S.L. 437.82 Art. 8Record the graph paper of the zones the extract, for the formula: using the (UL) and the s and s in the 4 8 join them to UH and join For practical (UH-UL) do and s8 may be to give be satisfied. noted on the (log A) of upon whether three For four (c) log A = For three (d) log A = or (d) log A = Activ

EVALUATION Measure the diameter of the zones of inhibition to the nearest 0,1 mm. mean measurements for each concentration on semi-logarithmic showing the logarithm of the concentrations in relation to the diameters of inhibition. Plot the best fit lines of both the standard solution and example as below: Determine the ‘best fit’ point for the standard lowest level (SL) using

7s + 4s +s - 2s 1 2 4 8 SL = 10

Determine the ‘best fit’ point for the standard highest level (SH) formula:

7s + 4s +s - 2s 8 4 2 1 SH = 10

Similarly calculate the ‘best fit’ points for the extract lowest level extract highest level (UH) by substituting u , u , u and u for s , s , 1 2 4 8 1 2 above formulae. Record the calculated SL and SH values on the same graph paper and give the ‘best fit’ line for the standard solution. Similarly record UL and them to give the ‘best fit’ line for the extract. In the absence of any interference the lines should be parallel. purposes the lines can be considered parallel if the values (SH-SL) and not differ by more than 10% from their mean value.

If the lines are found to be non-parallel either u1 and s1 or u8 discarded and SL, SH, UL and UH calculated, using the alternative formulae, alternative ‘best fit’ lines:

5s + 2s - s 5s + 2s - s 1 2 4 2 4 8

(a) SL = or 6 6

5s + 2s - s 5s + 2s - s 4 2 1 8 4 2

(b) SH = or 6 6

and similarly for UL and UH. The same criteria of parallelism should The fact that the result has been calculated from three levels should be final report. When the lines are considered as being parallel, calculate the logarithm the relative activity (A) by means of one of the following formulae, depending or four levels have been used for the assessment of parallelism.

levels (U + U + U + U - S - S - S - S ) × 0.602 1 2 4 8 1 2 4 8

U + U + S + S - U - U - S - S 4 8 4 8 1 2 1 2 levels

(U + U + U - S - S - S ) × 0.401 1 2 4 1 2 4

U + S - U - S 4 4 1 1

(U + U + U - S - S - S ) × 0.401 2 4 8 2 4 8

U + S - U - S 8 8 2 2 sample extract = activity of relevant standard × A (u = s × A) 8 8 relative activity is found to be outside the range of 0,5 to 2,0, then repeat the appropriate adjustments to the extract concentrations or, if this is not standard solutions.

relative activity cannot be brought into the required range, any result be considered as approximate and this should be noted on the final lines are considered as not being parallel, repeat the determination. If still not achieved, a satisfactory determination Has not been obtained. result in milligrams of zinc bacitracin per kilogram of feedingstuff.

REPEATABILITY difference between the results of two determinations carried out on the same same analyst should not exceed: mg/kg, in absolute value, for contents of zinc bacitracin up to 10 mg/kg, related to the highest value for contents from 10 to 25 mg/kg, mg/kg, in absolute value, for contents of 25 to 50 mg/kg, related to the highest value for contents above 50 mg/kg. DETERMINATION OF FLAVOPHOSPHOLIPOL BY DIFFUSION IN MEDIUM PURPOSE AND SCOPE

is for the determination of flavophospholipol in feedingstuffs, and premixes. The lower limit of determination is 1 mg/kg (1 ppm).

PRINCIPLE is extracted with diluted methanol by heating under reflux. After the extract is purified (where necessary) by treatment with ion resins and diluted. Its antibiotic activity is determined by measuring the flavophospholipol in an agar medium inoculated with Staphylococcus Diffusion is shown by the formation of zones of inhibition of the micro-diameter of these zones is taken to be in direct proportion to the the antibiotic concentration over the range of antibiotic concentrations

MICRO-ORGANISM: STAPHYLOCOCCUS AUREUS ATCC 6538 P 3.1. Maintenance of stock culture

Inoculate Staphylococcusaureus onto agar slopes of culture medium Incubate for 24 hours at 37ºC, store in a refrigerator at about 4ºC and every month onto agar slopes. 3.2. Preparation of the bacterial suspension*

Set aside two tubes containing the stock culture (3.1) and reinoculate weekly. Incubate for 24 hours at 37ºC and store in a refrigerator at about

24 hours before the assay, inoculate with this growth two to four tubes slopes of culture medium (4.1). Incubate for 16 to 18 hours at suspension of the growth in the sodium chloride solution (4.3). transmission of the suspension must be about 40 %, measured at 578 cell against sodium chloride solution (4.3).