Giemsa stain of thin blood smears - pale / faint

[Garz]

I have a question about the Giemsa staining of thin blood smears

I've been doing this for a few months now mainly studying red blood cells, white blood cells, platelets etc

The main problem I'm having is I'm finding it difficult to get the stain and dark enough especially to darkly stain the red blood cells using Giemsa stain

I'm currently making my own Giemsa stain solution from Giemsa powder from a microscopy stain supplier - and using about 1 g of Giemsa stain powder in 100 mL of pure methanol heating it and agitating gentility with glass beads for around 3-4hrs at 50-60 deg C then cooling and filtering it and using that solution neat in a Koplin Jar – immersing the methanol fixed slides for around 10minutes ( longer doesn’t seem to help ) before rinsing briefly in water ( if I rinse for more than around 3 seconds I get even less contrast – but any less than this and I get lots of stain artefacts).
staining is sort of okay but I just need ideally more contrast between the red blood cells and the background.
When viewed at 1000x under oil there is just a lack of contrast between the red blood cells and the background – and similar with white blood cells – white blood cells are stained kind of okay, the nucleus is visible - but again, they could be darker so it seems to be a general issue with the stain not being dark enough so am I hope you can help with that.

Any suggestions for increasing contrast especially in the red blood cells and any contents inside them

i will attach an image as an example - (the colour balance is doing something a bit off - but its not affecting the contrast much - just making the entire image more yellow than my last camera)



Much appreciated !

[toomanyhandles]

Aside from the color which you mention is probably a white balance thing for the photo.

Here's what Merck manual shows for a RBC prep with Wright-Giemsa stain:
https://www.merckmanuals.com/profession ... lood-smear

I am not sure your photo looks that different from a contrast comparison. Magnification is different of course. How do the WBC's come out? Even normal ones should look pretty complex/ have dark staining areas. If you don't expect inclusions in the RBCs, if you just want them darker, I would think about adjusting your washes post-staining.

I myself have very specific experience using only Wright-Geimsa looking for inclusions in RBCs way back. I am sure there are folks here that do this for a living so whatever someone else says probably is better for your purposes. That said:

Examples of what I would typically expect for RBC, background staining levels, kinda blah contrast on normal RBCs, and then with inclusions on some RBC:
https://eclinpath.com/hematology/infect ... per%20cell.

but again your RBC photo looks about like would expect for a normal RBC; they are kinda uniform.

Use of RO vs. DI water for the washes makes a difference I recall (use DI perhaps), and how many washes of the slide and degree of agitation to remove the stain can impact the degree of final RBC staining, for sure if you are someplace where tap water is chlorinated I wouldn't use the tap water.
RO vs DI: https://faseb.onlinelibrary.wiley.com/d ... 4.s1.06459#

I feel like the rinse I used to do was just a couple dips, very gentle, in a staining jar filled with just the wash water (DI).
Adjusting the wash- you could get things to be relatively purple-y.
Again that was Wright-Giemsa combo.

I hope this helps~

Brian.

[Garz]

Thanks very much for the input Brian

here is a white blood cell -

in this photo image you can see the cytoplasm - but by eye that is very hard to see
the contrast on the camera is somewhat better than the human eye
its nearly invisible through the scope - and the nucleus is more cerise than purple

here is an older image with the previous camera and a more normal colour balance - its higher magnification - but the same stain



by eye my slides of red blood cells look pale purple/blueish - something a bit odd going on with the camera - but its a side issue i will work on separately or just change it - contrast is my main issue here

ref the colour reference on the Merk site - thanks for that
i'm not 100% sure - but i think there is likely a difference between Wright-Giemsa vs straight Giemsa -
as the red/purple elements in Giemsa are i think from polychromed methylene blue - vs some different red stain in wright Giemsa
so i think this means that red blood cells stain a pretty deep pink with Wright-Giemsa - vs pale bluish purple with Giemsa

i am in the UK and finding it hard to source Wright-Giemsa - or any other suitable stain - which is why i ended up making my own Giemsa from powder

i am currently rinsing for less than 3 seconds by immersing in water briefly swirling the slide and quickly removing it
i did try super quick rinsing after staining - but the issue i ran into was lots of stain artefacts remaining on the slide - and uneven staining of the smear as the last droplets of water slide off the slide, persisting in some areas more than others.

i wonder if my 100% methanol stain - is perhaps causing fainter than usual staining - vs adding water to it prior to using it ( i had used the undiluted stain as a means of trying to get it darker)

i have heard talk of steaming the slides while staining - or even using a microwave to heat them to increase the stain
anyone have any experience of this or any other pointers?

[Polymerase]

Your pH is most likely too low. Increasing alkalinity will give a more intense colour uptake.
Take a look at this brief overview:

https://www.merckmillipore.com/NO/en/iv ... 20or%207.2.

[Polymerase]

Also - how do you fix your smears before staining?

[Garz]

Your pH is most likely too low. Increasing alkalinity will give a more intense colour uptake.
Take a look at this brief overview:

https://www.merckmillipore.com/NO/en/iv ... 20or%207.2.

thanks for this input - i will definitely give that a read -

i am currently fixing with 99.95% pure methanol for around 3 minutes and drying again fully before attempting to stain

[Polymerase]

This document will provide more details for you:

https://irp.cdn-website.com/240a28f1/fi ... ns-web.pdf

See especially p. 7-8

[Garz]

Thanks for the links to the documents - i have given those a read

so the main thing seems to be to experiment with different pH levels

but in a few places i have seen kind of indirect references to the way the staining process actually works -
in that some are indicating the the water content of the stain solution is actually critical to the stain process and that much of the staining takes place only in the presence of water

in fact for some specimens people are suggesting stain immersion times of up to 24hrs - but in quite dilute stain solutions - over 90% water

so far in my attempts to get darker stain, and being surprised how little of the stain powder actually dissolves in methanol even with hrs of agitating with glass beads in a water bath, i have actually been using what is essentially neat stock stain solution ( 0% water)
( i am not sure the pH is even present there as there is no water present....)

so this would seem to be the best place for me to start - ie try 20% stock stain - and 80% water and longer immersion times