STOCK CULTURE AND ITS MAINTENANCE
INTRODUCTION:All practicing microbiologists have felt the need to preserve the viability of microorganisms 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 or for an unlimited time period. 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 microorganisms have been developed. Here, it is to be noted that there exist different types of microorganisms (bacteria, viruses, algae, protozoa, yeasts and mould). Therefore, there are two criteria for selecting a method of preservation for a given culture. They are:1. The period of preservation desired, and 2. The nature of a culture to be preserved. Definition: - Stock cultures are those cultures of microorganisms that are stored or maintained for future use in such a fashion that their growth and productive capacities remains unaltered.There are two types of stock cultures: (i) working stocks and (ii) Primary stocks.· The working stock cultures are those which are used frequently and they must be maintained in a vigorous and uncontaminated condition. These cultures are maintained as agar slants, agar stabs, spore preparations or broth cultures and they are held under refrigeration. They must be checked constantly for possible changes in growth characteristics, nutrition, productive capacity and contamination.· Primary stocks are cultures that are held in reserve for practical or new fermentations, for comparative purposes, for biological assays or for possible later screening programs. These cultures are not maintained in a state of high physiological activity and they are delved into only rarely. Transfers from these cultures are made only when a new working-stock is required, or when the primary stock culture must be sub-cultured to avoid death of the cells. Thus, primary stock cultures are stored in such a manner as to require the least possible numbers of transfers over a period of time. Death of a high percent of cells in a primary stock culture is not particularly serious, if viable cells can still be recovered for subculture to fresh medium. Primary stock cultures stored at room temperature are maintained in sterile soil or in agar or broth that is provided with an overlay of sterile mineral oil. Agar and broth cultures without mineral oil also are refrigerated and cultures in milk or agar are maintained frozen at low temperatures. Finally, primary stock cultures are lyophilized or freeze-dried and stored at low temperatures. Often mote than one of these procedure is employed to insure against loss of the cultures or changes in the cells.There are three basic aims in maintaining and preserving the microorganisms. They are:- i. To keep culture alive ii. Uncontaminated and iii. As healthy as possible, both physically and physiologically, preserving their original properties until they are deposited in any major collections. iv. To have adequate stocks and appropriate sustems for replenishing stocks when necessary. 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 serial hyphae. The drying of 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.The maintenance of refrigerated stock cultures on agar or in broth is the least desirable of these procedures. Although the cultures may survive six months or more under refrigeration, usually they are transferred more frequently. These frequent culture transfers and the many cell generations accompanying these transfers allow the possible occurrence of and selection for undesired genetic changes in the organisms. Also the potential for contamination is markedly increased with frequent transfer of the cultures. Certain microorganisms, such as Blakeslea trispora used in b-carotene production, cannot be stored at refrigeration temperatures, because they die out relatively quickly at these temperatures. However, such cultures can be held as agar slants at room temperature with transfers being made to fresh medium when the culture have become nearly dried out. Overlaying cultures with mineral oil: Agar slant and stab cultures of many microorganisms will survive several years at room temperature if the growth is submerged under sterile mineral oil. The oil overlay provides dissolved oxygen, prevents drying of the agar and apparently decreases the metabolic activity of the cells to an almost negligible rate. However, genetic changes do occur in cultures stored in this manner.The steps involved in this method are:· Inoculation of agar salnt/stab with the culture to be maintained.· Inoculated agar slant/stab is subjected to incubation until good growth appears.· Using sterile technique, a healthy agar slant/stab culture is covered with sterile mineral oil to a depth of about 1 cm above the top of the agar slant. If a short slant is used, less oil is required.· Finally, oiled cultures can be stored at room temperature. But better viability is obtained when stored at lower temperatures (15 °C).Note: The depth of oil of 1 cm is fairly critical, as the oxygen transmission by layers of mineral oil in excess of 1 cm becomes less favorable. If less oil is used, strands of mycelium may be exposed which allows the cultures to dry out. If the bottles or screw capped tubes are used, the rubber liners should be removed form the caps as the oil tends to dissolve the rubber and this can be toxic to the culture. This method has following advantages: i. Practically all bacterial species or strains tested live longer under oul than in the control tubes without oil. Some bacterial species have been preserved satisfactorily for 15-20 years. ii. Transplants may be prepared when desired without affecting the preservatio of the stock cultures. iii. 