I have tried exactly the same protocol as mentioned in the paper given below:
http://www.nature.com/nprot/journal/v4/n12/abs/nprot.2009.198.html
"Synchronous culture of Plasmodium falciparum at high parasitemia levels"
Here is the protocol:-
Wright’s azur eosin methylene blue staining ● tIMInG15 min
(i) Completely cover the smear with Wright’s solution for 4 min.
! cautIonDo not exceed this time. This incubation time should be adjusted according to the weather; e.g., when working in temperate climates such as an environmental temperature of 36 °C at low humidity, 1 min is sufficient.
(ii) Add distilled or PBS (pH 7.2) until the slide is immersed.
(iii) Incubate for 9 min.
(iv) Clean both sides of the slide with water. A tissue paper soaked in methanol can be used to remove excess stain from the underside.
(v) Leave to dry before microscopic investigation. To assess the parasitemia level and growth stage, 1,000–1,200 cells are counted using PlasmoScore 1.3 software 46 in different image fields. Parasites are observed on a red cell background. Viability is confirmed by visualization of the characteristic P. falciparummorphology along its intraerythrocytic cycle,
which is shown in Box 2. The invasion window can also be estimated from the smears as described in Box 2.
I am able to observe the Pink colour RBC with blue condensed particles. There is no proper light blue colour of Plasmodium falciparum's cytoplasm.
Changing the incubation time in the Wright's stain didn't help.
Also, what is the role of incubation in distill /PBS for 9min in this technique?
I will be very thankful for your valuable suggestions. :)
Please list up the staining protocol for those, who have no access to nature.
In general. The Wright or Giemsa-stains can be influenced by the pH of the rinsing solution after the incubation in the dye-solution. The lower the pH the more pink will be the result. The higher the pH the more blue will be the result. Usually Giemsa-stains are differentiated at an approximately neutral pH. (Weise buffer pH 7,2 or acidified water pH 6,5). To lower the pH you can add few drops of acetic acid. This will also enhance the speed of differentiation.
Giemsa-stains are also sensitive for too long rinsing in diluted ethanols. The colour turns blue and more faint.
Thanks Gudrun Lang .
I have updated the protocol.
Pink colouration of RBC is clear in my slides but there is no differentiation between parasite cytoplasm (supposed to be blue colour) and RBC(pink colour). Parasites appearing as condensed dark particles which may be nucleus of parasite but proper blue cytoplasm is not there.
i have not used wright’s azur eosin methylene blue stain before. May be you should try Giemsa stain, with that you will be able to differentiate the colour of the cytoplasm from the RBC.
Try regular Giemsa technique, if you have any adequate Giemsa stock solution.
This is how I use it:
1. Fix air-dried smears for 5min in absolute methanol.
2. Drain the excess of methanol and leave the smears to dry out on air.
3. Put the smears directly into Giemsa stock solution (undiluted, just what you got in the bottle of "Giemsa stain") - keep them there for 1-3min.
4. Put the slides into buffered water or very diluted buffer of pH around 7.0 (6.8-7.2). The water level should be ABOVE the top of your glass slides.
You can "buffer" regular tap water by carefully adding drops of any acid (acetic, HCl, etc) or alkali (sodium hydroxide, or anything similar), until you reach the desired pH.
NOTE: Slides should be left in this first buffered water for 3-5min without any movement, vibrations or any mechanical disturbance! Otherwise, it might fail!
5. At the end of this period, you'll see metal-like shining squamous material at the surface of water/buffer. Use filter paper or regular nose tissue or any clean paper towel to collect this material.
6. Wash the slides in another two bath of buffered water for about 30sec with agitation, and finally in distilled water.
7. Dry the slides on air and mount them if necessary or watch them under the microscope. Do not use ethanol for dehydration!
Once the slides are completely dried on air, just put them into ethanol-free (!) new xylene for 1min and mount them using Canada balsam or DPX.
I don't want to interfere with any of the good hints Gudrun made.
Nobody was born as an experienced (self-staining) histologist / interpreter of blood smear slides... we all have to learn / are learning....
Just for eventually finding an other staing solution which perhaps would be more "efficient" or repeatable/reproducible I add here three of saved files out of my e-library collection... Enjoy or delete....(:-)) ....and yes, I haven't had the opportunity to stain anytime smears of blood containing malaria parasites....
best wishes and regards, Wolfgang
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