In this article we will discuss about how to get a pure culture of microorganisms. Learn about:- 1. Maintaining Cultures 2. Maintaining Purity.
Maintaining Cultures:
Slants and Broths:
Use of giants and broth cultures for maintaining primary stock cultures is probably the simplest method available. For the majority of microorganisms, mature cultures grown on slants or in broth may be refrigerated and thereby preserved for some time, usually for a few months. However, use of slants and broths presents some serious shortcomings and, in general, it is the least desirable method available. It necessitates the frequent transfer of cultures which greatly increases the chance of contamination and undesirable genetic change.
Many fungi, for example- grown under the artificial conditions of slants lose their ability to perform a desired biochemical process after several transfers. Bacteria, like fungi, may lose some of the desired properties such as virulence, antigenicity, or the ability to produce a particular metabolite.
Some of the problems may be minimized by altering the media in which the microorganisms are sub-cultured. This helps to prevent the natural selection for strains which grow well in a particular medium. Much attention has been given to the composition of media which may be used successfully to culture microorganisms but the selection must be tailored to each particular situation.
Oil Overlay:
Mineral oil may be used to overlay the mature growth of microorganisms on a slant; This process reduces water loss by evaporation, slows the exchange of gas, and allows sub-culturing without destruction of the primary stock culture. But use of the oil overlay method is subject to objections similar to those for the broth or slant method, such as loss of sporulation, loss of biochemical activity, etc. The technique is, however, particularly useful in maintaining fungi which do not produce spores.
Soil:
A third and relatively simple method of preserving cultures is the dry spore stock on sterile soil, this method has been used successfully for a variety of microorganisms including fungi and bacteria. The first consideration in using soil should be to note whether the soil is to be used strictly as a carrier or also as a growth medium. If used as a carrier, abundant spores of the bacterial or fungal species must be prepared in advance. The mature spores may then be placed in sterile soil and the resulting preparation may be dried in air or under vacuum.
For many fungi, maintenance by use of soil stocks is very useful. Moist soil may be inoculated and the fungus may be allowed to grow until sporulation has been completed. This type of culture may be used both as primary stock and as a working stock culture.
Freezing:
When the need for long time storage arises, it is necessary to reduce the metabolic activity of microbial cells to such a point that reproduction is halted. Presently two methods are employed to reach this goal. The biochemical activity of cells can be reduced to a state of “suspended animation” by reducing their temperature by the almost complete removal of water.
Freezing cells is a harsh process which can damage cells beyond repair. Methods have been developed which allow freezing with minimum damage to assure the recovery of live cells of many microorganisms. A considerable amount of information has been published on the events that take place during the freezing process and many opinions have been advanced on the best method to be used.
The following are a few generally accepted rules on the frozen storage of live cells:
(1) The temperature drop should be about 1°C per min to -20°C; then as rapid as possible to the storage temperature.
(2) Thawing should be as rapid as possible.
(3) Survival rates may be increased by the addition of glycerol, sugars, or other protective agents.
(4) Electrolytes should be kept at a minimum.
Liquid Nitrogen:
In recent years the use of liquid nitrogen for ultralow temperature storage of cells, including microbial cells, has become a very useful method for long term preservation. Assuming that cellular metabolism is completely stopped at temperatures of -130°C, organisms should be able to survive for an indefinite period of time provided they can withstand the cooling and thawing process. This process has been used successfully for a wide variety of cells including fungi, bacteriophages, protozoa, algae, mammalian cells, and bacteria.
The overall process for ultralow temperature preservation is similar to that for the freezing process. A cell suspension protected by such materials as glycerol is cooled at a rate of about 1°C per min until a temperature of -35°C is reached. At this point the temperature is allowed to drop at an uncontrolled rate.
Some differences of opinion exist as to the method by which the frozen cells should be thawed. Several studies have been conducted. Generally the tubes are removed directly from the low temperature storage and placed in a 37°-40°C water bath and agitated for rapid thawing.
Caution should be exercised during the thawing process. A cracked or otherwise faulty ampoule may have become contaminated with liquid nitrogen. As thawing proceeds the pressure created by the passage of this liquid nitrogen to the gas phase may cause an-explosion.
The use of liquid nitrogen storage of microbial cells has several advantages- No sub-culturing of cells; non-spore-forming microbes such as basidiomycetes may be preserved; no changes in the biochemical mechanism or the genetic equipment of the cells; no contamination problems once the ampoules are stored. The disadvantages are- The initial cost of the equipment; the need for a constant-supply of liquid nitrogen; and the difficulty in distributing cultures to other laboratories.
Lyophilization:
Lyophilization is probably the most satisfactory method for long term preservation of those organisms which will withstand the process.
Although it had been used earlier, the lyophilization process was developed into a valuable tool during the 1940s primarily by the Northern Regional Laboratory in Peoria, 111. Later work at this institute turned this process into a very useful and highly reliable method for the preservation of many types of microorganisms.
