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A preliminary investigation of some of the factors affecting the percentage germination of lyophilized… Anastasiou, Joan Diane 1954

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A PRKT.TMINARY INVESTIGATION OF  SOME OF THE FACTORS AFFECTING THE  PERCENTAGE GERMINATION OF LYQPHILIZED CONIDIA  OF ASPERGILLUS NIGER 'VAN TIES. by JOAN DIANE ANASTASIOU A THESIS SUBMITTED IN PARTIAL FULFILMEt^T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS AND SCIENCE i n the Department of BIOLOGY AND BOTANY We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS AND SCIENCE Members or tne Department of Biology and Botany THE UNTVERSITY OF BRITISH COLUMBIA Apr i l , 1 9 5 4 A PRELBIINARY INVESTIGATION OF SOME OF THE FACTORS AFFECTING THE PERCENTAGE GERMINATION OF LYOPHILIZED CONIDIA OF ASPERGILLUS NIGER 'VAN TIEG./ Abstract Variations of the techniques and materials used during lyophilization procedures are considered for their effect upon the percentage v i a b i l i t y of the conidia of Aspergillus niger (van Tieg.) after freeze-drying. Of the factors investigated, those affecting percentage survival are: (1) Age of the culture from which the conidia are harvested. At 24-26°C, maximum survival occurs after two to six days of incuba-tion. (2) The temperature at which the culture i s incubated. A temperature of 6°C. slows the loss of v i a b i l i t y after lyophilization. (3) The carrier i n which the conidia are dried. Solutions containing 20$ sucrose allowed the best survival of the diluents tested. 0*5% ascorbic acid depressed v i a b i l i t y immediately after drying and after 13 months' storage. (4) Degassing the spore suspension before freezing i t . This treatment increased survival after lyophilization. This i s not a surface phenomenon. (5) The length of time and the temperature at which the conidia are suspended before being frozen. A long period and a high temperature of spore suspension produces a rapid loss of v i a b i l i t y after drying. (6) The general method of lyophilization. The Edwards centrifuge method of freeze-drying produces better survival than either of the other two methods described. Those factors which do not significantly affect conidial survival are: (1) Concentration of conidia i n the drying suspension. Only a narrow range of dilutions were tested. (2) The length of time from 5 minutes to 10 hours that the suspension i s frozen at -50°C. before evacuating. ACKNOWLEDGMENTS The author wishes to express her gratitude to Dr. R. H. Haskinsl for his direct guidance during the f i r s t part of the investigations and generously informative correspondence afterwards, and to Dr. G. A. Ledingham^ who authorized f u l l cooperation extending to the construction of a small lyophilization apparatus which was used for a l l work at the University of Brit i s h Columbia. The author wishes to thank Dr. A. H. Hutchin-son^ and Dr. F. Dickson^- for their encouragement, suggestions, and organization of financial backing throughout the investigations on the West coast, and Dr. J. J. Stock^ who offered helpful criticism of the manuscript. She i s greatly indebted to her husband with-out whose encouragement, patience and help, this work would never have been completed. This investigation was financed by the National Research Council and a scholarship from the Br i t i s h Columbia Sugar Refining Company Limi-ted. 1 Mycologist, Prairie Regional Laboratory, National Research Council, Saskatoon, Saskatchewan. 2 Director, Prairie Regional Laboratory, National Research Council, Saskatoon, Saskatchewan. 3 Head of Department of Biology and Botany, University of British Columbia. 4 Professor, Department of Biology and Botany, University of British Columbia. 5 Professor, Department of Bacteriology and Immunology, University of British Columbia. C O N T E N T S Page INTRODUCTION: REVIEW OF FREEZE-DRYING 1 I History of freeze-drying 1 II Applications of freeze-drying 1 III Methods of freeze-drying 2 IV Freeze-drying as a method of preservation 7 V Freeze-drying of fungi 10 GENERAL PROCEDURES 13 I Organism and culture 13 II Apparatus 14 III Methods 18 EXPERIMENTS TO DETERMINE EFFECTS OF THE FOLLOWING FACTORS 23 ON PERCENTAGE SURVIVAL OF A. NIGER CONIDIA SUBJECTED TO LYOPHILIZATION Study I Source culture age 23 A. Germination of lyophilized conidia 23 from cultures incubated for various lengths of time after planting B. Correlation of germination of lyo- 2 7 philized conidia with conditions of growth medium C Germination of lyophilized conidia 28 from cultures of different ages which were stored at 6°C. for various lengths of time i v D. Survival curve of lyophilized conidia harvested from cultures of various ages and then stored over night at 6°C Comments on Study I Page 32 32 Study II Conidial concentration i n suspension Comments on Study II 3 4 35 Study III Diluent Study IV A. The germination of lyophilized conidia dried i n various menstrua and observed immediately after freeze-drying B. The germination of lyophilized conidia dried i n various menstrua after storage for one year at room temperature Comments on Study III Degassing the conidial suspension before freezing Comments on Study IV 35 35 44 47 47 49 Study V Maintaining conidia i n l i q u i d suspension 51 prior to freezing A. Germination of lyophilized conidia 51 maintained i n suspension for various lengths of time at room temperature before freezing B. Germination of lyophilized conidia 52 maintained i n suspension for one to two hours i n an ice bath before freezing Comments on Study V 54 V Page Study VI Maintaining conidia i n frozen suspension 56 below - 5 0 ° C for various lengths of time before evacuation A. Germination of lyophilized conidia 56 dried after storage i n a - 5 0 ° C chamber for lengths of time from five minutes to ten hours B. Germination of lyophilized conidia 5# after short time exposure to freez-ing temperatures by direct immersion at -&0°C. and by cold air cooling at - 6 0 ° C Comments on Study VI 63 Study VII Various lyophilization methods 63 A. Germination of conidia lyophilized 63 by three methods, germinated imme-diately after sealing B. Germination of conidia lyophilized 67 by three methods germinated after 13 months' storage " i n vacuo" at room temperature Comments on Study VII 70 SUMMARY 71 BIBLIOGRAPHY 73 T A B L E S Table ' Page The percentage germination of lyophilized conidia 25 of A. niger harvested from cultures of various ages The pH, titratable acidity, and reducing sugar 29 content of the medium of variously aged cultures of A. niger. Comparative germination percentages of lyophilized 31 conidia of A. niger from cultures incubated at 26°C. and those from cultures incubated at 26°C followed by a period at 6°C The percentage germination of lyophilized conidia 36 of A. niger which have been suspended in beef serum and variously diluted The effect of six diluents on the percentage 41 germination of lyophilized conidia of A. niger revived soon after drying The effect of the diluents glucose, sucrose, sucrose & 43 ascorbic acid, and sucrose & peptone on the percentage germination of lyophilized conidia of A. niger revived soon after drying 7 The loss of v i a b i l i t y of conidia of A. niger lyophi- 46 li z e d i n water, a sucrose solution, and a sucrose solution with added ascorbic acid after storage for 12 months. 8 The effect of wetting agent and pre-lyophilization 50 treatment on the percentage germination of A. niger conidia lyophilized i n beef serum by various tech-niques The effect on the percentage germination of the conidia of A. niger which have been lyophilized after being kept i n suspension at room tempera-ture and at 0°C for 1-2 hours. The effect of freezing at -56°C for various lengths of time before evacuation, on the percentage germination of lyophilized conidia of A. niger The effect on percentage germination of A. niger conidia lyophilized with a rapid pre-freeze by direct immersion in a - 80°C. bath and a slower pre-freeze by a i r cooling to -60°C. for two minutes and for ten minutes Comparisons of the survival of the conidia of A. niger lyophilized i n beef serum by various techniques The effect of three lyophilization methods upon the v i a b i l i t y of conidia of A. niger dried i n beef serum following storage "in vacuo" at room temperature in the ligh t for 13 months F I G U R E S Page Figure 1 15 Figure 2 17 Figure 3 26 Figure 4 33 Figure 5 53 INTRODUCTION RWISM O F F R E E Z E r - D R Y I N G I History of freeze-drying Freeze-drying i s no newcomer to the world of s c i e n t i f i c processes. It has been discussed many times and the history and extent of i t s development appears again and again i n print ( l l , 14, 52, 53, 54, 55, 56, 76, 77, 84, 85, 94)- In a l l the his t o r i c a l sketches, two names stand out - Wallaston and Shackell. Wallaston, i n 1813 (179) f i r s t demonstrated the principle of vacuum sublimation but i t was Shackell who, after a lapse of nearly a hundred years, f i r s t appreciated the po s s i b i l i t i e s of this early experiment. In 1909 he reported remarkable success with freeze drying as a method of preservation (152). Since the publication of Shackell*s paper, the preservation of bacteria, yeasts and viruses by freeze-drying has received much attention. The process has been extended and methods modified (81, 83) and remodified. During the last decade mold cultures have entered the picture and attempts to preserve them by lyophilization have proved successful (139). II Applications of freeze-drying The use of freeze-drying as a method of preservation i s 2 spreading rapidly. Its application to the study of micro-organisms has been mainly concerned with medicine. Bacterial investigation, although fairly extensive, has dealt almost exclusively with pathogenic organisms. Occasional exceptions include the preservation of the legume inoculant, Rhizobium (5, 6). Studies of virus preservation also have been generally restricted to medically important viruses such as the tumor producers (84). Freeze-drying has passed the laboratory stage in the medical world and has proved invaluable in the preservation of products for general medical use. Large-scale freeze-drying to stabilize sera for extended storage and easy shipment has shown its worth in recent years (107, 165, 166), when a supply of blood serum has meant the difference between l i f e and death. Enzyme preparations, too, have been improved and are now available in stable form (84, 115)' A medical service on a smaller scale but of vital importance is the recent establishment of local freeze-dried bone banks (1Q3). Freeze-drying has become a part of standard technique in laboratory and histological procedures. Extractions of unstable compounds are frequently only possible after a preliminary freeze-dry (63). Histological fixation by this method for light (19) and electron (132) microscopy has been proved superior in certain respects (85, p.202, 170) to older techniques. Its use has been of particular value in animal tissue studies (44, 130). I l l Methods of freeze-drying Since Shackell's paper in 1909, there have been many changes 3 in the methods of freeze-drying. The original apparatus has been improved and adapted to the needs of the worker and has been developed with the advance of knowledge. Freeze-drying is a process in which a frozen material is dried under vacuum. Sublimation occurs, and the resulting vapour is removed by some method to maintain a diffusion gradient for dehydration. The physical principles involved are beyond the scope of this thesis. For such information the reader is referred to Carman (30) whose work was verified by Kramers and Stemerding (102), and Dunoyer (43) • The cryochemical method—"dessication in vacuo from the frozen state by means of chemicals"(59)—was the fi r s t type of freeze-drying to be developed. Shackell's (152) use of EjS.O^ to absorb the water of sublimation was followed by workers for several years (81, 83). To obtain a more complete desiccation, a secondary trap of CaCl2 was added to the procedure in 1929 (146). Vfith the dis-covery of more efficient drying agents, the secondary dry is usually omitted. Successful desiccation is now achieved by the use of .