: 3% '.I- aka LN“ .09!. THESl \S This is to certify that the thesis entitled "Studies on the Preservation of Leptoapira ictarohemorrhagiae" presented by John M. Shigekatta has been accepted towards fulfillment of the requirements for M. S. degree in Bacteriology MYW [Major profeissor Date November 21:, 1951; 0-169 ICTEROHEMORRHAGIAL: BY FREEZING AND FREE ZE-DR YING BY JOHN MASACHIKA SHIG EKA WA A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of theiequirements for the degree of MASTER OF SCIENCE Department of Bacte riology 1954 THESlS A CKNOWLEDGMENT My sincere appreciation to Dr. Jack J. Stockton for his patient guidance and counsel during the course of this study and his assistance in the writing of this thesis. ii 344 08'? INTRODUCTION HISTORICAL REVIEW TABLE OF CONTENTS Low Tempe ratu re s ........................... Freeze -Drying MATERIALS METHODS AND RESULTS Freezing .................................. FreezeaDrying DISCUSSION .................................. SUMMARY..........L ....................... . BIB LIOG RAPHY iii 16 21 21 35 39 '43 44 STUDIES ON THE PRESERVATION OF LEPTOSPIRA ICTEROHEMORRHAGIAE BY FREEZING AND FREEZE-DRYING by JOHN MASACHIKA SHIGEKAWA This investigation consisted of studying the effects of freez- ing and freeze-drying on the viability and morphology of Leptospira icterohemorrhagiae. A comparison was made of various suspending menstrua and storage temperatures to determine which menstruum and under what storage conditions the organiSms best survived in the frozen state and whether the menstrua used could provide suitable conditions for the successful 1y0philization of the organism. Six to seven day-old cultures of E. icterohemorrhagiae grown in Korthoff's medium at 50°C were concentrated by centrifugation and re- suspended in various menstrua, chosen for this study because of their colloidal preperty. These were as follows: 1 percent starch, l and 5 percent gelatin, 0.25 percent agar, 1 and 2 percent casein, skim milk, normal rabbit and horse serum, egg yolk, and fresh and frozen allantoic fluid. The suspensions of organisms were tubed in "IyOphile" tubes and following freezing in a dry ice-alcohol mixture at -70°C, were stored in different deep-freeze units at -27°C, -47°C, and -7§°C. Test samples were reconstituted after storage of one week, two weeks, one month, and thereafter at monthly intervals. The contents of the tubes were inoculated into Korthoff's medium and the viability of the organ- isms was determined by dark-field microscOpy after one and two weeks' incubation at 50°C. The criterion used foe determining viability was motility of the leptospirae. Leptospirae suspended in the menstrua mentioned above and in infected tissues of young guinea pigs and hamsters were 1y0philized according to the technique of Flosdorf and Mudd. Following 1yophi1- zation, these cultures were checked for viability by incubation in Korthoff's medium.and subsequent darkbfield microscOpy. The results obtained in this study have shown that E, 122252- hemorrhagiae can be frozen and kept stable up to nine months in certain colloidal substances at subzero temperatures. The most effective of these substances were skim.milk and slightly hemolyzed normal rabbit and horse serum. There was very little variation in the results in the three storage temperatures (-2700, -47°C,,and -7§°O) employed. Freeze—drying as a method for preserving these organisms met with unsuccessful results. Although the leptospirae survive the preliminary freezing, death occurs during the drying process. The reason for this is still unknown. LIST OF TABLES TABLE Page I. Group I. Longevity of L. icterohemorrhagiag suspended in various menstrua and stored at -47°C ................................. 26 II. Group II. Longevity of L. icterohemorrhagiae suspended in various menstrua and stored at -47°C ................................. 28 III. Group III. Longevity of L. icterohemorrhagiae suspended in various menstrua and stored at —27°C ................................. 30 IV. Group IV. Longevity of I:_._ ict‘erohemorrhagiae suspended in various menstrua and stored at -73°C ................................. 32 iv IN TR ODUC TION The preservation of bacterial cultures by suitable methods provides the bacteriologist with readily available cultures of known characteristics. Satisfactory preservation obviates frequent manipu- lation of cultures, thereby lessening both the danger of contamina- tion and the possibility of variations or mutations. Preserved cul- tures also mean an economy of time, labor, space, and glassware. The method of preserving bacterial cultures must meet several criteria if it is to be accepted for use. Preservation should not only maintain the viability of the organism, but all its natural characteristics as well. There should be no alteration in morpho- logical, immunological, and biochemical characteristics, and no lessening of virulence if a pathogen. To place it in the realm of the practical and have it adopted for use in most laboratories, the procedures should be relatively simple, and the apparatus and equipment employed easy to operate and economical in cost and operation. Freezing and freeze-drying have proved efficacious in storage of many species of bacteria, some surviving for several years with little adverse effect and no apparent change in characteristics. However, bacteria vary in their susceptibility to freezing and to freeze-drying; while some species may be preserved for years by these methods, others may not withstand the conditions encountered in these processes. Preservation of the pathogenic spirochetes has been a prob- lem to the bacteriologist and one that has not yet been adequately solved. The importance of spirochete-produced diseases, both in man and animals, warrants efforts toward a solution to this problem. The present study was conducted in an attempt to employ methods previously used in the successful preservation of various bacteria for preserving one member of the Genus Lgptgspiga, Lepto