[Updated March 2007]
P R E A N A L Y T I C A L C O N S I D E R A T I O N S I. PRINCIPLE
II. SPECIMEN Concentrated sediment of fresh or formalin-preserved stool may be used. Other types of clinical specimens such as duodenal fluid, bile, or pulmonary (induced sputum, bronchial washings, biopsy specimens) may also be stained after centri-fugation.
III. MATERIALS A. Reagents (see Appendix 9.4.1–1) 1. Absolute methanol
2. 50% Ethanol
3. Kinyoun carbol fuchsin 4. 1% Sulfuric acid 5. Methylene blue B. Supplies
1. Disposable glass or plastic pipettes 2. Glass slides (1 by 3 in., or larger if
you prefer)
3. Coverslips (22 by 22 mm; no. 1, or larger if you prefer)
C. Equipment
1. Binocular microscope with 10⳯, 40⳯, and 100⳯ objectives (or the
approximate magnifications for low-power, high dry power, and oil immersion examination)
2. Oculars should be 10⳯. Some workers prefer 5⳯; however, over-all smover-aller magnification may make final organism identifications more difficult.
3. Tabletop centrifuge 4. Staining rack
A N A L Y T I C A L C O N S I D E R A T I O N S
A. A control slide of Cryptosporidium parvum from a 10% formalin-preserved specimen is included with each staining batch run. If the cryptosporidia stain well, any Isospora belli oocysts present will also take up the stain, as will C.
cayetanensis.
Observe standard precautions.
IV. QUALITY CONTROL
Cryptosporidium and Isospora species have been recognized as causes of severe diarrhea in immunocompromised hosts, but they can also cause diarrhea in im-munocompetent hosts. Oocysts in clinical
specimens may be difficult to detect with-out special staining. Cyclospora cayeta-nensis has also been reported to be acid fast. Modified acid-fast stains are recom-mended for demonstrating these
organ-isms. Unlike the Ziehl-Neelsen modified fast stain, the modified Kinyoun acid-fast stain does not require heating the re-agents used for staining and uses a mild decolorizer (1–3).
B. Cryptosporidia stain pink-red. Oocysts are 4 to 6 lm in diameter, and four sporozoites may be present internally. The background should stain uniformly blue.
C. Check the specimen (macroscopically) for adherence to the slide.
D. Record all QC results.
V. PROCEDURE A. Smear 1 or 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.
B. Fix with absolute methanol for 1 min. Allow to air dry.
C. Flood slide with Kinyoun’s carbol fuchsin, and stain for 5 min.
D. Rinse slide briefly (3 to 5 s) with 50% ethanol.
E. Rinse thoroughly with water.
F. Decolorize with 1% sulfuric acid for 2 min or until no more color runs from the slide.
G. Rinse slide with water. Drain.
H. Counterstain with methylene blue for 1 min.
I. Rinse slide with water. Air dry.
J. Examine using low-power or high dry power objectives. To see internal mor-phology, use oil immersion objective (100⳯).
VI. RESULTS A. With this cold Kinyoun acid-fast method, C. cayetanensis and the oocysts of Cryptosporidium and Isospora will stain pink to red to deep purple. Some of the four sporozoites may be visible in the Cryptosporidium oocysts. Some of the Isospora immature oocysts (entire oocyst) will stain, while in oocysts that are mature, the two sporocysts within the oocyst wall will usually stain pink to purple and there will be a clear area between the stained sporocysts and the oocyst wall. The background will stain blue. If Cyclospora oocysts are present (uncommon), they tend to be approximately 8 to 10 lm, they resemble C.
parvum but are larger, and they have no definite internal morphology; the acid-fast staining will tend to be more variable than that seen with Cryptosporidium or Isospora spp. Modified acid-fast stains stain the Cyclospora oocysts from light pink to deep red, and some of the oocysts will contain granules or have a bubbly appearance, often being described as looking like “wrinkled cellophane.”
Even with the 1% acid decolorizer, some oocysts of Cyclospora may appear clear or very pale. If the patient has a heavy infection with microsporidia (im-munocompromised patient), small (1- to 2-lm) spores may be seen but may not be recognized as anything other than bacteria or small yeast cells.
