Known positive microscope slides and photographs (reference books) should be available at the workstation.
6. Record all QC results; the laboratory should also have an action plan for “out-of-control” results.
Figure 3.23 Quality control slides for performing modified acid-fast stains (Cryptosporidium and Cyclospora) (Medical Chemical Corp.). doi:10.1128/9781555819002.ch3.f23
Procedure for Kinyoun’s Acid-Fast Stain
1. Smear 1 to 2 drops of concentrated specimen sediment 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. Fix with absolute methanol for 1 min.
3. Flood the slide with Kinyoun’s carbol fuchsin and stain for 5 min.
4. Rinse the slide briefly (3 to 5 s) with 50% ethanol.
5. Rinse the slide thoroughly with water.
6. Decolorize with 1% sulfuric acid for 2 min or until no more color runs from the slide.
7. Rinse the slide with water. Drain.
8. Counterstain with methylene blue for 1 min.
9. Rinse the slide with water. Air dry.
10. Examine the slide with the low or high dry objective. To see the internal morphology, use an oil objective (100×).
Results and Patient Reports from Kinyoun’s Acid-Fast Staining Method
The oocysts of Cryptosporidium, Cyclospora, 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) will stain, while those that are mature usually appear with the two sporocysts within the oocyst wall stained pink to purple and a clear area between the stained sporocysts and the oocyst wall. The background stains blue. If Cyclospora oocysts are present (uncommon unless in an outbreak situation), they tend to be approximately 8 to 10 µm, they resemble Cryptosporidium spp. 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. (Fig. 3.24 and 3.25). Some of the Cyclospora oocysts tend to look like “wrinkled cellophane,” with tremendous variation in color intensity ranging from clear to pink to red to deep purple to almost black. The stain intensity also depends on the thickness of the smear, the percentage of acid in the decolorizer (1% sulfuric acid recommended), and the length of time the smear is in contact with the decolorizer. 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 3.24 Cyclospora cayetanensis oocysts (8–10 µm). (Top) Autofluorescent oocysts; (bottom) oocysts stained with modified acid-fast stain. Note the range of color intensity; some oocysts resemble “wrinkled cellophane.” doi:10.1128/9781555819002.ch3.f24
Figure 3.25 (Left) Cyclospora (8 to 10 µm), Cryptosporidium (4 to 6 µm), and artifact (∼2 µm) stained with modified acid-fast stain. Note that one of the Cyclospora oocysts did not stain (modified acid-fast variable) and has the “wrinkled-cellophane” appearance, while the other oocyst displays the typical purple/red color; the Cryptosporidium oocyst and the artifact did stain modified acid-fast positive. These artifacts are frequently seen; it is very important to measure objects seen in the modified acid-fast smears to confirm that they are actual parasites and not artifacts. (Right) Cyclospora stained with a hot safranin stain (higher magnification than the image on the left). (Courtesy of CDC.) doi:10.1128/9781555819002.ch3.f25
There is usually a range of color intensity in the organisms present; not every oocyst appears deep pink to purple. The greatest staining variation will be seen with Cyclospora oocysts; do not decolorize too long.
1. Report the organism and stage (oocyst). Do not use abbreviations.
Examples: Cryptosporidium spp. oocysts
Cystoisospora belli 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 Kinyoun’s Acid-Fast Staining Method
1. Routine stool examination stains (trichrome and iron hematoxylin) are not recommended; however, sedimentation concentration (500 × g for 10 min) is recommended for the recovery and identification of the coccidia after the concentration sediment has been stained with one of the modified acid-fast stains. The routine concentration (formalin-ethyl acetate) can be used to recover Cystoisospora oocysts (wet sediment examination and/or modified acid-fast stains), but routine permanent stains (trichrome and iron hematoxylin) are not reliable for this purpose.
2. Preserved specimens containing PVA are not acceptable for staining with the modified acid-fast stain. However, specimens preserved in SAF, other non-mercury single-vial-system fixatives, or those preserved in Universal Fixative are perfectly acceptable.
3. Avoid the use of wet-gauze filtration (an old, standardized method of filtering stool prior to centrifugation) with too many layers of gauze that may trap organisms and not allow them to flow into the fluid to be concentrated. It is recommended that no more than two layers of gauze be used; another option is to use the commercially available concentrators that use plastic or metal screens instead of gauze.
4. Other organisms that stain positive include acid-fast bacteria, Nocardia spp., and the microsporidia (which 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 many to very few; therefore, the organisms are concentrated in the sediment, which is used to prepare the smears for subsequent staining.
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. (See Appendix 1.)
9. Concentrations of sulfuric acid of 1.0 to 3.0% are normally used. Higher concentrations remove too much stain. The use of acid-alcohol (routinely used in the Ziehl-Neelsen acid-fast staining method for the mycobacteria) decolorizes all organisms; therefore, one must use the modified decolorizer (1 to 3% H2SO4) for good results. In general, 1% acid is recommended; this approach will provide excellent staining results for all the coccidia.
10.
