for people all over the world

3.  Presented by :
 Muslim Khan
 Presented to :
 Dr. Momina

4. For antibiotics assay:
 Introduction
 Prep of Media
 Prep.. of Buffer solution
 Prep..of standard solution
 Prep.. Sample solution
 Methods (cylindrical method & turbidimetric)
For vitamin –D assay:
 Introduction
 Souces
 Stages of assay
 Line test

5.  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.

6. 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.

8.  Buffer solution
Prepare 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.

9. Buffer No. Dipotassium
Hydrogen
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

10.  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.

11.  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.
.

12. 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.

13.  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

14.  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.

15.  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.

16.  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.

17. BIOASSY OF VITAMIN-D

18.  Vitamin-D is a fat soluble vitamin
 Vitamin – D is a sterol, it contains steroid nucleus
 Forms of vitamin D:
 Vitamin D in the diet occurs in two forms
 Vitamin D2 (Ergocalciferol)
 Vitamin D3 (Cholecalciferol
Vitamin D

19.  Butter,
 Milk
 Chees
 It 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.

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

21.  Not more than 30 days
 Litter of rats taken
 Group of litter of are made 7-8
 They are given balance diet containing all nutrients
except vit-D.
 Weight the rats
 Weight should not <44 & >60gm
 Any physical sign of disease or damage should not be
present

22. ingredients Parts by weight
Whole yellow corn grounded 76
White glutein grounded 20
Caco3 3
Nacl 1

23.  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.

24.  Assay dose:
 Select two dose level
 Large dose to small dose ratio<1.5 & >2.5
 Duration of group assigning is 30 days
 Dose level of reference & sample is same

25.  The rats in two groups receive standard dose and other
two receive assay (reference) dose
 3rd & 4th day assay and standard dose is half
 Environmental condition should be uniform
 Avoid antiricket radiation (sun light)
 Select rat which don’t show change in weight
 Select rat which consume provided food in 24hrs
 Slaughter these rats and check weather desired level of
calcification is produced

26.  Take proximal end of tibia bone & distal end of radius
bone
 Disect & remove tissue attached with them
 Immersed 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