spira icte rohemorrhagiae. The choice of this spirochete as the test organism was prompted by the fact that, among the pathogenic spirochetes, it is probably the easiest to cultivate in artificial media. Also, the mem- bers of this genus are the cause of leptospirosis in man and ani- mals, a disease that has gained increasing attention in recent years because of growing awareness of its prevalence. Increased interest will demand more intense study of the disease and its etiological agents. Therefore, it is desirable that an adequate means of pre- serving these leptospirae be developed. This investigation consisted of studying the effects of freezing and freeze-drying on the viability and morphology of 1:; icterohemorr- hagiag. A comparison was made of various suspending menstrua and storage temperatures to determine which menstruum and under what storage conditions the organisms would best survive in the frozen state and whether the menstrua used could provide suitable conditions for the successful lyophilization of the organism. HISTORICAL REVIEW Low Temperature 5 Many observations have been presented by investigators on the effect of low temperatures and freezing on bacteria. Before the turn of the century, most bacteriologists considered cold a powerful germicidal agent, but later investigations and observations have shown that in many instances cold may, in fact, act as a preserver of microbial life. One of the earliest observations on the influence of low tem- peratures on microorganisms was made by Pumpelly in 1882, as cited by Hilliard and Davis (1918). They stated that when Pumpelly cut samples from the center of a block of ice and placed them in sterile beef broth, contamination soon became evident. Hilliard and Davis (1918) reported that the first extensive study of the effects of cold on bacteria was made by Prudden in 1887. He subjected Bacillus typhosus to temperatures ranging from 14° to 30°F, and found that. under these conditions, the organisms remained viable for 103 days. Ten microorganisms possessing varying degrees of resistance to environmental conditions were employed by Macfadyen (1900) in his study of the influence of the temperature of liquid air on bac- teria. Young cultures were eXposed to the temierature of liquid air (-190°C) for 24 hours and then thawed and examined. No im- pairment of vitality or functional activity could be detected. Mac- fadyen and Rowland (1900) later reported that a number of organisms, mainly of the gi‘ifll‘li group, subjected to the temperature of liquid hydrogen (-252°C) for 10 hours, remained unchanged in appearance and vigor of growth. Further studies by Macfadyen and Rowland (1902) gave similar results. McLean (1918) isolated four species of bac- teria that had lived dormant in ice, snow, and frozen algae for pro- longed periods. Luyet and Gehenio (1934) stated that other investi- gators--Brehme, Citovicz, Gladin, Kasansky, and Pictet--had found, that various types of bacteria were able to survive for considerable periods in the frozen state. Bacteria are known to vary in their resistance to cold and freezing. Smith and Swingle (1905) demonstrated this using twelve different bacteria. Represented in this group were saprophytes and plant and animal pathogens. Cultures 24 to 48 hours old were suspended in peptonized beef boullion and part of each culture was frozen in liquid air (-190°C) for 10 minutes to 24 hours and the remaining cultures in a salt and ice mixture (-17.8°C) for 2 hours. The cultures varied in their resistance to these temperatures, but even in case of the most sensitive bacteria, some individuals of each culture were able to withstand the temperature of liquid air. Hilliard, Torossian, and Stone (1915) observed a difference in the susceptibility of Bacillus coli and Bacillus subtilis to freezing in tap water for 3 hours. The results of other workers--Tanner and Wil- liamson (1927), Keith (1913), Smart (1935), and Haines (l938)--added supporting evidence to the findings of previous investigators. There are a number of variables that appear to have an ef- fect on the susceptibility of microorganisms to freezing. Among these are the temperature of the freezing mixture, the duration of the freezing, the abruptness of the temperature changes, and the nature of the suspending medium. To study the effect of the degree of cold used in the freez- ing mixture, Hilliard, Torossian, and Stone (1915) froze tubes of cultures and held them for 3 hours for comparison at temperatures of approximately -15°C and -Z°C. It was observed that the lower temperature had a considerably greater effect in the reduction of the number of organisms. Haines (1938) noted that the temperature or rate of freezing had little effect on the mortality of the cells. Studies of the effect of. repeated freezings and thawings on twelve different bacterial cultures by Smith and Swingle (1905) showed a gradual reduction in the living bacterial populations to zero. Hil- liard, Torossian, and Stone (1915) stated that freezing and thawing at intervals was considerably more fatal than continuous freezing. In contradiction to the findings of the two previously mentioned in— vestigations, Tanner and Wallace (1931) reported that alternate freezing and thawing was no more destructive to microorganisms than continuous freezing. The findings of Haines (1938) suggested that there is some correlation between storage temperatures and survival of micro- organisms. When frozen aqueous suspensions of bacteria were stored at temperatures ranging from -1°C to -20°C, the most rapid death rate took place near the highest storage temperature. A The medium in which the organisms are suspended seems to have an important bearing on how well they withstand the adverse effects of the initial freezing and subsequent storage at low tem- peratures. The formation of ice crystals or crystallization is thought to be one of the chief factors in the reduction of bacterial populations undergoing freezing, due to the mechanical