RE: [Histonet] Malarial smear

From:"Barnhart, Tammy"

We do a Wright's stain.

Tammy Barnhart, BS, HTL(ASCP)
Anatomic Pathology Manager
St. Alexius Medical Center
Bismarck, ND



Wright/Tzanks stain is a polychromatic stain.  The dyes present in the stain
produce multiple colors when applied to cells.  Wright stain is a mixture of
Methylene Blue, Azure B (obtained from oxidation of the Methylene Blue), and
Eosin Y dissolved in methanol.  
The quantities of the dyes used in preparing the Wright stain powder must be
carefully controlled to yield a neutral compound dye and optimum staining
results.  During the staining process, when the buffer solution is added to
the stain, ionization occurs, during which time the process of staining the
cells takes place.  The Eosin ions are negatively charged and stain the
basic component of the cells an orange to pink color.  The acid structures
of the cell are stained varying shades of blue to purple by the positively
charged Azure B.  Neutrophil granules are probably stained by the Azure
compounds.  There is a wide variability in the staining characteristics of
these stains from one lot to the next, which is most likely due to
contaminants present in the dyes.                            


Smears prepared on standard high quality glass slides.  Allow the smear to
air dry.


Internal patient control is used.

Wright's Stain
1.	500 ml of 100% Methanol is heated to 50C in a water bath.
2.	1.5 gm of Wright's stain powder is added to the methanol and mixed
3.	The solution is placed in the oven at 37C overnight.
4.	Filtered and placed in dark bottle
5.	Keep at room temperature.

Distilled Water


1.  Place air-dried smears with the smear side up on a stain rack.

2.  Flood slides with Wright stain.    
     Set timer for 2 minute.

3.  Buffer the slides with a small volume of distilled water 
     Mix the two reagents on the slide by gently blowing back and forth over
the solution.  A 
     metallic sheen should form on top of mixture.  Set timer for 7 minutes
for peripheral 
     smear and 10 minutes for Bone Marrow, FNA and Lymph Node imprint

4.  Rinse the slides thoroughly with a stream of distilled water.

5.  Wipe off the back of slide and stand on end in a rack to dry.
6.  Dip in xylene and coverslip.


A well-stained smear will show pink to orange red blood cells, dark purple
nuclei in lymphocytes and neutrophils, a lighter purple nucleus in
monocytes, bright orange granules in eosinophils, dark blue black granules
in basophils, and violet to purple 
platelet granules.  The cytoplasm of monocytes is a gray blue with fine
reddish granules.  The neutrophil has a light pink cytoplasm with lilac
granules, and the lymphocyte shows varying shades of blue cytoplasm.  
Many factors can alter staining.  During staining the buffer controls the pH
of the stain.  If the pH is too acid, those cells or cell parts taking up an
acid dye will stain pinker and the acid components that stain with the basic
dye show very pale staining.  If 
the stain-buffer mixture is too alkaline, the red blood cells will appear
grayish-blue and the white cell nuclei will stain very deeply purple.
Therefore to stain all cells well the pH of the buffer is critical.  The
staining rack must be level to guard against uneven staining of the smear.
Insufficient washing of the smears when removing the stain and buffer
mixture may cause precipitate on the dried smear.  Leaving water on the
smear after rinsing or prolonged rinsing causes the stain to fade.


Hematology:  Principles and Procedures, Barbara A. Brown, Sixth Edition,
pp. 100 - 102

-----Original Message-----
[]On Behalf Of Michelle
D. Moore
Sent: Thursday, December 28, 2006 9:45 AM
To: Histonet
Subject: [Histonet] Malarial smear

Good morning!
    I have a question I was requested to do a stain for malaria on a blood
smear. I have looked through my literature, I have many books, and I cannot
find a stain for a smear only for tissue. Could someone direct me to a
source or e-mail me a procedure? I would be greatly appreciate it, I am on
my last leg. Thank you in advance for your responses. Have a good day and a
better tomorrow :) 

Michelle D. Moore HT (ASCP)
785 Russell St
Craig, CO 81625
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