Principle
Antibiotics are chemical compound produced by one type of
microorganism which at low concentration selectively inhibit or kill another
organism. The sensitivity of microbes to antibiotics can be expediently tested
in the laboratory by lb broth diffusion method. In this method, the organism to
be tested is heavily inoculated on the liquid medium in Test-Tubes. All
Test-Tubes are filled with liquid media and varying concentration of
antibiotics. The inoculated tubes are observed after incubation for a definite
period of time forthe zone of inhibition. The diameters of zone of inhibition
are measured. The inhibition is due to the diffusion of antibiotic from the
media an area of high concentration to an area of low concentration. The
smallest amount of the antibiotic needed to inhibit the growth of microorganism
is called as MIC.
Requirements
- Lb broth media
- Sterile Test-tubes
- Ampicillin (antibiotics)
- Micropipette
- Bacteria culture
Procedure
1. Prepare serial dilution of a ampicillin antibiotic as fellow:
12.5mg + 25ml DDW -- 0.5mg/ml............(a)
5ml (A)+ 5ml DDW -- 0.20mg/ml..........(b)
5ml (B)+ 5ml DDW -- 0.125mg/ml....... (c)
5ml (C)+ 5ml DDW -- 0.0625 .............. (d)
5ml (D)+ 5ml DDW -- 0.03125..............(e)
5ml (E)+ 5ml DDW -- 0.015625.............(f)
5ml (F)+ 5ml DDW -- 0.0078125............(g)
5ml (G)+ 5ml DDW -- 0.00390625.......... (h)
5ml (H)+ 5ml DDW -- 0.001953125.........(i)
2. Arrange sterile tubes and no. Them (A) to (I)
3. Take Equal amount of lb broth media in test tubes (3ml).
4. Take 10ml bacterial sample and mixed it properly an all the test tubes (1ml in each test tube).
5. Ampicillin solution.
6. Incubate the tubes at 37ºC for 24 hours and observe it.
Observation
Zone of inhibition was observed around the well containing
varying concentration of ampicillin.
|
S.No. |
Test-Tube No. |
(OD) after Incubate
the tubes at 37ºC for 24 hours and observe it at 610nm, |
|
1. |
A |
0.904 |
|
2. |
B |
0.559 |
|
3. |
C |
0.712 |
|
4. |
D |
0.748 |
|
5. |
E |
1.153 |
|
6. |
F |
1.219 |
|
7. |
G |
1.122 |
|
8. |
H |
0.985 |
|
9. |
I |
1.029 |
Result
The minimum inhibitory concentration expressed as the lowest
concentration of ampicillin dilution for given bacterial culture.
No comments:
Post a Comment