Disk, paper, assays

Numerous oxyluminescences, which con be obtained in organic chemistry by rising ozone, open new possibilities for ozone assay, and preliminary results show that a suitable method may be found. Numerical values show the sensitivity of this technique. Small paper disks about the size of confetti and containing no more than 1 y of luminol permit measurements which take only a few minutes. Relatively simple electronic devices permit translation of th se luminescence phenomena into doses (percentages). 

Pumiacidins A, B, C, D, E, F and G (Fig. 5) were isolated by Naruse et al. in 1990 from Bacillus pumilus. Pumilacidins show no inhibitory activity against bacteria and fungi at l,000ng/ml by paper-disk assay. These compormds inhibit herpes virus typel and K -ATPase, and demonstrate anti-ulcer activity in rats ... 

For antimicrobial assays, there are several common methods employed. Due to its ease of operation, the most common method used is the disk diffusion method, which involves the application of a material onto a filter paper disk, and then the disk is placed onto solid medium previously seeded with the test microorganism of interest. Sometimes, the sample is dissolved in an appropriate solvent before application onto the paper disk. This method is very common in the evaluation of antibiotics and is the method adopted by the National Committee for Clinical Laboratory Standards (NCCLS). The method depends on the aqueous solubility of the antibiotics in order to facilitate diffusion through the solid medium. Essentials oils, however, are generally hydrophobic, do not readily diffuse through an aqueous medium and, therefore, the prevalence of false negatives or reduced activity might then be anticipated. 

Micronesia revealed that the source of 78 and 79 was not the sponge but rather the symbiont unicellular bacteria and filamentous eubacteria resident in the interior of T. swinhoei [80]. Swinholide A was reported to be toxic to L1210 (IC50 = 21 nM) and KB (IC50 = 28 nM) cell lines [81]. Theopalauamide inhibited the growth of Candida albicans in the standard paper disk assay at a concentration of 10 pg/disk. 

Fig. 1. Cephamycin A diffusion plate assay. This assay was performed versus P. vulgaris MB-838 in nutrient agar plus 0.2% yeast extract with 7-mm filter paper disks soaked in antibiotic solutions. Data from Stapley et al., 1972.

Figure 1.1. Representation of a typical disk susceptibility assay. A paper disk impregnated with a suspected antibiotic compound is applied to an agar plate seeded with a test microorganism and the area of inhibited microbial growth is indicated by a cleared zone surrounding the disk.

Test chemicals or chemical fractions can be dispensed from a variety of materials, depending on their volatility and solubility. Chemicals may be evaporated from such materials as glass rods or beads, metal surfaces, filter paper disks, cotton balls or wicks, rubber septa, polyethylene vials, glass capillary tubes, etc. It is important to ensure that the dispenser emits the compound at a fairly constant rate over the course of the assay. This may be more difficult for very volatile chemicals, thus, fresh dispensers uniformly prepared ahead of time may be required. This may be most important in the early stages of investigation where fractions of unknown composition and concentration are being used in a bioassay-driven fractionation scheme to isolate and, ultimately, identify specific chemical compounds eliciting particular behaviors. 

Two types of tests were utilized in the evaluation of the modified cellulose products. The paper disk assays were conducted with each test organism suspended in sterile water (10 spores per ml). Appropriate dilutions were made in Sabourand s dextrose agar and poured into Petri plates. The best confluent growth resulted when employing lO" spores per ml which is the concentration employed for subsequent studies. Protein analysis of the samples suspended by continuous vibration were conducted. Cold perchloric acid was added to each culture tube after seven days of growth. Pellets were collected by centrifugation and subsequently suspended in NaOH. 

Since the time Breitman (1963) and Furlong (1963) introduced the use of DEAE-paper in disk form for anion exchange and counting of radioactive nucleotides absorbed onto the disk, immersing it directly into the scintillation fluid, the method has found numerous applications in the assay of enzymes involved in ribo-, and deoxyribonucleotide metabolism (Bresnick and Karjala, 1964, Cheng and Prusoff, 1974, Ives et al., 1969, Patel et al., 1977, Stirpe and La Placa, 1971). 

A useful procedure for this assay is as follows Up to 40 /A of a reaction mixture is absorbed on a 2.4 cm circle of Whatman No. 3 MM paper and washed sequentially in 90% ethanol-10% TCA, 67% ethanol-33% CHCls, twice with 50% ethanol-50% ether and ether. The dried disk is then counted under 5 ml of a toluene-based scintillation fluid. This procedure avoids the high background obtained when solutions containing [ CjAPA-Br are treated with TCA and filtered through Millipore membranes. 

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