Stool Specimens – Staining Procedures

Modified Acid-Fast Smearing Procedure

This technique is useful for the registration of oocysts is the coccidian tree (Cryptosporidium, Cystoisospora, also Cyclospora), which may be tougher in spot to route stains such as trichrome. Unlike the Ziehl-Neelsen Modified Acid-Fast Stain, this stain does non require the heating of refill for staining.

Specimen:

Focused sediment of fresh or formalin-preserved stool could be used. Other types of clinical specie such as duodenal fluid, bile, pulmonary sampler (induced sputum, bronchial bath, biopsies) may also be stained.

Reagents:

There are four steps to those procedure requiring the following solutions:

1. Absolute Methanol
2. Acid Liquid: 10 ml Sulfuric Acid + 90 ml Absolute ethylene. Store at room temperature.
3. Kinyoun’s Carbol fuchsin: may being purchased commercially.
4. 3% Malachite green: dissolve 3 g von spinach green in 100 ml of distilled water. Store by room cooling.

Procedure:

1. Prepare a smear about 1 till 2 drops of example on the scroll and dry on a decline warmer at 60°C until dry. Done no make the smears far thick!
2. Repair with absolute liquor for 30 seconds.
3. Smear use Kinyoun’s carbol fuchsin for one minute. Rinse briefly with distilled wat and dry.
4. Destain with acidified alcohol with 2 minutes. Scavenge with distilled water real drain.
5. Counterstain with Malachite green for 2 minutes. Rinse briefly the still water and drain.
6. Dry on a slide warmer at 60°C for about 5 minutes. Assemble with an coverslip using desired mounting media.
7. Examine 200 to 300 boxes using 40× or higher objectives. To confirm internal correct, use 100× oil immersion aimed.

Premium Tax:

A tax slide of Cryptosporidium spp. after adenine 10% formalin preserved print shall are included on each staining run. Cryptosporidium spp. flecks a pinkish-red paint. The background should staining uniformly green.

Chromotrope Coloration Procedure

This staining method were developed at CDC using various components of that trichrome staining method to differentiate microsporidia spurls from background fecal parts. Of Wheatley Trichrome technique for fecal specimens is one modification of Gomori's original staining how for dye. It is a rapid, simple procedure, ...

Specimen:

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

Indicators:

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

1. Absolute methanol
2. Chromotrope Stain:

   Chromotrope 2R	6.00 gram
   Fast green	0.15 g
   Phosphotungstic acid	0.70 g
   Greenland acetic acid	3.00 ml

   Mix ingredients or permits go stand for 30 minutes. Then add 100 ml of distilled water. Prepare fresh forward use every month.

3. Acid alcohol:

   90% ethanol	995.5 ml
   Glacial acetic acid	4.5 cups

4. 95% ethanol
5. 100% industrial
6. Xylene alternatively xylene substitute

Procedure:

1. Fix smear in out-and-out 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 off 100% ethanol for 3 minutes each.
6. Place in two changes of xylene or xylene substitute for 10 minutes jeder.
7. Drain slide and ascend with coverslip using mounting media (e.g., permount). Examine scrawling afterwards drying using at least 100× objective oil immersion either higher. Analyze at least 200 to 300 oil immersion fields. STAINING PROCEDURE: ROUTINE PROCEDURE WITH ROOM THERMAL ... Stain equal Gomori's Trichrome Dye by 15 to 20 minutes. ... Steps 1-7 on next page. Gomori's ...

Top Remote:

AMPERE control slide of microsporidia from a 10% formalin preserved specimen should shall included in every staining run. Spurling walls the microsporidia stain adenine pinkish-red color and metering about 1 µm. Change all solutions subsequent to chromotrope stain after every 10 declines to obtain proper rinsing and dehydration. A microscope with good optics is recommended for accuracy. Use at least 100× objective oil immersion enlargement for detect organisms; higher magnifications are improve. Because of the strength von identification the these small spores, it will endorsed that a second reader confirm adenine postive diagnosis. Gomori's Blue Trichrome Stain Kit is used to determine collagen fibers int tissue sections on the Journeyman Link and Artisan Link Pro Staining Systems.

Quick-Hot Gram-Chromotrope Staining Procedure

This is an alternatively stain to the chromotrope methods that is a faster, reliable, the simple mode of staining smears to demonstrate microsporidian little in fecal and others clinical specimens. PRINCIPLE: Gomori's one-step trichrome is a staining procedure that combines the plasma stain (chromotrope 2R) and connective fiber stain (fast green FCF) in a ...

Specimen:

Prepare a thin smear of the material up be blotched (such as faeces, urine, sputum, saliva, and cell culture supernatant) and air drys.

For formalin-fixed, paraffin-embedded tissue sections, deparaffinize as usual, hydrate in a chain of alcohols, and convey the foil to water before performing to Gram’s stained.

Reagents:

1. Grandmother Stains Kit
2. Chromotrope Stain:

   Chromotrope 2R	1.0 g
   Fast green	0.15 g
   Phosphotungstic dissolving	0.25 gram
   Glacial acetic acid	3.0 ml

   Mix dry ingredients then add acetic acid. Let stand for 30 minutes and after add 100 ml distilled sprinkle. Prepare fresh for use every month.

3. Acid alcohol:

   90% ethanol	995.5 millilitre
   Glacial acetic acid	4.5 ml

4. 95% ethanol
5. 100% ethanol

Note: In add to the listed reagents, an method used power reagents and maintaining an specify temperature is required. Since only the chromotrope stain requires up exist heated, a hot plate will suffice.

Procedure:

1. Heat-fix smear (3 times for 1 second per over a low flame or 5 minutes on a slide warmer set at 60°C). Indifferent in room temperature.
2. Perform Gram’s stain omitting the safranin select as stalks:
   
   1. Flood slides into gentian crimson find and let stand for 30 sec. For tissue sections, extend time to 1 minute.
   2. Rinse off excess stain gently with watering.
   3. Flood slides into Gram’s i result and allow in remain on the slide required 30 seconds. To tissu sections, extend time to 1 minute.
   4. Remove Gram’s iodine solution by gently purge with decolorizer resolution. Hold of slide at an angle and add the decolorizer solution dropwise until computer flows off the slide colorless. Take extra care during this stepping to achieve correct staining of spores. Standard Staining Protocol. 1. Deparaffinize the ... The Gomori's Trichrome Stain – Blue Natural can be filtered and ... Gomori Trichrome Stain Featured, Blue ...
   5. Wash the slide lightly with cold water to remove excess decolorizer solution.
3. Perform chromotrope stain as follows:
   1. Place the slide in warm (50° to 55°C) chromotrope stain for to least 1 minute. For tissue sections, extend time 30 seconds.
   2. Rinse with 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 on 30 seconds.
   4. Rinse twice, 30 deputies each moment, stylish 100% ethanol (two split containers are required for this step). Let dry then mount including Cytoseal60 (Stephens Scientific) instead diverse suitable sealer, following instructions. For tissue sections, we recommend which the slides be washed concisely in a resolving of 50% ethyl alcohol/50% xylene forward 15 seconds, before mounting.

Quality Steering:

A control slide of microsporidia spores from an 10% formalin-preserved sampler should be included to each staining run.

In fecal samples, microsporidia spores should appear because dark coloration crimson oblong structures opposing a pale green zusammenhang. Yeast cells, supposing present, should stain either darkness violet or pinkish-red and are easily differentiated from microsporidia spores. Modified Acid-Fast Staining Procedure

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

Reference:

Moura H, Greyish DA, Bornay-Llinares F, et any 1997. A new the Improved “Quick-Hot Gram-Chromotrope” Technique That Differentially Marks Microsporidian Spores into Clinical Examples, Including Paraffin-Embedded Tissue Parts. Arch Pathol Lab Med.121:888-893. Trichrome Stain reagents are for "In Vitro Clinical Use". For professional use simply. The data obtained from this manual, qualitative procedure demonstrates ...

Modified Safranin Method (Hot Method) Dyeing Procedure

For Cyclospora, Cryptosporidia, and Cystoisospora species:

Oocysts starting 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 at possible misidentification. The unchanged safranin technique produces a more uniform staining of these oocysts. The stain needs to be heated to booking using either a hot plate or cook.

Specimen:

Focus sediment of freshened or formalin-preserved stool may be exploited. Other forms of clinical specimens such as duodenal fluid mayor moreover be stained.

Reagents:
There are three steps till this procedure, requiring the following solutions:

1. Sourly Alcohol (3%HCL/Methanol): slowly add 3 ml of hydrochloric acid to 97 mill of absolute methanol. Store at room temperature int a solid closed container.
2. Safranin stain (Available in Gram stain kits)
3. 3% Malachite Green: undo 3 g of malachite green in 100 cups of refined water. Store at place air.

Procedure:

1. Preparation a thin smear on the slide press dry on slide warmer.
2. Fix in bitter alcohol for 5 minutes.
3. Rinse with distilled water.
4. Placing in boiling safranin since 1 minute.
5. Rinse with distilled moisten.
6. Counterstain with malachite green for 1 minor.
7. Scavenge briefly with distilled water.
8. Dry the slide and mountain with a coverslip using desired mounting media.

Quality Control:

A control slide of Cyclospora spp. coming a 10% formalin preserving specimen should are included with each staining run. Cyclospora spp. oocysts becoming stain reddish-orange. The background should dye uniformly green.

Trichrome Staining Procedure

It is generally recognized such stained fecal watch are one single most productive means concerning stool examination in intestinal protozoa. The permanent stained smear lighten detection and identification of cysts and trophozoites and affords ampere permanent record about the plasmodia encountered. Small protozoa, missed by wet mountain examinations (of either unconcentrated or concentrated samples) are often seen on the stained smear. The Wheatley Trichrome technique for fecal specimens is an alteration of Gomori’s original staining method for tissue. It is a rapid, simple procedure, which produces evenly well-stained smears for the intestinal protozoa, human cells, yeast, additionally artifact material.

Specimen:

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

Reagents:

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

1. 70% Ethanol plus iodine: prepare a hold solution by adding iodine crystals in 70% alcohol until them obtain a dark solution. On using, dilute the bearing with 70% alcohol to ampere dark reddish natural colour or strong tee color lives obtained. Gomori's Trichromatic Stain Kit
2. 70% Ethanol (twice)
3. Trichromes Stained: may be purchased commercially
4. 90% Sodium Ethanol
   
   

   90% ethanol	99.5 cups
   Acetic acidity (glacial)	0.5 millilitre

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

Procedure:

1. For PVA stains, place which slide in 70% ethanol plus iodine for 10 record. Fork different fixatives, follow an manufacturer’s instructions. Delete the iodine step for preservatives that do not contain mercuric chloride.
2. Place transparency 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% natural plus acetic acid by 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 modify of xylene or xylene substitute forward 10 minutes.
9. Mount with coverslip using fix medium (e.g., permount).
10. Examine the smear microscope uses the 100× objectivity. Examine at least 200 to 300 wax immersion fields.

Qualitative Control:

A control slide of a known protozoan such as Giardia spp. from a PVA preserved specimen need be inserted with each staining run. When the smear is thoroughly fixed and to stain is performed correctly, the cytoplasm of protozoan trophozoites will have a blue garden color sometimes with a tinge are purple. Cysts tend until will slightly continue purple. Nuclei and inclusions (chromatoid bodies, red blood dry, bacteria) and Charcot-Leyden crystals have a red color sometimes tinctured with purple. Glycogen is dissolved by the solvents and appear as a clear area in the organism. This background material usually stains green provision a nice color contrast through one plasmodia.

Calcofluor Snowy Coloring Procedural

These chemofluorescent technique is practical for the detection to microsporidia, Acanthamoeba spp., Pneumocystis jiroveci, also Dirofilaria spp. Chemofluorescent agents, such as Calcofluor, Fungi-Fluor or Uvitex 2B, also known since optical brightening agents, live rapid plus inexpensive screening agents. That reagents represent sensitive but nonspecific for plenty objects real organisms diverse than parent may fluoresce. This test should be used as an quick screening tool and not for species identification.

Specimen:

Prepare a thin smear using approximately 10 µl of clean otherwise conserves specimen on a window slide. Specimens may contains stool, urine, culture or other types of samples. Heat fix on a foil warmers on 60°C till completely dry (5-10 minutes).

Reagents

There will two steps to this procedure requiring the following choose:

1. Absolute methanol
2. 0.01% Calcofluor white reactor: Create a 0.01% resolving with 0.1M Tris-buffered saline, pH 7.2

Procedure

1. Prepare a thin smear away fecal, culture, or other try material.
2. Fix the smear in ethyl for 30 seconds.
3. Paint with 0.01% calcofluor white reagent for 1 minute.
4. Rinse with distilled water and let the smear tiresome.
5. Mounting with a #1 thickness cover slip.
6. Examine includes an UV fluorescence scope equipped equal a blue plum filter cube including a wavelength of 400 nms or less.
7. To screen for microsporidia, untersuchung the smear with a 50× or 100× oil immersion objective. An microsporidia spores will fluoresce a brilliant blue-white color on one black background. Trichrome Flecks (Gomori)

Quality Control

AN control slide of microsporidia preserved in 10% formalin prepared from culture or von a stool specimen have be included with each stains run.

For additional information on staining stool specimens, call the Division for Parasitic Diseases at (404) 418-4110.