Production Strains PDF

# PRODUCTION STRAINS ## 1. INTRODUCTION The most important factor for the success of any fermentation industry is a production strain. It is highly desirable to use a production strain possessing the following four characteristics: * It should be a high-yielding strain. * It should have stable bi...

# PRODUCTION STRAINS ## 1. INTRODUCTION The most important factor for the success of any fermentation industry is a production strain. It is highly desirable to use a production strain possessing the following four characteristics: * It should be a high-yielding strain. * It should have stable biochemical characteristics. * It should not produce undesirable substances. * It should be easily cultivated on a large-scale. ## 2. SCREENING TECHNIQUES Both detection and isolation of high-yielding species from the natural source material, such as soil, containing a heterogeneous microbial population is called screening. There are many screening techniques. Usually screening programmes include primary screening and secondary screening. ### 2.1. Primary Screening This consists of some elementary tests required to detect and to isolate new microbial species exhibiting the desired property. With antibiotic producers, primary screening programmes serve to remove worthless micro-organisms on the basis of relatively simple, fundamental criteria. The important selection criteria are the activity of antibiotics in vitro, and possibly in vivo, against a small number of the most important test organisms. Primary screening is also needed in the case of other useful microbial species (e.g. micro-organisms capable of producing organic acids, amines, vitamins, etc). The evaluation of the primary screening in industrial research programmes may be made by citing some specific examples of screening procedures as under: #### The crowded plate technique The crowded plate technique is the simplest screening technique employed in detecting and isolating antibiotic producers. It consists of preparing a series of dilutions of the soil or other source material for the antibiotic producing micro-organisms, followed by spreading of dilutions on the nutrient agar plates. The agar plates having 300 to 400 or more colonies per plate are considered since they are helpful in locating the colonies producing antibiotic activity. The ability of a colony to exhibit antibiotic activity is indicated by the presence of a zone of growth inhibition surrounding the colony. Such a colony is subcultured to a similar medium, and purified by streaking, before making stock cultures. It is necessary to carry on further testing to confirm the antibiotic activity associated with a micro-organism, since the zone of inhibition surrounding the colony may, sometimes, be due to other causes. The crowded plate technique has a limited application, since it merely provides information regarding the inhibitory activity of a colony against the unwanted microbes that may be present by chance on the plate. Therefore, the technique has been improved upon by introducing the use of a 'test-organism'. In this modified technique, agar plates which give well-isolated colonies (roughly 100 to 300 colonies per plate) after incubation are flooded with a suspension of the test organism. Then the plates are subjected to further incubation to allow the growth of the test organism. The formation of inhibitory zones around certain colonies indicates their antibiotic activity. The diameter of the zones of inhibition are measured in millimeters, to obtain a rough approximation of the relative amounts of antibiotic(s) produced by various colonies. The colonies of the antibiotic producers must be isolated and purified before further testing. #### Auxanography This technique is largely employed for detecting micro-organisms able to produce growth factors (e.g. amino acids and vitamins) extracellularly. The two major steps of the technique are as under: **(A) Preparation of First Plate.** 1. A filter paper strip (1.5x12 cm) is put across the bottom of a petri dish in such a way that the two ends pass over the edge of the dish. 2. A filter paper disc of petri dish size is placed over paper strip on the bottom of the dish. 3. The nutrient agar (45°C) is poured on the paper disc in the dish and allowed to solidify. 4. Microbial source material such as soil, is subjected to dilution such that aliquots on plating will produce well isolated colonies. 5. Plating of aliquots of properly diluted soil sample is done. **(B) Preparation of Second Plate.** 1. A minimal medium lacking the growth factor under consideration is seeded with the test organism. 