Introduction
Vitamins B2 (riboflavin) and B12 (cyanocobalamin) are essential nutrients required for normal metabolic functions. Microbiological assays are widely used to determine their potency because certain microorganisms depend on these vitamins for growth. The response of these organisms helps to measure the concentration of the vitamin in a sample. These assays are simple, sensitive and suitable for routine use in pharmaceutical quality control.
Microbiological Assay of Vitamin B2 (Riboflavin)
Vitamin B2 plays a major role in energy metabolism. Deficiency can cause sore throat, inflammation of the mouth corners and a red painful tongue. In the assay, the growth of a selected microorganism depends on the amount of vitamin B2 present in the medium.
Principle
The method is based on the ability of the test organism to use vitamin B2 for growth. A known concentration of vitamin B2 promotes proportional microbial growth. By comparing the growth response of the organism to the test sample and to standard concentrations, the amount of vitamin B2 present can be determined.
Test Organism
Lactobacillus rhamnosus is used because it requires vitamin B2 for growth. Even a minor change in vitamin concentration affects its growth pattern, making it a suitable indicator organism.
Media
- The medium must contain all nutrients needed by the organism except vitamin B2.
- The organism is fastidious, so nutrients must be supplemented adequately.
- pH of the medium is maintained at 6.1 ± 0.2 at 25°C.
- Medium is sterilized by autoclaving at 121°C for 15–20 minutes.
Turbidimetric Method (Tube Assay)
This is the primary method for vitamin B2 assay.
- 10 ml of prepared medium is dispensed into sterile test tubes.
- Different concentrations of vitamin B2 (0.00–0.25 ng) are added to separate tubes.
- The test sample is diluted similarly and added to its designated tube.
- 100 µl of the test organism is inoculated into each tube.
- Incubate at 37°C for 24–48 hours.
- Growth is measured by optical density at 520 nm.
Standard Curve
After recording optical densities, a graph is plotted:
- X-axis: Vitamin B2 concentration
- Y-axis: Optical density
The growth increases with concentration, giving a straight line. The unknown sample concentration is read by extrapolating the test value on the curve.
Cup Plate Method (Alternative)
This method measures the zone of growth around wells in solid agar medium.
- Agar media without vitamin B2 is poured into plates and allowed to solidify.
- Lactobacillus rhamnosus is spread evenly on the surface.
- Cups (6–8 mm) are made using a sterile borer.
- Different concentrations of vitamin B2 and the test sample are added to each cup.
- Antibiotic diffuses into the agar; plates are incubated at 37°C for 24–48 hours.
- Zones of growth are measured in mm.
- A standard curve is plotted using vitamin B2 concentration (x-axis) vs. zone size (y-axis).
Microbiological Assay of Vitamin B12
Vitamin B12 is essential for cell growth and blood formation. Its assay also depends on microbial growth requirements.
Test Organism
Lactobacillus leichmannii (ATCC 4797) is used due to its stable growth response to vitamin B12. It requires similar nutrients as Lactobacillus lactis dorner but shows less tendency to disassociate.
Media
- Prepared as a dehydrated product by Difco Laboratories.
- Contains all nutrients except vitamin B12.
- Supports growth only when vitamin B12 is present.
Preparation of Standard and Test Solutions
- Standard and test samples are diluted to around 0.02 µg/ml.
- The organism responds well in the range of 0.01–0.10 µg per tube.
- Control tubes containing only basal medium must show no growth.
Turbidimetric Assay Method
- The inoculum is prepared from 24-hour growth and washed several times with sterile saline.
- Tubes containing medium plus different levels of vitamin B12 are inoculated.
- Tubes are incubated at 37°C for about 18 hours.
- Turbidity (optical density) is measured using a colorimeter or spectrophotometer.
- Growth occurs only when vitamin B12 is present.
Interpretation
- A standard curve is prepared using absorbance and known concentrations.
- The test sample concentration is determined by comparing its turbidity with the standard curve.
Detailed Notes:
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