Cephalosporinase Enzyme in Sterility testing of antibiotic

Microbiology
#1

Hi
As per USP it is recommended to use Cephalsporinase or penicillinase enzyme in sterility testing of antibiotic but in my case when I used rinsing buffer in memebrane filtration method the effect is also nullify so what’s the point of using enzyme if we rinse membrane with rinsing buffer? I conclude these results on the basis of experiment where I run following set of experiment with routine testing.
Negative control ( Culture+ Antibiotic product)
Positive Control ( Culture+Antibiotic product + Enzyme)

Growth should not be appeared in negative control but I found bacterial growth in both conditions which shows that after rinsing of product the effect of antibiotic is nullify as we perform in case of method suitability testing.( there is no case of contamination)
need your insight in this regard that is enzyme should be used only in direct method or what else?
thank you

#2

Hi!
Can I ask did you add the cephalosporinase enzyme to buffer together with the antibiotic, culture and then filter? then rinse with buffer?
In my case, I can’t do that because it will contaminate our sterility room. And we only have one set of manifold and funnels. And it was fixed only in the sterility room so what I did was I filter the samples (penicillin antibiotic) did not rinse at all cut the filter in half then placed into FT and TSB broth. Then proceed to Biosafety Hood in another separate room to inoculate 100 cfu/ml per organism to the samples. Then I put the beta-lactamase directly to the samples (antibiotic+culture). Then incubate. Based on the results it needs at least 1-1.5ml of the enzyme to neutralize the activity of the antibiotic.
I hope this helps you.