In which solution, there are minimum chances of bacterial death and control of the growth at the same time?
In my opinion Saline solution/water (0.8%) is the best diluent for cell count.
Peptone water: It supports growth and after utilizing the nutrients (peptone) bacterial cell die.
Deionized water: Devoid of ions, acts as hypotonic solution, cell will swell and burst.
Saline water: isotonic solution (no swelling or shrinking of cell), same as cell protoplasm, minimizes the chances of cell death.
Kunal
It depends on the background material that you have the E. coli in. I'm a Food Microbiologist, so often times you are plating samples of acidic foods, foods with antimicrobials, or swab samples that may have residual sanitizer and you would want to eliminate the antimicrobial residues in your early dilutions (if you have low counts) by diluting in buffered peptone water or DE Neutralizing broth, or some equivalent diluent. If you do enough food samples and do not do anything to address this, you may end up noticing, at times, low counts on a low dilution (i.e., 10e-1) and then higher counts as you plate a further dilution (10e-2) that doesn't make sense at first, but can be interpreted as possibly residual inhibitor preventing a full spectrum of counts to appear on the low dilution, but as the inhibitors are diluted, the organisms are more capable of showing up.....but that will depend on a sufficiently high microbial count in the sample to allow that to happen and if the inhibitor was bacterio-static instead of -cidal. So again, you use diluents dependent on the type of sample you are plating.
As long as the diluent is somewhat buffered and is not going to generate an osmotic shock, then it may not matter. If I make the dilution and plate immediately then I often use growth medium that the cells are currently in, like peptone broth or LB. If your dilutions are going to sit around for 30 min or an hour or longer, then you will have a problem with continued growth. So using buffered saline or something similar would be good.
PBS (phosphate buffer saline) or isotonic saline solution (0.85%) is the best, no doubt, for the reasons exposed above by Dr Kunal Garg
Hi all, could you further clarify whether the saline should be sterilized or salt should be added to sterilized deionized water? I am fearing that the evaporation of water from salt/water solution may increase the concentration of salt and thus alter the ability of the saline solution to provide a isotonic property. Apologies if the question sounds funny, as I have very limited microbiological background..
Yes, sterilization is crucial. It is best to sterilise the diluent and the tubes (with caps) separately and then sterilely dispense the diluent into the tubes. This removes the issue of water loss. I wouldn't wish to take issue with Kunal on the subject, as he may have more experience than I do, but my take on the matter is: small volumes of hot liquids evaporate; larger volumes may superheat and flash boil if disturbed.
Do you have access to an experienced microbiologist? There are aspects of sterile technique that can really only be learned by observation and imitation of someone with lots of lab experience.
Again, the answer of Dr. Kunal Garg is correct. Make an aqueous solution of NaCl 0.85% in water, ad sterilize by autoclaving. No more. There is no water evaporation.
Hi all, can you tell me if we should use hydrophobic cotton for auto clave of NaCl solution or will alluminium foil be sufficient. I am undergraduate student doing a project whose sub part is to approximate number of e.coli. Present.
A cotton cap, surrounded by alluminium for avoid evaporation....