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Tips for staining (light microscopy)

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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Tips for staining (light microscopy)

Postby animus » Tue Jun 19, 2012 2:14 am

Hello. I'm new at staining, and I was wondering if anyone could offer some tips.

For example: how can you minimize air bubbles when creating a wet mount?, how long should you let your stains sit on your specimen(s)?, how do you prevent your specimen(s) from being taken up by the paper towel when 'dragging' the stain across the slide and when clearing the stain from the slide?.

Thanks. :)
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Re: Tips for staining (light microscopy)

Postby PsycoCarrot » Fri Jul 06, 2012 11:03 pm

I'll try to reply to all these questions, but I'm probably not as experienced as some here.

1) The way I minimize bubbles on my wet mounts is by holding the coverslip at a 45° angle and placing the edge of the coverslip on the drop of liquid. This will draw the liquid fully across the edge of the slip. You can then slowly lower the coverslip down and bubbles should be slowly pushed out. Depending on how much liquid you use, it is sometimes a good idea to place a lab wipe at the edge of the coverslip, which will draw excess liquid out (helps stop the slip from sliding around under oil) and may help draw bubbles to the edge.

2)Staining time depends on the type of stain done, and which industry you work in. For example, standard lab micobiology Gram stain procedure may require staining for 1 minute for each stain. However, in some industries like wastewater treatment, the time standards are different. Wastewater Gram stains typically stain Crystal Violet and Gram's Iodine for 2 minutes, and Safranin counter-stain for 30 seconds.

3) I have no input on this subject, except to say that the stain should stick to the organism, and that excess stain can probably be rinsed off with DI water. Paper towels shouldn't be needed. As for specimens being absorbed into the paper towel, there are two things to consider: stains are normally dragged with a spare slide or coverslip, and you should consider heat-fixing your sample to the slide (or air-drying for more sensitive work).
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Postby animus » Sat Jul 07, 2012 5:52 am

Thanks a lot! I wasn't sure I was ever going to get an answer before you replied. Hah.

I will try the things you've mentioned. I read about heat-fixing not long ago and that worked with what I've done so far (a couple of bacterial samples from surfaces around the house). I also feel quite fancy heat-fixing specimens. ;)
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Postby triton » Wed Aug 22, 2012 8:33 am

my tip about reducing bubbles is. Use a needle. Hold the coverslip with your left hand, hold the needle with your right hand. Place the edge of a coverslip next to sample in sharp angle and support the oposite edge with a needle. Go down (slowly) with a needle, placing the coverslip onto sample. Needle os thin enough to do it slowly and precisly and you can control distrubution of liquid and bubbles.
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