistical analyses were performed to test the agree- ment between these two sets of D values, the values ob- tained with moist heat at 250 F and those for super- heated steam at 820 F using an initial population of 1.27 x 107 per ml were very comparable. At 235 F, the values from the survivor curves are higher and more vari- able than those calculated by the Schmidt method. The value for the 1.27 x 10'7 per ml spore concentration at 235 F was quite high when calculated from a survivor curve. However, it is believed that the variations in the aforementioned results are‘Within the experimental error. The survivor curve D value determined at 320 F in superheated steam for the lowest spore population TABLE 3 Comparison gfIQ'Values Cbtained by the Schmidt Kettod with Thos§_Cbtained from the Survivor Curves 48g D in Minutes Iemp., 0F. Spore Concentration Schmidt Survivor Method Curve Moist Heat: 235 1.25 x lOe/ml. 5.038 5.7 235 1.27 x lOZ/ml. 5.220 8.4 225 1.25 x ice/m1. 4.910 6.0 250 1.25 x 106/m1. '.270 0.305 250 .25 x 108/m1. 0.329 0.257 Superheated Steam: 520 1.25 x lOe/ml. 4.45 10.0 320 1.27 x 107 ml. 4.5 4.40 320 1.26 x 10 /ml. 4.02 5.05 was much higher than could be accounted for by normal variation. This probably resulted from the machine difficulties encountered and mentioned previously. 50. Percentage of necovered Spores from Cups by washings If the per cent of spores adhering to the cup after washing was large, then there would be an effect upon the survivor curve. Table 4 shows the results obtained after a series of washings. The number of Spores recovered after the first washing was found to vary above or below the theoretical number of Spores originally dispensed into the cup (washing to. l and cups a, B, and C). It is highly improbable that 100 per cent recovery of the theoretical number of Spores per cup could ever be obtained. The reason f0r the low number of Spores recovered compared to the theoretical number in Cup 3 and the high number of Spores recovered compared to the theoretical number in Cup 0 after the first washing may be due to errors involved in dispensing the original 0.01 ml. into the cups and/or to coun ing error. Even though the number of Spores recovered from the first washing varied some- what from the theoretical number per cup, these results provide an answer to the question of what effect the spores adhering to the cup have upon the survivor curve. Since the procedure involved the transferring of the same cup from one washing toaanother, there would 51. TABLE 4 gumber of Sgores Recovered After Successive hashings Cup Theoretical no. Uashings of spores/cup l 2 3 4 No./cup No./cup No./cug>ho.[cug A 12,800 9,680 480 106 38 B 127,000 64,800 2,280 218 142 (3 1,260,000 1,312,000 23,400 3,778 1,462 (31 (\3 9 always be some carry-over of the spores which had not been removed in the previous washing. If the number of Spores recovered from succeeding washings indicated a high percentage of those recOVered after the first washinb, then the initial reCOVeries would be much less than the theoretical number per cup. The nwhber of Spores recovered from the second washing of the cups range from 1.73 to 4.96 per cent of the number recover- ed from the first washing. The per cent of spores re- covered from washings three and four diminish rapidly. Considering that many of the spores recovered after the first washing would be due to carry-over, it is highly doubtful that there would be any effect upon the sur- vivor curves due to Spores adhering to the cups. 53. DISCUSSION hasoon (1926) stated that for accuratetic it and the theory of disin- tion. J. 8a0t3r101., 11, 1-25. - . , - - ,_ '7: x"! ._ A -.'