2. The seeded medium, is poured on the surface of a fresh petri dish. 3. The plate is allowed to set. The agar in the first plate, as prepared in step (A), is carefully and aseptically lifted out with the help of tweezers and a spatula and, placed, without inverting, on the surface of the second plate as prepared in major steps (B). The growth factor(s) produced by colonies present on the surface of the first layer of agar can diffuse into the lower layer of agar containing the test organism. The zones of stimulated growth, of the test organism around the colonies is an indication that they produce growth factor(s) extracellularly. Productive colonies are sub-cultured and are further tested. #### Enrichment Culture technique This technique was designed by a soil microbiologist, Beijerinck, to isolate the desired micro-organisms from a heterogeneous microbial population present in soil. Either medium or incubation conditions are adjusted so as to favour the growth of the desired micro-organisms. On the other hand, unwanted micro-organisms are eliminated, or develop poorly since they do not find suitable growth conditions in the newly created environment. Today, this technique has become a valuable tool in many screening programmes meant for isolating industrially important strains. Generally, it consists of the following steps: 1. Nutrient broth containing an unusual substrate (e.g. Cellulose powder) is inoculated with the microbial source material (e.g. soil) and incubated. 2. A small portion of inoculum from step (1) is plated onto a solid medium having the same composition. Well isolated colonies appear after incubation. 3. Suspected colonies from plate of step (2) are sub-cultured on fresh media and they are also subjected to further testing. An example of screening of enzyme producing micro-organisms may be cited. Micro-organisms excreting alkaline proteases may be detected from the soil as under: * Soil is subjected to serial dilution. * All soil dilutions are heated at 80°C. for 10 minutes. This treatment kills vegetative cells but spores remain unaffected. * The plating of heat treated samples is done by spreading the samples (usually 0-1 ml.) from dilutions onto the surface of nutrient agar containing casein at pH 10-12. * The colonies surrounded by a clear zone are sub-cultured. #### Use of an indicator dye The pH indicating dyes may be employed in some screening methods for detecting micro-organisms capable of producing organic acids or amines, since a pH indicating dye undergoes colour changes according to its pH. Such dyes (e.g. neutral red, bromthymol blue, etc.) are added to the poorly buffered nutrient agar media. The change in the colour of a dye in the vicinity of the colony suggests the capability of colonial cells to produce either organic acid(s) or amine(s), depending upon the nature of reaction. Such colonies are subcultured to make stock cultures. However, further testing is needed, since inorganic acids or bases are also potential metabolic products of microbial growth. In other words, these methods are not foolproof. This differential technique may also be employed in finding out whether micro-organisms are capable of certain microbial transformations or not. E.g. A-7 oestrogen formation by bioconversion. ### 2.2. Secondary Screening Secondary screening is strictly essential in any systematic screening programme intended to isolate industrially useful micro-organisms, since primary screening merely allows the detection and isolation of micro-organisms that possess potentially interesting industrial applications. Moreover, primary screening does not provide much information needed in setting up a new fermentation process. Secondary screening helps in detecting really useful micro-organisms in fermentation processes. This can be realized by a careful understanding of the following points associated with secondary screening: 1. It is very useful in sorting out micro-organisms that have real commercial value from many isolates obtained during primary screening. At the same time, micro-organisms that have poor applicability in a fermentation process are discarded. It is advisable to discard poor cultures as soon as possible, since such studies involve much labour and high expense. 2. It provides information whether the product produced by a micro-organism is a new one or not. This may be accomplished by paper, thin layer, or other chromatographic techniques. The compound under consideration is compared with previously known compounds. 3. It gives an idea about the economic position of the fermentation process involving the use of a newly discovered culture. Thus, one may have a comparative study of this process with processes that are already known, so far as the economic status picture is concerned. 4. It helps in providing information regarding the product yield potentials of different isolates. Thus, this is useful in selecting efficient cultures for the fermentation processes. 5. It determines the optimum conditions for growth or accumulation of a product associated with a particular culture. 6. It provides information pertaining to the effect of different components of a medium. This is valuable in designing the medium that may be attractive so far as economic consideration is concerned. 7. It detects gross genetic instability in microbial cultures. This type of information is very important, since micro-organisms tending to undergo mutation or alteration in some way may lose their capability for maximum accumulation of the fermentation products. 