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The effect of buffer composition on simultaneous PCR amplification of 16S rRNA gene fragments of five bacterial species was examined using a number of different buffer systems. Tris-based PCR buffers at final concentrations of 10 mM proved unreliable. However, when the final concentration of Tris was increased to 75 mM, all five samples were routinely detected. The use of other buffers, 3-[(1,1-dimethyl-2-hydroxyethyl) amino]-2-hydroxypropanesulfonic acid (AMPSO) and 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO), resulted in PCR amplification of five products even at low final concentrations (10 mM). The presence of certain proteins in the amplification reaction could overcome an inhibitory effect seen when soil suspension was present in the reaction, as might occur when testing field samples for the presence of bacteria. Bovine serum albumin was found to be the most effective additive tested in overcoming inhibition.

A range of rRNA‐targeted alkaline phosphatase‐labelled oligonucleotide probes was tested for use as culture confirmation reagents for the rapid identification of micro‐organisms. The probes were specific to clinically important bacteria (Helicobacter pyloriand Mycobacterium tuberculosis), fish and shellfish pathogens (Renibacterium salmoninarumand Vibrio vulnificus), food spoilage bacteria (Listeriaspp. and L. monocytogenes), for bacteria of biotechnological importance (Streptomycesspp.) and for bacteria associated with the oil industry (Sulphate‐reducing bacteria, SRB). A universal bacterial probe and a eukaryotic probe were included in the study as positive and negative controls, respectively. A total of 93 bacterial strains was screened. With the exception of a large number of cross‐reactions of the SRB probe (specificity value of 29·4%) and a single cross‐reaction of the R. salmoninarumprobe (specificity value of 97·7%), dot blot analysis indicated that each probe hybridized 100% specifically to the organisms tested. A simple culture confirmation method was then developed using these probes to enable the identification of bacterial colonies using a simple hybridization procedure.