staining blood with giemsa

[nigeldodd]

I am trying to stain a blood smear to reveal the different white blood cell types. So far I have not revealed any. The white blood cells are vastly outnumbered by the red cells but even with a wide field of view and manually scanning across the smear I can only see red blood cells.



Giemsa stain is supposed to stain the nuclei. Red blood cells don't have a nucleus. My technique has been to get a peripheral blood sample, smear it over the microscope slide, let it dry in air, fix in methanol, air dry, apply giemsa stain diluted 1 part to 10 (I have used lower dilutions) and leave for 15mins to 1 hour, rinse with tap water. I have tried variations on this spell.

The photo was taken with a phone looking down a compound microscope with total magnification about 400x. I apologise for the rubbish photo - the vignetting is probably due to me not getting the condenser set up properly. The many visible cells are red blood cells with typical toroidal shape, thick at the edges and thin in the middle. I cannot see anything that has taken up the stain.

Could anybody please offer constructive criticism and tell me what I am doing wrong, or whether I have false expectations?

[rjlittlefield]

Nigel, welcome aboard!

I cannot speak to the issue of stains.

However, I do notice that for me, your image displays as a just a large space with a big bold circle with a dash in the middle of it.

The same thing happens if I extract the image URL and try to open it in a separate tab of my browser.

I wonder if there is some permissions problem that does not allow public viewing of your image at googleusercontent.com .

--Rik

[nigeldodd]

thanks, Rik.

The photo is in Google Photos. I right clicked and got the url and it appeared ok in the preview. I will investigate further.

thanks
Nigel

[nigeldodd]

For now maybe you could look at

https://photos.app.goo.gl/kH3YuH3kZbStH8td8

which should show the photo. Does it?

Nigel

[rjlittlefield]

Yes, the photos.app.goo.gl URL opens a new tab and shows the image.

--Rik

[nigeldodd]

good, thanks for confirming you can see the image.

I wonder if anybody has experience with staining blood smears.

[Olympusman]

I'm surprised the condenser is vignetting at such a high magnification. Maybe you should rack it up closer to the bottom of the slide.

Mike

[nigeldodd]

The vignetting was only in the photo, not the view through the eyepiece.

I have not (yet) set up a proper photographic solution and the photo you see is taken with a phone, hand-held, using my late father's medical students' microscope. I would not have matched the exit pupil of the microscope to the entrance pupil of the phone. It was extremely Heath Robinson.

But the main issue is Why can't I see any white cells with stained nucleus?

[Ichthyophthirius]

Hi,

After washing off the stain, did you dry again? What is the colour of your dry, stained blood smear?

The smear should be purple (naked eye) and the RBCs should be red under the microscope. If they remain unstained, the staining didn't work. Frequent problems are expired Giemsa dye (which dye is it actually?) or 1:10 dilution with the wrong diluent or the staining solution getting too concentrated by evaporation of the open slide on air.

The ratio of RBCs to leukocytes is 600:1, so there are none to a few in every field of view.

[nigeldodd]

I tried a number of variations of the basic technique of

air dry blood smear
fix with methanol
stain with giemsa
wash with water
air dry

with different times and different concentrations of giemsa (diluted in water)

To the naked eye the colour of the dry, stained blood smear is a very pale purple but under the microscope the RBCs are not red - they are quite similar to how they appear in my photo: almost colourless. So it would fit with what you say, that the staining did not work.
Here is a photo of the bottle of stain
https://photos.app.goo.gl/z1VD4wfogeJH5Dcc9

and here
https://photos.app.goo.gl/gdny8fZVNk9fyJcT6
are the instructions.

Re-reading the instructions I have not followed part of stage 5 in that I did not leave for 3-4 minutes in buffered distilled water. I am not sure what "buffered" means in this context.

Thanks for your help.

[Thl05]

Hi,
as you may have seen in a topic nearby I am a bit new to all this so please excuse me I dont want to teach granny about eggs !
However, by coincidence, while exploring youtubes about my other problems, I came across this one by Oliver Kim (aka Microbehunter) in which he talks (8 minutes in) about staining white blood cells and not red, using methylene blue.
https://youtu.be/ckdKIb1ZE1Y
It was news to me, as you say, about reds not having a nucleus, and with no dna to take up the stain.
So, as Ichy has said about the ratio of white to red, I wondered if you might use the m'blue as a control to test against the gemisa, ie. if you can find them with m'blue (it looks simple in Olivers hands !) then it would suggest your giemsa has gone off !?

I didnt comment earlier as you were asking about giemsa of which I know nothing !
Again, forgive me if you have already been there/done that.

edit : it does help if one can get a ytube link in !!

[Ichthyophthirius]

I am not sure what "buffered" means in this context.

It's not immediately obvious what the problem is.

I do my blood smear stainings on a staining rack (slides horizontal and in the open) which doesn't use a lot of reagent if you only do a few slides.

I use a Giemsa buffer for diluting the staining stock. I dilute 1:10 and stain for 10 min. It's a dilute phosphate buffer, pH 7.2 - 7.4.

You can also try the "Quick Giemsa" protocol just to see if your stain is OK. https://www.sigmaaldrich.com/content/da ... 1/gs10.pdf But in addition fix with methanol before staining in case your Giemsa solution is faulty and contains no or too little methanol.

- smear, air dry
- fix by flooding with methanol, 1 min
- air dry (or hair drier)
- Giemsa undiluted, 2-4 min
- wash under running tap water, 5 sec
- stand vertical and air dry or blow dry
- add a small drop of immersion oil
- do microscopy: first with 10x, then change directly to 100x (usually, no coverslip is used), take care no to use a 40x as immersion oil would get on the front lens
[if you don't have a 100x immersion objective and have to use a 40x dry, cover the immersion oil with a coverslip!!]

[nigeldodd]

Thanks to both respondents.

There is work to be done to try out these ideas.

I once used a pipette, that had been used for water, to draw Giemsa from the bottle. Is it possible that the traces of water have contaminated the Giemsa? How sensitive is it to water contamination?

I will report back.

[Ichthyophthirius]

If you can do so, also measure the pH of your destilled water (whatever you're using) and your tap water. As you can see from the Magnacol trouble shooting instructions, the pH has a slight effect on the staining results. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC540181/

[billjanes1]

I am now retired but have had extensive experience in the hematology lab as a pathologist.

Your blood smear is very poor. Too thick with extensive Rouleaux formation (stack of coins). To make a good smear you put a small drop of blood near one end of the slide and then use another slide to make the smear. Place the second slide at about a 30 degree angle to the smear slide with the edge just ahead of the drop. Then back up the second slide until it just touches the drop of blood and smoothly push the spreader slide forward.
You should have a tapered edge towards the end of the smear and this is where you examine the blood cells. They should be evenly spread out and not touching.

Giemsa is often used for bone marrow and parasitology, but Wright's stain is easier to use. You simple place the undiluted stain on the slide and the methanol in the stain fixes the cells. You then add buffer and staining begins. Consult the manufacturer for timing. The whole procedure takes only a couple of minutes. In your slide I see only red cells and they do not show appreciable staining. An even quicker stain is the Diff Quik (do a google search).

Here are examples of Wright stained blood:
https://www.google.com/search?q=wright+ ... e&ie=UTF-8

Bill