^0^ (28, 36, 135, 144, 167, 169) or Calcium sulfate (59, 60, 171) as primary desiccants. The actual freezing may be accomplished by pre-freezing or by self-freezing. In the f i r s t case, the container with the material to be dried is placed in a cold chamber or a freezing bath (146, 167, l69)« Successful self-freezing is contingent upon evaporation. If this method is used, the evaporation must be suffi-ciently rapid to ensure the freezing of.the material without an 4 external freezing source (36, 59, 144)* The capacity of the cryochemical apparatus is usually small (ex 171) since i t is limited by the weight of desiccant required to absorb each gram of water. Drierite, for instance, used exclusively in the U.S.A. because of i t s ease in handling (unlike regenera-b i l i t y (water can be removed fifteen times before the Calcium sulfate must be changed), and its fairly good affinity for water, holds only 6.6$ of i t s weight in water (58). Lyophilization is the second and probably most widely known method of freeze-drying. Reichel (140), who first applied the term which means "solvent-loving", defined i t as a process in which material is frozen at -50°C or lower before the vapour is removed by low temperature sublimation and trapped by condensation. In the last twenty years, the term has broadened to encompass methods involving higher pre-freeze temperatures and, recently, self-freezing (85). In this paper the broadest concept of the word has been accepted and allows the inclusion of centrifuge freeze-drying (65, 75) in lyophi-lization methods. The pre-freezing, s t i l l used in most lyophile procedures, is usually carried out in small tubes or ampoules. These are attached to a manifold either before or after immersion in the cold bath (8, 58, 89, 90, 95, 151> 180). This use of small tubes was an adaptation by Flosdorf and Mudd (58) of the original Reichel apparatus (140) xvhich dried sera in bulk. If bulk is required to-day, shell freezing is common (47, 89). When a vacuum is applied to the frozen material the rate of 5 evaporation usually keeps i t frozen. Frequently heat i s applied externally to increase the temperature gradient and hasten the rate of drying (24, 165). Some material has a tendency to melt before dry-ing i s completed. I f this occurs, the material must be kept i n a cold bath throughout the procedure (12). Condensation* may be brought about by condensor coils (117, 165) or by a cold trap immersed i n a cold bath such as li q u i d a i r (181), l i q u i d Nitrogen (164) or dry ice mixed with acetone (30a), ethylene glycol (176), or methyl chloride (79). Occasionally a secondary drying period over Drierite or P2O5 i s added to the lyophile process to complete the drying (53, 117). Lyophiln zation has certain advantages over the cryochemical procedure. While the latt e r technique i s limited to the drying of small amounts, lyophilization can be carried out on both the small scale (31, 90, 95, 100, 143, 150) and the large. Secondly, lyophi-l i z a t i o n results i n a more complete desiccation. Although Flosdorf (55) claims that the moisture content of cryochemically dried material i s v i r t u a l l y zero, i t has been shown that more complete desiccation i s possible by lyophilization than by cryochemical drying. With a very efficient low temperature condensor immersed i n l i q u i d Nitrogen, the f i n a l content at 1 micron Hg. pressure i s 10"^ mgm. water per l i t r e , while at the same pressure using Drierite, the moisture content of the + Throughout the literature the t em'Sublimation" i s generally restricted to the formation of vapour from a solid. To avoid con-fusion i n freeze-drying discussions "condensation" has become the conventional way to refer to the formation of ice from vapour. 6 system i s l C f 5 mgm. per l i t r e (164). A third advantage of a cold trap i s i t s a b i l i t y to condense a l l types of vapours, not just water. This i s of particular importance when a McLeod gauge i s included i n the vacuum system. Mercury evaporates at low pressure and, were i t not collected i n the cold trap, would vaporize through the system, including the ampoules of drying material, perhaps causing denaturation of proteins and death to microorganisms on subsequent storage. The main disadvantage of this method i s the fact that a low pressure i s required for freezing to be maintained throughout the process. 700 microns Hg. i s the maximum pressure allowed, and 250 microns Hg. i s definitely preferred. The cryochemical procedure, on the other hand, requires less vacuum, 1 to 2 mm. Hg. pressure being sufficient (58). The Adtevac process (93) was developed as an adaption of the cryochemical process. It employs s i l i c a gel as the dehydrating agent and has the advantage of absorbing a l l the vapours, not just water. A fourth freeze dry method (48) employs a multiple steam jet which creates a vacuum and eliminates moisture from the system without the need of a desiccant or condensor. This method has proved efficient and economical i n large-scale industrial processes. The Desivac process (60) removes water vapour by diffusion i n a multiple stage o i l diffusion pump which maintains the o i l at a very low vapour pressure. A vacuum of only 4«5 mm. Hg. w i l l keep the material frozen and the moisture content i s at less than 1$ when the system reaches 100 microns Hg. It i s only practical on a small 7 scale sinc,e huge volumes of vapour are produced from small quantities of ice (59).- Larger scale models involve extremely expensive appa-ratus (165). IV Freeze-drying as a method of preservation Freeze-drying has proved of great value as a method of preservation despite the time and trouble involved i n the i n i t i a l preparation. It i s , i n many instances, preferred to less laborious and more inexpensive methods of preservation. This i s true when sensitive organisms or easily destroyed substances (ex-27 ) are to be stored for any length of time. In spite of the fact that at present fungus preservation i s limited to small, thick-walled spores, that a new preparation must be opened for each subculture and that the processing steps are time consuming and require a special apparatus, many laboratories, for example N.R.R.L. Peoria, 111., and P.R.L., Saskatoon, Sask., have incorporated the technique into their routine of herbarium mainte-nance. The commonly used methods of fungous culture preservation have been evaluated by Fennel, Raper and Flickinger (49). In their publication, the advantages of freeze-drying over the other methods are recognized. Some of these are included i n the following para-graphs. (l) There i s less concentration and aggregation of protein molecules, leading to denaturation (36, 58, 75, 84). Any denaturation 8 that does occur (42) i s dependent upon tissue hydration (170). (2) Oxidative (137) and other chemical changes are greatly reduced. The oxidation i s minimized not only by the low temperatures sustained during freeze-drying but also by the effect of low pressure resulting i n a low oxygen concentration. (3) Most writers report no alteration i n properties during - and after freeze-drying. This applies to animal tissue (19), yeasts (46, 47), other fungi (49), bacteria (127, 133, 134, 144, 163), viruses (84, 138), and enzymes (89) • The depression of the rate of metabolism (74) i s responsible for this lack of variation i n micro-organisms during storage. The a c t i v i t y of freeze-dried material i s not completely eliminated i n some cases. There are a few reports of variations during and after treatment. Dopter (41) found that though a l l other physiological and morphological characteristics of two yeast strains remained constant for one year, i n one Saccharomyces  ellipsoides the a b i l i t y to sporulate was l o s t . Atkin, et a l , (7) observed variation on 50% of the revitalized yeast cultures which had been lyophilized by the N.R.R.L. method (see below "methods"). Haskihs. recently reports (87) ' the coincident low spore survival with greater metabolic variations i n Ustilago cultures. It has also been noted that any process involving freezing and thawing w i l l s p l i t Streptococcus chains (91). Changes i n antisera (66) and enzymes (115) have also been reported. (4) The lyophyllic property of a l l properly freeze dried material i s a valuable asset to laboratory technique at the time of reconstitution. 9 (5) A low moisture content i s rapidly and easily attainable i n freeze-dried preparations. This i s due to the elimination of the hard outer case which forms about air-dried material. (6) When low temperatures are maintained throughout freeze-drying, the loss of volatiles i s at a minimum (172). Whether or not this i s of value during the drying of micro organisms has never been investigated... Its importance i n the food and drug industries i s , however, obvious. (7) The long-term maintenance of a c t i v i t y or v i a b i l i t y i s probably the foremost reason for the use of freeze-drying i n the preservation of cultures and organic substances. Although the i n i t i a l k i l l i n g during freeze-drying i s great, the subsequent death rate during storage i s comparatively slight under good drying and storage conditions. Some authors have even claimed no drop whatever i n potency of sera (56, 57). (9) Further storage advantages include laboratory con-veniences. The freeze-dried material takes up l i t t l e space and does not require special racks or low incubation temperatures. Unlike other preservative methods i n use, by dint of the vacuum seal, a l l contamination between the processing of the substance and i t s reconstitution i s eliminated. For comparative studies i n bacterial preservative methods, including freeze-drying see Holt (96), Morton and Pulaski (124) and Stamp (160). Of the three, only the lat t e r found lyophilized^cultures less viable than those preserved by other methods. He recommended a procedure involving slow drying at room temperature. Other workers 1 0 have not substantiated his conclusions ( 6 5 ) . Of the two groups of workers who quantitatively compared lyophilization with other methods of drying fungous spores, each found a different technique to give a higher survival. Sharp and Smith ( 1 5 3 ) obtained higher v i a b i l i t y of uredospores after drying at room temperature than after freeze-drying. The effect of this method on other fungous spores has not been reported. Mazur and Weston (122) found that a larger percentage of Aspergillus flavus spores survive spray drying than freeze-drying. Spray drying, unfortunately, requires a large apparatus and i s not readily adapted for drying a variety of organisms. V Freeze-drying of fungi Although there are many comparative investigations of bac-t e r i a l freeze drying, there has been a paucity of similar fungus studies. Most work to date has consisted merely of statements that one organism w i l l survive freeze-drying, while another w i l l not. Bushnell, i n 1 9 4 1 ( 2 6 ) , for instance, states that whereas a l l the Eubacteria that he tested survived, the "higher fungi which were tested f a i l e d to remain viable". Conant i n the same year wrote to Raper ( 1 3 9 ) of his "indifferent success" i n lyophilizing fungi. In 1 9 4 2 Wickerham and Andreasen ( 1 7 6 ) conducted very brief tests of survival on storage. Their percentage survival estimates were based not on spore survival but on those tubes which would produce a culture and those which would not. Raper and Alexander (139), concerned with 11 herbarium culture maintenance found that, of the fungi tested, a l l Penicillia and most of the Aspergilli, Mucorales, and Fungi imperfecti survived lyophilization, while none of the Entomophthorales withstood the extreme conditions. Five years later Fennel, Raper and Flickinger (49) added observations that most sporulating wood destroying and pathogenic (ex. Dermatophytes) fungi survived, whereas aquatic Phy-comycetes did not. Sporophores may be preserved for herbaria by lyophilization followed by wax imbedding (126). However no reference was made to spore viability. In the last two years a few workers have published compara-tive studies dealing with a single organism and its resistance under various conditions of lyophilization. Sharp and Smith (153) worked on the resistance of puccinia uredospores to lyophilization and other methods of drying, using various suspending vehicles. They studied storage loss and methods of germination. Haskins and Anastasiou (88), working with Aspergillus niger presented quantitative data on suspending media, preliminary degassing and comparative methods of lyophili zation. Unpublished as yet are the detailed studies of Weston and Mazur, which have been conducted mainly with Aspergillus flavus and reported at various meetings (121, 175), and of Haskins who has been concerned with the relation of cultural characteristics of fungi and their preservation in various diluents (87). This thesis is a continuation of the work of Haskins and Anastasiou {88) on Aspergillus niger. Some of the data presented are repetitive of the contents of the published article. . This work 12 represents the f i r s t part of a methodical investigation of the factors affecting the survival of lyophilized fungus spores, and a determination of the crucial steps i n the various procedures. GENERAL PROCEDURES I Organism and culture The test organism used throughout this series of experiments was Aspergillus niger (Van Tiegh'em • It was obtained November 11, 1 9 4 S , as an a i r contaminant i n Saskatoon and stored under o i l under the code number PRL 2 4 ' An Aspergillus was chosen because members of this genus are able to withstand conditions encountered during lyophilization. For many years i t has been known that sub freezing temperatures are not lethal to a l l Aspergillus mycelVum (109) especially that which i s old or submerged (108). The spores are even more resistant ( 1 0 , 15, 9 9 ) . Luyet and Gehenio (112) classify Aspergillus spores among the "organisms which, when dry, re s i s t extremely low temperatures." In the last seven years, several workers (122, 139) have reported the successful lyophilization of Aspergillus spores. A. niger was selected for i t s ease of culture, i t s unifor-mity of germination, and for the dark coloration of the conidia which makes them readily observed for direct microscopic counts from petri plates. The organism was grown on the sporulation medium of Gastrock et al.(68), the formula for which appears i n the Manual 14 of the Aspergilli (173)• II Apparatus In the course of these investigations, two types of apparatus were used. The centrifuge method (75) made use of an Edwards freeze-dry apparatus Model 3« c For the other techniques a small glass apparatus, identical to that of Haskins and Anastasiou (88) was used and i s illustrated i n Figures 1 and 2 . It consists of: Vt - a vapor trap, or condensor, which i s immersed during operation i n a bath of ethylene glycol, water and dry ice at a temperature between -60°C and -80°C The vapour produced by sublimation from the material in the drying tubes condenses as ice i n the base of the trap. The low temperatures used are not absolutely necessary (55) but they offer a margin of safety i n allowing for plug obstruction. Also, there i s a sl i g h t l y lower percentage of moisture remaining after desiccation (79)• Dc - The drying chamber, which has a maximum capacity of twenty-two lyophile tubes, was used for primary drying according to the PEL method. It i s to be noted that there are no restrictions i n the vapour passage from this chamber to the vapour trap. 15 F i g u r e 1. Table l y o p h i l e appara tus . M - The 6 tube m a n i f o l d has one s topcock f o r each s e t o f t h ree t ubes . Thus s e v e r a l l y o p h i l e tubes may be a t t ached to the m a n i f o l d and evacuated w i t h o u t d e s t r o y i n g the vacuum i n another group o f tubes which have been p r e v i o u s l y evacuated on t he o ther s e t o f th ree openings . These s topcocks , however, had a narrow bore and the passage o f vapour was r e s t r i c t e d . Thus, when the NRRL method was a p p l i e d , a c o l d b a t h o f about -10°C 16 was frequently necessary to keep the spore suspen-sions frozen. VR - The vacuum release i s necessary for introduction of a i r when removal of the tubes or disconnection of the parts i s required. It also allows for calibration of the McLeod gauge by furnishing a place of attachment for a second pressure gauge, which has been previously standardized. This stopcock may be used i n the ad-mission of various gases for storage tests. VG - A McLeod vacuum gauge was attached to the central piece (Fig. 2) of the apparatus by a ground glass joint. The calibration was approximate and recorded on a cardboard or plastic scale clipped to the glass. McLeod gauges have been used i n many apparatus (30a, 58, 181). Although i t i s not accurate at low pres-sures i n which water vapour i s present, i t proved inexpensive, convenient, and indicated the pressure i n the system sufficiently for the purposes of these experiments. In most larger apparatus, the Piranni gauge i s used (ex. i n the Edwards Centri-fuge model 3). Unlike the McLeod guage i t records total pressure and gives a continuous reading. VP - The outlet to the vacuum pump consists of a tube which extends almost to the base of the condensor. The end must extend well below the surface of the freezing mixture surrounding the vapour trap and 17 Figure 2. Central piece of table lyophile apparatus yet be above the anticipated surface of the ice collecting within the condensor. A rubber con-nection separates the top portion of the tube from the lower section allowing adjustment of length and f l e x i b i l i t y for attachment to the rest of the apparatus. The upper end of the tube i s connected by high pressure tubing to a vacuum pump. i d LT - The lyophile tubes were blown from 7 mm. external diameter pyrex tubing. Their finished length was four to six inches. It was found unnecessary to constrict tubes of this diameter before cutting them off on the manifold. To allow maximum free passage of vapour during dryingjCaps of cotton batting and cellulose tape (23) were used i n maintaining s t e r i l i t y u n t i l the tubes were attached to the manifold at which time they were removed. A Welch duoseal or a Cenco hivac pump was used to evacuate this apparatus. Lyophilization i s possible using a pressure of 700 microns Hg. (58) but a pressure of 100 microns to 200 microns i s recommended for most rapid drying. Lower pressures of 25 to 50 microns are precautionary against a leaking system or inefficient condensor (56). Throughout the experiments using this table model, the system was main-tained at pressures of between 50 microns and 100 microns Hg. A l l glass used i n the apparatus was pyrex. Anhydrous lanolin was found to give a most satisfactory seal. Although i t was necessary, to make new applications to a l l the connections between each run, i t was easy to work with and leakages i n the system were rarely encountered. A cross-fire Hoke oxygen torch was used to seal the tubes " i n vacuo". I l l Methods The three methods of lyophilization have been b r i e f l y described 19 ( 8 8 ) . 1. The NRRL method Essentially this i s the Wickerham and Andreasen (176) method modified by Raper and Alexander (139) and Fennel, Raper and Flickinger (49) i n the Northern Regional Research Lab. at Peonia, 111. to preserve fungous cultures for the herbarium. OA The lyophile tubes containing^rnl. of the spore suspension were attached to the manifold and pre-frozen by plunging the tubes into a bath of ethylene glycol, water and solid carbon dioxide, at -50 to -60°C. for one half to two minutes. When the vacuum was established, the bath with the tubes suspended i n i t was warmed to -10°C. ±5°C. where i t was kept for several hours or u n t i l the pellets i n the tubes appeared to be dry. The cold bath was then removed and drying was continued for 3 to 8 hours at room temperature. The tubes were then sealed off. This entire process of freeze-drying i s carried out while the tubes are attached to the manifold. Once the vacuum i s establish-ed, i t i s never broken u n t i l the tube i s opened for subculturing. 2. The PRL method This method i s frequently used for routine culture preser-vation at the Prairie Regional Laboratory of the National Research Council at Saskatoon, Sask. (106, 23). The tubes containing 0.1 ml. of the spore suspension were pre-frozen by exposing for at least 15 minutes to cold chamber tem-peratures of -31°C, -40°C. or, more often, -70°C. There was no 20 freezing by direct immersion i n a freezing l i q u i d . The tubes with their frozen contents were then as rapidly as possible placed i n the side drying chamber (DC), attached to the apparatus and evacuated. When apparently dry, the tubes were moved to the manifold, re-evacu-ated and sealed off i n vacuo. Thus freezing i s accomplished more slowly than i n the previous method and the vacuum i s broken between each step i n the procedure. A modification of this method was adopted for convenience i n Studies I, II, V, and VT. The tube contents were frozen either by direct immersion i n the condensor bath or by immersion of the side drying chamber (DC) containing the tubes i n the condensor bath. When the freezing was completed, connection was made to the apparatus and the procedure was continued according to the PRL method. When many tubes were to be lyophilized at once, the cotton caps were removed to make room i n the chamber . This probably had the effect of maintaining the material at a lower temperature and served as a precaution against melting. It did not greatly increase the removal of vapour, since the effect of the removal of the ob-struction was balanced by a lowered temperature gradient (53, 79)-3. The Centrifuge method Although this method of freeze drying does not comply with the original definition of lyophilization (140), the authority for i t s inclusion i n this group of procedures has already been stated. The procedure outlined here i s based on the original technique 21 of Greaves (75) and f u l l y described i n the handbook accompanying the apparatus ( 4 5 ) ' There i s no pre-freeze i n this method. The tubes containing the 0 . 