Tiêu đề 21. MICROBIOLOGICAL ASSAY OF VITAMIN B2 AND B12.pdf ✅

PHARMD GURU Page 1 MICROBIOLOGICAL ASSAY OF VITAMIN B2:  Vitamin B2 (Riboflavin) is an important component and essentially used in metabolism.  Riboflavin deficiency results in stomatitis including red tongue with sore throat and inflammation of the corners of the mouth.  Microbiological assay of vitamin B2 is performed with the aid of microorganisms.  Many therapeutic agents which either inhibit the growth of microorganisms or are essential for growth are standardized by microbial assays. PRINCIPLE: The main principle involved in the microbiological assay of vitamin B2 includes mainly growth sensitivity in which known concentration of vitamin B2 is compared with the test preparation. The main basis of this assay is to measure the ability of test organism to utilize the substance being assayed under nutritional conditions. The growth of test organism is proportional to the dose added to the medium. TEST ORGANISM: Selection of test organism is very important. In this assay “Lactobacillus Rhamnosue” is used. It is one of the fastidious organism and it requires elaborative nutrients. This organism is sensitive towards vitamin B2. Even a small fluctuation in the quantity of vitamin B2 will lead to a fluctuation in the growth of microorganism. MEDIA: The media used for this assay should contain all essential nutrients which helps for the growth of microorganism. As it is a fastidious organism it cannot synthesize nutrients by its own. So all the essential nutrients should be provided in the media or lyophilized cells can be used for conduction of a particular organism. Assay media contains all the ingredients except vitamin B2, pH of this media is 6.1 ± 0.2 at 25°C. The media is autoclaved at 121°C for 15-20 min and then cooled. METHOD:  The prepared media is cooled and transferred into test tubes. Then 10ml of media is transferred to 10 test tubes aseptically. MICROBIOLOGICAL ASSAY OF VITAMIN B2 AND B12
PHARMD GURU Page 2  The test tubes should be sterilized and cooled.  Then different vitamin B2 concentrations should be transferred into corresponding tubes (the corresponding concentrations of vitamin B2 in 10 test tubes is 0.00, 0.025, 0.05,...... 0.25 nanograms).  The test vitamin B2 solution is diluted as of standard and added in one of the tubes as vitamin B2 supplement.  In each test tube 100μl of test organism is incorporated.  The test tubes are incubated at 37°C for 24-48 hrs.  After incubation for a particular period, the growth of organism is observed.  The growth of microorganism is measured by optical density at 520nm.  There is maximum absorption at a particular wavelength.  All the test tubes are measured by optical density and so is the test sample. STANDARD CURVE: After observing optical density for all the samples, the graph is plotted. The standard curve is plotted by taking concentration of vitamin B2 on x-axis and observances on y-axis. At the beginning of concentrations, the growth is less and as the concentrations increases the growth also increases and so do the observances. The straight line is obtained and from this line, the concentration of unknown is measured. After extrapolation on x-axis the concentration of test is obtained. This method is known as turbidimetric method because turbidimetry is also measured in a liquid media. An alternate method is also present i.e. agar diffusion method and it is also called as cup plate method. CUP PLATE METHOD:  The vitamin B2 assay is alternatively done by this method.  A petri plate consists of agar media (solid media which contains all nutrients and agar -solidifying agent) except vitamin B2.  The agar media is autoclaved, cooled and transferred to petri plates and solidified in gel form.  To this agar media the test organism (lactobacillus rhamnosus) is transferred into surface of this media and spreaded with L-shaped glass rod.  After application of test organism cups are made by appropriate diameter (6-8mm) by using a borer.
PHARMD GURU Page 3  The various vitamin B2 concentrations are prepared and added to each cup from lower to higher concentrations.  One of the cups contains unknown concentration.  The cups are kept aside so that vitamin B2 diffuses into the media.  The plates are incubated In Incubator at 37 °C for 24-48 hrs.  After incubation period, the growth is observed around the cups because test organism is sensitive to vitamin B2.  The zone of growth (mm) is observed around the cups.  After observing all the results the standard curve is plotted by taking concentration of vitamin B2 on x-axis and zone of growth (mm) on y-axis.  The graph is extrapolated to know the unknown concentration. MICROBIOLOGICAL ASSAY OF VITAMIN B12:  Vitamin B12 is also called as cyanocobalamin; it helps in growth of microorganisms.  It has been demonstrated that lactobacillus lactis dorner (ATCC 8000) must be cultivated with special precautions to prevent disassociation when used in the microbiological assay of vitamin B12.  Lactobacillus leichmannii has been found to require nutrients similar to those needed by lactobacillus lactis dorner and to exhibit little tendency to disassociate.  The use of a stable test organism in the microbiological assay of vitamin B12 would be of distinct advantage.  The method herein outlined employs lactobacillus leichmannii (ATCC 4797) as the test organism.  The medium for the assay is a dehydrated product prepared by the Difco Laboratories.  The standard and test materials are diluted to a concentration of 0.02 y per ml. in distilled water.  The test organism responds most satisfactorily within the range of 0.01 to 0.10 y per tube levels.  Duplicate or triplicate tubes on each level give growth response in good agreement turbidimetrically.  Tubes containing only distilled water and the basal medium produce no growth in the absence of vitamin B12.
PHARMD GURU Page 4  The results of the assay are evaluated turbidimetrically after 18 hours growth at 37.  Inoculums for the test is prepared by washing four or five times in sterile saline; the cells produced from a 24 hour growth of Lactobacillus leichmannii in Difco micro inoculum broth.