PA) diluted 1:20 with phosphate buffer containing 0.01% Triton X-100.
4. Buffers prepared from stock buffers for the Giemsa stain:
A. Alkaline buffer NaHPO4 M/15 solution is 9.5 g of Na2HPO4 dissolved in 1 liter of distilled water.
B. Acid buffer NaHPO4 M/15 solution is 9.2 g of N Na2HPO4 · H2O dissolved in 1 liter of distilled water.
These buffers can be kept for 12 months. To prepare buffered water for the stain, add 39 ml of acid buffer and 61 ml of alkaline buffer to 900 ml of water.
Reagents for Rapid Methenamine Silver Stain (Microwave) (6)
Chromic Acid (10% Solution)
Solution is stable for up to 1 year.
Methenamine (3% Solution)
Solution is stable for up to 6 months.
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.
Sodium Bisulfite (1% Solution)
Solution is stable for up to 1 year.
Gold Chloride (1% Solution)
Solution is stable for up to 1 year.
Sodium Thiosulfate (5% Solution)
Solution is stable for up to 1 year.
Stock Light Green
Solution is stable for up to 1 year.
Working Light Green
Solution is stable for up to 1 month.
Ethanol, 100% (Absolute) and 95%
Methanol, 100% (Absolute)
Xylene or Xylene Substitute
All reagents can be stored at 25°C except for the silver nitrate, which must be refrigerated. Although stable for up to 6 months, silver nitrate may have to be discarded and replaced after storage for 1 month.
Procedure for Rapid Methenamine Silver Stain (Microwave) (11)
1. Place specimen smear slides and control slides on stain rack.
2. Add 10% chromic acid to cover smears, and let stand for 10 min.
3. During this time, prepare working methenamine-silver nitrate by placing the following in order in a plastic Coplin jar: 20 ml of 3% methenamine, 1 ml of 5% silver nitrate, 1.5 ml of 5% sodium borate, and 17 ml of distilled deionized water.
4. Wash slides with distilled deionized water.
5. Cover slides with 1% sodium bisulfite for 1 min.
6. Wash slides with water, place them in the plastic Coplin jar containing methenamine-silver nitrate, and cover with cap.
7. Place in microwave oven at 50% power for approximately 35 s, rotate 90°, and heat for another 35 s; leave slides in hot liquid for 2 min. Solution should turn brown (fresh reagent may appear almost clear if the stock methenamine is freshly prepared; with time and use, the working solution will turn more brown).
8. If a water bath is used, heat it to 80°C. Place plastic Coplin jar containing stain reagents in bath for 6 min prior to placing slides in jar, add slides, and leave jar in bath for an additional 5 min. Solution should turn brown (fresh reagent may appear almost clear if the stock methenamine is freshly prepared; with time and use, the working solution will turn more brown).
9. Remove slides; wash in distilled deionized water.
10. Dip slides up and down in 1% gold chloride, wash in distilled deionized water, and place on rack again.
11. Cover slides with 5% sodium thiosulfate for 1 min.
12. Wash in distilled deionized water.
13. Cover with 0.2% fast green in acetic acid, counterstain for 1 min; use working solution.
14. Wash with distilled deionized water, and stand slides on end to drain and dry.
15. Examine with oil immersion or mount slides.
16. Fill Coplin jar used for staining with bleach (full strength), and let stand for at least 1 h.
Reagents for Rapid Methenamine Silver Stain (No Microwave) (12)
In another rapid methenamine silver-staining procedure, a 5% (rather than 10%) chromic acid solution is used, the gold chloride is present at 0.2% (rather than 1%), the sodium thiosulfate is present at 2% (rather than 5%), and the microwave oven is not used (12). Another difference from the microwave method involves fixation of the smears prior to staining. Heat fix all smears at 70°C for 10 min on a heating block for specimens that could contain Mycobacterium tuberculosis. Place the air-dried smears in absolute methyl alcohol for 5 min. Fix a control smear (microsporidia, fungus, or P. jirovecii) at the same time. It is mandatory that control smears be used every time patient specimens are stained.
Chromic Acid (5% Solution)
Solution is stable for up to 1 year.
Methenamine (3% Solution)
Solution is stable for up to 1 year.
Sodium Bisulfite (1% Solution)
