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Date:Mon, 13 Sep 1999 11:19:52 -0400
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Date: 12 Sep 1999 01:34:28 -0500
From: "J. A. Kiernan" <> 
Subject: Re: new microtomes
On Fri, 10 Sep 1999, Donna Sitrin wrote:
> Could I get a little feedback on any new microtome purchases?  I'm looking 
> for good quality machines with reasonable service contracts.
  The prices of new microtomes are abominably high.
  I nearly typed "absurdly high," but this is a simple 
  instrument that is needed everywhere in the world.
  Older microtomes, if they have been carefully maintained 
  (meaning occasional cleaning, oiling, a spot of grease in the 
  right place, and keeping and reading the maker's manual) are 
  almost immortal, so the market for new ones cannot be very 
  large. This may be why they are so expensive.
  You could save thousands of dollars/pounds/etc by buying
  a second-hand microtome. Universities often sell off their 
  former capital assets very cheaply.
 John A. Kiernan,
 Department of Anatomy & Cell Biology, 
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1
Date: 12 Sep 1999 12:00:59 -0500
From: "Mitchell, Jeannette M." <> 
> -----Original Message-----
> From: Mitchell, Jeannette M. [] 
> Sent: Saturday, September 11, 1999 12:52 PM
> To:   '' 
> Subject:      ippexes
> Dear fellow histotechs,
> Our lab is having problems with mostly cytolgy ipexes.  The negative and 
> positive patient control is staining in the lymphocytes. The tumor cells 
> are staining.  There is also nonspecific staining in the tumor. On the
> negative surgical tissues we are also seeing a blush and staining at the 
> edges of the tissues.
>    We are currently using the Dako lsab+ kit,  we are blocking with
> methanol/hydrogen perxiode and using avidin/biotin  on these thin preps. 
> I discovered an insert in the Dako kit that mentions that there has been 
> minor modifications in the link component and that it would affect only 
> goat primary antibodies.  Has anyone experienced any of the above
> problems?? Please if you let me know.  I welcome any theories one may
> have.  We have been doing ipexes on the dako machine for quite a while and 
> have had no problems.
> Jeannette
Date: 12 Sep 1999 13:01:03 -0500
From: linda coscarelli <> 
Subject: incubators
We are going to be starting some manuel immunoperoxidase 
staining in the near future.  We are now in the process 
of looking at equipment for these procedures.  Can someone 
tell me what they are using for incubation of manuel staining.
Bixby Medical Center
Date: 12 Sep 1999 17:47:09 -0500
Subject: Twort's gram stain
Would anyone have a procedure for Twort's gram stain?
Date: 12 Sep 1999 19:45:33 -0500
From: "Hagerty, Marjorie A." <> 
Subject: RE: Twort's gram stain
This is our procedure:
Marg Hagerty
Supervisor, Anatomic Pathology
Eisenhower Medical Center
39-000 Bob Hope Drive
Rancho Mirage, CA 92270
PURPOSE:  To differentiate as well as demonstrate gram positive and gram 
negative bacteria in tissue and in smears.
PRINCIPLE:  Variants of the Gram stain are useful in the determination of 
whether an abscess or necrosis is bacterial in origin.  Gram positive fungal 
filaments of Nocardia and Actinomyces may also be shown.  This procedure 
involves the application of a crystal violet solution, followed by an iodine 
mordant to form a dye lake.  Both gram-positive and gram-negative organisms 
are colored blue-black after these 2 steps. Decolorization is the third major 
step, and its purpose is to render the gram-negative ones colorless while 
leaving the blue-black dye lake in the positive ones.  The decolorizer 
penetrates the entire cell, and the step is a relative rather than an 
absolute one.  If sections are exposed too long to the action of the 
decolorizing agent, even gram-positive cells will lose the dye lake and 
become colorless.  The final major step is counterstaining; Neutral Red and 
Fast Green are used for this purpose. Gram negative bacteria stain red and 
the background is green.
APPLICATIONS: This procedure will stain gram positive and negative bacteria 
without the toxic and dangerous chemicals picric acid and ether. 
SPECIMEN REQUIRED:  Formalin-fixed paraffin sections cut at 5 to 6 microns 
or smears.
1.      Crystal Violet Oxalate, working solution
        Newcomer Supply (cat#10422)
2.      Gram's Iodine (Made at EMC - use same as used in de-B5 set up)
3.      Acetone (Baxter)
4.      Twort Stain Set, Newcomer Supply, (cat# 14034)
        a.      Solution A, Neutral Red
        b.      Solution B, Fast Green
                Newcomer Supply, (cat# 14034)
QUALITY CONTROL:   Tissue containing both gram-positive and gram-negative 
1.      Deparaffinize and hydrate to distilled water.
2.      Using dropper technique, place Crystal Violet-Oxalate on slides for 
30 seconds.   
