Common Bacterial Culture Techniques

In this common bacterial culture techniques post we have briefly explained about streak culture, lawn culture, stroke culture, stab culture, pour-plate culture, shake culture and liquid culture.

Culture Techniques in Microbiology

Culture techniques in microbiology are commonly used in the clinical laboratory include streak culture, lawn culture, stroke culture, stab culture, pour-plate culture, shake culture and liquid culture.

Streak Culture

This method is routinely employed for the isolation of bacteria in pure culture from clinical specimens.

A platinum loop No. 23 SWG, 6.5 cm long, is charged with the specimen to be cultured. Owing to the high cost of platinum, loops for routine work are made of nichrome resistance wire, No. 24 SWG.

The loop is flat, circular and completely closed with 2-4 mm internal diameter mounted on a handle. One loopful of the specimen is smeared thoroughly over area A, on the surface of a well dried plate, to give a well-inoculum or ‘well’.

The loop is re-sterilized and drawn from the well in two or three parallel lines on to the fresh surface of the medium (B). This process is repeated as shown (C, D, E), care being taken to sterilize the loop, and cool it on unseeded medium, between each sequence.

At each step the inoculum is derived from the most distal part of the immediately preceding strokes. Plates are incubated in the inverted position with the lid underneath.

On incubation, growth may be confluent at the site of original inoculation (well), but becomes progressively thinner, and well separated colonies are obtained over the final series of streaks.

Bacterial Culture

Culture techniques in microbiology: Streak culture (streak plating) on solid media

Lawn Culture

Lawn cultures are prepared by flooding the surface of the plate with a liquid culture techniques in microbiology or suspension culture techniques in microbiology of the bacterium, pipetting off the excess inoculum and incubating the plate.

Alternatively, the surface of the plate may be inoculated by applying a swab soaked in the bacterial culture or suspension. After incubation, lawn culture provides a uniform growth of the bacterium.

It is useful for antibiotic susceptibility testing by disk diffusion method. For preparation of bacterial antigens and vaccines.

It may also be employed when a large amount of growth is required on solid media as, for instance, in the preparation of bacterial antigens and vaccines.

Stroke Culture

Stroke culture techniques in microbiology is made in tubes containing agar slope or slant. Slopes are seeded by lightly smearing the surface of agar with loop in a zig-zag pattern taking care not to cut the agar.

Stroke culture techniques in microbiology is employed for providing pure growth of the bacterium for slide agglutination and other diagnostic tests.

Stab Culture

The preparation of the stab culture techniques in microbiology a suitable medium such as nutrient gelatin or glucose agar is punctured with a long, straight, charged wire into the center of the medium and withdrawing it in the same line to avoid splitting the medium.

The medium is allowed to set, with the tube in the upright position, providing a flat surface at the top of the medium.

Stab culture techniques in microbiology is useful mainly for demonstration of gelatin liquefaction. Demonstration of oxygen requirement of the bacterium under study. For the maintenance of stock cultures. To study motility of bacteria in semisolid agar.

Pour-Plate Culture

Pour-plate culture techniques in microbiology is used for counting the number of living bacteria or groups of bacteria in a liquid culture or suspension.

A measured amount of the suspension is mixed with molten agar medium in a Petri dish. Either 1.0 ml or 0.1 ml of dilutions of the bacterial suspension is introduced into a Petri dish.

The nutrient medium, in which the agar is kept liquid by holding it in a water bath at 45-50°C, is poured over the sample, which is then mixed into the medium by gentle agitation of the plate.

When the agar solidifies, the plate is incubated inverted at 37°C for 48 hours, or most suitable for the species examined in pour-plate culture  techniques in microbiology.

After incubation, colonies will grow within the nutrient agar as well as on the surface of the agar plate and can be enumerated using colony counters.

Shake Culture

Shake culture  techniques in microbiology is made by melting nutrient agar in a test tube, cooling it to 45°C and inoculating it while molten from a liquid medium with a drop from a capillary pipette or a wetted straight wire, depending on the desired size of the inoculum.

Withdraw the pipette or wire and flame the mouth of the tube if it has a cotton-wool plug. Replace the cap or plug and discard the pipette into disinfectant or flame the wire.

Mix the contents of the tube by rotation between the palms of the hands before the agar solidifies. Incubate it at 37°C for 24 hours and look for the growth of the organisms.

Liquid Cultures

Liquid cultures in tubes, bottles or flasks may be inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes.

Blood culture and for sterility: Large inoculum can be employed and hence this method is adopted for blood culture and for sterility tests, where the concentration of bacteria in the inoculum is expected to be small.

Dilution in the medium for inoculum containing antibiotics and other inhibitory substances liquid cultures are preferable, as these are rendered ineffective by dilution in the medium.

Large yields: Liquid cultures are also preferred when large yields are desired, the yield being enhanced by agitation, aeration, addition of nutrients and removal of toxic metabolites (continuous culture methods).

Further Readings