I did a FISH method for gram positive bacteria using 1N HCl instead of lysozyme to lyse the bacterial cell wall. And the FISH result was good.
But i am confused that whether 1N HCl is effective or not? Because i search different articles using HCl to lyse the cell wall of gram positive bacteria in FISH method, but i did not found any articles. So would anybody help me whether my process is correct or not.
Dear Giri,
Practically, i think no. There is a specificity between lysosomes as enzyme and their receptor in the cell wall. Thus, it is difficult to use another like acid e.g., HCl. Secondly, i think no effect of HCl on a thick peptidoglycan layer which composed the higher content of Gram positive cell wall.
Hope the answer is benefit
Dear Mushtak T.S. Al-Ouqaili,
Thank you very much for your suggestions.
I repeat the same experiment twice and i get the same result. Its kind of weird because with 1N HCl result was good in comparision with the lysozyme. So, I have no idea. I just did the experiment as follows;
Taking 4 µl sample and spot on the well of the glass slide. Dry at oven 60 ºC for 10 minutes. Dip the glass slide on 50ml 1N HCl solution and kept at water bath (37 ºC for 30 minutes). Rinse with distilled water completely and air dry. Then dehydrate for 3 minutes each in 50, 85 and 100 % ethanol and air dry. Adding probe+hybridization buffer(1.5+13.5 µl) to the samples in each well and placing the slide into the polyethylene tube (horizontal position). Incubate at 46ºC for 3 hours. Rinsing the slide carefully with a bit of washing buffer, transferring the slide into washing buffer (50 ml in a polyethylene tube) and incubate for 15 minutes at 48ºC. Rinse slide with distilled water, air-dry. Mounting with 4µl DAPI solution and observe through the fluorescence microscope.
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