Histological

identification of
protozoans in tissues
Group 9
Group 9
08/18/2023 1
 Members
Name Reg No.
Musau Immanuel Afrika 2019/MLS/087/PS
Tungo Scovia 2019/MLS/177/PS
Nahamya Ambrose 2019/MLS/094/PS
Gimei Nicholas 2019/MLS/062/PS
Oyera Shamua 2019/MLS/178/PS
Aliruku Emmanuel 2019/MLS/039/PS

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 Objectives
• Introduction
• Protozoan diseases
• H&E
• Romanowsky stain for protozoans
• IHC
• PAS
• PTAH
• Best’s carmine
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 INTRODUCTION
• Protozoans are single-celled microorganisms that are functionally complex structures. The
means of locomotion provides the means of classification of the protozoans.
• Amebae move by using extensions of their cytoplasm (pseudopods), and other protozoans
move by means of flagella or cilia.
• Some medically important protozoans include Entamoeba histolytica, Giardia lamblia, and
Toxoplasma gondii.
• Until recently Pneumocystis jirovecii (formally P carinii) was classified as a protozoan, and it
has trophozoite and cyst stages; however, nucleic acid sequence analysis of the small subunit
ribosomal RNA gene has shown this organism to be most closely related to fungi in general
and yeasts in particular
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 Intro..
• The protozoa of medical importance belong to the following groups:
a) Sarcodina e.g. Entamoeba histolytica
b) Mastigophora e.g. Giardia lamblia, Trichomonas vaginalis,
•Trypanosoma species, Leishmania species
a) Ciliophora e.g. Balantidium coli
b) Coccidia e.g. Isospora belli, Cryptosporidium species, Toxoplasma gondii,
Plasmodium species
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 PROTOZOAN DISEASES
• Entamoeba histolytica, the organism causing amebic colitis or
dysentery, can be found in ulcers that occur in infected colon and in
amebic liver abscesses.
• They may be seen in granulation tissue within ulcers on routine H&E
staining, or in the luminal mucus overlying normal-appearing mucosa.
They are PAS positive; brief counterstaining in 1% aqueous metanil
yellow emphasizes the ingested red cells.

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 CONT..
• Toxoplasma gondii, a commonly encountered or ganism that is spread in cat litter,
causes an acute lymphadenopathy which is often subclinical. Affected nodes show non-
specific changes and no organisms.
• In AIDS and other immunosuppressed patients this protozoon causes systemic diseases,
including meningoencephalitis where encysted bradyzoites and free tachyzoites can be
seen in necrotic brain tissue.
• Cysts also occur in other tissues such as cardiac muscle, and measure up to 40 µm with
tachyzoites 4–6 µm, which can be seen on H&E.
• A Giemsa stain can also be used, but the use of labeled specific antiserum is
recommended.
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 CONT..
• Leishmania tropica is transmitted by sandfly bite and causes a chronic
inflammatory disease of the skin sometimes called cutaneous leishmaniasis.
• The injected parasite forms (2 µm), or amastigotes, are found in large numbers within
the cytoplasm of multiple swollen histiocytes that congregate in early lesions in the
dermis.
• A related organism, L. donovani, causes a systemic visceral infection, kala azar, in
which the organisms are seen within histiocytes in spleen, lymph nodes, liver, and bone
marrow.
• The organisms are hematoxyphilic and can be emphasized with a Giemsa stain.
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 Cont..
• Giardia duodenalis (lamblia)is a flagellate protozoon that is ingested in cyst form
from drinking water with fecal contamination;
• The trophozoites migrate to the duodenum where they may cause severe diarrhea
and malabsorption.
• These organisms can been seen on an H&E stain, where they appear as eosinophilic,
sickle-shaped flakes with indistinct nuclei resting on intestinal mucosa that may
show littleevidence of inflammation.
• When seen in a fresh Giemsa-stained duodenal aspirate, they appear kiteshaped,
11–18 µm in size, binucleate, and have faint terminal flagella.
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 Cont..
• Trichomonas vaginalis is a similar flagellate protozoon most
frequently seen in a Papanicolaou stain.
• Inflammatory cells and mildly dysplastic squamous cells often
accompany this parasite as it causes cervicitis in the female, and urethritis
in both sexes.

