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Participants on the one-day teaching course are provided with a subject-specific illustrated bench book. 

The following few pages are 'tasty tit-bits' extracted from the complete bench book.

[eyepiece calibration] [faecal concentration] [urine concentration] [iodine] 

[Field's] [Giemsa] [Cyclospora] [Blastocystis] [Dientamoeba] [Microsporidia] 

[The Entamoeba debate]

Calibration of eyepiece graticule. 

Please note: This procedure is very important. Microscopes must be recalibrated after every major service.

Worked example.

1.         The eyepiece scale is divided into 100 small divisions

2.         The stage micrometer (calibration slide) scale is 1 mm divided into 100 divisions, each division is equivalent to 10µm.

3.         Insert the eyepiece scale (round glass disc) into the eyepiece by removing the uppermost lens and placing the scale on field stop.

4.         Insert the eyepiece into the microscope.

5.         Place stage micrometer on microscope stage

6.         Focus low power objective on stage slide.

7.         Adjust stage and eyepiece scales until eyepiece scale (numbered) lies along the single line of the stage scale.

8.         Note number of eyepiece divisions and its appropriate stage measurement, eg.

            a) 50 eyepiece divisions = 125 µm

Therefore:

            b) 10 eyepiece divisions = 25 µm

9.         From this reading work out value for one eyepiece division.

            eg  a)  50 eyepiece divisions = 125 µm

Therefore:  1 eyepiece division = 125/50 µm = 2.5 µm

                  b)  10 eyepiece divisions = 25 µm

Therefore:    1 eyepiece division = 25/10 µm = 2.5 µm

10.            Repeat for all objectives, note readings and keep in a prominent place near the microscope.

It is recommended that a separate eyepiece be kept with the micrometer disc inside.

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Faecal concentration methods.

Ridley & Allen modification formol-ether concentration method for ova and cysts.

Materials.

1.        *10% formalin water (100ml formaldehyde + 900ml distilled water)

2.       **        ** Ether or ethyl acetate.

3.         40 mesh  (425µm) brass wire filter - 3" diameter (Endcott Sieves Ltd. Lombard Road, London SW19 3BR)  Nylon coffee strainers provide a low cost alternative.

4.         Small 3" porcelain or stainless steel dish.

Method.

1.         Using orange sticks, select a quantity of faeces (approx 1 g) to include external and internal portions.

2.         Place in a centrifuge tube containing 7 ml of 10% formalin.

3.         Emulsify the faeces in the formalin and filter through the brass/plastic filter into the dish.

4.         Wash the filter and discard any lumpy residue.

5.         Transfer the filtrate to a boiling tube - add 3 ml of **ether or 5 ml of ethyl acetate and mix well on a vortex mixer for 15 seconds or by hand for 1 minute.

6.         Transfer back to the centrifuge tube and centrifuge at 3,000 rpm. for 1 minute.

7.          Loosen the fatty plug with an orange stick and pour the supernatant away by quickly inverting the tube.

8.         Allow the fluid on the side of the tube to drain on to the deposit - mix well and transfer a drop to a slide for examination under a coverslip.

Use the x10 and x40 objectives to examine the whole of the deposit for ova and cysts.

* Formaldehyde is toxic by inhalation, ingestion and by skin contact. Repeated skin contact may cause sensitisation.

**The use of ether is to be discouraged due to its highly flammable nature and liability to form explosive peroxidases on exposure to air. Ethyl acetate is recommended as an alternative although this liquid is also highly flammable.

The zinc sulphate and saturated salt flotation methods are low cost alternatives. Methodology for these two techniques may be found in any reputable parasitology text book.

There are a number of commercial faecal concentration devices available to clinical laboratories. One advantage that many of them offer is an enclosed, odour free operating technique. All of these devices are broadly based on the original  Ridley-Allen modification formol-ether concentration technique.

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Concentration of urine for parasites.

There are no adult helminths that inhabit the urinary tract but ova of several species are found in the urine, mainly Schistosoma haematobium. Occasionally other parasites may be found in urine samples the most common being ova of Enterobius vermicularis and trophozoites of Trichomonas vaginalis. Rarely found are larvae of Strongyloides stercoralis and microfilaria of Wuchereria sp and Brugia sp.

1.         Centrifugation method.

A.     Collect a terminal urine sample and centrifuge at 1,500 rpm. for 2 minutes to deposit the ova. (at higher speeds the ova may be damaged and the miracidia will escape)

B.     Decant the supernatant and place a drop of the deposit on to a slide. Cover with a coverslip; examine the whole of the coverslip area for ova using the x10 objective. Identify any ova found under the x40 objective.

2.         Filtration method.

1.         Draw approx.10 ml of terminal urine or a larger volume of random urine into a  syringe.

2.         Connect the syringe to a Swinnex filter containing a *polycarbonate filter of pore size 12µm.

3.         Gently push the urine through.

4.         Draw up 20 ml of air and push this through the filter.

5.         Remove the top of the filter and using blunt forceps, place the membrane on to a microscope slide.

Add a drop of saline and a coverslip and observe under the microscope.

Polycarbonate membranes may be obtained from Corning Costar

Website: http://www.corning.com

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Staining methods.

Protozoa.

Temporary stains.

Stains for wet preparations following concentration by the formol-ether/ethyl acetate method.

Lugol's iodine solution.

Reagent 1

Potassium iodide          20 g

Iodine                             10 g

Distilled water               100 ml

Add potassium iodide to the distilled water; when dissolved, add the iodine crystals. Store in a brown bottle. This reagent remains stable for many weeks.

Reagent 2

25% glacial acid

Mix equal parts of reagent 1 and 2 for use.

Iodine stains glycogen brown and the nuclear chromatin of amoebic cysts brown/black.

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Permanent stains.

Field's stain.

Field's stain is made from two component stains. Field's stain A and Field's stain B

By using buffers of differing pH it is possible to stain a wide variety of clinical materials.

Rapid Field's stain.

This is a modification of Field's stain to enable rapid staining of fixed thin films of various clinical samples. This particular staining method is very useful for staining films of unformed faeces, faecal exudate, duodenal aspirates etc.

Method.

1.         Make a thin film of faeces/exudate. Allow to air dry.

2.         Fix in methanol for 1 minute.

3.         Flood slide with 1 ml of Field's stain B (diluted 1:4 with distilled water)

4.         Immediately add an equal volume of undiluted Field's stain A, mix well and allow to stain for 1 minute.

5.         Rinse well in tap water and drain dry.

Parasite nuclei and structures containing chromatin stain red.

Cytoplasm stains bluish-grey.

Leucocyte nuclei stain purple.

Yeasts and bacteria stain dark blue.

Giemsa stain

Giemsa stain can also be used to stain films of unformed faeces, faecal exudate, duodenal aspirates etc.

Method

1.         Make a thin film of faeces/exudate. Allow to air dry.

2.         Fix in methanol for 1 minute.

3.         Tip off the methanol and flood the slide with R66 formulation Giemsa stain diluted 1:10 with buffered distilled water pH 7.2 The diluted stain must be freshly prepared each time

4.         Stain for 20-25 minutes.

5.         Run tap water on to the slide to float off the stain and to prevent deposition of precipitate on to  the film. 

6.         Examine the film using the oil immersion objective.

Cytoplasm, parasites etc stain as described for Field's stain.

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Infrequently seen parasites.

Cyclospora cayetanensis.

Cyclospora cayetanensis is a coccidian protozoan that has been described in association with diarrhoeal illness in various countries. Most notably Nepal, India and Pakistan. In fresh stools they appear as spherical oocysts, measuring 8-10µm when examined by light microscopy in an unstained wet preparation, a refractile "cyst-like" structure with a central morula, containing several refractile spheres may be seen.

When incubated in potassium dichromate solution (5%) at room temperature the central bodies of the parasite differentiate to form two sporocysts. The sporocysts mature and rupture to release sporozoites. The complete lifecycle and mode of transmission remain to be described.

