Preservation is a process of maintaining pure culture for extended periods in a viable condition without any genetic changes. Once a pure culture has been obtained from the mixture, the microbes may be maintained in the laboratory over long periods. The most important factor of preservation is to stop microbial growth or at least suppress the growth rate.
Objectives of Preservation:
• To maintain isolated pure cultures for longer periods in viable
conditions.
• To avoid contamination.
• To restrict genetic changes.
Several techniques are used to preserve cultures viz. period
transfer in fresh medium or Subculturing, Storage in sterile soil, Saline
suspension, Refrigeration, Paraffin Method, Cryopreservation, and Lyophilization.
1. Subculturing: Strains are maintained by
periodically preparing a fresh culture from the previous stock culture. Culture
medium, storage temperature and the time interval at which transfers are made
vary with the species. Nutrient agar medium is commonly used where most of the
heterotrophs remain viable for several months.
2. Storage in sterile soil: The soil is prepared by
mixing enough sand with rich garden soil that makes it friable and easy to
handle. A small amount of calcium carbonate may be added and the mixture is
transferred to screw-capped tubes or tubes plugged with cotton. The tubes are
autoclaved until complete sterility. This soil is widely used in preservation
cultures that form "spores". A thick suspension of spores is added to the sterile soil to this. Moisture is removed by placing the cap and
maintaining reduced pressure over a drying agent.
3. Saline suspension: Sodium chloride in high
concentration is frequently an inhibitor of bacterial growth. Bacteria are
suspended in a 1% salt solution. The tubes are stored at room temperature.
Whenever needed the transfer is made on an agar slant.
4. Refrigeration: Pure cultures are also stored at
0-4°C either in refrigerators or in cold rooms. This method is applied for a short
duration (2-3 weeks for bacteria and 3-4 months for fungi) because the
metabolic activities of the microorganisms are greatly slowed down but not
stopped.
5. Paraffin method: This is a simple and economical
method of maintaining pure cultures of bacteria and fungi. In this method,
sterile liquid paraffin is poured over the slant (slope) of the culture and
stored above at room temperature. The layer of paraffin ensures anaerobic
conditions and prevents dehydration of the medium. This condition helps
microorganisms or pure culture to remain in a dormant state. Hence, the culture
is preserved for several years. This method is also known as Agar Slant Culture
Covered with Oil.
6. Cryopreservation: Cryopreservation (i.e., freezing
in liquid nitrogen at −196°C) helps in the survival of pure cultures for long
storage times. In this method, the microorganisms of the culture are rapidly
frozen in liquid nitrogen at −196°C in the presence of stabilizing agents
(glycerol). It prevents the formation of ice crystals and promotes cell
survival.
7. Lyophilization: It is also known as Freeze-Drying.
It is the method of removal of the frozen solvent by sublimation under vacuum
and unfrozen solvent by desorption. In this method, the culture is rapidly
frozen at a very low temperature (−70°C) and then dehydrated by vacuum. Under
these conditions, the microbial cells are dehydrated and their metabolic
activities are stopped; as a result, the microbes go into a dormant state and
retain viability for years. Lyophilized or freeze-dried pure cultures and then
sealed and stored in the dark at 4°C in refrigerators.
Cultivation of Anaerobes:
Anaerobic microorganisms are widely spread in the environment.
They do not require oxygen for their growth. Sometimes these organisms are very
toxic. There are two types of anaerobes namely, facultative anaerobes, obligate
anaerobes, and aerotolerant anaerobes.
• Facultative anaerobes: They are organisms that can
grow in the presence or absence of oxygen. They obtain energy by both
respiration as well as fermentation. Some examples of facultative anaerobic
bacteria are Staphylococcus spp. Streptococcus spp., Escherichia coli,
Salmonella, Listeria spp. and Shewanella oneidensis.
• Obligate anaerobes: They are organisms that can
grow totally in the absence of oxygen. Oxygen is toxic for their normal growth.
Examples: Actinomyces, Bacteroides, Clostridium, Fusobacterium,
Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Veillonella,
etc.
• Aerotolerant anaerobes: They are organisms that
cannot use oxygen for growth but are able to tolerate its presence. They have
superoxide dismutase and peroxidase but do not have catalase. Examples:
Streptococcus, Clostridium, and Actinomyces.
Principle: Reduce the O2 content of the culture
medium and remove any oxygen already present inside the system or in the
medium. Oxygen is ubiquitous in the air so special methods are used to culture
anaerobes. Four types of culture media are used likely:
1. Special Anaerobic Culture Media
2. Anaerobic Chamber
3. Anaerobic Bags or Pouches
4. Anaerobic Jars.
1. Special Anaerobic Culture Media: This media is
also known as pre-reduced media. During preparation of this media, the liquid
culture medium is boiled by holding in a boiling water bath for 10 minutes to
remove the dissolved oxygen. Liquid media soon become aerobic so to keep media
anaerobic, reducing agents such as cysteine 0.1%, ascorbic acid 0.1%, and sodium
thioglycollate 0.1% are added to lower the oxygen content.