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 Cont..
• Cryptosporidium is one of a group of protozoa (including Isospora
and Microsporidium) that causes severe and relentless outbreaks of
diarrhea among AIDS patients.
• Cryptosporidial gametes are seen on H&E stain as blue dots arranged
along the mucosal surface.
• Mature cysts are shed into feces and are acid fast in a ZN stain of fecal
smears.

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 Cont..

Human Trypanosomes
• They show strict geographical distribution:
• Trypanosoma cruzi: It is the causative agent of South American trypanosomiasis (also called as
Chagas’ disease) in man and transmitted by insect vector reduviid bug
• Trypanosoma brucei: It causes African trypanosomiasis, transmitted by tsetse fly.
• It has three important subspecies out of which only two of them infect humans
• Trypanosma brucei rhodesiense: It is the causative agent of East African sleeping sickness
• Trypanosoma brucei gambiense: It is the causative agent of West African sleeping sickness
• Trypanosoma rangeli: It is a nonpathogenic species that rarely infects humans in South America.
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 CONT..
• Plasmodium species cause malaria.
• Malaria is a febrile life-threatening mosquito-blood borne disease.
• There are more than 100 species of Plasmodium, which can infect many animal
species such as reptiles, birds, and various mammals.
• Four species of Plasmodium have long been recognized to infect humans in
nature.That is Plasmodium falciparum, P. vivax, P. vale and P. malariae
• In addition, P. knowlesi that naturally infects macaques (monkeys) has recently
been recognized to be a cause of zoonotic malaria in humans.
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 H&E
PRINCIPLE
• Hematoxylin is a basic dye that stains acidic components, such as the
nuclei of cells, blue-purple.
• Eosin is an acidic dye that stains basic components, such as cytoplasm
and connective tissues, pink or red.

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 procedure
• Dewax sections, hydrate through descending grades of alcohol to water.
• Stain in hematoxylin for 3 minutes.
• Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 5–10 seconds
• Wash well in tap water and ‘blue’ (10–15 minutes), or
• Blue by dipping in an alkaline solution (e.g. ammonia water), followed by a 5-minute tap water
wash.
• Stain in 1% eosin Y for 30 to 60 seconds
• Dehydrate through increasing concentrations of alcohols, clear in 2 changes of xylene , and
mount using DPX.
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 Results
• Nucleus……………………….blue black
• Cytoplasm……………………pink or red
• Giardia duodenalis (lamblia)……………..pink
• Toxoplasma gondii…………………………....blue black
• Cryptosporidium gametes…………………. blue dots

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H&E demonstrating the single-celled parasite, Toxoplasma gondii in the heart.
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 PAS
Principle
• Periodic acid oxidises 1,2 glycol group to form an aldehyde, the aldehyde
reacts with schiffs reagent to for a magenta colouration.
• The stain is used to stain the amylopectin granules contained
inbradyzoites and oocysts of Toxoplasma gondii .
• The cell membrane of the trophozoites of Entamoeba histolytica contain
glycoproteins, and therefore they can be stained using PAS

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 Procedure

• Deparaffinize
• Hydrate
• Flood with periodic acid for 5minutes
• Wash
• Stain with schiffs reagent for 15 minutes
• Wash and flood with water for 15 minutes for color development.
• Counterstain with haematoxylin for 30 seconds and wash and blue. Dehydrate, clear
and mount.
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 Results
• Nucleus………………………………blue black
• Mucin………………………………….magenta
• Glycogen………………………………magenta
• E. histolytica………………………….magenta/pink
• T. gondii…………………………………red