This process has many of the advantages of the liquid nitrogen process: It requires no sub-culturing of cells; there is no change in the biochemical reactions of the cells, and the cells are genetically stable; there are no contamination problems once the ampoules have been sealed; the finished ampoules can be easily shipped by mail; and the initial costs are much less than those for liquid nitrogen storage. The major disadvantage of the lyophilization process is simply that not all microorganisms can be preserved by this method.
Lyophilization or freeze-drying consists primarily of suspending propagative cells (bacteria or yeast cells, conidia, ascospores, etc.) in a protective medium, freezing, and the removal of water by sublimation under reduced pressure. The desiccated cultures are then sealed under vacuum and stored at low temperature (4°C).
The majority of molds (penicillia, aspergilli, Mucorales) survive well, whereas Pythicacea, Entomophtorales, large spored fungi, and mycelial forms seldom survive the initial treatment. Many other single celled organisms such as yeast and bacteria also can be preserved by this method. If the organisms survive the initial treatment, they are likely to remain viable for a period of 20 years or more.
The spores or cells may be suspended in various protective media such as bovine serum or media containing sugars. However, skim milk is probably the most desirable medium, and it is more easily obtained. The microbial suspension is dispensed into small ampoules using sterile techniques. The ampoules are then quick frozen at about -35°C and placed under a vacuum of at least 200 μm of Hg. After drying, the ampoules are sealed under vacuum.
Ampoules for lyophilization may be prepared by cutting pyrex glass tubing into lengths of approximately 11 cm. One end of the tube is then sealed by rapidly rotating it in an oxygen-natural gas flame. Care must be used to seal the end completely. The other end is then fire polished to smooth the rough edges. Before lyophilization the tubes should be snugly stoppered with cotton and sterilized. If the cotton stoppers are too tight, the frozen cell suspension will melt during the process, resulting in an unusable preparation.
A suspension medium prepared from a mixture of 1 part fresh skim milk and 1 part distilled water should be sterilized. A cell suspension is prepared by introducing the sterile skim milk-water mixture into a fresh fungus culture about 2 days after sporulation has been completed. A highly concentrated suspension of fungal spores increases the chance of recovery of the culture after lyophilization.
Approximately 3 drops of the cell suspension should be placed into each ampoule with a sterile Pasteur pipette. The excess cotton should be cut from the stopper leaving a stopper about 8-10 mm long which is pushed to within 10 mm of the medium. A small tag showing the culture identification on one side and the date on the other side is also placed inside the ampoule.
The preparation may be frozen in a beaker of ethanol with chips of dry ice at a temperature of -35° to -40°C. It is important that the temperature not rise above -35°C during freezing. Temperatures below -35°C are usually not damaging to the culture, while temperatures above -35°C may result in poor viability.
Once the preparations are frozen the tubes should be subjected to the vacuum. A vacuum of 200 μm Hg is usually satisfactory. The actual vacuum is not really critical. The important point is that the frozen preparation not melts during the process. Often it is necessary to maintain the frozen preparation in an ice bath for the first few minutes of drying.
After the cultures are dry, the tubes are sealed under vacuum with an oxygen gas torch. Finished cultures may be stored at room temperature. However, it is advisable to store the ampoules in the dark since light can reduce viability; and storage in the refrigerator is recommended.
Several methods may be used to open the tubes, but sterility must be maintained: Thump the tube with the fingers to break up the pellet. Wipe the surface of the tube with a sterilizing solution (70% ethanol or Chlorox 1:1) after making a file scratch across the center of the cotton plug. Apply a red hot glass rod to the scratch to crack the glass or simply apply
pressure as one would to break glass tubing. Use care in opening the ampoules as the contents are under vacuum. Allow time for air, filtered by the plug, to seep into the ampoule. Otherwise, when the pointed end is snapped off the plug will be drawn to one end.
Hasty opening may release live particles of the dried organism into the air of the laboratory. Since the cotton plug and the tube may contain spores or cells they should be auto- claved after use. If the tube does not have a cotton stopper make a file scratch on the sealed tube, sterilize the outer surface with ethanol or some other disinfectant, and break open inside a wrapping of sterile cotton.
Re-Culturing:
One of several methods may be used for re-culturing. Regardless of the method, the resulting culture should be compared with a description of the culture originally lyophilized.
Method A:
After opening the tubes, introduce a small volume of sterile water equal in volume to the pellet, and replace the cotton plug. Allow the ampoule to stand for 30 min, and then streak the suspension on agar medium. For bacteria, blood agar should be used since some bacteria require haemin for re-cultivation.
Method B:
Prepare 50 ml of a suitable culture medium, place in a 250 ml Erlenmeyer flask, stopper, and sterilize. The contents of the ampoule may then be dumped into the flask. This method does not allow for the possibility that contamination may have occurred (as does the streaking method). Patience must be used in re-culturing. Often several days to a week are required for growth to appear.
A medium suitable for the growth of each individual microorganism should be used for re-culturing lyophilized cultures. Some investigations have shown that malt extract increases the chance of satisfactory results in re-culturing fungi, and malt extract is generally included in re-culturing media.
