are very difficult to find and identify even when they appear to be acid fast).
5. It is very important that smears not be too thick. Thicker smears may not adequately destain.
6. Concentration of the specimen is essential to demonstrate organisms. The number of organisms seen in the specimen may vary from numerous to very few. Therefore, the sensitivity of the method is enhanced if the stains are performed on concentrated sediment.
7. Some specimens require treatment with 10% KOH because of their mucoid consistency. Add 10 drops of 10% KOH to the sediment, and vortex until homogeneous. Rinse with 10% formalin, and centrifuge (500 × g for 10 min). Without decanting the supernatant, take 1 drop of the sediment and smear it thinly on a slide.
8. Commercial concentrators and reagents are available.
9. Do not boil the stain. Gently heat until steam rises from the slide. Do not allow the stain to dry on the slide.
10. Various concentrations of sulfuric acid (0.25 to 10%) may be used; the destaining time varies according to the concentration used. Generally, a 1 or 3% solution is used. The use of acid-alcohol (routinely used in the Ziehl-Neelsen acid-fast staining method for the mycobacteria) decolorizes all organisms; therefore, the modified decolorizer must be used for good results.
11. 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.
12. Specimens should be centrifuged in capped tubes, and gloves should be worn during all phases of specimen processing.
Procedure Limitations for the Modified Ziehl-Neelsen Acid-Fast Staining Method
1. Light infections with Cryptosporidium or Cyclospora may be missed (small number of oocysts). When available, fecal immunoassay methods for Cryptosporidium 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. The identification of both Cyclospora organisms and microsporidia may be difficult. Cyclospora may be suspected if the organisms appear to be Cryptosporidium but are about twice the size (about 8 to 10 µm). 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.
4. Often, artifact material may be seen in these stained smears (Fig. 3.25). The artifacts may resemble the oocysts of Cryptosporidium or Cyclospora; therefore, it is very important that any “parasites” seen in the stained smears be measured for confirmation.
There are three other stains that can be used for the coccidia, although they may not be as common as the Kinyoun or hot acid-fast method. They are the carbol fuchsin negative stain, the safranin stain, and the auramine O stain.
Carbol Fuchsin Negative Stain for Cryptosporidium (from W. L. Current)
1. Mix thoroughly an equal volume (3 to 10 µl) of fresh or formalin-fixed stool and Kinyoun’s carbol fuchsin on a slide.
2. Spread out as a thin film, and allow to air dry at room temperature.
3. Add immersion oil directly to the stained smear, and then cover with a coverslip.
4. Observe under bright-field microscopy (×400). Everything but the oocysts stains darkly. The oocysts are bright and refractile because they contain water and everything else is oil soluble.
Rapid Safranin Method for Cryptosporidium (36)
1. Smear fresh or formalin-fixed feces on a slide, and allow the film to air dry at room temperature.
2. Fix briefly with one pass through the Bunsen burner flame.
3. Fix for 3 to 5 min with 3% HCl in methanol.
4. Wash with tap water (brief rinse).
5. Stain with 1% aqueous safranin for 1 min (heat until steam appears) (the authors indicate by personal communication that boiling may be beneficial).
6. Rinse in tap water.
7. Counterstain with 1% methylene blue for 30 s (0.1% aqueous crystal violet was almost as good, but malachite green was unsatisfactory).
Rapid Safranin Method for Cyclospora, Using a Microwave Oven (40, 44)
Another rapid safranin method uniformly stains Cyclospora oocysts a brilliant reddish orange. However, the fecal smears must be heated in a microwave oven before being stained. This stain is fast, reliable, and easy to perform (40) (Fig. 3.25).
1. Using a 10-µl aliquot of concentrated stool, prepare the smear by spreading the material thinly across the slide.
2. Allow the smear to dry on a 60°C slide warmer.
3. Cool the slide to room temperature before staining.
4. Place the slide in a Coplin jar containing acidic alcohol (3% [vol/vol] HCl in methanol), and let it stand for 5 min.
5. Wash off excess acidic alcohol with cold tap water.
6. Place the slide(s) into the Coplin jar containing the safranin solution in acidified water (pH 6.5), and microwave at full power (650 W) for 1 min. (Place the staining jar in another container to catch the overflow of stain because of boiling.)
7. Wash off excess stain with cold tap water.
8. Place the slide(s) for 1 min in a Coplin jar containing 1% methylene blue.
9. Rinse gently with cold tap water.
10. Air dry.
11. Add a coverslip to the slide by using Cytoseal 60 or other mounting medium; the immersion oil mounting method can also be used.
12. Examine the smear with low-power or high dry power objectives. To see additional morphology, use the oil immersion objective (100×).
Auramine O Stain for Coccidia (from Thomas Hänscheid)
Coccidia are acid-fast organisms and also stain well with auramine O (phenolized auramine O). The size and typical appearance of Cryptosporidium, Cyclospora, and Cystoisospora oocysts enable auramine O-stained slides to be examined at low power using the 10× objective. The entire sample area can usually be examined in less than 30 s. The low cost of the reagents, the simple staining protocol, and the rapid microscopic examination also make this staining method suitable for screening unconcentrated fecal specimens.
Concentrated sediment from fresh or non-PVA-preserved stool may be used. Other stool samples may also be used, such as unconcentrated stool submitted for culture in a bacteriology transport medium. However, to increase the sensitivity of the test, small numbers of oocysts are more easily detected using concentrated stools.
Auramine O Stain
Solution 1
Solution 2