Single Agar Layer Research Articles

Improved Specificity in Detecting F-Specific Coliphages in Environmental Samples by Suppression of Somatic Phages

Analysis of environmental waters for F-specific coliphages on the genetically constructed host Salmonella tvphimurium strain WG49 is hampered by the detection of Bomatic Salmonella phages. To improve the specificity of F-specific coliphage detection, two agents were tested for selective inhibition of Salmonella phage infectivity: heat-killed cells of female (F) S. tvphimurium WG45 and chemically extracted S. tvphimurium lipopolysaccharide (LPS). When both treatments were tested on pools of environmental isolates of Salmonella phages and the FRNA coliphage MS2, Salmonella phage infectivity was reduced by >90% with no change in MS2 detection. When environmental waters were tested by single agar layer or membrane filter methods, LPS increased the selectivity of F-specific coliphage detection from 28% to >90%, with no loss of sensitivity. The results of this study indicate that LPS effectively inhibits Bomatic Salmonella phage infectivity when added directly to a water sample or to molten agar medium or when used to treat filters on which phages have been concentrated.

Granulocyte/macrophage colony-forming units from cord blood of premature and full-term neonates: its role in ontogeny of human hemopoiesis.

It is still uncertain whether the yolk sac is the only origin of hemopoietic stem cells during fetal development. We studied the level of circulating granulocyte/macrophage colony-forming unit (CFU-GM) from the 23rd week of gestation to fullterm, trying to delineate its role in the ontogeny of human hemopoiesis. Cord blood samples were collected from 45 healthy premature neonates and 91 healthy full-term neonates. CFU-GM assays were performed using a single agar layer system with human placental conditioned medium as a source of colony stimulating activity. The mean numbers of colonies produced from the cord blood samples of the premature and full-term neonates were 116 +/- 185 (SD) and 96 +/- 113/2 x 10(5) mononuclear cells, respectively. There was no statistical difference between the numbers of colonies of the two groups (Mann-Whitney test, p greater than 0.05). These results indicate that the level of circulating CFU-GM from the 23rd week of gestation to full term is constantly high and add credence to the assumption that CFU-GM are produced not only from the yolk sac but also from other hemopoietic sites.

Development of a field test kit for coliphage detection in natural waters

AbstractColiphages may be suitable alternative indicators of fecal pollution in natural waters. The relationship between coliphage and fecal coliform numbers may be expressed by log10 transformed linear regression equations in surface waters (correlation coefficient, r2 = 0.5872; p < 0.001) and in well waters (r2 = 0.4767; p < 0.001). Using the American Public Health Association 919C single agar layer method, coliphages can be enumerated easily and at less cost and more rapidly than coliform enumeration methods. In adapting this test for use in a field test kit, however, further modifications and simplifications are required.The use of 2,3,5‐triphenyl tetrazolium chloride (TPTZ) to enhance plaque visibility while helpful, was not essential. Matched‐pair t‐test analysis showed no significant difference between the plaque numbers enumerated with or without TPTZ to help plaque visibility.The Escherichia coli C host bacteria can be maintained either as a frozen preparation or dried on to paper disks with protective milk colloids. In the latter procedure, a resuscitation period of 18–24 h in broth at 30°C was found to be satisfactory in giving plaque numbers in the test that were not significantly different than when frozen host was used.A portable field kit has been designed to carry out a total of eight tests. The kit includes the media, the bacterial host, petri dishes, syringes, receptacles, and a camping gas cylinder. It measures 38 cm by 25.5 cm by 33.5 cm and weighs about 6.4 kg. Using the kit, the test procedure for a water sample can be carried out in under 15 min.

Factors affecting the enumeration of coliphages in sewage and sewage-polluted waters.

The count of coliphages in naturally polluted waters was found to be dependent on many experimental factors. If Escherichia coli C was used as a host strain, consistently higher counts were obtained than with other strains (B,K-12-derivatives). This could be explained partly by the absence of a restriction system in C. A nutrient medium (modified Scholtens' agar, MSA) was developed with optimal concentrations of calcium- and magnesium-ions. MSA was compared with other media used for phage work and was found to give higher counts than all but one medium, Phage Assay Agar (PAA), which performed equally. If plating was done in a single agar layer in a large-size Petri-dish, higher counts were found than with the well-known double-agar-layer method.