How do you do this. l hear some saying they got above 80% recovery but at best l have got 35% recovery and that was when l swabbed the inoculum while it was still wet on stainless stell surface. Gets worse when l let it dry (to mimic actual conditions). l’m putting an inoculum of 1000cfu per swab area of 25sq cm(5x5cm) so l have btw 10-100 pre squre cm then recovering the organisms from the swab in 2 ml saline. We buy pre-made agar plates so use spread plating and am actually plating out 500ul on 2 plates to add up to 1 ml. ls there anything wrong with my method and if so what do you recommend baring in mind that l am using spread plate not pour plate so cant really plate out 1ml unless l use petrifilm hence the 2 ml not 10ml for the swab recovery.
the specification of 80% recovery applies only to chemical cleaning validation, not to microbiological. 35% recovery is outstanding, usual are recoveries around 1 - 10%, and some companies even state only “growth” as the recovery requirement. Take into account that there is one factor affecting these recoveries that is normally no issue in chemical cleaning validation: the survival of the microorganisms. Additionally, the recovery strongly depends from how you kept the plates, and the microorganism. In our case we determined the recovery for different aerobic microorganisms, for moulds and for yeasts, and the worst recovery obtained was 2%. We then calculated the limits with the following data:
Worst case product/equipment train combination
Lowest specification for product contamination (10³ CFU/g aerobic bacteria, 10² CFU/g moulds and yeasts) (solids)
10% contribution to the total count by the equipment (90% of the contamination coming from materia prima)
0,1 safety factor.
The limits we got were 40 CFU/100 cm² for aerobic bacteria and 4 CFU/100 cm² for moulds and yeasts, which are pretty in line with usual microbial cleanliness criteria. Obviously this depends from you specific combination of product/process/specification.
Thanks a lot. Thats the problem with most validation methods, they are written up with chemistry in mind. Most litreature supports the 10-35% recovery so l guess now l will work out the specs.
Hello, please help me too. I am from Georgia and in our country we have very poor knowledge in this field. I’m seeking literature by the internet but there are less information about microbiological aspects of cleaning validation. I don’t know how to determine microbiological limits. If we know pharmacopeian limits for nonsteril product mainly are 10³ CFU/g aerobic bacteria, 10² CFU/g moulds and yeasts and due to your dialogue recovery must be from 1-10%(I need to know also from where are taken these datas) then limit must be less then recovery or how to determine it. Please help me, may be you have corresponding mateials about this them or you can prompt to me where I can find it. My E-mail is tmaghlakelidze@hotmail.com
Thank you in advance
as far as I know there are no regulatory requirements for microbial limits or recovery, for cleaning validation. The recovery of 1 - 10% is an internal requirement based on recovery tests performed on various strains. Take into account that microbial contamination found by sampling in the equipment is only loosely related to the actual contamination, because (and in opposition to chemical contamination) microorganisms may disappear (die), stay in latent life, or multiply (grow). This is the reason why the requirement for recovery in microbiological cleaning validation is not as strict as in chemical cleaning validation, and for the same reason a lot of microbiological acceptance criteria are based more on historical values and trending than on fixed figures. The limit is basically not related to the recovery, but you can set your acceptance criteria including the recovery factor.
The calculation of the accepted microbiological limits in the equipment is quite easy because the calculation is exactly the same as for the chemical cleaning validation. With the specification of the accepted contamination (in your case 10³ CFU/g aerobic bacteria and 10² CFU/g moulds and yeasts) and the smallest batch size in the equipment train (in weight!) you can calculate the whole overall microbial contamination in that train. With the surface of the train you can calculate the contamination limit per unit surface: 10³ CFU/g, batch size 250 kg = 250000 g, equipment train surface 150000 cm², (arbitrary) safety factor of 0,01. Then the contamination is 250000000 CFU/batch, and thus 250000000*0,01/150000= 17 CFU/cm². This would be your limit. To calculate the acceptance criterion including the recovery you may consider a worst case recovery factor of 1%, and a sampled surface of 100 cm, and get a result of 17 CFU/100 cm², which is fairly in line with the limit of 20 CFU/cm² used for equipment cleanliness (although not cleaning validation).
