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Staining Procedures
Permanently stained smears provide laboratories with a permanent record and a specimen that can be reexamined as needed.  In addition, when organisms with unusual morphology are encountered, or when identification is difficult, the slides can be sent to a reference laboratory.  For the above reasons, the permanent stained smear is recommended for use with every stool specimen submitted for a routine parasite examination.  Below is a list of staining techniques that are being used at CDC.

Modified Acid-Fast Staining Procedure
This technique is useful for the identification of oocysts of the coccidian species (Cryptosporidium, Isospora, and Cyclospora), which may be difficult to detect with routine stains such as trichrome.  Unlike the Ziehl-Neelsen Modified Acid-Fast Stain, this stain does not require the heating of reagents for staining.

Specimen:
Concentrated sediment of fresh or formalin-preserved stool may be used.  Other types of clinical specimens such as duodenal fluid, bile, pulmonary samples (induced sputum, bronchial wash, biopsies) may also be stained.

Reagents:
There are four steps to this procedure requiring the following solutions:

1. Absolute Methanol
2. Acid Alcohol: 10 ml Sulfuric Acid + 90 ml Absolute ethanol.  Store at room temperature.
3. Kinyoun's Carbol fuchsin: may be purchased commercially.
4. 3% Malachite green: dissolve 3 g of malachite green in 100 ml of distilled water.  Store at room temperature.

Procedure:

1. Prepare a smear with 1 to 2 drops of specimen on the slide and dry on a slide warmer at 60°C until dry.  Do not make the smears too thick!
2. Fix with absolute methanol for 30 seconds.
3. Stain with Kinyoun’s carbol fuchsin for one minute.  Rinse briefly with distilled water and drain.
4. Destain with acid alcohol for 2 minutes.   Rinse with distilled water and drain.
5. Counterstain with Malachite green for 2 minutes.  Rinse briefly with distilled water and drain.
6. Dry on a slide warmer at 60°C for about 5 minutes.  Mount with a coverslip using desired mounting media.
7. Examine 200 to 300 fields using 40× or higher objectives.  To confirm internal morphology, use 100× oil immersion objective.

Quality Control:
A control slide of Cryptosporidium spp. from a 10% formalin preserved specimen should be included with each staining run.  Cryptosporidium spp. stains a pinkish-red color.  The background should stain uniformly green.

Chromotrope Staining Procedure
This staining method was developed at CDC using various components of the trichrome staining method to differentiate microsporidia spores from background fecal elements.

Specimen:
Prepare a thin smear using approximately 10 µl of 10% formalin fixed stool suspension (unconcentrated) on a glass slide.  Formalin concentrates may also be used but the number of organisms will be essentially the same as before concentration.  Heat fix on a slide warmer at 60°C until completely dry (5-10 minutes).

Reagents:
There are six steps to this procedure requiring the following solutions:

1. Absolute methanol
2. Chromotrope Stain:
   

   Chromotrope 2R	6.00 g
   Fast green	0.15 g
   Phosphotungstic acid	0.70 g
   Glacial acetic acid	3.00 ml
   Mix ingredients and allow to stand for 30 minutes.  Then add 100 ml of distilled water.  Prepare fresh for use every month.

3. Acid alcohol:
   

   90% ethanol	995.5 ml
   Glacial acetic acid	4.5 ml

4. 95% ethanol
5. 100% ethanol
6. Xylene or xylene substitute

Procedure:

1. Fix smear in absolute methanol for 5 minutes.
2. Place in chromotrope stain for 90 minutes.
3. Destain in acid alcohol for only 1 to 3 seconds.
4. Rinse in 95% ethanol by dipping.
5. Place in two changes of 100% ethanol for 3 minutes each.
6. Place in two changes of xylene or xylene substitute for 10 minutes each.
7. Drain slide and mount with coverslip using mounting media (e.g., permount).  Examine smear after drying using at least 100× objective oil immersion or higher.  Examine at least 200 to 300 oil immersion fields.

Quality Control:
A control slide of microsporidia from a 10% formalin preserved specimen should be included with each staining run.  Spore walls of microsporidia stain a pinkish-red color and measure about 1 µm.  Change all solutions subsequent to chromotrope stain after every 10 slides to obtain proper rinsing and dehydration.  A microscope with good optics is recommended for accuracy.  Use at least 100× objective oil immersion magnification to detect organisms; higher magnifications are better.  Because of the difficulty of identification of these small spores, it is recommended that a second reader confirm a positive diagnosis.

Quick-Hot Gram-Chromotrope Staining Procedure
This is an alternative stain to the chromotrope procedure that is a fast, reliable, and simple method of staining smears to demonstrate microsporidian spores in fecal and other clinical specimens.

Specimen:
Prepare a thin smear of the material to be stained (such as feces, urine, sputum, saliva, and cell culture supernatant) and air dry.

For formalin-fixed, paraffin-embedded tissue sections, deparaffinize as usual, hydrate in a series of alcohols, and bring the slides to water before performing the Gram’s stain.

Reagents:

1. Gram Stain Kit
2. Chromotrope Stain:
   

   Chromotrope 2R	1.0 g
   Fast green	0.15 g
   Phosphotungstic acid	0.25 g
   Glacial acetic acid	3.0 ml
   Mix dry ingredients then add acetic acid.  Let stand for 30 minutes and then add 100 ml distilled water.  Prepare fresh for use every month.

3. Acid alcohol:
   

   90% ethanol	995.5 ml
   Glacial acetic acid	4.5 ml

4. 95% ethanol
5. 100% ethanol

Note: In addition to the listed reagents, a method for heating reagents and maintaining a specific temperature is required.  Since only the chromotrope stain needs to be warmed, a hot plate will suffice.

Procedure:

1. Heat-fix smear (3 times for 1 second each over a low flame or 5 minutes on a slide warmer set at 60°C). Cool to room temperature.
2. Perform Gram’s stain omitting the safranin step as follows:
   

1. Flood slides into gentian violet solution and let stand for 30 seconds.  For tissue sections, extend time to 1 minute.
2. Rinse off excess stain gently with water.
3. Flood slides into Gram’s iodine solution and allow to remain on the slide for 30 seconds.  For tissue sections, extend time to 1 minute. 
4. Remove Gram’s iodine solution by gently rinsing with decolorizer solution.  Hold the slide at an angle and add the decolorizer solution dropwise until it flows off the slide colorless.  Take extra care during this step to achieve correct staining of spores.
5. Wash the slide gently with cold water to remove excess decolorizer solution.

3. Perform chromotrope stain as follows:

1. Place the slide in warmed (50° to 55°C) chromotrope stain for at least 1 minute.  For tissue sections, extend time 30 seconds.
2. Rinse in 90% acid-alcohol for 1 to 3 seconds.  Take extra care during this step to achieve correct staining of spores.
3. Rinse in 95% ethanol for 30 seconds.
4. Rinse twice, 30 seconds each time, in 100% ethanol (two separate containers are required for this step).  Let dry then mount with Cytoseal60 (Stephens Scientific) or other suitable sealer, following instructions.  For tissue sections, we recommend that the slides be washed briefly in a solution of 50% ethyl alcohol/50% xylene for 15 seconds, before mounting.

Quality Control:
A control slide of microsporidia spores from a 10% formalin-preserved specimen should be included for each staining run.

In fecal samples, microsporidia spores should appear as dark staining violet ovoid structures against a pale green background.  Yeast cells, if present, should stain either dark violet or pinkish-red and are easily differentiated from microsporidia spores.

In cytologic preparations, microsporidia spores should stain deep violet to pink violet and may contain gram-positive granules.  Occasionally, spores will demonstrate a prominent equatorial belt-like stripe.

Reference:

Moura H, Schwartz DA, Bornay-Llinares F, et al. A new and Improved “Quick-Hot Gram-Chromotrope” Technique That Differentially Stains Microsporidian Spores in Clinical Samples, Including Paraffin-Embedded Tissue Sections. Arch Pathol Lab Med 1997;121:888-893.

Modified Safranin Technique (Hot Method) Staining Procedure
For Cyclospora, Cryptosporidia, and Isospora species:
Oocysts of Cyclospora in clinical specimens are routinely demonstrated using modified acid-fast stain (cold).  However, with that technique, the oocysts stain variably from nonstaining to full staining leading to possible misidentification.  The modified safranin technique produces a more uniform staining of these oocysts.  The stain needs to be heated to boiling using either a hot plate or microwave.

Specimen:
Concentrated sediment of fresh or formalin-preserved stool may be used.  Other types of clinical specimens such as duodenal fluid may also be stained.

1. Prepare a thin smear on the slide and dry on slide warmer.
2. Fix in acid alcohol for 5 minutes.
3. Rinse with distilled water.
4. Place in boiling safranin for 1 minute.
5. Rinse with distilled water.
6. Counterstain with malachite green for 1 minute.
7. Rinse briefly with distilled water.
8. Dry the slide and mount with a coverslip using desired mounting media.

Quality Control:
A control slide of Cyclospora spp. from a 10% formalin preserved specimen should be included with each staining run.  Cyclospora spp. oocysts will stain reddish-orange.  The background should stain uniformly green.

