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<DIV><FONT face=Arial size=2>Alcian yellow is not being made anymore,
anywhere.</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>ANATECH, Ltd. has a substitute, called HP Yellow,
if I remember correctly. The usual TB/AY staining procedure has to be modified
slightly, in order for this yellow dye to bind, but according to the procedure I
read, it's just one extra solution. 1-800-ANATECH. (No, I don't work for
them. Just passing the information along.)</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>We don't do the Tol. Bl/Al Y stain. We do the
Diff-Qwik - the same one that is done in cytology. It's fast, reusable (we use
it about 1 week, then throw it out, but then we're staining 20-30
slides/day).</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>The directions say to run down to abs. reagent
alcohol, then go into the DQ fixative (methanol). If you run your slides down to
water, it's OK. Just put them directly into the DQ fix before staining them. I
have tried to go from abs. directly into the stain, skipping the methanol. (I
figured, alcohol is alcohol.) Well, it doesn't stain right if you skip the
methanol fixative. Don't know why. Just thought I'd pass this on.</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>Our procedure is below.</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>Peggy A. Wenk, HTL(ASCP)SLS</FONT></DIV>
<DIV><FONT face=Arial size=2>William Beaumont Hospital</FONT></DIV>
<DIV><FONT face=Arial size=2>Royal Oak, MI</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><B><U><FONT face="Times New Roman" size=5>
<P align=justify>MICRO-ORGANISM - DIFF-QUIK </P></B></U></FONT><FONT
face="Times New Roman" size=3>
<P align=justify></P>
<P align=justify>PREPARED BY: Peggy A. Wenk, BS, HTL(ASCP)SLS</P>
<P align=justify></P>
<P align=justify>ADOPTED BY: _______________________________ DATE:
<U></P></U></FONT>
<TABLE borderColor=#000000 cellSpacing=1 cellPadding=8 width=624 border=1>
<TBODY>
<TR>
<TD width="15%"><FONT face="Times New Roman"></FONT><FONT
face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P>DATE</FONT></P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P>INITIAL</FONT></P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P>DATE</FONT></P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P>INITIAL</FONT></P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P>DATE</FONT></P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P>INITIAL</FONT></P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P>DATE</FONT></P></TD>
<TD width="14%"><FONT face="Times New Roman" size=2>
<P>INITIAL</FONT></P></TD></TR>
<TR>
<TD width="15%"><FONT face="Times New Roman" size=2>
<P>REVIEWED</FONT></P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="14%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD></TR>
<TR>
<TD width="15%"><FONT face="Times New Roman" size=2>
<P>REVIEWED</FONT></P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="14%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD></TR>
<TR>
<TD width="15%"><FONT face="Times New Roman" size=2>
<P>REVIEWED</FONT></P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="14%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD></TR>
<TR>
<TD width="15%"><FONT face="Times New Roman" size=2>
<P>REVIEWED</FONT></P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="12%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="9%" bgColor=#ffffff><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD>
<TD width="14%"><FONT face="Times New Roman" size=2>
<P><FONT face=Arial></FONT></FONT> </P></TD></TR></TBODY></TABLE><FONT
face="Times New Roman">
<P align=justify><STRONG>SUPERSEDES:</STRONG></P>
<P align=justify></P></FONT><B><U><FONT face="Times New Roman" size=4>
<P align=justify>PURPOSE</B></U>:</P></FONT><FONT face="Times New Roman" size=3>
<P align=justify>This stain demonstrates <I>Helicobacter pylori</I>, which is
present in many gastric biopsy specimens with active gastritis. It can also be
used to non-differentially stain bacteria and for the demonstration of some
parasites.</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>PRINCIPLE</U></STRONG>:</P></FONT><FONT
face="Times New Roman" size=3>
