Important Note:Merck Millipore reco

Important Note:Merck Millipore recommends performing the different test series in parallel(filter alternately Fluo L1 replicate, Fluo L2 replicate, Fluo L3 replicate and Pos. Control replicate).
EZ-Fluo™ sample with test organism and with product
As the test method with neutralization and/or rinsing steps should already be developed and verified, use the internal SOP for sample processing.
If the test product cannot be inoculated directly, the inoculum can be introduced into the filtration funnel.
If the volume of test product is less than 50 mL, pre-wet the membrane with 30 mL sterile rinse fluid, pour the test product and make up the volume in the funnel to 100 mL with sterile rinse fluid.
Repeat the Sample Preparation procedure to obtain the desired number of replicates for each test product lot to be tested.
EZ-Fluo™ sample with test organism but without product
Proceed following the internal SOP, but replace the test product by the same volume of sterile rinse fluid and add the inoculum directly into the filtration funnel.
Repeat the Sample Preparation procedure to obtain five replicates.
EZ-Fluo™ negative control without test organism but with test product
Proceed following the internal SOP, replacing the inoculated test product by non-inoculated test product.
Obtain one negative control per test product lot tested.
EZ-Fluo™ negative control without test organism and without test product
Proceed following the internal SOP, replacing the test product by the same volume of sterile rinse fluid.
Obtain at least one negative control.
Traditional method positive control with test organism but without product
Repeat the Sample Preparation procedure to obtain the desired number of replicates.
Traditional method negative control without test organism and without test product
Incubate the EZ-Fluo™ samples with the membrane facing down, at the temperature specified for the medium and micro-organism, for the period of time validated for the test product.
Re-incubate the EZ-Fluo™ samples on the appropriate solid media plate, with the membrane facing down, at the temperature specified for the medium and micro-organism, to achieve the longest period of time specified in the applicable pharmacopeia.
After incubation period specified in the applicable pharmacopeia, count the colonies on the EZ- Fluo™ membranes; these counts are called Re-Incubation counts.
Repeat the complete test procedure for each additional micro-organism to be tested, and for each
Test Product to be tested.
Calculate the average counts for each sample set, and calculate the percentage(%)Recovery as follows:
Comparison of the EZ-Fluo™ method with the traditional method:for each product lot tested with the EZ-Fluo™ method, calculate the% Recovery in comparison with the Traditional Method samples without test product.
See example of% Recovery Calculation for first test product lot:
% Fluo L1 Recovery=
Average Fluo L1 Counts
Average Pos Control Counts
This% Recovery is important to verify that the micro-organisms can be recovered after re-incubation.
% Re-incubation L1 Recovery=
Average Re-incubation L1 Counts
Statistical study for the Interference of Test Product
The interference(if any)of the test product with the EZ-Fluo™ fluorescent reaction could have two different impacts:
Decrease the fluorescent signal, causing false negative results,
Increasing the fluorescent signal, causing false positive results.
To validate the product has no interference with the fluorescent reaction it is possible to perform a statistical analysis on the microbiological counts.
Use a relevant statistical test to compare the Fluo counts obtained with the three product lots to the Fluo counts obtained without product.
Refer to section 5.9:Statistical Data Analysis Methods, or check with your internal regulatory affairs/validation department for clarification.
Important Note:the acceptance criteria apply to test product that is sterile.
If testing non-sterile test product, the acceptance criteria based on numbers of CFU are no longer applicable.
No fluorescent spot is observed on the EZ-Fluo™ Negative Control.