Aim: To perform spread plate method/Lawn method
Introduction: The spread plate is a technique used to separate the microorganisms from a mixed sample which is spread over the surface of an agar plate. Samples are diluted for separation and isolation of individual bacterial colony. The spread plate is routinely used in enrichment, selection and screening experiments.
Requirements:
Media: Nutrient agar
Apparatus: L-shaped bent glass rod, beaker, busen burner, wax marking pencil
Chemical: 95% alcohol
Bacterial Culture: Staphylococcus album, Chromobacterium indica, Bacillus subtilis
Instruments: Colony counter with a magnifying glass, Autoclave, Hot air oven, Incubator.
Procedure:
- Prepare the nutrient agar plate and label it with respective organisms like Staphylococcus aureus, Staphylococcus album, Bacillus subtilis.
- Aseptically transfer a loopful of bacterial culture from suspension in respective Petri plate containing media.
- Take 95% alcohol into the beaker and sterile the bent portion of the glass rod and then pass on the flame of a bunsen burner.
- Cool the rod for 15-20 seconds.
- Tilt the lid of the plate and lightly touch the sterile rod to the agar surface and uniformly spread the bacterial suspension.
- Immerse the bent rod in alcohol and reflame to sterilize it on the flame of a bunsen burner.
- Repeat the same steps and inoculate the bacterial sample in the remaining two plates.
- Incubate the all plates in an inverted position at 25°C for 24 to 48 hours.
Observation: Few colonies may be separate while the rest of the colonies are maybe in a bunch. Record the result of colonies their form, elevation, pigment formation by colonies, and their size.
Results: Isolated colonies have appeared on the surface of the nutrient agar plate.
Advantage:
- The spread plate method is useful for isolation of aerobic microorganisms.
- Bacterial culture is not exposed to high temperature as compare to pour plate method.
Disadvantage:
- The spread plate method allows the growth of other microbes along with desired microbes.
- The spread plate method allows the growth of obligate anaerobic microorganisms.
- Accidental contamination and hence the growth of undesired microbes may be possible.
Application:
- The incubator is the most commonly used equipment in cell culture, tissue culture, biochemical studies, food processing, haematological studies, fermentation studies etc.
- It also finds application in the study of tissue culture.
- Recently advanced technology made it possible to use incubators in genetic engineering
Precautions:
- The volume of sample that to be spread on media should not be more than 0.1 ml.
- The culture was never exposed to 45°C temperature.
- The surface of the plate must be dry.