Trichrome Staining Procedure
It is generally recognized that stained fecal films are the single most productive means of stool examination for intestinal protozoa.  The permanent stained smear facilitates detection and identification of cysts and trophozoites and affords a permanent record of the protozoa encountered.  Small protozoa, missed by wet mount examinations (of either unconcentrated or concentrated samples) are often seen on the stained smear.  The Wheatley Trichrome technique for fecal specimens is a modification of Gomori’s original staining procedure for tissue.  It is a rapid, simple procedure, which produces uniformly stained smears of the intestinal protozoa, human cells, yeast, and artifact material.

Specimen:
The specimens usually consist of fresh stool or stool fixed in polyvinyl alcohol (PVA) smeared on microscope slides and allowed to air dry or dry on a slide warmer at 60°C.  Stool preserved in sodium acetate-acetic acid-formalin (SAF) or some of the one-vial fixatives can also be used.

Reagents:
There are seven steps to this procedure, requiring the following solutions:

1. 70% Ethanol plus iodine: prepare a stock solution by adding iodine crystals to 70% alcohol until you obtain a dark solution.  To use, dilute the stock with 70% alcohol until a dark reddish brown color or strong tea color is obtained.
2. 70% Ethanol (twice)
3. Trichrome Stain: may be purchased commercially
4. 90% Acid Ethanol
   

   90% ethanol	99.5 ml
   Acetic acid (glacial)	0.5 ml

5. 95% ethanol
6. 100% ethanol (twice)
7. Xylene or xylene substitute (twice)

Procedure: 

1. For PVA smears, place the slide in 70% ethanol plus iodine for 10 minutes.  For other fixatives, follow the manufacturer's instructions.  Omit the iodine step for preservatives that do not contain mercuric chloride.
2. Place slide in 70% Ethanol for 5 minutes.
3. Place in second 70% Ethanol for 3 minutes.
4. Place in Trichrome stain for 10 minutes.
5. Destain in 90% ethanol plus acetic acid for 1 to 3 seconds.
6. Rinse several times in 100% ethanol.
7. Place in two changes of 100% ethanol for 3 minutes each.
8. Place in two changes of xylene or xylene substitute for 10 minutes.
9. Mount with coverslip using mounting medium (e.g., permount).
10. Examine the smear microscopically utilizing the 100× objective.  Examine at least 200 to 300 oil immersion fields.

Quality Control:
A control slide of a known protozoan such as Giardia spp. from a PVA preserved specimen should be included with each staining run.  When the smear is thoroughly fixed and the stain is performed correctly, the cytoplasm of protozoan trophozoites will have a blue green color sometimes with a tinge of purple.  Cysts tend to be slightly more purple.  Nuclei and inclusions (chromatoid bodies, red blood cells, bacteria) and Charcot-Leyden crystals have a red color sometimes tinged with purple.  Glycogen is dissolved by the solvents and appears as a clear area in the organism.  The background material usually stains green providing a nice color contrast with the protozoa.

Calcofluor White Staining Procedure
This chemofluorescent technique is useful for the detection of microsporidia, Acanthamoeba spp., Pneumocystis jiroveci (previously Pneumocystis carinii), and Dirofilaria spp.  Chemofluorescent agents, such as Calcofluor, Fungi-Fluor or Uvitex 2B, also known as optical brightening agents, are rapid and inexpensive screening agents.  These reagents are sensitive but nonspecific since many objects and organisms other than parasites may fluoresce.  This test should be used as a quick screening tool and not for species identification.

Specimen:
Prepare a thin smear using approximately 10 µl of fresh or preserved specimens on a glass slide.  Specimens may include stool, urine, culture or other types of samples.  Heat fix on a slide warmer at 60°C until completely dry (5-10 minutes).

Reagents
There are two steps to this procedure requiring the following solutions:

1. Absolute methanol
2. 0.01% Calcofluor white reagent: Prepare a 0.01% solution in 0.1M Tris-buffered saline, pH 7.2

Procedure

1. Prepare a thin smear of fecal, culture, or other sample material.
   
2. Fix the smear in methanol for 30 seconds.
3. Stain with 0.01% calcofluor white reagent for 1 minute.
4. Rinse with distilled water and let the smear dry.
5. Mount with a #1 thickness cover slip.
6. Examine with an epifluorescence microscope equipped with a blue violet filter cube with a wavelength of 400 nm or less.
7. To screen for microsporidia, examine the smear with a 50× or 100× oil immersion objective.  The microsporidia spores will fluoresce a brilliant blue-white color on a black background.

Quality Control
A control slide of microsporidia preserved in 10% formalin prepared from culture or from a stool specimen should be included with each staining run.

For additional information on staining stool specimens, call the Division of Parasitic Diseases at 770-488-4474.