<P align=justify>Diff-Quik is a modified Giemsa/ Romanowsky stain. Diff-Quik
solution I contains eosin, which gives a pink color to cytoplasm. Diff-Quik
solution II contains Azure A and Methylene Blue. The methylene blue is an impure
dye, and will oxidize into azure A, azure B, and methylene violet, thereby
giving a wider range of colors. Both solutions contain buffers, which establish
the best pH for staining.</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>FIXATION</U></STRONG>:</P></FONT><FONT
face="Times New Roman" size=3>
<P align=justify>Formalin fixed tissues preferred.</P>
<P align=justify>Hollandes fixed tissues may stain false negative, due to the
Hollandes solution creating holes in the walls of the bacteria. Steiner and
Steiner is recommended for Hollandes-fixed tissues.</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>TECHNIQUE</U></STRONG>:</P></FONT><FONT
face="Times New Roman" size=3>
<P align=justify>Cut routine paraffin sections at 5 um.</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>CONTROL</U></STRONG>:</P></FONT><FONT
face="Times New Roman" size=3>
<P align=justify>Gastric tissue with <I>H. pylori</I> preferred.</P>
<P align=justify>Any tissue with small, gram negative bacteria may also be used,
such as <I>E. coli</I>.</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>QUALITY CONTROL</U></STRONG>:</P>
<P align=justify></FONT><FONT face="Times New Roman" size=3>1. Do not use this
stain with Hollandes fixed tissues. Steiner and Steiner will demonstrate
these.</P>
<P align=justify>2. Use a microscope to determine the assess quality of
staining.</P></FONT><FONT face="Times New Roman" size=4>
<P align=justify></P>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><U><STRONG>EQUIPMENT</STRONG>:</U></P></FONT><FONT
face="Times New Roman" size=3>
<P align=justify>Erlenmeyer flask, graduated cylinders, forceps.</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>CAUTION</U></STRONG>:</FONT><FONT
face="Times New Roman" size=3> <I>FOLLOW STANDARD SAFETY PROCEDURES WHEN
PREPARING STAINS</I>.</P><B>
<P align=justify>DIFF-QUIK Fixative</B> contains methanol which is poisonous.
May be fatal or cause blindness</P>
<P align=justify>if swallowed. Cannot be made nonpoisonous. Liquid and vapor are
flammable. Harmful if </P>
<P align=justify>inhaled or absorbed through skin. May cause skin and eye
irritation.</P>
<P align=justify></P><B>
<P align=justify>DIFF-QUIK Solution I </B>- Contains Eosin, Buffer and
0.001% sodium azide.</P>
<P align=justify></P><B>
<P align=justify>DIFF-QUIK Solution II </B>- Contains Azure A, Methylene
Blue, and a buffer.</P><B>
<P align=justify></P>
<P align=justify>ACETIC ACID</B> is an acid. Add drop by drop to solution. May
cause skin/eye burns.</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>REAGENTS</U></STRONG>:</P></FONT><B><FONT
face="Times New Roman" size=3>
<P align=justify>DIFF-QUIK FIXATIVE</P></B>
<P align=justify></P><B>
<P align=justify>DIFF-QUIK SOLUTION I</B> </P>
<P align=justify></P><B>
<P align=justify>DIFF-QUIK SOLUTION II</B> (Baxter catalog #B4132-12)</P>
<P align=justify></P><B>
<P align=justify>ACID-ALCOHOL SOLUTION</P></B>
<P align=justify>95% alcohol 368.0 mL</P>
<P align=justify>Distilled water 132.0 mL</P>
<P align=justify>Acetic acid (HCOOH) 1.25 mL</P>
<DIR>
<DIR>
<P align=justify>Mix together alcohol and water. Slowly add, drop by drop,
acetic acid to solution. Store at room temperature in a plastic or glass
container. Good for several months.</P>
<P align=justify></P></DIR></DIR></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>PROCEDURE - Diff-Quik</U></STRONG>:</P>
<P align=justify>1. </FONT><FONT face="Times New Roman" size=3>Deparaffinize and
place in several changes of absolute alcohol. </P>
<DIR>
<DIR>
<P align=justify></P>
<P align=justify>NOTE: If possible, proceed to Step 2 after the absolute
alcohol. However, if slides have already been run in water, go to Step 2 and
extend the time in Step 2 to 2 minutes.</P>
<P align=justify></P>
<P align=justify>NOTE: If a frozen section or cytology specimen, follow
directions under "PROCEDURAL NOTES."</P>
<P align=justify>2. Place slides directly into Diff-Quik Fixative Solution 1
minute</P>
<P align=justify></P>
<P align=justify>3. Place slides directly into Diff-Quik Solution I 2
minutes</P>
<P align=justify></P></DIR></DIR>
<P align=justify>4. Place slides directly into Diff-Quik Solution II 4
minutes</P>
<P align=justify></P>
<P align=justify>5. Rinse quickly in 2-3 changes of distilled water 1-2 seconds
each</P>
<P align=justify></P>
<P align=justify>6. Pour on, pour off quickly Acid-Alcohol Solution 1-2
seconds</P>
<P align=justify></P>
<P align=justify>7. Rinse in distilled water, 2-4 changes 5 seconds each</P>
<P align=justify></P>
<P align=justify>8. Allow slides to air dry completely 2 minutes or longer</P>
<P align=justify></P>
<P align=justify>9. Coverslip with synthetic mounting media</P></FONT><FONT
face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>RESULTS</U></STRONG>:</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=3>
<P align=justify>Bacteria, including <I>Helicobacter pylori</I> deep-blue</P>
<P align=justify>Fungus deep-blue</P>
<P align=justify>Nuclei blue</P>
<P align=justify>Cytoplasm, collagen, muscle, cytoplasm varying shades blue and
pink</P>
<P align=justify></P>
<P align=justify><FONT face=Arial size=2></FONT> </P></FONT><B><U><FONT
face="Times New Roman" size=4>
<P align=justify>PROCEDURAL NOTES</B></U>:</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=3>
<P align=justify>1. Check slides after water rinse (Step #6). Slides may be
restained, if necessary.</P>
<P align=justify>2. If tissues have been fixed in Hollandes, use the Steiner and
Steiner procedure, as the bacteria may be false negative is stained with the
Diff-Quick.</P>
<P align=justify>3. Slides may be left in Diff-Quik Fixative solution (blue)
longer than 1 minute, without harm to the tissue.</P>
<P align=justify></P>
<P align=justify>4. IF SPECIMEN IS A FROZEN SECTION: Use the following
procedure:</P>
<P align=justify> Do <I>NOT</I> deparaffinize and
dehydrate (Step #1)</P>
<P align=justify> Place in Diff-Quik Fixative solution
(blue) 1-5 minutes</P>
<P align=justify> Go directly to Step 2.</P>
<P align=justify></P>
<P align=justify>5. IF SPECIMEN IS A CYTOLOGY SPECIMEN: Use the following
procedure:</P>
<P align=justify> Deparaffinize and hydrate through
absolute alcohol.</P>
<P align=justify> Place in Diff-Quik Fixative solution
(blue) 1 minute</P>
<P align=justify> Go directly to Step 2.</P>
<P align=justify>6. If specimen must go through the Diff-Quik Fixative solution,
tissue may be left in solution longer than 1 minute, without harm to the
tissue.</P>
<P align=justify></P></FONT><FONT face="Times New Roman" size=4>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><STRONG><U>REFERENCES</U></STRONG>:</P></FONT><FONT
face="Times New Roman" size=3>
<P align=justify></P>
<P align=justify>Manufacturer's notes that come with Diff-Quik Solutions</P>
<P align=justify></P>
<P align=justify>Ray Skipper and Don B. DeStephano: A Rapid Stain for
<I>Campylbacter pylori</I> in gastrointestinal sections using Diff-Quik. J.
Histotechnol 12:303-304, Dec. 1989</P>
<P align=justify><FONT face=Arial size=2></FONT> </P>
<P align=justify><FONT face=Arial size=2></FONT> </P></FONT></DIV>
<BLOCKQUOTE
style="PADDING-RIGHT: 0px; PADDING-LEFT: 5px; MARGIN-LEFT: 5px; BORDER-LEFT: #000000 2px solid; MARGIN-RIGHT: 0px">
<DIV style="FONT: 10pt arial">----- Original Message ----- </DIV>
<DIV
style="BACKGROUND: #e4e4e4; FONT: 10pt arial; font-color: black"><B>From:</B>
<A title=jcox90@yahoo.com href="mailto:jcox90@yahoo.com">Jill Cox</A> </DIV>
<DIV style="FONT: 10pt arial"><B>To:</B> <A
title=histonet@lists.utsouthwestern.edu
href="mailto:histonet@lists.utsouthwestern.edu">histonet@lists.utsouthwestern.edu</A>
</DIV>
<DIV style="FONT: 10pt arial"><B>Sent:</B> Wednesday, December 03, 2003 11:58
AM</DIV>
<DIV style="FONT: 10pt arial"><B>Subject:</B> [Histonet] Stain for
H.Pylori</DIV>
<DIV><BR></DIV>
<DIV>Hello everyone,</DIV>
<DIV> Has anyone done a Toluidine blue/Alcian yellow stain for H. Pylori?
If so how do the Doctors like it? Or, what other stains are you
doing for that. I am trying to get away from the Genta!! Thanks in
advance</DIV><BR><BR>
<DIV>
<DIV>Jill Cox HT (ASCP) </DIV>
<DIV>Histology Supervisor<BR>Seattle Histology Lab</DIV></DIV>
<P>
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