1 ml.' of suspension were angle centrifuges to prevent bubbling in a chamber at 26°C. while being evacuated. Self-freezing occurred at about 1 mm. Hg. ( 6 5 , 75) or 1.5 mm. Hg. (79) and usually took place i n three or four minutes. After freezing occurred, the centrifuge was stopped and heat externally applied bringing the chamber to almost 37°C« The condensor became equilibrated at -20°C. or lower, while the pressure reached 120 microns Hg. or usually less. Although the apparatus i s equipped for a secondary drying over ^2^5 sealing off may be accomplished on this manifold, for purposes of uniformity of storage conditions, the tubes with their dried contents were transferred to the manifold of the table apparatus for re-evacuation and sealing off. Reconstitution of the dried suspension The revival method of Sharp and Smith (153) involving a moisture chamber seemed unnecessary for our purposes. The sealed tubes were snapped open on ra f i l e mark. This invites contamination, and i f A. niger were a less vigorous organism, precautions of slow opening by heat cracks ( 3 5 ) , or covering with antiseptic cloths ( 5 7 ) , would undoubtedly have been necessary. Sterile water ( 0 . 1 ml.) was added by the same type of hand drawn pipettes that were used to dispense the original suspensions. When the dried product was apparently dissolved, the drops were plated on Potato Dextrose agar. 22 Incubation at 26°C. followed and the procedure was concluded with direct counts of spore germination on the plates. These direct counts eliminate the errors i n percentage survival encountered during bacterial studies due to " s p i H u which occurs during the centrifuge method just before the completion of the primary drying (35)• Unless otherwise stated, at least 1000 spores were counted from each tube. Statements of significance are based on those of McCallan and Wilcoxon (114). Controls consisted of spore germination counts of unlyophi-l i z e d material which had been treated with the dried spores up to the point of freezing when they were plated. The control percentage germinations are quoted but not used i n the calculations of lyophilized spore germination. EXPERIMENTS TO DETERMINE EFFECTS OF THE FOLLOWING FACTORS ON PERCENTAGE SURVIVAL OF A. NIGER CONIDIA SUBJECTED TO LYOPHILIZATION Study I Source culture age A. Germination of lyophilized conidia from cultures incubated for various lengths of time after planting Introduction Quantitative comparative studies on source culture age as i t affects lyophilization survival are limited to a few reports regarding bacteria. , Naylor ( 1 2 5 ) , Pry and Greaves ( 6 5 ) obtained maximum sur-v i v a l i n 18 to 24 hour old cultures. Proom and Hemmons ( 1 3 5 ) , on the other hand, state that very young cultures 5 to 9 hours old are best able to withstand freeze-drying. In most bacterial studies of freeze-drying, young cultures were used without preliminary investigation. The age of the culture from which the dried cells were obtained i s not mentioned i n articles dealing with fungous lyophilization. Procedure Two experiments were carried out using different batches of medium ( 6 8 ) , 20 ml. aliquots being used i n one series, and 40 and 10 ml., i n the second. For more than a month, individual flasks were planted at irregular intervals. During the la s t two weeks before 24 drying, one flask containing 10 ml. and one containing 40 ml. were planted at the same time. At the time of lyophilization, cultures were available ranging in age from less than three days to over forty. Within each of the two series, conidia from each culture were suspended as rapidly as possible i n random order and placed for an hour or more i n an ice bath to eliminate as much variation as possible due to suspension time (see Study VI A). A l l the tubes i n a series were placed simultaneously i n the side drying chamber, which had been previously immersed in the condensor bath. The spores were revived immediately after primary drying. Results and discussion The figures obtained from germination counts appear i n Table 1 and the graph, Figure 3« - From the data, i t i s apparent that the culture age did affect the germination rate of the lyophilized conidia. Judging from the control figures obtained i n the 10 and 40 ml. series, the conidia had lost their v i a b i l i t y during incubation of the culture. The control figures of the series grown on 20 ml. of medium, on the other hand, indicated that the lyophile survival curve was produced by differences i n the resistance among those conidia which were viable when harvested. Further study, with careful reference to controls w i l l indicate whether the survival curve i s the result of changes i n degree of resistance to lyophilization. There appears to be a peak of spore survival at between two and six days of incubation at 24.-25°C, depending on unknown factors. The increment of the r i s e to this peak i s great, while the Table 1. The % germination of lyophilized conidia of A. niger harvested from cultures of various ages. Those grown i n 10 and 40 ml. of medium were dried at the same time. Volume of culture medium 10 ml. 20 ml. 40 ml. Culture age (days) 1.9 3.1 3.9 4-9 % germination of spores dried 83.0 93.1 85.0 89-3 control 99.1 96.4 68.8 88.6 Culture age (days) 2.3 5.1 5-5 9.1 11.1 12.9 14-2 26.3 32.5 40.5 % germination of spores dried 21.2 30.0 47.0 40.4 34-2 32.0 23.9 26.2 15-5 7.1 control 99.0 98.6 99.1 97-3 98.0 97.4 99-2 85-4 75.9 89-3 Culture age (days) 1.9 3.1 3- 9 4- 9 12.0 15.1 22.1 28.2 32.0 36.2 % germination of spores dried 77.2 77.8 89.8 74.6 66.5 78.7 54.9 69-9 65.2 64-4 fig. 3. EFFECT of CULTURE AGE on SURVIVAL of CONIDIA. 27 loss of viablity of lyophilized conidia after this time is gradual. From these results, there is no evidence that the volume of the growth medium has an effect on conidial survival of lyophili-zation or the age of the culture at which the survival peak occurs. B. Correlation of germination of lyophilized conidia with the conditions of the growth medium Introduction In order to determine some reason for the effect of the age of the culture on the percentage germination of lyophilized conidia, i t is obvious that f i r s t the age of the individual spore must be considered. Such a study involves obtaining accurate measurements of a great many spores at regular short intervals. The spores must be isolated, since germination measurements in mass spore suspension are deceptive. (This has been indicated previously by Fry and Greaves (65) who found that the long lag phase, observed throughout the course of these experiments and reported by other workers on bacteria (135, 174), 'was due, not so much to dormancy, as to a lower concentration of germinating cells.) Such properties as degree of hydration of- conidia (no method exists at present sufficiently precise for this determination (3, 61, 182)) and spore wall thickness should be considered with spore age. Studies of these factors have not been included in this paper. Instead, an attempt was made to correlate lyophilization survival and culture age with some chemical factor of the culture medium. If some relationship could be found, i t would prove useful in developing a medium which would produce the most satisfactory 28 growth of spores for freeze-drying. To this purpose, pH, titratable acidity, and reducing sugars were tested. Procedure The day following the harvesting of spores from the 40 ml* cultures, the l i q u i d substrate was tested for pH, titratable acidity, and reducing sugars. pH readings were carried out on a Beckman meter model G; titratable acidity was determined by t i t r a t i n g with NaOH to the neutrality point of phenolphthalein, and reducing sugars were estimated using the Somogyi-Schaffer-Hartman method. Results and discussion The results of the various determinations appear i n Table 2. L i t t l e information was gleaned from these brief tests. No correlation was noted between percentage germination of dried spores and pH, reducing sugars or titratable acidity. The peak of survival, however, roughly coincided with the slowing of metabolism, indicated by the leveling off i n c i t r i c acid production (155)-C. Germination of lyophilized conidia from cultures of different ages which were stored at 6°C. for various lengths of time Introduction Frequently those cultures from which spores are to be har-vested for drying are placed i n a refrigerator' after sporulation has occurred "satisfactorily" (usually after about one week's growth). The effect of this treatment on spore counts of lyophilization survival was determined. Table 2. The pH, titratable acidity, and reducing sugar content of the medium of variously aged cultures of A. niger» Conidia were harvested for lyophilization 22 hours before these determinations were made. Culture age pH Equivalents of Reducing sugar Volume of (days) titratable (mgm./ml.) medium acid/mgm. (ml.) 0 5.45 .064 .76 2.8 2.22 •37 .81 3.9 2.18 •36 .81 4.8 2.11 .46 .83 . 40 5.8 2.05 .49 .75 13.8 1.99 .74 .67 15-9 1.93 .75 .73 23-0 1.92 .94 .73 29-0 1.97 .72 .82 32.8 1.97 .83 •74 38.0 1.90 1.08 .69 48.9 1.90 .78 .83 2.8 2.26 .35 .92 20 3.9 2.20 .43 .84 (spores were 4.8 2.15 .46 .91 not dried) 5.8 2.10 •58 .79 2.8 2.30 not tested 1.1 10 3.9 2.13 because of .77 4.8 2.11 small volume 1.06 5.8 2.11 of medium .97 30 Procedure Some of the flasks containing 20 ml. of medium, prepared for Study I A were planted i n pairs or threes. One flask was main-tained at 26°C. u n t i l lyophilization, while the other was placed, after a period of growth at 26°C, in a 6°C. temperature chamber u n t i l lyophilization. A l l spores were freeze-dried simultaneously. Results and discussion The data regarding the germination of the conidia appears in Table 3« The survival of spores from cultures maintained at refrige-rator temperatures was, i n every case but one (line 5, Table 3), higher than those maintained at room temperature. Even in this case, when the recorded germination rates are converted to a percen-tage of the control, the percentages of germination are changed from a ratio of 26.2^:35.6^ to 30.7^:36.3^. This difference, though s t i l l significant, i s reduced from a difference having a margin of significance of h»5% to that having one of ,9% at p = .02 (114, Table 3)« On the other hand, from these data, one cannot state that the germination was as high as i t would have been i f the spores had been dried immediately after they were harvested. When i t i s necessary to store cultures for more than two weeks before conidia are harvested for lyophilization, i t i s advisable to remove the culture from room temperatures to cooler at approximately the time of maximum survival. Under the conditions encountered during this experiment, i t would appear that this should be done after about six days of growth. Table 3. Comparative germination percentages of lyophilized conidia of A. niger from cultures incubated at 26°C. and those from cultures incubated at 26°C. followed by a period at 6°C. Total age of Time of incubation Time of incubation % germination of conidia culture (days) at 26°C. (days) at 6°C. (days) after drying before drying 14.2 14.2 23.9 99.2 14.2 8.7 5.5 27.1 98.3 8.7 # 8.7 - (42) 26.3 26.3 26.2 85-4 26.3 12.1 14.2 20.9 97.0 26.3 4-3 22.0 35*6. 98.0 12.1 # 12.1 - (33) 4-3-# 4.3 - (43) 32.5 32.5 _ 15.5 78.6 32.5 36.2 26.3 57.6 92.4 6.2 # 6.2 - (47) 40.5 40.5 7.1 89-5 40.5 8.1 32-4 36.0 87.4 s.i # 8.1 — (48) # The figures for % germination are approximate and are obtained by consulting the graph, figure 3. For example, i f one had inoculated 20 ml. of the medium 8.7 days before freeze-drying, maintained i t at a temperature of 24-26°C, then lyophilized the spores produced, with the others i n the series, one should expect, from the graph, germination of about k2$. 