The MIF solution can be used for staining protozoan cysts and trophozoites in wet-mount preparations and as a preservative for these stages and helminth eggs and larvae (Sapero and Lawless, 1953). Slightly different formulas are recommended for these two

uses.

PREPARATION

1. Wet-Mount Preparations

Lugol's solution (see below)

Formaldehyde (USP).

Tincture merthiolate, No. 99, 1:1,000 (Lilly)*

0.10 ml

0.15 ml

0.75 ml

One ml of stain is sufficient for 25 to 30 fecal preparations. The stain is prepared fresh each day. The amounts of the 3 ingredients may be varied for different purposes. The greater

*Tincture of Merthiolate, No. 99, 1:1,000 (Lilly) (Eli Lilly & Co., Indianapolis, Indiana) must be used, since this contains eosin, the stain ingredient in the second stage.

the iodine strength, the more immediate the staining reaction; however, the cysts and trophozoites are more easily detected at a lower iodine strength. Cytoplasmic detail is more distinct at a greater formaldehyde strength.

Mix solutions and store in brown bottle.

B. Lugol's Iodine Solution (Prepared Fresh Every 3 Weeks)

50 ml

5 ml

40 ml

1 ml

5 gm

10 gm

100 ml

Dissolve the potassium iodide in the distilled water. Add the iodine crystals slowly and shake until dissolved. Filter. Store in a brown bottle.

Mix solutions A and B together as indicated in item 2 below to prepare the MIF preservative.

TECHNIQUE

1. Wet Mounts

a. Place 1 drop of distilled water on a slide using a standard medicine dropper.

b. Add 1 drop of MIF stain (prepared for wet mounts).

c. Comminute a portion of the fecal specimen in this mixture. Have the suspension of the correct density for a satisfactory mount. d. Mount with coverslip, seal, and examine. The preparation may be examined several hours later without loss of staining or details.

2. Preservation of Specimens

a. Add 2.35 ml MF stock to 0.15 ml Lugol's solution immediately before use because prior addition of the iodine causes a dense precipitate to be formed and the iodine fails to act satisfactorily on the protozoa.

b. Thoroughly mix a portion of the feces about the size of a pea in the MIF solution (approximately 1 part of feces to 2 or 3 parts of preservative).

Do not use too much feces. For larger samples, use increased amounts of the MIF stain in the same proportions of MF stock and iodine solutions.

c. To examine, take a drop of fluid from the top layer of the sedimented feces and place on a slide. A preparation roughly comparable in density to a direct smear should be obtained.

STAIN CHARACTERISTICS

The staining reaction in both direct smears and preserved specimens is divided into two phases: 1) an iodine phase in which cysts and trophozoites stain yellow-green or yellow-brown and 2) an eosin phase which replaces the iodine phase and is permanent.

In the iodine phase, nuclei stain dark brown and in the eosin phase, dark red to jet black. Cytoplasm of the trophozoites and cysts changes from yellow-green or brown in iodine to an eosin (pink or red) color in the second stage. Occasionally, refractory mature cysts are encountered which fail to stain in the eosin phase. Nuclear elements of all species except Dientamoeba are fairly well defined. Glycogen appears as a dark brown area in the iodine stage and as a colorless area in the eosin stage. Chromatoid bodies are characteristic in appearance. Flagella may be detected on flagellate trophozoites. Helminth eggs also stain and retain their normal characteristics. Fecal debris stains darker brown than parasites or blood and tissue cells. No distortion occurs, but for good contrast, a blue filter should be used in the illumination system.

QUENSEL'S STAIN SOLUTION

The nuclei of amebae trophozoites are indistinct or entirely invisible in saline preparations. Iodine solution is unsatisfactory for staining this stage, since it may distort the entire organism. Quensel's solution is recommended for the study of trophic amebae in temporary preparations (Svensson, 1935) and may be used with unpreserved feces or sediment from cultures.

PREPARATION

A. Stock Solutions

1. Sudan III Saturated Alcoholic Solution

Sudan III powder

80% ethyl alcohol.

1.6 gm

.. 100 ml

Add the stain to the alcohol. Shake thoroughly. Let stand for a few hours or overnight to be sure the solution will be saturated. If all of the stain dissolves, add more powder until no more will go into solution. Filter or decant to remove the excess powder and store in a screw-cap or glass-stoppered bottle. 2. Methylene Blue, Saturated Aqueous Solution Methylene blue powder, medicinal ...

Distilled water..

3.5 gm

100 ml

Add the stain to the water and shake thoroughly. Let stand for a few hours, shake at intervals. If all of the stain goes into solution, add more. Filter. Store in a screw-cap or glass-stoppered bottle.

Dissolve the cadmium chloride crystals in the water. Store in a screw-cap or glass-stoppered bottle.

1. Mix the Sudan III and methylene blue.

2. Add the mixture to the cadmium chloride in an Erlenmeyer flask.

3. Gently shake the mixture now and then for 15 to 20 minutes. A voluminous flocculent precipitate develops and the fluid becomes almost colorless.

4. Filter. Note: The precipitate can be more easily removed from the paper (Step 7) if the material is filtered so that the residue (precipitate) is collected at the bottom of the filter cone rather than spread over a large area.

5. Remove all excess liquid from precipitate by placing the filter paper with the precipitate upon another filter paper. Leave overnight.

6. Transfer precipitate to a fresh filter and rapidly pour through 25 to 30 ml of distilled water.

7. Dissolve the washed precipitate in 250 ml of distilled water.

8. Filter in a few days if fine crystals of cadmium chloride precipitate.

TECHNIQUE

A small amount of the fecal sample or culture sediment is picked up with an applicator stick, toothpick, or pipette (culture) and thoroughly comminuted in a drop of Quensel's solution. The mixture is covered with a coverslip and sealed.

STAIN CHARACTERISTICS

After about 10 to 20 minutes, the amebae trophozoites are stained a pale blue with their nuclei a deeper blue shade. The stained nuclei present the same morphologic characteristics that they do in a permanently stained hematoxylin preparation. Food inclusions within the cytoplasm are also stained. After 1⁄2 to 1 hour, the organisms become overstained and can no longer be identified. Occasionally, organisms fail to stain. The nuclei of Dientamoeba fragilis do not stain well, but the presence of 1 or 2 nuclei may be discernible. The use of a warm stage may aid in the staining of Dientamoeba. Blastocystis hominis stains beautifully, but ciliates, flagellates, and living cysts do not stain. Cysts preserved in Formalin will stain, however, but details are not always easily seen.

•BUFFERED METHYLENE BLUE SOLUTION

The pH of the stain solution has been found the deciding factor in bringing out the morphologic details of the nuclei of protozoan trophozoites in wet mounts. Satisfactory results can be obtained by dissolving biological dyes, such as methylene blue, in an appropriately buffered solution (Nair, 1953). The buffered methylene blue stain can be substituted for Quensel's. Staining results are the same and the buffered methylene blue solution is easier to prepare than Quensel's stain. For staining trophozoites of E. histolytica, the exact