BACTERIAL COUNT

PROCEDURES

Procedures of preparing nutrient media

1. Mix peptone, beef extract, agar and distilled water in 600mL beaker and boil it.
2. Cool the agar up to 45-50oC.
3. For the Spread Plate test, pour the nutrient media into half of the six petri plates.

Note: All the agar preparation procedures should be performed under laminar flow to keep the
samples sterile. Use gloves to prevent contamination of the samples

Dilution procedures

1. Use a clean, sterile, dry pipette to remove 0.1mL from the bacteria sample and blow it
into the 9.9mL of dilution fluid (normally deionized/distilled water) in tube#1 and mix
thoroughly by blowing lots of bubbles with the pipette for a couple seconds. Discard the
pipette into the used jar for later cleaning. Notice tube#1 now contains 1/100 the
concentration of bacteria in the original sample because 0.1mL is 1/100 of 10mL. Since
nearly 0.1mL of liquid may cling to the outside of the pipette, you must wipe the pipette
with Kleenex or toilet paper before inserting the pipette into tube#1.
2. Using another clean, sterile, dry pipette remove 0.1mL from tube#1, wipe pipette, blow
contents of pipette into tube#2, continue blowing bubbles for a second or two for good
mixing.
3. Using another clean, sterile, dry pipette remove 0.1mL from tube#2, wipe, blow
contents of pipette into tube#3, continue blowing bubbles for a second or two for
mixing.
4. Keep applying the same procedures until tube#6. Refer the below diagram for better
understanding.
5. Label your tubes with the dilution factor as to notice the bacteria content in the tubes.

Note: There are many types of pipettes, and you are advised to use blow out pipette, that is
indicated by a frosted ring on the pipette at the top end.
 0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mL

test tubes
contain 9.9 mL
water dilution fluid
sample test tubes
1/10 1/100 1/103 1/104 1/105 1/106 contain 9.0 mL
dilution fluid
water
sample
1.0 mL
1.0 mL
1.0 mL
1.0 mL
Spread Plate test method 1.0 mL
1.0 mL
1. Using clean, dry, sterile pipette to remove 0.1mL of diluted sample from each test1/10tube
6

into six different petri plates contain sterile agar. 1/10 4

2. Close the petri plate. 1/105

3. Place all the petri plates inside the incubator for 18-24 hours with a temperature of 3
1/10
37oC. water
sample
Pour Plate test method

1. Using clean, dry, sterile pipette to remove 0.1mL of diluted sample from each test tube
into six different petri plates.
2. Pour the agar into the plates. Wait until the agar to solidify.
3. Close the petri plates.
4. Place all the petri plates inside the incubator for 18-24 hours with a temperature of
37oC.

Methods of counting bacteria

1. After being incubate for 1 day, take out the petri plates.
2. Place the petri plate on the counting chamber.
3. Count the bacteria colonies on the culture.