Division Principle

When the FISH test is used as the first screening test and found to be positive, the IF test must be performed as a second compulsory screening test. When the FISH test is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.

The following procedure should preferably be performed on freshly prepared sample extract but can also be successfully performed on sample extract that has been stored under glycerol at -16 to -24 °C or -68 to -86 °C.

As negative controls, use aliquots of sample extract that previously tested negative for C. m. subsp. sepedonicus.

As positive controls prepare suspensions containing 10⁵ to 10⁶ cells per ml of C. m. subsp. sepedonicus (e.g. strain NCPPB 4053, or PD 406) in 0,01M phosphate buffer (PB) from a three to five day culture (preparation see Appendix 2). Prepare separate positive control slides of the homologous strain or any other reference strain of C. m. subsp. sepedonicus, suspended in potato extract, as specified in Appendix 2.

The use of the FITC-labelled eubacterial oligo-probe offers a control for the hybridisation process, since it will stain all eubacteria that are present in the sample.

Test control material in an identical manner as the sample(s).