Wash the slide in running tap water (constant stream of water into container) for 1 min.
C. Place the slide in acid-alcohol decolorizer for 2 min.
D. Wash the slide in running tap water (constant stream of water into container) for 1 min.
E. Continue the staining sequence with step 9 (iron hematoxylin working solution).
Procedure Notes for Modified Iron Hematoxylin Stain (Carbol Fuchsin Step)
1. The first 70% alcohol step acts with the Mayer’s albumin to “glue” the specimen to the glass slide. The specimen may wash off if insufficient albumin is used or if the slides are too thick and are not completely dry before being stained.
2. The working hematoxylin stain should be checked each day of use by adding a drop of stain to alkaline tap water. If a blue color does not develop, prepare a fresh working stain solution.
3. The picric acid differentiates the hematoxylin stain by removing more stain from fecal debris than from the protozoa and removing more stain from the organism cytoplasm than from the nucleus. When properly stained, the background should be various shades of gray-blue and the protozoa, with medium blue cytoplasm and dark blue-black nuclei, should be easily seen.
Polychrome IV Stain
Polychrome IV stain can be used in place of trichrome for staining fecal smears by the MIF, PVA, or SAF fixative method. Both the stain and staining directions are available commercially (Devetec, Inc., P.O. Box 10275, Bradenton, FL 34282). Another source for the stain is Scientific Device Laboratory, Inc., P.O. Box 88, Glenview, IL 60025. Polychrome IV stain has been used primarily to stain permanent smears prepared from MIF-preserved fecal specimens.
Chlorazol Black E Stain
Chlorazol black E staining, developed by Kohn (9), is a method in which both fixation and staining occur in a single solution. This approach is used for fresh specimens, but it is not recommended for preserved material containing mercuric chloride (6) because it does not include an iodine-alcohol step, which is used to remove the mercuric chloride compound found in Schaudinn’s fixative (with and without PVA) prepared with mercuric chloride. The optimal staining time must be determined for each batch of fixative-stain. The length of time for which the fixative-stain can be used depends on the number of slides run through the solution within a 30-day period. If the slides appear visibly red, the solution must be changed. This stain is not widely used.
Modified Kinyoun’s Acid-Fast Stain (Cold Method)
Cryptosporidium spp. and C. belli have been recognized as causes of severe diarrhea in immunocompromised hosts but can also cause diarrhea in immunocompetent hosts (33). Oocysts in clinical specimens may be difficult to detect without special staining (34). Modified acid-fast stains are recommended to demonstrate these organisms. Unlike the Ziehl-Neelsen modified acid-fast stain, the modified Kinyoun’s stain does not require the heating of reagents for staining (35). With additional reports of diarrheal outbreaks due to Cyclospora spp., it is also important to remember that these organisms are modified acid-fast variable and can be identified by this staining approach. Although the microsporidial spores are also acid fast, their size (1 to 2 µm) makes identification very difficult without special modified trichrome stains or the use of immunoassay reagents (36–41).
Concentrated (500 × g for 10 min) sediment of fresh, formalin-preserved, or other single-vial fixative-preserved stool may be used. Other types of clinical specimens such as duodenal fluid, bile, and pulmonary specimens (induced sputum, bronchial washings, or biopsy specimens) may also be stained (Fig. 3.22).
Figure 3.22 Work flow diagram for fecal specimens (coccidia). The most important part of the procedure would be the concentrate (Cytoisospora sp.) and the permanent stained smear, using one of the modified acid-fast techniques (Cryptosporidium and Cyclospora). doi:10.1128/9781555819002.ch3.f22
50% Ethanol
1. Add 50 ml of absolute ethanol to 50 ml of distilled water.
2. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label.
Kinyoun’s Carbol Fuchsin
1. Dissolve 4 g of basic fuchsin in 20 ml of 95% ethanol (solution A).
2. Dissolve 8 g of phenol crystals in 100 ml of distilled water (solution B).
3. Mix solutions A and B.
4. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label.
1% Sulfuric Acid
1. Add 1 ml of concentrated sulfuric acid to 99 ml of distilled water.
2. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label.
Loeffler Alkaline Methylene Blue
1. Dissolve 0.3 g of methylene blue in 30 ml of 95% ethanol.
2. Add 100 ml of dilute (0.01%) potassium hydroxide.
3. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label.
Quality Control for Kinyoun’s Acid-Fast Stain
1. A control slide of Cryptosporidium from a 10% formalin-preserved specimen is included with each staining batch run. If the Cryptosporidium slide stains well, any Cystoisospora or Cyclospora oocysts present will also take up the stain, although Cyclospora oocysts tend to be acid-fast variable (Fig. 3.23).
2. Cryptosporidium spp. stain pink-red. Oocysts measure 4 to 6 µm, and four sporozoites may be visible within some oocysts. The background should stain uniformly blue.
3. The specimen is also checked for adherence to the slide (macroscopically).
4. The microscope should be calibrated (within the last 12 months), and the objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. The calibration factors for all objectives should be posted on the microscope for easy access (multiplication factors can be pasted on the body of the microscope). If the microscopes receive adequate maintenance and are not moved frequently, yearly recalibration may not be necessary.