3 2 D. Survival curve of lyophilized conidia harvested from cultures of various ages, suspended, and then stored over night at 6°C. Introduction It i s frequently desirable to store tubes containing a sus-pension before freezing. The effect of this treatment on the survival of lyophilized spores from cultures of various ages was determined. Procedure At the time that the spores from the 20 ml. cultures of Study I A were suspended, duplicate tubes were f i l l e d . One set was placed at 6°C. for 16 hours while the other (recorded i n part A of this study) was lyophilized within 1^ hours of suspending. Results and discussion The difference i n the survival curves of those spores lyophilized immediately after suspension and those suspended and stored over night at 6°C. appears in the graph, Figure 4* The storage of spores suspended i n beef serum over night at 6°C. appeared to eliminate the i n i t i a l phase preceding the ma-vimim survival peak. The temperature of 6°C. i s well above that of -6 , 1 -8 .9 , and -10°C, at which mold growth has been observed ( 2 0 , 158). Some development, therefore, seems quite possible i n the suspended conidia of A. niger at this temperature. Whatever processes are involved i n development of lyophile resistance, a temperature of 6°C. does not appear to stop them. Comments on Study I The study of the effect of cultural conditions on A. niger f ig.4. EFFECT of PROLONGED SUSPENSION on LYOPHILIZED CONIDIA (6°C. ) 3 4 conidial resistance to lyophilization is anything but .complete. It is suggested that more detailed investigations be made to determine the factors influencing the survival curve, with special reference to metabolic rates, medium content, and conidial maturity. Such a study would be of practical value in hastening or delaying spore formation, and sustaining the maximum spore, survival. Study II Conidial concentration in suspension Introduction Reports have been meagre and conflicting regarding the effect of the concentration of suspended cells on their resistance to freeze-drying. Fry and Greaves ( 6 4 , 6 5 ) report that the highest survival rate of bacteria was obtained after drying dilute suspensions. This effect was noted only in i n i t i a l counts and was eliminated after 8 months' storage. Campbell-Renton (28), on the other hand, found that phage concentration during freeze-drying did not affect sur-vival. There have been no reports on the effect of concentration of fungous spores on lyophilization. Procedure Two sets of dilutions were lyophilized on successive days. Dilutions of the.initial conidial suspension were made in beef serum to 1/5, 1/25, 1/125, 1/375, and 1/1125 concentration. Because of the narrow range of spore concentration which could be counted by direct observation, i t was necessary to abandon the higher dilutions. unfortunately no hemocytometric observations were made to determine the actual cell concentrations obtained in this study. 35 Results and discussion The observations of the survival rate are recorded i n Table 4-No significant effect of dilution was noted within the narrow range of dilutions tested. Comments on Study II A wider range of dilutions than those used i n this study should be tested. More concentrated suspensions could be prepared using a young culture, which gives a large number of spores per loopful and a higher survival, and by adding a detergent to the diluent to s p l i t the spore clumps. In Study IV, "Vel" and "Dreft" were not found to have a significant effect on conidial survival. Thorn and Raper (173) recommend Sodium lauryl sulphonate for dilution purposes. Dilution series could be readily made to compare the effects of very low concentrations of conidia i n the menstruum. A product of the Geigy Co., Switzerland, "Tinovetine B", has been recommended for use, as i t restricts radial growth but has no effect upon spore germination (118). Study III Diluent A. The germination of lyophilized conidia dried i n various menstrua and observed immediately after freeze-drying (88) Introduction More studies on the effect of the suspending vehicles have been conducted with a l l types of.micro-organisms, than on any other Table 4 . The % germination of lyophiilized conidia of A. niger which have been suspended i n beef serum and variously diluted. Dilution of spores i n beef serum % germination of conidia Set I Set II dried control dried control 1 20.7 81 23.6 # 88 1/5 21.1 60 18.6 92 1/25 21.3 # 75 24.4 -1/125 - - 24.4 87 # With the exception of these tubes, each % of germination after drying i s based on figures obtained by counting at least 1000 spores from each of three tubes. These two data represent the % survival average of two tubes. The control figures are based on spore counts of 500. ON 37 phase of the freeze-drying procedure. Yet very l i t t l e is known, and no uncontested generalizations can be made. It has long been recognized that the carrier has an important effect on the survival of dried organisms ( l , 50, 62). Early studies were made on the varying effectiveness of different diluents on the preservation of bacteria. In 1908 (51), i t was found that drying vehicles, in order of bacterial protection, were milk, serum, bouillon saliva, distilled water, physiological saline, and urine. Since that time, with the development of freeze-drying, similar protection is provided by colloids to bacteria and viruses. It has been claimed that "the nature of the protective colloid...may make some differences to the i n i t i a l survival rate but in our experience these are not significant." (135). This statement has not been corroborated by other workers. In rebuttle, i t was pointed out that, not only was the choice of material important, but even the proportions used determined whether certain sensitive bacteria would survive (65). Until recently most bacterial carriers have consisted of naturally occurring complex colloids. Because of i t s availability and low cost, milk has been extensively used as a diluent (23, 26, 57, 181). Proom (134), however, claims that i t is only one third as effective as broth. Serum was found, by comparative bacterial studies to be very effective also (91, 96, 131). Special menstrua have been prepared more recently and found to be very beneficial for certain bacteria. Naylor and Smith (125) developed a nutrient gelatine which they considered gave maximum 38 survival. They attributed the effectiveness mainly to the gelatine itself, although others point out that,, alone, gelatine does not allow good survial, either upon immediate germination after drying ( 1 6 0 ) or after a storage period ( 6 5 ) . Fry and Greaves'"mist desiccans" ( 6 4 , 6 5 ) is another example of a recently devised protective colloid mixture. Similarly, in virus studies, the protective action of col-loids has been demonstrated with respect to freeze-drying ( 3 9 ) and to other adverse conditions such as air drying ( 3 4 ) and shaking ( 2 ) . In comparative studies of both liquid storage ( 1 6 ) and storage after freeze-drying ( 3 2 ) gelatine, peptone, and horse serum were preferred to other media including glucose, saline, distilled water and albumins. Gum acasia was found to be a good diluent (38, ICO.) although its effect was not always apparent until after storage ( 1 4 9 ) . Mucin ( 3 8 ) , egg yolk, and lecithins ( 1 4 7 ) have also been recommended as stabilizers of certain viruses. Various explanations have been offered-1 concerning the protective action of colloids for micro-organisms. It has been correlated with nutrient properties ( 1 0 5 ) . Some claim that, since 3% gelatine is known t° reduce the rate of crystal formation to 1 / 3 5 0 of water (136), the protective action is due to less crystal formation in i n i t i a l freezing. (Crystals are generally considered to be des-tructive to living cells ( 1 7 , 1 1 2 ) . Several theories account for this (82, 1 2 0 ) . ) The protective action of sugars during freezing has been known for some time. It was f i r s t thought to be a result of 39 depression of the freezing point (-2.775°C/G.M. (67)) below that of other media, such as mammalian serum, which freezes at -.5 to -.7°C (120). Kaiser (98), however, considers this to be of secondary-importance. Sugar solutions are thought to prevent complete desic-cation, which effect some workers believe to be beneficial. The substitution of another moisture retaining substance by these workers, however, depressed the survival rate (64, 65). High moisture content i n freeze-dried sugar ampoules was seriously questioned by Flosdorf (85, p«51), who added that "the danger (to survival) l i e s i n removing too l i t t l e water" (also 178). He considered that less than 1% was necessary and .5% moisture content was safe for successful storage of freeze-dried material. Ascorbic acid added to the carrier increases the survival of some bacteria considerably (47, 125, 134). Tests of various concentrations of ascorbic acid i n gelatine showed that .25-.75$ gave the best survival (160). Its protective action was attributed to i t s reducing properties. Cystein (125), thiourea (47),' and l i p i d antioxidants such as l e c i t h i n (148) have been successfully substituted for use with bacteria and viruses. Fry (85, p.164), hbwever, found that ascorbic acid did not improve the survival of his bacterium and, with the addition of Sodium hydrosulphite, another reducing agent, the survival was appreciably lessened. Early experiments on the fungi were carried out on the assumption that the carriers used for bacteria would be the most satisfactory for the molds. Thus, although sucrose has long been known to protect A. niger spores at freezing temperatures (10), most 40 preliminary fungous preservation was done i n horse serum (176) or beef serum (139)• It was found, however, that yeast germination was only .02% using horse serum and many variations appeared (7) . Sharp and Smith (153) obtained "no measureable survival" when conventional d i -luents of blood serum, gelatine, or sucrose were used and concluded that no carrier was preferable to any of these when storing uredo-spores. A mixture of algin and sucrose was found to give a good germination of 93$ when used as the suspending medium for freeze-drying. Aj;_f/L3vus (125). Smut spores have also given best lyophile survival when a mixture of sucrose and a protein i s used as a carrier (87). Mazur (121) states that higher survival was obtained after freezing and thawing when the spores were suspended i n a Calcium chlo-ride solution than when only water was used. Glycerol gave even lower survival than water. These results are completely different from any obtained with bacteria or tissue, i n which cases glycerol was protective (129) and salt solutions were deleterious to freezing ce l l s (162). Procedure Conidia of A. niger were suspended i n various media and lyophilized by the centrifuge method. In addition to the diluents appearing i n the tables, milk suspensions were also prepared. How-ever, spore counts had to be abandoned since the conidia could not. be located among the particles of dried milk on the plate. The f i r s t three runs included a comparison of the six diluents l i s t e d in. Table 5« These were st e r i l e beef serum (Difco), Table 5« The effect of six diluents on the % germination of lyophilized conidia of A. niger revived soon after drying. Diluent % germination Lyop l i l i z e d conidia Controls run 1 run 2 run 3 run 4 average Kelgin (l#) 15 52 41 - 36 91 Water 27 56 34 32 37 91 Case m i hydrolizate 15 63 48 52 44 94 Beef serum 41 39 49 - 43 87 Peptone (l(#) 25 76 50 34 46 96 Sucrose 70 85 85 84 81 92 42 20% sucrose i n water, ~L0% bacto peptone i n water, vitamin