-. .. " f Irde, no .u, (W11 gd‘u‘bGll, Lo Lo, 01. lbw? C3h3.h0n105‘ arltnmit rate 01 trelnnl -cvplubulQ‘ of spores of nadillus Goa; ulans. “$31. licrooio1., 5, 248-213. fl . .- ’:’ ‘v‘ n v '1 \ '1 V. 1 r N.“ 1...‘ .3 C ‘ f 7. .‘. ‘ -’ '— 3 r1 ‘3“ 11.0)] , V . A.— . R" J(— L; Hr; ’ A.) . 7~J . L'J' ‘3 :3 aka ‘J—il .4 ‘ L tel: com. I p A ‘ . . _. -~ ‘ \ -.~-; ~ \ '3- ~ --4 tent o1 Ve;etat1ve anJ $101: fo1ms of b:—ct=11a. J. Bacter1ol., 83, 29—108. f“ . q \ ‘~ s“ 01- n ,‘q r A ‘.‘ ; " .- '0 ' . " -. a -, ' '4 -‘ ulLlCQLJ, 1. a. 1940 The heat resistance of the spores q n R f‘ w 3 o - O 1 l‘ ‘ ' r \ v1 .- -. I o: trexnophilic bacteria. I. Introcucto1g. and. “v. 1-.., ° . r, n , -. . H :1.» . x?- ». heft. F1u1t and Ee;. Ireth. 8th., Jaipoeh, 40-51. -rlverson, E. t., anC lie ler, L. n. 1988 Application of statistics in bacteriolo: y. I. A means of determin- ing bacterial 3 Opulation by tze dilution method. J. cacteriol., 25, 101-121. Lea lee, K. R. 1281 Thermal We ath po oint. III Spores of Q19§t31”iun melchii. J. Ir fec t ious 81seases, as, 3:3‘3280 A. 1987 hater contert of Eenry, L. 8., and i . So to ’jBCteriOlo, 33, 323‘3290 \r r ‘_\ ' A. ‘ ‘1 __"‘ 0 .- I .- r- ‘ __ a ‘ Isaacs, L. L. 8.5 1 e31; 01 1 sense an re t e§;;t_ic , -‘f‘ - " fl - ‘ ‘1' fl a -~.-.( .~ A V H “‘3: _' "‘ 1 -Al. 0“ Vex, 1. £0, gnarleo IJAHAJD, 89r'1wl 11€LH8 IlLO Faplen, a. 1., Lichtenstein, 2., and he Jnolds, E. 1953 ‘he initial dev1ation fr 10m lluewllt 01 the therual death rate curve of a 1:uut1e:octive anaerobe. J. Easteriol., g1, Zeb-Zée. , GMDIUL261, Lo A0, obj $LPCE;, Lo 3- 1843 ion of tle15ecima1 reductioi timd orihciple a ‘ f the resist nce of colif orm bR teria to asteurization. J. Bact31iol., fig, 265—278. (‘ Knasvi, a. 1980 Disiniect on IV. Do Lagteria 3' ~- 07- q A T 8 c- O tio us Licenses, €23 8 -'\ r1 , A _._ ' -. A . ' .1. .o ,.. ' _.. _ ~. 1r 1, ,1 . w, ,. Lam3111na, o. 1842.1e1¢.1ohsh p 01 maX1mum LPOntfl t8“-bla- tuie to res: stance to heat. J. Secteriol., 1a, 29-85. Le Llano, F. h.,. N'e lin, K. n., and 8tu2 bo, 3. L. 1958 unti" t'c s Ar food rrere1at10h. I. T1 e influence of subtilin or t1 tne"2:l re ista1wc of s ores of Jm of Clo: tcidium tot linom ard ahe putrefactive eneerobe 86 Foo-a ‘1‘ecil-cl., Z, 181- 59.. Lewis, J. 8., Iichenei, E. 3., btumbc, C. 8., and Titus, 8. 5. 18:4 “deitiv ves accelerating death of spores by LfiOlSt hest to u. 11;”. T9051 35-81779, 2:, 9980 Levis , J.b 8. 1856 The estimation of decimal reflection tinm 5331. Licro1iol., 5, 211—221. La5oon, C. A., l 2 Tnermal resls c .1 -« ~L 7‘1... ment. J. DQ 6 Studies upon bac te rial spor es. II. tzice as effectefl by age wlo environ- DC) O'Brien, 8. T., and Titus, D. S. 1855 The effect of subti- line on hes -activ;tei and severely heated sgores of a putre1act1Ve anaerobic bacterium. J. Bacteriol., 293 4“ ‘7’45u o Pernins, J. C. 1957 Eacter10105icol ani surLical steril- ization by heat. 5535552t1cs, 51855§§g§55555 £81 1- gl§§§5_5§fi cgor1ce; gggth;SICsl sterilization. 