Assay Organism Research Articles

Factors Influencing Optimization of Diffusion Assays for Antibiotics

Agar nutrient content, cylinder charge volume, thickness (volume) of the agar layer, and incubation temperature were 4 factors varied to determine their effect(s) on the optimization of the cylinder-plate diffusion assay. Chlortetracycline was the pilot antibiotic and Bacillus cereus was used as the assay organism. Zones of inhibition were larger when the incubation temperature was lower than that which was commonly used and/or when the nutrient level was decreased; the zones were smaller when the incubation temperature was raised and/or when an increased nutrient level was used. The thickness (volume) of the assay layer played the most important role; the thinner the layer the less the effect the cylinder charge volume had on the zone diameter. The slopes of the response lines were minimally affected by cylinder charge volume. For a 7 mL assay layer per standard Petri plate, cylinder charge volumes ranging from 150 to 250 microL had little effect on zone diameter. The linearity of the response line was unaffected by assay layer thickness (volume), nutrient level, temperature of incubation, or cylinder charge volume. As long as the conditions for the assay were standardized, there were no discernible effects on recoveries or potencies.

A sensitive bioassay for teicoplanin in serum in the presence or absence of other antibiotics

A teicoplanin bioassay has been developed that is accurate, sensitive, and reliable. A linear relationship is obtained between the diameter of the zone of inhibition and log 10 teicoplanin concentration in human serum over the range of 0.15 or 1.25 to 96 μg/ml using wells or paper filter disks, respectively. The assay medium devised consists of 50 g BBL Mueller-Hinton II Agr, 30 g NaCl, 8 g CaCl 2, and 1.0 g citric acid (monohydrate) per liter of deionized water (resulting pH 5.1 ± 0.1) and the assay organism Bacillus subtilis ATCC 6633. This system allows the assay of teicoplanin in the presence of commonly used aminoglycosides and in the presence of β-lactams after inactivation by β-lactamase. Additionally, it has the potential to be used in the presence of rifampin by using a rifampin-resistant strain of B. subtilis.

Algal assay of relative abundance of phytoplankton nutrients in northeast United States coastal and shelf waters

Regulation of phytoplankton growth by chemical water quality, in the coastal and shelf region between Cape Hatteras and the Gulf of Maine, was assessed by enrichment bioassay. Water sample collections, made in five surveys over an annual cycle, were assayed in the laboratory. The assay results, based on growth of a centric diatom, Thalassiosira pseudonana (clone 3H) Hasle and Heimdal, suggest that nitrogen, potentially, was the most important growth-limiting nutrient region-wide and year-round. The results also suggest that phosphorus had considerable importance and that silicon and vitamin B 12 occasionally were limiting. The potential regulatory importance of phosphorus apparently was less consistent, temporally and spatially, than that of nitrogen. Phosphorus limitation was detected primarily in just two of the collections, from March and July surveys. In the March collection it was evident predominantly in the inshore samples, but in the July collection its incidence was approximately the same among samples from inner, mid and outer shelf. Inhibitory water quality was prevalent in two of the collections, particularly in samples from inshore locales. The cause of this inhibition has not been identified but it was not associated with nutrient concentration. It is possible that adverse water quality was an important growth regulator in the region, at least for species having sensitivities analogous to that of the assay organism.

Freshwater Sediment Toxicity Bioassessment: Rationale for Species Selection and Test Design

The rationale and conceptual basis for the use of sediment toxicity assays are discussed in relationship to their use in sediment evaluations employing faunal surveys, toxicity assays, and chemical analyses. The disadvantages and advantages of various species from the major classes of aquatic organisms for use as sediment toxicity assay organisms are presented. Relative sensitivities of selected species and their ease of laboratory culture and utility as assay organisms are used to rank assays and propose a battery of assays for the rapid screening evaluation of sediment toxicity. The usefulness of a battery of assays for the screening evaluation of sediment toxicity and statistical considerations which are important in the development of study designs and the analysis of results from the battery of proposed assays are discussed. The assays recommended for inclusion in the screening battery for evaluation of sediment toxicity are Microtox ®, an algal assay, the Chironomus tentans 10-d growth assay, and the 48-h Daphnia magna acute assay.

