I am having problems with my method of fixation. I am attempting to fix my bacteria (M. tuberculosis) to poly - l - lysine slides. My cells are grown in 98 microtiter plates (used for antimicrobial investigations) and one well contains ~ 200 ul of 1 x 10^5 CFU/ml in media. I have attempted centrifugation to obtain a pellet, but no luck. So my last attempt was to remove the 200 ul of cells in the media and place them in fixative (2.5 % v/v FA and GA for AFM and 4 % v/v FA for confocal in 1 x PBS). I place ~ 50 - 100 ul of the sample on a slide and allow to fix to for ~ 30 min at room temperature. I then rinse with 1 x PBS and dH2O.
Almost no cells are being fixed to slide and I can't understand why?
If there are any suggestions, that would be great.
Would it be better to avoid this method of fixation and rather use heat? I thought I could allow the solution to dry to the slide, heat on a slide heater, then pass it through a flame, similar to preparation for ZN staining? Morphology is important in my investigations, so a I can't use a method that will damage the cells too much.
Thanks
In my opinion heat treatment (just like ZN staining) will be a better option as it won't deteriorate the morphological features of the bacterium.
If one centrifuges the bacterial suspension at a high speed 10,000 rpm for 30 minutes the pellet of bacteria will be formed. Supernatant can be aspirated slowly and the pellet can be resuspended in the remaining fluid and the suspension can be smeared on the slide and can be easily fixed by air drying followed by passing over the flame (light warming).
Hi, If your cells are moving and not getting fix on poly - l - lysine slides then you can use 1% agarose to fix your cells. If you are centrifuging cells from 96 well plate you might not see any pellet but cells will remain at bottom. If you growing Mycobacteria in microtiter plate can you please provide me a detail of plate and also want to know, is it Mycobacteria still remain as a suspension or it form clumps?
Amol, I am using 96 well plates from Eppendorf, polypropylene with lids, sterile, non tissue cultured treated with max working volumes of 400ul. Be sure to use flat bottom plates. The bacteria do sometimes form clumps, but when you add 0.05 % v/v Tween 80 to your media, it helps control this issue.
Dear Shane,
Excuse me, before answering, I would ask you two questions: 1. what kind of cells are you using, if they adhere to the bottom of the plate wells. If so, are you sure they are removed and present in the 200 ul? 2. Once fixed on the slide, what will you do? a stain?
1) H37Ra, they don't adhere, when I remove the media and centrifuge it, I can see a small pellet. I am sure they are present in the 200ul. Once fixed, no staining for AFM analysis and DRAQ7 staining for confocal analysis.
Dear Shane,
Excuse me, I though you referred to eucariotic cells, from cellular culture. I have not experience in confocal microscopy. On staining mycobacterial cells for diagnosis, we use slides previosly treated with a solution of 10% gelatin (immersing the slide few seconds in the solution and let it dry). This treatment allow us a better fixation (not miracles). Moreover, if you allow the smear to dry and heat the smear, for example igniting few drops of etanol over it, it is sure the fixation will improve. Maybe you should test and decide if the cell structure is to affected for your purposes.
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