Dear Guerrino

I saw, When the person ate antibiotics, this phenomenon happens.

Dear Guerrino

I saw, When the person ate antibiotics, this phenomenon happens.

Depending on the type of samples, you might have a huge number of dead cells and few or no live cells at all. Which could perfectly explain your results. In a recent study I found symptomatic plant material harvested months after the onset of symptoms showing about 10^9 dead cells of the pathogen per gram of sample. However, I couldn't detect live cells (if present, they were below the detection limit of the technique). It is also relatively frequent to obtain positive PCR detection of pathogens when analyzing samples of asymptomatic material, either because bacterial cells were dead, or live cells were in the VBNC state or in very low numbers. Sometimes, just because of sample manipulation, bacterial cells can get stressed and one of the first responses is stop growing on solid media. In this case, usually adding pyruvate or catalase to culture media improves the recovery of stressed cells. Everything depends on the pathogen you are working with. Liquid enrichments of the samples would also help you to determine if you had live cells in your samples. In some cases, VBNC cells become culturable again in liquid medium. Depending on the pathogen, you might need changing incubation temperatures or using specific media to improve the recovery.

In case you are interested in the molecular detection of live cells, I have recently been working with propidium monoazide (PMA; and an improved commercial version, PMAxx), which is less toxic than reagent D or ethidium monoazide (EMA), for the selective detection of live cells by PCR. The use of these viability-PCR dyes requires a previous optimization of staining/photo-activation conditions to avoid an underestimation (typically occurrying when using EMA) or an overestimation of live cell numbers (that is the case of PMA). Although the optimization of these protocols can be challenging, it probably would help in the discrimination between live and dead cells in your samples. Depending on how homogeneous your samples are, a simple viability staining with syto9/propidium iodide (you have a commercial version of this, Baclight live/dead bacteria viability kit, Invitrogen) or nalidixic acid + acridine orange (kogure method) might help you distinguishing live and dead cells by simple observation with an epifluorescence microscope.

Also, you cannot discard the possibility of an inespecific detection of something else in your samples that amplifies with your primers.

this phenomenon is well known in efforts to diagnose bacterial infection in animals [inculding humans]. Commonly used culture media and incubation conditions are not allways suitable to grow bacterial cells. Examples comprise Sstreptobacillus moniliformis; Haemophilus'Bordetella.

Only 1% of microbial population could be cultured in lab environment that is why researchers try to develop another technique to investigate microbial community in a given sample. Metagenomics is one of approach used to study non-cultureable microbes. Then other omics integrated into this approach. Some researcher tried coculturing techniques, culture-independent microbial isolation and characterization.

If you want to confirm that the PCR product of your specific gene comes from existing at this moment, you should perform CARD-FISH using oligonucleotide that targets microbes of your interest.

I agree with Dr Ricardo Delgado Santander . But I'd try to grow st. using catalase-supplemented plates

This might be due to the dead cells you have in your sample. Because DNA is there so result is positive and perhaps the cells are dead so they can't grow.