The retention testing of sterilising grade membranes with Pseudomonas diminuta

Membranes with a pore size rating of 0.2μm are recommended for the sterilisation of liquids by filtration and are validated for this purpose by a retention test with Pseudomonas diminuta. Practices for retention testing were found to vary among the membrane manufacturers and only one type of commercial 0.2μm rated membrane was found to reliably retain P. diminuta. The retention for P. diminuta given by experimental grafted membranes was studied and was sometimes higher than that given by non-grafted membranes due to obstruction of the pores by graft material. The dimensions for individual cells of P. diminuta was studied by scanning electron microscopy and a rapid electronic method. Bacteria of larger dimensions than the pore size rating of experimental membranes were found in test permeates. It was shown that cells from an aerated P. diminuta culture were larger than cells from a similar but stationary culture. A retention test procedure for 0.2 μm rated membranes using cross-flow filtration was developed. The procedure simulated process conditions and enabled tubular ceramic monolithic membranes and flat-sheet membranes to be retention tested with P. diminuta. It is feasible that a standard retention test using cross-flow filtration can be developed. The time needed for results from current retention test procedures to become available is a consequence of using traditional cultural techniques for permeate analysis. Test procedure were developed using three popular methods for the rapid detection and enumeration of bacteria (ATP luminescence, impedance microbiology and the DEFT) for the detection and enumeration of P. diminuta in retention test permeates. The method using ATP luminescence was found to be the most applicable. The development of a bioluminescent strain of P. diminuta through genetic engineering will enable the rapid, sensitive and straightforward retention testing of 0.2 μm rated membranes. Retention tests using a bioluminescent strain of Escherichia coli containing the structural genes for bacterial luciferase indicated that the proposed test is feasible. Developments were made towards cloning the same genes into P. diminuta. The use of all bioluminescent micro-organisms for membrane retention testing is the subject of a patent application and a proposal for a three year SERC research grant.