Participants on the one-day teaching course are provided with a subject specific illustrated bench book.
[Field's] [Giemsa] [Cyclospora] [Blastocystis] [Dientamoeba] [Microsporidia]
note: This procedure is very important. Microscopes must be recalibrated after
every major service.
The eyepiece scale is divided into 100 small divisions
The stage micrometer (calibration slide) scale is 1 mm divided
into 100 divisions, each division is equivalent to 10µm.
Insert the eyepiece scale (round glass disc) into the eyepiece by
removing the uppermost lens and placing the scale on field stop.
Insert the eyepiece into the microscope.
Place stage micrometer on microscope stage
Focus low power objective on stage slide.
Adjust stage and eyepiece scales until eyepiece scale (numbered) lies
along the single line of the stage scale.
Note number of eyepiece divisions and its appropriate stage measurement,
eyepiece divisions = 125 µm
10 eyepiece divisions = 25 µm
From this reading work out value for one eyepiece division.
a) 50 eyepiece divisions = 125
eyepiece division = 125/50 µm = 2.5 µm
10 eyepiece divisions = 25
eyepiece division = 25/10 µm = 2.5 µm
Repeat for all objectives, note readings and keep in a prominent place
near the microscope.
is recommended that a separate eyepiece be kept with the micrometer disc inside.
Ridley & Allen modification formol-ether concentration method for ova and cysts.
formalin water (100ml formaldehyde + 900ml distilled water)
2. **** ** Ether or ethyl acetate.
40 mesh (425µm) brass wire filter - 3" diameter (Endcott Sieves
Ltd. Lombard Road, London SW19 3BR) Nylon
coffee strainers provide a low cost alternative.
Small 3" porcelain or stainless steel dish.
Using orange sticks, select a quantity of faeces (approx 1 g) to include
external and internal portions.
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.
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
1100g or 3,000 rpm. for 3 minutes.
7. Loosen the fatty plug with an orange stick and pour the supernatant away
by quickly inverting the tube.
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.
the x10 and x40 objectives to examine the whole of the deposit for ova
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.
See the PHE web site pages; UK Standards for Microbiology Investigations (SMI B31 Investigation of Specimens other than Blood for Parasites)
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
are no adult helminths that inhabit the urinary tract but ova of several species
are found in the urine, mainly Schistosoma haematobium.
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)
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.
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.
Gently push the urine through.
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.
a drop of saline and a coverslip and observe under the microscope.
membranes may be obtained from Corning Costar®
for wet preparations following concentration by the formol-ether/ethyl acetate
potassium iodide to the distilled water; when dissolved, add the iodine
crystals. Store in a brown bottle. This reagent remains stable for many weeks.
equal parts of reagent 1 and 2 for use.
Iodine stains glycogen brown and the nuclear chromatin of amoebic cysts brown/black.
stain is made from two component stains. Field's stain A and Field's stain B
using buffers of differing pH it is possible to stain a wide variety of clinical
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.
Make a thin film of faeces/exudate. Allow to air dry.
Fix in methanol for 1 minute.
Flood slide with 1 ml of Field's stain B (diluted 1:4 with distilled
Immediately add an equal volume of undiluted Field's stain A, mix well
and allow to stain for 1 minute.
Rinse well in tap water and drain dry.
nuclei and structures containing chromatin stain red.
nuclei stain purple.
Yeasts and bacteria stain dark blue.
stain can also be used to stain films of unformed faeces, faecal exudate,
duodenal aspirates etc.
Make a thin film of faeces/exudate. Allow to air dry.
Fix in methanol for 1 minute.
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
Stain for 20-25 minutes.
Run tap water on to the slide to float off the stain and to prevent
deposition of precipitate on to the
film. Allow to drain dry
Examine the film using the oil immersion objective.
Cytoplasm, parasites etc stain as described for Field's stain.
is a coccidian protozoan that has been described in association with diarrhoeal
illness in various countries. Most notably Nepal, India and Pakistan.
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.
oocysts can be successfully concentrated by the formol-ether technique.
UV microscopes set up for FITC and auramine microscopy only
wavelength) will fail to detect the autofluorescence of Cyclospora.
-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.
Oocysts of Cyclospora
do not stain well with Phenol - Auramine
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.
of Cyclospora cayetanensis infections.
Disease Report. volume 7. Number 37. 12th September 1997.
Brumpt first described Blastocystis hominis, an inhabitant of the human intestinal tract, in 1912.
This organism has finally been classified as
part of a diverse group of organisms called Stramenopiles.
Life cycle and morphology.
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
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.
organisms should be quantitated; this is normally expressed as a numerical value
per high power (x 1000 oil immersion) field.
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,
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.
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
Life cycle and
life cycle and mode of transmission are not known, although transmission via
helminth eggs such as Ascaris and Enterobius has been postulated.
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.
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 reliably
these organisms by stained faecal smears.
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.
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.
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
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.
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.
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.
of microsporidia reported from humans.
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.
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.
Entamoeba histolytica or
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.
The commensal or non-invasive luminal form where the parasite induces no signs
or symptoms of disease.
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.
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.
bit of parasitology history.
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".
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
(1978) conclusively proved that invasive and non-invasive strains of
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.
of amoebic cysts by the tried and tested methods of microscopy and micrometry
has been complicated by recent developments in molecular biology. Laboratories
E. histolytica/E. dispar?
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
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.
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
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.
Real time PCR technology is now available to aid differentiation between E. histolytica and E. dispar.