TITLE

Determination of Bacterial Number in Water and Wastewater

1.0 OBJECTIVE

To determine bacteria number in water and wastewater sample

2.0 MATERIALS & METHODOLOGY

a. Four sterilized saline tube: Saline water is prepared by diluting 0.9 g of sodium chloride
and 100 ml of distilled water.
b. Six petri dishes containing tryptic soy agar:
Tryptic soy agar (1 L) is made by mixing 17 g of tryptone, 5 g of NaCl, 2.5 g of
dipotassium hydro phosphate, 2.5 g of yeast extract and 15 g of agar, autoclave it at
121°C for 20 min. After reach 60°C, pour into petri dish and let it until solidified.
c. Two spreader
d. Biosafety cabinet
e. Incubator 35°C

1. Label the bottom of six petri dish 1 – 6. Label four tubes of saline 10-1, 10-2, 10-3, and 10-4.
The bottom of xix petri dishes are well labelled with 1 – 6 and four saline tube also has been
labelled with 10-1, 10-2, 10-3, and 10-4 respectively.

2. Using aseptic technique, the initial dilution is made by transferring 1 ml of water and
wastewater samples to a 9 ml sterile saline blank.
The initial dilution has been made where 1 ml of water and wastewater samples are
transferred to a 9 ml sterile saline blank by using aseptic technique.

3. Immediately after the 10-1 dilution has been shaken, uncap it and aseptically transfer 1 ml
to a second 9 ml sterile saline blank. This second blank represents a 10-2 dilution of the
original sample. Repeat the process two mores to produce a 10-3 and 10-4 dilution.

4. Shake the 10-2 dilution again and aseptically transfer 1 ml to one petri dish and duplicate it.
Do the same for the 10-3 and 10-4 dilutions.

5. Incubate all the petri dish at 35°C for 24 h.

6. At the end of incubation period, select all of the petri dishes containing between 30 and 300
colonies. Petri dish with more than 300 colonies cannot be counted and are designed too
many to count (TMTC). Petri dish with fewer than 30 colonies are designed too few to count
(TFTC). Count the colonies on each dish.

7. Calculate the number of bacteria (CFU) per milliliter by using the following formula;

1
 CFU/mL = Colonies counted x Dilution
Amount plated

3.0 RESULTS

Water Wastewater
Dilution
No. of colonies
-2
10
10-3
10-4

4.0 DISCUSSION

1. Identify the sources of bacteria in wastewater.

2. What are the typical bacteria number in water and wastewater? Compare the experimental
result with typical value.
3. Explain the role of microbe in the wastewater.

5.0 REFERENCE

Kumar S. (2012). Textbook of microbiology. JP Medical Ltd, London. 754 p.