Can bacteria be quantified from a 14 day abscess? - (May/27/2014 )

The aim of my experiment is to show the anti-bacterial efficacy of my compound. The study is based on proving antibacterial activity based on a cutaneous infection model. Several papers i have read, report that they are able to quantify bacteria (CFU) from 12-14 day abscess by homogenizing the abscess in selected broths. I have tried to quantify 14 day abscesses but without homogenizing them as I do not have a homogenizer. I had used a syringe and vortexed the tissue as best as I could. I did not get any bacteria growing on my plate.

My question is, aren't abscesses (especially mature ones) made up of dead cells and therefore non-quantifiable?

Or is there something I'm doing wrong?

Please kindly advise.

Hi,

It is unlikely that all of your bacteria have died, may be the growth of them has inhibited.(it definitely depends on your compound).but two things are important:1- you have to use a negative control (I mean an abscess without treatment with the antibiotic) 2- you could enhance the bacteria( both sample and negative control in the same amount) by inoculation in the nutrient broth and incubation them for 24h or more in a favorite Tm for the bacteria and then take it on the plate. After growing you could compare between neg-cont and sample and count them. (if you have enough time and facilities the q-PCR for both negative and sample is possible when you use the drug before 14 days since this method evaluate both live and death bacteria). 

Ensyeh on Wed May 28 14:10:10 2014 said:

Hi,

It is unlikely that all of your bacteria have died, may be the growth of them has inhibited.(it definitely depends on your compound).but two things are important:1- you have to use a negative control (I mean an abscess without treatment with the antibiotic) 2- you could enhance the bacteria( both sample and negative control in the same amount) by inoculation in the nutrient broth and incubation them for 24h or more in a favorite Tm for the bacteria and then take it on the plate. After growing you could compare between neg-cont and sample and count them. (if you have enough time and facilities the q-PCR for both negative and sample is possible when you use the drug before 14 days since this method evaluate both live and death bacteria). 

This is a weird option.

How are you going to "count" after a 24hour enrichment....

with making a serial dilution and take them on the several plates. isn't possible? Myself didn't have an experiment like that, but I think if the antibiotic has been effective , compare between them would be possible.

Hello Ensyeh,

Thank you for taking the time to consider the question. I did conduct a serial dilution of the ruptured abscess and tried to enumerate them on several plates. But i did not get any growth after 24 or 48hr incubation. Enriching them with media may not be possible as I won't be able to determine the amount of bacteria isolated purely from the abscess. My confusion or difficulty is actually enumerating the negative control (ie untreated abscess). Would anyone be able to tell me if abscess, especially a 14 day abscess is made up of only dead bacteria or should I be able to recover live ones and enumerate them?

Thank you in advance once again.