POUR PLATE TECHNIQUE

POUR PLATE TECHNIQUE

AIM

          To isolate and enumerate the bacteria present in Soil sample by Pour plate method.

PRINCIPLE   

          Pour plate technique is the method of choice for counting the number of bacteria present in a sample.  In this method, fixed amount of inoculum (generally 1 ml) from a sample is placed in the center of sterile Petridish using a sterile pipette. Sterile Nutrient agar (approximately 20 ml) is then poured into the Petridish containing the inoculum and mixed well.  After the solidification of the Nutrient agar, the plate is inverted and incubated at 37 °C for 24 hours. Microorganisms will grow both on the surface and within the medium. Colonies that grow within the medium generally are small in size and may be confluent; the few that grow on the agar surface are of the same size and appearance as those on a streak plate. Each (both large and small) colony is carefully counted. Each colony represents a “colony forming unit” (cfu/ml).

MATERIALS REQUIRED

• Soil sample
• Erlenmeyer flask
• Pipettes
• Test tubes
• Petridishes
• Distilled water
• Nutrient agar plates
• Incubator

PROCEDURE

I.  Serial Dilution Technique

a)    Prepare a series of 6 test tubes containing 10 ml of sterile Distilled water.

b)  Prepare the Master dilution by adding 1 gram of Soil sample in the Erlenmeyer flask containing 100 ml of Sterile Distilled water.

c)    Mix the contents well by shaking the Master dilution flask for few times.

d)   From the First tube (10^-1 Dilution), take 1 ml of the sample and transfer to the Second tube (10^-2 Dilution).

e)   Repeat the procedure with all the remaining tubes labeling them until 10^-6 Dilution.

f)    Discard the final 1 ml from the 10^-6 Dilution.

 

II. Pour Plate Technique

a) Pipette out 1 ml from the appropriate desired dilution series onto the center of the surface of the sterile Petridishes.

b)  Sterile Nutrient agar (approximately 20 ml) is then poured into the Petridish containing the inoculum and mixed well. 

c)  After the solidification of the Nutrient agar, the plate is inverted and incubated at 37 °C for 24 hours.

d)  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.

 

Calculation

Colony forming unit (cfu/ml) = (Number of colonies × Dilution factor)/Volume of the sample

*Dilution factor – Reciprocal of the Dilution (For example, 10^-1 is the Dilution and 10¹ is the Dilution factor)

OBSERVATION AND RESULTS

        After incubation, count the bacterial population by using Quebec colony counter, Calculate using cfu/ml calculation and express the number of bacterial colonies as cfu/ml.