OBJECTIVE Apply the techniques of bacterial count using the pour plate and the spread plate methods.

s. Calculate the cell numbers in CFU/ml (colony forming units).

INTRODUCTION Aerobic organisms thrive at oxygen levels greater than 5 percent (fresh air is approximately 21 percent oxygen). They are the preferred microorganisms for composting because they provide the most rapid and effective breakdown or organic materials. Anaerobes thrive when the compost pile is oxygen deficient--referred to as an anaerobic condition. Anaerobic microorganisms: Anaerobic microorganisms are undesirable in a compost pile because they create unpleasant odors. Anaerobic processes can generate acids and alcohols that are harmful to plants. Aerobic microorganisms: Among all the microorganisms at work in a compost pile, the aerobic bacteria are the most important initiators of decomposition and temperature increase in the compost pile. A pour plate is an alternative method for using agar plates to obtain isolated colonies. Pour plates are used when it is necessary to know the number of organisms present per unit volume of specimen or other sample. When a specific aliquot is placed in the Petri dish, a count of the colonies that grow after incubation reveals their concentration in the original sample. Pour plates are used commonly in the bacteriologic examination of milk, or could also be used to determine whether sufficient bacterial numbers are present in urine samples to signify the patient has a urinary tract infection. The number of bacteria in solution can be readily quantified by using the spread plate technique. In this technique, the sample is appropriately diluted and a small aliquot transferred to an agar plate. The bacteria are then distributed evenly over the surface by a special streaking technique. After colonies are grown, they are counted and the number of bacteria in the original sample calculated. Streaking in this technique is done using a bent glass rod. 0.1 mL of bacterial suspension is placed in the center of the plate using a sterile pipet. The glass rod is sterilized by first dipping it into a 70% alcohol solution and then passing it quickly through the Bunsen burner flame. The burning alcohol sterilizes the rod at a cooler temperature than holding the rod in the burner flame thus reducing the chance of you burning your fingers. When all the alcohol has burned off and the rod has air-cooled, streak the rod back and forth across the plate working up and down several times. Unlike streaking for isolation, you want to backtrack many times in order to distribute the bacteria as evenly as possible. Turn the plate 90 degrees and repeat the side to side, up and down streaking. Turn the plate 45 degrees and streak a third time. Do not sterilize the glass rod between plate turnings. Cover the plate and wait several minutes before turning it upside down for incubation. This will allow the broth to soak into the plate so the bacteria won't drip onto the plate lid.

PROCEDURE A. Procedure for the preparation of the 6 fold sample dilution The universal bottles containing 9 ml of sterile distilled water was arranged. 1.0 ml of the cell suspension (stock culture) of E. coli transferred aseptically into the first bottle using the pipettes with sterile tips. The content of the dilution was mixed by shaking. The tips were discarded and the first dilution was labeled as 10-1. Aseptically, transfer 1.0 ml from the content of dilution as 10-1 into the second bottle and the label this dilution as 10-2. Prepare the same manner as described above for further dilution up to 10-7 (Figure 1).

1. Pour plate method samples by a combination of horizontal and circular movement for 5 -10 seconds. The procedure consists of 5 right angle movements, 5 clockwise circular movements and 5 anticlockwise circular movements. The procedure will ensure complete dispersal of the sample in the agar. Take care not to splash the agar. Allow the Petri dishes to set at room temperature, then seal them using parafilm and label at the bottom. dilution and calculate the cell number. Repeat the process for dilution 10-4 and 10-6. Transfer 1.0 ml of the dilution 10-2 into an empty sterile Petri dish. Make another duplicate.

2. Spread plate method Transfer 0.1 ml of the dilution 10-2 onto a PDA. Make another duplicate. The sample is then spread evenly over the surface of the agar using an L-shaped glass rod. Allow the samples to dry then seal them using parafilm, and then label at the bottom. dilution and calculate the cell number. Repeat the process for dilution 10-4 and 10-6. Determine the number of colonies formed on each plate at different dilution.

RESULTS DISCUSSION CONCLUSION REFERENCES