Diagnostics of infectious diseases
Lab: 6
Antimicrobial Susceptibility Testing
Minimal Inhibitory Concentration (MIC) Test
2.9.1.1
Principle
The agar dilution technique is used to measure qualitatively the invitro activity of an
antimicrobial agent against the test bacteria. In this method, graded amounts of
antibiotics are incorporated in agar plates and inoculated in spots with the organisms
under study. If the organism under study is susceptible to the incorporated antibiotic,
no bacterial growth is expected in agar plates with higher concentrations of the drugs.
Bacterial growth is observed as the antibiotic concentration in the agar plate
diminishes. Inhibition of growth at the minimum or lowest concentration of antibiotic
is regarded as the end point.
2.9.1.2
Procedure for MIC
The minimal inhibitory concentration (MIC) test was performed using MuellerHinton Agar (MHA), which is the best medium and gives satisfactory growth of most
bacterial pathogens. The media was made by adding 3.8 gram of Muller Hinton Agar
in 100 ml of distilled water. The agar was mixed thoroughly in distilled water and
kept in autoclave for 45 min at 121ºC. After 45 minutes the media was kept outside
and allowed it to cool to the extent that it should not solidify. After cooling the
calculated amount of media was withdrawn from the flask. The formula used to
calculate the volume of the media to be replaced by equal amount of stock solution of
the drug is given in Figure 2.9.The amount of media that was taken out from the flask
was calculated according to the different concentrations which I had selected to find
out the range of MIC. The amount of media that was withdrawn and the amount from
stock solutions of drugs that was added in the media are shown in Appendix-B (Table
2.1, Table 2.2, Table 2.3 and Table 2.4) for drugs during the process of calculating
MIC.
After adding the calculated drug from stock solution in MHA, the media was poured
in sterile petri dishes. These petri dishes were labeled according to the names and
concentrations of drugs made. These plates were kept at room temperature and
allowed to solidify. When the media was solidified then the plates were placed in
incubator at 37º C for 24 hrs.
Formula Used To Make Different Concentrations Of Drug: C1V1=C2V2
FIG
URE
2.9
C1 = Concentration of Media that is selected to calculate MIC e.g. 2000, 2500 etc
For
mula
used
V1 = Volume of Media that is needed for MH agar plates
to
calcu
late
C2 = Concentration of Stock Solution
the
volu
V2 =
Volume of Stock Solution that is added after removing same volume from media to
achieve desired concentration of media
me
of
the
medi
a
2.9.1.3
Preparation of Antimicrobial Agent Stock Solution
Stock solution was made by dissolving specified amount of drug in pararticular solvent. The
drugs which were used for calculating MIC in this procedure were commonly used antibiotics for
UTI. The list of these antimicrobial agents according to their solvent is given in Table 2.5.
2.9.1.3.1
Preparation of Stock Solution of Sulphamethoxazole
Stock solution of Sulphamethoxazole was made by dissolving 0.5g of the drug in 2.5ml of
Sodium Hydroxide, when the drug was dissolved then the distilled water was added to make total
volume up to 10ml in a sterile falcon tube. This stock solution was kept in refrigerator for use.
2.9.1.3.2
Preparation of Stock Solution of Trimethoprim
Stock solution of Trimethoprim was made by adding 0.5g of the drug in 2.5ml of Acetic acid,
when the drug was dissolved then the distilled water was added as a diluent to make total volume
up to 10ml.
2.9.1.3.3
Preparation of Stock Solution of Cefotaxime
Stock solution of Cefotaxime was made by adding 0.1g of the drug in 10ml of distilled water.
2.9.1.3.4
Preparation of Stock Solution of Ciprofloxacin
Stock solution of Ciprofloxacin was made by adding 0.1g of the drug in 10ml of distilled water.
