Microbial Growth Curve of Bacteria << Back

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  1. To study the growth dynamics of a bacterial culture
  2. To study the different phases of a bacterial growth curve
  3. Use of spectrophotometer in microbial studies
  4. To calculate bacterial growth and turbidity
  5. To plot the growth curve and determine the generation time of a culture of E. coli

 

 

Materials:

1.       Tryptic soy broth

2.       Brain-heart infusion

3.       21 99-ml saline saline blanks

4.       Temperature controlled shaker incubator

5.       Spectrophotometer

6.       13 × 100 mm cuvettes

7.       Culture flasks

8.       Colony counter

9.       Petri plates

10.   1-ml and 10-ml sterile pipettes with pipettor

11.   Bunsen burner

12.   Wax pencil or Glass marker

13.   1,000-ml beaker

14.   Ruler

Procedure:

A.      Classical Method

1.       Prepare the saline solution separate it into 21 tubes and autoclave it

2.       Divide saline tubes into three groups and labelled them accordingly using wax pencil or glass marker. (lable each set as time of incubation (t = 0, t = 30, t = 60, t = 90, t = 120, t = 150, and t = 180) and dilution in each blank ((10–2, 10–4, and 10–6).

3.       Label 7 petri plats, the time of incubation and the dilution to be plated as above.

4.       Melt three tryptic soy agar tubes into water bath and cooled at 45 °C.

5.       Using a sterile pipette, transfer 5 ml of bacterial culture to the flask containing 100 ml of brain-heart infusion broth. Labelled it with alphabet A, and time, and date (absorbance (A) of this broth should be about 0.1 at 550 to 600 nm).

6.       Record the initial A, aseptically transfer 1 ml of culture to 99 ml of saline blank bottle and labelled it 10–2 and continue to serially dilute to 10–6

7.       Incubate the culture flask in shaker incubator at 37 °C and 120 rpm.

8.       Plate the 1.0 ml and 0.1 ml of zero time dilution into petriplate and labelled accordingly. Pour 15 ml of melted agar into each plate and mix gently.

9.       After 30 minute intervals, determine the A at 550 to 600 nm (suspend the culture thoroughly before taking sample).

10.   Repeat step 1 for 10–2 saline blank, perform step 2 (Serial dilution and labelling), and add melted agar (Step 8).

11.   Waite for media hardening and place them as an inverted passion in incubator at 37 °C for 24 hours.

12.   After 24 hour’s incubation take the petriplate out and count the colonies.

13.   Record the measurements, and plot the graphs of Log absorbances v/s incubation times, Log10 values of the bacterial counts v/s incubation times, and calculate generation time and mean growth rate constant.

14.   Report the results.