I have problem with antibiotic carry over in my work with Actinobacillus pleuropneumoniae and the antibiotic tulathromycin. I have tried to use a full agar plate for each sample (100µl) (to dilute ab conc) but still have problems. I have also tried to centrifuge bacteria but my recovery is about 2 % for 10^4 cfu/ml and 13 % for 10^6 cfu/ml (without added ab). Do you have any suggestions? I have read about filtering but can't find any easy equipment for that.
Could you maybe try to just let the tube sit until gravity settles your bacteria at the bottom of the tube?
Thank you for your answer. How long do you think I have to wait for enough sedimentation? I think the antibiotic in the sample will kill alot of the bacteria during that time. But now when you say this, maybe if I dilute my sample really much and then let it stand..
I am not sure if I understood well. Why is so low your recovery after centrifugation?
I have only experience with E. coli, but If you want to filter you could try a microcon or centricon device. You have a lot of pore sizes and for different volumes. Millipore is a good choice.
Have you try to use your ab more diluted?
Hi Rosa, I don't know that but I have tried several times and centrifugation is not well tolerated by my bacteria, it seems. I have also tried with centrifugal filters but it didn't increase my recovery. I was thinking of some kind of sterile syringe filter that I can disassemble after filtering and take out the filter paper to put on an agar plate..
Maybe you can use the bottles with filter used in cell culture to sterilice buffers and culture media . They have two parts, the upper part is a funnel with a 0.2 micr filter and the lower part is the bottle were you recover the liquid. It works with vacuum not centrifugation.
Or maybe I can just filter my sample through a syringe filter and then "flush" the filter with non antibiotic solution from the other side. Do you think the bacteria will come off the filter, is it worth trying?
Good idea Rosa, I will look into that :)
Henry: I'm using an eppendorf bench top centrifuge. I have tried 4000RPM and 6000 RPM in 5 minutes and can't see any difference. It is only in the 10^6 CFU/ml samples where I actually can see a sediment after centrifugation.
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