Preservation

Whenever specimens cannot be examined immediately after collection, a portion or the whole sample can be preserved for later examination. This procedure maintains parasites in their existing morphological form and structure. There is no perfect fixative and the method of preservation is usually determined by the particular organism a. Formalin. While protozoan trophozoites are destroyed by formalin, cysts, ova, and larvae are well preserved. The stool specimens should be preserved within one hour after collection. A five percent formalin is used for protozoan cysts, for ova of Hymenolepis species, and for larvae. The 10 percent solution is used for the other organisms.

suspected.

Neutral Buffered Formalin

Formaldehyde

Distilled water

Sodium phosphate monobasic

Sodium phosphate, dibasic, anhydrous

90.00 ml 95.00 ml

Carefully mix the reagents and swirl vigorously. Store in an aspirator bottle or in a one liter glass-stoppered bottle.

(2) Procedure.

Mix a portion of feces with three times the amount of formalin.

Allow to settle for at least one hour and store in screw-cap bottles.

If shipment or prolonged storing is required, dip the top portion of the tightly fastened container, to include the cap, two or three times into a hot paraffin bath. This will prevent spillage and reduce the rate of evaporation.

(a) Formalin is the most commonly employed reagent for fixing and preserving specimens. Formaldehyde solution is a near saturated solution of gas and water. Such a solution contains from 37 to 40 percent formaldehyde with nine volumes of water as 10 percent formalin. This 10 percent formalin dilution contains approximately 4.1 to 4.5 parts formaldehyde gas.

(b) Formalin preserved specimens stored for reference material should have the preservative changed every six months. The formalin volume should be maintained at all times.

b. Polyvinyl Alcohol (PVA). This is a mixture of fixative and water-soluble resin that is specifically used to fix and preserve trophozoites of intestinal amoebic organisms. These trophozoites are very fragile and will become distorted or disintegrate completely within a few hours after stool passage. This fixative will preserve trophozoites for long periods of time and will make it easier to identify them. PVA is primarily used for preserving fresh specimens to be shipped to central laboratories. Permanently stained films can be prepared from the preserved material. The solution serves as an adhesive as well as a preservative and prevents the loss of organisms during staining. This is advantageous when preparing smears from liquid specimens.

(1) Preparation of reagents. Polyvinyl Alcohol Fixative (PVA) Modified Schaudinn's Fixative.

Glacial acetic acid 5.0 ml

Glycerol 1.5 ml

Schaudinn's fixative (two parts saturated aqueous mercury bichloride to one part 955 ethyl alcohol) 93.5 ml

Polyvinyl alcohol powder (PVA) 5.0 gm

(a) While constantly stirring at room temperature, slowly add the 50 grams of PVA to 1,000 milliliters of modified Schaudinn's fixative.

(b) While stirring, heat gently to about 75 degrees centigrade until the powder dissolves and the solution clears.

(c) Upon cooling, the solution should be clear. Solutions prepared from some lots of PVA may be slightly turbid and contain some precipitate. Unless these are excessive, the solution is satisfactory.

(2) Procedure.

(a) Polyvinyl alcohol (PVA) fixation-preservation in vials.

STEP 1 : Stool specimens should be immediately submitted to the laboratory or fixed at the site of collection.

STEP 2: With applicator sticks, thoroughly mix in a labeled bottle one part (five grams) feces with three parts (15 grams) of PVA fixative. The fixative should be employed at room temperature or up to 50° C. This step should be performed immediately on receipt of the specimen.

STEP 3: Dip the screwcap and top portion of the bottle two or three times into a hot, melted paraffin bath to prevent leakage and evaporation upon storage.

(b) PVA fixation-preservation on microscope slides.

STEP 1: Stool specimens should be immediately submitted to the laboratory.

STEP 2: With an applicator stick, immediately mix one part of the specimen with three parts of PVA-fixative on a microscope slide. Three drops of mixed, bottled, PVA-fixed feces may also be spread onto a microscope slide. (The best yield of trophozoites or cysts is from the finely dispersed upper layer of the sediment).

STEP 3: Spread (do not smear) the mixture over one-third of the slide. To prevent later peeling, extend the specimen to the edges of the slide.

STEP 4: Allow the mixture to dry thoroughly (preferably overnight at 35° C). Dry films remain satisfactory for staining for several months.

(a) Commercially available vials and bottles are available.

(b) If the specimen has been stored for long periods in PVA and become jelled, it can be liquified by heating in a water bath. (Set at 37 to 50° C.)

(c) Specimens preserved in PVA may be maintained for record or for reference study material.

(d) When ordering polyvinyl alcohol powder, specify its use. All grades of PVA are not satisfactory for preserving fecal specimens.

(e) If dehydration occurs, add more fixative.

c. Merthiolate-Iodine-Formaldehyde Fixative (MIF). This solution can be used as a fixative, as a stain for wet preparations, and as a concentration procedure.

(1) Stock reagents.

(a) Merthiolate-Formaldehyde (MF)--Solution A.

Distilled water 250.0 ml

Formaldehyde (saturated) 25.0 ml

Tincture of merthiolate (Lilly 1:1,000) 200.0 ml

Glycerin 5.0 ml

Store in a stoppered brown glass bottle.

(b) Lugol's Iodine Solution (I)--Solution B.

Iodine crystals (powdered) 5.0 gm

Potassium iodide 10.0 gm

Distilled water 100.0 ml

Dissolve the potassium iodide in water. Add the iodine crystals slowly and shake until dissolved. Filter and store in a brown bottle. This solution is stable only for three weeks.

(2) Working reagent. Add 18.0 milliliters of solution A to 1.2 milliliters of solution B immediately prior to use. (For smaller or larger volumes, decrease or increase both components proportionately). Mixing the two solutions too far in advance causes a precipitate to form, thus reducing the staining properties.

(3) Procedure.

(a) Stool specimens should be immediately submitted to the laboratory or fixed at the collection site.

(b) With an applicator stick, thoroughly mix one part feces with 10 parts MIF in a labeled 23 milliliter screwcap bottle. This should be performed immediately on receipt of the specimen.

(c) Dip the screwcap and top portion of the bottle two or three times in a hot, melted paraffin bath. This will prevent leakage and reduce the rate of evaporation.

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Responses

  • jean
    What is the preservation that used in parasitology?
    3 months ago

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