Assay for biotin in the presence of dethiobiotin with Lactobacillus plantarum

At concentrations greater than approximately 0.5 μ dethiobiotin can cause the bioassay for biotin, which employs Lactobacillus plantarum, to overvalue the actual biotin level. This can be as much as 30-fold at 10 μ m dl-dethiobiotin and 5 p m biotin. Dethiobiotin does this by exerting a sparing effect on the biotin response by the assay organism. We demonstrate one way to determine the actual biotin concentration in the presence of interfering levels of dethiobiotin.

An Improved Microbiological Assay for Chlortetracycline in Avian Plasma

Bacillus cereus was used as the assay organism for the quantification of chlortetracycline (CTC) in avian plasma. Antibiotic medium #8 gave significantly larger zones of inhibition than nutrient agar 1.5% when used as the assay medium (P less than or equal to 0.05). When the CTC concentration was measured in serum, citrated plasma, heparinized plasma, and oxalated plasma, no significant differences were found between the inhibition zone diameters produced on antibiotic medium #8. However, there was a significant decrease in the zone diameters produced on this medium when citrated whole blood, oxalated whole blood, and heparinized whole blood were used instead of plasma. The length of incubation of assay plates was inversely related to the inhibition zone diameter, with an incubation time of 8 hr giving the largest and most distinct zones. Storing prepared assay plates at 4 C for 24 hr before use appeared to decrease the variability of the inhibition zone diameters. Storage of citrated plasma at 4 C for 48 hr and at -60 C for 9 days resulted in no significant decreases in CTC concentrations.

Microbiological Assay for Chloramphenicol Residues

Procedures for the assay of chloramphenicol in milk, urine, serum, and muscle tissue are presented. The procedures specify an assay design with all standards as well as samples present on each plate, oxytetracycline in the buffer-diluent for greater sensitivity, a minimal medium to enhance the inhibitory effect of chloramphenicol on the assay organism, and a tetrazolium dye to improve the ability to measure the zones of inhibition. Recoveries of unbound chloramphenicol from bovine urine were 90.8%, from serum 88.3%, from milk 79.3%, from swine muscle 71.3%, and from beef and chicken muscle 61.0 and 61.4%, respectively. The lower levels of measurement in urine and serum were 0.25 microgram/mL, 0.025 microgram/mL in milk, and 0.10 microgram/g in muscle tissue.

Simplified bioassay method for measurement of flucytosine or ketoconazole.

A simple agar-well diffusion bioassay suitable for measurement of flucytosine or ketoconazole was developed by using Candida pseudotropicalis ATCC 46764 as the assay organism. A test medium composed of (per liter) 7 g of Trypticase peptone, 7 g of YNB (yeast-nitrogen base), 15 g of glucose, and 15 g of agar was seeded with an inoculum which had been grown to no. 2 McFarland turbidity after 4 to 6 h in YNB-glucose broth. Determinations of flucytosine or ketoconazole were performed without necessity of heating or diluting of serum samples to alleviate amphotericin B interference. A linear relationship between zone diameters and log10 concentration of the drugs was observed over the pharmacologically relevant ranges of 25 to 160 micrograms/ml for flucytosine and 0.5 to 20 micrograms/ml for ketoconazole. The mean coefficient of variability for samples measured on 5 separate days was 2.4% for flucytosin and 4.0% for ketoconazole. This assay represents a significant improvement over previous bioassay methods in that a single test system may be used for measurement of either flucytosine or ketoconazole, no serum dilution or pretreatment is required, inoculum preparation is accomplished entirely on the day of the assay, and sharp, clearly defined zones of inhibition are obtained with both drugs.

