RE: Chlamydia

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From:Roberta Horner <rjr6@psu.edu>
To:Christine Lee <c.lee@mailbox.uq.edu.au>, histonet@pathology.swmed.edu
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This is the procedure we use for Chlamydia.
Roberta Horner HT
Animal Diagnostic Lab
Penn State University

PIERCE VANDER KAMP MODIFIED GIMENEZ STAIN (PVK)

Purpose:	To demonstrate rickettsia and Chlamydia 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  chlamydia.  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

Reagents:

Basic Fuchsin Stock
	36 ml	Distilled water (H2O)
	0.4 ml	Phenol
	0.2 gm	Basic Fuchsin
	4 ml	95% Alcohol (C2H5OH)
Store in a tightly capped glass bottle, incubate 48 hrs at 37 C.  Filter.
Usable when freshly made.
shelf life:	3 months
storage:	room temperature

Phosphate Buffer pH 7.5
	11.65 gm	Sodium Phosphate Dibasic (Na2HPO4)
	2.47 gm	Sodium Phosphate Monobasic (NaH2PO4)
	100 ml	Distilled water (H2O)

Basic Fuchsin Working
	8 ml	Basic Fuchsin Stock
	40 ml	Phosphate Buffer
Mix let stand 10 minutes, and filter
shelf life:	Make fresh

0.5% Citric Acid Solution
	0.5 gm	Citric Acid (H3C6H5O7)
	95 ml	Distilled water (H2O)
shelf life:	2 months
storage:	room temperature
Fast Green Stock
	0.2 gm	Fast Green
	100 ml	Distilled water (H2O)
	0.2 ml	Glacial Acetic Acid  (CH3COOH)
shelf life:	3 months
storage:	room temperature

Fast Green Working
	10 ml	Fast Green Stock
	40 ml	Distilled water (H2O)
shelf life:	Make fresh

Procedure:
1.	Deparaffinize and hydrate to distilled water.

2.	Stain in Working Fuchsin for 10 minutes.

3.	Rinse well in tap water.

4.	Differentiate in 0.5% Citric Acid solution until no more red runs out.

5.	Rinse well in water.

6.	Counterstain in Working Fast Green for twenty dips.

7.	Dehydrate in 95% alcohol, 100% alcohol, and clear in xylene, 2 changes
each.

8.	Mount with Permount.

Results:
		Chlamydia	bright red
		Background	green

I would be grateful if any of the Veterinary pathology labs could let me
know if you have any sucessful light microscopy stains for Chlamydia in
parrafin sections apart from fluorescent techniques. Thanks.

Christine Lee
Vet. Pathology,
University of Queensland






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