free casein * hydrolyzate,distilled water, and 1$ Kelgin + i n water. Two tubes of each suspension were germinated i n these t r i a l s . The fourth run consisted of a comparison of four media and only one tube was opened to obtain each figure. The l a s t three runs, reported i n Table 6, include a com-parison of the effect of suspending the spores i n 20% sucrose, 20% glucose, 20% sucrose and 10% peptone,and 20% sucrose and »5% ascorbic acid a l l i n d i s t i l l e d water. Only one tube of each kind was counted i n each of these assays. Results and discussion The data derived from these experiments appear i n Tables 5 and 6. Among the six diluents f i r s t tested, there were no consis-tent significant differences except' i n the case of the 20% sucrose solution. In every case, this diluent gave considerably greater protection than any other. Colloids were not found to improve the survival rate over water. The addition of ascorbic acid i n the second set of assays did not provide the protective effect observed i n many of the inves-tigators mentioned i n the introduction to this part of Study III. According to these results, ascorbic acid significantly depressed the rate of conidial survival. In a concentration of 20%, sucrose was found to be significantly more protective than glucose. The + An algin produced by the Kelco Co., Los Angeles, California. Table 6. The effect of the diluents glucose, sucrose, sucrose & ascorbic acid, and sucrose & peptone the % germination of lyophilized conidia of A. niger revived soon after drying. % germination Diluent Lyophilized conidia Controls run 1 run 2 run 3 average 1 20% sucrose & .5% ascorbic acid - 72 70 71 99 20% glucose 88 75 - 81 90 20% sucrose & 1.0% peptone 99 89 57 81 95 20% sucrose 97 86 84 89 97 addition of peptone to a surcrose solution gave a slight but s i g n i f i -cant advantage i n two out of three t r i a l s . The thi r d run, however, showed a very considerable drop i n survival. Obviously, this com-parison of the value of sugar and sugar & protein must be repeated. B. The germination of lyophUized conidia dried i n various menstrua after storage for one year at room temperature Introduction Carrier efficiencies during lyophilization are frequently quite different from those during storage. For instance, a mixture of tryptic digest broth serum and glucose gave excellent i n i t i a l bacterial survival which was not evident after a period of storage ( 6 4 , 6 5 ) . There are reports of bacterial survival of storage being greatly improved by the addition of reducing agents, i n particular ascorbic acid ( 1 2 5 , 1 6 0 ) . These reports prompted a test for viabi-l i t y of A. niger conidia which had been lyophilized i n a diluent containing ascorbic acid, then stored for 1 2 months. Water lyophilized conidia were tested at the same time. Procedure Conidia of A. niger were suspended i n three diluents, 20% sucrose, 20$ sucrose & 0.5% ascorbic acid, and water, then lyophilized simultaneously. The tubes were sealed off " i n vacuo", i n groups of three. Each group contained one suspension of each of the three carriers. One group was plated immediately, while the other two were stored at room temperature with no attempt to shield the tube 45 contents from light, claimed by some to possess a deleterious action ( 1 2 3 ) but to have a negligible effect by others ( 6 5 ) . Before the tubes were opened, the vacuum was tested i n each with a high tension c o i l spark tester, and i t was found to be f a i r l y uniform. The pairs of tubes were then opened, and the spores plated. Results and discussion The data obtained from the spore counts are recorded i n Table 7-The survival of spores i n sucrose was f a i r l y well maintained, the drop i n v i a b i l i t y being about 10% over the one year period. With the ascorbic acid added, however, spore v i a b i l i t y after storage was almost negligible. The spores dried i n water also gave very poor storage survival, the drop i n v i a b i l i t y of 98% being only s l i g h t l y less than that of sucrose & ascorbic acid. It w i l l be noted that a comparison of these results with those of Study VII B indicates that less drop i n survival would be expected when the carrier i s beef serum. However, since the con-ditions of lyophilization were not comparable, no conclusions on this point can be made from these results. Fry and Greaves (64, 6 5 ) state that the drier the medium i s beyond an unknown point, the greater i s the drop i n survival on storage. I f this i s a correct observation, the ineffective storage i n water may be explained by over-drying, since ice sublimes more rapidly than other more complex liquids. (For instance, i t sublimes to dryness i n 1/4 to 1/5 of the time that does plasma which contains 18% peptone (14 ) 0 Table 7« The loss of v i a b i l i t y of conidia of A. niger lyophilized i n water, a sucrose solution, and sucrose solution with added ascorbic acid after storage for 12 months. % germination of conidia Diluent Before drying (av. 1000 spores) Soon after drying (av. 1000 spores) 12 months after drying (av. 2000 spores) Water 95 32 0.8 20% sucrose & 0.5% ascorbic acid 98 70 0.05 20% sucrose 98 84 20.1 47 Comments on Study III It i s recommended that a systematic study be i n i t i a t e d on the suspending medium as i t affects the survival of fungous spores during and after lyophilization. Special attention should be directed to osmotic pressures and nutrient properties. No recom-mendations have been made with respect to the pH optimum for fungous lyophilization. It i s known that a low pH increases bacterial cold sensitivity (162) and that the loss of Carbon dioxide during drying increases the pH (52). The effect of this, however, has only been postulated with respect to bacteria (134, 160). Nothing is mentioned i n the literature regarding the possible effects of pH changes on fungous survival during freeze-drying. It i s imperative for both practical and theoretical purposes that future diluent studies include investigations of the reduction of v i a b i l i t y during storage. I f time is limited, short term storage at temperatures above room temperature i s recommended (116, 135). Heller (91), i n addition to correlating bacterial survival with nutrient, postulated that the drop i n v i a b i l i t y of dried bacteria during storage i s dependent upon the gold number (I64) of the carrier. This i s the a b i l i t y of one c o l l i d to protect another from the precipi-tating action of an electrolyte. Whether this i s applicable to fungi or not i s unknown. Study IV Degassing the conidial suspension before freezing Introduction In preliminary degassing, frothing, due to the liberation of 48 dissolved gases, i s induced by water pump suction. When these gases are removed, a quiescent l i q u i d results which w i l l not bubble on evacuation to low pressures (86). Degassing has been used to i eliminate the need for pre-freezing bacterial suspensions i n se l f -freeze-drying procedures which do not include centrifuging (59, 78, 79). This preliminary treatment, although convenient, i s not used as extensively as i t might be, since i t limits the amount of li q u i d that can be dried to one half of the material possible when a pre-freeze i s employed (60). I f the samples are too small, moreover, part may dry during the degassing. Under any conditions, when the vacuum i s imposed, there i s the danger that freezing w i l l occur err a t i c a l l y and, unless the degassing has been thoroughly carried out, small internal explosions may cause the loss of most of the material being dried (75). Weston (175) reports that the survival of A. flavus i s increased during freeze-drying i f preliminary water suction i s applied to the suspension before freezing. When testing for a similar effect o n A. niger i t was thought that the protection might be a result of the elimination of tiny gas bubbles at the interface of the diluent surrounding each spore. The effect of increasing the contact of spore and menstruum by degassing should be duplicated by the action of a detergent. Procedure Conidia of A. niger were suspended i n two test tubes of beef serum. To one was added a few grains of a detergent. The contents of each tube were then dispensed to a pair of lyophile tubes. 49 One tube of each pair was placed i n a desiccator connected to a water suction pump, while the other was placed on the bench beside the desiccator. The four tubes were then lyophilized according to the methods indicated i n Table 8 . Results and discussion The data appear i n Table 8 (reproduced from 8 8 ) . Regardless of the method used or the detergent added, a significant increase i n conidial survival was obtained when the sus-pensions were given a preliminary degassing. When the set of tubes was lyophilized according to the NRRL method, the use of a detergent increased the percentage survival in addition to the degassing. Because the experiment was not repeated, and since no other runs were made by either of the other two lyophi-l i z a t i o n procedures showed any significant increase i n survival when a detergent was added, this set of results awaits verification and may be merely experimental error. Since the detergent did not consistently increase the sur-vi v a l of the untreated spores, the function of degassing does not appear to establish an intimate contact of carrier with spore. The phenomenon may be related to the beneficial effect of washing uredospores before germinating i n order to remove any inhibit-ing substances ( 8 7 ) • Comments on Study IV The effect of: degassing before lyophilization on Aspergillus spores deserves investigation. Is i t due to a change i n acidity or Table 8. The effect of wetting agent and pre-lyophilization treatment on the % germination of A. niger conidia lyophilized i n beef serum by various techniques. Treatment % germination NRRL PRL Control Centrifuge Control Average Range without wetting agent without pre-evacuation 11.8 23.1 91.7 51.3 35-62 with pre-evacuation 22.9 45.2 56.9 49-68 86-89 with wetting agent without pre-evacuation 21.8 20.4 • 97.2 45.1 30-51 87-88 with pre-evacuation 34.5 42.1 59-5 46-65 wetting agent "Vel" " Qreft" "Dreft" number of runs 1 1 6 51 to the removal of denaturing volatiles? I f there i s dehydration involved, the process of v i t r i f i c a t i o n may occur, rather than c r y s t a l i -zation. On the other hand, i f metabolism i s slowed under reduced pressure, the i n i t i t a l survival drop among suspended spores, apparent i n Study V, might be delayed. It was, i n fact, noted on one occasion that the beneficial effects of pre-evacuation were markedly reduced when the suspension was kept at room temperature and pressure for 45 minutes instead of the usual 3-5 minute interval between turning off the suction and freezing. Study V Maintaining conidia i n li q u i d suspension /•. A. Germination of lyophilized conidia maintained i n suspension for various lengths of time at room temperature before freezing Introduction When lyophilizing cultures, i t i s frequently convenient to prepare the spore suspension some time before the freeze-drying i s to be carried out. It was necessary to know i f this delay would alter the preservation of A. niger conidia. Procedure i n beef i t r u m Conidia of A. niger were suspended, at various intervals up to 2 hours before freezing. Within each series, the volume of the diluent was constant and every effort was made to keep the techniques of flaming, spore harvesting and mixing as constant as possible. The contents of a l l tubes i n a given series