on the Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa paper by skindersoe et. al, they have used skimmed milk.
i am searching for a test for protease and elastase. im going to use a plant extract of tannins, is it possible that i use that method to?
how can that test (skim milk test) prove the effectivity of my sample? or how what is the manifestation of a postive result for this test?
skimmed milk is used because in turbid milk agar made from full fat milk part of the agar turbidity would be due to emulsified fat droplets. A halo (clear zone) around a colony therefore could indicate lipase as well as protease activity . The skim milk agar test is at best semiquantitative, a clear halo and its dimension around a colony indicating protease activities. I can not give an answer for the remaining questions.
skimmed milk is used because in turbid milk agar made from full fat milk part of the agar turbidity would be due to emulsified fat droplets. A halo (clear zone) around a colony therefore could indicate lipase as well as protease activity . The skim milk agar test is at best semiquantitative, a clear halo and its dimension around a colony indicating protease activities. I can not give an answer for the remaining questions.
They have used skimmed milk because it is devoid of fat and good source of protein.
You can use egg yolk agar for testing protease (zone of clearing around colonies in positive protease). Bovine serum albumin can also be used.
I don't know about tannins.
I agree with Mike for the protease part. As for the elastase assay, the mode of action is that they can degrade elastin, which you can take this to determine their activity.
you can try to test the effect of tannins on Pa by combining them together and see the appearance and diameter of halos.
Other way to try is to use qRT-PCR to check the expression of those virulence factors at transcriptional levels.
Try making a plate with a thin layer (2 or 3 mm) of skim milk in agar. Punch holes and fill them with your extracts; any protease diffusing out will make clear halos.
I would suggest to use the elastin congo red assay for the determination of elastase activity as it is a very defined method with easy quantification.
1) 100 µL of supernatant of PAO culture + 900 µL ECR buffer; 2) incubate 3 hours at 37°C and 900 rpm; 3) Centrifuge and measure absorbance at 495 nm; 4) use medium as reference
ECR buffer: 0.147 g CaCl (1mM), 12.114 g Trizma Base (100 mM), pH 7.5, ad 1 l water; add 22.5 mg Elastin Congo Red to 1 mL ECR buffer
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