The main cause of failure to recover lyophilized cultures is poorly sealed tubes which lose their vacuum. A few days after lyophilization, the vacuum in the tubes should be checked. This can be done by using an induction coil (spark coil tester, high frequency coil, or generator, Tesla type). The tubes may be lined up side ‘by side on a table top at room temperature. Touching the end of the tube with the discharge tip of the tester results in a white to purplish glow in the tube. Absence of this glow indicates a loss of vacuum.
Many factors influence the survival rates of microorganisms preserved by lyophilizaton. These have previously been reviewed. The most important generalization about lyophilization is simply this: If the culture withstands the original process of lyophilization, it is likely to survive for many years as long as the culture remains under vacuum.
Maintaining Purity:
Antibiotics:
A wide variety of antibiotics and other chemicals are useful in obtaining pure cultures during isolation procedures and for maintaining purity in culture collections some common and easily available compounds and their applications are listed in Table 3.1.
Mites:
Invasion of fungal cultures by mites has long been a problem which is more easily prevented than cured. Mites feed on fungal spores and travel from one culture to another and spread contamination throughout a collection. A collection which is heavily infested by mites can usually be saved only by a re-isolation program. This requires long time periods, and even then many cultures may be lost.
A catastrophe caused by an invasion of mites can usually be prevented by observing the following guidelines. Primary cultures should preferably be stored by, a low risk method such as mineral overlay, lyophilization, or liquid nitrogen storage. Cultures which have completed sporulation should be stored under refrigeration.
This will not prevent mite growth but will reduce the rate of growth and reproduction of mites so that an entire collection will not be lost before mites are detected. Samples of vegetation should not be placed in close proximity to the culture and collection storage area. Vegetation, particularly dried samples such as hay, are noted as carriers of mites and should not be allowed in the same room as culture collections.
Clean Room:
Microbiologists find it important to have available a “transfer” or “clean” room. This should not be confused with a microbiological laboratory whose functions are primarily plate counts, assays, media preparation, and the like. A “transfer” or “clean” room is used in instances when critical work of culture transfer or culture preservation is done.
Some brief examples are given:
Transfer Cultures:
In many instances an inoculum is needed for a large scale industrial process. This usually requires growing of the microorganism in several stages of ever increasing volume to obtain an inoculum large enough for the final commercial stage. This propagation often requires several transfers over a period of several days. If contamination is introduced at an early stage, the final product will suffer severely m quality.
Strain Research:
Research on strain selection often requires that a clean area be available that can be occupied by the investigator for an extended period of time without interruption by other individuals. This is necessary to minimize the danger of contamination.
Storage:
In some instances, for example for nutritional work, several days may be required for treated cultures to grow. It is often necessary to prepare and incubate such cultures in an area where little air contamination exists.
Extended Culture:
Some fermentation work requires a very long growth period Just as for storage, an area with extremely low microbial contamination is required to complete such an investigation successfully.
Requirements of a Clean Room:
If a transfer room is to be used for critical work, it should have the following characteristics:
Limited Access:
It is important that during the process of transferring cultures no one enter or leave the room. This reduces air currents and the movement of air-borne bacterial and yeast cells and fungal spores.
Supervision and Responsibility by One Individual:
It is necessary to assign responsibility for such a critical area to one individual. He/she should be capable of caring for the area and should have the authority to maintain it in a proper way.
Clean Room should be Small:
The clean room should be small in size for proper cleaning and maintenance. Cleaning should be done frequently and thoroughly.
Filtered Air Supply:
A filtered air supply is essential to maintain a low level of air contamination after sanitation.
Cleanable Surfaces:
Surfaces must be able to withstand the action of harsh chemicals. Equipment which cannot be easily and completely cleaned should be excluded. A wide variety of agents are available for use as disinfectants on bench tops and other surfaces of a clean room. Ultraviolet light of a wavelength of 253.7 nm is an effective agent for destroying microorganisms in close proximity to the UV source. The value of ultraviolet radiation for disinfecting a large area such as a bench top is almost ml. After use, the source of a UV lamp will continue to glow long after it has ceased to emit germicidal radiation.
Ethanol, isopropanol, and other alcohols are often diluted to 70% with water and used as a general disinfectant; Use for bench top disinfecting is limited since some time is required for alcohols to become effective. Use for soaking of instruments is of value.
Formaldehyde is an effective agent against non-spore-forming organisms. It is available as a solution of about 40% formaldehyde in water (formalin), and it is used generally in concentrations of 0.1 to 0.5% formalin in water.
Hypochlorites are used as general germicides. They are effective against a wide range of microbes. A 1:1 mixture of household bleach is often used. Iodine in aqueous or alcoholic solution is a good general purpose disinfectant. It is effective against bacteria, spores, and viruses. A 1% solution in 70% ethanol is commonly used as a skin disinfectant.
Fumigation:
In some cases the air and bench contamination level will reach a point where fumigation of the entire room is required. For this reason the air of the clean room should be exhausted into a system not connected with an occupied area. Fumigation may be accomplished by several methods. Commonly, formalin is boiled to release the gas formaldehyde. Approximately 0.5 ml of formalin should be used for each 0.03 m3 (1 ft3) of air space.