FACULTY OF APPLIED SCIENCES

KUALA LUMPUR CAMPUS

Semester:     [ ✓ ] May [ ] September [ ] January (please tick “✓”)

Academic Year: 2019  
Course Code & Title: AACB 3233 MICROBIOLOGY
Programme: Diploma in Science (Chemistry and Biology )
Student’s Name (Registration Number): 1. Chee Yong Chun (18WLD01499)
2. Chin Jia
Yue (18WLD03979)
3. Ho Zhi
Wei (18WLD03928)
4. Ng Xue Qi
(18WLD02318)
5. Wong Pui
Mun (18WLD03931)

Submission Date:
 
Signature(s):_________________________________________________________________

Name(s):____________________________________________________________________
 Date: ___________________

Experiment 6
Title: Bacterial Growth
Objectives:
1) To learn how to calculate and prepare bacterial dilution.
2) To identify why and when bacteria counting might be needed
3) To learn how to operate a spectrophotometer.
4) To learn how to construct and evaluate a standard curve using optical density vs. number
of cells in suspension.

Material and apparatus:

Broth culture of E.coli, 4 nutrient agar plate, 4 test tube, cuvette, conical flask, pipette,
spectrophotometer, nutrient broth, saline solution, bunser burner, incubator, test-tube rack.

Procedure:
Part A- Turbidimetric Measurement of Growth
1. 5 cm3 of E.coli was inoculated into the conical flask with 20 cm3 of nutrient broth.
2. 3 cm3 of sterile nutrient broth was poured into another cuvette as blank before measuring
the absorbance of the culture.
3. After mixing, 3 cm3 of the culture was pipetted into the cuvette to measure the
absorbance by using spectrophotometer and the absorbance was recorded in table 1.
4. The culture in the cuvette was poured back to inoculated conical flask every time after
measure the absorbance.
5. The inoculated conical flask was incubated in 37 °C incubator.
6. Step 2 to 4 were repeated at every 15 minutes until 1 hours.
7. The culture was placed back into the incubator between the reading.

Part B- Dilution Culturing

1. 4 test tubes were labeled A, B, C and D.
2. 9.9 cm3 of saline solution was added into each of the test tube.
3. 0.1 cm3 of original inoculum of E.coli was added into test tube A.
4. The test tube was shaken gently to mix the inoculum with the saline solution.
5. 0.1 cm3 of the inoculum from test tube A was pipetted and added into test tube B.
 6. The test tube was shaken gently to mix the inoculum with the saline solution.
7. 0.1 cm3 of the inoculum from test tube B was pipetted and added into test tube C.
8. The test tube was shaken gently to mix the inoculum with the saline solution.
9. 0.1 cm3 of the inoculum from test tube C was pipetted and added into test tube D.
10. The test tube was shaken gently to mix the inoculum with the saline solution.
11. For each of the test tube, 0.1 cm3 of the inoculum was pipetted to perform spread plate
method by using nutrient agar to culture the bacteria in each of the tube with performing
aseptic technique.
12. The inoculated plates were incubated at 37 °C for 24 hours.

Result:
Table 1 the number of CFUs for each plate.

Plate CFU Total Dilution Factor(DF)(ml) Stock conc. (CFU x DF)

(CFU/ml)
1 TMTC 1 x 10-2 NA
2 TMTC 1 x 10-4 NA
3 71 1 x 10-6 7.1 x 108
4 2 1 x 10-8 NA

Table 2 the absorbance of E.coli at 600nm at each of the following time point.

Time (minutes) Abs. 600nm

0 0.223
15 0.255
30 0.310
45 0.347
60 0.367
 Graph 1: E. coli Growth Curve
Discussion:
1. What is CFU?
CFU is colony-forming unit, a unit used to estimate the number of viable bacteria or
fungal colonies in a sample (Tortora et al. 2010).
2. Is the E. coli growth linear or exponential ?
Linear.
3. Based on the results from the graph above what is the approximate generation time
of E. coli during the logarithmic growth phase in this experiment ?
Y = mx + c, m = 0.0025, c = 0.2244
Y = 0.0025x + 0.2244
When Y= 0.4, x = 70.24 min
When Y= 0.2, x = -9.76 min
Generation time of E.coli
= 70.24min - (-9.76min)
= 80 min
The approximate generation time of E. coli during the logarithmic growth phase is 80 min.

Reference:
1. Tortora, G.J., Funke, B.R., Case, C.L. (2010). Microbiology An Introduction. 10th
edition. San Francisco, CA: Pearson/Benjamin Cummings.