13 replies to this topic

[lemonjoy]

Hi,
I am trying to do enrichments from some seawater that I have. I would like to pellet down cells from my triplicates and add them to my media. I was going to centrifuge about 50mL at 3000g. Does this sound right to anyone? I don't want to hurt the cells so it needs to be gentle. I also was wondering if I should do 50mL of each of my triplicates then add the three pellets or a combined 50mL of the water. How can I get the pellet out without hurting the cells? Could I resuspend the pellet in the media and add it to the remaining media? THanks for any help

[leelee]

As someone answered this same question of yours yesterday, you don't need to be gentle with bacterial cells at all. They are particularly hardy and will withstand centrifugation speeds of much higher than what you are suggesting here. I would go with what was suggested to you yesterday. At the speed you have suggested, you will need to centrifuge for an extended period of time, and you may not necessarily recover all bacteria in your sample.

Resuspend in media or whatever you like with a normal pipette or a transfer pipette if you are really worried.

Also, have you considered filtering your seawater to collect your bacteria? You could then wash the filter into whatever media you want?

[Phil Geis]

This is incorrect.  There are a number of reports of centrifugation injury of bacteria - loss of viability due to centrifugation.   For filtration, please consider the change in immediate environment of the filtered cells - esp osmolality, etc. as you rinse and subsequently treat filtered cells.
Can you tell us the fluid matrix from which you expect to physically enrich?

Loss of salt-tolerance and transformation efficiency in Escherichia coli associated with sub-lethal injury by centrifugation.

Lett Appl Microbiol. 1994 Nov;19(5):312-6.


Centrifugation injury of gram-negative bacteria.
J Antimicrob Chemother. 1991 Apr;27(4):550-1

[leelee]

My bad, I guess I'm too used to the bugs I've used before

(I'd still be worried about recovery with centrifugation though. In a class I taught we used a vacuum apparatus to filter our water samples through a thin filter paper membrane, which could then be placed directly onto a plate for culture. I was thinking that gently rinsing them with growth medium, or even a smaller amount of the original sample water, would dislodge the bacteria.)

[lemonjoy]

I am going to be adding to an artificial seawater media. It will be liquid. I am enriching for methanotrophs from the water leftover from an experiment I did, so it will need to be liquid with a methane headspace.

[Phil Geis]

Are these from near surface waters?

[pito]

Phil Geis, on 20 March 2013 - 03:22 AM, said:

This is incorrect.  There are a number of reports of centrifugation injury of bacteria - loss of viability due to centrifugation.   For filtration, please consider the change in immediate environment of the filtered cells - esp osmolality, etc. as you rinse and subsequently treat filtered cells.
Can you tell us the fluid matrix from which you expect to physically enrich?

Loss of salt-tolerance and transformation efficiency in Escherichia coli associated with sub-lethal injury by centrifugation.

Lett Appl Microbiol. 1994 Nov;19(5):312-6.


Centrifugation injury of gram-negative bacteria.
J Antimicrob Chemother. 1991 Apr;27(4):550-1

I can understand this and agree, however I wonder: would it matter that much?
If you lose some cells or the efficienty drops a bit.. would it really matter?
(I have not been able to read the papers, but still, of some of the cells die you should still have enough left in the end..)

[Phil Geis]

Not sure Pito.  If enrichment is needed, the count could be limited so that some members will be missed, esp. if of differential sensitivity.

Think I have a hard copy of at least one - want me to email it?

[pito]

Phil Geis, on 20 March 2013 - 12:57 PM, said:

Not sure Pito.  If enrichment is needed, the count could be limited so that some members will be missed, esp. if of differential sensitivity.

Think I have a hard copy of at least one - want me to email it?

I see your point, you are right, in certain cases its indeed not wanted if you lose (some) cells.

Yes, I would appreciate a copy of that text.
If possible you could perhaps upload it here, so other can read it too? But perhaps the file is too big to host here.
I will send you a private message with my emailadres.

[El Crazy Xabi]

I partially agree. Deguchi et al. (2011) reported little or no difference in the growth curves of actively growing cultures under hypergravity conditions (7500g and below). The pressure itself does not produce mechanical damage

Talking about cell damage, you cannot compare competent cells with environmental samples, when just pippeting vigorously kills competent cells. And I given that plating with Drigalski damages cells(Hedderich et al., 2011) I'd be cautious with that article.

By other hand it is tru that if you expose the cells after centrifugation under non-ideal conditions or change the culture media, they will get stressed due the loss of eg. extracellular polysaccharides. This will be specially affected by pH, salinity, biocides, osmolarity, etc. But there is no ideal method. With a low speed will need a very long time to recover a decent ammount of cells (if you are looking for prokaryotes) without any huge bias due cell shape and size. Filtration will also wash out the EPS



Deguchi, S., Shimoshige, H., Tsudome, M., Mukai, S., Corkery, R. W., Ito, S. & Horikoshi, K. (2011). Microbial growth at hyperaccelerations up to 403,627 × g. PNAS. http://www.pnas.org/...027108.abstract

Hedderich, R., Müller, R., Greulich, Y., Bannert, N., Holland, G., Kaiser, P. & Reissbrodt, R. (2011). Mechanical damage to Gram-negative bacteria by surface plating with the Drigalski-spatula technique. International Journal of Food Microbiology 146, 105–107. http://edoc.rki.de/o...ESoOQNcEIn2.pdf

Attached Files

•  Hedderich et al. - 2011 - Mechanical damage to Gram-negative bacteria by sur.pdf   151.5K   27 downloads
•  Deguchi et al. - 2011 - Microbial growth at hyperaccelerations up to 403,6.pdf   1.22MB   32 downloads

Edited by El Crazy Xabi, 20 March 2013 - 09:06 PM.

[lemonjoy]

They water was originally taken as deep water. I have incubated it under methane for 3 months at various concentrations. I am now transfering cells to media to grow them in ASW under methane headspace. I'm not too concerned about losing too many cells just want to make sure that I get enough and if I should be able to see them when I pellet them. The previous comment seems as if it is going to take a really long time to pellet the cells.

Edited by lemonjoy, 21 March 2013 - 08:10 AM.

[Phil Geis]

Deep sea origin but cultivated at atmospheric pressure?

[lemonjoy]

Yes. well they are from in the water column they are aerobic. If I try filtering also, we do vacuum filtration. Wont this be hard on the bugs?

[Phil Geis]

Aerobic and deep sea origin?  Are you saying they're facultative?