2.9.1.4
Preparation of Inoculum
Inoculum was prepared from a pure 18-24 hour bacterial culture, 4-5 isolated colonies (to
minimize the risk of picking bacteria which have lost their resistance) was picked and subculture
to a tube having 3 ml of nutrient broth. This nutrient broth was made
TABLE 2.5
List of Solvents and Diluent
List of Solvents and Diluents needed for the Preparation of Stock Solutions of
Antimicrobial Agents
S/NO
ANTIMICROBIAL AGENT
SOLVENT
DILUENT
SODIUM HYDROXIDE (NaOH)
DISTILLED
1
SULPHAMETHOXAZOLE
2
TRIMETHOPRIM
ACETIC ACID 2.5 ml
3
CEFOTAXIME
DISTILLED WATER
4
CIPROFLOXACIN
DISTILLED WATER
2.5ml
WATER
DISTILLED
WATER
DISTILLED
WATER
DISTILLED
WATER
by adding 0.3 gram of beef extract and 0.5 gram of peptone in 100ml of distilled water .Then this
mixture was autoclaved for 45 minute at 121ºC.
Inoclum was made in glass test tubes by adding few drops of 24 hrs bacterial culture in 1ml of
Nutrient broth .This was kept in incubator for 24 hrs at 37ºC for growth of bacteria.
2.9.1.5
Preparation of antimicrobial agar plates
The empty sterile plates were labeled in order to identify the antimicrobial agent and their
concentrations. The label was placed on the upper portion of the bottom side of the petri dish to
ensure that the plate is inserted at the correct point. A scheme was drawn to locate each bacterial
strain on the outer layer of bottom of petri dish. Before putting the drop of inoculum on drug
containing MHA plates the Turbidity of these test tubes were checked by matching it with
standard Macfarland solution in a test tube. When the inoculums in test tubes were found turbid
as compared to Macfarland solution, then they were diluted by adding more Nutrient broth into
it. A drop of 5 µl of fresh inoculums (prepared 24 hrs before) was placed on drug containing
MHA plates. This drop was placed on the specified spot as labeled on the plate according to the
sample number. The whole procedure was carried out under aseptic condition near the flame.
There was at least two replicate of each concentration. Then these plates were kept for drying so
that drop of inoculum may not spread around, otherwise it become difficult to read the result.
The method of labeling and labeled plate is shown in Figure 2.10 & 2.11, respectively.
FIGURE 2.10.
Method of Labelling
FIGURE 2.11.
Labelled Plate
FIGURE 2.12
2.9.1.6
MH Agar Plates of Different Concentrations for MIC
Control agar plates/Drug-free agar plates
Prepare control agar plates by pipetting 10 ml of MHA into a sterile petri dish. No antimicrobial
agent was added into it. MH agar plates of different concentration for MIC are shown in Figure
2.12.
2.9.1.7
Inoculation sequence
The plates were inoculated according to the concentrations made, starting from lowest to highest
concentration.
2.9.1.8
Incubation
The inoculated agar plates were kept at room temperature until the moisture in the inoculum spot
was absorbed by the agar or until all spots became dry. The plates were incubated in an inverted
position at 370C for 24 hours (The incubation time is extremely important to obtain reliable end
points when reading the results).
2.9.1.9
The
Reading of MIC Values
plates
were
observed
after
24
hours
to
read
the
results.
When the growth was seen at the specified spots, bacterial growth was again cultured on MHA
plates. The whole process was repeated again. When there was no growth on the labeled spots it
was considered the MIC value in relation with the particular samples. Bacterial growth was
observed in control plates as well (Note: if there would have been no growth, then test would
have to be repeated). MIC values were recorded at lowest concentration of antimicrobial agent.
2.9.2 Calculation of Minimum Bactericidal Concentrations
Once an MIC has been performed a minimum bactericidal concentration (MBC) can subsequently be
determined. The MBC is set up with subcultures made from each MIC well that appeared visually clear.
These subcultures from MIC plates were mixed in 1 – 2 ml of nutrient broth in test tubes and kept in
incubator for 24 hrs at 37ºC for the growth of bacteria. After 24 hrs when these test tubes were
observed, when no growth was seen it means that the concentration of that particular plate was MBC
value not MIC value. But if growth was seen in this subculture it was suggesting that it was MIC value
not MBC value. So to calculate MBC further concentration was made to check the exact value.
MIC values cannot be determined without following up with an MBC test. After incubation, the MBC
dilution tubes will either be turbid with growth or they will be clear. A tube without turbid growth will
result from one of two possible events. Either the challenge microorganisms were killed by the test
product, or they were inhibited by the test product. A subculture tube that becomes turbid after
incubation indicates that the test product was inhibitory at that dilution for that particular organism. If
the subculture tubes remain negative after incubation, this indicates that the test product is lethal at
that concentration for that particular organism.