Assessment of serum bactericidal activity after administration of cefoperazone, cefotaxime, ceftizoxime, and moxalactam to healthy subjects

Bactericidal activity in serum produced after administration of 1-g intravenous doses of cefoperazone, cefotaxime, ceftizoxime, and moxalactam was ascertained in six healthy subjects. The assay organisms were a strain of Staphylococcus aureus which was moderately susceptible to the drugs (MBC, 2 to 8 micrograms/ml) and an isolate of Escherichia coli which was highly susceptible (MBC, 0.08 to 0.3 microgram/ml). Drug concentrations and bactericidal titers were measured from samples taken for up to 12 h after the dose. No bactericidal activity against the S. aureus strain was found at 4 to 6 h and beyond for any of the drugs. Ranking of the in vivo bactericidal activity of the drugs was cefoperazone = cefotaxime greater than ceftizoxime = moxalactam. Against the E. coli isolate, bactericidal activity was present for 8 h for cefotaxime, and for 12 h for the other drugs. Ranking of the drugs in terms of extent and duration of in vivo bactericidal activity versus E. coli was moxalactam = ceftizoxime greater than cefoperazone greater than cefotaxime. After administration of 1-g doses of these new beta-lactams, bactericidal activity in serum was maintained for 12 h against highly susceptible bacteria. More frequent (6 to 8 h) or higher (greater than or equal to 2 g) dosing appears to be necessary to achieve prolonged serum bactericidal activity against less susceptible isolates (MBC, greater than or equal to 2 to 8 micrograms/ml).

Human pharmacokinetics and antimicrobial activities of the temocillin epimers.

The pharmacokinetics of the epimers of temocillin were investigated in 4 healthy male subjects following intravenous administration of 1g of temocillin disodium (free acid) which contains a R : S epimer ratio of approximately 65 : 35. The R epimer had a 2-fold greater total plasma clearance, a 23% larger volume of distribution and a shorter beta half-life than the S epimer. Intermediate values were obtained for total temocillin (R + S) from high pressure liquid chromatography (HPLC) data. In each plasma sample, the unbound fraction of the R epimer was generally 2-fold higher than that of the S epimer, which is suggested as the reason for the differences in the pharmacokinetic properties of the epimers. The temocillin pharmacokinetic parameters obtained from the microbiological assay data reflect most closely those for the R epimer derived from HPLC data. The resolved R epimer exhibited twice the potency of the S epimer against the microbiological assay organism Pseudomonas aeruginosa NCTC 10701. However, in tests for antibacterial susceptibility, for instance minimum inhibitory concentration (MIC) determinations involving prolonged incubation, there was little difference in the inhibitory activities of the resolved R and S epimers compared with temocillin (R + S), presumably as a consequence of the epimerization of the individual epimers. In contrast, in rapid tests for bactericidal activity, which minimise the effect of epimerization, the R epimer exhibited greater bactericidal activity than the S epimer.

2'-Chloropentostatin: discovery, fermentation and biological activity.

2'-Chloropentostatin (2-CP) is a new nucleoside antibiotic produced by Actinomadura sp. ATCC 39365. A selectively sensitive assay organism, Enterococcus faecalis PD 05045 (MIC 0.005 micrograms/ml) was instrumental in the discovery of this compound. 2-CP is a tight-binding inhibitor of adenosine deaminase (Ki = 1.1 X 10(-10) M).

Relative Activity of the Nonphosphorylated B-6 Vitamers for Saccharomyces uvarum and Kloeckera brevis in Vitamin B-6 Microbiological Assay

Accuracy in the microbiological assay of vitamin B-6 is dependent, in part, on equivalent response to each form of the vitamin by the assay organism. Recently, Kloeckera brevis (ATCC 9774) has been suggested as an alternative to Saccharomyces uvarum (ATCC 9080) because of its reported equivalent response to pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM) in turbidimetric yeast growth assays. Evaluation of the merits of these yeasts in our laboratory yielded contrasting results. As widely reported, S. uvarum exhibited greater growth response with PN than with equivalent molar concentrations of PL or PM. K. brevis exhibited an even greater disparity. Pronounced concentration dependence was observed for the response differences among vitamers with each organism. On the basis of the variable specificity of K. brevis, this organism is not preferable to S. uvarum for the microbiological determination of total vitamin B-6.vitamin B-6 Saccharomyces uvarum Kloeckera brevis

Toxicity of Seven Heavy Metals On Panagrell Us Silusiae: the Efficacy of the Free-Living Nematode as an in Vivo Toxicological Bioassay