The oocysts can be successfully concentrated by the formol-ether technique. The oocysts auto-fluoresce with a blue colour in UV light.  

NB. UV microscopes set up for FITC and auramine microscopy only (450-500nm wavelength) will fail to detect the autofluorescence of Cyclospora.

Iodine-quartz sourced microscopes must be fitted with a 340-380nm excitation filter to demonstrate autofluorescence.

The oocysts are variably acid-fast when stained with the modified Ziehl-Neelsen method.

Regrettably, the oocysts of Cyclospora do not stain well with Phenol - Auramine

Environmental data suggest that Cyclospora cayetanensis is a water-borne parasite. Patients from whose stools Cyclospora have been isolated have reported nausea, vomiting, lethargy, weight loss and explosive watery diarrhoea. Flatulence and bloatedness are associated symptoms. The site of infection is the small bowel.

Further reading.

Diagnosis of Cyclospora cayetanensis infections.

Communicable Disease Report. volume 7. Number 37. 12^th September 1997.

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Blastocystis hominis.

Introduction.

Brumpt first described Blastocystis hominis, an inhabitant of the human intestinal tract, in 1912. Over the years, this organism has been classified as an organism related to Blastomyces, the cyst form of a flagellate and yeast of the Schizosaccharomyces. Due to extensive work of Ziert and others, this organism has finally been classified as a protozoan. It has been suggested that it be placed in a new class, Blastocystea, and a new order, Blastocystida.

Life cycle and morphology.

B. hominis is capable of pseudopod extension and retraction, reproduces by binary fission and has a membrane bound central body (previously called a vacuole) that takes up approximately 90% of the cell. The function of the central body has not been determined. The organism is strictly anaerobic, normally requires bacteria for growth, and is capable of ingesting bacteria and other debris. The classic form that is usually seen in the human stool specimen varies tremendously in size from 6 - 40µm and is characterised by the large central body and a thin peripheral rim of cytoplasm containing 1 - 5 irregularly distributed “nuclei”. The amoebic form can occasionally be seen in diarrhoeal samples but may be extremely difficult to recognise. Generally B. hominis will be identified on the basis of the more typical round form with the central body.

Laboratory diagnosis.

Routine stool examinations are very effective in recovering and identifying B. hominis. If present in very large numbers they can easily be seen on a direct saline wet preparation. A note of caution, the trophozoite or amoebic form of the parasite is easily lysed by water. In unstained formol - ether concentrations the parasite is usually seen as small refractile bodies, not unlike a partially lysed red cell. In iodine stained preparations the central body can be seen evenly stained yellow with a very pale cytoplasmic rim. However, the permanent stained smear is the procedure of choice. Romanowsky stains such as Giemsa or rapid Field’s are highly recommended for a routine diagnostic laboratory.  Longer and more complicated staining methods such as trichrome and iron haematoxylin stains are more popular in USA laboratories.

The organisms should be quantitated; this is normally expressed as a numerical value per high power (x 1000 oil immersion) field.

Clinical disease??

The precise role of B. hominis in causing human disease is not clear and many workers are currently pursuing this field of clinical research. Generally speaking, when this organism is present in large numbers in the absence of other disease causing agents it may be the cause of diarrhoea, abdominal pain and cramps, nausea, fever and vomiting and require therapy. In those symptomatic patients in whom no other aetiological agent has been identified, B. hominis should be considered as the possible causative agent.

Epidemiology.

From present information, it appears that B. hominis is transmitted via the faecal-oral route through contaminated food or water. The cystic stage is probably the infective form of the organism, although this is unproven.

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Dientamoeba fragilis.

Introduction. 

Dientamoeba fragilis was first seen in 1909 but not described in detail until 1918. Based on modern electron microscopy studies it has been reclassified as an amoeba-flagellate rather than an amoeba and is closely related to Histomonas and Trichomonas. It has a cosmopolitan distribution and past surveys provide incidence rates from 1.4% to 19% depending on the type of population surveyed. Much higher incidence figures have been reported for mental institutions, prison inmates and nursery school children.

Life cycle and morphology.

The life cycle and mode of transmission are not known, although transmission via helminth eggs such as Ascaris and Enterobius has been postulated.

There is no cystic stage. The trophozoite is characterised as having one (20-40%) or two (60-80%) nuclei. The nuclear chromatin is usually fragmented into 3-5 granules and there is normally no peripheral chromatin on the nuclear membrane. The cytoplasm is usually vacuolated and may contain ingested debris as well as some large uniform granules. The cytoplasm can also appear uniform and clear with few inclusions. There can be considerable size and shape variation among organisms, even on a single smear. D. fragilis lives in the lumen of the caecum and upper colon.

Laboratory diagnosis. 

There is no cystic stage; these organisms will not be seen in formol - ether/ethyl acetate concentrations. The trophozoite stage is extremely difficult to detect in wet saline preparations. Therefore, it is only possible to detect these organisms by stained faecal smears.

Freshly passed or preserved faeces are essential for accurate identification because survival time in terms of morphology is limited. Romanowsky stains such as Giemsa or rapid Field’s are highly recommended.  

Clinical disease??

This is a controversial area. The organism has been reported in association with mucous diarrhoea, abdominal pain and tenderness. The most common symptoms appear to be intermittent diarrhoea and fatigue. The precise role of this organism as a cause of disease remains to be determined.

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Microsporidia - general characteristics. 

All are obligate intracellular parasites. The vast majority of species are in invertebrates, especially insects, lower vertebrates and fish. Only a few have been reported from warm-blooded vertebrates.

They are considered to be primitive organisms. Their evolutionary history has been predicted from their prokaryote-like ribosomal characteristics - the absence of a separate 5.8S rRNA and the nucleotide sequence of the small subunit (16S) rRNA. They have no mitochondria. The infective stages are highly resistant spores. These are very uniform in size for a given species.

When a new host ingests spores, the cells are penetrated by means of an apparatus known as the polar tube. When this is fully extended the sporoplasm passes through the tube, to be inoculated into the cytoplasm of the host cell.

Following infection there follows a phase of multiplication by binary or multiple fission (merogony). The transition to the spore producing stage (sporogony) is heralded by the secretion of an electron dense surface coat - this will form the future exospore layer of the spore wall. The primary sporogonic cells are sporonts, which divide into sporoblasts, which mature into spores, which are released when the host cell ruptures.

Common species of microsporidia reported from humans.

Infections of the gastro-intestinal tract and urinary system can be detected by the presence of spores in faeces or urine. Spores from these sites can be visualised by staining them with the modified trichrome stain.

The spores of microsporidia are very small - 1 x 0.5 µm  

Enterocytozoon bieneusi is the most common microsporidial species found in AIDS patients. In the main, AIDS patients with a CD4 T-lymphocyte count below 100 x 10⁶ /L are more likely to become infected.

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The great Entamoeba histolytica debate.

Entamoeba histolytica or Entamoeba dispar?                   

There are large numbers of species of amoebae which parasitise the human intestinal tract. Of these Entamoeba histolytica is the only species found to be capable of causing disease. The parasite has been observed to be the causative agent of two very differing clinical presentations.

1. The commensal or non-invasive luminal form where the parasite induces no signs or symptoms of disease.

2. The pathogenic or invasive form where the parasite invades the intestinal mucosa and produces dysentery or amoebomas and through bloodborn spread gives rise to extraintestinal lesions, mainly in the liver.

Entamoeba histolytica is conservatively estimated to infect 500 million people world-wide. However, only about 10% develop clinically invasive disease. For many years wide varieties of workers have posed the questions of why do some people and not others develop invasive disease? Is this due to a variety of reasons such as host response, nutrition, and geographical location or is Entamoeba histolytica in fact two separate species.

A bit of parasitology history.

In 1918 Dobell proposed his "Promethean hypothesis" in which he stated quite eloquently "Entamoeba histolytica is, unlike most parasitic amoebae - a tissue parasite. It lives in and upon the living tissues of its host and it can exist in no other way. The ideal conditions for host and parasite alike is a state of equilibrium, like that between Prometheus and the eagle - the former regenerating sufficient tissue each day to compensate the ravages of the latter". Dobell proposed that there was the state of equilibrium between man and parasite, which he named the "carrier state" and the "dysenteric state" where the "amoebae devour more tissue than man can regenerate, man no longer living at peace with his parasites, but engaged in a life and death struggle with them".

Addressing the dichotomy in the course of infection, Brumpt (1925) proposed the existence of two morphologically indistinguishable species, one a potential pathogen, the other a harmless commensal. Unfortunately, his theory gained few supporters!

Sargeaunt and Williams (1978) conclusively proved that invasive and non-invasive strains of Entamoeba histolytica could be differentiated by isoenzyme electrophoresis.

After more than 70 years of intermittent debate over the true relationship between the "pathogenic" and "non-pathogenic" forms of Entamoeba histolytica molecular biological techniques have finally yielded an unambiguous answer. Molecular biologists studying amoebic genomic DNA and ribosomal RNA now conclude that there are two distinct genetic entities that just happen to be morphologically indistinguishable. In fact, it has been estimated that the genetic distance between the rRNA of these two organisms is comparable to that between human and mouse. The pathogenic or invasive species is to retain the name E. histolytica. The non-pathogenic or non-invasive species to be named E. dispar.

Laboratory identification.

Identification of amoebic cysts by the tried and tested methods of microscopy and micrometry has been complicated by recent developments in molecular biology. Laboratories identifying E. histolytica/dispar cysts in clinical samples are advised to refer samples containing cysts from cases of clinically suspected amoebic disease to a reputable reference facility. Conversely, if their regional/area demand warrants it, to purchase a kit specifically designed to differentiate between the two species.

E. histolytica/E. dispar?

E. histolytica and E. dispar are intestinal parasites that infect approximately 500 million people worldwide annually. Only malaria and schistosomiasis are believed to be more prevalent parasitic causes of morbidity and mortality. Of the huge number of persons infected, most are infected with E. dispar, which has not been associated with disease. Infection with E dispar rather than E. histolytica is believed to explain, at least in part, the low rate of disease considering the high rate of infection. Approximately 10% of the 500 million people infected each year are infected with E. histolytica . These individuals become symptomatic and develop colitis and liver abscess, resulting in a mortality rate estimated between 40,000 and 120,000 persons annually.

It is important to distinguish between these two species because E. dispar is not associated with colitis or liver abscess. Inaccurate diagnosis may result in unwarranted and unnecessary drug treatment.

Patients infected with E. histolytica may exhibit a wide range of conditions. Many are completely asymptomatic. These persons shed millions of cysts daily and represent a potential reservoir for dissemination. Some patients may show mild diarrhoea that develops into bloody diarrhoea with abdominal cramps, eventually resulting in fulminant colitis. Because of the tissue damage that can occur, perforation of the intestine may result and the amoebae may disseminate to other parts of the body. Approximately 10% of persons with invasive amoebiasis develop liver abscess.

E. histolytica exists either as a trophozoite or as a cyst. Humans serve as the primary reservoir, with organism being spread through ingested food and contaminated water, or by venereal transmission. The cyst is very environmentally stable. Once it enters the intestine, it begins to divide into trophozoites that bind to the intestinal mucosa via a galactose or N-acetyl-D-galactosamine-binding lectin referred to as the galactose adhesin. Once the trophozoites have attached, they release tissue-damaging enzymes and proteins that lyse the mucosal cell. The galactose adhesins of E. histolytica and E. dispar cross-react serologically but contain distinct epitopes. The adhesin is antigenically conserved but monoclonal antibodies can be used to distinguish the adhesin from E. histolytica and E. dispar.

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