Hi there,
i rescently opened a 3 new stocks of Lactobacillus brevis
and streaked them in a seperate MRS Agar.
after two days of incubation at 37 degree celcius i found single colonies and gram stained it.
Flame fix.
60 sec crystal violet.
wash with running water
60 sec iodine
wash
20sec acetone
wash
45sec safranin O
wash
then observe under OIO
and i found both gram negative chained bacilli, and gram positive chained bacilli on all of the stocks.
considering that i took SINGLE COLONY form the agar plate and how come there is gram negative and gram positive bacteria co-exist together in one single small colony?
i presume that there needs an adjustment for my protocol for this brevis.
I adjusted some protocols like Acetone 20sec-->10sec.
it did decreased gram negative bacilli, but there are some.
perhaps should i change the gram staining protocol for this Lactobacillus brevis? any recommendations?
Hi
You should use 70% Ethanol as decolorizing agent instead of acetone.Secondly increase the staining time of primary dyes to one minute and 40 seconds for that of decolorizer...
Hope this help
Shahid Nawaz I tried making smear in little volume so that it could be flame fixed quickly with out disrupting cell wall.
increased crystal violet staining time to 1min and 15 sec.
then I only briefly rinsed crystal violet for about 5 sec indirectly, and this really worked. i could see dark violet color of smear even with naked eyes
thanks to thy help. God Bless.
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