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What OD600 is suitable for assaying Ecoli viability

OD600 VIABILITY ECOLI BACTERIA STEADY STATE GROWTH

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9 replies to this topic

#1 Priya Tiwari

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Posted 28 February 2012 - 02:27 PM

Hi everyone,

I have WT and few mutants of a gene in Ecoli. I want to know if someone has done viability assays before which simply saying is:
O/N cultures-> Subculture to a OD600 -> dilute 10^-4, 10^-5, 10^-6 and plate 100ul on selection plates. Number of colonies next day tells you number of viable cells a culture has at that OD aka cfus/ml.
I have been doing this at OD600 = 0.3 - 0.4 but there has been variation. My question is what OD is suitable for this kind of study and by suitable I mean where the cells are the healthiest, happiest; and more importantly I really wanna know why that OD?

I am not looking for a correlation between OD and cfus/ml; I will be doing viability profiling for each of my strains; all I wanna know is what is the OD600 I should grow them upto.
Another question, is it ok to have a range like 0.3-0.4 for everything right from WT, null, mutant or should I normalize all my strains' cultures to a fixed number like 0.35.

Can you point a reference/article/book my way too?


PS: I am sure this has come up before; I'd be thankful if someone can direct me to the earlier discussion if any.

#2 Bluefunk

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Posted 28 February 2012 - 09:31 PM

Cells are the most healthy during mid-log phase. The range for mid-log variea depending on the media (stationary phase in minimal medium is od600= 2, in turbo broth is 8). 0.4 od should be fine and it is not necessary to normalize to exactly the same OD, as long as you take this into account when youre calculating CFUs

#3 Phil Geis

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Posted 29 February 2012 - 02:35 AM

Be aware that optical density per se does not measure viability or viable (healthy or happy) cells. By protocol as blue says one can expect that the cells quantified by OD would be viable. In any case it is a rouigh measure of CFU - I assume you're using it as a ranage finder and are doing viable cunts.

#4 pito

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Posted 29 February 2012 - 03:51 AM

Most has been said, but maybe this might help you to understand what/why OD is: http://www.exptec.co...rowth media.htm

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 Priya Tiwari

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Posted 29 February 2012 - 12:58 PM

Thank you very much for the help and prompt replies;
I will be diluting and plating and counting the colonies; I am confused about what OD600 to use because I got handed down 3 protocols where
1.) uses .3-.4
2.) lets grow upto 0.5+ and brings everything down to 0.46
3.) uses .4-.5

for the same experiment, same bacteria MG1655 with RECA ON PLASMID and same LB broth cultures.
So then I guess the way I asked my question was wrong.
I know log phase is where the cells are growing exponentially and are healthy with sufficient nutrients but my question is, is it better to test for viability at beginning of log phase [which would be 0.3] or late log which would be 0.5 or more?

#6 Bluefunk

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Posted 29 February 2012 - 01:43 PM

There should be no difference in baterial viability in coli growing in LB media at 0.3 or 0.5 OD600. Therefore, the answer to your question is neither is better or worse

#7 jarannihc

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Posted 29 February 2012 - 07:16 PM

Hi
Either exponential phase (log phase) or stationary phase can be used to check the viability of the bacteria.
The most preferred phase is the exponential phase as the name states the bacteria and their cellular constituents, cell mass increases exponentially with balanced and regular growth due to binary fission.ex:1,2,4,8,16....etc. Traditionally turbidity test is being employed.But you can also check the weight of the culture.And the doubling time of E.coli is 15-20 mins.
Before starting your actual experiment i suggest you perform an pilot experiment calculating the OD vs time of your culture every 15 mins (have 2 controls and one test).(Draw a growth curve-using the average of the three tubes).
Once the OD value doesnt change you will know that the culture has reached the stationary phase. Then it will be easy for you do determine which OD to use for viability count.

Do the pilot expression for Wt and mutants.You might find it bit tedious but its worth optimising the experiment urself based on how your cells grow (since they are mutants) instead of relying on other literature sources.

#8 Phil Geis

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Posted 01 March 2012 - 05:02 AM

Please pay heed to jarannihc's reponse, esp the last sentence. Responses here can be instructive but please build rigor into your protocol and its exection by answering your own question with relevant data.

#9 Priya Tiwari

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Posted 01 March 2012 - 08:29 AM

@jarannihc I'll go ahead and do a growth curve for WT, null and a mutant. I'd much rather bear with the tediousness now than suffer later.
Thanks a lot for the advice and help everyone.


#10 pito

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Posted 01 March 2012 - 11:46 AM

This has nothing to do with what you are doing now, but it might come in handy later: if you (for example) want to do conjugation experiments you need to use cells in the exponential growth phase because conjugation then works best.

A lot of the experiments need growing cells.
And like said: a lot depends on optimalisation of protocols.. its up to you to find the best "protocol" based on what kind of protocols are out there.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.






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