Paper disc assay

Tani, et al. (32) have described a paper disc assay method to screen the biosynthetic intermediates of vitamin Bg. 

The cells on the paper disc are viable and may be used in autoradiographic studies. Alternatively, they may be lysed by freeze-thawing and incubated in enzyme assays. 

Waksman and Reilly have summarized the factors which have a bearing upon the choice of the method to be employed in measuring quantitatively the activity or potency of an antibiotic substance. They have cited literature references to the more commonly used methods. Loo and coworkers have described a suitable method for the quantitative determination of streptomycin by the filter paper disc, agar plate diffusion technique using Bacillus subtilis as the test organism. The procedure proved satisfactory in the assay of surface and submerged culture beers and of preparations obtained in isolation and purification... 

Using casein as substrate, cyclic AMP has been measured directly in crude tissue extracts by the stimulation of the rate of phosphorylation of casein catalysed by skeletal muscle protein kinase [158]. By using high concentrations of casein and [y- P]ATP, the interference with the protein kinase activity by materials present in the tissue extracts is minimised and preliminary purification is not necessary. The phosphory-lated casein is isolated on filter paper discs. As little as 0.5 pmol of cyclic AMP can be measured. The assay is rapid and simple. 

In the commonest form of microbiological bioassay used today, samples to be assayed are applied in some form of reservoir (porcelain cup, paper disc or well) to a thin layer of agar seeded with indicator organism. The drug diffuses into the medium and after incubation a zone of growth inhibition forms, in this case as a circle around the reservoir. All other factors being constant, the diameter of the zone of inhibition is, within limits, related to the concentration of antibiotic in the reservoir. 

If ferrous ions are present during assays of DNA and RNA polymerases in which radioactive newly synthesized polynucleotides are precipitated on to filter paper discs in trichloroacetic acid, spuriously high results are obtained, apparently due to acid-insoluble complexes of Fe with substrate nucleotides precipitating on the paper. Clearly ferrous ions should be avoided in assays of this type. The complex Al. ATP is a potent inhibitor of hexokinase, and neutron activation analysis of many commercial samples of ATP has shown that Al is ubiquitous, and the most common metal contaminant It is best removed by passing the ATP preparation over a cellulose polyphosphate column at pH 5. Chelates of ATP with divalent metal ions have been separated from non-chelated ATP using reverse-phase h.p.l.c. ... 

Test specimens were immediately placed on the freshly inoculated agar plates and aerobically incubated for 18-24 hours at 37°C. Each plate contained 7 disc samples - two per material and a paper disc soaked with chlorhexidine at the center as the control. This assay was performed in quadruplets. After 18-24 hours, the diameters of the zone of inhibition of bacterial growth around the discs were measured using a caliper by two independent observers. 

Microbiological methods employ the paper disc-plate assay. For rifomycin B, Siz y-lococcus aureus ATCC 6538 is used as the test microorganism. The agar medium is buffered at pH 5.0, since a low piH enhances the sensitivity of the test, fevoring the transformation of the inactive rifamycin B into ri mycins O and S. The dose-response curve... 

The reaction with phenyl acetyl chloride in the presence of sodium bicarbonate is readily adapted to the detection of 6-aminopenicillanic add on paper chromatograms, which are sprayed with the appropriate reagents before plating on agar seeded with a sensitive bacterium in the usual way. This conversion of 6-aminopenicillanic acid to benzylpenicillin on paper strips also provides a convenient method of assay, being similar to the well known paper disc method... 

A standard curve is prepared by diluting the standard solution to the following concentrations 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 /ig. per milliliter. Use three plates for the determination of each point on the curve. Filter paper discs (No. 740E, Schleicher Schuell, Keene, N. H.) serve as reservoirs for the antibiotic. The standard curve and sample calculations are the same as those used in the plate assay of penicillin (Section 11,1). 

The amount of antibiotic in broths and solutions was estimated according to a modification of the paper-disc plate method of Loo et al. (1945), using either spores or a viable culture of Bacillus subtilis. Neomycin B sulfate was diluted to appropriate concentrations with 0AM phosphate buffer (pH 7-9) and used as standard. Solutions to be assayed were serially diluted with the same buffer solution. 

The solution is placed in contaet with agar, which is already inoculated with the test organism and after ineubation zones of inhibition observed. The solution may be placed in a small eup sealed to the agar surfaee (a method used widely in anhbiotic assays) in a well cut from the agar with a sterile cork-borer, or applied in the form of an inpregnated disc of filter paper (Fig. 11.5 A). 

Second instar C. puncticollis larvae (11 days old) were obtained by allowing adult mated weevils to oviposit on the roots for 24 hours. The age of the weevils was recorded from the first day of incubation. One larva was placed into each diet burrow with 5 burrows on each Petri dish. The Petri dishes were then covered with a disc of filter paper to absorb excess moisture from the diets and the lids were replaced and the bio-assay left to stand for 10 days. The percentage larval mortality was recorded. 

By utilizing commercially available antibiotic filter discs, which are slightly more resistant to cellulase activity and have a higher degree of crystallinity than conventional filter paper, an improved assay for cellulase activity has been developed. ... 

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