Culture sensitivity tests are used to determine the effectiveness of antibiotics against bacteria or fungi causing an infection. A sample is taken from the infected area and any germs grown in culture. Discs containing different antibiotics are placed on the culture and the size of the zone where no germs grow shows how sensitive the germs are. This helps doctors choose the most effective antibiotic treatment. Results may show germs as susceptible, intermediate, or resistant to each antibiotic tested. Sensitivity tests are important for guiding treatment decisions and monitoring antibiotic resistance in communities.

1. CULTURE SENSITIVITY TESTS
By: AYESHA ASIF ALI
PHARM-D
SULTAN-UL-ULOOM COLLEGE OF PHARMACY,
HYDERABAD
GUIDED BY:
Dr. S. P. SRINIVAS NAYAK,
ASSISTANT PROFESSOR, SUCP , HYD.

2. INTRODUCTION
CULTURE TEST
A culture test is performed to find germs (such as bacteria or a fungus) that
can cause an infection. It is done by using a culture media for their growth.
SENSITIVITY
Sensitivity measures how often a test correctly generates a positive result for
people who have the condition that’s being tested for (also known as the “true
positive” rate). A test that’s highly sensitive will flag almost everyone who has
the disease and not generate many false-negative results.

3. CULTURE SENSITIVITY TESTS:
These are in-vitro procedures used for the
● Detection of antimicrobial resistance in individual bacterial isolates .
● Monitoring emergence and spread of resistant microorganisms in a
population .
● Determining the sensitivity of a colony of bacteria to an antibiotic.
● Determining the efficacy of an antibiotic by it’s inhibitory effect on
microorganisms.

4. SENSITIVITY ANALYSIS
Sensitivity analysis is also called susceptibility testing.
A sensitivity analysis is a test that determines the “sensitivity” of bacteria to an
antibiotic and the ability of the drug to kill the bacteria.
It detects the most effective antibiotic to kill the infection causing
microorganism.
Sensitivity testing to determines the right antibiotic treatment for an infection
and to monitor changes in bacterial resistance to antibiotics.

5. PURPOSE OF CULTURE SENSITIVITY
● To guide the clinician in selecting the best drug for an individual patient
● To control the inappropriate use of drugs in clinical practice
● To accumulate epidemiological information on the resistance of
microorganisms for public health importance in a community
● To reveal the changing trends in local isolates
● To overcome microbial drug resistance

6. HOW IS SENSITIVITY ANALYSIS PERFORMED?
Sensitivity analysis starts with a bacterial sample , by swabbing the infected area.
Samples may be taken from:
Blood
Urine
Sputum
A pus-containing wound

7. The sample is sent to a laboratory, where it is spread on a special growing
surface.
The grown bacteria is known as a culture and bacteria in the culture will grow
and multiply.
The bacteria forms colonies, or large groups of bacteria, that will each be
exposed to different antibiotics.

8. Responses:
These colonies can be susceptible, resistant, or intermediate in response to
the antibiotics:
Susceptible means they can’t grow if the drug is present. This means the
antibiotic is effective against the bacteria.
Resistant means the bacteria can grow even if the drug is present. This is a
sign of an ineffective antibiotic.
Intermediate means a higher dose of the antibiotic is needed to prevent
growth.

9. What Are the Risks of a Sensitivity Analysis?
Rare risks of taking a blood sample include:
● lightheadedness or fainting
● hematoma (a bruise where blood accumulates under the skin)
● infection (usually prevented by the skin being cleaned before the needle is
inserted)
● excessive bleeding (bleeding for a long period afterwards may indicate a
more serious bleeding condition )

10. RESULTS OF SENSITIVITY ANALYSIS:
These results determine the best antibiotic to treat the infection.
Susceptible : meaning it can fight the bacteria.
Intermediate : A drug from the “intermediate” group is prescribed If there are no known
drugs available in the susceptible group. A higher dosage and for a longer time period is
taken for a drug from the intermediate group. The patient may also experience
medication side effects.
Resistant : Shows no response to the antibiotic . An antibiotic that bacteria, fungi, or
another microorganism has shown resistance to , shouldn’t be used to treat your
infection.

11. METHODS FOR CULTURE SENSITIVITY
1. DILUTION METHOD
2. E-TEST
3. DISC-DIFFUSION METHOD
4. AUTOMATED METHODS
5. MECHANISM - SPECIFIC TESTS
6. GENOTYPIC METHODS

12. DILUTION METHODS
Performed to determine the minimum inhibitory concentration (MIC) of an
antimicrobial agent.
MIC is defined as the lowest concentration of an antimicrobial agent that
inhibits the growth of organisms.
It includes :
1. BROTH DILUTION METHOD
2. AGAR DILUTION METHOD

13. BROTH DILUTION METHOD
Broth microdilution is done using microtiter plates and is considered the “gold standard.”
Quantitative method for determining the MIC of an antimicrobial agent that inhibits the
growth of organisms in vitro.
In this method, the antimicrobial agent is serially diluted in Mueller–Hinton broth by
doubling dilution in tubes and then a standard suspension of the broth culture of test
organism is added to each of the antibiotic dilutions and control tube.
This is mixed gently and incubated at 37°C for 16–18 hours.
An organism of known susceptibility is included as a control.
The MIC is recorded by noting the lowest concentration of the drug at which there is no
visible growth as demonstrated by the lack of turbidity in the tube.

14. The main advantage of this method is that this is a simple procedure for testing
a small number of isolates. The added advantage is that using the same tube,
the minimum bactericidal concentration (MBC) of the bacteria can be
determined.
The MBC is determined by subculturing from each tube, showing no growth on
a nutrient agar without any antibiotics.
Subcultures are made from each tube showing no growth into the nutrient agar
plates without any antibiotics.
The plates are examined for growth, if any, after incubation overnight at 37°C.
The tube containing the lowest concentration of the drug that fails to show any
growth on subculture plate is considered as the MBC of the antibiotic for that
strain.