were frozen simultaneously. t 52 The ages of the cultures were 3 days, 7 days, and 17 days. Results and discussion The data of this part of the study are recorded i n the graph, Figure 5« In every series, i t was noted that there was a marked reduc-tion i n v i a b i l i t y as the length of suspension time increased. The increment was greatest i n the f i r s t half hour and was only slight during the second hour at this temperature. No morphological changes are apparent during the f i r s t hour when the greatest survival drop occurs. Measurements of suspended spores from both young and old cultures show that swelling does not begin at room temperatures u n t i l at least the third or fourth hour. B. Germination of lyophilized conidia maintained i n suspension for one to two hours i n an ice bath before freezing Introduction It was considered of practical value to determine whether storage of the spore suspension at a lower temperature would lessen the detrimental effect of lyophilization observed i n part A. of this study. Procedure Conidia of A. niger were harvested on four occasions from variously aged cultures and suspended i n beef serum. Each suspension was dispensed to four lyophilization tubes, two of which were placed in an ice water bath, for an hour or more, while the other two were kept at room temperature (20-22°C.) for the same length of time. The i i I 1 i i i — n — i — i — i — r m 30 DAYS . (Culture O 1 I I I I I I I I I I I 10 20 30 40 50 60 120 SUSPENSION TIME (MINUTES) fig. 5. EFFECT of SUSPENSION TIME on LYOPHILIZED CONIDIA 54 four tubes i n each set were simultaneously frozen and then dried. The conidia were germinated at the conclusion of drying. The interval from harvesting to suspension i n the ice bath was less than five minutes i n every case. Results and discussion The data appear i n Table 9. I f the drop i n resistance of conidia i s associated with some preliminary germinative process, the slowing of the rate of germination by lowering the temperature would easily explain the results obtained. Cn the other hand, the dropping of the temperature to 0°C. before freezing may somewhat reduce "shock" similar to that observed among bacteria (154), resulting i n increased survival during freezing. Comments on Study V Part A. of this study should be carried out over a period of six hours or more to determine the effect of the different stages of germination on the loss of resistance to lyophilization. It i s further recommended that more extensive experiments be conducted regarding time intervals. It i s desirable that a determination of the resistance curve be made. Does i t merely drop more slowly as germination proceeds, or are other factors involved? Any investigation of spore condition should be related to this study and to Study I, and include other factors such as studies on metabolism, osmotic pressure, pH, and hydration. Table 9. The effect on the % germination of the conidia of A. niger which have been lyophilized after being kept i n suspension at room temperature and at 0°C for 1-2 hours. Age of source culture (days) Period of suspension (hours) % germination of conidia Held at 20-22°C. Held at 0°C dried control # dried control # 4 2 44.7 98.6 49.2 99.4 4 2 44.9 99-4 63.2 97.8 5 2 47.0 83.5 52.6 -2i m 1.3 51.6 85-3 58.8 94.0 # Only 500 spores were counted to obtain each of the figures for the controls. ## In addition to the two treatments recorded, one tube, lyophilized at the same time, contained spores that had been suspended for only 5 minutes before being frozen. When revived, the spores germinated at the rate of 63.7$ (control, 90.2$). This i s significantly greater (@ p - .05) than that obtained from the spores stored 1.3 hours at 0°C 56 Study VI Maintaining conidia i n frozen suspension below -50°C for various lengths of time before evacuation A. Germination of lyophilized conidia dried after storage i n -50°C.chamber for lengths of time from 5 minutes to 10 hours Introduction Because storage of spores at room temperature, at 6°C, and at 0°C. was found to alter survival of the lyophilized spores, i t was necessary to find another point i n the procedure at which spores might be kept, yet s t i l l allow simultaneous drying of materials which have had different preliminary treatments. The effect of long and short periods of pre-freezing was then investigated. It has been reported thai^ although killed.at higher tem-peratures (80), bacteria are maintained without: deterioration at -35°C or lower (181) and that length of time at -79°C. does not affect the potency of tumor tissue (130). Trypanosomes, on the other hand, are able to withstand l i q u i d a i r temperatures for only a short time (69)• Procedure Conidia of A. niger were suspended i n beef serum, shaken, dispensed to lyophile tubes, then frozen,•three minutes after the time of harvesting, i n the side drying chamber of the table apparatus. This was carried out at intervals before evacuation which are recorded i n Table 10. The source culture used was six weeks old and duplicate tubes were prepared and dried. A l l the tubes were evacuated simul-taneously. Table 10. The effect of freezing at -56°C for various lengths of time before evacuation, on the % ger-mination of lyophilized conidia of A. niger. Time of freezing at -56°C. 10 hrs. 5 hrs. 3 hrs. 1. hr. 30 min. 15 min 10 min. 5 min. 2 min. % germination of lyophilized spores (av. of 1000 spores from each of two tubes) 19-2 17.1 21.1 18.2 20.8 24-9 17.5 24.3 32.2 58 Results and discussion The results are recorded i n Table 10. The data show no significant differences (not attributable to unavoidable variations i n suspension techniques) among the survival of the spores frozen from 5 minutes to 10 hours before drying. The warming of the drying chamber as much as 16°C. with the addition of each new pair of tubes, did not appear to affect the v i a b i l i t y of the conidia. This temperature range has no effect on A. flavus (121) or bacteria (181). The only notable difference i n germination rate occurred i n the tubes which were the last to be frozen. On the addition of this pair of tubes, the side drying chamber warmed to -39°C. i n one minute. At this point the drying chamber containing a l l the tubes was connected to the lyophile apparatus and evacuation was effected one minute later. It i s doubtful that the contents of the two tubes reached -39°C before they were evacuated, since the temperature was s t i l l r i s i n g when la s t observed. B. The germination of lyophilized conidia dried after short-time exposure to freezing temperatures by direct immer-sion at -80°C. and by cold a i r cooling at - 60°C Introduction This part of the study was conducted to confirm the s i g n i f i -cant difference between the germination of spores from tubes frozen for two minutes and those frozen for five minutes or longer, as recorded i n part A. of this study. It was desirable to know whether 59 the time of contact with the low temperature was important, or whether some other factor, such as extent of freezing, or temperature reached during freezing, was responsible for the survival differences. Procedure Conidia from a six day old culture of A. niger were suspended and dispensed to six tubes. Three of these were directly immersed in the condensor freezing mixture for rapid freezing to -80°C while the others were frozen more slowly, as i n part A. The inside of the drying chamber was at -60°C. These six tubes were frozen 10 minutes before evacuation. Eight minutes after the f i r s t suspension was made, a second was similarly made and dispensed to six more lyophile tubes. These tubes were treated as the f i r s t but exposed to cold for only 2 minutes before they were evacuated with the others. The temperature of the drying chamber, immersed i n the bath, rose to -56°C. 1 minute after the second group of three tubes were placed i n i t . It was s t i l l r i s i n g at this time when the chamber was connected to the apparatus for evacuation. Results and discussion The data appear i n Table 11. These observations support those of the previous part of the study. Brief two minute exposure to cold a i r temperatures of -50 to -60°C. allows higher survival of A. niger conidia after subsequent drying than longer preliminary freezing in the same chamber. This effect i s not due directly to the length of time of the exposure since two minutes at -80^. did not give a significant Table 11. The effect on % germination of A. niger conidia lyophilized with a rapid pre-freeze by direct immersion in a -80°C. bath and a slower pre-freeze by a i r cooling to -60°C for two minutes and for ten minutes. Length of time of freezing (minutes) Controls % germination (average of 600 spores) Method of freezing Dried spores % germination (average of 1000 spores i n each of three tubes) 10 76.0 direct immersion a i r cooling 23-5 28.2 2 72.7 direct immersion air cooling 20.3 42.7 61 improvement over 10, at the same temperature, and since 2 minutes i n the freezing mixture gave a lower rate of survival than 2 minutes i n the drying chamber. The difference between the results from the shorter freezing time i s not merely the result of a lower freezing temperature, since the survival after 10 minutes at -80°C. was not significantly less than that at -60°C. Results of other single experiments have i n d i -cated that a difference i n the temperature of freezing does not affect the lyophilization survival of A. niger conidia. One pair of tubes was frozen at -30°C and at -40°C. i n cold chambers then dried by the PRL method. The revived conidia germinated at a rate of 12% and 13% respectively. Thus no significant difference was observed when a difference of 10°C. was applied at this range (although Mazur reported that the greatest destruction to A. flavus spores occurs at above -35°C). Cn another occasion, two tubes were frozen by direct immersion, one at -27°C and one at -57°C, for one minute, followed by evacuation and drying by the NRRL method. Again, no significant difference was observed when this 30°C. difference i n freezing temperatures was applied. The contents of the tube held at -27°C gave a germination rate of 38%, while the others produced 37% germination. However, the less extreme temperature of the tube contents i n this study may have been responsible for giving the highest survival. This is borne out by a report that a -15°C. pre-freeze allows better survival of lyophilized Ustilago cells than lower temperatures (87). Indirectly, the temperature of freezing affects the speed 62 of drying. The closer the temperature of the frozen material i s to that of the condensor, the slower i s the drying ( 5 3 )• Ice, for instance, at -40°C. may evaporate ten times more rapidly than ice at - 6 0 ° C (18). Thus i t would be expected that the tubes which were frozen more slowly for only 2 minutes and which, apparently, never did reach as low a temperature as the other tubes, would be i n i t i a l l y desiccated more rapidly than any of the other tubes. Rapid drying has been reported more beneficial than slow during the freeze-drying of bacteria (128) and sera ( 5 3 ) . However, although no experiments were conducted i n this series of studies dealing directly with the speed of drying as i t affects lyophilization survival, the experiments cited i n the preceding paragraph indicate that any effect of speed of drying i s offset by other factors and i s not apparent under these conditions. Another explanation involves sub-cooling. Mazur (121) reported that A. flavus spores are more resistant to low temperatures when the menstruum is sub-cooled than