CULTIVATION OF ANAEROBIC BACTERIA:

Stringent anaerobes can be grown only by taking special precautions to exclude all atmospheric O2 from the medium.

1.  Prereduced media: During preparation, the culture medium is boiled for several minutes to drive off most of the dissolved O2. A reducing agent (e.g.,0.05% cysteine, 0.1% thioglycolate, 0.1% ascorbic acid ) is added further to lower the O2 content. Oxygen- free N2 is bubbled through the medium to keep it anaerobic. The medium is then displaced into tubes which are being flushed with O2 free N2, stoppered tightly, and sterilized by autoclaving. Such tubes can be stored for many months. During inoculation, the tubes are continuously flushed with oxygen free CO2 by means of a cannula (inoculum is also added), restoppered , and incubated. This method is also called 'roll tube method’.

2.  Anaerobic chamber: This refers to a plastic anaerobic glove box.

·     Glove parts and rubber gloves allow the operator to perform manipulation within the chamber.

·     Air lock with inner and outer doors. Media are placed within the air lock with the inner door remaining sealed; air is removed by a vacuum pump connection and replaced with N2 through the another pipe.

·     The inner door is opened and the media are placed within the main chamber, which contains an atmosphere of H2 + CO2 + N2.

·     A circulator circulates the gas atmosphere through pellets of palladium catalyst, causing any residual O2 in the media to be used up by reaction with H2.

·     After media have become completely anaerobic they can be inoculated and placed in an incubator located within the chamber.

3. Gas Pack Jar (Anaerobic Jar):

Non stringent anaerobes can be cultured within an anaerobic jar. Media are inoculated and then placed in the jar. Water is added to the gaspack generator envelope causing the evolution of H2 and CO2. The H2 reacts with O2 on the surface of the palladium catalyst, forming water and establishing anaerobic conditions. The CO2 aids the growth of fastidious anaerobes which sometimes fails to grow, or grow only poorly, in its absence. An anaerobic indicator strip (a pad saturated with methylene blue solution) changes from blue to colorless in the absence of O2.

MAINTENANCE AND STOCKING OF PURE CULTURES:

A large collection of strains, frequently referred to as a stock culture collection. These organisms are needed for laboratory classes and research work, as test agents for particular procedures or as reference strains for taxonomic studies. The strains are used for screening of new, potentially effective chemotherapeutic agents; as assay tools for vitamins and amino acids; as agents for the production of vaccines. To maintain strains alive and uncontaminated and to prevent any change in their characteristics, following are the methods for maintenance:

1.  Strains can be maintained by periodically preparing a fresh stock culture from the previous stock culture. The temperature and type of medium chosen should support a slow rather than a rapid rate of growth so that the time interval between transfers can be as long as possible. The transfer method has the disadvantage of failing to prevent changes in the characteristics of a strain due to the development of variants and mutants.

2.  By overlaying cultures with mineral oil: Many bacteria can be preserved by covering the growth on an agar slant with sterile mineral oil. The oil must cover the slant completely; to ensure this, the oil should be about half in above the tip of the slanted surface. This method of maintenance has the unique advantage that you can remove some of the growth under the oil with a transfer Needle, inoculate a fresh medium, and still preserve the original culture. But changes in the characteristics of a strain can still occur.

3.  By lyophilization (freeze drying) : Most bacteria die if cultures are allowed to become dry, although spore- and cyst- formers remain viable for many years. However, freeze drying can satisfactorily preserve many kinds of bacteria that would be killed by ordinary drying. In this process, a dense cell suspension is placed in small vials and frozen at -60 to -78 C. The vials are then connected to a high vacuum line. The ice present in the frozen suspension sublimes under the vacuum, i.e., evaporates without first going into a liquid water phase. This results in dehydration of the bacteria with a minimum of damage to delicate cell structures. The vials are then sealed off under a vacuum and stored in a refrigerator.

Advantages: (1) Many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years.

(2)  Only minimal storage is required; hundreds of lyophilized cultures can be stored in a small area.

(3) The small vials can be sent conveniently through the mail to other microbiology labs when packaged in special sealed mailing containers.

(4) Lyophilized cultures are revived by opening the vials, adding liquid medium and transferring the rehydrated culture to a suitable growth medium.

4. Ultra freezing (storage at low temperature):

In this process, cells are prepared as a dense suspension in a medium containing a cryoprotective agent such as glycerol or dimethyl sulfoxide (DMSO), which prevents cell damage due to ice crystal formation during the subsequent steps. The cell suspension is sealed into small vials are then stored in a liquid nitrogen refrigerator either by immersion in the liquid N2 (-196 C) or by storage in the gas phase above the liquid N2 (-150 C).

Advantage: (i) Many species which cannot be preserved by lyophilization can be preserved by this method.

(ii) Most species can remain viable under these conditions for 10 to 30 years without undergoing change in their characteristics.

Disadvantage: This method is relatively expensive, since the liquid N2 in the refrigerators must be replenished at regular intervals to replace the loss due to evaporation.

METHODS OF ISOLATING PURE CULTURES:

Streak- Plate technique: By means of a transfer loop, a portion of the mixed culture is placed on the surface of an agar medium and streaked across the surface. This manipulation “thins out” the bacteria on the agar surface so that some individual bacteria are separated from each other. When streaking is properly performed, the bacterial cells will be sufficiently far apart in some areas of the plate to ensure that the colony developing from one cell will not merge with that growing from another.

The pour plate and spread plate technique:  In both of these methods, the mixed culture is first diluted to provide only a few cells per milliliter before being used to inoculate media. Since the number of bacteria in the specimen is not known beforehand, a series of dilutions is made so that at least one of the dilutions will contain a suitably sparse concentration of cells.

In pour plate method, the mixed culture is diluted directly in tubes of liquid (cooled) agar medium. The medium is maintained in a liquid state of temperature of 45 C to allow through distribution of the inoculum. The inoculated medium is dispensed into Petri dishes, allowed to solidify and then incubated.

Disadvantages: (a) Some of the organisms are trapped beneath the surface of the medium when it gels, and therefore both surface and subsurface colonies develop.

(b) The organisms being isolated must be able to withstand temporary exposure to the 45 C temperature of the liquid agar medium. Hence, it would be unsuitable for isolating psychrophilic bacteria.

In the spread plate method, the mixed culture is not diluted in the culture medium; instead it is diluted in a series of tubes containing a sterile liquid usually water or physiological saline. A sample is removed from each tube, placed onto the surface of an agar plate, and spread evenly over the surface by means of a sterile, bent glass rod. On at least one plate of the series the bacteria will be in numbers sufficiently low as to allow the development of well separated colonies. In contrast, to the pour plate technique, only surface colonies develop; moreover the organisms are not required to withstand the temperature of liquid agar. Unlike the streak plate technique, the pour plate and the spread plate technique may be performed in a quantitative manner to determine the number of bacteria present in a specimen.