Question

Exercise 6: Determination of growth of E.coli

At the conclusion of this exercise you should be able to:

• determine the growth of an E.coli suspension over time using turbidometric analysis and viable count method.
• determine the generation time of the growth of E.coli.
• understand the phases of growth of E.coli over a period of time.

Description of the experiments

This experiment will determine the growth of E.coli over a four hour time period. In order to record this growth, we will be using two methods of determining growth: turbidometry and viable count methods. To begin this experiment, a 1.0 ml sample of an overnight culture of E.coli was placed in a 100 ml flask containing sterile nutrient broth. To determine an initial timepoint (time zero), a 2.0 ml sample was removed, 1.0 ml of which was used to examine turbidometry (Absorbance) at 600nm wavelength and 0.1 ml of the sample was applied to a nutrient agar plate and spread onto the plate to determine viable counts.

The flask was subsequently placed in a 37⁰ C shaking water bath to allow the cells to grow. The flask was removed to obtain an hourly sample of culture, where 2.0 ml was aseptically removed from the flask to perform turbidometric and viable plate count methods (times 1 to 4 hours). To perform the viable counts over time, serial ten-fold dilutions of sample were made by adding 0.1 ml of sample to 9.9 ml of sterile water (this dilution is know as 1/10, or in scientific terms a 10¹ dilution). For this experiment, dilutions were made up to 10⁷ for each subsequent timepoint.

A 0.1ml sample was removed from dilutions and added to a nutrient agar plate. The sample was subsequently spread onto the surface of the plate covering all quadrants of the plate (this is known as a lawn inoculum). After allowing plates to dry, plates were inverted upside down and placed in an incubator at 37⁰C and removed after 18-24 hours. After incubation, plates showing colony numbers of 300 or less were counted for each time point. These are known as colony forming units (CFUs).

For a visual demonstration of how to perform serial dilutions and observe the appearance of colonies formed using this technique, watch How to Quantify Bacterial Culture Concentrations Using Serial Dilution and Plate Counts:

Results

Turbidometric recordings

Time (hours)	Optical Density (Absorbance) OD600 (nm)
0	0.005
1	0.03
2	0.21
3	0.29
4	0.35

Viable count (CFU) after 18 hours

Time (h)	# Colonies on plate	Dilution used	# cells/ml *	Log #cells/ml
0	10	10-3
1	184	10-2
2	179	10-5
3	56	10-6
4	64	10-5

*Use proper scientific notation

1. Tabulate the number of viable cells and record in the table above. Remember that each colony is equivalent to 1 colony forming unit (CFU) or 1 cell. Because you plated only 0.1 mL of sample per plate, you must also take into account a plating factor (PF) of 10. To calculate the number of cells per ml use this formula :

# colonies on plate X 1/dilution X PF = #cells/ml e.g. 33 cells on the 10-5 plate would be:

33 X 105 X 10 =3.30 X 107 cells/ml (33 000 000 cells/ml)

2. Provide a graph, plotting the turbidity recorded and the log number of viable cells/ml for each time point. A graph pad is provided below. NOTE: You can hand draw and insert a picture or insert excel graph

3. Determine the generation time by using the following formula :

Generation time =∆ t log 2

Log n – log N

Where: N = number of bacteria at a particular time point during log phase

n = number of bacteria at a second time point during log phase

∆t = time

Answer 1

ANSWER 1 :-

TIME (in hours)	Number of colonies on plate	Dilution used	Number of cells/ml	Log(Number of cells/ml)
0	10	10-3	1 x 105	11.51
1	184	10-2	1.84 x 105	12.12
2	179	10-5	1790 x 105	19
3	56	10-6	5600 x 105	20.14
4	64	10-5	640 x 105	17.97

Cells/ml at 0 hour = (10) x 103 x 10 = 1 x 105 cells/ml

Cells/ml at 1 hour = (184) x 102 x 10 = 1.84 x 105 cells/ml

Cells/ml at 2 hour = (179) x 105 x 10 = 1790 x 105 cells/ml

Cells/ml at 3 hour = (56) x 106 x 10 = 5600 x 105 cells/ml

Cells/ml at 4 hour = (64) x 105 x 10 = 640 x 105 cells/ml

ANSWER 2 :-

NOTE :- Respected Sir/Madam, please specify the formula for generation time so that the last question can be answered as according to me, both the generation number and the change in the microbial population over time is important to be considered. Please provide an upvote if the answer seems satisfactory.