3 microscopic views of pus

[discomorphella]

So that nobody gets disgusted, these are all smears....no picture of the actual "source" of the specimen or anything...there are 3 different stains here...the first is the traditional Gram stain (crystal violet, iodine, safranin 0). You can see the neutrophil's nuclei stained red w/ Safranin O, and their cytoplasm stained purple by the crystal violet. The staphylococci are stained purple (Gram positive). The second picture is stained with Wrights stain (eosin and methylene blue and analogs). You can see the neutrophils cytoplasm is full of red (eosinophillic) graunules, these are seriously pissed off neutrophils...the staph stain blue with methylene blue, as do the nuclei. I think its a bit pale because I stained it using the time for a thinner blood smear... The third view is using a stain that all amateurs can access, using only common household (aside from the dyes) chemicals. Its Phloxine B/ Methylene Blue. Both dyes are very safe, and can be purchased from a pharmacy in the US....I'll put the reference for it in later...but as you can see you get almost as good a result as the "pro" stains and it needs only the dyes, 95% ethanol and a bit of 5% vinegar and weak borax or carbonate solution....
Optics: 0.9 Achr condenser set for brightfield Koehler, 1.4 NA / 100X objective, 10X Periplan GW HP relay and Kodak 5 MP camera on an orthoplan...
I will post the third stain recipe and instructions separately.

--David

[Thomas Ashcraft]

Discomorphella wrote: "The staphylococci are stained purple (Gram positive).'

David,
Quite interesting. So the smallest purple clusters in the first photo are staphylococci?

Thanks for posting.

Tom

[beetleman]

Nice slides David.....do you do this for work? A few words I understand in your post, most I will have to look up I was hoping to see the gross parts (only kidding).

[Ken Ramos]

Doug said:

I was hoping to see the gross parts (only kidding).

Yeah Doug, red, throbbing and inflamed, squirting, bleeding pustules. Boy can we get gross with this post.

Now that, thats! out of our system, some pretty interesting stuff here Dave. I have thought in the past about ordering some dyes or stains but even if I did, I would not have the sense enough to know how to properly use them.

[discomorphella]

Thanks Guys--

Yes, the purplish dots are the staph, about 1 um in diameter. The clusters are characteristic.
Ken, the Methylene Blue / Phloxine B technique is very simple, cover your section or smear or whatever with 0.25% Phloxine B in 0.25% acetic acid (1/20 dilution of white vinegar will work just fine) for a minute, then rinse, and cover with 0.25% Methylene Blue in 0.25% sodium borate (borax) for ca. 1 min...then destain the blue a bit in more 0.25% acetic....then dip a few times in 95% ethanol, and mount as you like....just some glycerin jelly or immersion oil will work for a temporary mount if nothing else is available.
At work I would have used a completely different method, some nifty fluorescent labeled antibodies/lectins to stain the bacteria and a fluorescent nuclear dye to stain the neutrophils...not things most would have at home...
But as an amateur microscopist I like staining things, the same way I used to like developing film and slides and prints in the darkroom, and I think a lot of our fellow posters and readers might like to have some simple staining techniques at their disposal so they can see details of many specimens they might otherwise miss, even with DIC and phase contrast and all the other impressive illumination techniques we see displayed here on a regular basis...which is not to denigrate those techniques either, I use/enjoy them too, but having a few easily employed stains on the bench is a good idea too.
--David

p.s. Doug, you would be disappointed, the source wasn't all that bad...bad enough I suppose....

[Ken Ramos]

Well I for one will settle for the images from the slide I may look in to those stains, as you said it would not hurt to have a couple around and they may just come in handy, thanks.

[Kite]

you know, i was about to post this, I did EXACTLY the same thing today lol

my dad works as a pathologist on the nation health service, and as such, steals me my slides and cover slips, and recently, a nice collection of stains

meh, you got better shots than I did lol

[discomorphella]

'Tis the season for infections I suppose, or suppurate, or something...since we already saw Wright's stain, if you have some you might try Giemsa, I didn't have time but I bet it would stain the bacteria and the neutrophils the best...and be careful, staph can live a long time even dried...so best to immediately plunge these slides into something pretty strong to fix them and decontaminate them. We should warn other readers that pathological material is something to be careful with...
I fixed / stored these slides in 95% alcohol immediately after, well, lancing my finger actually...there's something about us microscopists that compels us to put everything possible that will fit under the scope.

--David