PHR 252

MICROBIOLOGY II
WEEK 2
Evaluation of antimicrobial agents:
Antimicrobial agents may be antibiotics or non-antibiotics
I. Qualitative evaluation of antibiotics:

Antimicrobial spectrum.

Antibiotic sensitivity (Antibiotic Disk Susceptibilities).

Combined antimicrobial activity.
II. Quantitative evaluation:
1. Of Antibiotics:

MIC by broth dilution.

MBC by broth dilution.

MIC by cup-plate technique (agar diffusion).

Antibiotic assay.
2. Of Non-antibiotics:

Evaluation of oral antiseptic by determination of killing
(extinction) time.

I.Evaluation of the bacteriostatic activity by

Quantitative method
Determination of MIC by Broth dilution method
How to express dilution:
A two-fold dilution reduces the concentration of a solution by a factor
of two that is it reduces the original concentration by one half. A
series of two-fold dilutions is described as two-fold serial dilutions.
Example: if we have a 1% solution
1%

2fold dilution

0.5%

2fold dilution

0.25%

2 fold serial dilution

1

2fold dilution

0.125%

..

fold serial dilution:

Fold dilution

Agent : Diluent

2 fold

: 1

3 fold

: 2

10 fold

: 9

n fold

: no of fold-1

Examples:
If we want to make 2 fold dilution of a 1% solution with a final
volume which equals 2 ml
2 fold means 1 : 1 or 2 :2
N.B. the final volume = the volume of the diluent
i.e. we will add 2 ml of the diluents to 2 ml of the agent.
If we want to make 5 fold dilution of a 1% solution with a final
volume equals 8 ml
5 fold means 1: 4 or 2: 8
i.e. we will add 8 ml of the diluents to 2 ml of the agent.

Determination of MIC by Broth dilution method

The tube dilution test is the standard method for determining
levels of resistance to an antibiotic. Serial dilutions of the
antibiotic are made in a liquid medium which is inoculated with
a standardized number of organisms and incubated for a
prescribed time.
The lowest concentration of antibiotic
preventing appearance of turbidity is considered to be the
minimal inhibitory concentration (MIC).

 Additionally, the minimal bactericidal concentration (MBC) can

be determined by subculturing the contents of the tubes onto
antibiotic-free solid medium and examining for bacterial growth.
Principle:
After preparation of different concentrations of the antimicrobial agent
in nutrient broth (by using the broth dilution method), we inoculate
them with the tested organism. Then after incubation we can
determine the MIC by choosing the lowest concentration in which no
growth occurs.

Practical
Determine the MIC of 1% phenol using the broth dilution method with
a total final volume of 2 ml.
Note: MIC determination is always done by 2 fold serial dilution of the
antimicrobial agent.
2 fold serial dilution, i.e. the ratio is 1 :1
And because we need the final volume = 2 ml so the ratio can be 2 :2
i.e. we add 2 ml phenol to 2 ml diluent (broth) to produce 2 fold
dilution, then we repeat this step
Here, we will prepare different concentrations of phenol by 2
fold serial dilution, i.e. each concentration is half the preceding
one.
(1%
0.5%
0.25%
0.125%
..etc.)
Volume of the agent in test tube = volume transferred + volume
of diluent.

Procedures:

2 ml

2 ml

2 ml

2 ml

2 ml
2 ml

P
Discard 2 ml

1%
Bacterial
suspension

0.5 %

1%
phenol

0.25 %
0.125 % 0.0625 %
2 ml nutrient
broth

inoculation

No

1. Number sterile capped test tubes 1 through 5. All of the

following steps are carried out using aseptic technique.
2. Add 2 ml of 1% phenol to the first and second tube.
3. Tubes from 2-5 contain 2 ml nutrient broth.
4. Transfer 2 ml from the second tube to the third tube.
5. Mix the contents of this tube and transfer 2 ml to the fourth
tube.
6. Continue dilutions in this manner to tube number 5.
7. Remove 2 ml from tube 5 and discard it.
8. Inoculate each test tube by using the given bacterial
suspension.
9. Incubate all tubes at 37oC.
10. Observe the results.
Unit of MIC:
Unit of MIC must be in (g/mL), any other given unit must be
converted to this universal unit.
If MIC = 0.25% i.e. 0.25g/100ml
1g =106 g
0.25g/100ml = 0.25X 106 g/100ml
=0.25X 104 g/ ml
N.B.
The lower the MIC, the higher the activity of the agent against
the organism. i.e. the efficiency of the agent increases with the
decrease of the MIC.
Drawbacks of the broth dilution method:
1- Although the tube dilution test is fairly precise, the test is
laborious because serial dilutions of the antibiotic must be
5

made and only one isolate can be tested in each series of

dilutions.
2- Since it is liable for contamination (which could not be detected
by the naked eye), this can act as a source of error.
2) Determination of MBC by broth dilution method:
MBC can not be determined except after determination of MIC.
Principle:
The minimal bactericidal concentration (MBC) can be determined by
subculturing the contents of the tubes of MIC showing no growth onto
antibiotic-free liquid medium and examining for bacterial growth.
Procedure:
1) We the take the clear test tubes of MIC.
2) Transfer one loopful from each test tube into 5 mL sterile
nutrient broth. (This relatively large volume is used to eliminate
the effect of the antimicrobial agent).
3) Incubate the test tubes at 37C for 24 hours.
4) Record the results after incubation.
Figure (1): shows 3 clear test tube, indicating the presence of a
bactericidal agent (MIC=MBC).

Figure (2): the third test tube in the figure became turbid due to the
refreshment of the organism after the removal of the effect of the
antimicrobial agent (MBC > MIC) indicating the presence of a
bacteriostatic agent.

MBC

PHR 252

Microbiology I
Week 5 Practical
Determination of MIC by Broth dilution method
Student Name:

Group:

UID:

Determine the MIC and the MBC of the given antimicrobial agent.
MIC =..%
MIC in g/ml=.

MBC=.%
MBC in g/ml=.

Comment: