Hi HistoNetters

I have two good microwave staining procedures for the demonstration of
pneumocystis carinii.  The first was developed with the help of colleagues
at Sigma (hi, Skip!) for use with wattage-controlled microwaves.  The second
was developed in Dr. Boon's lab for temperature-controlled microwaves.

Silver Stain, Mofidifed GMS, Sigma Diagnostics Procedure No. HT100
For the demonstration of Fungi, Basement Membrane, and some opportunistic
Organisms.
SUMMARY
Silver methenamine borate procedures are well documented. Generally, these
require elaborate solution prepration prior to test performance. Solution
stability is limited and results vary remarkably due to capricious nature of
metal impregnation and photographic development. The Silver Stain
incorporates a stable silver methenamine salt along with a buffer, toning
reagent, and developer. This provides the laboratory with a unique package
for visualizing fungi, basement membrane, and opportunistic organisms such
as Pneumocystis carinii. Microbial cell wall and membrane polysaccarides are
oxidized to aldehydes by treatment with periodic acid. The aldehyde group,
at alkaline pH, reduces silver ion to metallic silver. Rinsing with gold
salts forms a more stable gold complex and excess silver is removed by
sodium thiosulfate rinse.
REQUIRED REAGENTS:
Periodic Acid Solution
Periodic acid (1 g/dL), in deionized water.
Borax Solution
Borax, 5 g/d L, in deionized water. During shipment refrigeration, Borax
Solution may form crystals. Should this occur, bring solution to room
temperature and redissolve crystals. Best results are obtained using a stir
bar and stir plate. After opening bottle, store at room temperature
(18-26°C)
Silver Methenamine Reagent
Silver methenamine (110 mg).
Gold Chloride Solution
Gold Chloride (200 mg/d L) in deionized water.
Sodium Thiosulfate
Sodium Thiosulfate (2 g/d L) in deionized water.
Reagent Alcohol
Specially denatured alcohol. 3A (95 parts by volume) and isopropyl alcohol
(5 parts by volume).
Xylene
OPTIONAL REAGENTS
Fungi TISSUE-TROL
Tissue containing fungi (cryptococcus, histoplasmosis, blastomycosis, or
aspergillus).
Pnuemocytis TISSUE-TROL
Tissue containing opportunistic organism Pneumocystis carinii.
Light Green SF Yellowish, Certified
Tartrazine Solution
Eosin Y Solution, Aqueous
Hemotoxylin Solution, Harris, Modified
REAGENT PREPARATION
Working Silver Methenamine Solution is prepared by adding 8 ml of Borax
Solution, and contents of one vial Silver Methenamine Reagent, to 100 ml of
deionized water. Mix until dissolved. Use once and discard.
Note: To increase tests per vial to two, Silver Methenamine may be dissolved
in 100 ml deionized water and stored tighly capped (2-8°C) for 1 month. When
ready to use, take 50 ml of Silver Methenamine Solution and add 4 ml of
Borax Solution. Return unused portion of Silver Methenamine Solution to
refrigerator.
PROCEDURE:
1.    Deparaffinize slides and hydrate to deionized water.
2.    Place slides in 40 ml of Periodic Acid Solution in plastic Coplin jar.
Loosely cover jar with lid before placing in microwave oven, or use lids
with holes drilled in them. Microwave on 800 Watts for 10 seconds.
Periodic Acid may be reused if used at room temperature for 5 minutes for
fungus, or for basement membranes. Periodic Acid incubation time is 11
minutes at room temperature.
3.    Remove slides from Periodic Acid Solution and rinse in 6 changes of
deionized water.
4.    Place slides in 40 ml of Working Silver Methenamine Solution contained
in a plastic Coplin jar.
5.    a. For fungal organisms
Microwave on 600 Watts for 35 seconds. Gently mix Solution with a beral
pipet or applicator stick. Let incubate for 1-3 minutes. After no longer
than two minutes, place slides in warm deionized water and check development
microscopically. If organisms are not sufficiently developed, return slides
to Working Silver Methenamine Solution and let sit for 30 seconds to 1
minute.
Note: Suggested developing time for the following fungal organisms are as
follows:


Blastomyces    1-2 minutes
Pneumocystis    2-3 minutes
Aspergillus    2-3 minutes




5.    b. For basement membrane
Microwave on 600 Watts for 35 seconds. Gently agitate solution. Let slides
incubate for 5 minutes. Microwave slides and solution a second time at 600
Watts for 10 seconds. Let slides develop for 2 minutes. Check slides
microscopically. If slides are not sufficiently developed, repeat last step
until desired tone is achieved.
6.    Rinse slides in 6 changes of deionized water.
7.    Tone slides in Gold Chloride Solution for 30 seconds at room
temperature.
8.    Rinse slides in 6 changes of deionized water.
9.    Place slides in Sodium Thiosulfate Solution for 2 minutes at room
temperature.
10.    Wash well in running tap water.
11.    Counterstain according to personal preference with Light Green SF
Yellowish, Tartrazine or Hematoxylin and Eosin Solution.
12.    Dehydrate in alcohols and clear to xylene. Coverslip and mount
slides.

EXPECTED OBSERVATIONS

Fungi     Purplish-brown to black
P. carinii    Purplish-brown to black
Basement Membrane    Black
Background    Dependent upon counterstain

Grocott's Methenamine Silver Nitrate Method
The Grocott's Methenamine Silver Nitrate method is a metallic-staining
method widely used to demonstrate fungi and pneumocystis carinii.
SOLUTIONS NEEDED:

Methenamine Silver stock solution:
400 ml    3% Hexamethylene Tetramine (Methenamine) (C6H14N4)
20 ml    5% Aqueous Silver Nitrate (AgNO3)
Keep in refrigerator at 4°C
10% Chromic Acid solution (CrO3)
Methenamine Silver working solution:
50 ml    Methenamine silver stock solution
4 ml    5% Borax solution (Na2B4O7ˇ10H2O)
50 ml    Distilled water
0.1 % Gold Chloride solution (HAuCl4ˇ3H2O, 49% pure)
1% Sodium Metabisulfite solution (Na2S2O5)
5% Sodium Thiosulfate (Na2S2O3ˇ5H2O)
PROCEDURE:
1.    Deparaffinize and bring to water
2.    90 sec / 60°C / 50 ml of 10% Chromic Acid solution
3.    Rinse in tap water
4.    60 sec / clear slides in 1% Sodium Metabisulfite solution
5.    Rinse in tap water followed by several changes of distilled water
6.    60 sec / 95°C / 16 ml of Methenamine Silver working solution
7.    Rinse in distilled water and evaluate microscopically. If additional
staining is required, the slides may be placed back in the hot working
solution for 30 sec until the desired intensity is reached
8.    Rinse in distilled water
9.    4 min / tone in 0.1% Gold III Chloride solution
10.    Rinse in distilled water
11.    60 sec / drop 5% Sodium Thiosulfate on the sections
12.    Rinse in tap water
13.    Counterstain with Hematoxylin and Eosine, 1% Light Green, or Nuclear
Fast Red
14.    Rinse in tap water
15.    Dehydrate and mount

RESULTS:

Fungi    sharply delineated in black
Pneumocystis carinii    sharply delineated in black
NOTES:
Beware of chromic acid and fumes. They are both caustic and toxic!
Use coated slides.


best regards,
Steven Slap

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Marketing Manager/Microwave Product Specialist
Hacker Instruments & Industries, Inc.
http://www.hackerinstruments.com
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