BACTERIAL COUNT

PROCEDURES Procedures of preparing nutrient media 1.

2.
3.

Mix peptone, beef extract, agar and distilled water in 600mL beaker and boil it. Cool the agar up to 45-50oC. 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

water sample 1/10 1/100 1/103 1/104 1/105 1/106

Spread Plate test method

test tubes contain 9.9 mL dilution fluid test tubes contain 9.0 mL dilution fluid water sample 1.0 mL 1.0 mL 1.0 mL 1.0 mL 1.0 mL 1.0 mL 1/106 1/104 1/105 1/103 water sample

1. Using clean, dry, sterile pipette to remove 0.1mL of diluted sample from each test tube into six different petri plates contain sterile agar. 2. Close the petri plate. 3. Place all the petri plates inside the incubator for 18-24 hours with a temperature of 37oC. 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. 2. 3. After being incubate for 1 day, take out the petri plates. Place the petri plate on the counting chamber. Count the bacteria colonies on the culture.