when i t i s frozen at the same temperature. It i s possible that the beef serum containing A. niger conidia, when slowly cooled to a comparatively high temperature, did not actually freeze before the tubes were evacuated. However, no bubbling was observed when the vacuum was applied. The speed of freezing as the cause of these results of this experiment i s not the whole explanation. Although slow freezing i s said to protect cells against low temperatures ( 2 5 , 104, 129, 162), the spore counts of the suspensions frozen for 10 minutes, although frozen at different speeds, were not significantly different. Probably more than one factor i s involved i n producing 63 higher survival after a brief freeze by a i r cooling. Comments on Study VI It i s recommended that detailed studies be made on the effects of the temperatures of freezing, speed of freezing, and speed of drying, on conidial survival. Study VII Various lyophilization methods A. Germination of conidia lyophilized by three methods, germinated imme-diately after sealing (88) Introduction Most culture preservation u n t i l recently has involved com-paratively small apparatus, such as the table model used i n many of these studies. Large-scale culture maintenance has however become increasingly important. The Edwards centrifuge freeze-drier has been extensively used for this purpose (ex. 32, 37). It was of value to determine what effect, i f any, the various methods i n common use have on the survival of A. niger conidia. Procedure Three common methods of lyophilization, the PRL, the NRRL, and the centrifuge (see "Methods" i n the "General procedures") were selected for study. Because only two sets of tubes could be sealed off at a time, each run included a comparison of two methods. In each run, the spores were suspended simultaneously and frozen-or spun, when the 64 centrifuge machine was used—within a minute of each other. Results and discussion The results of this study appear i n Table 12. The NRRL and the PRL methods did not d i f f e r significantly i n effective spore preservation. The centrifuge method, however, was consistently superior to both of the other two methods i n i t s a b i l i t y to maintain conidial survival throughout the process of freeze-drying. The breaking of the vacuum during freeze-drying by the PRL method does not appear to have an effect on conidial v i a b i l i t y . I f there is any, i t i s offset by the effects of the other variable factors involved i n the PRL and NRRL methods. . Studies of the effect of degree of vacuum, and of the speed of evacuation were not possible on the apparatus available, so were omitted i n this paper. Reports are available, however, that discuss the effects of varying external heating, pressure, and condensor tem-perature on the drying time (53, 72, 79). Observations of bacterial survival related to drying speed have been conflicting. Some say that slow drying i s better than rapid (97), while others claim the reverse (128). Probably the slowest drying method i s the NRRL procedure, which gave lower germi-nation than did the centrifuge method, since the lat t e r apparatus has a large condensor with a wide passage from the drying chamber and maintains the drying material at a rela t i v e l y high temperature. No consistent difference was observed between the survivals after the PRL and the NRRL methods, although the NRRL drying is so retarded by Table 12. Comparisons of the survival of the conidia of A. niger lyophiliz techniques. ed i n beef serum by various Technique No. of runs % germination Average Range (a) NRRL PRL Controls 7 7 7 30.7 34-1 93-4 10-77 15-78 83-98 (b) NRRL Centrifuge Controls 5 5 5 32.1 54-3 95-4 16-58 44-61 92-98 (c) PRL Centrifuge Controls 7 7 5 53.8 67.8 84-4 26-75 47-87 72-98 66 the small diameter of the manifold (a rapid vapour flow from the mate-r i a l to the condensor i s dependent upon a large diameter more than on a short distance (13, 15, P'53) that cold must be supplied exter-nally to keep the suspension frozen). The speed of freezing was not measured quantitatively during any of these procedures. Freezing by the centrifuge method requires several minutes since snap freezing occurs at approximately 1 mm. Hg. The NRRL method of freezing i s the most rapid of the three. However, i t i s improbable that v i t r i f i c a t i o n , considered desirable for freeze-drying cells (21, 22, 70, 71, 110, 111, 113, 157, 136), has occurred at temperatures of -50°C. to - 60°C. When animal cells are being preserved and v i t r i f i c a t i o n does not occur, slow freezing i s prefer-able to rapid (25, 104, 129, 162), although colloids are preserved with a more normal structure i f rapidly frozen (82, 161). There was no difference between the survival of lyophilized conidia quick frozen by the NRRL procedure and those frozen more slowly by the FRL method. Further observations of the lack of effect of speed of freezing i s recorded i n Study VT B. A possible explanation for the high percentage survival of conidia dried by the centrifuge method may l i e i n the fact that, before freezing can occur during this procedure, 20$ of the l i q u i d must be evaporated (9). This would induce sub-cooling, which has proved favourable to A. flavus survival (121). In addition, t h i s pa r t i a l dehydration allows A.niger spores to withstand colder tempe-ratures than otherwise possible (112). Recently, Haskins reports that the beneficial effect of the 67 centrifuge method on Ustilago cells i s due to the temperature of freezing. A pre-freeze of -15°C. produces as high v i a b i l i t y after lyophilization by the NRRL method as that obtained after treatment by the centrifuge method (87) • B. Germination of conidia lyophilized by three methods germinated after 13 months storage"in vacuo" at room temperature Introduction The three lyophilization methods involve such different conditions that i t was questioned whether sufficient drying had oc-curred during a l l three procedures, or whether those tubes showing highest survival on immediate germination had been incompletely desic-cated. Collier (32) reports that lyophilization by the No. 3 Edwards apparatus, used i n this study, gives faster deterioration than other freeze-drying methods. Procedure Two sets of suspensions were prepared on two different occasions. One set was dried to compare the centrifuge method with the PRL, and the other was a comparison of the centrifuge and the NRRL methods. A l l tubes were lyophilized i n t r i p l i c a t e . Two tubes of each treatment were opened and revived immediately after being sealed off. The contents of the third were revived after storage for 13 months i n the_ l i g h t and at room temperature. Satisfactory vacua were found i n a l l the stored tubes when they were tested by a high voltage c o i l tester one year after being 68 sealed o f f . Results and discussion The data pertaining to this part of the study i s recorded i n Table 13. These results indicate that, although the PRL and the NRRL lyophilization methods do not allow as good i n i t i a l survival of the conidia as the centrifuge method does, the drop i n v i a b i l i t y during storage i s less rapid when either of the former procedures are f o l -lowed. Collier attributes the drop i n v i a b i l i t y after centrifuge freeze-drying to three things: (1) The faster snap freeze on the Edwards centrifuge. (How-ever, of the three methods compared i n this study, the centrifuge method i s slower than at least one of the other methods.) (2) No secondary drying. (None of these methods included the type of secondary drying referred to by Collier. The secondary drying, such as i t was, was carried out on the table apparatus regard-less of the method of freezing or primary drying.) (3) The lower vacuum of the Edwards machine (.015 mm. Hg. as compared with .001-.003mm Hg. obtained using the other method). (In the series of experiments i n Study VII, the centrifuged tubes were dried at a higher pressure than those dried by the other methods. However, the pressure difference for most of the primary drying period was relatively slight. The chamber of the Edwards machine gradually reached pressures of 80-120 microns of Hg., while the smaller system was rapidly evacuated to pressures i n the range of 50-100 microns Hg. Table 13 • The effect of three lyophilization methods upon the v i a b i l i t y of conidia of A. niger dried i n beef serum following storage i n vacuo at room temperature i n the l i g h t for 13 months. Method % germination of conidia % drop i n v i a b i l i t y of dried spores after storage for 13 months before drying immediately # after drying 13 months U after drying Centrifuge 72 79.6 41.2 48.2 PRL 57-1 38.2 33-1 Centrifuge 56.5 37-4 33-9 NRRL 98 45.4 32.1 27.1 # Average of 1000 spores from each of two tubes. ## Average of 1000 spores from one tube. ON NO 70 The pressure, excluding water vapour pressure, during storage should be, within each run, constant since the f i n a l evacuation for sealing off was done i n the same system.) Comments on Study VII Before further work i s done on comparisons of methods "in toto", each factor involved must be dealt with separately. Studies should include the speed and temperature of freezing, the thoroughness and length of drying, rapidity, degree and period of evacuation, drying temperatures and temperature differentials. Investigations of storage v i a b i l i t y loss should accompany each observation of immediate germi-nation. SUMMARY Seven studies are included i n this thesis with suggestions for further research. Each group of experiments within a study i n -cludes an introduction and a discussion of results. According to the materials and methods employed, and accor-ding to the experimental conditions under which this investigation has been carried out, the following facts were noted: (1) The age of the culture from which conidia of A. niger are harvested has a significant effect upon the percentage germination of these spores when they are lyophilized. When incubated at 2 4 - 2 6°C, maximum survival occurs i n 2 to 6 day old cultures. According to certain stated conditions, this peak of maximum survival approximately coincides with the conclusion of the greatest metabolic ac t i v i t y as determined by pH. The drop i n percentage germination i s retarded by incubation of the culture at 6°C. following a period of growth at higher temperatures. The processes leading to maximum survival are not arrested when spores from very young cultures are harvested and suspended at this temperature over night. ( 2 ) Within the narrow range of dilutions tested, c e l l con-centration has no effect on lyophilization survival of A. niger conidia. (3) Solutions containing 20$ sucrose give better protection to A. niger conidia during lyophilization than any other carrier tested. 72 The addition of 0»5% ascorbic acid to 20% sucrose s l i g h t l y depressed i n i t i a l survival and greatly depressed storage survival of the conidia. (4) Degassing the spore suspension by water suction just before freezing improves the conidial survival after lyophilization. This effect does not appear to be a result of a more intimate contact between c e l l and diluent. (5) Conidia of A. niger rapidly lose their resistance to lyophilization i n l i q u i d suspension at room temperature. The loss i s considerably diminished at 0°C-(6) The length of freezing time, within the range of 5 minutes to 10 hours, at -50°C. does not affect the survival of A. niger conidia after subsequent lyophilization. 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