8. It gives information about the number of products produced in a single fermentation. Additional major or minor products are of distinct value, since their recovery and sale as by-products can markedly improve the economic status of the prime fermentation. 9. Information about the solubility of the product in various organic solvents is made available. This knowledge is useful in the recovery and the subsequent purification of the product. 10. Chemical, physical and biological properties of a product are also determined during secondary screening. Moreover, it reveals whether a product produced in the culture broth occurs in more than one chemical form. 11. It reveals whether the culture is homofermentative or heterofermentative. 12. Determination of the structure of the product is done. The product may have a simple, complex, or even a macromolecular structure. 13. With certain types of products (e.g. antibiotics) determinations of the toxicity for animal, plant or man are made if they are to be used for therapeutic purposes. 14. It reveals whether micro-organisms are capable of a chemical change or of even destroying their own fermentation products. For instance, micro-organisms that produce the adaptive enzyme, decarboxylase, can remove carbon dioxide from the amino acid, leaving behind an organic amine. 15. It tells us something about the chemical stability of the fermentation product. Thus, secondary screening gives answers to many questions that arise during the final sorting out f industrially useful micro-organisms. This is accomplished by performing experiments on agar plates flasks or small bioreactors containing liquid media, or by a combination of these approaches. A specific example of antibiotic producing Streptomyces species may be taken for an understandin the sequence of events during a screening programme. Those streptomycetes able to produce antibiotics are detected and isolated in a primary screening programme. These streptomycetes exhibiting antimicrobial activity are subjected to an initial secondary screening where their inhibition spectra are determined. A simple, 'giant-colony', technique is used to do this. Each of the streptomycal isolates is streaked in a narrow band across the centres of the nutritious agar plates. Then, these plates are incubated until growth of a streptomycete occurs. Now, the test organisms are streaked from the edges of the plates upto but not touching the streptomycete growth. Again, the plates are incubated. At the end of incubation, growth inhibitory zones for each test organism are measured in millimeters. Thus, the 'microbial inhibition spectrum' study extensively helps in discarding poor cultures. Ultimately, streptomycete isolates that have exhibited interesting microbial inhibition spectra need further testing. With streptomycetes suspected to produce anti-biotics with poor solubility in water, the initial secondary screening is done in some different way which is out of the scope of this book. Further screening is carried out employing liquid media in flasks, since such studies give more information than that which can be obtained on agar media. At the same time, it is advisable to use accurate assay techniques (e.g. paper disc-agar diffusion assay) to exactly determine the amounts of antibiotic present in samples of culture fluids. Thus, each of the streptomycete isolates is studied by using several different liquid media in Erlenmeyer flasks provided with baffles. These streptomycete cultures are inoculated into sterilized liquid media. Then, such seeded flasks are incubated at a constant temperature. Usually, such cultures are incubated at near room temperature. Moreover, such flasks are aerated by keeping them on a mechanical shaker, since the growth of streptomycetes and production of antibiotics occur better in aerated flasks than in stationary ones. Samples are withdrawn at regular intervals under aseptic conditions and are tested in a quality control laboratory. Important tests to be carried out include: * checking for contamination, * checking of pH, * estimation of critical nutrients, * assaying of the antibiotic, and * other determinations, if necessary. The result of the above tests points out which medium is the best for antibiotic formation, and at which stage the antibiotic yields are greatest during the growth of the culture on the various media. After performing all necessary routine tests in the screening of an actually useful streptomycete for a fermentation process, other additional determinations are made. They are: 1. Screening of fermentation media through the exploitation of which the highest antibiotic yields may be obtained. 2. Determination of whether the antibiotic is new. 3. Determination of the number of antibiotics accumulated in the culture broth is made. 4. Effect of different bioparameters on the growth of streptomycete culture, fermentation process and accumulation of antibiotic. 