(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
Combine solutions 1 and 2. Store in a dark bottle at room temperature for up to 3 months.
Destaining Agent (0.5% Acid-Alcohol)
Store at room temperature for up to 3 months.
Counterstain (0.5% Potassium Permanganate)
Quality Control for the Auramine O Stain for Coccidia
QC guidelines are the same as those for the Kinyoun’s acid-fast stain and are given on p. 53.
Procedure for the Auramine O Stain for Coccidia
1. Using a 10- to 20-µl aliquot of concentrated stool, prepare the smear by spreading the material across the slide.
2. Heat fix the slides either on a 65 to 75°C heat block for at least 2 h or with the flame of a Bunsen burner. However, do not overheat. Another fixation option would be to fix the slide in absolute methanol for 1 min, air dry, and then proceed with staining.
3. Cool the slide to room temperature before staining.
4. Flood the slide with the phenolized auramine O solution.
5. Allow the smear to stain for ca. 15 min. Do not heat.
6. Rinse the slide in water. Drain excess water from the slide.
7. Flood the slide with the destaining solution (0.5% acid-alcohol).
8. Allow to decolorize for 2 min.
9. Flood with counterstain (potassium permanganate) solution.
10. Stain for 2 min. The timing of this step is critical.
11. Rinse the slide in water. Drain excess water from the slide.
12. Allow the smear to air dry. Do not blot.
13. Examine the smear with a fluorescence microscope with a 10× objective and fluorescein isothiocyanate optical filters (auramine O: excitation max., ∼435 nm in water; emission max., ∼510 nm). Screen the whole sample area for the presence of fluorescent oocysts. Suspicious objects can be reexamined using a 20× or 100× objective.
14. Smears can be restained by any of the carbol fuchsin (modified acid-fast) staining procedures to allow examination with light microscopy.
Results and Patient Reports from the Auramine O Staining Method
Cryptosporidium and Cyclospora oocysts fluoresce brightly and have a regular round appearance (“starry sky” appearance with the 10× objective). In contrast to the large majority of fluorescent artifacts, the oocysts do not stain homogeneously. Thus, the fluorescence is heterogeneously distributed in the interior of the oocyst (Fig. 3.27). Cystoisospora oocysts fluoresce brightly with three patterns: (i) a more or less brightly but heterogeneously stained interior of the whole oocyst, (ii) one brightly staining sporocyst, or (iii) two brightly staining sporocysts within the oocyst wall.
Figure 3.27 Cryptosporidium spp. oocysts stained with auramine O fluorescent stain (using different filters). doi:10.1128/9781555819002.ch3.f27
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). These slides can be kept at room temperature in the dark, and the fluorescence remains stable for up to 3 to 4 weeks.
Procedure Notes for the Auramine O Staining Method
1. It is mandatory that positive control smears be stained and examined each time patient specimens are stained and examined.
2. For best results, examine the auramine O solution for deposits and remove them by filtration or centrifugation. This problem can also be avoided by preparing smaller volumes more frequently.
3. Slides should be observed as soon as possible after staining. However, they can be kept at room temperature in the dark, and fluorescence remains stable for up to 3 to 4 weeks.
Procedure Limitations for the Auramine O Staining Method
1. Light infections might be missed, particularly if unconcentrated stool is used; it is always recommended that concentrated stool sediment be used for staining.
2. Use of the 40× high dry objective often causes a blurred image (fluorescent “halo” around the image, hazy contours), which appears to be the effect of interfering fluorescence from the auramine O stain located outside the plane of focus (
