魚の組織に含まれるChlortetracyclineのPad plate microbioassayの一改良法

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TOMIYAMA et al4) reported that the sensitivity and recovery of their modified method for CTC assay in fish tissues were 0.02 mcg/cc and about 85%, respectively. A study has been made, therefore, of improvement of the extractant and the assay media in order to obtain higher recovery and sensitivity. From scrutinizing various factors governing the recovery and sensitivity, it has been found that the following modified method is quite feasible and reliable. A 20g aliquot of well comminuted and homogenized sample tissue is further mixed with 80cc of the extractant (35 parts of M/10 citrate (pH 5.2)+65 parts of acetone) for about two minutes. using Waring blender, followed by filtration with a dry filter paper. Each of three pad discs was dipped into the clear filtrate, excessive liquid being removed by touching on the glass wall, and then placed on an plate together with 0.02 mcg/cc reference. Four series of the standard CTC solution (0.08, 0.04, 0.01 and 0.005 mcg/cc of M/10 itrate (pH 5.7)+acetone) were placed on separate plates, three plates for each level. Each plate contains 3 discs of a standard solution plus 3 discs of the 0.02 mcg/cc CTC reference. All the plates were incubated overnight (15-18 his) at 30°C. The content of CTC can be calculated as follows: By employing the following formula, correction can be made of each standard set; Si=si+(R-r), where, Si is corrected value of si, si, average of nine readings for three dishes of standard set having i mcg per cc, R, average of 36 readings for all the reference on all the standard series, and r, average of nine readings for the reference placed on the three standard dishes. Draw the standard curve by plotting these corrected zone diameters, S, together with the average of the 36 readings, R, and the corresponding concentration of CTC (mcg per cc) on semilogarithmic paper, the concentration being plotted on the logarithmic scale. In the sample set, similarly calculate Pi, correced value of zone size for sample solution by using the following formula; P=p+(R-r), where P is average of nine readings for sample set, R and r are the same as the foregoing formula for Si. From the standard curve read the concentration, A mcg/cc which corresponds to the corrected value of zone size, P. Then, the antibiotic content of the sample tissue can be calculated by the following formula; C (mcg/g tissue)=A×(80+20), /20. The preparation of test organism, i.e., spore suspension of B. cereus No. 213 and of seed layer is the same as described by GROVES and RANDOLL.7) The method herein reported was shown to give recovery of 93% and the sensitivity of 0.005 mcg/cc.
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魚の組織に含まれるChlortetracyclineのPad plate microbioassayの一改良法

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