EXPERIMENT # 2

BACTERIAL GROWTH CURVE

Student #: 300769832
Course code: BI 303-061

Submitted by: Hitesh chavada

Submitted to: Marina Ioselevich
Date: 07th oct,2014

Result:
Table 1: measurement of absorbance with respect to time
Time (min)
0
5
15
25
35
45
55
65

Absorbance OD (600)
0.237
0.242
0.272
0.324
0.401
0.482
0.557
0.677

Table 2: measurement of cfu/ ml with respect to time (Group data)

Incubation Time
(min)
0
5
15
25

35
45

Colony counts (cfu/plate)

Cfu/ml

10-5
TNTC
TNTC
TNTC
TNTC

10-6
76 and 58
TNTC
TNTC
175

10-7
0
31 and 50
147
26

10-6

10-7

10-8

TNTC
TNTC

23
32

0
0

Count range for spread plate is 20-200 cfu/plate

6.7x 108
4.1x108
1.5x109
2.2x108

2.3x106
3.2x106

Calculation:
Time 0 min:
Cfu /ml = count X dilution factor
= (76 + 58)x 106
= 6.7x 108cfu/ml
Time 5 min:
Cfu /ml = count X dilution factor
= (31 + 50)x 106
= 4.1x108cfu/ml
Time 15 min:
Cfu /ml = count X dilution factor
= 147x 107
= 1.5x109cfu/ml
Time 25 min:
Cfu /ml = count X dilution factor
= (175x 106 + 26x107 )
= 2.2x108cfu/ml
Time 35 min:
Cfu /ml = count X dilution factor
= 23x 107 +
= 2.3x106cfu/ml
Time 45 min:
Cfu /ml = count X dilution factor
= 32x 107
= 3.2x106cfu/ml

Table 3: measurement of cfu/ml with respect to time (Class data)

Time (min)
0
10

Cfu/ ml
3.50x106
4.73 x 106

20
30
40
50

2.00 x 10+7
6.45 x 10+7
1.15 x 10+8
2.05 x 10+8

Plot of absorbance Vs. Time

0.8

absorbance

0.7
0.6
0.5

Series1

0.4

Linear (Series1)

0.3
y = 0.0067x + 0.1934
R = 0.9604

0.2
0.1
0
0

20

40

Time(min)

60

80

Graph 1 : Linear graph of Time (minutes) Vs Absorbance OD (600 nm)

The linear graph of the absorbance against time is plotted. The absorbance shows a linear
increase in graph as the number of cells increases with time.

semilog graph of absorbance Vs. Time

absorbance

100

Series1
10

Linear (Series1)

1
0

10

Time(min)

Graph 2 : Semi log graph of Time (min) Vs absorbance OD (600 nm)

The absorbance readings are plotted against time in minutes. The semi log reading of
absorbance shows a steady increase with respect to time as the number of cells increases.

1.40E+08
1.26E+08
1.20E+08

1.15E+08

Colony Counts(cfu/ml)

1.00E+08
8.00E+07
6.45E+07

6.00E+07
4.00E+07
2.00E+07

2.00E+07
4.73E+06

3.50E+06

0.00E+00
0

10

20

30

40

50

60

-2.00E+07
-4.00E+07

Time(mins)

Graph 3 : Linear graph of Time (min) Vs colony counts (cfu/ml)

The class data of cfu/ml is plotted against time in minutes is plotted. The class data shows a
steady growth in number of cfu/ml with respect to time.

1.00E+09
1.00E+08
2.00E+07

1.00E+07
4.73E+06

3.50E+06
Colony Counts(Cfu/ml)

1.26E+08

1.15E+08

6.45E+07

1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
0

10

20

30

40

50

60

Time(min)

Graph 4 : Semi log graph of Time (min) Vs colony counts (cfu/ml)

The semi log graph of cfu/ml is plotted against time in minutes. The class data shows
steady increase in the number of cfu/ml as the number of cells per ml increases with
respect to time.

3)Generation time:
Direct method
GT = t/ n
n= log N- log N0/0.301
where, N= number of cells at the end of time interval
N0= number of cells at time zero
= log (1.15 x 108) - log (6.45 x 107)
= 8.06-7.80 / 0.301
= 0.86
And
Gt= t/ n
= 14/0.86
= 16.27 min/ gen

Class data of absorbance at various time interval.

