The document details microbiological culture sensitivity tests, which analyze the susceptibility of microorganisms to antibiotics using various methods such as disc-diffusion and dilution techniques. It emphasizes the importance of these tests for guiding treatment choices and understanding antibiotic resistance. Additionally, it summarizes key methods, their principles, advantages, limitations, and clinical applications.

1. MICROBIOLOGICAL CULTURE
SENSITIVITY TEST
PREPARED BY:-
MALINI.R
PHARMACY PRACTICE
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2. CONTENTS
• DEFINITION
• DISC-DIFFUSION METHOD
• STOKES DISC DIFFUSION METHOD
• KIRBY-BAUER DISC DIFFUSION METHOD
• EPSILOMETER
• AUTOMATED METHODS
• BROTH DILUTION METHOD
• AGAR DILUTION METHOD
• SUMMARY
• REFERENCES
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3. MICROBIOLOGICAL CULTURE SENSITIVITY
TEST
• It is a method of multiplying microbial organisms in a predetermined culture media with favourable
conditions and test perform to check the sensitivity of organism towards the drugs.
PURPOSE OF CULTURE TEST
To guide the clinician in selecting the best drug for an individual patient.
To control the use of inappropriate drug in clinical practice.
 To accumulate epidemiological information on the resistance of microorganism of public health importance
within the community.
To overcome the resistance to antibiotics.
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4. HOW SENSITIVITY ANALYSIS IS DONE
Sensitivity analysis starts with a bacterial sample.
The sample is obtained by swabbing the infected area.
Cultures are taken from BLOOD, SPUTUM, PUS, URINE, FECES, THROAT SWAB, CSF.
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Selection of the appropriate method depends on:-
 degree of accuracy
Urgency
Convenience
bioavailability of sources
 cost
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5. DIFFUSION
• KIRBY-BAUER METHOD
• STOKES METHOD
DILUTION
• BROTH DILUTION
• AGAR DILUTION
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ANTIBIOTIC SENSITIVITY TEST
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E-TEST
DIFFUSION AND DILUTION

6. DIFFUSION TEST AND DILUTION TEST
DIFFUSION TEST
PRINCIPLE:-
• To allow the antimicrobial agent to diffuse through a solid medium so that the concentration is highest
near the site of application of antimicrobial agent and decreases with distance.
DILUTION TEST:-
• PRINCIPLE:-
Dilution tests are performed to determine the minimum inhibitory concentration (MIC) of an
antimicrobial agent.
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7. DISC-DIFFUSION METHOD
• It is the most simple and easy method, hence most commonly used.
PRINCIPLE:-
• This method involves the addition of known amount of an drug to a antibiotic discs measuring 6 mm in
diameter.
• The test bacterium is inoçulated on the medium and these antibiotic discs are applied.
• Sensitivity to the drug determined from the inhibition of bacterial growth around the disc.
• USES:-
• Disc diffusion tests are most widely used to determine the susceptibility of isolates of pathogenic bacteria
to antibiotics that are likely to be used in the treatment.
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8. DISC-DIFFUSION METHOD
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9. STOKES DISC DIFFUSION METHOD
• The test bacterium is inoculated on the central one third and control on upper and lower thirds of the plate.
• Antibiotic discs are applied between the standard and test innocula .
• A maximum of six antibiotic discs can be applied on a 100 mm diameter plate.
• The plates are then incubated at 37°C for l6-18 hours.
• The reporting of results is done by comparing the zones of inhibition of control and test bacterium.
• Measure the zone size i.e. the A distance in mm from edge of the disc to the zone edge It is interpreted as follows:
• Sensitive The zone of test bacterium is equal to, or larger than that of control strain.
• If zone size of test bacterium is smaller than that of control, the difference between the two should not be more than
3 mm.
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10. STOKES DISC DIFFUSION METHOD
• Intermediate sensitive The zone size of the test bacterium should be at least 2 mm and the difference between
the zone of test and control strain should be at least 3 mm.
• Resistant The zone size of the test bacterium is smaller than 2 mm.
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11. KIRBY-BAUER DISC DIFFUSION METHOD
• Dip a cotton swab into inoculum.
• Inoculate the Mueller Hinton agar plate by streaking the swab three times over the entire agar surface.
• Allow 3-5 minutes for the surface of agar to dry before applying the antibiotic discs, using either sterile
forceps.
• On a plate of 100 mm diameter, seven discs may be applied, one in the centre and six in the periphery.
• The plates are then incubated at 37°C for 16-18 hours.
• The interpretation of zone size into sensitive, intermediate or resistant is based on interpretation .
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12. KIRBY-BAUER DISC DIFFUSION METHOD
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• Control strains of Staph. aureus, Esch. coli, aeruginosa etc. should be tested each time when a
new batch of discs or agar is used.

