Help with new goodies

[AGreyMan]

Hi Folks:

Well, I got my hemacytometer, and I have some questions for the lab-type folks. The instruction booklet (printed in '73), speaks of using potassium Oxalate, ammonium oxalate, etc. Is this for anticoagulation? Our purple top tubes at work contain EDTA. Is this an acceptable alternative to use when using the hemacytometer?

Also, it says that the dilution fluid is 1% Glacial Acetic Acid. What is that, do I really need it, and where can I find it.

Thanks for any help you can give.

Stay Safe,
AGreyMAn

[tangent]

see my "sources for chems" post in this forum for that answer.

don't have time right now to find the other answers for you, and there are others more knowlegable here that could give you a better answer. I do recall that one of the med lab techs did discuss anticoagulants in the long "equiping a med lab" thread, again in this forum.

good luck and if you get no joy, I'll try to look into it for you. I do have a couple of books that discuss this.

be interested in what you find out. please share whatever you learn.

have fun with the new toy!

-t

[AGreyMan]

Ok, I found out that "Glacial acetic acid" means essentially acetic acid that is ~99% pure. I also found out that 1) vinegar is mostly a dilute acetic acid and 2) Photo developing stores sell glacial acetic acid.

Now I need to know 1) why the heck am I using acetic acid anyway, and 2) so I get some glacial acetic acid: What do I use to dilute it to 1%? My guess is either sterile water or saline. I am leaning to saline because sterile water is hypotonic, and I'll get cell destruction.

Any ideas? I may fool around with diluting vinegar to see if that's an acceptable austere substitute.

Stay Safe,
AGreyMan

[tangent]

ok - lets see...

acetic acid (ML 94a) A colorless liq w/ a strong smell used to test stools for occult blood, 10.11, for testing the urine for protein, 8.3, and for making white cell diluting fluid, 3.45

seems like your looking for section 3.45

3.45 Making White Cell Diluting Fluid - 100 ml.

Add 2 ml of glacial acetic acid or 1 ml concentrated hydrochloric acid to 100 ml of water. Add two or three drops of crystal violet stain to color the fluid a pale violet. Keep the fluid in in the bottle. Some people like to keep it in a bottle with a 1 ml pipette going through the cork.
This fluid is used for counting white cells (see section 7.30)

think that covers your questions.

-t

info from A Medical Laboratory for Developing Countries by Maurice King - excelent book! - british spelling has been converted to US spellings.

[AGreyMan]

Tangent:

Thanks for your help. I don't mean to pester anyone in particular, I was just posting for anyone who had the secret knowlege to help. Turns out you are the guy.

Thanks again.

Stay Safe,
AGreyMan

[AGreyMan]

P.S. The section Tangent gave to me mentions using crystal violet stain. Is this the same as gentian violet/solution? Or methyl violet?

Thanks everyone.

Stay Safe,
AGreyMan

[tangent]

haven't a clue - sorry

-t

[Reasonable Rascal]

Gentian Violet contains the stain Crystal Violet but apparently is not identical to same. Gentian Violet is a 1% solution. Besides use as a topical fungicide it is also used in fingerprint development on adhesives. Crystal Violet appears to be a much more concentrated solution.

Methyl Violet seems to be another compound entirely and does not cross reference to either of the other two.

RR

[AGreyMan]

Thanks Guys.

I may have to get a little more agggressive in locating the "crystal violet stain"...Or just try it withthe gentian and see if it works!

Stay Safe,
AGreyMan

[RESQDOC]

Ammonium oxalate is used for counting platelets.

Acetic acid, plain or as "WBC diluting solution" is used for counting white blood cells.

There are other solutions that can be used but these are the most common.

EDTA is the best anticoagulant, avoid heparin.

You can also count red cells with your chamber, using different solutions, however this is really a waste of time for most things.

Gentian violet is fine, don't bother hunting for crystal violet.

All of these solutions are really prone to fungus growth and clumping, keep sealed out of the light and filter frequently.

[AGreyMan]

Resqdoc:

Thanks for the info. Do you know WHY acetic acid and crystal violet is used in the proscess? Does it somehow stain the WBCs?

Thanks.

Stay Safe,
AGreyMan

[AGreyMan]

I found this at: http://www.tpub.com/corpsman/250.htm an online Navy Corpsman manual. It was in the urinalysis section:

· One drop of 5% acetic acid added to the urine sediment disintegrates any red cells, but it does not affect the white cells (except that the nuclei become more distinct).

So the acetic acid may make the nuclei more distinct!!

FYI

Stay Safe,
AGreyMan

[RESQDOC]

Acid of any sort destroys the RBC's, thus getting them out of the way making WBC counting easier. The stain is just that, staining to make the morphology easier to study.

[AGreyMan]

Excellent.

I think I am beginning to understand some of this. Thanks to all!

Stay Safe,
AGreyMan

P.S.: Microscope came today, packaged VERY poorly. I hope it's not damaged...

[tangent]

if it takes, this is the white blood cell count section of Kings Med lab book. rename from .zip to .pdf to view.

if it works, I'll get the section on the microscope scanned in.

-t

nope - it bombed... wasn't that big eithor - just over 1 MB...
I'll e-mail it to you.

[AGreyMan]

Thanks for the attempt!

I think the 'scope will be OK. The field of view has some little specks in it, but not too bad, The guy had taken off the occular, and upended his paper shredder in the box. There was bits of paper and paper dust EVERYWHERE. I got some canned air and blew all I could out, then used a lens brush to get stuff off the lenses. I need to make a light source for it yet, but then I'll look at a few slides and see how it goes. In case it is still way messed up, does anyone have the name of a reliable microscope service place? Hopefully inexpensive?

Thanks.

Stay Safe,
AGreyMan

[AGreyMan]

Hi Folks:

Well, I got the microscope working, after building a light source for it. I got some Wright's stain smears from work and practiced doing a few manul differentials. It has become obvious that this 'scope needs some repairs. It seems to drift out of focus, and the ocular is REALLY messed up. When I rotate the ocular while looking at the slide, about 75% of the crap in the visual field turns!

So, has anyone had experience with a microscope repair place? Or knows where I can pick up a Olympus 23mm 10X ocular cheap? I expected a few repais on this scope since it was so inexpensive, and if pressed, it would work "as-is."

Well, so go the travails of the newbies!

Stay Safe,
AGreyMan