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 Entamoeba histolytica trophozoites from a liver abscess

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 IHC
• IHC utilises specific antibodies to target protozoan antigens in tissue sections. It is valuable for
the detection and identification of protozoan parasites like toxoplasma gondii and
cryptosporidium species.
• Common IHC antibodies for protozoan identification include:
1. anti-cyst wall protein antibodies: for cyst of giardia and entamoeba.
2. anti-hemozoine antibodies: for malaria parasite(hemozoin).
3. anti-amoebapore antibodies: used to identify amoebic trophozoites of entamoeba hystolytica
4. anti-toxoplasma gondii antibodies: to detect toxoplasma gondii
5. Anti-cryposporidium antibodies: for crptosporidium.
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 markers
• CD1a………amastigotes of leshmania

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 procedure

• Tissue preparation: the tisue samples suspected to be having protozoan infections is

fixed, processed and embedded in parafin wax to create a parafin block, and sectioned
• Antigen retrieaval: since formalin fixation masks antigenic sites, antigen retrieval is
done to unmask epitopes and improve antibody binding. This done by subjecting
tissues to high temperatures or enzymatic digestion.
• Blocking: nonspecific binding sites on the tissue is done to prevent fales positive
• Primary antibody incubation: tissue sections are incubated with specific primary
antibodies targeting the protozoan antigens of interest.

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 Cont..
• Secondary antibody incubation: after washing off the unbound primary antibodies, tissue
sections are incubated with secondary actibodies which are conjugated with enzymes or
fluorochromes allowing for vizualisation and amplificatyion of the signal
• Signal develpomement: a chromogenic substance is added to the secondary enzyme
conjugated antibody leading to development of a visible color. For fluorochrome conjugated
antibodies, we use a flourescence microscope.
• Counterstain: to enhance contrast and visualization of tissue morphology, haematoxylin may
be used
• Mount: the stained sections are cover slipped with xylene to preserve the specimen and
examine them
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• Immunohistochemical staining demonstrating the single-celled parasite Toxoplasma gondii in heart

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 Best’s carmine
solutions
A. Nuclear counterstain C. Working staining solution
An iron–haematoxylin • Stock solution (above): 15 ml
B. Stock solution of carmine • Ammonium hydroxide: 12.5 ml
• Water: 60 ml • Methyl alcohol: 12.5 ml
• Carmine: 2.0 g D. Best’s differentiator
• Potassium carbonate (K2CO3): 1.0 g • Absolute methyl alcohol: 40 ml
• Potassium chloride (KCl): 5.0 g
• Absolute ethyl alcohol: 80 ml
• Water: 100 m
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 Principle
• staining is by hydrogen bond formation between OH groups on glycogen
content of protozoans and and hydrogen atoms of the carmic acid.

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 Procedure
(1) De-wax the slides and hydrate.
(2) Stain the nuclei with weigert’s iron haematoxylin 5minutes.
(3) Wash in water
(4) Decolorise with with 0.5% hydrochloric acid in 70% alcohol for 10 seconds
(5) Wash in water
(3) Stain in the working solution of carmine (C) for about 30 minutes.
(4) Differentiate for 10 seconds.
(5) Dehydrate, clear and mount
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 Results
Glycogen contents of protozoans………………..bright crimson.
Fibrin, mast cell granules and some mucus……… pink.
Nuclei ……………………………………………. Blue or black

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 Romanowsky staining of protozoans

Principle
Romanowsky stains are neutral stains composed of a mixture of oxidized
methylene blue(azure) dyes and eosin Y.
The azures are basic dyes that bind acid nuclear and result in a blue to purple
color
The acid dye eosin is attracted to the alkaline cytoplasm producing red
coloration.
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 Giemsa stain for parasites

Sections
• Fixative is not critical, but B5 or Zenker’s is preferred; thin (3 μm)
paraffin sections.
• If Zenker’s is not used, post-mordant in Zenker’s in a 60°C oven for 1
hour before staining.

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 Cont..
Solutions
• Giemsa stock (commercially available) or Giemsa stain powder 4 g
• Glycerol 250 ml
• Methanol 250 ml
• Dissolve powder in glycerol at 60°C with regular shaking.
• Add methanol, shake the mixture, and allow to stand for 7 days. Filter before use.

Working Giemsa for parasites

• Giemsa stock 4 ml
• Acetate buffered distilled water, pH 6.8 96 ml

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Method
1. Deparaffinize and rehydrate through graded alcohols to water.
2. Rinse in pH 6.8 buffered distilled water.
3. Stain in working Giemsa, overnight.
4. Rinse in distilled water.
5. Rinse in 0.5% aqueous acetic acid until section is pink.
6. Wash in tap water.
7. Blot until almost dry.
8. Dehydrate rapidly through alcohols, clear, and
mount.
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Results
• Protozoa and some other microorganisms……………………….dark blue
• Background …………………pink-pale blue
• Nuclei……………………….. blue

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 Other Romanowsky stains that can be used
• Leishman`s stain
• May-Grunwald stain
• Jenner`s stain
• Wrights
• Field stain

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 PTAH
• It stands for Phosphotungstic Acid Hematoxylin staining and it is
commonly used to visualize and identify tissue cysts of some protozoan
parasites belonging to phylum apicomplexa.
• Protozoans like toxoplasma gondii and sarcocystis species can be detected
using this method.
• It selectively stains the cyst walls making them stand out from the
surrounding tissues.

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 Principle
• The amount of phosphotungstic acid in the staining solution is far greater than the
amount of hematein (20:1), and it is believed that tungsten binds all available
hematein to give a blue lake.
• This metal-hematein lake stains selected tissue components blue, while the
phosphotungstic acid is thought to stain the red-brown components.
• This stain has been referred to as a polychrome stain because 1 solution gives 2
major colors. The components colored red-brown will lose this color with water or
prolonged alcohol washes, and therefore dehydration of the section after staining
must be rapid.
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 Procedure
• Fixative
• Zenker solution is preferred, but 10% neutral-buffered formalin may be used
• Solution A
• Hematein 0.8 g
• Distilled water 1 ml
• Grind the 0.8 g hematein to a paste with 1 ml distilled water. (The paste should be chocolate brown. Lighter colors are
usually indicative of an unsuitable batch of hematein and should be discarded.)
• Solution B
• Phosphotungstic acid 0.9 g
• Distilled water 9 ml
• Mix solution A and solution B, bring to the boil, then cool and filter.

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 method
1. Dewax, rehydrate through descending grades of alcohol to water.
2. Treat with acid permanganate for 5 minutes.
3. Rinse in tap water.
4. Bleach with 5% aqueous oxalic acid.
5. Wash well in tap water.
6. Stain in PTAH solution for 12–24 hours at room temperature.
7. Wash in distilled water.
8. Dehydrate rapidly, clear, and mount.
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 Results

• Muscle striations, neuroglia, fibers, fibrin and amoebae………..dark blue.

• Nuclei, cilia, red blood cells…………………………blue.
• Myelin ……………………………………………….lighter blue.
• Collagen, osteoid, cartilage, elastic fibers……………deep brownish-red.

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 Other techniques that can be used
• PAP
• PCR

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 References
• Brar, A.P.S., Sood, N.K., Singla, L.D. et al. Validation of Romanowsky
staining as a novel screening test for the detection of faecal
cryptosporidial oocysts. J Parasit Dis 41, 260–262 (2017). https://
doi.org/10.1007/s12639-016-0788-z
• Histological andHistochemical Methods by J. A. Kiernan Theory and
practice
• Bancrofts theory and practice of histological techniques, seventh edition.

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 YOU HAVE BEEN THE BEST
CLASSMATES

WE MEET IN THE FIELD

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 Thank you for listening

Questions????

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