3.      Rinse quickly in distilled water.
4.      Treat with Gram's Iodine for 20 seconds.
5.      Rinse quickly is distilled water.
6.      Decolorize by gently dipping in acetone in a coplin jar until no 
more purple color comes off. (2 quick   dips)
7.      Rinse quickly in distilled water.
8.      Twort Stain for 2 minutes. Prepare immediately before use. Combined 
stain must be used within       10 minutes of preparation. Combine in a  50 
mL coplin jar:
        a.      9 mL of solution A, Neutral Red
        b.      1 mL of Solution B, 1.0% Fast Green 
        c.      30 mL distilled water
9.      Rinse quickly in distilled water, shake excess off and carefully 
blot dry on paper towels.
10.     Agitate quickly in coplin jar of clean acetone to dehydrate.
11.     Two changes of xylene and mount.
Gram positive bacteria                      dark blue Gram negative 
bacteria                                  red Filaments of 
Norcardia and Actinomyces    blue
Nuclei                                                          red 
Background                                              light green
RBCs                                                             green to 
greenish blue
1.      Crystal Violet Oxalate solution is stable and may last up to two 
years, requring only occasional         filtering.      
2.      Gram positive organisms seem to stain exceptionally dark, making it 
harder to overdifferentiate with        acetone.
3.      Do not reuse Twort stain once it is mixed. The green loses its 
effectiveness within a half hour.
4.      Do not place slides in alcohol after Twort stain. The neutral red 
will be removed.        
1)      Bancroft and Stevens, "Theory and Practice of Histologic Technique", 
Third Ed., St. Louis,   980, The        C.V. Mosby & Company.
        With modifications developed at VAMC, Madison WI.
- -----Original Message-----
From: gary powers [] 
Sent: Sunday, September 12, 1999 3:43 PM
Subject: Twort's gram stain
Would anyone have a procedure for Twort's gram stain?
Date: 12 Sep 1999 21:00:26 -0500
From: Victoria Baker <> 
Subject: Re: Twort's gram stain
Ollett's Modification of Twort's Stain 
pgs. 393-5, Culling, 3rd edition
Gram-negative Bacteria
"Gram-negative bacteria may usually be demonstrated by staining 
sections with Leishman or Geimsa stain in addition to "control" 
sections stained by Gram's stain. Organisms present in the Leishman 
slide which are not Gram-positive are assumed to be Gram-negative. In 
practice this method works well.
There are a number of other methods described for the differentiation 
of Gram-positive bacteria, but of these only the following has given 
consistent results.
Ollett's Modification of Twort's Stain
This method (Ollett, 1951) is simple and gives reasonable contrast 
between Gram-positive bacteria, Gram-negative bacteria and tissues. The 
light green in Twort's stain is replaced by fast green F.C.F to avoid 
Special Reagent Required
Modified Twort's stain
0.2 per cent alcoholic neutral red (C.I. No. 825).....90ml 
0.2 per cent alcoholic fast green F.C.F.......10ml
For use, dilute one volume of the above stock solution with three 
volumes of distilled water.
The pH of this stain, like that of Twort's stain, is 4.9; it may depend 
on the dye samples used, but uniformity can be secured by diluting the 
stock solution in M/5 acetate buffer instead of distilled water.
The proportions of the dyes given are only approximate, and the optimum 
formula will depend on the dye content of the samples used.  The stock 
alcoholic solution should be of reddish-magenta tint; too much green 
will weaken the red bacterial staining; the total dye concentration is 
less critical.
1.  Fix material in 5 per cent formol saline, pass through the alcohols 
and embed in paraffin.
2.  Cut sections at 3 microns in thickness. 
3.  Bring sections to distilled water.
4.  Stain in aniline crystal violet for 3-5 minutes.
5.  Pour off stain and wash quickly in distilled water. 
6.  Treat with Gram's iodine for 3 minutes
7.  Pour off the iodine, wash quickly in distilled water and blot dry. 
8.  Decolorize with 2 per cent acetic acid in absolute alcohol until no 
more colour comes away - the section should be a dirty straw colour at 
this stage.
9.  Wash quickly in distilled water.
10. Counterstain in the modified Twort's neutral red-fast green stain, 
diluted 1 part with 3 parts of distilled water, or pH 4.9 buffer, for 5 
11. Wash quickly in distilled water.
12. Differentiate with 2 per cent acetic acid alcohol until no more red 
stain (neutral red) comes away (15-30 seconds).