disruption of cells. There- fore, most investigators are of the opinion that those substances which are colloidal in nature give the greatest degree of protection. Keith (1913) has compared the survival time of Iéggiiu_s__c_o_li in dif- ferent suspending media. One group of cultures was suspended in milk and the other group in tap water and frozen. Results of this study proved milk more efficacious in its protection than tap water. These findings were substantiated by Hilliard and Davis (1918), Prucha and Brannon (1926), and Tanner and Wallace (1931). Studies on the effect of freezing on spirochetes have been of relatively recent date and the number of publications on this subject have been few. Turner (1938) reported preserving two species of treponemes in the frozen state at —78°C for as long as one year. As Haines (1938) had previously noticed with other types of bacteria, Turner (1938) observed a difference in survival times of treponemes held at different temperatures. Those stored at -20°C were found to survive for rnuch shorter periods than those kept at -78°CL Treponemes held at -10°C had lost their motility and many appeared shrunken and distorted. In the course of their work, they noted that the temperature during the initial freezing had little effect on the organisms. This indicated that the damage to the cells occurred not at the time of the initial freezing but during the maintenance period. In a later publication, Turner and Fleming (1939) stated that the viability and virulence of various types of spirochetes were main- tained after storage at -78°C for periods up to 3 years. One strain of L. icterohemorrhagiae, isolated from rats and propagated in guinea pigs, remained active after storage for 10 months at -78°C. Stavitsky (1945) was able to maintain the viability and viru- lence of L. icterohemorrhagiae in infected whole guinea pig liver blocks, frozen and kept at -20°C, for 100 days. Freeze- Drying Freeze-drying was first described by Shackell (1909), who suggested several practical applications of this process to biologics. Martin (1896) had previously reported a simple method of obtaining dried sterile serum from the liquid state and Morton and Pulaski (1938) stated that Kitasato, Ficker, Kirstein, and Hein were successful in preserving several species of bacteria by various methods of desiccation. Freeze-drying, as described by Shackell (1909), was the in- troduction of freezing as a preliminary step to desiccation. His method consisted of placing the frozen material in a desiccator over absorbing sulphuric acid which was thoroughly mixed by occasional 10 rotation to facilitate absorption and drying. The vacuum was pro- duced by the use of a ”Geryk" vacuum pump which was capable of reducing the air pressure in the desiccator to less than 1 mm. of mercury in 2 minutes. Employing Shackell's method of freeze-drying, Harris and Shackell (1911) successfully dried brains and cords of rabies-infected rabbits. They observed no destruction of virulence of the virus. The preservation of bacteria by freeze-drying was first ac- complished by Hammer (1911). Later, Roger (1914) applied the principle of freezing and drying to the preservation of bacterial cultures on a large scale. A comparative study of the survival of several non-spore— forming bacteria was made by Shattock and Dudgeon (1912), employ«- ing two methods of desiccation: desiccation in air and drying in a liquid air vacuum. In the case of some of the bacteria there was little difference in viability and survival time, but with others, the difference was very marked in favor of freeze-drying. Stock cultures of streptococci and pneumococci were main- tained by Swift (192.1) for periods from 2 to 4 years after freeze- drying. He noted that the physical state of the dried material had much to do with the viability of the organisms. The recovery of ll organisms was greatest in those tubes showing a ”dry-foam" con- dition while in those appearing gummy, recovery was not possible in most cases. The results of his studies indicated that it was. necessary to maintain the frozen state until drying was complete. This led Swift to devise a technique in which the tubes containing the specimen were immersed in glycerol contained in the lower part of the desiccator which was then placed in the salt-ice mixture. The glycerol acted as the medium for the conduction of cold from the salt-ice mixture to the material which was kept frozen until drying was complete. Bacteria preserved in this manner were observed to retain all their desirable characteristics. The preservation of yellow fever virus by freeze-drying was reported by Sawyer, Lloyd, and Kitchen (1929). Brown (1932) main- tained thirty-eight strains of bacteria 4 to 12 years following drying £1113qu from the frozen state. The efficacy of this method for preserving the more sensitive bacteria was shown by Rake (1935), who successfully maintained several strains of meningococci over a period of many months. Elser, Thomas, and Steffen (1935) made significant contribu- tions to the improvement of techniques and apparatus involved in the freeze-drying process. They described methods and apparatus 12 for freeze-drying biological products and microorganisms which dif- fered from previously reported procedures in that theirs involved the use of manifolds and individual containers in the drying process. Both chemicals and refrigerants were employed as condensers to dispose of water vapor arising during the high-vacuum desiccation. The use of refrigerants brought this method into the realm of the practical for the drying and preservation of large volumes of thera- peutic products. Chemical desiccants were limited in their capacity to absorb water and therefore were limited in the volume of product that could be processed. Therapeutic agents preserved, using the techniques and apparatus described by these workers, retained their clarity, porosity of texture, and solubility. They did not de- teriorate on standing and exhibited no