CONSISTING OF A SHEET OF GLASS WITH A FACED ALUMINIUM OR PLASTIC RING PLACED ON TOP, 200 MM IN DIAMETER AND 20 MM HIGH .
POUR INTO THE PLATES A QUANTITY OF THE MEDIUM ( 4.2 ) INOCULATED AS IN POINT 7.1, TO GIVE A LAYER ABOUT 2 MM THICK ( 60 ML FOR A PLATE OF 200 MM DIAMETER ), ALLOW TO SET IN A LEVEL POSITION, BORE THE HOLES AND PLACE IN THEM EXACTLY MEASURED VOLUMES OF ASSAY AND STANDARD SOLUTIONS ( BETWEEN 0,10 AND 0,15 ML PER HOLE, ACCORDING TO THE DIAMETER ). APPLY EACH CONCENTRATION AT LEAST FOUR TIMES SO THAT EACH DETERMINATION IS SUBJECT TO AN EVALUATION OF 32 ZONES OF INHIBITION .
7.3 . INCUBATION
INCUBATE THE PLATES FOR 16 TO 18 HOURS AT 30 2 C .
8 . EVALUATION
MEASURE THE DIAMETER OF THE ZONES OF INHIBITION TO THE NEAREST 0,1 MM . RECORD THE MEAN MEASUREMENTS FOR EACH CONCENTRATION ON SEMI-LOGARITHMIC GRAPH PAPER SHOWING THE LOGARITHM OF THE CONCENTRATIONS IN RELATION TO THE DIAMETERS OF THE ZONES OF INHIBITION . PLOT THE BEST FIT LINES OF BOTH THE STANDARD SOLUTION AND THE EXTRACT, FOR EXAMPLE AS BELOW :
DETERMINE THE BEST FIT" POINT FOR THE STANDARD LOWEST LEVEL ( SL ) USING THE FORMULA :
1.2(A ) SL =
7S1 + 4S2 + S4 - 2S8 10
DETERMINE THE BEST FIT" POINT FOR THE STANDARD HIGHEST LEVEL ( SH ) USING THE FORMULA :
1.2(B ) SH =
7S8 + 4S4 + S2 - 2S1 10
SIMILARLY CALCULATE THE BEST FIT" POINTS FOR THE EXTRACT LOWEST LEVEL ( UL ) AND THE EXTRACT HIGHEST LEVEL ( UH ) BY SUBSTITUTING U1, U2, U4 AND U8 FOR S1, S2, S4 AND S8 IN THE ABOVE FORMULAE .
RECORD THE CALCULATED SL AND SH VALUES ON THE SAME GRAPH PAPER AND JOIN THEM TO GIVE THE BEST FIT" LINE FOR THE STANDARD SOLUTION . SIMILARLY RECORD UL AND UH AND JOIN THEM TO GIVE THE BEST FIT" LINE FOR THE EXTRACT .
IN THE ABSENCE OF ANY INTERFERENCE THE LINES SHOULD BE PARALLEL . FOR PRACTICAL PURPOSES THE LINES CAN BE CONSIDERED PARALLEL IF THE VALUES ( SH-SL ) AND ( UH-UL ) DO 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 AND S8 MAY BE DISCARDED AND SL, SH, UL AND UH CALCULATED, USING THE ALTERNATIVE FORMULAE, TO GIVE ALTERNATIVE BEST FIT" LINES :
(A' ) SL =
5S1 + 2S2 - S4 6
OR
5S2 + 2S4 - S8 6
( B' ) SH =
5S4 + 2S2 - S1 6
OR
5S8 + 2S4 - S2 6
AND SIMILARLY FOR UL AND UH . THE SAME CRITERIA OF PARALLELISM SHOULD BE SATISFIED . THE FACT THAT THE RESULT HAS BEEN CALCULATED FROM THREE LEVELS SHOULD BE NOTED ON THE FINAL REPORT .
WHEN THE LINES ARE CONSIDERED AS BEING PARALLEL, CALCULATE THE