A microbial bioassay is a testing procedure in which the biological activity of a substance or product to stimulate or inhibit the growth of a microbial test organism is estimated. In contrast to common physical or chemical methods, a microbial bioassay results in detailed information on the true activity of a substance. Over the last decade, this type of assays has become increasingly important for quality control and product development.
Types of Microbial Bioassay
There are two types of microbial bioassay as per USP.
- Cylindrical Plate Assay
- Turbidimetric Assay
Cylindrical Plate Assay
This method depends upon the diffusion of antibiotics through a solidified agar layer in a Petri dish to an extent that the growth of microorganisms is prevented in a circular area known as the zone of inhibition, around the cavity containing antibiotic solution.
Turbidimetric Assay
The turbidimetric method is a method to determine the antimicrobial potency of an antibiotic, based on the measurement of the inhibition of growth of a microbial culture in a fluid medium. The inhibition of the growth of a test organism is photometrically measured as changes in the turbidity of the microbial culture.
Procedure
In this article, I will discuss in detail the procedure of the cylindrical plate method of microbial bioassay.
Reagents
Buffer pH 8.0
Dissolve 1.70gm of KH2PO4 and 0.491gm of NaOH in distilled water and make volume to 500ml with distilled water. Adjust pH with 0.1N NaOH or 0.1N HCl if required. Sterilize this buffer in the autoclave for 15 minutes at 121 C and 15 PSI pressure.
Preparation of Innoculum
Grow pure organism Staphylococcus epidermidis ATCC 12228 for 18 hours in Antibiotic Agar # 1 pH 6.6 ± 0.1 at 32 — 35°C. This growth is transferred with 3ml sterile saline to the surface of 250ml Antibiotic Agar # 1 pH 8.3 ± 0.1 and incubate it for 24 hours at 32 — 35°C. Prepare the stock suspension by collecting the surface growth in 50ml of sterile saline.
Preparation of Standard Dilutions
The quantity (mg) of standard is calculated as below and weighed exactly to prepare standard stock solution of 1000 IU / ml.
100,000/ T1
Where T1 = Std. concentration in IU /mg
Place the weighed Standard in a 100ml volumetric flask.
Add approx. 80ml of sterile phosphate buffer solution pH 8.0 ± 0.1.
Mix with a magnetic stirrer and make-up the volume to 100ml with sterile phosphate buffer solution pH 8.0 ± 0.1.
Make standard high and standard low dilution as follows.
i- Standard High: 1ml stock solution volume to 50 ml with sterile phosphate buffer pH 8.0 (i.e. 20 IU / ml).
ii- Standard Low: 0.5ml stock solution volume to 50ml with phosphate buffer pH 8.0 (i.e. 10 IU / ml).
Preparation of Sample Dilutions
Dissolve equivalent sample powder in 100ml sterile phosphate buffer pH 8.0. Filter stock sample solution after 30 minutes stirring before proceeding for dilutions.
Prepare sample high and low dilutions as in case of standard.
Preparation of Media Plates
Prepare Antibiotic Agar # 1 as directed by the manufacturer in 200ml quantity. Adjust pH to 8.3 ± 0.1 and then divide it into 150ml and 50 ml in the separate flask and then autoclave them.
Give base layer by pouring 21 ml each into six sterilized Petri plates with the help of sterile wide tip 25ml pipette let it solidify.
Add 0.2ml of prepared organism suspension to 50ml of prepared media while at 45°C swirl to mix suspension evenly and give seed layer of 4ml with the help of a 5ml wide tip sterile pipette. Leave the plates for solidification of the seed layer.
Make four cavities 6 — 8 mm with the help of a sterilized borer and spatula and mark as SL, SH, STL, STH.
S = Sample
ST = Standard
L = Low Dosage
H = High Dosage
Fill cavities by pouring 100µl of prepared low dilution (1 IU / 100µl) and High dilution (2IU / 60ml).
Incubation
Allow the transferred solution to absorb in the media for 1 — 2 hours then incubate the plates in the incubator at 36 — 37.5°C for 18 hours.
Observations
After incubation period observe the plates for zones of inhibition
Measure the zones on zone magnifier with the help of vernier caliper.
Four out of six plates should give reproducible results.
Calculations
Calculate the content as follows.
| Potency (%) = | Antilog | (SH + SL) — (STH + STL) ____________________ x 0.301 (SH – SL) + (STH – STL) | x 100 |