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. iv. This method also appears to be an ideal method of storage for a busy laboratory with limited funds and a relatively small collection. Soil culture (Soil stock):Sterile soil has found wide use for the stock culture maintenance of microorganisms that form spores. This method is particularly applied for the preservation of sporing microbes specially fungi. In fact, microorganisms that do not form spores also will survive in sterile soil, bur they may die out unexpectedly after a period of time. Soil stocks are prepared as follows: i. A mixture of soil (20 %), sand (78%) and calcium carbonate (2%) is prepared and distributed into tubes (a few grams per tube). They are sterilezed for 8-15 hours at 130°C and then cooled. ii. A small volume of thick suspension of spores or of an actively growing culture is then added to the sterile soil and incubated till good growth. iii. The inoculated tubes are kept in desiccators under vacuum. The reason behind this is to evaporate the excess water. Then the tubes are sealed. iv. Soil stocks, thus prepared are stored at room temperatures with cotton plugs or screw caps protected from dust. These cultures can be stored in refrigerators at about 5-8 °C temperature. Lyophilization or freeze-drying: Lyophilization is the most satisfactory method of long-term preservation of microorganisms. It is universally used for the preservation of bacteria, viruses, fungi, sera, toxins, enzymes and other biological materials. Lyophilization is the most popular form of suspended metabolism. It consists of drying of cultures or a spore suspension from the frozen state under reduced pressure. This can be accomplished in several ways. The major steps involve in this techniques are: i. A thick cell or spore suspension is prepared in a suitable protective medium (10% skimmed milk or bovine serum, 5% inositol in distilled water). ii. Using sterile techniques, this thick suspension is distributed in small quantities into glass ampoules. iii. These ampoules are subjected to deep-freezing by keeping the cultures at lower temperature (-20°C). iv. Then the chilled 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). v. The vacuum pump is turned on and the ampoules are evacuated till drying is complete. vi. Freeze dried ampoules are then immediately sealed off and stored under refrigeration. If properly prepared and stored, most lyophilized cultures will remain viable for long periods (> 20 yrs.), without the occurrence of genetic changes. When needed, the cultures are recovered from the ampoules by suspending the lyophilized cells in a minimal amount of growth medium and then incubating. Advantages of lyophilization: i. As the ampoules are sealed there is no risk of contamination of infection with mites. ii. The prepared ampoules are easily stored, they are not readily broken and most species remain viable for many years. iii. There is less opportunity for the cultures to undergo changes in characteristics (i.e. they remain unchanged during storage period). iv. Owing to the small size of glass ampoules, hundreds of lyophilized cultures can be stored in a small storage space. In addition to this, the ampoules size makes them ideal for postage. v. Lyophilization cuts down the number of transfers. Liquid nitrogen storage: This method is also called as Cryogenic storage. It is like lyophilization, a satisfactory method for the long-term preservation of microorganisms. It has also been successful with many specimens which cannot be preserved by lyophilization. The maintenance of microbes is done by suspended metabolism. Life is regarded as “Stand still” at –130°C and below, so at the temperature of liquid nitrogen (-196°C), provided the cultures survive the treatment, the period of preservation should be indefinite. Thus, long-term preservation without any change in the cultural characteristics is now attainable. Major steps involved in the methods are: i. The culture to be maintained is suspended (thick suspension) as a cell or spore suspension in 10% glycerol. ii. This thick suspension in glycerol is distributed into ampoules (resistant to cold-shock). iii. Ampoules filled with a culture suspension are frozen (at the rate of about 1°C per minute to –20 to –35°C) and are sealed. iv. The frozen ampoules are then clipped on metal (aluminium) canes, one above the other and six to each cane. The canes in turn are packed in metal boxes or canisters (aluminium), which holds about 20 canes. These are perforated to allow the free running of the liquid nitrogen. v. The cultures are revived by removing form the container rapidly thawing and culturing them in the usual manner. Advantages: i. It is effective method of preservation ii. No subculturing is required iii. The cultural characteristics remain unchanged. iv. The ampoules are not open to contamination or infection by mites, since they are sealed. v. The living material of a type, which would not normally grow in a culture and would not be preserved in a culture collection, can be retained in a viable state. Regardless of the method or methods chosen for preservation of primary stock cultures, it is of utmost importance that good, descriptive records be kept on these cultures and that the cultures be well labeled. If little is known or recorded about a newly isolated microbial strain, we cannot hope to be able to recognize changes that may have occurred in that culture after prolonged storage period.