16. AGAR DILUTION METHOD
Quantitative method for determining the MIC of antimicrobial agent against the
test organism.
Mueller–Hinton agar is used in this method.
Serial dilution of the antibiotic are made in agar and poured onto Petri dishes.
Dilutions are made in distilled water and added to the agar that has been melted
and cooled to not more than 60°C.
One control plate is inoculated without antibiotics.
Organism to be tested is inoculated and incubated overnight at 37°C.

17. Plates are examined for presence or absence of growth of the bacteria.
The concentration at which bacterial growth is completely inhibited is
considered as the MIC of the antibiotic.
The organisms are reported sensitive, intermediate, or resistant by comparing
the test MIC values with that given in CLSI guidelines.
The main advantage of the method is that a number of organisms can be
tested simultaneously on each plate containing an antibiotic solution.

19. EPSILOMETER TEST (E Test)
Based on the principle of disc diffusion, is an automated system for measuring
MIC of a bacterial isolate. In this method, an absorbent plastic strip with a
continuous gradient of antibiotic is immobilized on one side.
MIC interpretative scale corresponding to 15 twofold MIC dilutions is used on the
other side.
The strip is placed on the agar plate inoculated with the test organism with the
MIC scale facing toward the opening side of the plate.
An elliptical zone of growth inhibition is seen around the strip after incubation at
37°C overnight.

20. The MIC is read from the scale at the intersection of the zone with the strip.
The end point is always read at complete inhibition of all growth including
hazes and isolated colonies.
E test is a very useful test for easy interpretation of the MIC of an antibiotic.

21. DISC-DIFFUSION METHOD
The disk diffusion test, or agar diffusion test, or Kirby–Bauer test (disc-
diffusion antibiotic susceptibility test, disc-diffusion antibiotic sensitivity test,
KB test), is an antibiotic susceptibility test.
It uses antibiotic discs to test the extent to which bacteria are affected by
those antibiotics.
If an antibiotic stops the bacteria from growing or kills the bacteria, there will
be an area around the sample where the bacteria have not grown enough to
be visible.This is called a zone of inhibition.

22. PROCEDURE:
The media used in Kirby–Bauer testing must be Mueller-Hinton agar at only 4 mm
deep, poured into either 100 mm or 150 mm Petri dishes.
The pH level of the agar must be between 7.2 and 7.4.
1. Using an aseptic technique, place a sterile swab into the broth culture of a
specific organism and then gently remove the excess liquid by gently pressing
or rotating the swab against the inside of the tube.
2. Using the swab, streak the Mueller-Hinton agar plate to form a bacterial lawn.
3. To obtain uniform growth, streak the plate with the swab in one direction,
rotate the plate 90° and streak the plate again in that direction.

23. 4.Repeat this rotation 3 times.
5.Allow the plate to dry for approximately 5 minutes.
6.Use an antibiotic disc dispenser to dispense discs containing specific
antibiotics onto the plate.
7.Using a flame-sterilized forceps, gently press each disc to the agar to ensure
that the disc is attached to the agar.
8.Plates should be incubated overnight at an incubation temperature of 37 °C
(98.6 °F)

25. AUTOMATED METHODS
Most automated antimicrobial susceptibility testing systems provide
automated inoculation , reading and interpretation.
These systems have the advantage of being rapid and convienient , but one
major limitation for most laboratories is the cost entailed in initial purchase ,
operation and maintenance of the machinery

26. MECHANISM - SPECIFIC TESTS
These include Beta-lactamase detection test and Chromogenic Cephalosporin
test.
Resistance may also be established through tests that directly detect the
presence of a particular resistance mechanism.
For example . beta lactamase detection can be accomplished using an assay
such as the chromogenic cephalosporinase test.

27. GENOTYPIC METHODS
Although nucleic acid-based detections systems are generally rapid and
sensitive , it is important to remember that the presence of a resistance gene
does not necessarily equate to treatment failure , because resistance is also
dependant on the mode and level of expressions of these genes
Some of the most common molecular techniques utilized for anti-microbial
resistance detection are PCR and DNA hybridization methods

28. POLYMERIZATION CHAIN REACTION:
It is one of the most commonly used molecular techniques for detecting
certain DNA sequences of interest.
This involves several cycles of denaturation of sample DNA , annealing of a
specific primers to the target sequence and the extension of this sequence
as facilitated by a thermostable polymerase leading to replication of a
duplicate DNA sequence , in an exponential manner, to a point which will be
visibly detectable by gel electrophoresis with the aid of a DNA - intercalating
chemical fluoresces under UV light.

29. DNA hybridization method
This is based on the fact the DNA pyrimidines (Cytosine and thymidine)
specifically pair up with purines (guanine and adenine; or Uracil for RNA).
Therefore , a labelled probe with a known specific sequence can pair up with
opened or denatured DNA from the the sample, as long as their sequences
complement each other .
If this hybridization occurs , the probe labels this with a detectable radioactive
isotope , antigenic substrate , enzyme or chemiluminescent compound .
Whereas if no target sequence is present or the isolate does not have the
specific gene of interest , no attachment of probes will occur ,and therefore no
signals will be detected.

30. APPLICATIONS
To provide a reliable prediction of whether an infection caused by a bacterial
isolate will respond therapeutically to a particular antibiotic treatment.
This may be utilized as guidelines for chemotherapy , or at the population
level as indicator of emergence and spread of resistance based on passive or
active surveillance

31. THANK YOU