Rapid methods for testing the efficacy of sterilising grade filter membranes
Current filter validation methods require 48 hours culture for results to become available, which creates time delays within the manufacturing process and quality control back-logs The thesis compares alternative methods for the production of filter challenge test data Within 24 hours to the desired test sensitivity, using bioluminescent and fluorescent genetically engineered strains of the test organism Brevundzmonas dzmznuta The recombinant strains were produced using a Tn5 transposon system, using a filter mating method. The genes cloned into the bacterial chromosome were the biolummescence lux_ genes, taken from the marme bacteria, Photorhabdus lummescens or Vzbno harveyz, and the gene encoding green fluorescent protein taken from the marine jelly fish Aequoria victoria The cloned strains were found to show no difference to the w1ld type strain With respect to their surface hydrophobicity, according to a bacterial adherence to hydrocarbons assay, and surface charge, according to an electro-static interaction chromatography method. Furthermore, the cell size according to Transmission Electron Microscopy was not significantly different to the wild type strain, which had cell dimensions of 1 05 x 0 52 Jlm The retention of cells by 0 45 mtcron rated filters, was shown to be not significantly different to the wild type All strains were retained by 0 2 Jlm filters These data confirmed that the cloned strains were suitable for challenge testing Four methods were used to detect microcolonies of the recombinant strains on filters. The advantage of the microcolony detection system was that it showed that the cells detected downstream of the filter were viable and culturable. The best detection method was with an epifluorescent microscope and the fluorescent strain after 24 hours, for which the sensitivity was 98.1 %. Two CCD camera systems were used to detect the bioluminescent strains on filters. The sensitivity of these systems were 80.1% and 83 9%, for the Nucleovision and Nightowl CCD camera systems, respectively, after 24 hours In addition, the Bwprobe photomultipherbased system was shown to achieve the detection sensitivity of one microcolony after 24 hours. Also, steps were made to study transcription Initiation signals for gene expression in fluorescent recombinant Brevundzmonas dzmmuta. Various putative promoter sequences were Identified in one fluorescent strain, using a DNA sequencmg method. These sequences showed homology to previously identified E colr and Brevundzmonas promoter sequences. Finally, an attempt was made to produce recombinant fluorescent and bioluminescent Acholeplasma lazdlawu, however this was unsuccessful and further work will be required to achieve this objective.