loss in potency even after long periods of time. Meningococci and gonococci, normally sensi- tive to adverse conditions, were successfully preserved but attempts to preserve the spirochete Spirochaeta duttoni by this method proved discouraging. The organism was recovered only in a very few in- stances following freeze—drying. The development of the freeze-drying apparatus to what it is today was largely due to the efforts of Flosdorf and Mudd (1935). The increased efficiency of their so-called ”lyophile" apparatus I l3 enabled them to preserve large amounts of labile biological products, bacterial cultures, and viruses without any deleterious effects. These dried products were porous and reconstituted with ease. Sera so de— hydrated were designated "lyophile" ("liquid-loving"), a word first coined by Reichel, as cited by Flosdorf and Mudd (1935). Flosdorf and Mudd (1938) later described a method by which biologics could be preserved more conveniently and economically. This method involved the use of calcium sulfate as the desiccant in the condenser. The calcium sulfate was specially prepared and could be easily regenerated by heat for further use. The word ”cryochem" was coined to describe this process. Swift (1937) stated that colloidal substances, such as serum, appeared to give greater protection to microorganisms undergoing drying. Studies by Heller (1941) showed that the death rates of lyophilized bacteria suspended in crystalline substances were greater than those of bacteria suSpended in colloids. Bauer and Pickels (1940) reported that yellow fever virus, ordinarily labile to adverse conditions, could be preserved and kept active for years if suspended in a medium rich in proteins and prop- erly desiccated. They observed that it was essential to maintain the material in the frozen state until desiccation was complete. l4 Lyophilization has not yet proved to be a satisfactory method for preserving spirochetes. Results of most workers have been discouraging. Turner, Bauer, and Kluth (1941), employing the ap- paratus and techniques used by other investigators in the successful preservation of bacteria and viruses, were unable to recover two species of treponemes after freeze-drying. The spirochetes were suspended in tissue juices and serum and dried for 3 to 5 days after freezing. Large quantities of this dehydrated material was then injected into normal rabbits. Results were completely negative, indicating that the lyophilized organisms had lost viability. Stavitsky (1945) reported that, after lyophilization of cultures of 1:493:52- hemorrhagiae and infected guinea pig livers, no leptospirae could be recovered. Samples of the lyophilized material were checked at various intervals for viability, variability, and virulence by direct dark-field examination, subculturing, and inoculation into young guinea pigs, but in all instances the results were negative. At variance with reports by earlier workers, Hampp (1947) reported the successful preservation of spirochetes by freeze-drying. The test organisms used were Borrelia vincenti and various strains of Treponemna pallidum which were grown 5 to 7 days in a men- struum consisting of equal parts of Huntoon's "hormone" broth 15 and 5 percent gastric mucin and enriched with ascitic fluid and glutathione. ‘This medium was used both for cultivation and suspen- sion of the organisms for freeze-drying. Lyophilization was done according to the method of Flosdorf and Mudd, using the "cryochem" apparatus. The lyophilized cultures were tested for viability both microscopically and by subcultures The cultures were positive with only one exception. Hampp (1951) later reported the maintenance of viability and pathogenicity of Nichol's rabbit strain of T. pgllidum in lyophilized infected rabbit testes. Brunner and Meyer (1950) employed lyophilization as a method to kill leptospirae for preparation of a vaccine for the immunization of hamsters and dogs against experimental leptospirosis. They found that lyophilization had no effect on the antigenic quality and the vac- cine so produced provided excellent protection against the homologous type of L. icterohemorrha iag. MA TE RIALS Members of the Genus Leptospira are readily cultivable on artificial media. Many different media have been used for the cul- tivation and propagation of these organisms, most of them consisting of a solution of various salts, enriched with blood, serum, or various other substances. Babudieri (1943), while studying the survival times of nineteen strains of various leptospirae as determined on different blood and serum media, found that serum media were superior to blood media. Of all the media tested, Korthoff's me- dium gave the best results. Serum is an essential ingredient in leptospira culture media. Rosenfeld and Greene (1941), investigating the effect of growth factors in stimulating the growth of leptospira species in the presence of se- rum, observed that no factor or combination of factors was capable of maintaining leptospiral growth in the absence of serum. Accord- ing to Babudieri (1943), sera which were slightly hemolyzed gave much better growth than those that were not hemolyzed. This was also noted by Ringen and Gillespie (1954), and was true in our own experience. 16 l7 Korthoff's leptospira medium was used in this study, both for maintaining the stock cultures and for subculturing organisms taken out of ”lyophile" and those reconstituted from the frozen state. This medium consists of one part of 1 percent Bacto-Tryptose to eight parts of buffer. The buffer was made up of solutions of the following salts: NaCl 2.5% ............. 11.2 ml. NaHCO3 0.1% ........... 4.0 ml. KCl 0.1% .............. 8.0 ml. CaClZ 0.1% ............ 8.0 ml. KHZPO4 2.5% ........... 1.44 ml. Nazi-1130‘1 2.5% .......... 7.68 ml. Distilled water q.s ........ 