I inoculated E.coli O104 in lettuce and did cfu counting after spreading in LB plate containing cefotaxime as well as MacConkey containing cefotaxime parallaly but in both case I found lot of of background flora growing including O104. So I am worried that what can I do to prevent background flora growth for proper cfu counting of my target bacteria, E.coli O104 in this situation, if anybody has some suggestions, please give me.
if you can use a selective media in which you can count your target microorganism is the best option (i.e. chromocult)... the use of a selective media will affect the sub-lethal damaged cells but i thibk will be less than the effect of antibiotic. The addition of more than 1 antibiotic increase the stress to the sub-lethal damaged cells, decreasing the counts, take into account that the antibiotic can be considered a hurdle, so as more hurdles less microorganisms.
If I understand you correctly, you are putting the E. coli on the lettuce, leaving it for a defined period of time, then performing colony counts to see how it has behaved on the lettuce (ie. did it grow). Presumably the strain is cefotaxime-resistant. But the problem you have is that are other species of bacteria (and fungi?) growing which makes the counting difficult. Is that right?
Assuming I have you right, there are at least three things that you can do. One, determine what other antimicrobials your strain is resistant to, and incorporate that in the media. Two, use a higher concentration of antibiotic. Three, clean the lettuce first (bleach) to remove the microbiota, rinse well with sterile water, and then go ahead with the experiment.
@ Higgins, Thank you very much your answer. I would like to inform you that first of all you got my point. 2nd. Before iI inoculation of O104 in Letttuce, I washed the letttuce in DW followed by hypochlorite solution to remove the background flora. 3rd. For counting cfu,I used agar media with along with cefotexime,i also used 2 antibiotic to which O104 is resistant. Still normal flora in lettuce still growing with O104. if you have any additional suggestions, please give me.
Where do you incubate the lettuce and for how long? Have you performed a few test cultures; unwashed lettuce to see what is the natural flora, and also after washing with hypochlorite to confirm that the leaf is sterile? If it is impossible to remove all background flora, try another antibiotic combination, and combine it with something like MacConkey agar whereby you can identify the E. coli.
Alternatively you could try a PCR-based approach to quantify the number of bacteria, but that will not necessarily tell you that the cells are living and viable.
Dear Azimun Nahar
Cefotaxime are third generation cephalosporins (Broad spectrum beta lactam antibiotic). The orgainisms are capable to grow even in the presence of Cefotaxime that could third generation cephalosporin resistant. The organism which are resistant to cefotaxime they are capable of producing Extended Spectrum Beta lactamase (ESBL) enzyme. For these resistance you have to go for combination antibiotic (i.e. beta lactam with beta lactamase inhibitor). The combination of Cefotaxime with clavulanic acid could control the these type of ESBL-producing.
For your additional information
You can also isolate these type of organism from media and if you study their mechanism of resistance really it sounds higher in the field of medicine
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques