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The purpose of this experiment is to observe the characteristics of eukaryotic pathogens, including protozoa, fungi and helminthes; to be able to distinguish between different organisms, and apply the results in the field of microbiology.
Protozoa
The identification of protozoa is based on their morphological and stainingcharacteristics. Saline wet mount is used for detection ofBalantidium coli, Entamoebahistolytica and Giardia Lamblia. Thick and thin blood smears are used for detection ofPlasmodium, Toxoplasma gondii and Trypansomabrucei.Trypansomabrucei can also be detected by microscopic examination of lymph node aspirate or chancre biopsy. Trichomonasvaginalis can be detected through a wet preparation of the sample, and can be cultured in commercially available Diamond’s medium with pH 7.0 +/- 0.3.
Balantidium coli have trophozoite and cyst stages. In trophozoites, two nuclei are visible. In cysts, only the macronucleus and sometimes cilia are visible. Also, cysts have a tough cyst wall. Their size is about 30 µm under magnification 400.
Trichomonasvaginalis exists only in a trophozoite stage, and its size is about 22.5 µm under magnification 1000. It ispear shapedwith five flagella; four extend outside the cell together, whereas the fifth wraps backwards.
Entamoebahistolytica exist in trophozoite stage and cyst stage. Their size is about 36 µm under magnification 1000. The cell body of the trophozoite is divided into ectoplasm and endoplasm, with a single pseudopodium and a nucleus showing a central karyosome and a uniform ring of peripheral chromatid granules.
Plasmodium species exist in four developmental stages in humans (hepatic schizonts,intraerythrocytictrophozoites, schizonts and gamonts). Amorphous multinucleate schizonts measure about 6 µm under magnification 1000.
Giardia Lamblia has trophozoite and cyst stages. The trophozoiteis pear shaped, with two nuclei, two median bodies and four pairs of flagella. The cyst measures about 3 µm under magnification 1000, and it contains double the organelles.
Toxoplasma gondii have four stages include the tachyzoites, merozoites, bradyzoites, and sporozoites. The tachyzoite measures about 4.5 µm under magnification 1000, crescent shapedwith a pointed anterior end and a rounded posterior end.
Trypansomabruceiare about 20 mm long, slender, witha centrally positioned nucleus, a kinetoplast towards the posterior anda flagellum.
Fungi
Fungi can be detected using various stains.They are stained dark blue with Lactophenol blue stain. In Periodic Acid Schiffreaction, the fungal elements adopt a bright red color. Regarding the pre-treated specimen with alkali, it should be neutralized with 10% lactic acid and adjusted to pH 3-5. Using the Gomorimethenamine silver (GMS) stain, the fungi are seen as brown to black on a light green background.
Saccharomgcoscereuisae are spherical to ovoid, with no or simple pseudohyphae. They are cultivated on Malt agar withmedium sized white convex circular colony.
Candida Albicans are spherical to ovoid, 3.0-5.5 x 4.0-9.0 um; pseudohyphae and hyphae may be formed. Their colony on Malt agar is white to cream color, smooth, glabrous, fringed border.
Rhizopusunder microscope are about 1500 um in length and 18 um in width, smooth walled, simple or branched. Sporangia appearpowdery,globose, grayish black, up to 175 um in diameter.
Aspergilliusunder microscopeappear as conidiophores end with a sac, phialides are attached to this sac or on cells called metula (biserate), and conidia attach to phialides in chains.
Rhizopusfills the petri dish, and may overgrow and inhibit other fungi. Some structures are visible to the naked eye like sporangia as black dots in the white, cottony mycelia.
Aspergilluson the petri dish, the surface of the colony is yellow-green and the reverse is yellow, brownish.
Helminthes
Parasitological diagnosis of helminthes depends on detecting the different stages of life cycle by microscopic examination. Examples for the methods used include Kato-Katz thick smear, McMaster egg counting method and Mini-FLOTAC.
Schistosoma male measures about 4500 µm under magnification 40. It has a mouth and an oral sucker. Also, it has a deep ventral groove along its bodyfor the female to live in.
Schistosomahaematobium egg measures about 150 um in length. It has a prominent terminal spine. Schistosomamansoni egg has a large, lateral spine.
Fascioba hepatica measures about 1800 µm under magnification 100. It is leaf-shaped, with pointed posteriorand wide anterior ends. It has a powerful oral sucker and a larger sucker, the acetabulum.
Clonorchissinensis measures about 2250 µm under magnification 40. It is flattened, leaf-shaped. It has a small oral sucker and a poorly developed ventral sucker. It has two tubes representing the digestive and excretory tracts.
Dipylidiumcaninum egg measures about 60 µm under magnification 100. The eggs are spherical and form an egg-ball.
Taeniaproglottid measures about 4500 µm under magnification 40. The gravid uterus shows 7-12 lateral branches in Taeniasoliumand more lateral branches in Taeniarhynchussaginatus.
Taeniascolex measures about 4500 µm under magnification 40. InTaeniasolium, it has 2 circles of hooks and 4 suckers. InTaeniarhynchussaginatus, it lacks the hooks.
Dipylidiumcaninumlength measures 1 cm up to 70 cm. The head measures about 0.5 mm and has hooks and suckers. The body has between 50 and 150 proglottids. The gravid segments are about 1 cm long, shed with feces.
Taenialength measures about 4 cm. It has no body cavity, and its body is white in colour and flattened. The scolex has suckers that surround the rostellum. The elongated body is called the strobila, covered by a tegument, and divided into proglottids.
Ascaris male measures about 4500 µm under magnification 40. It is slender (2-4 mm), and characterized by a curled posterior end.
Ascarisfemale measures about 4500 µm under magnification 40. It is thicker than male (3-6 mm), and characterized by a genital girdle.
Ascaris egg measures about 45 µm under magnification 100. The outer surface is mamillated and stained brown from bile.
Ascaris female worms measure 20 to 35 cm in length, but males do not exceed 30 cm. They appear creamy-white, occasionally with a pinkish cast.
Ascaris digestive tract is simple but complete. It consists of a mouth, a pharynx, an intestine, and an anus.
Enterobiusvermicularis egg measures about 64 µm under magnification 400. It hasa thin shell and one side is flattened.
Enterobiusvermicularis adult measures about 225 µm under magnification 40. Female has a long, pointed tail, and male has a curved, blunt posterior end.
Generally, protozoa, fungi and helminthes could be distinguished from each other by their morphology and arrangement.
Protozoa are unicellular eukaryotes that can move by pseudopods, cilia and flagella.They have many shapes. They reproduce asexually through encystment. Many of them are normal flora, but some are pathogenic such as plasmodium.
Fungi are eukaryotes that exist as unicellular yeasts or larger multicellular moulds. They lack chlorophyll and have a rigid cell wall.Yeasts reproduce asexually by budding and sexually by formingascospores.Mouldsshow asexual and sexual reproduction by producing spores.
Helminths are multicellular eukaryotes, which can be classified into nematodes, cestodes and trematodes. They have complex life cycles, with at least one stage living in its main host where it achieves sexual maturity and reproduce.
Aothermetod of differentiation is through the use of EosinMethyleneBlueAgar for lactose fermentation. Vigorouslactosefermenter turns the media to green/black, less aggressive turns it to pink to purple, whereas non fermenter maintains normal colour. Also, yeasts are alcohol fermenter producing ethanol, whereas lactic acid fermentation occurs in humans.
Finally, the importance of differentiation of different eukaryotes is mainly related to the clinical field of microbiology. That enables identification of specific pathogen affecting the patient, and consequently giving the proper treatment. Also, more understanding of different organisms and their life cycles; enables getting more benefits of harmless organisms, and developing effective treatments for harmful ones.
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