13. Clear in xylol and mount in D.P.X. or H.S.R
Cytoplasm......................................Light green 
Red blood corpuscles...................Green
Gram-positive bacteria..................Dark blue 
Gram-negative bacteria.................Pink"
Sorry to say, I'm not all that familiar with this stain.  When this 
stain was developed Formol saline was considered a common fixative, 
it's still used but only in specialized procedures.  Perhaps someone 
else in this server might be able to give you better information.  If 
you're working in a clinical lab, you may not have a lot of choice with 
fixation and processing.  I don't know of a more update version on this 
procedure.  The pH most likely plays a major part in this procedure 
given the results.  
Best of luck.
- --- gary powers  wrote:
> Would anyone have a procedure for Twort's gram 
> stain?
> Thanks,
> Marian
Do You Yahoo!?
Bid and sell for free at
Date: 12 Sep 1999 22:15:41 -0500
From: "J. A. Kiernan" <> 
Subject: Re: Twort's gram stain (Ollett's variant etc)
On Sun, 12 Sep 1999, gary powers wrote:
> Would anyone have a procedure for Twort's gram stain? 
  Twort's mixture is actually the counterstain, for showing 
  Gram-negative organisms (and also the tissue if you do it 
  on sections). It's quite a nice general-purpose stain if 
  you like red nuclei and green collagen.
Twort's stain was originally made by mixing saturated aqueous solutions 
of neutral red and light green, collecting the resulting precipitate, 
dissolving it in 80% alcohol to make a stock solution, and diluting 
this before use with an equal volume of water. A modification by Ollett 
(1951) is easier to make, and light green is replaced by fast green 
FCF, which is less prone to fading. The working solution contains red 
cations and green anions, which impart their colours simultaneously to 
different components of the tissue. Usually the neutral red staining is 
too strong, so it is differentiated in acidified alcohol.
This method was originally devised as a counterstain for Gram-positive 
bacteria that had been selectively stained with crystal violet. The 
nucleic acids in Gram-negative bacteria and in nuclei of eukaryotic 
cells take up the neutral red cations, and most of the background of 
cytoplasm and collagen is green.
Solutions required.
  Ollett's modified Twort stain
   Stock solution
    Neutral red (C.I. 50040):  0.36 g
    Dissolve in 180 ml of 95% alcohol.
    Fast green FCF (C.I. 42053): 0.04 g
    Dissolve in 20 ml of 95% alcohol.
    Mix these two solutions and store in a screw-capped bottle. 
    It is stable for at least a year.
  Working solution
    To one volume of stock solution, add three volumes of either 
    distilled water or 0.2 M acetate buffer, pH 4.9. This mixture is 
    used only on the day it is made.
  2% acetic acid-alcohol
    Absolute ethanol:     200 ml
    Glacial acetic acid:    4 ml
    Usually this is made up as needed, but it can be stored for several 
    weeks without deterioration. It is used only once.
  1. Dewax and hydrate paraffin sections. Remove mercury deposits if 
  2. Immerse in the working solution of Twort's stain for 5-10 minutes. 
  3. Wash quickly in distilled water.
  4. Immerse slides in 2% acetic acid-alcohol for about 15 seconds,
     with agitation.
  5. Complete the dehydration in two changes of 100% alcohol.
  6. Clear in two changes of xylene and examine. If there is too much
     red in the sections, repeat Step 4.
  7. Apply coverslips, using a resinous mounting medium.
 Nuclei, mast cell granules and sites of high RNA concentration are 
 red. Cytoplasm and collagen are green. Purple colours are seen in some 
 sites, such as the matrix of cartilage and bone, that take up both 
 dyes. The staining is influenced by the fixative and the thickness of 
 the sections. Don't expect too much of a procedure that allows little 
 visual control of the staining achieved by simultaneously applied dye 
  In a variant of this method (Monroe & Frommer, 1967) the sections are 
  placed in 1% phosphotungstic acid (PTA) for 2-3 minutes and rinsed in 
  water before staining, and the acetic acid-alcohol at Step 4 is 
  replaced by 2-3 minutes in 70% alcohol. The PTA treatment introduces 
  large negatively charged ions into collagen and most cytoplasms, so 
  that these, like nuclei, are stained in shades of red and pink, 
  whereas striated muscle, erythrocytes and keratin are green.
Monroe, C.W. & Frommer, J. (1967). Neutral red-fast green FCF, a
  single stain for mammalian tissues. Stain Technol. 42: 262-264. 
Ollett, W.S. (1951). Further observations on the Gram-Twort stain.
  J. Path. Bact. 63: 166.
 John A. Kiernan,
 Department of Anatomy & Cell Biology, 
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1
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