2-Spread Plate Technique PDF

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Spread Plate Technique •

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A variety of techniques has been developed for the isolation of microorganism, mainly the bacteria, from the specimen or from the sample cultures. The spread plate method is a technique to plate a liquid sample containing bacteria so that the bacteria are easy to count and isolate. A successful spread plate will have a countable number of isolated bacterial colonies evenly distributed on the plate. Spread plate culture technique is among the most widely used culture technique for isolating the bacteria.

Principle of Spread Plate Technique In this technique, a serially diluted specimen containing 2 or more bacteria or microbe (Mixed culture) is used which is spread over the solidified agar media plates as a thin layer with the help of a sterile L-shape glass rod (Spreader) while the media plate is spun on a turntable. The principle behind this method is that when the Media plate is spun, at some stage, single cells will be deposited with the bent glass rod (Spreader) onto the surface of the Agar media. Some of the cells present in the specimen / diluted specimen will be separated from each other by a distance sufficient to allow the colonies that develop to be free from each other.

Procedure of Spread Plate Technique 1. Make a dilution series from a sample. 2. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate. 3. Dip the L-shaped glass spreader into alcohol. 4. Flame the glass spreader (hockey stick) over a Bunsen burner. 5. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petridish underneath at the same time. 6. Incubate the plate at 37°C for 24 hours. 7. Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample.

Applications of Spread Plate Technique • • • • • • • •

The Spread Plate Technique, in conjunction with serial dilutions, is a valuable research tool. To demonstrate the cultural characteristics of the bacteria (e.g. color, texture, size, elevation etc.). To isolate the bacteria in discrete colonies from the specimen containing more than 1 bacterium. For determining the Sensitivity and/or Resistance of bacterium towards the particular Drug/Antibiotics or Test substances. To obtain the sufficient growth of the bacterium for various biochemical and other tests. To estimate the viable counts of the bacteria in the specimen. To maintain the stock cultures. To transport or short-term storage of the specimen (e.g. stab culture).

Advantages of Spread Plate Technique 1. Heat sensitive microbes are not affected. 2. No subsurface colonies appear in spread plate so isolation of the organism is easy.

Limitations of Spread Plate Technique • • •

Strict aerobes are favored while microaerophilic tends to glow slower. Crowding of the colonies makes the enumeration difficult. Accurate dilutions using pipettes should be made.

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Volume no greater than 0.1 ml can be spread on the nutrient agar plate because it would not soak well and may result in colonies to coalesce as they form....