83. by teddisb, 8. n., 2nd ed. Lea and Febiger, Inilar delphia. E1lu5, I. 8. £18 kselen, J. 8. 1858 Development and appli- CatiOD.Of'cIl%é n1aratils for studv of thermal resistance of baCterial s3 ores and triamine at temoe atures above (Lg-20 8"“. .51.}06. rec 1111.010, 1, «Lad/‘- 4.4.1.1. Pflng, I. J. and Esselen, J. B. 1954 Observations on the thermal resistance of putre efactive anaerobg ;o. 8579 /\ "‘j spores in tne teinerature rance of ESQ-88's 2. Food 1.88801‘011, .19 92-9 7. Pflng, I. J., and Esselen, I. 3. 1855 Heat transfer into open metal thermoresistoeeter cu ps. Food gesearch, 42:3, 237 -246. Pflug, I. J. 1957 Thermal resistance of vegetative cells and Spores of microorganisms. anuol1sico retort on Iroj ect 88;, for yea r 1957. Kichi5en ~tete University. Powell, J. F. and Stran5e, j. 8. 1858 Biochemical charges occurr Hi 3 during the :ermination of bacterial Spores. biochemu Jo, 54, 205-209. Powell, J. r. and Str 1;e, R. E. 1856 81ocne11cal cha1 es cccu1r1n5 on; ini sporulation in §5cillus sget cies. Biochem. J., 68, 661-668. (‘>-' Or 0 | Rahn, C. 1928 Incomplete sterilization of food products due to heavy syrups. Janning Age, é, 705-706. Hahn, 0. 1929 The size of bacteria as the cause of the logarithmic order of‘death. J. Gen. Physiol., LE, 179-205. Rahn, 0. 931 The order of death of organisms larger than bacteria. J. Gen. Physiol., 1%, 315-337. fiahn, 0. 1032 Physiology g£_bacteria. The Blakiston 00., new YbrK, K. Y. Rahn, 0., and Schroeder, a. R. 1941 Inactivation of enzymes as the cause of death of bacteria. Biodynamica, a, 199-208. Rahn, O. 1943 The problem of the logarithmic order of death in bacteria. Biodynamica, 2, 41-130. hahn, 0. 1945a Physical methods of sterilization of microorganisms. Bacteriol. Revs. 2, 1-47. Rahn, 0. 1945b Death of bacteria by chemical agents. BiOd‘Ynamica, i, 1.116. Reed, J. M., Bohrer, G. W., and Cameron, E. J. 1951 Spore destruction.rate studies on organisms of significance in the processing of canned foods. Reed, L. J. 1936 Biological effects 9: radiation. Ed. by Bugger, B» M., thraw-Hill Book 00., Inc., New Ybrk. Reynolds, H., Kaplan, A. K., Spencer, F. 5., and Lichten- stein, H. 1952 Thermal destruction of Cameron’s putrefactive anaerobe 3679 in food subtrates. Food Research, 12, 153-167. Reynolds, H., and Lichtenstein, E. 1952 Evaluation of heat resistance data for bacterial spores. Bacteriol. BJBVSO, é, 1.26-2350 Schmidt, C. F. 1950 A.method forcietermination of the thermal resistance of bacterial spores. J. Bacteriol., 29-, 433.437 0 c“ - '5' a *1 c — . y- : .—_- we- . : JCI'ml‘ t, v. .3 o 1% U4 £11,158; LC"). (4-1-311itt‘30tdntSL 1?fo .L" . -. p. n . Hr _‘ -- “:73 "TTTI’T 7'" "“7“: ":3 '7' 77",: , ‘3 (:1er c.1.;j_.1_ CL;S-__£QQL one: £!.L;blgi;_ 5 Ct; £11301“ I. "Jag. bv H5H=~f a F “t a T L es“ s 2‘ a» ri'l-% l u'. J -XVJ. .Lb ‘, \J. O, Q 8‘“. Led. (JILL) AeL'lCIJI , ~¢l 0V6 9 lcho » ' : '1 '1 2- ‘ “ z" 7,» v a v. " C I: ‘ A- - O‘Cl:flllut’ v0 1* o , .LJOC K U o 1- o , CIA} .L..O Delg, U o no 111’ou .Ll ACTJJQ reelstance determinations in steam using thermal death time retor s. Fool hesearch, 20, 606-613. - _O'_fi: f" Tfi ‘- n‘ 0" v o A‘ :‘.° .-,-.._. f .. -2 a Scnmiut, .. r. 1957 antiseptits,_dieiifect«nts,_fongicidesl ang_cheric:1 and physical s'erilizatior. J“. by “ed- | o .** , J1 .‘ A‘ " «f r u. '1 ‘.. "_‘ ’ _ ‘_ ‘ _ t‘ “ " .- r . cisn, a. i., 2nd e ., hea and leoiger, :hil—celphia. ': \ . (‘- ‘- T ' ‘ v» r” 'F‘ 3 :2: r ,‘u J" '. ‘ secrist, a. h., aid stumbo, o. n. lUUB acne rectors in- fluenCing‘tlerwal realstance velu 8 obtained by he tn rm resistcneteJ~ netlod. Food “esearch, Ea, Sl’bOo Segnefest 1., Says, a. 1., .heaton, 3., and :engamin, H. A. lee? Effect of pH on hermal process require- ments of canned food. Food nesearch, la, éOQ-a16. corner, 3. I. 1933 Heat resistance of spores of §_g§- tridium botglinpm. J. Infectious Diseases, 46, €35-le o Stern, J. A., and IrOthr, B. 3. 1954 a micro-method and apparatus for the multiple determination of rates of destruction of bacteria and bacterial spores sucgec- ted to heat. Food Technol., g, 139-le3. Stumbo, C. 4., Gross, C. 3., and Vinton, C. 1945 Bacteri- ological stuCies relating to thermal processing of canned meats. Food nesearch, 10, 260-372. Stumbo, C. A. lees A technique for studying r si tance e s of bacterial Spores to temperatures in the hi range. Food Technol., g, 226-240. Stwnbo, 3. i. see Further considerations relating to thermal processes for foods. Food Technol., Q, 126-121. Stumbo, 3. 4., hurphy, J. 3., and Cochran, J. E of thermal death time curves of PA.3679 and dium botulinum. Food Technol., g, 821-326. Sugiyama, E. 1951 Studies on factors affecting the heat resistance of spores of giggtridium :gtulinum. u. Gait-9:31.01. , .2, 81.960 , . , 3sty, J. 3., and Baselt, F. C. 1 at reels tar ce studies on srores of futref re ‘ s s in: eL tion to determination of or Gain led foo. Food 1e search, a, Virtanen, A. I. 1934 Ch the enzymes of bacteria and bac- terial metabolism. J. Bacteriol., 28, 447-460. Jaldham, D. G. and Palvcrson, 1. C. 1954 Studies on the relationship between equilibrium'vapor pressure and moisture content of bacterial endospores. nipl. Licrobiol., é, 333-338. Reiss, h. 1921 The heat res1 st;m_1ce of Spores with Special reference to the Spores of :' octulinus. J. In- fectious Diseases, 28, 70-92. Jillians, 3. C. ., herrill, C. E., and Cameron, E. J. 1937 Afparatus f’or the determination of spore destruction rates. Food Research, 2, 369-375. .illiams s, Fred T. 1936 Attempts to increase the heat resistance of bacterial spores. J. Bacteriol., Jilliams, C. B. 1929 The heat resistance of spores. J. Infectious nisea see, 44, 421-465. gilliams, 0. 5., and Campbell, L. L., Jr. 1951 The ef- fect of subtilin on thermophilic flat sour bacteria. Food Research, 16, 347-352. Jilliams, 0. 8., and Fleming, T. 3. 1952 Subtilineand the spores of Clostridi um botulinum. Antibiotics and Chemotherapy, a, 75-78. dilliams, C. 8., and fiobertson, w. J. 1954 Studies on heat resistance. VI. Effect of temperature of in- cubation on heat resistance of aerooic thermophilic Spores. J. Bacteriol., Q1, 377-8. fiilliams, C. 8., and hennessee, A. D. 1956 Studies on heat resistance of spores of 8. stearothermo ohilus. Food Research, 21, 112-116. Yesair, J. and Cameron, E. J. 1942 Inhibitive effect of _ curing agents on anaerobic Spores. Canner, 94, 89-92. Youland, G. C. and Stumbo, C. R. 1953 Resistance values reflecting the order of death of Spores of B, coagu- lans subjected to moist heat. Food Technol., 2, 286-288. HICHIGQN STQTE UNIV. LIBRQRIES IIIll|||l§|||l|||||||||||l1||||l||||||||l||\Hllllllllllllllllll 31293014040426