5. Solubility picture of antibiotic in various organic solvents. Also, it is to be determined whether antibiotic is adsorbed by adsorbent materials (eg. ion-exchange resins or activated carbon). This knowledge is essential in the recovery and purification of an antibiotic from the fermented broth. 6. Toxicity tests are conducted on mice or other laboratory animals. An antibiotic is also tested for the adverse effects if any, on man, animal or plant. 7. The streptomycete culture is characterized and is classified upto species. 8. Further studies are made on a selected individual streptomycete culture. For example, mutation and other genetic studies for strain improvement are carried out. In conclusion, tests are designed and conducted in such a way that production streptomycete strains may be obtained with least expenses. Similar screening and analytical techniques could be employed for the isolation of microbial isolates important in the production of other industrial chemical substances. ## 3. STRAIN DEVELOPMENT It is highly desirable that the industrial fermentation process should be made more and more economical. This largely depends upon the efficiency of the production strain involved in the fermentation process. Therefore, a person interested in starting a fermentation industry or in competing with other industries must procure an efficient strain. Thus it is clear that, the use of a high-yielding strain in any fermentation process is the most critical factor. Usually, newly isolated strains obtained by screening techniques are not so efficient as could be used in industrial fermentation processes. Therefore, such strains require improvement, so far as the yield of a particularly desired compound is concerned. This is accomplished by producing the mutant fermentation strains with the help of physical or chemical methods. These mutants may be grouped into two major categories: * auxotrophic mutants, and * mutants resistant to analogues. Micro-organisms, usually, have regulatory mechanisms that control the amount of metabolites synthesized. Therefore, micro-organisms cannot synthesize excess of the metabolites overlimiting the cells' requirements. Obviously, suppression of these regulatory mechanisms is necessary to develop the strains for higher yields of the desired metabolites. Microbial cultures which have multivalent mechanisms, concerted repression or feed-back inhibition may be used for strain improvement. Subsequently, a search is made for mutants which have lost the ability to synthesize one of the end products capable of feed-back inhibition or repression. This may be explained by considering a situation where three end products (E.P.1, E.P.2, and E.P.3) are synthesized via a branched biosynthetic pathway from an intermediary metabolite (A) as shown in Figure 3.1. There are two main regulatory mechanisms that differ from each other. There may be three distinct iso-enzymes (a, b and c) capable of effecting the first reaction in the pathway (A to B). And each may be inhibited or repressed by one of the three end products. With the multivalent or concerted regulatory mechanism, repression is only apparent if all the three end products are present together as illustrated in Figure 3.2. Here, it is to be noted that, there is only a single enzyme for the reaction A to B. It should also be noted that there is sometimes a cumulative action of the three end products as shown schematically in Table 3.1. Different types of industrially important mutants have been summarized as under: **(a) A mutant strain of Corynebacterium glutamicum can excrete about 60 g. of lysine per litre in a a medium based on glucose and minerals. This mutant strain needs homoserine. On the other hand, wild strain of this bacterium does not need homoserine and fails to excrete lysine. This discrepancy can be well explained by the schematic illustration as shown in Figure 3.3.** In the case of a wild strain, there is a common biosynthetic pathway to the biosynthesis of lysine and threonine, for the first few reaction steps. This pathway is subject to feed-back inhibition by a mixture of lysine and threonine controlling the activity of aspartate kinase. But a mutant strain requiring homoserine can no longer synthesize threonine. Moreover, feed-back inhibition no longer occurs, and lysine gets accumulated in the medium. Optimum production of lysine takes place in a medium containing 400 µg of homoserine per ml., and a high concentration of biotin (20 µg/l). It may be shown that inhibition due to threonine is increased by methionine. Methionine reacts competitively with a regulatory site on aspartate kinase. The ratio of threonine to methionine also plays an important role as shown in Table 3.2. **(b) It should be noted that in the wild strains of Escherichia coli, the biosynthetic pathway to lysine is the same, but its regulatory mechanism is different. In this case, there are three aspartate kinases, cacn separately controlled by either lysine, methionine or threonine. This regulatory system may be represented by Figure 3.1., E.P., being lysine. There is also feed-back inhibition of dihydropicolinate synthetase by lysine.