B. There is usually a range of color intensity in the organisms present.
P O S T A N A L Y T I C A L C O N S I D E R A T I O N S
A. Report the organism and stage (oocyst or C. cayetanensis). Do not use abbre-viations.
Examples: Cryptosporidium parvum oocysts or Isospora belli oocysts or Cyclospora cayetanensis oocysts
B. Call the physician when these organisms are identified.
IV. QUALITY CONTROL (continued)
VII. REPORTING RESULTS
Modified Kinyoun’s Acid-Fast Stain 9.4.1.3
VIII. PROCEDURE NOTES A. Routine stool examination stains are not recommended; however, the sedimen-tation concentration is acceptable (500⳯ g for 10 min) for the recovery and identification of Cryptosporidium and Cyclospora spp. Routine concentration (formalin-ethyl acetate) can be used to recover Isospora oocysts, but routine permanent stains are not reliable for this purpose.
B. Polyvinyl alcohol-preserved specimens are not acceptable for staining with the modified acid-fast stain. However, specimens preserved in SAF are perfectly acceptable.
C. 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 or-ganisms and prevent them from flowing 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 no gauze but instead use plastic or metal screens.
D. Other organisms, such as acid-fast bacteria and some Nocardia spp., stain posi-tive.
E. It is very important that smears not be too thick. Thick smears may not ade-quately destain.
F. Concentration of the specimen is essential for demonstrating organisms (500⳯ g for 10 min). The number of organisms seen in the specimens may vary from numerous to very few.
G. Because of their mucoid consistency, some specimens require treatment with 10% KOH. Add 10 drops of 10% KOH to the sediment, and vortex until ho-mogeneous. Rinse with 10% formalin, and centrifuge (500⳯ g for 10 min).
Without decanting the supernatant, take 1 drop of the sediment and smear it thinly on a slide.
H. Commercial concentrators and reagents are available (see Appendix 9.10.6–1 at the end of this section).
I. Weak concentrations of sulfuric acid (1.0 to 3.0%) are normally used. Stronger concentrations will remove too much stain.
J. There is some debate about whether organisms lose their abilities to take up the acid-fast stain after long-term storage in 10% formalin. Use of the hot modified acid-fast method might eliminate this problem (1).
K. Centrifuge specimens in capped tubes, and wear gloves during all phases of specimen processing.
L. Currently, no commercial immunoassays are available for C. cayetanensis.
However, several reagents are in the research phase.
IX. LIMITATIONS OF THE
PROCEDURE A. Light infections (low number of oocysts) may be missed. Immunoassay methods for C. parvum are more sensitive.
B. Multiple specimens must be examined, since the numbers of oocysts in the stool will vary from day to day. A series of three specimens submitted on alternate days is recommended.
REFERENCES 1. Garcia, L. S. 2001. Diagnostic Medical Par-asitology, 4th ed., p. 723. ASM Press, Wash-ington, D.C.
2. Ma, P., and R. Soave. 1983. Three step stool examination for cryptosporidiosis in 10 ho-mosexual men with protracted diarrhea. J. In-fect. Dis. 147:824–828.
3. Miller, J. M. 1991. Quality control of media, reagents, and stains, p. 1203–1225. In A. Bal-ows, W. J. Hausler, Jr., K. L. Herrmann, H. D.
Isenberg, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 5th ed. American So-ciety for Microbiology, Washington, D.C.
APPENDIX 9.4.1–2
(Top) C. parvum oocysts; sporozoites are visible within some oocysts. (Bottom) I. belli immature oocyst; note that the entire oocyst stains with modified acid-fast stain.
Include QC information on reagent container and in QC records.
APPENDIX 9.4.1–1 Reagents
䊓 Indicate the expiration date on the label and in the work record or on the manufacturer’s label.
A. 50% Ethanol
1. Add 50 ml of absolute ethanol to 50 ml of distilled water.
2. Store at room temperature. Stable for 1 year.
B. Kinyoun 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 together.
4. Store at room temperature. Stable for 1 year.
C. 1% Sulfuric acid
1. Add 1 ml of concentrated sulfuric acid to 99 ml of distilled water.
2. Store at room temperature. Stable for 1 year.
D. Methylene blue
1. Dissolve 0.3 g of methylene blue in 100 ml of 95% ethanol.
2. Store at room temperature. Stable for 1 year.
9.4.2.1