This document summarizes an experiment on bacterial evolution in response to antibiotic exposure. The experiment exposed E. coli bacteria to increasing concentrations of tetracycline antibiotic over multiple generations. It was found that after 5 days of growth, the bacteria developed resistance and were able to grow in higher concentrations of tetracycline compared to the original bacteria. Their ability to survive and reproduce in the presence of antibiotics increased over generations through genetic changes, demonstrating how bacteria can evolve resistance to drugs over time through exposure.

1. Evolution in
Bacteria by the
use of antibiotic
Swarnaprava Behera
10MS84,
Sujeet Kumar Choudhary
10MS54
02-04-2012

2. Project Overview
 Bacterial evolution-
 Effect of antibiotic – it acts on three
parts of the cell
• Bacterial protein synthesis
• Nucleic acid replication
• Cell wall biosynthesis
How tetracycline acts: protein
synthesis inhibition. T-RNA to M-RNA
ribosome complex(30s).

3. How cells becomes resistant
 When exposed, most dies, some
survives having genetic recombination
processes.
 They reproduce & all the descendants
will have same resistance power.
 How bacteria responds to it?
1)enzymatic
inactivation (tetracycline)
2)Ribosomal protection
3)Efflux

4. Key terms
o E.Coli :- Gram negative, rod shaped.
• Why we choose ?
well developed(lab environment)
easily available
• Life cycle- 20 min.
o E. coli- efflux & ribosomal protection.
o Tetracycline :-subclass of polyketides
• MIC- lowest conc. Of antibiotic which
inhibits visible growth after overnight
incubation.

5. Tetracycline
Click to add text
Picture Courtesy - http://www.chm.bris.ac.uk/motm/tetracycline/mol.htm
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6. Statement of the Problem
o Expectation :
When bacteria are exposed to harsh
condition(bacteria may die) repeatedly,
after some generation they can have
developed the capability to survive
under same situation.

8. Materials Required
Reagents
o Media – LB and LB Agar
Composition :
 0.5 % Peptone
 0.3 % beef extract/yeast extract
 1.5 % agar
 0.5% NaCl

9. Materials Required
 Reagents:
o Antibiotic – Tetracycline(1mg/ml)
o Bacteria- E. Coli
Glass and Plastic Apparatus:
 Beaker
 Conical Flask (250 ml)
 Test Tubes
 Measuring Cylinder

10. Materials Required
 Glass and Plastic Apparatus:
 Quartz cuvette
 Pipette (of diff. range)
Others – cotton, aluminum foil, rubber
band etc.
Electrical Equipments:
 Spectrophotometer

11. Materials Required
Electrical Equipments:
 Incubator (37’c,150 rpm)
 Autoclave Machine(37’ c.45 min.)
 Laminar Air Flow

12. Procedure
Day-1
Fresh culture of overnight grown
bacteria were taken and OD was
measured .
1 OD @ 600 nm ~ 1 X 109 CFU/ml
Bacteria were grown at 5 different
concentrations of antibiotics.
 Concentration of bacteria was kept
equal (~ 1 X 106 CFU/ml) throughout
the experiment.

13. Procedure
Each test tubes were duplicated.
10 Petridis plates were prepared with
LB Agar media.
Fig.1-Test tubes with media only Fig.2- Petridis plates inside Laminar Air Flow

14. Data set-1
Media
[NB
Media]
(ml)
Antibiotic
(Tetracycline)
(ug/ml)
Bacterial
concentration
(E.Coli)
(CFU/ml)
Sl.
No.
5 0.1 1 X 106 1
5 0.5 1 X 106 2
5 1.0 1 X 106 3
5 2.0 1 X 106 4
5 5.0 1 X 106 5
OD of the fresh culture of
Bacteria
1.44

15. Procedure
Day-2
Growth were observed and MIC was
determined.
MIC = 0.5 ug/ml
Fig.3- Grown culture at lowest concentration(0.1 ug/ml)

16. Procedure
OD was measured(0.448 &0.450) of
the grown bacteria at lowest antibiotic
concentration(0.1 ug/ml).
At lowest antibiotic concentration, the
previously grown bacteria at
0.1ug/ml(antibiotic concentration)
were grown overnight. [Named M1 and
P1].
Again the bacterial concentration was
~ 1 X 106 CFU/ml

17. Procedure
P1 was the name of duplicate test
tube.
 The amount of media was same for
M1 & P1
Fig.4- Grown bacteria in M1 and P1

18. Procedure
Day-3
Bacteria were taken from M1and P1
and OD were observed.
 Again at lowest concentration it was
grown overnight and named it M2 And
P2.
Like this the same procedure was
repeated for 5 days.(For further
generations)

19. Data Set-2
Bacteria
taken
from
LB Media
(ml)
Antibiotic
(ug/ml)
Bacteria
(CFU/ml)
Name Name Day Sl.No.
OD of M1 = 0.645 OD of P1 = 0.639
M1 5 0.1 1 X 106 M2 P2 3 1
OD of M2 = 0.457 OD of P2 = 0.448
M2 5 0.1 1 X 106 M3 P3 4 2
OD of M3 = 1.630 OD of P3 = 1.600
M3 5 0.1 1 X 106 M4 P4 5 3
OD of M4 = 1.604 OD of P4 = 1.590

20. OD of different generations
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
1 2 3 4 5 6 7
Absorbance
No. of days

21. Procedure
Bacteria were taken from M4 and OD
was measured.
Again this bacteria were grown
overnight at 5 different concentration
of antibiotic.
Data set is same as Data Set-1.
Next day growth of the bacteria were
observed.

22. Final observation
Fig.4 – Growth of the bacteria at higher concentration(0.5 ug/ml)

23. Pictorial Comparison
Growth of bacteria before
evolution
Growth of bacteria after
evolution
Fig.3 Fig.5
0.1
ug/ml
0.1
Ug/ml
0.1
ug/ml
0.1
ug/ml
0.5
ug/ml
0.5
ug/ml

24. Observations
Growth of bacteria at higher
concentration of antibiotic.
Increment in MIC.(From 0.5 to 1.0
ug/ml)
Bacteria has developed resistant
power.
 Increased OD.

25. Conclusion
 Bacteria were evolved over time.
 From observation we get the expected
result.
 Difference is due to 3 day gap.
 2nd one is for they have developed a
high resistance power.

26. How related to Social issues.
 Diseases are not cured.
 Bacteria getting resistant.
 Without prescription only 1 dose will
do them resistant.
 2 or 4 dose according to the Dr. can
kill the bacteria(when life time Is
known).
Super germs.
MRSA-Multidrug resistant
staphylococcus Aureus.

27. Limitations
Our convention 1 OD @ 600 nm ~ 1 X
109 CFU/ml is an approximate value.
 Many generation were kept for three
days.
We took only one species of bacteria.
Triplication

28. More Information
Related readings
• Evolutionary Biology by Douglas J.
Futuyma
• Useful Web sites
http://www.chm.bris.ac.uk/motm/tetracycline/m
ol.htm

29. Acknowledgement
We are sincerely thankful to Dr. Anuradha
Bhat and Dr. Annagiri Sumana for
encouraging us to do this project.
We are also thankful to Gregor P. Jose (PhD
Student) and Shudhansu Da for their kind
help in doing our experiment.

30. THANK YOU !

31. Questions ?