200.00 ml. After the medium was prepared, the pH was adjusted to 7.4 by addition of approximately 0.1 N NaOH solution drop by drop with continuous stirring until the desired pH was obtained, as indicated by a Beckman (model H) pH meter. The medium was then tubed in 9 ml. amounts in 25 ml. screw-cap test tubes and autoclaved for 15 minutes at 121°C. These were kept at refrigeration temperature until ready for use. At the time of use 1 ml. of normal serum was added aseptically to each tube containing 9 ml. of the base medium. 18 The serum used was of rabbit and horse origin. Subsequent to bleeding, the blood was allowed to clot and the cells were sep- arated from the serum by centrifugation at approximately 1600 r.p.m. in an International, Size 2, centrifuge. After collection of the serum, it was clarified by passing it through clarifying filters and then sterilized by Seitz-filtering employing No. 3 and No. 6, ST-3, Seitz type, Hercules sterilizing pads. The’serum was collected in sterile 30 ml. vials, capped, and inactivated in a water bath at 56°C for 30 minutes. This serum was stored under refrigeration till the time of use. At the time of bleeding or during the separation of serum from the cells, some of the red blood cells were purposely lysed in order to add hemoglobin to the serum. The test culture employed in this study, L. icterohemorrhaiiae, strain 871, was obtained from Dr. John P. Newman, Michigan State College, and was originally isolated from a human case of lepto- spirosis at Walter Reed Army Hospital, Washington, D. C. It had been shown by several investigators--Hilliard, Toros- sian, and Stone (1915), Hilliard and Davis (1918), and Heller (194l)-- that the menstrua in which bacteria were suspended during freezing or freeze-drying had a decided effect on their survival. The results 19 of these investigations indicated that colloidal substances gave greater protection against mechanical damage than substances which were crystalline in nature. For this reason, various colloidal materials were used to suspend the test organisms in preparation for freezing and lyophilization. These are listed as follows: 1 percent starch, normal rabbit serum, normal horse serum, egg yolk, fresh allantoic fluid, frozen allantoic fluid, 1 and 3 percent gelatin, 1 and 2. percent casein, 0.25 percent agar, and skim milk. With the exception of the serum, egg yolk, and allantoic fluid, these substances were adjusted to a pH of 7.4 and then sterilized by autoclaving at 121°C for 15 minutes, prior to use. Reports by a number of workers--Harris and Shackell (1911), Hampp (1951), Turner (1938), and Stavitsky (l945)--mentioned that the freezing and freeze-drying of experimentally infected tissues of susceptible laboratory animals sometimes resulted in the maintenance of both the viability and virulence of the infecting microorganism. Morton (1942) and Larson (1944) had found that young guinea pigs and young ’golden hamsters were most susceptible to experimentally produced leptospirosis. The greatest concentration of infective leptospirae appeared in the kidneys and liver due to the affinity of these organisms for those particular tissues. An attempt was made 20 in this study to preserve L. icterohemorrhagiae by lyophilizing in- .0 fected tissues of young guinea pigs and golden hamsters. METHODS AND RESULTS Freezing Stock cultures of L. icterohemorrhagiae were maintained in Korthoff‘s medium contained in 25 ml.. screw-cap test tubes in 10 ml. amounts. These were transferred weekly into fresh medium and incu- bated at 30°C. An inoculum of 0.25 ml. of actively growing culture was used to seed each tube of fresh medium. The test cultures that were subjected to "sharp" freezing and subsequent storage at subzero temperatures were seeded fromthe stock cultures and incubated for 6 to 7 days at 30°C. At the end of the incubation period the cultures were observed macroscopically, using transmitted light from an intense light source, for evidence of growth as indicated by a slight cloudiness of the medium. Prior to suspension in the various menstrua, the organisms _were concentrated by centrifugation in an International, PR-l, refrig- erated centrifuge (40° to 45°F) at a R.C.F. of approximately 1000 x G for 45 minutes. The organisms in each suspending menstruum repre- sented the pooled growth from five tubes. 21 22 The first group of cultures frozen were Suspended in only eight of the eleven menstrua previously mentioned. These were: 1 percent starch, l and 3 percent gelatin, 0.25 percent agar, l and 2 percent casein, skim milk, and normal rabbit serum. Ten ml. of each medium was used to suspend the concentrated organisms in each tube. For the other groups of test cultures, egg yolk and fresh and frozen allantoic fluid were used in addition to the suspending men- strua listed above. In two of the groups, normal horse serum was used in place of rabbit serum. The suspended organisms in these groups represented a greater concentration of organisms since only 5 ml. of each menstruum was used for suspension of the organisms. After the organisms were well suspended in each menstruum, they were tubed in approximately 0.5 ml. amounts in sterile pyrex ”lyophile” tubes using sterile capillary pipettes, and were then re- plugged with cotton. Before freezing the tubes were sealed with “parafilm.” Some investigators had observed that the rate at which bac- teria were frozen had a considerable effect on the number of organ- isms that withstood the freezing process. They observed that the reduction of bacterial populations was less after ”sharp" freezing 23 at very low temperatures than that of those subjected to slow freez- ing at 0°C or at temperatures just below the freezing point of water. For this reason, this work was conducted using a freezing mixture of solid C02 and 95 percent ethyl alcohol, at a temperature of ap- proximately -70°C, to assure rapid freezing of the specimens and decrease the percent reduction due to this factor. The suspended organisms in each ”lyophile” tube were “bleb” frozen in the dry ice-alcohol mixture for a period of 5 to 10 minutes, after which they were placed in suitable containers and stored in freezing units at different subzero temperatures. According to Turner (1938), another factor that influenced the survival of spirochetes in the frozen state was the temperature at which these organisms were held during the storage period. To determine the optimum temperature range for the storage of these leptOSpirae, as indicated by survival times, tubes of sus-5 pended organisms were kept in various freezing cabinets at different temperatures. These groups of test cultures, held at the various freezing temperatures, will be designated hereafter as Group I, Group II, Group III, and Group IV, for convenience. The suspended cultures in Groups I and II were both stored in the same deep-freezing unit at a temperature of approximately 24 -47°C. The difference between these two groups, as previously stated, was that in Group I only eight of the eleven suspending men- strua were employed, and the suSpensions of organisms were less concentrated than those in the other three groups. The tubes in Group III were held in a freezing unit at -27°C, and those in Group IV in a dry ice chest at approximately -73°C. To examine the. frozen cultures for viability, tubes were re- moved from the storage units, thawed at room temperature, and the contents placed into Korthoff's medium. At the end of one and two weeks' incubation at 30°C, these subcultures were examined by dark- field microscopy for viability, with motility of the leptospirae as the criterion. A rough estimation of the degree of growth was made and recorded, with one plus indicating little growth, and four plusses indicating very heavy growth. Prior to freezing, organisms suspended in each of the sus- pending menstrua were subcultured in Korthoff's medium to ascer- tain the viability of the test cultures. Immediately following the initial freezing in the dry ice- alcohol mixture, one culture in each of the suspending menstrua was thawed and inoculated into the cultivation medium to determine whether or not the organisms survived this initial freezing at -70°C. 25 Subsequently, sets of cultures from each of the four groups were reconstituted at the end of one and 'two weeks‘ storage, and there- after at monthly intervals. These were all examined following sim- ilar procedures as described above. The results of this study are shown in Tables I to IV. The cultures in Group I were checked over a period of nine months. Of the eight menstrua used for suspending the test organ- isms, five of them still yielded viable organisms at the end of nine months' storage at a temperature of -47°C. These were: 3 percent gelatin, 1 and 2 percent casein, skim milk, and normal rabbit serum, with the rabbit serum appearing to be the most effective. Very good growth followed thawing and subsequent incubation in Korthoff's me- dium, and the spirochetes appeared normal and were actively motile. Casein, l and 2 percent, gave heavy growth, but approximately 50 percent of the organisms were feebly mbtile and those in 2 percent casein appeared somewhat granulated. Growth of organisms suspended in skim milk was not quite as heavy as growth of the organisms sus— pended in l and 2 percent casein and in normal rabbit serum. There was some granulation of the leptospirae, but they were actively mo- tile. Growth was slight in 3 percent gelatin, but the organisms were active and normal in morphology. Starch, 1 percent, supported no 26 TABLE I GROUP 1. LONGEVITY OF L. ICTEROHEMORRHAGIAE SUSPENDED IN VARIOUS MENSTRUA AND STORED AT ~47°C St a e T' e Starch Gelatin Gelatin or 1m g 1% 1% 3% Not frozen . ++++ ++++ ++++ Reconstituted * - .. ++ immediately ** - +++ ++++ One week * - ++++ ++++ ** - ++++ ++++ Two weeks * - - .. ** - - - One month * - - + ** - + +++ Two months * — - + ** - - ++ Three months * - - - 3101‘ — - - Four months * - .. - ** - — - Five months * - .. .. *3? _ - + Six months * - - .. ** - + ++ Seven months * - _ _ ** - - ++++ Eight months * — - ' + ** - - + Nine months * - - - ea - - + *One week incubation; **two weeks incubation; - no growth; +, ++++ estimated degree of growth. TABLE I (Continued) 27 . . . N a Agar Casein Casein Skim R0 2:13 . a 1 o .2507. 1% 2% Milk Se rum ++++ ++++ ++++ ++++ ++++ - ++ - ++ ++++ - ++++ ++++ ++++ - +++ - +++ ++++ + ++++ - ++++ ++++ ’ ++ ++ - ++++ - +++ +++ ++ ++++ - + ++ — ++++ - ++ +++ +++ ++++ _ + + ++++ _ + +++ ++++ - - - - + - - - - + - - + + + - - + ++++ + - + + - + - — +++ + + - - - - + - + +++ +++ - + - - ++ - ++++ - ++++ +++ - + - ++++ - ++++ ++ +++ - + + - +++ - ++++ ++++ ' ++ ++++ 28 TABLE II GROUP 11. LONGEVITY OF L. ICTEROHEMORRHAGIAE SUSPENDED IN VARIOUS MENSTRUA AND STORED AT -47°C St T' Starch Gelatin Gelatin Agar ora e 1me g 1% 1% 3% 0 .2570 lflotfrozen ++++ ++++ ++++ ++++ Reconstituted * + ++ +++ - immediately ** ++++ ++++ ++++ - One week * - — ++ ._ ** ++++ - +++ - Two weeks * - - +++ + .. ** ++++ ++++ — One month * 1 - .. ++ _ ** — - +++ - Two months * - - + _ ** - - +++ - Three months * - + - _ aw _ + _ — Six months * - - + _ aunt: - - ++ - ‘—‘_’ J fr * One week incubation. ** Two weeks incubation. - No growth. +, ++++ Estimated degree of growth. TABLE II (Continued) 29 Fresh Frozen . . . Normal Casein Casein Skim Horse Egg Allan- Allan— 10/0 2% Milk Yolk toic toic Se rum . . . Fluid Fluid ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ ++ ++++ ++ ++ ++ ++++ +++ ++++ ++++ ++++ +++ +++ ++ ++ ++ ++++ ++ - - +++ +++ ++++ ++++ ++++ +++ + ++ ++ +++ ++++ ++ + - +++ +++ ++++ ++++ +++ ++++ ++++ - - — ++ +++ - _ + + +++ +++ ++++ +++ - + + - - +++ - - +++ +++ +++ - - +++ ++ + + ++ ++ +++ - - ++ +++ ++++ - ++++ - +++ - + - ++ ++ - - ++ ++++ ++++ ++++ ++ - - 30 TAB LE III GROUP 111. LONGEVITY OF L. ICTEROHEMORRHAGIAE SUSPENDED IN VARIOUS MENSTRUA AND STORED AT -27°C , Starch Gelatin Gelatin Agar Storage T1me 1% 1% 3% 0.25% Not frozen ++++ ++++ ++++ ++++ Reconstituted * +++ ++ H, + immediately ** +++ +++ ++ + One week * + + ++ _ ** +++ ++++ ++++ - Two weeks * ++++ ++++ ++++ + ** ++++ ++++ ++++ ++++ One month * + - + _, ** +++ - +++ - Two months * - +++ + - ** +++ +++ +++ - Three months * - +++ - _ ' ** +++ +++ +++ +++ Four months * + - ++ .. ** +++ +++ +++ - Five months ’1‘ - - ++ _ ** ++ ++++ ++++ - t t * One week incubation. ** Two weeks incubation. - No growth. +, 1+++ Estimated degree of growth. 