Introduction
Microbial culture sensitivity testing is a laboratory method used to identify microorganisms in a sample and determine which antimicrobial drug is most effective against them. It helps clinicians select appropriate therapy, avoid unnecessary antibiotic use and monitor drug resistance trends in the community. Samples commonly used include blood, urine, sputum and wound swabs.
Purpose of Culture Sensitivity Testing
- To identify the organism present in the patient sample.
- To check which antibiotic can inhibit or kill the organism.
- To improve treatment outcomes and prevent antimicrobial misuse.
- To study changing patterns of bacterial resistance in an area.
Minimum Inhibitory Concentration (MIC)
MIC is the lowest concentration of an antimicrobial agent that can stop visible growth of a microorganism after overnight incubation. It is used in diagnostic laboratories to confirm whether a microorganism is sensitive or resistant to a drug. Lower MIC values usually indicate better drug activity.
Agar Dilution Method
This method is used to determine MIC by preparing serial dilutions of an antibiotic in agar plates. The test organism is swabbed onto the agar surface. The lowest antibiotic concentration where no growth occurs is recorded as the MIC.
Disc Diffusion Method (Kirby–Bauer)
A commonly used method for sensitivity testing:
- Mueller–Hinton agar plate is evenly seeded with a standardized bacterial inoculum.
- Antibiotic-impregnated discs are placed on the surface.
- Antibiotics diffuse into the agar, forming a concentration gradient.
- Sensitive organisms show a clear zone of inhibition around a disc.
The diameter of the zone is measured and interpreted as sensitive, intermediate or resistant based on standard charts.
Automated Sensitivity Testing
Modern automated systems perform inoculation, reading and interpretation of results electronically. These systems provide quicker, more accurate and standardized results.
Microbiological Assays
Microbiological assays measure the potency or concentration of antibiotics, vitamins and amino acids by observing their effect on microorganisms. These assays are required under pharmacopeial standards such as IP, BP and USP.
Applications of Microbiological Assays
- To determine potency of antibiotics.
- To support dose selection in antimicrobial therapy.
- To provide reliable in-vitro and in-vivo correlations.
- To perform simple, accurate and cost-effective analyses.
Methods of Microbiological Assays
Two main methods are used:
- Cylinder Plate (Cup Plate) Method
- Tube Assay (Turbidity) Method
Method A: Cylinder Plate Method
This method depends on the diffusion of antibiotic from a cavity into agar. The growth of the test organism is inhibited around the cavity where antibiotic concentration is effective.
Principle
An antibiotic solution diffuses through solid agar and forms a zone of inhibition. The size of this zone is proportional to the antibiotic concentration.
Procedure
- Nutrient agar is melted, cooled and poured into sterile Petri plates.
- 0.2 ml of test inoculum is spread uniformly over the agar surface.
- Cylindrical holes are made with a sterile borer.
- Different volumes of antibiotic (0.2, 0.4, 0.6, 0.8 and 1.0 ml) are placed in the cups.
- Plates are incubated at 37°C for 24 hours.
- Zone of inhibition is measured from the backside of the plate.
Types of Cylinder Plate Assays
One-Level Assay
Zone diameters of standard and test solutions are compared to calculate potency.
Two-Level Factorial Assay
Parallel dilutions of standard (S1, S2) and unknown (U1, U2) solutions are prepared. After incubation, the potency of the unknown sample is calculated from statistical formulas. Assay is invalid if potency is below 60% or above 150% of the standard.
Method B: Tube Assay Method (Turbidity Method)
This method measures the ability of an antibiotic to inhibit microbial growth in liquid medium. Turbidity of the culture reflects the number of microorganisms present.
Principle
The turbidity (cloudiness) of the medium decreases when microbial growth is inhibited by the antibiotic. The absorbance at around 530 nm is measured using a colorimeter.
Procedure
- Five different concentrations of standard antibiotic solution are prepared.
- Unknown sample is diluted to match the test range.
- 1 ml of each standard and test solution is transferred into duplicate tubes.
- 9 ml of nutrient medium is added to each tube.
- Control tubes are prepared: inoculated medium, blank control and uninoculated medium.
- Incubate at 37°C for 3–4 hours.
- Add 0.5 ml dilute formaldehyde to stop growth.
- Measure absorbance of each tube and compare with standard curve.
Advantages
- Requires only 3–4 hours for incubation.
- Simple and rapid method.
Disadvantages
- Results may be affected by solvent residues or inhibitory substances.
- Not suitable for turbid or cloudy samples.
Interpretation of Results
- Large zone of inhibition or low MIC: Organism is sensitive to the drug.
- Small or no zone, high MIC: Organism is resistant.
Key Points
- Culture sensitivity testing guides clinicians to choose the right antibiotic.
- MIC is the lowest concentration of antibiotic that stops visible growth.
- Cylinder plate and tube assay methods help determine drug potency.
- Automated systems improve accuracy and reduce testing time.
Detailed Notes:
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