The efficacy of the free-living nematode Panagrellus silusiae as a toxicological assay organism was studied using nickel, cadmium, lead, chromium, mercury, copper and zinc as test substances. Acute lethal toxicity values (LC50 values) were obtained for each heavy metal and Panagrellus was found to be highly resistant. When the LC50 values are ordered and correlated, there was no difference between rankings for juvenile (J2) and adult worms. Juvenile worms tend to be slightly more sensitive than adults but this trend was not significant. Testing of the chronic effects of the seven heavy metals revealed that (1) copper, chromium and cadmium effectively blocked growth at all stages of development, (2) the highest soluble concentrations of lead and nickel were without effect on growth of the worms, (3) high concentrations of zinc (>500 mg/l) partially blocked growth and (4) mercury was either lethal (20 mg/l) or ineffective in blocking growth (10 mg/l). When compared with the toxicological data that have been obtained for a number of other organisms, Panagrellus may have some fundamental shortcomings as a model in vivo bioassay organism if the present results with heavy metals pertain to other kinds of toxic substances.

Rapid chemotaxis assay using radioactively labeled bacterial cells.

A rapid chemotaxis assay is described in which radioactively labeled cells of the assay organism are used to detect the number of cells trapped in capillaries containing attractant. The sensitivity and reproducibility of the radioactive technique is comparable to that of the dilution plating procedure of Adler (J. Adler, J. Gen. Microbiol. 17:77-91, 1973), but is faster and also permits the results of the assay to be determined on the day that the assay is run. The method could be particularly useful for environmental studies and for field experiments, since it does not rely on sterile techniques for dilution plating.

The systematics and ecology of soil algae

Algae occur in nearly all terrestrial environments on earth and are invariably encountered both on and beneath soil surfaces. The algal flora of the soil includes members of the Cyanochloronta, Chlorophycophyta, Euglenophycophyta, Chrysophycophyta, and Rhodophycophyta. Thirty-eight genera of prokaryotic and 147 genera of eukaryotic algae include terrestrial species, the majority of which are edaphic. Whereas systematic nomenclature of blue-green algae adheres to traditional classification based upon morphological features, proper taxonomic treatment of eukaryotic soil algae is predicated on standard methods of culture and interpretation of physiological attributes, plant mass characteristics, and morphological properties of axenic clones. While knowledge of the physiological ecology of soil algae is primarily based on laboratory evidence, it is well established that solar radiation, water, and temperature are the most important abiotic factors governing their distribution, metabolism, and life history strategies. Biotic interactions are often as important, whereas ionic factors (including pH), oxidation-reduction potential, and soil texture are less important, if only because their influences are less well understood. Algae play an important role in primary and secondary plant community succession by acting as an integral part of the colonial synusium. The principal functional attributes of algal communities in soil include primary production, dinitrogen fixation, and stabilization of aggregates. Although as many as 108 algae per gram have been documented, soils commonly support between 103 and 104 per gram. As an avenue for the incorporation of carbon and nitrogen and for minimizing erosion through stabilization of aggregates, algae are valuable in agriculture. Although manipulation of edaphic algal populations in temperate countries is of novel occurence, their successful use in India as a means for reclaiming saline soils and as a source of fertilizer nitrogen is well documented. Soil algae are affected by synthetic pesticides and pollutants. In general, most herbicides, fungicides, and soil fumigants are detrimental to soil algae while most insecticides are not. Algae have been utilized as biological assay organisms for anticipating crop response to both fertilizers and pesticides because of their biochemical similarity to higher plants and their quick generation time. As future research with soil algae leads to a greater awareness of their importance in soil, it might be anticipated that manipulation of populations in agricultural systems of temperate regions will become more widespread with consideration of the potential benefits of soil algae made by scientists in other disciplines.

Improved Microbiological Procedure for Determining Erythromycin in Milk and Some Milk Products

An improved microbiological assay for erythromycin in milk and some milk products was developed. Acidification of the samples to pH 4.5 resulted in precipitation of milk solids which were separated by centrifugation. The pH of the aqueous supernatant solution was readjusted to pH 8.0 to obtain maximum sensitivity. Th sample cleanup and the use of a single, thin, seeded agar layer with Microsoccus lutea as the assay organism permitted the mesurement of 0.05 micrograms/mL milk with recoveries averaging 84.0%, 0.50 micrograms/g nonfat dry milk with recoveries averaging 55.4%, 0.10 micrograms/g cream cheese with recoveries averaging 100.2%, and 0.05 micrograms/mL buttermilk with recoveries averaging 44.0%. The improved procedures showed superior recoveries and improved the ability to detect and measure lower levels when compared with commonly recommended assay procedures for erythromycin residues.

