Practical No. -1- - الصفحات الشخصية | الجامعة الإسلامية ...site.iugaza.edu.ps/ahindi/files/2011/10/Diagnostic...-Normal saline solution (9.85% sodium

Adnan I. AL-Hindi Islamic University of Gaza 2005-2006 Diagnostic Parasitology

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Practical No. -1- -Objectives:

-To familiarizes the student with the most widely used technique for detection of parasites. -To be able to identify the parasite stages (adults, larvae, ova, or cysts) . -To learn the- students, how to deal with risk samples. -Materials and reagents needed:

-Microscope, clean slides and coverslip, wooden applicators. -Normal saline solution (9.85% sodium chloride “ Nacl in distilled water). -Pens or markers for labeling.

The Microscope:- The Microscope is the parasitologist’s main tool. If possible the Microscope-

should be binocular, most suitable objectives are the x10, x40, and x100. -The Microscope must be covered and immersion oil removed from the lens -with xylene or ether when not in use.

Calibration of the Microscope Eyepiece Micrometer: (Fig.1,2 ) -On many occasions measuring the size of suspected parasites in faeces is helpful for identification. A measuring eyepiece (eyepiece micrometer) is used, most often in combination with x40 objective, but the eyepiece micrometer must first be calibrated against a standard stage micrometer. The procedure is as follows:

1. Focus the x40 objective on the stage micrometer scale and using the mechanical stage position the stage micrometer and the eyepiece micrometer so that they are parallel and almost overlapping.

2. Examination of the two scales will show two points where the divisions on both scale coincide exactly (A and B on the following diagram).

3. Determine the number of eyepiece micrometer divisions (em) between A and B and the corresponding number of stage micrometer divisions (sm).

4. The stage micrometer measures exactly 1.00mm long and is divided into 100 subdivisions, each 10um. The eyepiece micrometer also has 100 subdivisions. Each of which measures sm microns. In the example, sm=890 divisions and em=70um, therefore one eyepiece micrometer division at that magnification and on that microscope is equal to 890um/70 = 12.7um Calibration should be carried out for each objective likely to be used and will vary between microscopes according to the precise optical equipment in use.

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Laboratory Methods For Parasites In Faeces No technique is 100% successful in detecting parasites by a single stool examination, and at least three serial stools must be examined before a patient can be considered free from infections in which stages of parasites would be expected to be found in the faeces. Whilst clinical symptoms or a case history may provide clues as to which parasites may be present, each faecal specimen should be treated as an unknown, as parasite stages unrelated to the clinical picture may be present. -Faecal specimens: -Faecal specimens are examined for the presence of protozoa and helminthes larvae or eggs. -The stages of protozoa found in stools are trophozoites and cysts. The stages of helminthes usually found in stools are eggs and larvae, though whole adult’s worms or segments of worms may also be seen. Adult worms and segments of tapeworms are usually visible to the naked eye, but eggs, larvae, trophozoites, and cysts can be seen only with the microscope. In order to see these structures, the faecal material must be properly prepared and examined. -Collection of faecal specimens: 1. Because of the fragile nature of many intestinal parasites, and the need to maintain their morphology for accurate identification, reliable microscopic diagnosis can’t be made unless the stool is collected properly. 2. Approximately 10 gm of fresh faeces uncontaminated by urine, oil, water, dyes or radio-opaque into a clean plastic container. 3. The container should be free from antiseptics and disinfectants. 4. Label all samples clearly with the patient’s name, reference number, date, and time of collection. 5. All samples should be accompained by a requisition form from the physician giving relevant clinical details and recent travel history. 6. Samples and forms from patients with a confirmed or suspected diagnosis of certain infectious diseases such as AIDS or hepatitis should be clearly labeled with “ Risk of Infection” or “ Biohazard” 7. Most viable parasites are susceptible to desiccation or temperature variation. If time lapse between collection and observation is considerable, i.e. more than 4 days, it may be necessary to add some form of preservative to the faeces to retain the morphology as near to the original as possible. 8. Formed samples can be kept in a refrigerator at + 4c for a short while, but not in incubator. 9. Any whole worms or segments passed should be placed in a separate container.





