MICROBIOLOGICAL ASSAY: C & BE : NCP

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DEFINITION: Microbiological assay is a type of biological assay performed with the aid of microorganisms. Many therapeutic agents, which either inhibit the growth of microorganisms or are essential for the growth of them are standardized by microbial assay. Principles of microbial assay were developed in 1920s. MICROBIOLOGICAL ASSAY OF VITAMINS

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MICROBIOLOGICAL ASSAY OF VITAMINS : PRINCIPLE : Vitamins and amino acids are essential for the growth of microorganisms. The basis of this assay is to measure the ability of test organism to utilize the substance being assayed under a proper nutritional condition. The organisms require these growth factors (vitamins & amino acids) in micro or nano grams.

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The response (growth of test organism) is proportional to the dose (amount of factor) added to medium. Materials required for Microbial assay of vitamins & amino acids: A stock solution. A inoculum media. Assay medium. A standard curve.

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MICROBIAL ASSAY OF VITAMIN B 12 : About VitB 12 : Also known as cyanocobalamin. Its a water soluble vitamin. Structure is similar to that of heme where the iron is replaced with cobalt as a centre of molecule. Its main sources are liver, eggs , milk meat & fish. VitB 12 deficiency causes Macrolytic anemia, pernicious anemia. National Research Council, USA recommends a daily intake of about 5mg of vitB 12

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PRINCIPLE OF ASSAY: The test organism selected must be capable of utilizing free cyanocobalamin. Lactobacillus Liechmannii is found to satisfy the requirements. Gram negative bacilli, non-pathogenic, easy to culture & easily available. Isolated from milk, cheese, & other dairy products. Assay is performed by using either titrimetric or turbidimetric method.

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ASSAY PARAMETERS Culture : Lactobacillus Liechmannii Principle : microbial assay, titrimetric method Vitamin : cyanocobalamin Apparatus : test tubes, pipettes, conical flask, autoclave, refrigerator, hot air oven, incubator. Upper limit : 10picogram/ml Medium : Basal medium stock solution

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PRECAUTIONS: Great care must be taken to avoid contamination All the glass wares must be free from detergents and other chemicals. Glass wares must be heated to 250 0 C for atleast 1hr before use. The whole experiment must be carried out under proper aseptic condition.

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Composition of Basal medium stock solution: INGREDIENTS QUANTITY L-cystine 0.1gm DL-Tryptophan 0.1gm 1N HCl 10ml Adenine-Guanine-Uracil solution 5ml Xanthine solution 5ml Riboflavin-Thiamine-Biotin- 10ml Nicotinic acid solution p-Aminobenzoic acid-pyridoxine Pyridoxal-pyridoxamine solution 10ml Calcium Pantothenate-Folic acid solution 5ml Salt solution A 5ml Salt solution B 5ml Asparagine Solution 5ml Acid-Hydrolysed Casein Solution 25ml Dextrose 10gm Sodium Acetate 5gm Ascorbic acid 1gm Sorbitan Mono-oleate derivative solution 5ml

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Reagents Required : 1) Standard stock solution: An accurately weighed amount of Cyanocobalamin reference Standard is added to sufficient 25%ethanol (resulting in a solution containing 1.0 μg of cyanocobalamin per ml ). Stored in refrigerator. It should be used within 2months. Further dilutions of this stock solution (1 μg/ ml) are made as follows: Add 1 ml stock solution to 99 ml purified water (1 ml =10 ng). Add 1 mL of the above solution to 199 ml purified water (1 ml = 0.05 ng).

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2) Test solution to be assayed : Accurate amount of Vitamin to be assayed is taken & dissolved in water . Dil HCl or NaOH is added to adjust pH at 6.0 Make up to volume with water

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3)Preparation of Inoculum : Transfer a loop full of Lactobacillus Liechmannii from a recent sub-culture into two tubes each containing 10ml of sterile culture medium. Composition of culture medium :( pH 6.8) Yeast extract - 0.75gm Peptone - 0.75gm Dextrose - 1gm Pot dihydrogen phosphate - 0.2gm Tomato juice filtrate - 10ml Sorbitan mono oleate solution - 1ml Water up to - 100ml

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Incubate for 18 to 24hrs at 37 o C. Centrifuge the culture . Decant the supernatent fluid, under aseptic condition. Suspend these cultured cells into 10ml of sterile suspension of Basal medium stock solution. Again centrifuge & decant off supernatent fluid. Repeat this for atleast 3times to avoid contamination. Finally suspend the cells uniformly in 10ml of sterile medium. Aseptically transfer 1ml of the suspension of cells to 10ml of sterile medium & mix. This resulting cell suspension is taken as inoculum.

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Procedure for assay : Clean ten test tubes & add to it 0, 0.5, 1.0, 2.0, 2.5, 2.5, 3.0, 3.5, 4.0, 4.5, & 5ml of standard cyanocobalamin solution. To each tube add 5ml of Basal medium solution Volume of each is adjusted to 10ml by water In another 4 test tubes add 1, 2, 3, 4ml of test solution which is to be assayed . To each of this also add 5ml of Basal medium stock solution & adjust volume to 10ml with water.

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Sterilize all test tubes in autoclave at 121 o C for 15mins. Cool the test tubes at room temperature. Inoculate a drop of inoculum prepared of lactobacillus liechmannii. Incubate the test tubes for 64 to 72hrs at temperature range of 30 to 37 o C. After incubation period titrate contents of each test tube with 0.05N NaOH using bromothymol blue as indicator until green colour. Record all the titre readings clearly.

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Interpretation of results: Determine the average of titration values of each level of both standard & test solutions. Plot a graph considering average titration values (in ml) of 0.05N NaOH against concentration of standard cyanocobalamin solution. A linear graph is obtained. By interpolating the standard curve determine the concentration as activity per ml of vit B 12 . From the graph the concentration of test solution of cyanocobalamin is found & reported.

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Assay of cyanocobalamin can also be performed by turbidimetric method By using a un inoculated medium as blank & adjusting transmittance to 640nm at 100% in a photoelectronic colorimeter. The other procedure is carried out as like done in titrimetric method. Plot the graph by considering transmittance v/s standard cyanocobalamin solution.