13. INTERPRETATION
• For Interpretation of the results of Stokes and Kirby-Bauer diffusion methods, the following points should
be considered in certain conditions.
1. Penicillinase producing strains of Staphylococcus sometimes fail to form enough enzyme to neutralise
penicillin close to the disc.
• it will show a zone of inhibition but colonies at the edge are large and there is not gradual fading away of
growth towards the disc.
It should be reported resistant, irrespective of the zone size.
2. When trimethoprim and sulphamethoxazole discs containing both drugs are used, it is impossible to know
whether the bacterium is sensitive to both or ONLY to one of these.
• To overcome this problem, each drug should be tested separately.
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14. INTERPRETATION
3. Methicillin resistant Staphylococcus aureus (MRSA) will often appear fully sensitive when tested in
ordinary way.
• Many of these organisms grow slowly in the presence of methicillin.
• The growth will only appear within the zone when the incubation is continued for 48 hours.
• Overcome either by incubating the plates at 30°C or by using 5% salt agar and incubating at 37°C.
4.Proteus mirabilis and Proteus vulgaris may swarm on agar surface resulting in a thin veil that may
penetrate into the zones of inhibition.
• To overcome this problem, the zones of swarming should be ignored and the outer margin, which is
clearly outlined, should be measured.
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15. INTERPRETATION
5.Polymyxin diffuses poorly in agar hence these zones are small.
Interpretation is as follows:
Sensitive- Many of these organisms diffuses slowly in the agar medium.
Resistant-The zone size of the test bacterium is smaller than that of the control strain.
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16. INTERPRETATION
6. Large zones are seen around ciprofloxacin discs with the sensitive control strains.
Interpretation is as follows:
• (a) When Staph. aureus or Ps. aeruginosa is used as sensitive control.
• Sensitive-Inhibition zone of the test bacterium is equal to, greater than, or not more than 7 mm
smaller than that of control.
• Intermediate sensitive--Inhibition zone of the test bacterium is more than 2 mm but is smaller than
that of the control by more than 7 mm.
• Resistant-Inhibition zone of the test bacterium Is 2 mm or less.
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17. INTERPRETATION
• (b) When Esch. coli or H. Infiuenzae is used as sensitive control.
• Sensitive- Inhibition zone of the test bacterium is equal to, greater than, or not more than 10 mm smaller than
that of the control.
• Intermediate sensitive-Inhibition zone of the test bacterium is more than 2 mm but is smaller than that of the
control by more than 10 mm.
• Resistant-Inhibition zone of the test bacterium is 2 mm or less.
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18. EPSILOMETER OR ANTI MICROBIAL
GRADIENT METHOD
• Epsilometer or E-test is antimicrobial sensitivity test to detect minimum inhibitory concentration (MIC) of
antibiotic.
• The antimicrobial gradient method combines the principle of dilution methods with that of diffusion methods.
• It uses an E-STRIP with a known gradient of antibiotic concentration.
• When the strip is placed on the agar plate inoculated with the test organism, the antibiotic diffuses into the
medium.
• Incubate it at 37°C for overnight.
• The minimum inhibitory concentraion (MIC) is recorded as the lowest concentration which inhibits the
growth of the microorganism.
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19. EPSILOMETER
• MIC value is determined at the intersection of the strip and the zone of inhibition ellipse.
ADVANTAGE –
• Easy to perform
• Combination of antibiotics can be tested.
• Does not require expertise.
• Does not require special technologies.
LIMITATION-
• automated systems are available for reliable determination of MIC.
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20. AUTOMATED METHODS
Most automated antimicrobial sensitivity testing systems provide automated inoculation, incubation,
reading, and interpretation.
ADVANTAGE
rapid and convenient
LIMITATION-
laboratories is the cost entailed in
initial purchase
Operation
 and maintenance of the machinery.
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21. BROTH DILUTION METHOD
• Serial dilutions (e.g. 1, 2, 4, 8, 16 and 32 µg/mL) of the drug in Mueller-Hinton broth are taken in tubes and
a standardised suspension of the test bacterium inoculated.
• An organism of known sensitivity should also be titrated.
• Incubate at 37°C for 16-18 hours.
• For determination of MIC of methicillin incubate at 30°C.
• The minimum inhibitory concentration (MIC) is read by noting the lowest concentration of the drug at
which there is no visible growth.
• The minimum bactericidal concentration (MBC) is determined by subculturing from each tube showing
no growth on a nutrient agar plate without any antimicrobial agent.
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22. BROTH DILUTION METHOD
• Incubate the plates and examine them for growth, if any.
• MIC inhibits the bacterial growth while MBC kills the bacterium.
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23. AGAR DILUTION METHOD
• The agar dilution method involves the incorporation of varying desired concentrations of the
antimicrobial agent into an agar medium (molten agar medium).
• serial two-fold dilutions
• inoculation of a defined microbial inoculum onto the agar plate surface.
• The MIC value is determined.
• ADVANTAGES -
• This method is more convenient.
• several strains are to be tested at the same time.
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24. APPLICATIONS
• Antibiotic sensitivity tests are very useful for clinician in diagnostic bacteriology.
 To provide a reliable prediction of whether an infection caused by a bacterial isolate will
respond therapeutically to a particular antibiotic treatment.
It acts as a indicator for the spread of resistance to a antibiotic.
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25. SUMMARY
1. Antimicrobial sensitivity test are two types:- diffusion tests and dilution tests.
2. Stokes disc diffusion tests and Kirby-Bauer disc diffusion method are examples of diffusion tests.
3. Dilution tests include broth dilution method and agar dilution method.
4. Epsilometer or E-test combines the principle of dilution methods with that of diffusion methods.
5. Minimum inhibitory concentration (MIC) inhibits the bacterial growth while the minimum bactericidal
concentration (MBC) kills the bacterium.
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26. REFERENCES
• Textbook of Microbiology fourth edition – BY prof. CP BAVEJA.
• The short textbook of medical microbiology eighth edition –BY SATISH GUPTE
• Medical microbiology and parasitology preparation manual for undergraduates third edition- BS NAGOBA and ASHA PICHARE
• Textbook of Microbiology fourth edition – BY DR. ARORA BRIJ BALAARORA
• https://www.ncbi.nlm.nih.gov/books/NBK539714/
• https://pubmed.ncbi.nlm.nih.gov/30969536/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762448/
• https://emedicine.medscape.com/article/2103786-overview#a1
• https://medlineplus.gov/lab-tests/antibiotic-sensitivity-test/
• https://www.webmd.com/a-to-z-guides/what-to-know-about-antibiotic-sensitivity-testing
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