Note:Merck Millipore recommends per

Note:Merck Millipore recommends performing the EZ-Fluo™(Fluo tests)and Traditional Method(Control tests)in parallel(filter alternately Fluo replicate and Control replicate).
Repeat the Filtration procedure to obtain the desired number of replicates for each inoculum concentration and method(EZ-Fluo™ and traditional methods)tested.
Filtration of 0 CFU and negative control samples
For the 0 CFU series:repeat the Filtration procedure to obtain the desired number of replicates for each method(EZ-Fluo™ and Traditional methods)tested; identify and label the test samples with the method tested, and replicate number.
For the negative control processed at the beginning of each series(5 CFU to 100 CFU), identify and label the test sample with the method tested and the corresponding series.
EZ-Fluo™ samples
Incubate the samples corresponding to the concentrations tested(0 CFU to 100 CFU)with the membrane facing down, at the temperature specified for the medium and micro-organism, for the period of time validated during Incubation Time Robustness test.
Traditional method samples
Incubate the samples corresponding to the six concentrations tested(0 CFU to 100 CFU)with the membrane facing down, at the temperature specified for the medium and micro-organism, for the period of time specified in the applicable pharmacopeia.
EZ-Fluo™ Staining Step Staining step processing
When the incubation period of the EZ-Fluo™ samples is achieved, remove the samples from the incubator and bring them in the controlled environment area.
Proceed to the fluorescent staining step as detailed in section 6.1.3.5.
After the Staining step remove the stained Fluo samples from the incubator.
Count the EZ-Fluo™ samples as detailed in section 6.1.3.6.
If required, re-incubate the EZ-Fluo™ samples to determine Accuracy of the EZ-Fluo™ method after re-incubation.
Make a copy of Appendix 9 to record the results.
After incubation period specified in the applicable pharmacopeia, count the colonies on the traditional Method samples.
The complete test procedure should be repeated with each of the micro-organisms to be tested.
Calculate the average counts for each sample series, and calculate the Recovery Percentage(%)for the EZ-Fluo™ samples:
% Re - Incubation Recovery=
Report Results in the copy of Appendix 9.
Statistical study for the Accuracy
Accuracy of the EZ-Fluo™ method is the closeness of the results obtained with EZ-Fluo™ method to the results obtained with traditional method.
The closeness can be determined with different manners, such as Recovery Percentage calculation or statistical study demonstrating the equivalence of the counts(UFC)obtained with the two methods.
The latter is more stringent that the Recovery Percentage calculation.
To compare the counts obtained with the two methods, it is possible to use an ANOVA test or Student’s T-test after having verified the distribution of the microbiological counts.
The choice of the statistical method(and related acceptance criterion)might depend on the level of closeness that needs to be reached in the company’s laboratory.