Microbiological assay of Vitamines.

 

Introduction:

• The microbiological or microbial assay is a type of biological assay in which the relative potency of activity of a compound is determined by measuring the amount required for producing the predicted effect on a suitable test organism under standard conditions. 

• Vitamins are essentially required for the growth and multiplication of microorganisms (which are highly sensitive even to small amounts of growth factors). 

• These test microorganisms can synthesise the factor being assayed which forms the basis of microbiological assay of vitamins and amino acids. 

•  The test microorganisms used for the standardisation of water-soluble vitamins are given in table 1.1.  

Table 1.1 Test Microorganisms and Conditions for the Microbial Assay of Vitamins

Sr No.	Vitamin	Test Microorganism	Incubation Temp (℃)	Assay pH
1	Vitamin B12	Lactobacillus leichamannii ATCC 7830	37	6.1
		Poteriochromonas stipitata ATCC 11532	30	5.5
2	Vitamin B6	Saccharomyces uvarum ATCC 9080	30	4.5
		Tetrahymena thermophila ATCC 30008	30	6.1
3	Riboflavin	Lactobacillus casei ATCC 7469	37	6.8
		Tetrahymena thermophila ATCC 30008	30	6.1
4	Thiamine	Lactobacillus viridescens ATCC 12706	30	6.0
		Ochromonas danica ATCC 30004	30	5.5
5	Biotin	Lactobacillus plantarum ATCC 8014	37	6.8
		Ochromonas danica ATCC 30004	30	5.5
6	Niacin	Lactobacillus plantarum ATCC 8014	37	6.8
		Tetrahymena thermophila ATCC 30008	30	6.1
7	Pentothenic Acid	Lactobacillus plantarum ATCC 8014	37	6.7
		Tetrahymena thermophila ATCC 30008	30	6.1

Microbiological Assay of Cynocobalamin (Vitamin B12):

• Microbiological assay of Vitamin B12 is can be performed by using one of the following methods,

• Tritrimetric Method.

• Turbidimetric Method.

Tritrimetric Method:

Preparation of Standard Cynocobalmine stock solution:

• The standard stock solution is prepared by adding a sufficient amount of 25% alcohol to an accurately weighed quantity of U.S.P. cyanocobalamin RS. 

• The solution obtained having a known concentration of 1.0µg/ml is stored in a refrigerator for not more than two months.

Preparation of Basal Medium Stock Solution:

• Composition of basal medium stock solution is as follows,

Table 1.2: Basal Medium Ingredients  

Ingredient	Quantity
L-Cystine	0.1gm
L-Tryptophan	0.05gm
1N Hydrochloric acid	5 ml
Adenine-guanine-uracil solution	5ml
Xanthine solution	5 ml
Vitamin solution I	10 ml
Vitamin solution II	10 ml
Salt solution A	5 ml
Salt solution B	5 ml
Asparagine solution	5 ml
Acid-hydrolysed casein solution	25 ml
Dextrose (anhydrous)	10 gm
Sodium Acetate (anhydrous)	5 gm
Ascorbic Acid	1 gm
Polysorbate 80 Solution	5 ml

• Cysteine and tryptophan are dissolved in hydrochloric acid and then the next 8 solutions are added. 

• To the resultant solution, 100ml water is added and mixed. 

• To this mixture, dextrose, sodium acetate, and ascorbic acid are added. 

• The solution is filtered and polysorbate 80 solution is added. 

• The pH is adjusted between 5.5 and 6.0 using 1 N sodium hydroxide, and volume is adjusted to 250ml with purified water.

Test Solution of the material to be assayed:

• Take an accurate amount of material to be assayed and dissolve it in water.

• Add dilute HCL or solution of sodium hydroxide to adjust pH (6.0) and add water to make up the final volume.

Preparation of inoculum:

• The inoculum is prepared by transferring cells from the stock culture of the organism to two sterile tubes, each containing 10ml culture medium.

• These culture tubes are then incubated for 16-24 hours at 30-40 ± 0.5°C temperature. 

• The incubated tubes are aseptically centrifuged, and the supernatant is decanted. 

• The cells from the culture are suspended and mixed with a 5ml sterile suspension medium.

• The volume is adjusted using sterile suspension medium so that 1 in 20 dilutions in saline TS produces 70% transmittance on a spectrophotometer at 530nm wavelength, and read against saline TS set at 100% transmittance. 

• 1 in 400 dilution of the adjusted suspension is prepared using basal medium stock solution to use for the test inoculum.  

Procedure of Titrimetric method:

• Clean ten test tubes and add 0.0 ml, 0.5 ml, 1 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3 ml, 4 ml, 4.5 ml and 5 ml respectively of standard cynocobalamine solution (1.0µg/ml).

• To each test tube add 5 ml of basic medium stock solution.

• Adjust final volume (10ml) using water.

• Take another four test tubes and add 1 ml, 2 ml, 3 ml, 4 ml respectively of test solution to be assayed.

• To each test tube add 5 ml of basal stock medium solution and make up final volume (10ml) with water.

• Autoclave all the test tubes @ 121℃ for 5 minutes.

• Cool all test tubes at room temperature and then inoculate one drop of inoculum ( Lactobacillus leichmanni ATCC 7830).

• Incubate all tubes at temperature between 30-37 ℃.

• Titrate contents of each test tube electrometrically or using Bromothymol blue as an indicator to obtain average titration values.

• Plot the graph of average titration values vs std cynocobalamine solution and determine concentration of activity per ml of test solution.

Turbidimetric Method:

• Everything is the same as the Titrimetric method; additionally it contains two test tubes to which neither standard  nor a test cyanocobalamin solution is added.

• Incubate all test tubes at 30-37 ℃ for 16 - 24 hours.

• By using an “Uninoculated Blank Tube” adjust transmittance at 640 mµ to 100% in the photoelectric colorimeter.

• Thoroughly mix contents of each test tube and record transmittance.

• Plot graph of transmittance against concentration of cyanocobalamin solution.

• Draw a smooth curve and calculate the concentration of test solution of cynocobalamine.