Machiavello stain revisited

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From:"Renee Escalona" <>
To:"Histonet" <>
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For those of you who missed out on the Machiavello stain responses, here
they are.  I think the problem of things not making it out on the listserve
is people forgetting to carbon copy to Histonet.  I know because I have
caught myself on occassion.  But those of you who expressed concern are
right.  We need to make sure that EVERYBODY has access to the information
that is flying back and forth.

Renee Escalona, BS, HTL(ASCP)
Veterinary Laboratory Technologist
TX Vet. Med. Diag. Lab
College Station, TX

We use Pinkerton's Adaption of Machiavello for paraffin blocks.  I will
attach our procedure for you.  If you have any problems with the attachment
let me know and I will send it differently.
Roberta Horner HT
Animal Diagnostic Lab
Penn State University


Purpose: To demonstrate rickettsia on tissue sections

Fixation: Any well fixed tissues may be used.

Microtomy: Cut paraffin sections at 4 to 5 microns.

Quality Control: Control material consists of a slide made from tissue known
to contain  rickettsia.  One known positive slide is run with each set of
patient unknowns.  If the control slide results are negative, the stain will
be repeated.

Equipment and supplies: graduated cylinders, flasks, pipettes, balance,
coplin jars, filter paper, funnels.

Health and safety precautions: see MSDS manual


1% Methylene Blue
 1.0 gm Methylene Blue (C.I.52015)
 100 ml Distilled water (H2O)
shelf life: 3 months
storage: room temperature

0.25% Basic Fuchsin
 0.25 gm Basic Fuchsin
 100 ml Distilled water (H2O)
shelf life: 1 month
storage: room temperature

0.5% Citric Acid Solution
 0.5 gm Citric Acid (H3C6H5O7)
 95 ml Distilled water (H2O)
shelf life: 2 months
storage: room temperature

1. Deparaffinize and hydrate to distilled water.

2. Stain in 1% methylene blue solution overnight.

3. Decolorize in 95% alcohol.

4. Wash quickly in distilled water.
 5. Counterstain in 0.25% basic fuchsin solution for 30 minutes.

6. Decolorize rapidly in 0.5% citric acid solution for 1 or 2 seconds, never
more than 3 seconds.

7. Differentiate rapidly in absolute alcohol.

8. Dehydrate in 95% alcohol and absolute alcohol 3 changes each.  Clear in
xylene, 3 changes.

9. Mount with Permount.

  Rickettsia bright red
  Nuclei blue


Luna, Lee G.:  Histopathologic Methods and Color Atlas of Special Stains and
Tissue Artifacts ; American Histolabs, Inc., Publications Division,
Gaithersburg, MD, pg. 236.

> >
> > We do a modified Geminez (sp?) for rickettsia. I am at home right now,
> > but will look up the procedure and fax it to you if you would like. You
> > can call me at 309-344-2451 and give me your fax number. We regularly
> > stain for chlamydia with this stain.

Rae Ann Staskiewicz
> > Galesburg Animal Disease Lab
> > Galesburg, IL

Years ago we did the Machiavello to demonstrate Chlamydia in paraffin
tissue. We changed to a stain called Gimenez- now IHC has replaced
both Machiavello and Gimenez in our lab.
                Thank you
 Kathy Oprea
 University of Georgia
 College of Veterinary Medicine
 Athens, GA 30602-7388

Dear Renee

We use the macchiavello to stain rickettsiae and chlamydia.  It works well
for us.  It's a little long but it can be shortened and performed in the
microwave. Our reference is from the big Selected histochemical and
Histopathological Methods by Samuel Wesley Thompson's book (pp 998-1000). We
also do veterinary tissues.  Good Luck!

Barbara Stancel, HTL (ASCP)
Lead Technician, Pathology Section
USDA, FSIS, Eastern Laboratory
RRC, 950 College Station Road
Athens, Georgia 30605

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