Solution is stable for up to 1 year.
Sodium Thiosulfate (Hypo) (2% Solution)
Solution is stable for up to 1 year.
Stock Light Green
Solution is stable for up to 1 year.
Silver Nitrate (5% Solution)
Solution is stable for 1 to 6 months at 4°C.
Sodium Borate (Borax) (5% Solution)
Solution is stable for up to 1 year.
Gold Chloride (0.2% Solution)
This solution is stable for 1 year; however, when toning begins to fail and the organisms come out too black, it should be changed.
Note The 1% gold chloride solution is made from ampoules and is diluted as specified in the accompanying directions (dilute with tap water).
Stock Methenamine-Silver Nitrate
A white precipitate forms but immediately dissolves on shaking. Clear solutions remain stable for months at refrigerator temperature (4°C).
Working Light Green
Solution is stable for up to 1 month.
Working Methenamine-Silver Nitrate
Caution The working methenamine-silver nitrate solution must be prepared fresh each time the stain is run. Do not try to reuse, even for a stain run immediately following one previously performed.
Procedure for Rapid Methenamine Silver Stain (No Microwave) (12)
1. While slides are fixing, fill one Coplin jar (with a lid) with 5% chromic acid. In a second, screw-cap Coplin jar, prepare the working methenamine-silver nitrate solution.
2. Place fixed slides into chromic acid, and put both Coplin jars into the 80°C water bath. Warm the jars and their contents in a stream of hot water for approximately 30 s before placing them in the water bath, to prevent the jars from cracking. Place smears into the heated 5% chromic acid, and incubate them in the water bath for 2 min (oxidation step).
3. Wash slides briefly in running tap water.
4. Place slides into 1% sodium bisulfite for 30 s.
5. Rinse slides in three changes of distilled water.
6. Place slides into the jar of working methenamine-silver nitrate solution in the 80°C water bath for 4.5 min (reduction step).
7. Rinse slides with three changes of distilled water. Wipe backs of slides with a paper towel to remove excess methenamine-silver nitrate.
8. Tone in 0.2% gold chloride for 30 s.
9. Rinse in three changes of distilled water.
10. Place slides into 2% sodium thiosulfate for 30 s (removes reduced silver).
11. Rinse in three changes of distilled water.
12. Counterstain in working light green solution for 30 s.
13. Rinse in three changes of distilled water.
14. Dehydrate and clear for 30-s intervals in two changes each of 95% ethyl alcohol, 100% ethyl alcohol, and xylene or xylene substitute, respectively.
15. Mount slides in Permount.
The fungi generally stain gray to black; P. jirovecii exhibits a delicately stained wall, usually brownish or grayish, which is somewhat transparent. Structures described as “parentheses” are usually seen and are stained dark gray or black (Fig. 6.4). If the organisms are too dark, it is probably time to change the solutions. It is also important to prepare the smears so that the material is thin and evenly spread on the glass slide. Glycerin, red blood cells, and mucin stain rose taupe to dark gray. The inner parts of mycelia and hyphae stain “old rose.” The background usually appears pale green. Microsporidial spores stain dark gray to black; the horizontal or diagonal “stripe” or polar filament is not visible in every spore (Fig. 6.4). Various background colors are visible, depending on the stain used, thickness of the specimen, and tissue source of the specimen.
Figure 6.4 (Upper) Pneumocystis jirovecii. Note that the “parentheses” are visible within the cell wall (methenamine silver stain). (Middle) Microsporidial spores in a stool specimen stained with silver stain (methenamine silver stain). Note the spore with a polar tubule (horizontal line through the spore) within the black box; there are other examples of spores containing the polar tubule throughout the image. (Bottom) Microsporidian spores within an ocular biopsy specimen (methenamine silver stain). In this image the spores are clustered together and polar tubules are not visible. doi:10.1128/9781555819002.ch6.f4
Note The examination of specimens for the diagnosis of microsporidia is not often considered to be a STAT procedure and may be handled in the anatomic pathology division. The stained smears can be difficult to read and interpret, hence the need for available experienced personnel. However, special modified trichrome stains for the microsporidia are often performed in the microbiology laboratory; the procedure is not difficult, but examination of the stained smears is very labor intensive.
Rapid Giemsa Stain (Diff-Quik or Giemsa Plus)
1. Keep stain solutions in dropper bottles. Place 1 or 2 drops of red stain (solution 1) on specimen smear and control slide (normal blood film), hold for 10 s, and drain.
2. Add drops of blue (solution 2), hold for 10 s, drain, and rinse very briefly with deionized water.
3. Stand slides on end to drain and air dry.
4. Slides must be examined with oil or mounted with mounting medium.
Giemsa Stain
Make Giemsa working solution fresh each day. Discard and make new solution after 10 slides have been stained in a Coplin jar. Additional directions can be found in chapter 7.
1.
