In this article we will discuss about the Minimum Inhibitory Concentration of Antibiotic for Bacteria.
Principle:
Antibiotic is a chemical compound produced by some microorganisms, which in low conc. affects the growth of other sensitive microorganisms. The minimum inhibitory conc. (MIC) of an antibiotic is that conc. which just inhibits the growth of a susceptible organism. This conc. primarily depends on the age of the organism concerned.
Materials:
(i) 24 hours broth culture of
(a) E. Coli,
(b) Staphylococcus aureus,
(c) Bacillus cereus.
(ii) Antibiotics:
(a) Penicillin,
(b) Chloramphenicol,
(c) Streptomycin.
(iii) Sterilised distilled water, culture tubes, pipettes.
(iv) Nutrient broth
Procedure:
A. Nutrient Broth Preparation:
Composition:
Beef extract – 6 gms.
Peptone – 10 gms.
Distilled water – 1000 ml.
350 ml. of nutrient broth was dispensed in 70 tubes, each containing 5 ml. of medium. The tubes are plugged and autoclaved.
B. Preparation of Antibiotic Solution:
Penicillin:
10 mg. of penicillin is dissolved in 10 ml. of sterilised distilled water in a tube and mixed in a vortex mixer. 2 ml. of this solution is taken by a sterilised pipette and then mixed with 98 ml. of sterilised distilled water in a flask, so that the conc. of penicillin becomes 20 µgm./ml. The whole thing is done aseptically.
Streptomycin:
10 mg. of penicillin is dissolved in 10 ml. of sterilised distilled water in a tube and mixed in a vortex mixer. 2 ml. of this solution is taken by a sterilised pipette and then mixed with 98 ml. of sterilised distilled water in a flask, so that the conc. of penicillin becomes 20 µgm./ml. The whole thing is done aseptically.
Chloramphenicol:
10 mg of chloramphenicol is dissolved in a small amount of alcohol and then the volume is made up to 10 ml by adding sterilised distilled water. Then the process is repeated as in penicillin.
C. Addition of Antibiotics in Media:
The tubes containing media of 5 ml. each are arranged in a test tube rack. They are labelled with 0 µgm., 1 µgm., 2 µgm……… 10 µgm. each of which designates the amount of antibiotic in that tube, containing 5 ml. media. Since the original antibiotic solution is of 20 µgm./ml. concentration, the required conc. of antibiotic is attained by addition of water in the following way.
D. Inoculation:
0.1 ml. of broth culture is added to each tube. Inoculation of one set of tubes, containing a particular antibiotic, with a particular organism is previously determined and it is decided from the fact that Staphylococcus aureus and B. cereus are susceptible to penicillin; E. coli and B. cereus are sensitive to streptomycin.
E. coli and S. aureus are sensitive to chloramphenicol etc. After, addition of the microorganism, it is mixed thoroughly in a vortex mixture and then is incubated at 37°C for 48 hours.
Observation:
After proper incubation, the tubes are checked to find out, in which tube growth has occurred and in which it has not. The conc. of antibiotic in which growth has just stopped gives the MIC of the antibiotic for that organism.
Result:
A. MIC for Penicillin:
B. MIC for Streptomycin:
C. MIC for Chloramphenicol:
