Giardia trophozoite stained with Trichrome stain: Introduction, Principle, Procedure and Result Interpretation
Giardia Trophozoite in Trichrome stain
Trichrome stained slide of stool specimen showing trophozoite of Giardia lamblia observed at 10X eye piece and 100X objective under microscope as shown above figure also with two clear nuclei. Size of trophozoite is nearly 14 μm long by 7 μm broad. When viewed flat, the shape of the trophozoite is like that of a tennis or badminton racket and when viewed side on it resembles a longitudinally split pear. This stain is Wheatley modified Gomori’s technique by addition of fixation and dehydration steps resulting in a simple and rapid staining procedure for intestinal protozoan parasites cysts and trophozoites. Gomori developed Trichrome stain originally for staining tissue sections and cytological smears. The trophozoite of Giradia as shown above image has purple-red nuclei and blue-green with a faint purple tinge cytoplasm.
Importance of Wheatley’s Modification of the Gomori Trichrome stain
Identification of cysts and trophozoites in fecal specimens is very useful for diagnosis of intestinal parasitic infections conformation. Since smaller protozoans often go undetected in direct wet mount and concentration methods, the identification of intestinal protozoa depends on examination of a permanent stained smear. The Wheatley’s Modification of the Gomori Trichrome stain provides excellent detail and contrast with preserved specimens. For example trophozoite of Giardia as shown above image
Principle of Trichome stain
Chromotrope 2R has an affinity for chromatin material. Nuclear
chromatin, karyosomes, chromatoid bodies,parasite eggs and larvae,
bacteria, and ingested erythrocytes stain red to purple-red. Light
green and fast green dyes stain the cytoplasm of preserved cysts,
trophozoites, and cellular constituents blue-green. The Trichrome
Stain results in excellent contrast and visualization of cellular details
that help in the identification of protozoa.
Requirements
- Wheatley Trichrome Stain: It contains following ingedients and they are-
Phosphotungstic Acid : 7.0 g
Chromotrope 2R: 6.0 g
Light Green SF: 1.5 g
Fast Green FCF: 1.5 g
Glacial Acetic Acid : 10.0 ml
Demineralized Water: 1000.0 ml - Xylene
- Ethanol 95%
- Ethanol 70%
- Acid Ethanol 90%:
- Lugol’s Iodine Ampules
- Permount
- Coverslip
- Clean and grease free slides
- Warmer
- Gloves
- Times
- Applicator stick or sterile bamboo stick
- Absorbent paper( paper towels)
- Coplin jars
- staining rack
- forceps
- Pipettes
- Microscope
- Immersion oil (cedarwood oil)
Quality Control
A control slide of a known protozoan parasite e.g. Giardia species from a Polyvinyl alcohol (PVA) preserved specimen should be included with each staining run.
Procedure of Wheatley’s Modification of the Gomori Trichrome stain
Smear preparation
- Wear gloves when performing this procedure.
- Allow stool specimens preserved in polyvinyl alcohol to fix at least 30 minutes where as mix fresh specimens received in the laboratory with PVA (1 part
feces to 3 parts fixative) and allow to fix for 30 minutes. - Thoroughly mix the specimen and the PVA. Pour a small
amount of the mixture onto a paper towel to absorb excess
fixative. Allow the fixative to soak into the paper towel for 3
minutes before preparing slides. - With an applicator stick transfer some of the stool material from the paper towel to 2 clean glass slides. Spread the mixture to the edges of the slide so the specimen will adhere to the slide during staining. Note: The amount of material applied to the slide should be thin enough that newsprint can be read through the smear.
- Allow the slides to dry overnight at room temperature or 60ºC for 4 minutes in warmer or an hour at 35-37ºC .
Staining
- Immerse slides prepared from fresh specimens in ethanol-Iodine for 1 minute, where as immerse PVA-preserved, air-dried smears in ethanol-Iodine for 5-10 minutes.
- Place slides in 70% ethanol for 5 minutes. Drain excess liquid.
- Place slides in a second jar of 70% ethanol for 3 minutes.
- Place slides in Wheatley Trichrome stain for 10 minutes.
- Place slides in acid ethanol for 1-3 seconds. Immediately proceed to the next step. Do not allow the slides to remain in contact with this solution longer than 3 seconds.
- Dip slides several times in 95% ethanol .
- Place slides in two changes of 95% ethanol for 3 minutes each.
- Place slides in two changes of Xylene for 5-10 minutes each.
- Apply mounting medium (e.g. permount) to the smear and cover with a No. 1
thickness cover-slip. - Allow the smear to dry overnight at room temperature or for 1 hour at 35-37°C.
- Examine the slide microscopically, using the oil immersion
objective for nuclear detail. At least 200-300 oil immersion fields
should be examined.
Result Interpretation of Wheatley’s Modification of the Gomori Trichrome stain
Fixatives are responsible for variation in staining characteristics and the typical staining reactions with Trichrome Stain are as follows:
- Nuclear chromatin, chromatoid bodies, ingested erythrocytes,
and bacteria : They stain red to purple-red. - Cytoplasm: It stains blue-green with a faint purple tinge.
- Macrophages, leukocytes, and yeast cells: They vary in staining
reactions. - Background material: It stains green providing a nice color contrast with the protozoa/
- Glycogen : It is dissolved by the solvents and appears as a clear area in the organism.
References
- Garcia, L.S. 2001. Diagnostic Medical Parasitology. 4th ed. ASM Press,
Washington, D.C. - Isenberg, H.D. 2007. Clinical Microbiology Procedures Handbook. 2nd ed. update ASM Press, Washington, D.C.
- http://med-chem.com/pages/lab_procedure /pdf/wheatleys_trichrome_stain.pdf
- http://tools.thermofisher.com/content/sfs/manuals/IFU40217.pdf
- https://www.cdc.gov/dpdx/diagnosticprocedures/stool/staining.html
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86497/