A scientific presentation that describes the steps to perform antimicrobial sensitivity assay for water insoluble compounds and the type of data that can be obtained.

1. Dr. Leena Abdulaziz Muhamed
OIU - Faculty of pharmacy
Department of Pharmacology
Microbiology experiments

2. Content
 Introduction
 Materials and equipments
 Experimental steps
 Presenting results

3. Introduction
 Discovery of new antimicrobial agents is one of the most
important strategies for fighting antibiotic resistance.
 Many of the synthesized compounds and natural extracts
suggested to have antimicrobial activity are highly water
in soluble and large in structure.
 Being large in structure and water insoluble results in
precipitation in aqueous media and hinders the diffusion
through the agar solid media (which represent a problem
for scientists when performing sensitivity test).

4. Introduction
 Microdilution assay is an antimicrobial sensitivity test
that can be effective even with water insoluble compounds
and those with large molecular weight.
 The procedure of the experiment is described in guidelines
such as CLSI’s standards and EUCAST’s standards.
 Some modifications were applied to the procedure by
scientists regarding results reading process.

5. Introduction
 According to the modifications applied to the procedure there
can be different ways to perform microdilution assay:
 The colorimetric method
 The fluorometric method
 The simple visual method
 This presentation will demonstrate modified fluorometric
microdilution assay method.

6. Materials and Equipments
Chemicals
Equipments
Apparatuses
Nutrient agar
Micropipettes and
disposable tips
Autoclave
Nutrient broth
Syringe filters size 0.22 µm
Incubator
Barium Chloride
96 well microtiter plates
Refrigerator
Sulfuric acid
disposable Petri-dishes
PCR cabinet
Dimethylsulfoxide
(DMSO)
Glass wares (beakers, flasks,
test tubes and measuring
cylinders)
ELISA reader
Normal saline
Cotton and cotton swabs
Microtiter plates shaker
Ethanol
permanent marker pens
Sensitive balance
Tested drugs
Standard dugs

7. Experimental steps
1. Preparation of tested compounds stock solutions
(standard and tested drugs)
2. Preparation of the inoculum
3. Preparation and inoculation of the microtiter plates
4. Incubation and reading of the plates
5. Calculation of bacterial growth inhibition %

8. 10240 µg/ml stock solution
Of each tested compound
Positive control
(Standard drug)
Negative control
(solvent used to prepare
tested compound solution)
 Tested compounds stock solutions could be prepared by dissolving
10.24 mg of each compound in 1mL DMSO to obtain final concentration
of 10240µg/mL (if the compounds shows higher activity the
concentration levels could be reduced).
 Concentrations of standard drugs stock solution should be determined
according to standards and literatures information.
 Pure DMSO will be used as negative control.

9. MF
Select colonies from a
24 h subculture of the
tested microorganism
and suspend them in
normal saline
Compare the optical density of the
bacterial suspension with 0.5 McFarland
standard (prepared by adding 0.5 mL of
the BaCl2 solution to 99.5 mL of the
H2SO4 stock solution)

10. Inoculums
suspension
Dilute the 0.5 McFarland adjusted inoculums suspensions in normal saline in a
ratio of 1:100 (within 15 minutes of preparing the 0.5 McFarland equivalent
bacterial suspnsion and before inoculating the prepared microtiter plates).

11. Nutrient
broth
Add 90 µL nutrient
broth to wells of row
A and 50 µL to wells
of row B-H in the
microtiter plate
Add 10 µL from the stock solution prepared (tested compounds,
standard drugs, and solvent (DMSO)) to row A.
Transfer 50 µL of the solution in row A to the row B, repeat the
process with the following rows to carry out two fold serial
dilution to obtain certain range of concentration.
Fill the wells of one
coloumn with 100 µL
nutrient broth to
perform as sterility
(uninoculated) wells
(e.g. column 11).
Fill the wells of
another column with
50 µL nutrient broth
so as to serve as
growth control wells.
(e.g. column 12).

12. Inoculums
suspension
Add 50 µL of the
standardized inoculum
within 15 minutes of
preparation to each well
of the microtiter assay
plate except for the
sterility control wells

13. Note:
For highly water in soluble compounds a microorganism-
free microtiter plate (contain only medium and drug
concentrations) should be processed in the same manner
as the inoculated plates to be used as background control
for the tested compounds
Inoculated microdilution
assay plate
uninoculated background
control plate

14. To check the purity of the inoculums and the colony count for each inoculated
microtiter plate:
-Transfer 10 μL from the growth control well to 10 mL saline (1:1000 dilutions).
-Then transfer 100 μL of the diluted solution to a nutrient agar plate and spread it
over the entire agar surface with a sterile loop.

15. 1. Shaking
3. incubation
Measure the optical density
of each microtiter plate
immediately after inoculation
using microtiter plate reader
at wave length 620 nm.
Then incubate the microtiter
plates and colony count
plates at 35 ± 2°C for 16 to 20
hours.

16. Agitate the microtiter plates before measuring the
optical density at wave length 620 nm.
After 16 – 20 h incubation

17. Calculation of bacterial growth inhibition %:
 Growth inhibition % =
OD ≡ Optical Density … GC ≡ Growth Control
Note: The (OD of treated wells after incubation) for highly water insoluble
compounds is calculated by subtracting the optical density of the respective
well of each concentration in the uninoculated background plate from the
optical density of the treated inoculated well.

18. Sterility control
Growth control
v
v
1. Check the sterility control
2. Check the count of CFU
3. Calculate growth inhibition % for
each well
4. Determine MIC
5. Concentration – response curve
simulation
Colony count plate
minimum inhibitory concentration (MIC) will be considered as
the concentration that results in 90% growth inhibition%
(equivalent to the lowest concentration of drug that inhibit the
visible growth of the microorganism).

19. Presenting results
 Construct a concentration response
curve by plotting the growth
inhibition % of each concentration
against the natural logarithm (ln)
of the concentration (more
preferably the molar concentration.
 Determine the EC50 (potency) and
the Emax (efficacy) of the drug from
the curve.
 Use the obtained parameters (MIC,
EC50 and Emax) to compare between
the activity of drugs.
E.g.:
Emax
EC50 (ex)