S.L. 437.63 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 (b') SH = and similarly The fact that final report. When the relative whether three For four (c) Log A For three (d

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

7 + 4 + - 2 S1 S2 S4 S8

(a) SL = 10

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

7 + 4 + - 2 S8 S4 S2 S1

(b) 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:

5 + 2 - 5 + 2 -S1 S2 S4 S2 S4 S8 (a') SL = OR 6 6 5 + 2 - 5 + 2 -S4 S2 S1 S8 S4 S2 OR 6 6

for UL and UH. The same criteria of parallelism should be satisfied. the result has been calculated from three levels should be noted on the lines are considered as being parallel, calculate the logarithm of the activity (log A) by means of one of the following formulae, depending upon or four levels have been used for the assessment of parallelism. levels

(U + U + U + U - S - S - S - S ) ꞏ0ꞏ602 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 - S ) × 0ꞏ401 1 2 4 1 2 4 8 = U + S - U - S 4 4 1 1

(U + U + U - S - S - S ) × 0ꞏ401 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. When the relative activity cannot be brought into range, any result obtained must be considered as approximate and this noted on the final report.

lines are considered as not being parallel, repeat the determination. If is still not achieved, the determination must be considered as result in milligrams of virginiamycin per kilogram of feedingstuff.

Repeatability difference between the results of two parallel determinations carried out on sample by the same analyst should not exceed: mg/kg, in absolute value, for contents of virginiamycin up to 10 mg/kg, related to the highest value for contents of 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.