** **(c) Those mutant strains in which one of the enzymes of a biosynthetic pathway is missing are also valuable strains, since they may be employed in the production of an intermediary metabolite of that particular pathway. This may be exemplified by a mutant strain of Corynebacterium glutamicum. The biosynthesis of amino acid, arginine, occurs by a biosynthetic pathway as illustrated in Figure 3.4.** Now, a mutant strain which has lost the enzyme acting on ornithine will excrete that amino acid so long as just sufficient arginine is provided for growth, without enough being present to cause feed-back inhibition. Optimum production of ornithine occurs in a medium containing 200µg of arginine/ ml. and 5 µg of biotin per litre. Moreover, this medium should be rich in glucose and ammonium salts. **(d) There are some mutant strains with enzymes that offer resistance to feed-back control. Looking to the regulatory mechanism of feed-back inhibition, interaction between the end product and the regulatory site of an enzyme changes the enzyme configuration. Subsequently, the enzyme becomes non-functional. A mutant strain may be produced having the enzyme with an altered regulatory site. Such an altered regulatory site fails to interact with the inhibitor. Therefore, feed-back inhibition does not take place.** **(e) It is also possible to use an analogue in the selection of industrially important strains. An analogue can interact with the regulatory site associated with feed-back inhibition. Such an analogue often exerts toxic effect. And, this toxicity eliminates all sensitive mutant cells in a population. For example, a-amino ß-hydroxyvaleric acid is the analogue of threonine. By the use of this antimetabolite, the selection of a mutant strain may be done at the following two stages:** * The analogue of an amino acid, threonine, is added during the preparation of a nutrient agar medium that is poured into a sterile petri-dish. Then, the medium is allowed to solidify at an angle as shown under: When the wedge has set, a second layer of the same medium, without analogue is poured onto it and allowed to set with the plate level as under: After some time, diffusion of an analogue into the upper layer of the medium takes place. As a result of this, there is development of a concentration gradient at the surface. Now, a culture, previously treated with a mutagen, is spread on the surface of this medium. Then, selection of any mutants offering resistance to high concentrations is done. * Lastly, a search is to be made to find out resistant mutants capable of producing threonine. This may be accomplished by inoculating the mutants, as point cultures, onto an agar medium seeded with a threonine dependent culture. The growth of seeded culture (i.e., threonine requiring culture) around each colony of threonine excreting mutant strain may occur. The diameter of the zone of seeded culture growth depends upon the quantity of threonine produced by the mutants. Thus, analogue-resistant mutant strains excreting higher yields of threonine may be obtained. Using the above technique, a mutant strain of Brevibacterium flavum capable of excreting threonine upto 12-6 g. per litre is obtained. **(f) Mutant strains may, sometimes, undergo reversion since mutations are not always stable. As a result of this, revertants may develop in the microbial population of a mutant strain. The revertant strain possesses an enzyme different from that which has been lost due to the previous mutation. Also, the enzyme is not sensitive to feed-back inhibition. This may be exemplified by the threonine deaminase of a revertant strain of the bacterial genus, Hydrogenomonos. The situation is represented as under:** * The wild strain did not require the amino acid, isoleucine. Moreover, it did not produce this amino acid. * The auxotrophic mutant required isoleucine for growth and multiplication. * The revertant strain did not require isoleucine. In addition to that, it also produced this amino acid as shown in Figure 3.5. **(g) Constitutive mutants are also important in a fermentation industry, since they may be used to produce increased yields of particular enzymes. These mutants produce particular enzymes in the absence of inducing substrates or other substrates that offer resistance to catabolite repression. There are numerous techniques for selecting these mutants. Some simple methods have been briefly discussed here:** * The microbial cells are cultured on a medium containing a carbon source with the following two characteristics: * It should not act as an inducing substrate for a particular enzyme. * It should serve as a substrate for the same particular enzyme. For example, phenyl ß-galactoside may be used for selecting constitutive mutants for the excretion of B-galactosidase. By limiting the concentration of an inducing substrate (Lactose) during the continuous cultivation of a culture, a constitutive mutant of Escherichia coli has been obtained. This mutant strain produces the enzyme, f-galactosidase, as 25 per cent of its total protein. * The