Note: Development of an external quality assurance scheme for the detection and enumeration of Pseudomonas aeruginosa from water and comparison of results using modified King's A broth and a commercial agar

Two trials of the isolation and enumeration of a given strain of Pseudomonas aeruginosa from water are reported. In each trial participants received concentrated samples from two batches, one with low and one with high counts, to be diluted to 500 ml in sterile distilled or deionized water and examined for Ps. aeruginosa by membrane filtration. Membranes were incubated at 37°C for 48 h on pads soaked in modified King's A broth (MKAB) and Unipath Pseudomonas Agar plus CFC supplement (PCFC). The first trial involved eight Public Health Laboratories (PHL) and the organizers provided media from single batches. The second trial, involving 50 PHL, examined the feasibility of a large scale external quality assessment (EQA) distribution. Participants were invited to use the same two media and their usual medium if different. Average counts were close to expected and the spread of results was comparable to that observed from the EQA scheme for indicator organisms. From the results of the two trials a better isolation of the strain of Ps. aeruginosa under consideration was noted with PCFC compared with MKAB.     

The crystal state binding of dithionite to deoxy-hemoglobin.

The crystal state binding of sodium dithionite to deoxyhemoglobin is reported. Dithionite has been used extensively to deoxygenate hemoglobin and myoglobin and there has been considerable interest among users of dithionite about its effect on protein structure and binding site(s). We have determined that dithionite binds to deoxygenated hemoglobin crystals at the interface of two molecules in the crystal lattice. Specific residues involved in hydrogen bonds or salt interactions with dithionite include His116 and His117 of the beta 2 subunit and Lys16 of the alpha 1 subunit of the adjacent hemoglobin molecule. No binding was observed at the symmetry related His116 and 117 beta 1 residues. We have shown that dithionite does not affect the native hemoglobin structure or the binding of several allosteric inhibitors to hemoglobin and can be used to mount T state crystals in the air.     

Direct sequencing and bidirectional allele specific polymerase chain reaction of the bovine beta-casein B variant.

We have used the polymerase chain reaction (PCR) to amplify exon VII of the bovine beta-casein gene. The mutations responsible for the B variant were identified by direct sequencing of the amplification products. A bidirectional allele-specific PCR method (BAS-PCR) has been developed using oligonucleotides overlapping the mutation site at their 3' ends. This new procedure allows a rapid and reliable discrimination between the B and non-B alleles of beta-casein.