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-Preservation methods for faecal material:

Preservation allows faecal samples to be examined after a delay in delivery or postage or testing. Many methods for the preservation of stool samples and permanent staining procedures have been described in the literature. The most common fixatives are: Polyvinyl alcohol (PVA), Merthiolate-iodine-formaline (MIF), Sodium Acetate Acetic Acid Formalin (SAF), Formalin and Bayer’s solution. From the permanent stains the Trichrome, Chlorazol Black, and Iron Haematoxylin are the most frequently used procedures. The preservatives used have differing effects on the various stages of the parasites. The Fixatives: Formalin: Formalin (4%) has been used for many years as an all-purpose fixative that is appropriate for helminth eggs and larvae and for protozoan cysts. The preservation of the protozoan trophozoites is not as good as with e.g. PVA or SAF. The fixative can easily be prepared by simply diluting the commercially available Formaldehyde 40% solution with distilled water. The fixative has a long shelf life. Concentration methods, like Formalin-Ether concentration according Ridley and Hawgood, can be performed from the preserved stool samples without loss of “concentration abilities’. The major disadvantage of this fixative is that permanent staining procedures can not be performed from Formalin preserved stool samples. PVA This fixative is recommended for the preservation of the trophozoite and cyst stages of the intestinal protozoa. PVA is also suitable for helminth eggs and larvae. The preservation of the two stages of the protozoa is excellent. The fixative contains PVA powder, Ethyl alcohol, Mercuric Chloride, Acetic acid, and Glycerin. The PVA is a plastic resin that serves as adhesive for the stool material; e.g. when the stool-PVA mixture is spread onto the glass slide prior to staining. The fixative is difficult to prepare in the laboratory. It has a long shelf life (months to years), although it may turn white and gelatinous when dehydration occurs or when refrigerated. Concentration methods (e.g. Formalin Ether concentration) can not be performed from specimens preserved in PVA.. The greatest advantage of this fixative is that a permanent stain can be prepared from the specimen; giving excellent results with the Trichrome staining. The protozoan morphology is unclear when the Chlorazol Black (CB), Iron Haematoxylin Kinyoun (IHK), or mod. Ziehl Neelsen staining procedures (Cryptosporidium spa, and Isospora belli) are performed. Specimens





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preserved in PVA cannt be used with the Immunoassay kits (e.g. G. lamblia specific EIA). Emulsify 1 part faeces in 3 parts of PVA solution. This method will preserve ova, larvae and trophozoites well but cysts may show some distortion. Before staining, the slides must be placed in 70 per cent ethyl alcohol containing 5-10 drops of Lugol’s iodine to remove the mercuric chloride. SAF This fixative is composed of Sodium acetate, Acetic acid, and Formalin and is a good routine fixative for protozoan cysts and trophozoites, helminth eggs, and larvae. SAF is inexpensive, easy to prepare in the laboratory, and has a long shelf life (months to years). The preserved stool samples permits concentration techniques, most monoclonal detection kits (e.g. G. lamblia and Cryptosporidium spp specific EIA, not the E. histolytica/E.dispar EIA), and permanent staining to be used. However, unlike the PVA, the SAF fixative has poor “adhesive properties”; when SAF preserved specimens are used to prepare permanent stained smears, there may be some difficulty in getting material to adhere to the slide. Mayer’s albumin has been recommended as an adhesive. The combination of SAF-preserved material and CB, IHK, and mod. Ziel Neelsen provides excellent staining of protozoans whereas staining of SAF-preserved material with Trichrome gives poor results. MIF This fixative was originally developed as a screening procedure for intestinal parasitosis in field surveys. MIF combines preservation and staining for most kinds and stages of parasites found in faeces; it contains Merthiolate, Iodine, and Formalin. The preserved material permits concentration techniques. The major disadvantages are the short shelf life (due to the Iodine), and the fact those permanent stained smears cannot be prepared from MIF-preserved material. Store in a brown bottle. Add approximately 1 gm faeces to MIF solution and emulsify well. Ova, larvae, cysts and trophozoites are preserved for several months.





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Table1: Fixatives used for the preservation of stool specimens: an overview of the advantages and disadvantages.

Formalin 4% PVA SAF MIF

Toxicity +++ (due to Hg)

+/- +/- +/-

Shelf life Long (months)

Long (months/years)

Long (months/years)

limited

Preparation at laboratory

Easy Difficult Easy Easy

Quality of fixation

Eggs: ++ Cyst: ++ Troph’s: +/-

Eggs: ++ Cyst: +++ Troph’s +++

Eggs: ++ Cyst: +++ Troph’s +++

Eggs: ++ Cyst: ++ Troph’s ++

Formalin Ether concentration

Possible Not possible Possible Possible

Permanent Stained Smear

Not possible Only trichrome IHK CB

Ziel Neelsen

Not possible

SAF, the best compromise? The different advantages and disadvantages of Formalin, PVA, MIF, and SAF are summarized in table 1. Specific advantages of the use of SAF are:

- SAF preserved material can be used for concentration techniques and permanent stained smears (CB, IHK).

- SAF preserved material can be used for some immunoassay methods. - The fixative is easy to prepare and has a long shelf life. - Unlike the PVA, the SAF fixative contains no mercury compounds. It is

therefore much less toxic than PVA. Bayer’s solution: Stock solution: Cu cl2 7 gm 20 percent v/v formaldehyde 11 glacial acetic acid 70 ml Dilute stock solution 1 in 10 with water before use. Mix 1 part faeces with 1 part Bayer’s solution. This technique was found to be very useful, particularly for cysts.





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Preservation of Worms: Cestodes. Wash in water to remove the mucus. Large tapeworms such as Taenia can be washed for several hours to relax the musculature, and can then be fixed in 10% formol saline between two glass slides to give flatter specimens. Specimens can be preserved in fresh 5% formalin. Prolonged staining with dilute Delafield’s or Ehrlich’s haematoxylin followed by decoloursing with acid alcohol and blueing in alkaline tap water is usually satisfactory. Trematodes. These should be treated in a similar manner to cestodes, and mounted with the ventral sucker uppermost. Aceto-carmine (or dilute Delafield’s or Ehrlich’s haematoxylin) will stain small flukes satisfactorily, but it is not so good for large specimens. Decolourising with acid alcohol is usually necessary and the haematoxylin stains require blueing in alkaline tap water. Nematodes. Adults are washed in saline to remove mucus. Worms up to about 7 cm in length ar fixed in hot 70% alcohol (60-70 C), which straightens out living worms, except those which have natural curvatures at the head or the tail. Alternatively, they can be fixed in hot 5% formalin. Large worms such as Ascaris can be fixed and preserved in cold 5% formalin. Adult worms are examined unstained, in lactophenol or glycerine. Larvae can be killed and stained with dilute Gram’s iodine.

-Examination: -Macroscopic examination of stool: -As soon as the specimen is received in the laboratory, check: 1. The consistency (degree of moisture) and write one of the following letters on the container: F (formed), S (soft), L (loose), or W (watery). (Fig. 3).





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2. Abnormal features:If mucus is present write M, and if blood is present write B. For example, a loose stool with blood and mucus would be recorded as L, B, M. The consistency, or degree of moisture, will be a guide as to whether the trophozoite stage or the cyst stage of protozoa is likely to be present. The various categories of stool and the appropriate techniques to be used are shown in Table 1. -If several specimens are received at the same time; those containing blood and mucus should be examined first, followed by liquid specimens. These specimens are the most likely to contain amoebic trophozoites (which die soon after being passed) and must be examined within 1 hour after passage. Formed specimens may be examined at any time during the first day, but should not be left overnight (cyst may disintegrate). 3-Parasites: -Species: worm eggs: give the common English name (together with the Latin name in the case of Schistosomes, flukes and tapeworms). protozoa: give the scientific Latin name. -Scientific first name (the genus): write with a capital letter, e.g. -name Schistosoma. second name (the species): write with a small letter, e.g. mansoni. -Stage eggs, larvae, vegetative forms, worm segments, etc. When describing E. histolytica, always specify whether it contains ingested red blood cells. -Quantity occasional (1-2 eggs per slide); a few (3-5); a moderate number (6-12); many (more than 12).

If no parasites are found, state: “No ova or parasites seen”, and specify whether this result was obtained by direct examination or by a concentration method (name method used). Never state categorically: “No parasites” -Note: The faeces may have adult helminthes or segments present such as Ascaris lumbricoides, Enterobius vermicularis or Taenia sp. Gravid Taenis segments are frequently motile for several days and may migrate to the top of the container. Dark or black stools occur when iron or bismuth is taken or when there is intestinal haemorrhage. Excessive bulky stools may indicate conditions such as giardiasis.





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-How to record the results of Stool Examination: -The following details should be given when recording the results of a stool examination: 1. Consistency of stool=======à 2. Abnormal features seen with the naked eye ===============à 3. Parasites found by microscopic examination specifying -Genus================à -Species===============à -Quantity===============à

Formed, Soft, Loose, Watery. Flakes of mucus present , blood. Giardia lamblia e.g. few 3-5 eggs per slide.

-Microscopic Examination of Wet Mount: -Wet mount is the simplest and easiest technique for the examination of faeces, and this method should be performed in all laboratories at the peripheral level. -A wet mount can be prepared directly from faecal material or from concentrated specimens. The basic types of wet mount that should be used for each faecal examination are saline, iodine, and buffered methylene blue. -The saline wet mount, is used for the initial microscopic examination of stools. It is employed primarily to demonstrate worms eggs, larvae, protozoan trophozoites, and cysts. This type of mount can also reveal the presence of red blood cells and white blood cells. -The iodine wet mount, is used mainly to stain glycogen and the nuclei of cysts, if present. Cysts can usually be specifically identified in this mount. -The buffered methylene blue (BMB) wet mount should be prepared each time amoebic trophozoites are seen in a saline wet mount, or when their presence is suspected. BMB stains amoebic trophozoites, but does not stain amoebic cysts, flagellate trophozoites, or flagellate cysts. BMB stain is appropriate only for fresh unpreserved specimens. It is not used on preserved specimens in which the organisms have been killed. -Direct saline and iodine mounts: (Fig.4. ) 1. With a wax pencil writes the patient’s name or number and the date at the left-hand end of the slide.





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2. Place a drop of saline in the center of the left half of the slide and place a drop of iodine solution in the center of the right half of the slide. Note: If the presence of amoebic trophozoites is suspected, warm saline (37c) should be used. 3. With an applicator stick (match or tooth pick), pick up a small portion of the specimen (size of a match head) and mix the drop of saline. Note: -Formed stool: take the portion of stool from an area to include inside and outside parts of the specimen. -Stool with mucus: if mucus is present, label a second slide with the patient’s name or number. Put a drop of saline on the slide, pick up a small portion of mucus and mix with the saline. Trophozoites, if present, are sometimes more readily found in mucus than in the solid parts of the stool. -Loose watery stool: if mucus is not present, pick up a small portion of the stool (any part) and mix with the saline. 4. Similarly, pick up a small amount of the stool and mix with the drop of iodine, to prepare an iodine mount. If a wire loop is used, flame it after making the mount. If applicator sticks are used, discard them. 5-Cover the drop of saline and the drop of iodine with a coverslip. Hold the coverslip at an angle, touch the edge of the drop, and lower gently on to the slide. This will reduce the chance of including air bubbles in the mount. -Examination: 1. Put the slide with the mounts on the microscope stage and focus on the mount with the x10 or low-power objective. 2. Regulate the light in the microscope field with the substage diaphragm. You should be able to see objects in the field distinctly. Too much or too little light is not good. 3. Examine the entire coverslip area with the x10 objective; focus the objective on the top left-hand corner and move the slide systematically backwards and forwards, or up and down. 4. When organisms or suspicious material are seen, switch to the high-dry objective, and increase the light by opening the substage diaphragm to observe the detailed morphology. -This is a systematic examination. If mounts are examined in this way, any parasites present will usually be found. If the mount is not examined systematically, parasites may be missed. Examine each microscope field carefully, focusing up and down, before moving to the next field.

REMEMBER EXAMINE MOUNTS SYSTEMATICALLY





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Fig.4. Making Direct Smear Microscopy





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Practical No. -1- - الصفحات الشخصية | الجامعة الإسلامية ...site.iugaza.edu.ps/ahindi/files/2011/10/Diagnostic...-Normal saline solution (9.85% sodium