Hello, if your cultures are bacilli and you have contamination with non spore forming bacteria you can easily clean your culture by heating cell suspension at 80'C for 20 min than use sterile nutrient media for isolation. If you have contamination with other bacilli so you can perform microscopy to see some cell morphology differences and you can use serial decimal dilutions to reach the dilution where you will have till 50 colonies and see the morphology and appearance of colonies. If you see differences you can separate the cultures, if no - perform microscopy to ensure that you receive pure culture.

Hi. You must know the colony morphology of the bacteria. I recommend you to perform serial dilutions and picking single bacterial colony. You can perform microscopy to select the bacteria.

Thanks for your suggestions but I already have the pure colony cultures of bacteria. I have recorded their colony morphology and confirmation of pure culture through gram staining. The main problem I am facing is during identification of bacillus members through 16SrRNA sequencing upto species level. My cultures do not have any contamination during sequencing as clean peaks throughout the sequences. But during online database analysis, 60% of them belong to such Bacillus species whose colony morphology and biochemical tests are not available online. So I am not able to predict them. Biochemical tests which I have done are not helpful. Please suggest some online databases for both biochemical and 16SrRNA sequences where i can predict them upto species level.

Hello,

which program you use after 16S RNA sequencing? 60 % similarity to certain species of bacilli or genera of bacilli??

If you have spore forming bacteria it means it can be Bacilli, Sporolactobacillus, Sporosarcina or Clostridium:

If your bacteria is aerobic, so we can exclude Clostridium.

Sporosarcina is very different by appearance and you can easily distinguish them from bacilli.

If your culture spore-lactobacillus I can say that you are very lucky its very rear microorganism. However, I not think so, you should first perform catalase test for that your culture should be catalase-negative, do not reduce nitrates to nitrites, and do not form indole. Lactic acid is production without liberation of gas from glucose, fructose, mannose, sucrose, maltose, trehalose, raffinose, inulin, mannitol, sorbitol, and alpha-methylglucoside.

So if your culture catalase positive it’s Bacillus not for 60 but for100%.

Do you perform BLAS or similar analysis after sequencing did you compare our sequence data with gene bank ?

Hello Karen,

I already used NCBI gene bank data. From that data most of them belong to Bacilli and after comparing them together they are almost 95-99% similarity in sequences. few of their species name I have never heard of. So I am unable to find any data about their biochemical and colony characters. and yes all are catalase positive, nitrate positive, oxidase negative and indole negative and most of them produce acid from sugar.

I also have results from other biochemical test like urease, citrate , vp, arginine dihydrolase, starch, esculin and gelatin hydrolysis but when i ve tried to assemble this data on ABIS online identification software I came to dead end. I dont know whats wrong. for e.g. I do not find any data about Bacillus nakamurai.

And all the culture have drastic difference in their colony morphology, cell size and arrangements. I have to incorporate them in my thesis. I did not find any online data especially about bacilli to confirm it.

Hello, 95-99% similarity in sequences can be some culture with strain differences. You can use old but reliable Bergey's Manual of Systematic Bacteriology, you can identify your culture by old nomenclature than you can find out new nomenclature. You need to perform several biochemical tests for this case.

Thanks Karen, I'll get through the bergeys manual.

Your Welcome!