a brief summary of antibacterial assays performed in lab studies to find out the dosing and efficacy of drugs and compounds on bacterial effect. different methods used are described alongwith imaged to give you a good idea.

1. Antibacterial Assay
Dr. Mahesh Kumar | Salim Habib University

2. Assay
• Any investigative procedure for
determination of quality or quantity for
any given substance.

3. Antibacterial Assay
• Assay performed to test microbial
susceptibility for any given antimicrobial
agent.
• Antimicrobial susceptibility testing can
be used for drug discovery,
epidemiology and prediction of
therapeutic outcome.

4. Methods of Antibacterial Assay
1. Diffusion Method.
2. Agar or Broth Dilution Method.
3. Thin Layer Chromatography (TLC)-bioautography
4. Time Kill Test
5. ATP Bioluminescence assay
6. Flow Cytofluorometric Method.

5. Diffusion Methods.
• Developed in 1940.
• The official method used in many clinical microbiology laboratories for routine
antimicrobial susceptibility testing.
• Can be performed :-
1. Agar Disk Diffusion.
2. Antimicrobial Gradient Method (Etest).
3. Agar Well Diffusion.
4. Agar Plug Diffusion.
5. Cross Streak Method.
6. Poisoned Food Method.

6. Diffusion Methods.
1. AGAR DISK DIFUSSION.
The disk diffusion method of
susceptibility testing allows the
categorization of most bacterial
isolates as susceptible,
intermediate, or resistant to a
variety of antimicrobial agents.

7. Diffusion Methods.
1. AGAR DISK DIFUSSION.
• The disk diffusion test has several
advantages:
• It is technically simple to perform and very
reproducible.
• The reagents are relatively inexpensive.
• It does not require any special equipment.
• It provides susceptibility category results
that are easily understood by clinicians.
• It is flexible regarding the selection of
antimicrobial agents for testing.
• Major DISADVANTAGE, only provides
qualitative not quantitative results. Only
approximation can be done of MIC.

8. Diffusion Methods.
2. ANTIMICROBIAL GRADIENT
METHOD.
• The antimicrobial gradient method
combines the principle of dilution
methods with that of diffusion
methods in order to determine the
MIC value.
• The Etests (BioMérieux) is a
commercial version of this
technique.
• This method is used for the MIC
determination of antibiotics,
antifungals and antimycobacterials.

9. Diffusion Methods.
3. AGAR WELL DIFFUSION
METHOD.
• Agar well diffusion method is
widely used to evaluate the
antimicrobial activity of plants or
microbial extracts.
4. AGAR PLUG DIFFUSION.
• Agar plug diffusion method is
often used to highlight the
antagonism between
microorganisms and the
procedure is similar to that used
in the disk-diffusion method.

10. Diffusion Methods.
5. CROSS STREAK METHOD.
• Cross streak method is used to
rapidly screen microorganisms
for antagonism The microbial
strain of interest is seeded by a
single streak in the center of the
agar plate. After an incubation
period depending upon the
microbial strain, the plate is
seeded with the microorganisms
tested by single streak
perpendicular to the central
streak.

11. Diffusion Methods.
6. POISONED FOOD METHOD.
• Poisoned food method is mostly
used to evaluate the antifungal
effect against molds.
• Generally, when standardization
of the method used fails, the
researcher must carry a positive
control with known antimicrobial
molecule to compare the results
found and assert the
experimental approach right.

12. Dilution Methods.
• Dilution methods are the most
appropriate ones for the
determination of MIC values, since
they offer the possibility to estimate
the concentration of the tested
antimicrobial agent in the agar (agar
dilution) or broth medium
(macrodilution or microdilution).
• MIC value recorded is defined as
the lowest concentration of the
assayed antimicrobial agent that
inhibits the visible growth of the
microorganism tested, and it is
usually expressed in mg/mL or
mg/L.
• Can be done by Broth Dilution or
Agar Dilution.

13. Thin Layer Chromatography (TLC)
• TLC–bioautography is a simple, effective and inexpensive technique for the separation of a
complex mixture.
• It localizes the active constituents on the TLC plate. Therefore, it can be performed both in
sophisticated laboratories and small laboratories which only have access to a minimum of
equipment.
• method of choice for a final clean-up of extractive fractions to obtain pure compounds.
• the TLC–bioautography offers a rapid technique for the screening of a large number of
samples for bioactivity and in the bioactivity-guided fractionation.
• It can be used for detection of antimicrobials in environmental and food samples as well as
for searching for new antimicrobial drugs.
• Three bioautographic techniques, i.e., agar diffusion, direct bioautography (Most coomonly
used) and agaroverlay assay, have been described for the investigation of antimicrobial
compounds by this approach.

14. Time Kill Test
• Time-kill test is the most
appropriate method for
determining the bactericidal or
fungicidal effect.
• It is a strong tool for obtaining
information about the dynamic
interaction between the
antimicrobial agent and the
microbial strain.
• The time-kill test reveals a time-
dependent or a concentration-
dependent antimicrobial effect.

15. ATP bioluminescence assay
• ATP bioluminescence assay is based on the capacity to measure adenosine
triphosphate (ATP) produced by bacteria or fungi.
• The rapidity is the major advantage of this technique that provides
quantitative results.
• Indeed it has been demonstrated that this technique can provide results in 3–
5 days for antimycobacterial tests in comparison with the conventional
dilution technique, which requires 3–4 weeks of incubation.
• Bioluminescence assay also has the advantage of being used for
antimicrobial testing in vivo or in situ.

16. Flow cytofluorometric method
• The rapid detection of damaged cells by this approach depends on the use of
appropriate dyes staining.
• Propidium Iodide (PI), a fluorescent and intercalating agent, is widely used as DNA
stain.
• Flow cytofluorometric method allows the detection of antimicrobial resistance and
estimates the impact of the molecule tested on the viability and cell damage of the
tested microorganism.
• It gives reproducible results rapidly (2–6 h compared to 24–72 h for the microdilution
method).
• The widespread use of this methodology for antimicrobial susceptibility testing
currently appears unlikely due to the inaccessibility of the required flow cytometry
equipment in various laboratories.

18. References
• Review Paper:
Methods for in vitro evaluating antimicrobial activity: A review
Mounyr Balouiri n , Moulay Sadiki, Saad Koraichi Ibnsouda

19. THANK YOU