31 TABLE III (Continued) Fresh Frozen . . . No rmal Casein Casein Skim Rabbit Egg Allan- Allan- l% 2% Milk Se rum Yolk toic toic Fluid Fluid ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ ++ +++ ++++ + + + ++ +++ ++++ ++++ + - +++ + ++ ++ +++ + - - - +++ +++ ++++ +++ - - +++ +++ +++ + + - - ++++ ++++ ++++ ++++ + - - _ .. + .. - .. - + + ++++ - +++ - - - _ + _ + - - ++++ ++++ +++ - + - - + - ++++ + — - - + .. _ .. - - _ ++++ + +++ - - - - - +++ ++++ — - - - 32 TABLE IV GROUP IV. LONGEVITY OF L. ICTEROHEMORRHAGIAE SUSPENDED IN VARIOUS MENSTRUA AND STORED AT -73°C ‘—: mr‘t— T St a Ti Starch Gelatin Gelatin Agar or e me g 1% 1% 3% 0.25% liotfrozen ++++ ++++ ++++ ++++ Reconstituted * + + ++ - immediately ** ++ + ++ .. Two weeks * ' — - .. - ** + - - - One month * - + + .. ** + ++++ ++++ - Two months * - - _ _ ** ++++ - - - Three months * - - - .. ** +++ +++ - Four months * - - .. - #31: .. - - .. f * One week incubation. ** Two weeks incubation. - No growth. +, ++++ Estimated degree of growth. TABLE IV (Continued) 33 Fresh Frozen . . . Normal Case1n Case1n Skim H 5 Egg Allan- Allan- , or e . . 1% 2% Milk Ser m Yolk tom tom u . . Fluid Fluid ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ ++ +++ ++ +++ + +++ +++ +++ ++++ +++ +++ - +++ _ — ++ ++++ +++ - +++ + + ++++ ++++ ++++ - ++++ +++ + +++ ++++ +++ +++ ++++ ++++ ++++ +++ ++++ ++++ ++++ ++++ +++ +++ +++ ++++ ++ - - ++++ +++ +++ +++ - - - ++ ++++ ++ ++++ - - + +++ ++++ ++++ ++++ + - +++ _ .. - + .. .. _ _ +++ ++++ ++++ - - - 34 life at all, and 0.25 percent agar gave negative results after one week. Gelatin, 1 percent, gave erratic results, all being negative with the exception of those cultures reconstituted after one and six months. These cultures showed very little growth. Of the eleven menstrua used in Group 11, normal horse serum, skim milk, and 2 percent casein showed heavy, active growth after storage of six months at -47°C. Egg yolk and 3 percent gelatin gave moderate growth, and the leptospirae were actively motile. Growth was observed in cultures suspended in fresh allantoic fluid and frozen for two months, in frozen allantoic fluid for three months, and in 1 percent gelatin after two weeks; however, one culture, sus- pended in 1 percent gelatin, reconstituted after three months' storage showed actively motile leptOSpirae. The organisms in 1 percent starch were sluggishly motile after a month in the frozen state. It was noted that in each case where growth was observed, a con- taminating mold-was present. Cultures suspended in 0.25 percent agar were completely negative. The Group III cultures were stored at -27°C. The latest set of cultures reconstituted were frozen for five months. The greatest amount of growth was found from test samples suspended in skim milk and l and 3 percent gelatin. Gelatin, 3 percent, showed a 35 number of feebly motile leptospirae, whereas those from skim milk and 1 percent gelatin appeared actively motile. Starch, 1 percent, and 2 percent casein also supported viability of the leptospirae. Organisms that were suspended in these menstrua were active and normal in appearance. Leptospirae kept in a dry ice chest at approximately -73°C were observed for viability and morphological changes over a four- month period. After four months in the frozen state, only those organisms suspended in 2 percent casein, skim milk, and normal horse serum were still living. However, after storage for three months at this temperature, the organisms suspended in all menstrua but 0.25 percent agar and fresh allantoic fluid gave growth after being subcultured in Korthoff's medium. Freeze-Drying s that were subjected to freeze-drying were The organism first frozen following similar procedures as described in the section ‘ ‘ ' sed in on freezing. In addition to the eleven suspending menstrua u the study of the effects of freezing and storage at various subzero temperatures, infected tissues of young guinea pigs and young golden ham ste rs we re lyophilized. 36 Different groups of animals were inoculated intraperitoneally with l to 2 ml. of an actively growing eight- to fourteen-day-old cul- ture of L. icterohemorrhagiag. The animals were observed daily for symptoms of leptospirosis and death. If the animals did not die within a week's time, they were sacrificed and the tissues harvested. As soon as possible after death, an autopsy was performed, and the kidneys, liver, and, in some cases, the spleen, were removed. The tissues were removed aseptically and placed in a sterile mortar. Sterile allundum was added as an abrasive and the tissues were emulsified with a pestle, taking care to avoid contamination. Just enough normal horse serum was added as a diluent to facilitate pipetting the ground tissues into lyophile tubes for freezing and drying. To make certain that the tissues to be lyophilized were in- fected with the leptospirae,.dark—field examinations of saline suspen— sions of the minced tissues were done and some of the same material was also inoculated into Korthoff's medium, incubated, and observed for evidence of growth. In many instances, the leptospirae could not be isolated from either the animals that died, or from those that were sacri- ficed. Only those tissues that were infected, as determined by dark: field microscopy. were used for lyophilization. 