MCB 101 Lab - PART UNO

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MCB 101 Lab LE Coverage

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1. Lab Ex2 - Antimicrobial Susceptibility Assays

2. Lab Ex3 - Collection, Identification, and Culture of Fungi

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Bacteria:

Learning Outcomes

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1.Prepare standardized inoculum

2.Practice serial dilution and spread plate methods

3.Perform basic assays to test antimicrobial activity

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Define McFarland Standards

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-chemical solution of BaCl2 and H2SO4, whose reaction produces a fine precipitate = Barium Sulfate

-when shaken well, its turbidity is visually comparable to a bacterial suspension of known concentration

-used to standardize the number of bacteria in a liquid suspension by comparing the turbidity of the test suspension with that of McFarland standard

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most commonly used standard in clinical microbiology which is prescribed for antimicrobial susceptibility testing and culture media performance testing

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0.5 McFarland Standard

0.05 1%BaCl2

9.95 1%H2SO4

1.5 x 10^8 approx. bacterial suspension

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How to prepare inoculum using McFarland Standards?

PROCEDURE

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1. 1. Mix the standard on a vortex or manually swirl/mix prior to examination.

2. 2. Make sure it is aliquoted into a tube that is the same size and diameter as the tube used to prepare the test suspension.

3. 3. Prepare a test suspension (obtain fresh pure culture of test org and inoculate in a suitable broth)

4. 4. Visually compare the turbidity by comparing the clarity of the lines on the Wickerham card in good lighting.

5. 5. If test suspension is too light, inoculate with additional organisms or incubate tube until turbidity matches that of standard.

6. 6. If dilution is needed, use sterile pipette and add sufficient broth or saline to obtain matched turbidity.

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why same size and diameter for both standard and test suspension?

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for accuracy and consistency

-if diameters differ, depth of liquid in tubes also differ

optical consistency (the way light passes through a liquid can be affected by tube's diameter and shape0

-avoid systematic errors during standardization

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McFarland Standard Precations

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1.Sensitive to air and light so tubes should be closed tightly and kept in dark

2.level of standards should be checked occasionally to ensure that evaporation has not occurred.

3. 3.should be vigorously agitated on a mechanical vortex before each use and inspected for a uniform turbid appearance. If large particles appear or if clumping is apparent discard the standard

5. 4.Actual numbers of viable bacteria present in an adjusted suspension depends on the size, viability, and clumping of the particular bacterium used

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Preparation of test compounds

PROCEDURE

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1. 1.Collect enough plant material (at least 200g -dry)

2. 2. Cut plant material into small pieces for faster drying - can use oven or food dehydrator at low temp

3. 3. Macerate plant material in 70% methanol (2:1 w/v ratio) by soaking at least 24hrs.

4. 4.Filter extract and decant into a separate flask.

5. 5. Loosely cap the flask with a watch glass to allow solvent to evaporate for few days.

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Agar Well Diffusion

PROCEDURE

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1. 1.Using prepared standardized inoculum, prepare spread plates - spread inoculum evenly across the surface of the agar using cotton swab.

2. 2.Flame sterilize a metal borer (5-6mm) and punch holes into the agar to create wells.

3.Using aseptic technique, fill wells with your test extract (20-100mL) until it reaches the top of the well

4.Do not forget to include wells for a negative and positive control.

5.Incupate plates at 37* for 24 hrs. DO NOT INVERT THE PLATES.

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Determination of MIC and MBC

2 GENERAL PROCEDURE

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1. 2-Fold Microdilution using 96 well plates

Actual Determination of MIC and MBC