The document outlines microbiological assays for standardizing therapeutic agents such as antibiotics, vitamins, and amino acids, detailing two primary methods: the cylinder plate (cup plate) method and the tube assay method. The cylinder plate method involves measuring the zone of inhibition of an antibiotic in agar, while the tube assay method includes preparing multiple test tubes with varying concentrations of cyanocobalamin for titration. Both methods aim to assess the effectiveness of antimicrobial agents through bacterial growth inhibition.

1. Microbial Assay
Prepared by ,
Mr. Snehal Patel,
Assistant Professor,
Sumandeep Vidyapeeth,
Vadodara, Gujarat, India.

2. • Many therapeutic agents, which either inhibit the growth of
microorganisms (Antibiotics) or essential for their growth
(vitamins and aminoacids) can be standardised by
microbiological assays.
• Two general methods are used for microbiological assays
1. Method A: Cylinder plate method or cup plate method.
2. Method B: Tube assay method or titrimetric method.

3. Method A: Cup Plate or Cylinder plate Method
Principle:
• This method depends on the diffusion of an antibiotic from a vertical cavity or
cylinder, through the solidified agar layer in a petri plate. The growth of test
microorganism is inhibited entirely in a circular area or zone around the cavity
or cylinder containing a solution of antibiotic.
Procedure:
• The nutrient agar is melted, cooled suitably, poured into petri dish.
• Spread 0.2 ml of known concentration of inoculum on the surface of the
solidified agar ( Spread Plate Technique).
• Cups or cavities are made by using a sterile borer.
• Now 0.2,0.4,0.6,0.8,1.0 ml of antibiotic is poured into the cups of agar plate
and then incubated at 370C for 24 hr.
• If the antibiotic has any anti-bacterial effect it will show the zone of inhibition.

4. 2. Cup Plate or Cylinder plate Method
Zone of Inhibition

6. Assay of Cyanocobalamin ( Vitamin B12)

7. Method B: Tube assay method / Titrimetric method
• Clean 10 test tubes and add 0.0ml, 0.5ml, 1.0ml, 1.5ml, 2ml, 2.5ml,
3ml,4ml,4.5ml and 5ml respectively of standard cyanocobalamin
solution.
• To each test tube add 5ml basal medium stock solution.
• Adjust final volume 10ml by using water.
• To other four test tubes, add 1ml, 2ml,3ml,4ml respectively of the test
solution to be assayed.
• To each test tube add 5ml basal medium stock solution.
• Adjust final volume 10ml by using water.

8. • Sterilize all test tubes in autoclave at 1210C for 5 minutes. After
sterilization, cool all test tubes upto room temperature and inoculate
with one drop of inoculum (bacterial culture). Incubate the test tubes
for 64 to 72 hours at any chosen temperature with in the range of 30-
37OC.
• Titrate the contents of each tube with 0.05N NaOH using 0.1%w/v
bromothymol blue as an indicator (converts to green colour).
Determine the average of the titration values for each level of
standard and test sample used.
• Plot the graph of titration value versus Std. Cyanocobalamin solution
concentration.