3..

0 METHODOLOGY
3.1 QUANTIFICATION OF BACTERIAL CELLS USING POUR PLATE METHOD
Preparation of Culture Medium
Preparation of nutrient agar: 23g of nutrient agar powder was weighed out. It added to 1L
of distilled or deionized water in a 1L Schott bottle. Sterile at 121°C for 20 minutes. It cooled
to 50°C.

1. All the eight sterile petri dishes labelled with your name and lab section and
the date.
2. 9.0 ml salt solution (saline)/distilled water was transferred into each of 12 test
tubes using sterile pipettes (10 ml) with aseptic technique.
3. The test tubes was labelled with 10-1, 10-2, 10-3, 10-4, 10-5till 10-12. Then, the sterile
petri plate was labelled with 10-8, 10-9, 10-10, 10-11 and 10-12.
4. Using a sterile pipette (1 ml), transfer 1 ml of bacteria suspension with
aseptic technique into 10-1 test tube.
5. After that, a new pipette was used to mix the test tube 10-1 properly. With the
same pipette, 1 ml solution of test tube 10-1 was transferred into test tube 10-2.
6. Next, continued the dilution with aseptic technique for the test tube 10-3 till 10-
12
. The step was similar to step 4 and 5.
7. With a new pipette, 1 ml solution was transferred from test tube 10-8 with aseptic
technique to the sterile petri plate of 10-8. Mixed the plate well before sampling.
8. Step 7 repeated for the sterile petri plate of 10-9, 10-10, 10-11 and 10-12. Replicated
the sample.
9. Remove the test tube contains 14 ml of dilute nutrient agar from water bath.
The test tube outer surface was dried by using adsorbent paper. Transferred
the agar to petri plate and mix properly with aseptic technique.
10. Let the petri plate cold down for few minutes on the table until the agar freeze.
All the important data recorded at the back of the plate.
11. Incubated it at 30oC for 48 hours.
12. Examined and quantified the colony present. Determined the total cell growth in 1 ml
of original suspended solution.
3.2 QUANTIFICATION OF BACTERIAL CELLS USING SPREAD PLATE
METHOD
Preparation of Culture Medium

Preparation of agar plates: Poured 15-20mL of a warm sterile nutrient agar per petri plate.
Allowed the nutrient agar to harden.
1. Repeated step 1 to step 6 from the method in section 3.1 in order to prepare dilution of
10-1 to 10-12.
2. With the pipette, 1 ml of dilute solution 10-8 was transferred to the middle of the agar
which been labeled as 10-8with aseptic technique. Then, the petri plate cap was opened
and spread properly the suspended solution at the surface of the agar using the sterile
glass spreader. Replicated the sample.
3. The glass spreader sterilized by pass it through the flame burner before place it in the
alcohol and pass it through the flame burner again.
4. Repeated step 2 for the dilution of 10-9, 10-10, 10-11 and 10-12
5. All the plate was labelled and all the important data was recorded before incubate it at
30oC for 48 hours.
6. Examined and quantified the colony present. Determined the total cell growth in 1 ml
of original suspended solution.