ANTIMICROBIAL SUSCEPTIBILITY TEST

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ANTIMICROBIAL SUSCEPTIBILITY

The Kirby-Bauer Disk Diffusion Method

1 Aim

To determine the in vitro susceptibility of pathogenic bacteria to various pharmacological antibiotics using the standardized Kirby–Bauer disk diffusion method.

2 Principle & The Diffusion Gradient

The Kirby-Bauer test is the global gold standard for evaluating antibiotic efficacy. It relies on the principle of antibiotic diffusion through a solid agar matrix.

When a filter paper disk impregnated with a specific concentration of an antibiotic is placed on an agar plate uniformly seeded with bacteria, the antibiotic immediately begins to diffuse outward radially. This creates a concentration gradient—the antibiotic concentration is highest right next to the disk and decreases the further it travels.

Zone of Inhibition (ZOI)

If the bacteria are sensitive to the antibiotic, they cannot grow in the area where the drug concentration is sufficiently high. This results in a completely clear, circular halo around the disk called the Zone of Inhibition. The edge of this zone represents the Minimum Inhibitory Concentration (MIC) of that specific antibiotic.

The Gold Standard Parameters

To ensure reproducible results globally, the CLSI mandates three strict parameters: Mueller-Hinton Agar (MHA) poured to exactly a 4mm depth, a bacterial inoculum matching a 0.5 McFarland standard, and incubation at 37°C for exactly 16-18 hours.

3 Materials Required

Media & Reagents

• Mueller-Hinton Agar (MHA) plates (150mm or 100mm)
• Pure bacterial culture (e.g., S. aureus, E. coli)
• Sterile physiological saline (0.85% NaCl)
• 0.5 McFarland Turbidity Standard
• Commercially prepared Antibiotic Disks (e.g., Penicillin, Tetracycline, Gentamicin)

Apparatus

• Laminar airflow cabinet
• Sterile non-toxic cotton swabs
• Sterile fine-tipped forceps or disk dispenser
• Bacteriological Incubator (37°C)
• Precision Ruler or Vernier Caliper (mm)

A. Procedure: Inoculum Preparation & Lawning

Fig 1: Creating a "Bacterial Lawn". The plate is swabbed entirely, rotated 60 degrees, and swabbed again to ensure absolutely no gaps in growth.

1. Using a sterile loop, touch 3–5 morphologically similar, well-isolated colonies from a fresh culture plate.
2. Emulsify the colonies in 4 mL of sterile saline.
3. Vortex and compare the turbidity of your suspension to a 0.5 McFarland standard against a white card with black lines. Add more bacteria or more saline until the cloudiness matches exactly.
4. Dip a sterile cotton swab into the suspension. Press the swab firmly against the inside wall of the tube to squeeze out excess fluid.
5. Streak the swab over the entire surface of the MHA plate. Rotate the plate 60° and streak again. Rotate another 60° and streak a third time. Finally, run the swab around the outer rim of the agar. This guarantees a uniform confluent bacterial lawn.
6. Leave the lid slightly ajar for 3–5 minutes to allow surface moisture to absorb before applying disks.

B. Procedure: Disk Placement & Measurement

1. Sterilize fine-tipped forceps with ethanol and a flame.
2. Aseptically retrieve an antibiotic disk and place it firmly onto the agar surface. Tap it lightly with the forceps to ensure 100% contact (if it doesn't touch perfectly, diffusion will be uneven).
3. Rule of Spacing: Disks must be placed at least 24 mm apart (center to center) and no closer than 15 mm from the edge of the Petri dish.
4. Once a disk touches the agar, do not move it! The antibiotic begins diffusing instantaneously.
5. Invert the plates and incubate them at 37°C for 16 to 18 hours.

Fig 2: Measuring the diameter of the clear Zone of Inhibition against a dark background using a ruler. Measure edge-to-edge right through the center of the disk.

5. Observation & Interpretation (CLSI Guidelines)

You cannot simply look at a zone and guess if it is "Sensitive." A 15mm zone might be considered Sensitive for one drug, but Resistant for another! You must compare the millimeter diameter to the standardized CLSI (Clinical and Laboratory Standards Institute) Performance Standards Chart.

Antibiotic Disk	Zone Measured (mm)	Resistant (R)	Intermediate (I)	Sensitive (S)	Conclusion
Ampicillin (10µg)	28 mm	≤ 13	14 - 16	≥ 17	Sensitive
Tetracycline (30µg)	16 mm	≤ 14	15 - 18	≥ 19	Intermediate
Penicillin G (10 U)	8 mm	≤ 14	--	≥ 15	Resistant

6. Troubleshooting False Results

Observation	Root Cause & Explanation
Falsely small zones (False Resistance)	1. Inoculum too heavy: You used a 1.0 McFarland standard instead of 0.5. Too many bacteria overwhelmed the antibiotic. 2. Agar too thick: You poured 6mm of agar instead of 4mm. The antibiotic diffused downwards instead of outwards.
Falsely large zones (False Sensitivity)	1. Inoculum too light: You didn't swab enough bacteria onto the plate. 2. Agar too thin: The antibiotic couldn't diffuse down, so it spread entirely outwards, creating a massive false halo.

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🧠 Interactive Viva Quiz

Test your clinical knowledge! Click on the questions below to reveal the correct answers.

1. Why is Mueller-Hinton Agar (MHA) strictly used for this test instead of Nutrient Agar?

✅ Answer: It is highly standardized and non-inhibitory.

MHA shows excellent batch-to-batch reproducibility. Crucially, it is very low in sulfonamide, trimethoprim, and tetracycline inhibitors (like PABA). If you used Nutrient Agar, the rich ingredients might neutralize the antibiotics, falsely making the bacteria look resistant.

2. Does a larger Zone of Inhibition automatically mean the antibiotic is "stronger" or "better"?

✅ Answer: No!

Zone size is heavily dependent on the molecular weight of the antibiotic. A small, lightweight drug will diffuse rapidly and create a huge zone. A large, heavy drug (like Polymyxin or Vancomycin) diffuses slowly and creates a small zone, even if it is incredibly lethal to the bacteria. You can only compare zone sizes against the CLSI charts, not against other antibiotics.

3. What exactly is a "0.5 McFarland Standard"?

✅ Answer: A visual standard representing ~1.5 x 10⁸ CFU/mL.

It is a chemical mixture (usually Barium chloride and Sulfuric acid) that creates a specific level of cloudiness (turbidity). By matching your bacterial suspension to this cloudiness, you guarantee that you are swabbing approximately 150 million bacterial cells per milliliter onto the plate, standardizing the test.

4. What do you do if you see individual colonies growing INSIDE the clear Zone of Inhibition?

✅ Answer: Report the bacteria as Resistant, or suspect a mixed culture.

Those are mutant, highly resistant clones that survived the antibiotic gradient! Even if the main zone is huge, the presence of resistant colonies inside the halo means the infection could rebound if treated with that drug. The diameter must be measured at the inner-most resistant colony.