Shake culture technique

Shake culture technique:

A tube in molten cooled (45⁰C) agar medium is inoculated with the test organism. The tube is shaken, cooled rapidly and incubated. Upon incubation, different oxygen requirements of organisms may be manifested as follows:

1. Strict anaerobes grow in the deeper portions of the culture.
2. Facultative anaerobes are found throughout the presentation.
3. Microaerophiles grow near the surface of the medium.
4. Aerobes grow at the surface of the medium.
5. Aerotolerant grow throughout the medium.

A redox dye as resazurin may be added to the medium because the dye changes colour in the presence of oxygen and thereby indicates the degree of penetration of oxygen into the medium. It is also possible to add a reducing agent that reacts with oxygen and reduces it to H₂O. a good example is a thioglycolate which is added to a medium called thioglycolate broth. After thioglycolate reacts with O₂ throughout the tube, oxygen cane penetrates only near the top of the tube where the medium contacts air. Shake culture technique may be slightly modified by preparing series of dilutions too. The technique is then known as dilution shake culture.

A layer of sterile petroleum jelly and paraffin may be added on the surface of the medium before incubation to prevent access of air to the agar column. In shake culture, the colonies develop deep in the agar column and thus are not easily accessible for transfer. To make a transfer, the seal (if used) is removed with a sterile needle, and the agar column is extruded from the tube into a sterile Petri dish by gently blowing a stream of gas through a capillary pipette inserted between the tube wall and the agar. The column is sectioned into discs with a sterile knife to permit examination and transfer of colonies.

Procedure:

1. Take a nutrient agar deep and label it.
2. Melt the nutrient agar in the test tube.
3. Allow it to cool to 45⁰C
4. Inoculate the test organism into molten, cooled medium aseptically.
5. Shake the tube by gently striking it with your fingers.
6. Cool, the tube rapidly under running cold water to solidify the medium.
7. Incubate the tube at 37⁰C for 24-48 hours.
8. Repeat the same process for other test organisms also.

Wright’s tube method:

This method is also called pyrogallic acid sodium hydroxide method. To grow anaerobes on a nutrient agar slant, one creates anaerobic conditions within the tube by using pyrogallol and sodium hydroxide. When activated with sodium hydroxide, pyrogallol acts as a powerful reducing agent to remove the oxygen from the tube. The outer plug of the tube should significantly be rubber or cork and be air tight. Inoculation of a control tube enables us to determine the relative oxygen needs of different organisms and the effectiveness of a Wright’s tube.

Procedure:

1. Take a nutrient agar slant and label it.
2. Aseptically, inoculate slant with the test organism by streaking the agar surface.
3. Replace the cotton plug.
4. Ignite the plug by passing it through the Bunsen flame .alternatively, cut off the cotton plug outside the tube with a pair of scissors.
5. Using a glass rod, push the cotton plug into the tube until it nearly touches the slant.
6. Add pyrogallol (pyrogallic acid crystals) to fill the space between the cotton and top of the tube.
7. Add few ml of sodium hydroxide (4%) on the top of pyrogallol.
8. Apply a rubber stopper immediately and force it down to the tube
9. Invert the tube quickly to prevent seepage of the solution down onto the agar slant.
10. Incubate the tube in the inverted position at 37⁰C for 24-48 hours.
11. Repeat the same procedure for control i.e. strict anaerobe as clostridium sporogens.

Roll tube technique:

The role tube procedure, developed by R.E. Hungate, is used to obtain pure cultures of strict anaerobes by distributing cells in a thin layer of agar on the walls of a test tube, where they develop into isolated colonies. The test contains few ml of the medium that has been reduced chemically to remove dissolved oxygen and tightly stoppered with a butyl rubber bung (small but lethal quantities of oxygen diffuse through ordinary rubber). The molten agar is inoculated with the appropriate dilution of organism inserting through the rubber stopper with a sterile syringe.

The tubes are then laid on their sides in ice and rolled until the agar solidifies in a thin layer on the wall of the tube. After the period of incubation when colonies become visible, the bung is removed and isolated colonies picked from the agar with a needle or capillary tube. Whenever a tube is unstopped, entry of air has is prevented by continuously passing a stream of O₂ free gas (normally CO₂and N₂) into the tube. To ensure that some entry of air has not inadvertently occurred, it is usual to include in the medium the redox dye, resazurin.

Paraffin plug method:

This method is usually employed to create the anaerobic environment in broth culture. After the inoculation of organism into suitable liquid or semisolid medium, immediately a layer of sterile paraffin oil used to cover the medium. The culture tube is then stoppered (cotton plug) and incubated. Paraffin plug method is widely applicable in studying the metabolic activities of the organisms. For example, during oxidation fermentation test of an organism, duplicate media tubes are used. One of them is covered with a layer of paraffin during incubation for the anaerobic metabolism (e.g. fermentation) of the organism if present.

Pre-reduced media preparation:

The culture media is boiled for several minutes to drive off most of the dissolved lower the oxygen. A reducing agent eg. Cysteine, H₂S is added to further lower the oxygen content. Oxygen free N₂ is bubbled through the medium to keep it anaerobic. The medium is then dispensed into tubes, which are being flushed with O₂-free N₂, stoppered tightly and sterilized by autoclaving. Such tubes can be stored for many months before being used. During inoculation, the tubes are continuously flushed with oxygen free CO₂ by means of a cannula, restoppered and incubated. Inoculation is best carried out by using Pasteur’s pipette.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.