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Discussion Starter · #1 ·
So I'm trying to do my first gram stain, and I feel nauseous and my hands are all purple.

Am I basically looking for rods and cocci only? What about "blobs"? Do they count as bacteria, or what are they? What about rods of differening lengths??

I don't see many distinct rods at all on the slide; mostly blobs. Did a very thin smear, so I don't think it's a matter of the feces being too thick on the slide.

Any suggestions?

Thanks,
Jennifer
 

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The worst part for me has always been fixing the slide to get it to colorize properly. You really want to see both red and blue elements in the field so that you'll know that you're somewhere in the right ballpark with your technique. The pictures that we take are almost never as good as what we physically see with our own eyeballs through the scope, too.

Dang it!

The straight rod in the top picture might probably be an artifact--most long rod bacteria have curves in them like a slightly winding road. Are these pictures taken with your 100x objective running oil?

Pidgey
 

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Discussion Starter · #3 ·
Are these pictures taken with your 100x objective running oil?
Yep. I saturated with crystal violet, iodine about 60 seconds each, rinsing in between, then decolorized and rinsed, then safranin 60 seconds with an ending rinse.

I have a hard enough time viewing through the eyepieces without being distracted by seeing a reflection of my own hair or eye floaters.

And, yes, I know the pictures kind of suck. Maybe I should invest in a camera attachment, if one exists.

What about those blobs?

Still yellow urates, sometimes diarrhea in this bird. I can't find any budding yeast, so I don't know if the Nystatin has taken care of it, or if I'm not finding it for some other reason.

Thanks,
Jennifer
 

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Well, I just use my regular digital camera, probably like you. It takes some getting used to, taking pictures that way and hand-holding the camera besides. Anyhow, I think I can tell from your picture that you don't have a "Plan Achromat" objective lens, so you're going to be concentrating on what's primarily in the middle of the field generally because the focus will get fuzzier out towards the edges due to the nature of the lens. That may be why in the second picture that everything in the middle seems sharper than what's out further outboard.

Anyway, in the second image I can see some rods that look dark, which might be really blue from the crystal violet staining. There's red stuff, too, from the safranin, but I don't see as many red things that look like bacteria. Here's one that shows both blue and red ones:



Here's a link to that one in full size:

http://image61.webshots.com/61/5/13/64/2087513640073664377yLTmgN_fs.jpg

It's not often that they turn out that good, but I got lucky that time.

Pidgey
 

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Ya' know... there's more than one way to do the Gram stains. I've usually used this one:

1. Air dry and gently heat fix slide
2. Cover with crystal violet (1 min.)
3. Gently rinse in tap water
4. Cover with Gram’s iodine (1 min.)
5. Gently rinse in tap water
6. Decolorize with 95% ethyl alcohol (15 to 30 sec.)
7. Gently rinse in tap water
8. Cover with safranin (1 min.)
9. Gently rinse in tap water (air dry)

...out of AVIAN MEDICINE: PRINCIPLES AND APPLICATION, Cytology section. I set up the bottles, droppers and my watch at the sink in the bathroom to do it as best I can. I just drop a drop or two of each stain and the iodine onto the actual smear and hold it flat for its part of the sequence. If I didn't fix it well enough at first, everything just washes away. If I've fixed it too much, the colors don't work properly.

Pidgey
 

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When you clicked on that link, a separate browser window probably popped up with a "The website declined to show this webpage" message. When you see that, try clicking the Refresh button on that browser window and see if it does what it's supposed to.

Yeah, "Plans" are better and cost more to boot. You don't need them, but they've got that Cadillac feel to them. They're designed to basically have the entire field in focus at the same time. You can go to eBay and search on "100x Plan" to see some examples. I found an old set cheap for my AO and got 'em all for my scope on eBay a couple of years ago.

Incidentally (and you've probably already discovered this on your own), one technique for studying the entire field is to work the fine focus knob to bring the outer ring into focus. You'll see the center area go out of focus when doing that. Or you can just work the mechanical stage knobs to bring items of interest into the center.

Pidgey
 

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Discussion Starter · #9 ·
I get "Forbidden: You don't have permission to access /61/5/13/64/2087513640073664377yLTmgN_fs.jpg on this server" when I click the link.

I have been using the fine focus and the mechanical stage knobs to try to scan the whole slide. After some time, I got nauseated and had to lie down, which is why it took me so long to respond. :eek:

Am doing a second slide just for "fun" right now.

Jennifer
 

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If the Refresh button won't get it, then you can try copy-and-pasting the link directly into a browser's address bar and going that route.

If you're getting nauseated looking into a binocular microscope, then you might try beer or wine before you start the session.

Pid <hiccup!> gey
 

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Discussion Starter · #11 ·
Ah, that did it.

So do you actually count the gram pos vs. gram neg per field or just eyeball it? Are those lighter pink round spots cocci? Overall (incl. those lighter pink cocci), looks like about 50% neg and 50% pos?

Jennifer
 

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Pardon me for butting in, but proper heat fixing of the slide seems to be everything. Otherwise it just washes away or you get cinders.
 

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I just eyeball it. One of the things you don't want to see is mostly Gram(-) long rods in fecal smears. Sometimes you'll actually see strepto- (string of pearls) chains. That's okay, by and large.

I sometimes do swabs down the throat for a wet-mount, looking for extremely motile bacilli, too--no stain.

Pidgey
 

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Discussion Starter · #15 ·
OK, so here's a new picture.

Those brown circular things are yeast, I guess? The ones that are doubled up (and I see quite a few) would be budding yeast?

Today's day 5 of Nystatin. I don't know if I should still be seeing budding yeast at this point. He acts pretty good, though.

Do you smear the whole slide, or just a cover slip's worth?

I've been told to hit fix for 3 seconds. So far (and this is my 4th attempt), I haven't had any wash away. What do cinders look like?

To do a crop stain, would you have to swab the swab against the wall of the crop? I wouldn't know how to tell when I'm "there."

Jennifer
 

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I can't make out anything in particular on that one with any certainty. There's more debris than I'd like. I usually do a fecal swab by spinning a Q-Tip in some poop, and then dab it on the slide in such a way as to only leave a light film, like a cloudy window. If there's a fleck of real debris, I wipe it off before ever I fix the slide. I only make a smear about the size of the head of the Q-Tip sideways instead of a huge, coverslip-sized spread.

Pidgey
 

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Discussion Starter · #17 ·
You think a cover-slip's worth is huge? The way I was taught was to smear the entire slide (because then the chances of finding something are greater) and then scan the whole thing.

I think I discovered the reason for my nausea.

Jennifer
 

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Sounds like an awful lot of "more of the same". Some folks do smears to find coccidial oocysts and worm eggs, too. I use the float to concentrate them for that.

Take two glasses of wine and call me in the morning...

Pidgey
 
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