Bacteremic infection and malnutrition in patients with solid tumors. Investigation of host defense mechanisms

Eighty-five patients with solid tumors without neutropenia were studied in order to determine host characteristics associated with bacteremic infection. Twenty patients without neoplastic disease who had bacteremia served as controls. Bacteremia frequently occurred in patients with tumors of the gastrointestinal and urinary tract, who were over 51 years of age, and had evidence of malnutrition. Peripheral blood lymphocyte dysfunction in vitro was commonly noted in infected patients with cancer. Polymorphonuclear leukocyte (PMNL) dysfunction was common in older bacteremic patients with and without cancer. The PMNL abnormality appeared to be cellular, it was not corrected with use of pooled normal serum, and was manifested by poor uptake of the assay organisms.

BEHAVIORAL AND BIOCHEMICAL EVIDENCE FOR SPECIES DISTINCTIVENESS IN THE FIDDLER CRABS, UCA SPECIOSA AND U. SPINIZARPA.

The fiddler crabs (Genus Uca) have been widely used as experimental animals in several areas of biological research. Their behavior, especially social communication, has long attracted the attention of ethologists and evolutionary biologists. Recent progress in these fields was summarized by Crane (1975). There are over 60 known species, ranging through the tropics and the temperate zone. They face diverse problems of adaptation to different tidal regimes (Barnwell, 1968, 1976; Powers, 1975), temperature patterns (Vernberg, 1969), salinity and substrate composition (Teal, 1958). The crabs have also been used in endocrinological (Fingerman, 1974), genetic (Selander et al., 1971), and neurophysiological (Hyatt, 1975; Salmon and Horch, 1976) investigations, and as assay organisms for evaluating the long-term effects of pollutants upon behavior and survival in marine habitats (Krebs and Burns, 1977). An important generalization emerging from these studies is that there are many species-typical differences in behavior and physiological processes. It therefore becomes necessary to identify species accurately so that valid correlations and future comparisons can be made. The systematics of the group, based primarily upon morphological characters (e.g., Rathbun, 1918; Crane, 1975), may not be able to provide an accurate assessment of species distinctiveness, at least for those forms

Standardization of a rapid microbiologic assay for aminoglycosides using Enterobacter cloacae.

The standardization of a rapid serum aminoglycoside assay using Enterobacter cloacae is described. This includes the sensitivity testing of the organism and its performance on various media, with Mueller-Hinton agar being the medium of choice. The precision and reproducibility of the assay, as measured by the within-run and between-run coefficients of variation, were 5.0 and 5.9, respectively. A significant positive correlation was obtained between the microbiologic assay for gentamicin and a 125I-labeled gentamicin radioimmunoassay with the use of both normal and uremic sera. When known amounts of gentamicin, tobramycin, and amikacin were added to antibiotic-free sera from normal persons, recovery rates of 80.0% to 97.9% were found. In the case of gentamicin, recovery rates of 85.0% to 97.9% were found with the use of sera from patients undergoing either hemodialysis or peritoneal dialysis. There were no effects on the recovery rates of the aminoglycosides from normal serum if high concentrations of clindamycin, methicillin, ampicillin, penicillin G, cephalothin, cefamandole or cefoxitin were also present in the sera. The newer cephalosporins, cefamandole and cefoxitin, had no in-vitro effect on the Kirby-Bauer sensitivity patterns of gentamicin, tobramycin, or amikacin, when tested against the assay organism.

Improved Microbiological Assay for Penicillin Residues in Milk and Dairy Products

The microbiological assay for penicillin residues in milk was improved. Acidification of milk with phosphoric acid to pH 4.5, centrifugation to remove precipitated proteinaceous matter, adjustment of pH to 6.0, a second centrifugation, and the use of a 2% agar base layer resulted in the consistent measurement of 0.01 unit penicillin activity/ml milk. Sarcina lutea was used as the assay organism. Recoveries in milk averaged 94%, in nonfat dry milk 84%, in cottage cheese 97%, and in cream cheese 94%.