I think your trouble is incomplete technique which should conclude one more step. First step is to use selective medium (like you did) with TCBS or Chromagar Vibrio. The step means to select suspicious colonies. Then, second step is colony accumulation applying enrichment medium sodium chloride 1,5 % within (e.g. alkaline agar with sodium chloride 1,5 %).

Thank you for your answer. I have already done that and it has worked fine. Still I can not find why in TCBS the colonies appear so harsh, making sometimes even impossible to pick them up.

That's unusual.   I have seen less mucoid colonies on CHROMAGAR but rarely on TCBS.    When you say they a positive on CHROMAGAR do you mean they are VV or VP positive?

Thank you for your answer. They are not mucoid they are rigid and only on TCBS. Stil, they are VP.

I used to work with the v.p, but i never had this problem with TCBS media. but you can try to re streak just one isolated colony using small and sharp loop on fresh TCBS plate and see how il looks.

Hi, I do got some colonies like that. they appear green in color its hard to pick as they are sticky in nature. How did you confirm them as VP. Have you done any molecular characterization as the green colonies doesnt always be VP.

Thank you for your answers. Except from TCBS I am using Chromagar Vibrio in which these colonies appear mauve as suggested by the manufacturer and as shown by my positive controls. Also I am performing toxR PCR and therefore I am quite confident that these strains are VP

CHROMagar Vibrio is best to confirm VP..... I am also working with VP