microbial culture is cultivated in a cyclic manner, alternatively with and without an inducing substrate. For example, the microbial culture may be grown in the presence of glucose and then of lactose in a cyclic manner. After a certain number of cycles, the bacterial population will contain an increased proportion of constitutive mutants. * Use of inhibitors of the inducer in a medium may also be made in the selection of constitutive mutants. For example, 2-nitrophenyl ẞ-fucoside is an inhibitor of lactose and it may be used in selecting mutants constitutive for the production of ẞ-galactosidase. Thus, mutants of Escherichia coli produce ẞ-galactosidase without induction, provided the medium contains lactose as the sole source of carbon. * It is possible to isolate constitutive mutants which offer resistance against toxic substances. The enzyme, for which the mutants are constitutive destroys the toxic substances present in the environment. For example, when a culture of the photosynthetic bacterium, Rhodopseudomonas spheroides, is repeatedly exposed to 0-1 M H₂O₃, 25 per cent of the surviving mutants are constitutive mutants for the production of catalase. * Use of toxic antimetabolites for selecting constitutive mutants having ability to produce an increased yield of enzymes involved in the bio-synthesis of the metabolite concerned, may be made. For example, mutants of Lactobacillus casei resistant to dichloroamethopterin excrete eighty times more thymidylate synthetase than those of the parent culture. Apart from different methods for inducing high-yielding mutant strains, there is also another method for obtaining high-yielding strains. In this case, the genetic constitution of the microbial cells is changed. This is accomplished by transferring all, or part of the DNA to the recipient culture from the donor culture. Again, there are many techniques for the transfer of genetic material. They have been listed as under: * Transformation * Transduction * Lysogeny * Conjugation, and * Parasexuality. ## 4. PRESERVATION OF MICRO-ORGANISMS All practising microbiologists have felt the need to preserve the viability of micro-organisms with which they work. In addition, all the cultural characteristics of a culture, as they were at the time of preservation, must be conserved. The nature of work being done will determine whether the preservation requirement is only very short-term (e.g. a few days) or for an unlimited time period (e.g. many years). Long-term preservation of a culture is required if a culture is to be deposited in one of the service culture collections with a view to preserving something of scientific value "for perpetuity". Many methods of preservation for micro-organisms have been developed. Here, it is to be noted that there exist different types of micro-organisms (bacteria, viruses, algae, protozoa, yeasts and moulds). Therefore, there are two criteria for selecting a method of preservation for a given culture. They are: * The period of preservation desired, and * The nature of a culture to be preserved. With the increasing importance of micro-organisms to industry (e.g. in biochemical and antibiotic production, bio-assay, as spoilage microbes, and the like), human, animal and plant pathologists, geneticists, taxonomists and teachers have felt the need for culture collections. There are several large public service collections. These serve as repositories for cultures and as sources of their distribution. The best known of these are the Central Bureau voor Schimmelcultures (C.B.S.), founded in 1906, the American Type Culture Collection (A.T.C.C.), founded in 1925 and the collection of the Commonwealth Mycological Institute (C.M.I.), founded in 1947. Several other countries are developing their own national collections, and there are large collections belonging to industrial concerns as well as specialized government departments. However, any biologist dealing with living material must at least temporarily maintain his own cultures during the course of his studies and preserve them until they are ready for depositing them in one of these major collections. This depositing of important strains is most desirable as in the past, many organisms which have been the subject of intensive investigation, have been discarded at the end of the work or on the death of the biologist. Thus, much valuable material has been lost. There are three basic aims in maintaining and preserving the micro-organisms. They are: * to keep cultures alive * uncontaminated, and * as healthy as possible, both physically and physiologically, preserving their original properties until they are deposited in any major collections (i.e. unchanged in their properties). For very long-term preservation, involving stocks of the strains (as opposed to single specimens of each strain) and where withdrawals from stocks are regularly made, a fourth aim is to have adequate stocks and appropriate systems for replenishing stocks when necessary. This fourth aim is very much the concern of service culture collections, of course, but the