There is some debate whether organisms lose their ability to take up the acid-fast stain after long-term storage in 10% formalin. Some laboratories have reported this diminished staining.
11. Specimens should be centrifuged in capped tubes, and gloves should be worn during all phases of specimen processing.
Procedure Limitations for Kinyoun’s Acid-Fast Staining Method
1. Light infections with Cryptosporidium spp. may be missed (small number of oocysts). The fecal immunoassay methods are more sensitive.
2. Multiple specimens must be examined, since the numbers of oocysts present in the stool vary from day to day. A series of three specimens submitted on alternate days is recommended.
3. Cyclospora may be suspected if the organisms appear to be Cryptosporidium but are about twice the size (about 10 µm) (Fig. 3.25). The microsporidial spores are extremely small (1 to 2 µm) and will probably not be recognized unless they are very numerous and appear to have a somewhat different morphology from the bacteria in the preparation.
Modified Ziehl-Neelsen Acid-Fast Stain (Hot Method)
Cryptosporidium and Cystoisospora have been recognized as causes of severe diarrhea in immunocompromised hosts but can also cause diarrhea in immunocompetent hosts. Oocysts in clinical specimens may be difficult to detect without special staining. Modified acid-fast stains are recommended to demonstrate these organisms. Application of heat to the carbol fuchsin assists in the staining, and the use of a milder decolorizer allows the organisms to retain their pink-red color (37). With continued reports of diarrheal outbreaks due to Cyclospora, it is also important to remember that these organisms are acid fast and can be identified by using this staining approach (42, 43). Although the microsporidial spores are also acid fast, their size (1 to 2 µm) makes identification very difficult without special stains or the use of monoclonal antibody reagents.
Concentrated sediment of fresh, formalin, other non-mercury single-vial fixative-preserved stool, or those preserved in Universal Fixative 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.
Carbol Fuchsin
1. To make basic fuchsin (solution A), dissolve 0.3 g of basic fuchsin in 10 ml of 95% ethanol.
2. To make phenol (solution B), dissolve 5 g of phenol crystals in 100 ml of distilled water. (Gentle heat may be needed.)
3. Mix solution A with solution B.
4. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label.
1–3% Sulfuric Acid
1. Add 1 to 3 ml of concentrated sulfuric acid to 99 or 97 ml of distilled water.
2. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label.
Methylene Blue
1. Dissolve 0.3 g of methylene blue chloride in 100 ml of distilled water.
2. Store at room temperature. The solution is stable for 1 year. Note the expiration date on the label.
Quality Control for the Modified Ziehl-Neelsen Acid-Fast Staining Method
QC guidelines are the same as those for the Kinyoun’s acid-fast stain and are given on p. 53.
Procedure for the Modified Ziehl-Neelsen Staining Method
1. Smear 1 to 2 drops of specimen on the slide, and allow it to air dry. Do not make the smears too thick (you should be able to see through the wet material before it dries). Prepare two smears.
2. Dry on a heating block (70°C) for 5 min.
3. Place the slide on a staining rack, and flood it with carbol fuchsin.
4. With an alcohol lamp or Bunsen burner, gently heat the slide to steaming by passing a flame under the slide. Discontinue heating once the stain begins to steam. Do not boil.
5. Allow the specimen to stain for 5 min. If the slide dries, add more stain without additional heating.
6. Rinse thoroughly with water. Drain.
7. Decolorize with 1–3% sulfuric acid for 30 s. (Thicker slides may require a longer destain.)
8. Rinse the slide with water. Drain.
9. Flood the slide with methylene blue for 1 min.
10. Rinse the slide with water, drain, and air dry.
11. Examine with low or high dry objective. To see internal morphology, use the oil objective (100×).
Results and Patient Reports from the Modified Ziehl-Neelsen Acid-Fast Staining Method
The oocysts of Cryptosporidium and Cystoisospora spp. stain pink to red to deep purple. Some of the four sporozoites may be visible in the Cryptosporidium oocysts. Some of the Cystoisospora immature oocysts (entire oocyst) stain, while those that are mature usually appear with the two sporocysts within the oocyst wall stained a pink to purple color and a clear area between the stained sporocysts and the oocyst wall (Fig. 3.26). The background stains blue. If Cyclospora oocysts are present (uncommon), they tend to be approximately 8 to 10 µm, they resemble Cryptosporidium oocysts but are larger, and they have no definite internal morphology; the acid-fast staining tends to be more variable than that seen with Cryptosporidium or Cystoisospora spp. If the patient has a heavy infection with microsporidia (immunocompromised patient), small (1- to 2-µm) spores may be seen but may not be recognized as anything other than bacteria or small yeast cells.
Figure 3.26 (Upper) Immature Cystoisospora belli oocyst stained with modified acid-fast stain. (Lower) C. belli immature oocyst (left) (note that the entire oocyst retains the stain) and C. belli mature oocyst containing two sporocysts (right). doi:10.1128/9781555819002.ch3.f26
There is usually a range of color intensity in the organisms present; not every oocyst appears deep pink to purple. The greatest staining variation would be seen with Cyclospora organisms.
1. Report the organism and stage (oocyst). Do not use abbreviations.
Examples: Cryptosporidium spp. oocysts
Cystoisospora belli oocysts
Cyclospora cayetanensis oocysts
2. Call the physician when these organisms are identified.
3. Save positive slides for future reference. Label prior to storage (name, patient number, organisms present).
Procedure Notes for the Modified Ziehl-Neelsen
