Amplification of fluorescently labelled DNA within gram-positive and acid-fast bacteria.
Representative organisms from a variety of Gram-positive genera were subjected to varying regimes in order to optimise the intracellular amplification of DNA. The bacteria were subjected to treatments with paraformaldehyde, muramidases and mild acid hydrolysis to discover which regime made each organism permeable to the amplification reagents yet allowed retention of the fluorescein-labelled amplified products within the cell. Scanning electron micrographs were used to corroborate the effectiveness of the treatments, as seen by fluorescent photomicrographs, with the damage caused to the bacterial walls. A combination of mutanolysin and lysozyme was found most effective for Bacillus cereus, whereas permeabilisation of Streptomyces coelicolor, Lactococcus lactis and Clostridium sporogenes was most effective when exposed to lysozyme only. Surprisingly, direct amplification with no pre-treatment gave the brightest fluorescence in Mycobacterium phlei. Comparing the techniques of whole cell PCR, primed in situ labelling (PRINS), and cycle PRINS showed that under the conditions used the strongest intensity of fluorescence was obtained with in situ PCR; only L. lactis and M. phlei produced signals with cycle PRINS, fluorescence was not seen for any of the organisms with PRINS.
Vaid
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A
School of Chemical and Life Sciences, University of Greenwich, London, UK.
Bishop
A H
AH
D004269:DNA, Bacterial / Q000032:analysis*; D006094:Gram-Positive Bacteria / Q000737:chemistry* / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D010539:Permeability; D020401:Primed In Situ Labeling / Q000379:methods*; D015203:Reproducibility of Results
D004269:DNA, Bacterial