Mohi-ud-Din Islamic Institute ofPharmaceutical Sciences.

Presented by :Muslim KhanPresented to :Dr. Momina

BIOLOGICAL ASSY OF ANTIBIOTICS & VITAMIN-D

For antibiotics assay: Introduction Prep of Media Prep.. of Buffer solutionPrep..of standard solution Prep.. Sample solutionMethods (cylindrical method & turbidimetric)For vitamin –D assay: IntroductionSouces Stages of assayLine test

Contents

The potency of a sample of an antibiotic is determined by comparing the dose which inhibit the growth of micro-organism with the dose of standard preparation of that antibiotic.

Two methods used.Cylinderical plate method.Turbidimetric method.

Introduction:

Prepare the media required for the preparation of test organism inoculam from the ingredients .Dissolve the ingredients in sufficient water to produce 1000 ml and Add sufficient 1 M sodium hydroxideor 1 M hydrochloric acid, as required so that after sterilization the pH is as given in monogram.

MEDIA:

Media: Quantities in g of ingredients per 1000 ml

Buffer solutionPrepare by dissolving the given quantities of dipotassiumhydrogen phosphate and potassium dihydrogen phosphatein sufficient watert produce 1000 ml after sterilisation, adjusting the pH with phosphoric acid or potassium hydroxide.

Buffer No. DipotassiumHydrogen Phosphate,        K2HPO4(g)

Potassium Dihydrogen phosphate,KH2PO4(g)

pH adjusted after sterilization to.

1 2.0 8.0 6.0±0.1

2 16.73 0.523 8.0±0.1

3 16.73 13.61 4.5±0.1

4 20.0 80.00 6.0±0.1

5 35.0 - 10.5±0.1*

6 13.6 4.0 7.0±0.2

Table: Buffer Solutions

To prepare a stock solution, dissolve a quantity of the Standard Preparation of a given antibiotic

then dilute to the required concentration as indicated. Store in a refrigerator and use within the period indicated.

On the day of assay, prepare from the stock solution five or more test dilutions, the successive solutions increasing stepwise in concentration, usually in the ratio 1:1.25 for Method A or smaller for Method B.

Use the final diluent specified in monogram.

standard solution:

Assign & assumed potency per unit weight or volume, and on this assumption prepare on the day of the assay a stock solution and test dilution as specified for each antibiotic in monogram but with the same final diluent as used for the Standard Preparation.

.

Sample solution

CYLINDER‐PLATE METHOD: Inoculate a previously liquified medium appropriate to the

assay quantity of suspension of the micro organism add the suspension to the medium at a temperature

between 40 and 50 and immediately pour the inoculated medium into the petri dishes or large rectangular plates to give a depth of 3 to 4 mm.

Ensure that the layers of medium are uniform in thickness. Using the appropriate buffer solutions prepare solutions of known concentrations of the

antibiotic to be examined.

Procedure:

Apply the solutions to the surface of the solid medium in sterile cylinders or in cavities prepared in the agar.

The volume of solution added to each cylinder or cavity must be uniform and sufficient almost to fill the holes when these are used

Leave the dishes or plates standing for 1 to 4 hours at room temperature

Incubate them for about 18 hours at the particular temperature.

Accurately measure the diameters or areas of the circular inhibition zones

The method has the advantage of a shorter incubation period for the growth of the test organism (usually 3 to 4 hours) but the presence of solvent residues or other inhibitory substances affects this assay

Prepare five different concentrations of the standard solution from the stock solution of the Standard Preparation of the antibiotic & increasing stepwise in the ratio 4:5.

Select the median concentration & dilute the solution of the substance being examined (unknown) to obtain approximately this concentration.

TURBIDIMETRIC /TUBE ASSAY METHOD:

Place 1 ml of each concentration of the standard solution and of the sample solution in each of the tubes in duplicate.

To each tube add 9 ml. of nutrient medium At the same time prepare three control tubes,

one containing the inoculated culture medium (culture control), another identical with it but treated immediately with 0.5 ml of dilute formaldehyde solution (blank) and a third containing uninoculated culture medium.

Place all the tubes, randomly distributed or in an incubator or water-bath and maintain them at the specified temperature, for 3 to 4 hours. After incubation add 0.5 ml of dilute formaldehyde solution to each tube.

Measure the growth of the test organism by determining the absorbance at about 530nm of each of the solutions in the tubes against the blank.

BIOASSY OF VITAMIN-D

Vitamin-D is a fat soluble vitaminVitamin – D is a sterol, it contains steroid

nucleusForms of vitamin D:Vitamin D in the diet occurs in two formsVitamin D2 (Ergocalciferol)Vitamin D3 (Cholecalciferol

Vitamin D

Butter,MilkCheesIt can be made by the body when the skin is

exposed to ultraviolet light.Deficiency of vit-D in childrens cause rickets

disease.Excess of vit-D cause irretability ,loss of

weight,& occcasionally death.

Sources:

There are three stages of assay.1: Preliminary period.2: Assigning rats for assay groups .3: Assay period.

Assay:

Not more than 30 daysLitter of rats takenGroup of litter of are made 7-8They are given balance diet containing all

nutrients except vit-D.Weight the rats Weight should not <44 & >60gmAny physical sign of disease or damage

should not be present

Preliminary period.

ingredients Parts by weightWhole yellow corn grounded 76

White glutein grounded 20Caco3 3Nacl 1

Rachitogenic Diet:

Choose rats which should wobbly rachitic gait and joint become larger than normal

X-ray examination is done to confirm rickets.The rats are divided into four groups.To each group one or more assay dose & not

<2 dose of standard should be provided. Difference of wt B/w two groups should not

be greter than 8mg.

Assigning rats for assay groups .

Assay dose:Select two dose levelLarge dose to small dose ratio<1.5 & >2.5Duration of group assigning is 30 daysDose level of reference & sample is same

The rats in two groups receive standard dose and other two receive assay (reference) dose

3rd & 4th day assay and standard dose is halfEnvironmental condition should be uniformAvoid antiricket radiation (sun light)Select rat which don’t show change in weightSelect rat which consume provided food in

24hrsSlaughter these rats and check weather

desired level of calcification is produced

Assay Period:

Take proximal end of tibia bone & distal end of radius bone

Disect & remove tissue attached with themImmersed 24hrs in 4% w/v formaldehyde

solution in water Cut by longitudinal section than immersed in

1.5% w/v sol. of silver nitrate for a few minutes and transferred into water

Exposed to sun light it will cause reaction & produced desired level calcification

Line Test: