Microbial Assay/Antibiotic Assay:-

 

Definition: microbiological or microbial assay or BioAssay is a kind of biological test that determines the potency of a compound by determining the amount needed to produce the expected effects on a test organism.

1.0 OBJECTIVE:

To establish a procedure for performing the microbial assay.

2.0  SCOPE :

This standard operating procedure is applicable for microbiology assay in Microbiology Department

3.0 RESPONSIBILITY:

3.1              Microbiologist

3.2            Head Microbiology

4.0 ACCOUNTABILITY:

4.1              Head – Quality Assurance & Quality Control

5.0  PROCEDURE:

5.1          Materials Required:

5.1.1        Laminar air flow

5.1.2        Incubator

5.1.3        Autoclave (double door autoclave)

5.1.4        Balance

5.1.5        Culture tubes

5.1.6        Measuring cylinder

5.1.7        Petriplate

5.1.8        SS borer (8mm)

5.1.9        Conical flask

5.1.10    Buffer peptone water

5.1.11    Micro tips 100µl

5.1.12    Zone Reader

5.2    Test Organisms:

Solvent Details of Antibiotic Microbial Assay

5.3      Testing Method:

a)            Prepare of Culture Suspension

1. 1. Take a freshly prepared slant of staphylococcus Staphylococcus aureus (ATCC 6538) or its equivalent

   2. Add 3 ml of normal saline, Mix the growth surface by sterile loop & vortex for homogenous solution.

   3.  cultivate/enumerate the organisms by using 250 ml antibiotic assay medium No A in roux bottle.

   4. Wash the growth from the nutrient surface using 50 mL saline solution.

   5. Take 1 ml suspension in 39 mL of normal saline store at 2 – 8 °C.

b)           Media Preparation in Antibiotic Microbial Assay.

1. 1. Dissolve 3.05 g of antibiotic assay agar no.  in 100 ml  purified water.

   2. Mix it properly & sterilize the media as per respective SOP.

c)            Buffer Preparation in Antibiotic Microbial Assay

1. 1. Dissolve 16.73 g of dipotassium hydrogen phosphate & 0.523 g of potassium dihydrogen phosphate in 1000 mL of purified water.

   2. Adjust the pH of solution to 8.0 ± 0.1

d)           Standard Preparation in Antibiotic Microbial Assay

1. 1. Take the weigh accurately gentamicin sulfate eq. to gentamicin about 50 mg in volumetric flask & make up to 50 ml with B2 buffer solution.

   2. Take 2 ml of this solution in volumetric flask and make up to100 ml with B2 buffer solution.

   3.  Take 1 ml of this solution in volumetric flask and make up to100 ml with B2 buffer solution.(SH = 0.2 mcg/mL).

   4. Take 25 ml of this solution in volumetric flask & make up to 100 ml with B2 buffer solution.(SL=0.05 mcg/mL)

e)            Sample Preparation in Antibiotic Microbial Assay 

1. 1. Take 2.5 ml of gentamicin injection in volumetric flask make up to 100 ml B2 Buffer solution.

   2. Take 2 ml of this solution in volumetric flask & make up to 100 ml with B2 Buffer solution.

   3. Take 1 ml of this solution in volumetric flask & make up to 100 ml with B2 Buffer solution.(TH = 0.2 mcg/ml).

   4. Take 25 ml of this solution in volumetric flask & make up to 100 ml with B2 buffer solution.(TL = 0.05 mcg/ml)

f)             Inoculums Preparation in Antibiotic Microbial Assay:

1. 1. Prepare & sterilize the media.

   2.  Cool the media at 40 °C to 50 °C

   3.  Inoculate the 3 ml culture suspension in media & mix well.

   4. Immediately pour the approx 25 ml media in petri plates to obtain 3-4 mm layer.

   5.  Ensure that the layers of medium are uniform in thickness & allow to solidifying.

   6. With the help of sterile cork borer (Diameter 6 mm ± 0.1) cut four agar blocks at equidistance from the four corners to get the four wells in each plates.

   7.  Mark the wells as standard High(SH), Standard low (SL), Test High(TH), Test Low (TL).

   8. Add 0.1 mL of diluted standard & test solution in specified wells.

   9.  Leave the plates in Laminar air flow for even diffusion of samples in medium for 1- 4 hours.

1. 1. Incubate the plates at 30 to 35 °C for about 18 – 24 hours.

   2. After incubation measure the zone of inhibition. Sum the diameter of zones for each dilution & calculate

   3.   the potency from following formula:

               

• TH and TL are the sum of zone diameter with solutions of sample of high & low concentration.

• SH and SL are the sum of zone diameter with solutions of sample of high & low concentration.

l = Ratio of high & low concentration

 

Antibiotic Microbial Assay

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