Oxygen-free N2 is bubbled through the medium to
maintain anaerobic conditions. The medium is then dispensed into tightly
stoppered tubes and autoclaved for sterilization. Such tubes are stored for
many months before being used. During inoculation, the tubes are continuously
flushed with oxygen-free CO2 with a gas cannula, re-stoppered, and
incubated (Fig).
Fig: Pre-reduced culture media
The inoculum of the bacterium is inserted deep in the medium in contact with the meat. Meat particles are placed in 30 ml bottles to a depth of about 2.5 cm and covered with about 15 ml broth.
Cooked meat broth is suitable for growing anaerobic bacteria
in air and also for the preservation of their stock cultures which is also
known as ‘Robertson’s bullock-heart medium’. Thioglycollate broth is also a useful
medium for anaerobes. Some other media such as Brucella blood agar, Bacteroides,
bile, aesculin agar, phenyl ethyl alcohol agar, kanamycin blood agar, etc. are
used for recovering anaerobes. Vitamin K, haemin, and yeast extract are used as
essential nutrition for all the anaerobes media.
2. Anaerobic Chamber: An Anaerobic chamber is an
ideal anaerobic incubation system, which provides an anaerobic environment for the
inoculation of media and the incubation of cultures. The chamber is made up of
a plastic anaerobic glove box that contains an atmosphere of H2, CO2,
and N2. Glove ports and rubber gloves are used during
experimentation to perform manipulations within the chamber. There is an
air-lock with inner and outer doors. Culture media are placed within the
air-lock with the inner door. The air of the chamber is removed by a vacuum
pump connection and replaced with N2 through the outer doors. The
culture media are then transferred from the air-lock to the main chamber
containing H2, CO2, and N2. A circulator
fitted in the main chamber circulates the gas atmosphere through pellets of
palladium catalyst causing any residual O2 present in the culture
media to be used up by reaction with H2. The existence function of
CO2 in the chamber is because of many anaerobic bacteria for their
optimum growth. Hence, when the culture media becomes completely anaerobic,
they are inoculated with bacterial culture and are placed in an incubator in
the chamber (Fig).
Fig: Anaerobic chamber Vacuum pump connection
3. Anaerobic Bags or Pouches: Anaerobic bags or
pouches make convenient containers when only a few samples are to be incubated
anaerobically. They are available commercially. Bags or pouches have an oxygen
removal system consisting of a catalyst and calcium carbonate to produce a CO2-rich
atmosphere. One or two inoculated plates are placed into the bag and the oxygen
removal system is activated and the bag is sealed and incubated. Plates are
examined for growth without removing the plates from the bag. These bags are
also useful in the transport of biopsy specimens for anaerobic cultures.
4. Anaerobic Jars (or GasPak Anaerobic System):
Fig: Anaerobic Jar
These anaerobic jars are required for obtaining surface
growth of anaerobic bacteria in anaerobic conditions. The most reliable and
widely used anaerobic jar is the Melntosh-Fildes’ anaerobic jar. It is a
cylindrical vessel made of glass or metal with a metal lid, which is fitted by
a clamp. The lid possesses two tubes with taps, one acting as a gas inlet and
the other as an outlet. On its under-surface, it carries a gauze sachet
carrying palladium pellets, which acts as a room-temperature catalyst for the
conversion of hydrogen and oxygen into water. Palladium pellets act as catalysts
(Fig).
Inoculated culture plates are placed inside the jar and the
lid is clamped tightly. The outlet tube is connected to a vacuum pump and the
air inside is evacuated. The outlet tap is then closed and the gas inlet tube is
connected to a hydrogen supply from where hydrogen is drawn rapidly. After 5
minutes inlet tube is further opened and the catalyst creates a reduced
pressure within the jar due to the conversion of hydrogen and leftover oxygen
into water. The jar is left connected to the hydrogen supply for about 5
minutes and then the inlet tube is closed and the jar is placed in the
incubator.
The gasPak is now the method of choice for preparing an anaerobic
jar. The gasPak is commercially available as a disposable envelope containing
chemicals that generate hydrogen and carbon dioxide when water is added. After
the inoculated plates are kept in the jar, the gasPak envelope with water is
added and is placed inside and the lid screwed tight. The culture plates should
be removed from the jar and placed in the oxygen-free holding system. From
there culture plates should be removed one by one for rapid microscopic
examination of colonies, and then quickly returned to the holding system.