37 Following the preliminary "sharp" freezing in the freezing mixture, the ”lyophile" tubes containing the frozen‘, suspended organ- isms were placed in a jar which was stoppered with a one-hole rubber stopper. By means of glass and rubber tubing, the bottle was connected to one of the outlets in the manifold of the lyophile apparatus and dried according to the technique of Flosdorf and Mudd (1938). The lyophile apparatus consisted of a manifold with sixteen outlets which was connected to the condenser containing "Drierite" (anhydrous calcium sulphate) as the desiccant. The vacuum was produced by a Cenco ”megavac" vacuum pump which was capable of producing a vacuum of approximately 100 1microns as measured by a Stokes-McLeod vacuum gauge. The cultures were allowed to dry for approximately seventy- two hours under reduced pressure. At the end of this period, the tubes were taken from the jar and attached to the outlets of the manifold by means of pressure tubing. The vacuum was applied again and when the pressure was sufficiently low, the tubes were hermetically sealed, using a cross-fire oxygen torch. After seal- ing, the tubes were kept at refrigeration temperature (40° to 45°F) until they were reconstituted. 38 The dried cultures were reconstituted by inoculation into K0 rthoff's medium and incubating them at 30°C. These subcultures we re examined at the end of one and two weeks' incubation by dark- field microscopy for evidence of active leptospirae. In no instance could the organisms be recovered after lyophilization. DISCUSSION Freezing and subsequent storage at subzero temperatures shows promise as a means of preserving members of the Genus Lgptospire for periods from several months to possibly several years. Frozen cultures of L. icterohemorrhagiae suspended in various colloidal substances and stored in a deep freeze at -47°C for nine months were thawed and, after inoculation into Korthoff's medium and subsequent incubation, were observed using dark-field micro- scopy. The leptOSpirae from several of these suspending menstrua appeared actively motile and normal in morphology. From these observations, it appeared that, if the original leptospiral population were great enough and a suitable storage temperature employed, the organisms would survive for considerable periods of time in the frozen state. The ability of the leptospirae to survive freezing temperatures seemed to vary somewhat with the type of menstruum in which they were suspended and the storage temperature at which they were held. Certain menstrua were consistent in supporting viability, while others consistently gave negative results regardless of the tempera- ture at which they were kept. . 39 40 The effectiveness of a few of the suspending menstrua varied with the storage temperature; for example, 1 percent starch at a temperature of -47°C did not support viability after a storage period of three months, whereas those leptospirae kept at -27°C and —73°C were observed to be actively motile after incubation in Korthoff's medium. Although there was some variation of results in the cultures held at different temperatures, the difference did not seem great enough to be significant, especially in those suspending menstrua that consistently gave positive results. The suspending menstrua that gave the most consistent results in supporting leptospiral life during initial freezing and subsequent cold storage were 3 percent gelatin, 1 and 2 percent casein, skim milk, and slightly hemolyzed normal rabbit or horse serum. It was observed during this study that the leptospirae that had been growing well in Korthoff's medium containing normal horse serum grew very poorly when subcultured into medium containing normal rabbit serum and that it took several series of transfers before good growth was obtained. The same was true with organisms growing in medium with normal rabbit serum subcultured into medium containing normal I horse serum. The poor maintenance of leptOSpirae suspended in 41 normal rabbit serum in Group 111 may possibly have been due to this factor since they had been grown in Korthoff‘s medium contain- ing horse serum prior to suspension and freezing. Skim milk and serum gave the most consistent results of heavy growth and motility with no apparent adverse effects on the morphological characteristics of the organisms. Therefore, these would be the suspending menstrua of choice. Freezing presents a practical method of preserving cultures. The procedure is not only simple as far as operation and materials are concerned, but is economical, representing economy of time, labor, glassware, and space. This method must be investigated further, to prove its worth for the leptospirae, but the results of this study showed that there are possibilities. Since Shackell (1900) first introduced freeze-drying and sug- gested a number of applications of its use for biologics, Elser, Thomas, and Steffen (1935), Flosdorf and Mudd (1935), and other investigators have developed the techniques and apparatus to such a Point of efficiency and practicality that it has probably become the most effective method for preserving bacterialcultures. Attempts to lyophilize cultures of 1:. icterohemorrhagiae, however, have resulted in death of the organism. Stavitsky (1945) 42 could not recover the organisms from infected guinea pig livers that were subjected to freeze-drying. Similar work by other inves- tigators also gave discouraging results. This study was conducted using procedures and apparatus similar to those described by Flosdorf and Mudd (1935), and the same suspending menstrua as those used in the experiments on freez- ing. Infected animal tissues were also lyophilized, but the organisms could not be recovered in any instance. Under specified conditions these organisms can withstand freezing, but attempts at freeze-drying have thus far been unsuccess- ful. The factor, or factors, involved are still unknown, so there is need for further research. SUMMARY This study has shown that L. icterohemorrhagiae can be frozen and kept viable without too great adverse effects as to activity and morphology for periods up to nine months or longer when suspended in certain colloidal substances. The most effective of these sub— stances were skim milk and slightly hemolyzed, normal rabbit or horse serum. There was very little variation in the three storage temperatures (-27°C, -47°C, and -73°C) employed. 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