About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.
12 posts • Page 1 of 1
i have some problems on my work.
i have isolated e coli from water distribution system and i studied it.
my bacteria show pink colonies on MaConkey's Agar.
give greenish golden metallic sheen on EMB Agar and
and correct IMViC biochemical pattern, +,+,-,-
but i think they are little bit fat than usual size of e coli.
can it be possible? or am i wrong?
if it is right , is there a variety on size and why?
please give me some advice.
Usually Colony size and morphology in many bacteria, including E. coli can greatly vary from strain to strain on the same media. The outer aspect of a colony is not a good guide for identification. I would say that if your biochemical test confirm the identification, it is correct.
If you really do not trust your test, it's up to you to start 16S RNA seuencing or other molecular method, but it will probably only confirm what you already know.
thank you all,
i have correct biochemical pattern of my e coli.
is IMViC pattern enough?
but i am unable to perform 16s rRNA typing as my lab is not equipped enough to do so now. but i wish i could do later.
i am now trying to extract plasmids from these e coli and i think i am able study these plasmids. ( but only a start )
i have one question please, why LB medium is used before plasmid extraction.
and why not other enrichment medium, such as Nutrient Medium.
can others be used? if not, is there a difference?
Probably. In our lab were we are working with thousands of environmental isolates, we usually even do not perform all those 4 tests (we look for Betaglucuronidase+, indole+, lactose+ strains)
16S typing is probably not very useful anyway. If you really have doubts, tests like API20E should be more than enough.
I have always been culturing my E. coli on LB, so I never even tought about it. But be careful to use LB Lennox (or L medium, with 5g/l of NaCl) rather than LB Miller (10g/l NaCl) since it reduces supercoiling and give better plasmid yield.
Reading threads like this make me want to go directly back to the books....
thank you for your replies,
for plasmid, i have heard that it is good to extract plasmids in Log Phase of the bacteria, is that right?
what is the best time to extract plasmids?
should i know the growth curve of my e coli?
if so, how should i draw the curve, apart from using Spectrophotometer?
the Spect: in our lab is out of service, and so i can't use it.
is there some way to do this?
It's true that it is usually better to extract DNA in log phase. But you can still do it at saturation, it is just harder to obtain a good quality of extraction.
How to know when you are in log phase without a spec? Hmmm, I don't really know... As a desperate mesure I would suggest this:
start a culture, compare it to Mc Farland Standard (0.5, 1, 2, 3, 4...) and plate each time you are close to the standard. You will end up with a very rough growth curve, that you could use next time (say you know you are at the end of the log phase at McFarland 3 that is when you are going to harvest cells).
Since saturatiom happens at very different cell densities for environmental strains, I think it is hard to ignore the growth curve.
thank you for your reply,
i have made a 12-hour growth curve of one of my isolates.
it reaches log phase at about 4 hour and reaches stationary phase at about 8 hour.
does it mean that i have to make plasmid extraction between 4 and 8 hours after incubation?
can i know the standard curve of e col?
In fact it seems according to all the protocol I read that you do not really need to extract plasmids in the log phase. So Why bother?
But if you really need to, Now that you have a nice curve try to make the extraction as late as possible in the log phase, but before entering the saturation plateau. In your case it seems that it would probably be around 10 to 11 hours of culture.
I do not know of any "standard" growth curve, but you can probably find plenty illustration of the classical curve on the net, the maximum cell density and time will vary with the different strains though.
12 posts • Page 1 of 1
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