other three are the concern of any maintenance and preservation programme. The running of the collection and the methods of maintenance used are designed to minimize the following hazards to which cultures are exposed: * By repeated transfer selection can occur, either of a mutant strain or of a purely vegetative non-sporulating form. The transfer should, therefore, be done as far as possible by an expert with an eye for the wild strain. However, the fewer transfers made, the less is the risk. * Some strains, sometimes, tend to become attenuated under the artificial conditions of culture. Others deteriorate to wet slimy disintegrated mycelium or spores. Simmons (1963) suggests that this may be due to virus infections and there is considerable evidence to support his theory. * The maintenance processes to which the micro-organism (e.g. the fungus) is subjected are selective, and only adaptive strains survive. These may have somewhat a typical characteristic. * Cultures are subject to contamination, infection with mites and adverse conditions of temperature, light, humidity, etc., are responsible for their contamination. The latter may arise through breakdown of apparatus, or by incomplete understanding of the organism. * Adequate documentation of the strains must be made. In a culture collection of long standing the strains may well survive several generations of microbiologists, so to assist in maintaining them in their original condition a clear description of the cultural characteristics supported by dried cultures should be provided at the time of depositing them. ### 4.1. Serial Subculture This is the simplest and most common method of maintaining microbial cultures. Microbes are grown on agar slants and are transferred to fresh media before they exhaust all the nutrients or dry out. An exception to this is aerobic Streptomyces spp. where drying-up of the medium has been found successful, provided the initial growth showed the production of aerial hyphae. The drying-up of the medium appeared to encourage good sporulation and the preserved specimen became simply a dried out strand of agar coated with spores which remained viable for a few years at room temperature. For some microbial cultures, no other methods have been found satisfactory, but for the majority of species other methods are available. There are several factors to be borne in mind while choosing a suitable medium. Solid media should be chosen in preference to liquid media, as growth of a contaminant can be more readily observed. However, bacteriophages are often successfully maintained as suspensions in liquid media. Also, anaerobes, especially Clostridium spp., are frequently maintained in a liquid medium (e.g. Robertson's cooked meat). Some technicians prefer stab cultures for maintenance. But there do not appear to be any published data to show these to be any better than slope cultures. Obviously, if the micro-organism is oxygen sensitive, a stab culture would be suggested as an extra safeguard while handling on the bench. While a rich medium may give the best initial growth for heterotrophs, it may also run the risk of accumulating toxic end-products of metabolism. Therefore, the best medium for growth may not necessarily be the best for maintenance and preservation of micro-organisms. Besides a suitable substratum, other factors affect the growth of cultures for storage. They are: light intensity, tempera-ture, humidity, standard growth conditions, method of transfer, culture vessel and storage. For further details of these factors, the reader is requested to refer to Methods In Microbiology, Vol. 4 (1971), edited by C. Booth. The time period appropriate for subculture may range from a week to even a few years (Table 3.3). Under normal conditions cultures have to be re-grown at fairly frequent intervals (e.g. every four, six or eight months). With a large collection, this requires much labour. Moreover, there is a risk of occurring hazards as discussed previously, everytime a culture is handled. To cut down the frequency of handling of the cultures, it is, therefore desirable to prolong the intervals between subculturing. There are various means to accomplish this (e.g. cold storage and mineral oil storage). ### 4.2. Preservation by overlaying cultures with mineral oil This method of preservation is a modification of serial subculture technique. It was first extensively used by Buell and Weston (1947). Of 2000 fungus strains maintained under oil for 10 years at the C.M.I., only forty-five were lost. This method is cheap and easy, since it does not require special skills or apparatus such as a centrifuge, dessiccator, or vacuum pump. The steps involved in this method are: * First of all, inoculation of the agar slant contained in a screw-cap tube with a given culture is practised. * Inoculated agar slant is subjected to incubation until good growth appears. * Using sterial technique, a healthy agar slant culture (from above step) is covered with sterile mineral oil to a depth of about 1 cm. above the top of the agar slant. If a short slant of agar is used, less oil is required. * Finally, oiled culture from step (iii) can be stored at room temperature. But better viability is obtained when stored at lower temperatures (e.g. 15°C.). The oil used should be of good quality, British pharmacopoeia medicinal paraffin oil of specific gravity 0-865 to 0-890 is quite satisfactory. Sterilization of oil at the C.M.I. is done in McCartney bottles for 15 minutes at 15 lb/in². However, Fennel (1960) insists that the oil be autoclaved at 15 lb/in². for 2 hours and then dried in the oven at 170°C. for 1 to 2 hours. Simmons (1963) also stresses the need for high quality oil, initial sterility and dryness. The covering of the culture with oil prevents drying out. The oil allows slow diffusion of gases so growth continues at a reduced rate. This may induce change due to adaption to growth in oil. Some fungus isolates appear stable and survivals of over 20 years have been obtained at the C.M.I. Others change rapidly, producing a typical culture in a few months (e.g. Fusarium species). The depth of oil of 1 cm. is fairly critical (Fennel, 1960),' as the oxygen transmission by layers of mineral oil in excess of 1 cm. becomes less favourable. If less oil is used, strands of mycelium may be exposed which allows the cultures to dry out (Dade, 1960; Fennell, 1960). If the McCartney bottles are used the rubber liners should be removed from the metal caps as the oil tends to dissolve the rubber and this can be toxic to the cultures. This method has the following advantages: * Practically all bacterial species or strains tested live longer under oil than in the control tubes without oil. Some bacterial species have been preserved satisfactorily for 15 to 20 years. * Transplants may be prepared when desired without affecting the preservation of the stock cultures. * The method is especially advantageous when working with unstable variants where occasional transfers to fresh media or growth in mass cultures results in changes in the developmental stages of the strains. This method also appears to be an ideal method of storage for a busy laboratory with limited funds and a relatively small collection. Many workers have reported their experiences with the oiled fungus culture maintenance method (Sherf, 1943; Norris, 1944; Wernham, 1946; Buell and Weston, 1947; and others). ### 4.3. Lyophilization or freeze-drying Lyophilization is the most satisfactory method of long-term preservation of micro-organisms. It is universally used for the preservation of bacteria, viruses, fungi, sera, toxins, enzymes and other biological materials. While, it offers a convenient technique for preserving a large number of cultures, it is by no means the perfect method for storing yeasts with completely unchanged characteristics. The process of lyophilization was first applied to microfungi on a large scale by Raper and Alexander in 1942. They were successful in processing the cultures at the N.R.R.L. (Northern Regional Research Laboratory) at Peoria and presented their work in 1945. Lyophilization is perhaps the most popular form of suspended metabolism. It consists of drying cultures or a spore suspension from the frozen state under reduced pressure. This can be accomplished in several ways. There are various kinds of equipment available to do this. Major steps involved in this technique are (Fig. 3.6): * A cell or spore suspension is prepared in a suitable protective medium (at the Commonwealth Mycological Institute 10% skimmed milk and 5% inositol in distilled water is found suitable). * Using a sterile technique, the suspension from (i) is distributed in small quantities into glass ampoules. * The ampoules are connected with a high vacuum system usually incorporating a desiccant (e.g. phosphorous pentoxide, silica gel or a freezing trap), and immersed into a freezing mixture of dry ice and alcohol (-78°C.). * The vacuum pump is turned on and the ampoules are evacuated till drying is complete, after which they may be sealed off. The details of the methods used vary from one laboratory to another. Relatively simple apparatus can be constructed for processing a few ampoules, though quite complex machines are available for larger scale work. At the C.M.I. a centrifugal two-stage freeze dryer is used. In this the cooling is by evaporation and no freezing mixture is required. After drying, the small vials can be filled with sterile dry nitrogen instead of sealing under vacuum. Dewald (1966) stressed the importance of the elimination of air and moisture from lyophilized cultures prior to sealing of the ampoules. Similar results were reported by Nei et al. (1966). The methods of revival vary from one laboratory to the other. In case of fungi, dry pellets may be transferred to a suitable liquid and allowed to dissolve before it is streaked out on agar. At the C.M.I., a volume of sterile water equal to the original volume of the spore suspension is placed in the ampoule at room temperature. Then, the ampoule is left for about 20 to 30 minutes for the water to be absorbed slowly before streaking