Bioassay; Purpose
Bioassay; To determine the potency of antibiotics of raw materials & products which are specified in different Pharmacopeia.
Bioassay; Scope
This SOP is applicable for Biological Assay of Antibiotics in Microbiology Section at XX Pharmaceuticals Limited.
Definitions
Microbial Assay
In Microbial assay the potency or concentration of a chemical substance (especially antibiotics) may be determined by its effect on the growth of a defined microorganism.
Responsibilities
Executive/Senior Executive, Microbiology
Assay plate preparation, assay dilution, inoculation, zone reading and report preparation.
Manager, Microbiology
Ensure that all activities of Biological assay, document preservation & application of sound technical information.
Checking SOP that the relevant technical information is applied.
Bioassay Procedure
Instructions
- Standard microorganisms are usually non-pathogenic but those are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if required).
- Advised to move softly at Test area not vigorously.
- After completion of sub-culture or transfer of pellets, disinfect the outer surface of vial or test or plate with 70% IPA.
- Remove all used items those are directly contacted with standard micro-organisms. Leave it at designated containers safely.
- Ensure waste container is properly capped until autoclaving.
- Don’t touch apparatus directly in open hands those are used.
- To lessen the contamination, make sure that all personal ornaments, cell phone are left before enter into the test room/area’.
Sterilization of Apparatus & Glassware:
- Sterilize all glassware including Latin Square Plate and others heat stable apparatus at 2000C for 60 minutes in hot air oven using a validated process.
- Use the glassware when the temperature reduce below 400′
Preparation of Culture Media:
- Select the media & diluents as per instruction of BP or USP.
- Prepare 300 ml of particular media as per indication by manufacturer instructions.
- Bring to boil for dissolve completely.
- Sterilize at 1210C for at least 15 minutes.
- Cool media approximately to 450C to 500C
- Allow to solidify the agar media to prepare the slope.
Preparation of Test solution
- Prepare different concentrations of Test Solution to determine of the lowest dose for detectable zone of inhibitions.
- Select & prepare two concentrations of the Test solution as “High Dose” and “Low Dose”.
Preparation of Standard solution
- Prepare different concentrations of Standard Solution to determine of the lowest dose for detectable zone of inhibitions.
- Select & prepare two concentrations of Standard solution as “High Dose” & “Low Dose”.
Preparation of Microorganisms Suspension:
For Spore suspension preparation
- Prepare 140ml of Nutrient agar medium.
- Aseptically Pour the medium on a petri plate (190mm).
- Allow the medium to solidify.
- Keep plate in refrigerator for 30 minutes.
- After 30 minutes take out plate & streak whole plate with the desired organism aseptically.
- Keep plate for incubation at 350C for 7 days.
- After incubation period take out the culture with the aid of sterilized glass beads & pre-sterilized saline water by rotating plate.
- Pour culture suspension along with few of those glass beads in a 100ml flask containing 50ml of sterilized saline.
- Heat culture suspension at 700C for 30 minutes in a water bath for spore formation.
- Cool suspension & then keep inside a refrigerator between (2 to 8)0
- Don’t use the spore suspension more than 60 days.
For Maintenance of Sub-culture of Vegetative bacteria
- Before using a culture maintained in a slant, subculture the organism in another slant containing a specified medium.
- Keep slant for incubation at (30 to 35)0C for 24 to 30 hours.
- Store the slant in the refrigerator at not more than 7 days.
Method: Plate Diffusion:
- Select Latin Square Plate of 12” X 12” size.
- Place plate on a leveled surface after sterilization.
- To the medium( 45 to 50)0C add the organism mentioned in above chart for a particular antibiotic in required amount.
- Shake flask gently to distribute organism throughout the medium.
- Pour medium on plate & allow it to stand for 30 minutes before placing lid in position.
- Transfer plate into refrigerator.
- When required for use, cut cups in agar by means of a sterile cork borer of 8mm diameter.
- Remove each disc of agar with a “spear” so that the surrounding is not lifted.
Application of solution to Assay Plate :
- Enter details of the sample numbers, weight & dilutions on the assay report form, after diluting the standard and test solutions, There is an assay report form for each plate.
- Concentrated solutions are coded with H (High dose) and the lower concentrated solutions are coded with L (Low dose).
- Apply using a standard (100 ±1) µL the solutions to the assay plate in the order of the design.
- Starting at top left hand corner & working from left to right across the rows down to the bottom right hand corner.
- Once started, plating out should be continuous, as it is important that solutions are placed in cups at regular interval.
- Keep the plate about one hour for proper diffusion.
- After diffusion lid the plate with glass lid.
Incubation of Assay Plate
Incubate assay plate for (16 to 20) hours at 370C for antibiotic or 250C for antifungal.
Measurement of the Zone Diameters
- Place the assay plate on a photographic light box.
- Measure zone of inhibition using Varnier calipers.
- Start at top left hand corner and measure the diameter of the zone accurately.
- Continue measuring zones from left to right on row 1, then right to left on row 2.
- Repeat the procedure until all 64 zones have been properly measured.
Bioassay; Calculation of Potencies:
- Sum high & low doses for each standard ( S1&S2)
- Sum high & low doses for each test sample (T1& T2)
- Substrate test treatment totals from standard treatment total. This will give a plus or a
Minus figure = D, D = (T1 +T2)-(S1 +S2).
- B = (Sum of high doses of test, T1 – Sum of low doses of test solution, T2) + (Sum of high doses of
Standard, S1– Sum of low doses of standard, S2)
- Calculate the “Dilution factor of high-test sample (F)” by dividing “Weight of sample” taken by “Total
Volume of dilution” up to high doses.
- Log ratio of dilution (I) = Log (High dose concentration ÷ Low dose concentration)
Actual weight
Calculate “Potency of High Standard (H) = ———————– X. High dose concentration Theoretical weight
Potency (P) = Antilog (D/B x I) x F x H.
Report the result in Biological Assay Report, Annexure-I.