Group Name : Rupal

Table 1: measurement of absorbance with respect to time
Time (min)
0
5
15
25
35
45

Absorbance OD (600)
0.238
0.283
0.420
0.508
0.600
0.750

Group Name : Kathan

Time (min)
0
5
15
25
35
45
55
65

Absorbance OD (600)
0.252
0.271
0.293
0.364
0.449
0.530
0.615
0.715

Group Name : Neha

Time (min)
0
10
20
30
40
50
60
70

Absorbance OD (600)
0.257
0.283
0.330
0.406
0.502
0.622
0.666
0.755

4) Question:
A) The growth curve studies about the growth of viable bacteria when grown under
optimum conditions of pH, temperature and broth. Under such conditions, the bacteria will
reproduce and result in a generation. Therefore, this curve helps us to calculate the time
required for a growth cycle under regulated conditions. It gives the time required by the
bacteria to double in number (Growth curve, 2012).

b) Limitations: The exponential growth curve is only a part of the life cycle of bacteria, it
does not represent the pattern in which the bacteria will grow in environment. It gives the
results of those only when grown under controlled conditions. It takes lot of time and
samples are collected every half-an-hour. The limitation in lab procedure was that samples
were collected at shorter intervals due to time scarcity (Todar, Growth of Bacterial
Populations, 2009).

Discussion:
Bacteria can under goes to cell division by binary fusion. The experiment is performed
using overnight culture of E.coli (A) to determine the bacterial growth curve. Therefore, the
bacteria passed their lag phase and now they are in their log phase. A series of dilution is
performed and culture is inoculated at of 0,5 ,15,25,35, and 45 min the interval of 10 min
time.
The graph potted of cfu /ml against time interval helps us to determine the generation
time. The exponential growth of bacterial culture is expressed as generation time which is
calculated as per the given equation.
Class data shows increase in absorbance which suggests exponential growth of bacteria.
Group data does not shows exponential growth of bacteria. The class data of absorbance
also shows a steady rise in the absorbance readings measured at different time intervals
0.10,20,30,40,50 and 60 minutes. Under favorable conditions E.coli proliferate and there is
increase in cell density leading to higher absorbance. Reason behind the incorrect data was
poor aseptic technique and poor skill of technician. Another reson may be poor dilution
schedule. The bacterial cells under favorable conditions doubles at regular intervals. The
growth is by geometric progression 1,2,4 etc or 20 ,21 ,22 . 2n (where n= number of
generations). This type of growth is called exponential growth.

Absorbance of the culture was taken by the spectrophotometer and absorbance reading
shows a steady rise with respect to the time. The bacterial growth count is expected to
increase exponentially as per the standard growth curve and thus the absorbance increases
due to more amount of cells present in the sample. The results of our group data shows
steady rise in the absorbance with respect to time. This suggests that there is exponential
growth of the bacterial count in the sample with respect to time in accordance to the
standard curve.

Conclusion:
The standard bacterial growth comprises of four stages lag phase, log phase, stationery and
death phase. The overnight E.coli culture expected to be in the log phase showing an
exponential growth.
As per the cfu/ml data the class data shows the exponential growth with respect to time.
But, the results of our group data does not show the exponential growth pattern .the
possible reason behind this may be due improper dilution practice and poor skills and
some contamination was observed in the plates it may be due to the poor aseptic
technique.

References:
Jones, T. (2004, January 1). The behaviour of log phase Escherichia coli at temperatures
that fluctuate about the minimum for growth. Retrieved October 14, 6, from
http://www.ncbi.nlm.nih.gov/pubmed/15287878
Lab Manual 302 Fall 2014.Retrieved from
https://e.centennialcollege.ca/d2l/le/content/158221/viewContent/1894929/Vie
w
The growth of bacterial population.Retrieved on oct 07,2014 from
http://textbookofbacteriology.net/growth_3.html
Todar, K. (2004). The Growth of Bacterial Populations. In Todar's online textbook of
bacteriology (p. 3). S.l.: Kenneth Todar :.