6 Streaking for Isolation:  Gram Staining

Now I’ll show you how
to perform gram stains. The first step is to
fix your smear with methanal. This is the female genital tract smear
and we’ll set it right there. “Fixing” means to
preserve the morphology of the cells. Then flood the slide with methanal. Let it sit on there for 10 seconds. After 10 seconds, pick up the slide
and shake off the methanal. And put the slide in your rack to dry. Okay, now I’ve put my smear here
next to my two controls a CFS control and a sputum control. And I’m going to flood them
with crystal violet for 30 seconds. So you just need to cover the area
that you’re staining. You don’t really need
to flood the whole slide. And then that stays
on there for 30 seconds. And I usually start timing
after I’ve added to the last slide. After the 30 seconds
with the crystal violet, then dump the crystal violet
in the sink, and rinse it off
until the crystal violet comes off. Don’t over-rinse, and you can shake off the excess water
just like that. I’m going to do that for all. The gloves are really useful for keeping your fingers
from getting stained. Now we’re going to add
the Gram’s iodine. And this fixes the crystal violet
in the cells. And that needs to stay on
for twice as long as the crystal violet,
so that’s 60 seconds. So then after the 60 seconds
with the Gram’s iodine, then rinse off the Gram’s iodine. Shake off the excess water. And now we’re going to decolorize. Decolorization
is the most critical step. So you’re going to hold your slide
at an angle like this, near the stream of water, and the timing is variable, depending on
the thickness of the smear. What you’re going to do
is just watch and as you see
the blue stop flowing out of the smear you rinse it off right away at that point. You want to keep the decolorizer continuously flowing over
the smear until that point. So, now we’re going to come
over with the decolorizer, and see how it streams out? And then just rinse
it off right away there. And then we’ll repeat the same process
with the sputum QC. And there you can see
the blue stopped flowing, and we rinse it off. And then our female genital tracts smear. It’s really important to shake off the excess water
after each step. Now I’ll cover each slide
with the safranin, the counterstain. And that will sit on for 30 seconds. It’s really important that you shake off the last water before this step
so it doesn’t get diluted. After 30 seconds with the safranin, I’m going to rinse off the safranin. Shake off the water. Then you can wipe
the back of the slide on a paper towel; make sure it’s the back and not the front and place it in your rack to dry. And repeat that with the sputum QC. And your female genital tract smear. So now we have finished
the gram stain procedure.

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