Use of C-banding for identifying radiation induced micronuclei

Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation.     

Efficacy of copper and silver ions with iodine in the inactivation of Pseudomonas cepacia.

Alternatives to chlorination of water have been sought for reasons which include trihalomethane formation, possible bacterial regrowth, the high concentrations of chlorine required in certain circumstances, and the taste, odour and bodily irritation in chlorine-treated water. Electrolytically generated Cu and Ag ions at low levels, in addition to very low chlorine concentrations, have been suggested as an alternative to routine chlorination. We have examined the combination of Cu and Ag ions with low levels of iodine. Pseudomonas cepacia was grown either in rich medium or under nutrient restriction prior to disinfection. Survival of the organism and its ability to regrow after treatment as well as the effects of varying buffers, metal ion and iodine concentrations were determined. Low concentrations of metal ions (100 ppb Cu and 11 ppb Ag) and iodine (200 ppb) were more effective than either metal ions or iodine alone against Ps. cepacia grown on rich agar or in low nutrient buffer. After iodination, buffer-grown suspensions recovered to their original cell concentrations within 7 d. When Cu and Ag ions were used with or without iodine, regrowth was prevented. The results show that low concentrations of Cu and Ag in combination with iodine permit effective disinfection of bacteria after cultivation on either rich media or under nutrient restriction. These results, along with published data, suggest that the combination of these metals with halogenation may have applications in the disinfection of both recreational and potable water.     

Factors Affecting the Determination of Respiratory Activity on the Basis of Cyanoditolyl Tetrazolium Chloride Reduction with Membrane Filtration

Deficiencies in traditional bacterial enumeration techniques which rely on colony formation have led to the use of total direct counting methods, such as the acridine orange direct count technique for the enumeration of planktonic bacteria. As total direct counts provide no information on the viability or activity of the organisms, demonstration of respiratory activity with the fluorochrome cyanoditolyl tetrazolium chloride (CTC) has been employed. We have modified this technique by performing filtration prior to CTC incubation. Cells captured on a polycarbonate membrane were incubated on absorbent pads saturated with medium containing CTC. Following counterstaining with DAPI (4(prm1),6-diamidino-2-phenylindole) total and respiring cells were enumerated by epifluorescence microscopy. Factors affecting CTC reduction by Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli K-12 were investigated. With K. pneumoniae, nutrient additions to the CTC medium did not increase the number of respiring cells detected. CTC reduction by all three organisms decreased in response to an increase of the pH of the CTC medium above pH 6.5. Increasing phosphate concentrations contributed to this inhibitory effect. CTC-membrane filter counts of K. pneumoniae, S. typhimurium, and E. coli K-12 and of bacteria in well water corresponded closely with plate counts (r = 0.987). The results show that careful attention should be given to the composition of CTC-containing media which are used to enumerate respiring bacteria. With an appropriate medium, reliable enumeration of respiring bacteria can be achieved within a few hours.     

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.