Parasitology

PARASITOLOGY PRESERVATION METHODS

Preservation of parasites in faecal samples is not only important for maintaining parasite structure during transportation but also as a means of preserving parasites for future quality control and training purposes.The following preservatives are most commonly used in clinical laboratories and have different effects on the various stages of parasites.

SCHAUDINNS FIXATIVE Intersep Product Code 1462 (250ml)

Saturated mercuric chloride (HgCl2) in distilled water.The Stock solution is mixed 2:1 in ethyl alcohol. For use: Add 5 ml glacial acetic acid to 95 ml of the stock solution. Prepare faecal smears without allowing the smears to dry and place them immediately in Schaudinns fixative for 1 hour. The reagents used in the preparation of this fixative are hazardous and should be handled with care.The working reagents should be prepared fresh for use. This fixative is particularly good for making permanently stained faecal smears of protozoan trophozoites.

10% FORMALIN FIXATIVE IN WATER Intersep Product Code 1463 (250ml)

Formalin (10%) in distilled water. This preservative is a good overall fixative and will fix both ova and cysts although it only preserves the internal morphology of the cysts for up to 6 months, after which the cytoplasm of the organism becomes granular with poor nuclear definition. Trophozoites do not preserve well in formalin and parasite morphology is not maintained adequately for a permanently stained faecal smear.

POLYVINYL ALCOHOL (PVA) Intersep Product Code 1466 (250ml)

95% ethyl alcohol in Glycerol with Schaudinns fixative and Polyvinyl alcohol powder. To use, emulsify 1 part of faeces in 3 parts of PVA solution. This method will preserve ova, larvae and trophozoites well, but cysts may show some distortion. However some ova and cysts do not concentrate well when preserved in PVA. Faecal smears made from the faeces/PVA mixture and allowed to dry can be used for the permanent staining of trophozoites. Before staining, the slides must be placed in 70% ethyl alcohol containing 10 drops of Lugols iodine to remove the mercuric chloride.

FORMALIN 10% NEUTRAL SALINE BUFFERED Intersep Product Code 1469 (250ml)
Formalin (10%) in 0.85% saline. This preserves the morphology of cysts better than that of 10% formalin solution. As with 10% formalin, trophozoites do not preserve well in formalin-saline and parasite morphology is not maintained adequately for a permanently stained faecal smear. It is recommended that 1 part of stool be mixed with 3 parts of formalin/formalin-saline preservative for the storage of bulk specimens.

BAYERS SOLUTION Intersep Product Code 1464 (250ml)

A mixture of CuCl2 in 20% v/v formaldehyde and Glacial acetic acid. Dilute stock solution 1 in 10 with distilled water before use. 1 part of faeces is mixed with 1 part of Bayers solution.This technique is useful for the preservation of cyst morphology.

SODIUM ACETATE-ACETIC FORMALIN ACID SOLUTION (SAF) Intersep Product Code 1461 (250ml)
Sodium acetate in Glacial acetic acid with 40% Formalin solution in distilled water. This preservative has the advantage of not containing mercuric chloride which both Schaudinns and PVA fixatives contain. This renders it less hazardous and more user friendly. This method preserves helminth eggs and larvae, protozoan cysts and trophozoites. This preservative can also be used for the formol-ethyl acetate concentration technique and for making permanent faecal smears.

MERTHIOLATE-IODINE-FORMALIN (MIF) Intersep Product Code 1465 (250ml)

A mixture of Glycerol, formaldehyde and thimerosal (or Tincture of Merthiolate 1:1000) in distilled water. For use the stock solution is mixed with Lugols iodine. Add approximately 1 gram of faeces to 5 ml of MIF solution and emulsify well. Ova, cysts and larvae can be preserved in MIF for several months.

Formol-ethyl acetate concentration methods can be performed on samples preserved by any of the above using Parasep

Parasitology
PARASITOLOGY STAINING METHODS
Temporary Staining Methods for Protozoa
Stains for wet preparations following concentration by the formol-ethyl acetate method.

Permanent Staining Methods

Faecal smears are made for the following reasons: Provide information on the exudate present. (Romanowsky stains) Helpful in accurately identifying flagellates. (Romanowsky stains, Iron haematoxylin) When parasites cannot be detected in either the direct wet preparation or concentrated deposit a permanent stain of a fresh faecal smear can reveal the presence of intestinal parasites. (Romanowsky stains,Trichrome stain, modified Ziehl-Neelsen) Useful in demonstrating the nuclear patterns of cysts and trophozoites thus facilitating identification. (Iron haematoxylin,Trichrome stain)

LUGOLS IODINE SOLUTION Intersep Product Code 1486 (250ml)

Reagent contains: Potassium iodine, Iodine, distilled water, glacial acetic acid. The addition of iodine to a stool concentrate highlights the internal inclusions of cysts; e.g. the nuclei and glycogen mass, thus aiding their identification. For example, the addition of iodine enhances refraction of the nuclei of Endolimax nana, stains the peripheral chromatin of the nuclei of Entamoeba species and demonstrates the welldefined glycogen mass which is a feature of pre-cysts or immature cysts of E. coli and cysts of Iodamoeba butschlii. Iodine does not stain the body of Entamoeba species. For further diagnosis of Entamoeba use Intersep Kit, product code 16219

1. ROMANOWSKY (FIELD A & B) STAINS 1.1 Modified Rapid Fields Stain Intersep Product Code 1482, 1483 (250ml)
This is a modification of Fields stain enabling rapid staining of fixed thin films of various clinical samples.This particular staining method is very useful for staining faecal smears, faecal exudate and duodenal aspirates. Method a) Make a thin film of faeces/exudate. Allow to air dry. b) Fix in methanol for 1 minute. c) Flood slide with 1 ml of Fields stain B (diluted 1:4 with distilled water) d) Immediately add an equal volume of undiluted Fields stain A, mix well and allow to stain for 1 minute. e) Rinse well in tap water and drain dry. Flagella, cilia and nuclei stain red and the cytoplasm stains blue.

ACRIDINE ORANGE Intersep Product Code 1480 (250ml)

Reagent contains: Acridine orange, glacial acetic acid, buffered water pH 6.8. Examine the deposit under UV light after 30 minutes. The addition of acridine orange to a faecal concentrate highlights the chromidial bars of Entamoeba coli, Entamoeba histolytica/dispar and Entamoeba hartmanni, which fluoresce bright green.

EOSIN/SALINE Intersep Product Code 1467 (250ml)

Reagent: 1% eosin in physiological saline Emulsify faeces directly in a warm 37C solution of eosin in saline.The amoebae are easily seen unstained against a pink background.The coarse, granular endoplasm can be differentiated from the clear, colourless ectoplasm. This stain is useful for the detection of motile trophozoites of Entamoeba species.

1.2

Giemsa stain Intersep Product Code 1484 (250ml)

Giemsa stain can also be used to stain films of unformed faeces, faecal exudate and duodenal aspirates. Method a) Make a thin film of faeces/exudate. Allow to air dry. b) Fix in methanol for 1 minute. c) Tip off the methanol and flood the slide with Giemsa stain diluted 1:10 with buffered distilled water. The diluted stain must be freshly prepared each time. d) Stain for 20-25 minutes. e) 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. f) Examine the film using the oil immersion objective. Flagella, cilia and nuclei stain red and the cytoplasm stains blue.

Parasitology
PARASITOLOGY PRESERVATION METHODS
A permanent Romanowsky stain (i.e. Giemsa or Rapid Fields stain) should be used for bloody diarrhoea and for semi-formed stools with blood and/or mucus. It provides information on: The exudate present. The presence of flagellate trophozoites. The presence of protozoa which are not readily detected or difficult to detect in the wet preparation eg. Dientamoeba fragilis and Blastocystis hominis. The presence of protozoa which are destroyed by the formol-ethyl acetate concentrate eg. Giardia lamblia.

Oocysts of Cyclospora cayetanensis

The oocysts of Cyclospora cayetanensis can be seen in formolethyl acetate concentrated stool samples. Alternatively they can be seen when stained with modified Ziehl-Neelsen where they exhibit variable staining; some cysts being acid fast whereas others appear as a round hole against the background Some are seen as glassy wrinkled spheres.

3. PHENOL- AURAMINE STAIN Intersep Product Code 1481 (250ml)

This stain can be used as an alternative to the modified ZiehlNeelsen stain for staining oocysts of Cryptosporidium parvum. Method a) Make faecal smears as for ZN and fix in methanol. b) Stain with phenol-auramine (Lemperts) for 10-15 minutes. c) Rinse thoroughly in tap water. d) Decolourise in acid alcohol. (as for ZN) e) Rinse thoroughly in tap water. f) Counterstain with 0.1% potassium permanganate for 30 seconds. g) Rinse thoroughly in tap water, allow to air dry. h) Do not blot dry as many brands of blotting paper will fluoresce. i) Observe the films with blue light under an incident light fluorescent microscope and low power followed by oil immersion x100 objective if oocysts are suspected. The oocysts of C ryptosporidium parvum appear as bright yellow discs against a dark background. The oocysts of Isospora belli and Cyclospora cayetanensis do not fluoresce well using the phenol-auramine stain.

2. MODIFIED ZIEHL-NEELSEN Intersep Product Code 1468 (250ml)

Use of the modified Ziehl-Neelsen stain for faecal smears has already been established for coccidian protozoa, in particular, oocysts of C ryptosporidium species, but it is also useful to confirm the presence of oocysts of Isospora belli and Cyclospora cayetanensis. Method a) Faecal smears are made either directly from the stool sample or from the concentration deposit. b) Allow to air dry. c) Fix in methanol for 3 minutes. d) Stain with strong carbol fuchsin for 15-20 minutes. e) Rinse thoroughly in tap water. f) Decolourise in acid alcohol (1% HCl in methanol) for 15-20 seconds. g) Rinse thoroughly in tap water. h) Counterstain with 0.4% malachite green (or methylene blue) for 30-60 seconds. i) Rinse thoroughly and air dry. j) Examine using x40 and x100 objectives.

4. TRICHROME STAIN Intersep Product Code 1490 (250ml)

The trichrome method for staining protozoa is especially recommended for identifying features of amoebic cysts and trophozoites. Solution A: Schaudinns fixative or SAF. See Preservation methods, paragraph (e). Solution B: Iodine alcohol. Solution C: Acid ethanol. Glacial acetic acid 0.5 ml 90% ethanol 100 ml Method a) Make a thin smear of the faecal material on a glass slide. b) While the smear is still wet, immediately place the slide in a coplin jar containing Schaudins fixative or SAF (solution A.) for 5 minutes at 50C or 1 hour at room temperature.

Oocysts of Cryptosporidium parvum

Oocysts of C ryptosporidium parvum concentrate well using Parasep but not with standard concentration techniques and are identified using various staining techniques. Using the modified Ziehl-Neelsen stain, the oocysts are acid-fast. However, staining within a smear and between specimens can vary from unstained, to partial red staining, to complete staining.

Oocysts of Isospora belli

The oocysts of Isospora belli can be demonstrated in faeces after formol-ethyl acetate concentration. Alternatively, they can be seen in a faecal smear stained by modified ZiehlNeelsen where the oocysts stain a granular red colour against a green background.

Parasitology
PARASITOLOGY STAINING METHODS
Staining Iodine alcohol (solution B) 10 minutes. 70% ethanol 3-5 minutes. 80% ethanol 3-5 minutes. Trichrome stain 10 minutes. Acid alcohol (solution C) two immersions of one second each 95% ethanol two immersions 95 % ethanol 5 minutes. 95% ethanol 5 minutes. 100% ethanol 3 minutes. Xylene 5 minutes. Mount with a coverslip using DPX - do not allow the xylene to dry on the slide. Nuclei, chromidial bars, chromatin, red cells and bacteria stain red. Cytoplasm stains blue-green Background and yeasts stain green The spores of Microsporidia are very small - 1.0 x 0.5 m, oval in appearance and frequently exhibit a central band like structure flanked on either side by non-staining areas. Microsporidia spores stain pinkish red against a greenish background (Figure 14).

6. FUNGUQUAL (UVITEX 2B OR CALCOFLUOR) Intersep Product Code 1469 (250ml)

This stain can be used as an alternative to the modified trichrome stain for the detection of spores of Microsporidia species Method a) Stain methanol fixed thin smears in 1% Fungiqual for 15 minutes for 5 minutes. b) Rinse in PBS. c) Dip momentarily in 0.5% Evans Blue. d) Wash in PBS. e) Mount f) Examine under blue light fluorescence (360-370nm). These stains bind to the chitin in the endospore layer of the spore wall and fluoresce brilliant blue-white. As other structures contain chitin, e.g. fungal spores, some experience is needed to differentiate these from Microsporidia - the small size of microsporidian spores helps and a modified Trichrome method will distinguish them from fungal spores. Immature spores with little chitin fluoresce faintly but mature spores fluoresce brightly.

5. MODIFIED TRICHROME STAIN Intersep Product Code 1489 (250ml)

A modified version of the trichrome stain is recommended, on thin faecal smears, for the detection of species of Microsporidia in immunocompromised patients with diarrhoea. Thus ill patients may not need to be subjected to invasive biopsy techniques in order to look for the organism. Reagent contains: Chromotrope 2R (Gurr), Fast Green , Phosphotungstic acid. Gloves must be worn when weighing the reagents. Mix the components together in 3 ml of glacial acetic acid and allow to stand for 30 minutes. Add 100 ml of distilled water. Mix well.

7. IRON HAEMATOXYLIN Intersep Product Code 1487, 1488 (250ml)

Staining with iron haematoxylin is useful to demonstrate the nuclear chromatin pattern and cytoplasmic inclusions of protozoan cysts

References (1984) A fluorescent technique for 1. Moody A.H.

demonstrating the chromatoid bodies and nuclei in the cysts of Entamoeba histolytica/dispar from faecal deposits ; J. Clin Pathol 37; 101-2 2. Moody A.H and Fleck S.L.(1985) Versatile Fields stain; J. Clin Pathol 38(7); 842-3 Casemore D.P. ACP Broadsheet 128, June 1991; Laboratory methods for diagnosing cryptosporidiosis Weber R et al (1992) Improved light-micriscopical detection of Microsporidia spores in stool and duodenal aspiarates. New Eng. J. Med 326 (3): 161 - 166

NB. the acetic acid does not dissolve the stain powder completely, it is quite normal to see a pink/red granular aggregate. Method a) Suspend unconcentrated stool in 10% formalin (1:3) b) Spread a small drop of stool suspension very thinly over 2/3 surface of a slide. c) Dry and fix in methanol for 5 minutes. d) Stain in Trichrome for 90 minutes at room temp. or stain for 10 minutes at 50C . e) Rinse in acid alcohol (4.5 ml glacial acetic acid in 95.5 ml of 90% ethanol) for 10 seconds. f) Rinse in 95 % ethanol until excess stain has washed off the slide. g) Dehydrate in 95% ethanol for 5 minutes. h) Dehydrate in 100% ethanol for 5 minutes. i) Clear in xylene for 5-10 minutes. j) Mount in DPX while still wet using a 22x40 coverslip and examine under oil immersion.

3. 4.

Parasitology
PARASITOLOGY PRESERVATION METHODS
All Intersep stains, reagents and fixatives are supplied prediluted ready for use.

Ordering Information
Product FIXATIVES
FORMALIN 10% NEUTRAL BUFFERED SAF FIXATIVE SCHAUDINNS FIXATIVE 10% FORMALIN FIXATIVE IN WATER BAYERS SOLUTION MERTHIOLATE-LODINE-FORMALIN (MIF) POLYVINYL ALCOHOL (PVA)

Pack Size
250ml 250ml 250ml 250ml 250ml 250ml 250ml

Ordering Code
1460 1461 1462 1463 1464 1465 1466

REAGENTS
MAYERS GLYCERIN/ALBUMIN PHYSIOLOGICAL SALINE TRITON X-100 SOLUTION ETHYL ACETATE ACETONE GRAMS DECOLOURISE 100ml 100ml 50ml 250ml 250ml 250ml 1470 1471 1472 1473 1475 1476

STAINS
EOSIN/SALINE MODIFIED ZIEHL-NEELSEN FUNGUQUAL (UVITEX 2B OR CALCOFLUOR) GRAM STAIN SAFRANIN NEUTRAL RED ACRIDINE ORANGE (ACETATE BUFFERED) AURAMINE PHENOL (LEMPERT) FlELD STAIN-SOLUTION A FIELD STAIN-SOLUTION B GIEMSA STAIN-RAPID IODINE-ALCOHOLIC LUGOLS IODINE-AQUEOUS IRON HAEMATOXYLIN-SOLUTION A IRON HAEMATOXYLIN-SOLUTION B TRICHROME FOR MICROSPORIDIA TRICHROME FOR PROTOZOA CRYSTAL VIOLET GRAMS FUCHSIN 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 250ml 1467 1468 1469 1491 1493 1494 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1492 1495

MOUNTING MEDIA
DPX MOUNTING MEDIA IMMERSION OlL MICROSCOPE SLIDES COVER GLASSES CARDBOARD SAMPLE SPOONS 100ml 50ml x100 x100 x50 1520 1521 1522 1523 1524