Bacterial culture in microcentrifuge tube? - Staphylococcus (Jul/22/2008 )
Hello everyone.
One question came to me today.
First, in our lab, we work extensively on Staphylococcus, and we always culture bacteria in 4ml BHI tubes(glass) and incubate overnight, and before performing experiments we always transfer like 1.5ml into microcentrifuge tubes, centrifuge them, and wash the pellet by 1x PBS, and resuspend in 1X PBS.
the question i have is that, can i just like get a 1.5ml or 2ml microcentrifuge tube and incubate the bacteria in it overnight and just like centrifuge it and do the following experiments?
there are some problems i've thought of about this method.
1. 2ml BHI as medium could become depletion of nutrients or build up of waste overnight if the bacterium grows too much?
2.Polypropene, PP, could have different effects on bacteria compared to BHI in glass tube?
3.lid-caped microcentrifuge tube may be a problem for providing aerobic environment, but may be using a screwed microcentrifuge tube and lossen the cap during incubation could solve this problem?
It just came to me that, if i use 4ml BHI glass tube to culture the bacteria, it often wastes 1 to 2ml cultured BHI medium and glass tube has to be autoclaved, washed with extensive scrubbing and ultrasonically washed.
And also, after the overnight incubation in 4ml glass BHI tube, i'm going to use a microcentrifuge tube to do the washing anyway.
Does anyone use microcentrifuge tubes to do culturing?
thanks alot
I don't use them for routine overnight cultures, mostly for the reasons you've pointed out (especially the difficulty in maintaining an aerobic environment). However, I do spin down more of the culture than I need, and freeze pellets of the bacteria for short-term later use. It really depends on what you're using the cultures for.
In order to get good aeration of the culture you need to grow a culture in a vessel that is at least 3X larger than the culture itself. For example, if you are going to grow 2ml of culture, you really need a vessel that will hold at least 6ml. This allows the culture to swirl and get good aeration. Your eppy would be full and when you put it on the rocker (especially with a loose lid) you'll get the culture everywhere and possibly get cross contamination from one sample to the other. Plus there just isn't enough room for the culture to swirl properly. An additional benefit to extra culture (I always grow 5ml for a miniprep) is that if something happens, such as dropping the eppy or accidentally pipetting into the wrong tube (we've all made stupid mistakes) you don't have to re-inoculate and wait a full day...you have extra! I'm not sure why your lab is using glass tubes but it might have something to do with the Staphylococcus strain that your lab works with. Ask around in the lab and see if there is a legitimate reason for the use of glass. Otherwise it could be an attempt at lower cost but you could make a strong argument for the time it takes to clean and autoclave the glass as actually costing more (you have to pay someone to do this). But, you don't have to order anything which makes it appear cheaper. Check out the Falcon pp round-bottom 14mL tubes and make a plea to the lab manager. They really aren't all that expensive and you may be able to work out a discount if you order multiple cases at once.
A 2 ml centrifuge tube filled with 1 ml of broth works pretty well in a rotator, and the broth does not stick in the conical end, as it does in 1.7 ml tubes. If you need aeration, Eppendorf makes gas permeable centrifuge tube lids. You can also grow cultures in 96 well deep well plates, which also hold around 2 ml. These can be covered with air permeable membranes and shaken.
Thank you
i think the major problem would be keeping a good aerobic environment.
i've been thinking about testing, but i couldn't think of what to test.
staining to see cell wall synthesis, phenotypic testing like coagulase, protease, lipase, or 2D PAGE?kind of expensive
anyway thanks alot
staph are facultative anaerobes so aeration is not much of a problem - growth kinetics are about the same.
However, I'm surprised none of you expressed a concern for contamination other than with other wells if you were to use microtiter type plates. Are these tubes sterile?
However, I'm surprised none of you expressed a concern for contamination other than with other wells if you were to use microtiter type plates. Are these tubes sterile?
The problem i have is that if the aeration is not the same compared with incubated in BHI tubes, maybe the phenotype of the staph would be different?
i was thinking about if i could use 2ml microcentrifuge tubes to culture bacteria, the tubes we always autoclave ourselves.
and as of the microtiter plates, currently we uses the plates from cornings, and the plates comes with lild already, and the package said it's presterilized, so we aren't really concerned about it.
(though we don't have the membrane mentioned by phage434, but i think that could properly sealed the plates, and isolate the different culture from different wells)
Thanks -I see the material is or can be sterilized. Will your procedures maintain that state? If you work with just medium (BHI), does it remain sterile through these techniques? And it's simple enough to run an OD or dilution plating side by side to answer the aeration question.
My bias here is (and forgive me if this doesn't fit) is that molecular work often engages folks with limited to no training in classical micro. I've seen some confide tly claim as E. coli cultures clearly contaminated with only the slightest idea how to determine, prevent that. The typical strategy, were that to be realized, is to ask after inhibitors/antibiotics rather than to pursue more rigorous aseptic technique.
My bias here is (and forgive me if this doesn't fit) is that molecular work often engages folks with limited to no training in classical micro. I've seen some confide tly claim as E. coli cultures clearly contaminated with only the slightest idea how to determine, prevent that. The typical strategy, were that to be realized, is to ask after inhibitors/antibiotics rather than to pursue more rigorous aseptic technique.
thank you jorge1907,
at first i didn't really understand what you mean, but now i see.
I work mainly on Staphylococcus spp., and you mean rather than verifying if the cultures are contaminated or not(since there's also possibility that i don't notice the contamination), i should find antibiotics added to growth media to keep my staphylococcus grow but inhibit other potential contaminants?
thank you for your precious questions
it really helps me to think more
thx alot