Fungus, GMS, Candida sp., Artificial

Controls Slides Histopathology Fungus, GMS, Candida albicans

Validation Stain: Grocott Methenamine Silver (GMS)
Other Applicable Stains: Gridley Fungus

 

Tissue: 
(+) Rat Lung / (-) Lung
Adhesive or Charged Slides: 
Superfrost +

Product Options:

Part # 4230A10/Set $46.00
Part # 4230B98/Set $330.00

Fungus, GMS, Candida sp., Artificial Control Slides contain sections of positive staining rat lung and negative staining human lung.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               

PRODUCT DESCRIPTION:

The enclosed positive control slides are intended to be used to verify histological techniques and reagent reactivity.  These slides are to be used for the qualitative purpose of determining positive or negative results, and are not intended to be used for any quantitative purpose.  The first serial section within the control box is stained and provided for your reference.  Before using the unstained slides, review the enclosed stained slide with your pathologist to ensure that this tissue source is acceptable.  Newcomer Supply will not accept a return with missing slides in the series. Newcomer Supply guarantees reactivity of these control slides for one year from the date of receipt.  Revalidate after one year to verify continued reactivity.  Store at 15-30°C in a light deprived and humidity controlled environment.

These Fungus, GMS, Candida sp., Artificial Control Slides were produced at the Newcomer Supply Laboratory under carefully controlled conditions.  The positive control sections are not human tissue.  The microorganisms were grown in pure culture, harvested, formalized and introduced in a freshly harvested rat lung.  No infective process occurred.  Candida albicans was used to produce these control slides, and was purchased from Remel Microbiology Products (R4601503 ATCC® 10231).  Reasonable measures are used to deliver quality control slides that are as consistent as possible.  However, characteristics of quality control slides may be dissimilar due to variations in the reagents, stains, techniques, laboratory conditions, and tissue sources used. Newcomer Supply Laboratory uses a manual method of performing quality control procedures, specifically avoiding automation, in order to provide reactive control slides for even less aggressive methods of staining that our customers may be using.

 

CONTROL SLIDE VALIDATION:          

With Fungus, Grocott Methenamine Silver (GMS) Stain Kit: Part 9121A/B  Individual Stain Solution
Solution A: Chromic Acid 5%, Aqueous 250/500 ml Part 10341
Solution B: Sodium Bisulfite 1%, Aqueous 250/500 ml Part 13821
Solution C: Silver Nitrate 125/250 ml Part 1142
Solution D: Methenamine Borate 125/250 ml Part 1142
Solution E: Gold Chloride 0.1%, Aqueous 250/500 ml Part 11285
Solution F: Sodium Thiosulfate 2%, Aqueous 250/500 ml Part 13888
Solution G: Light Green SF Yellowish Stain 0.02%, Aqueous 250/500 ml Part 12204

                                               

For storage requirements and expiration date refer to individual product labels.    

 

APPLICATION:

Newcomer Supply Fungus, GMS, Candida sp., Artificial Control Slides are for the positive histochemical staining of Candida sp. in tissue sections.

 

METHOD:

Fixation: Formalin 10%, Phosphate Buffered (Part 1090)

Technique:  Paraffin sections cut at 5 microns on Superfrost® Plus

Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

 

NEWCOMER SUPPLY VALIDATION PROCEDURE:

  1. All glassware/plasticware must be acid cleaned prior to use.
    1. See Procedure Notes #1 and #2.
  2. Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each.  Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each.  Wash well with distilled water.
    1. See Procedure Notes #3 and #4.
  3. Oxidize in Solution A: Chromic Acid 5%, Aqueous for 1 hour.

        Microwave Modification: See Procedure Note #5.

  1. Oxidize slides in a plastic Coplin jar containing Solution A: Chromic Acid 5%, Aqueous and microwave for 1 minute and 20 seconds at 60°C.
  1. Wash well in running tap water; rinse in distilled water.
  2. Place slides in Solution B: Sodium Bisulfite 1%, Aqueous for 1 minute. 
  3. Wash for 5 minutes in running tap water; followed by three to four changes of distilled water.
  4. Prepare Silver-Methenamine Working Solution and mix well:
    1. Solution C: Silver Nitrate                 20 ml
    2. Solution D: Methenamine Borate    20 ml
    3. Proceed to Step #10 for Microwave Modification.
  1. Preheat the Silver-Methenamine Working Solution to 45°C - 60°C.
    1. See Procedure Notes #6 and #7.
  2. Place slides in preheated Silver-Methenamine Working Solution and incubate in 45°C-60°C oven or water bath, or bench top/room temperature, for 12-18 minutes until sections appear paper-bag brown.  Periodically remove control, rinse in warm distilled water, check microscopically for adequate silver impregnation.  Fungi should be dark brown.  If organisms are not sufficiently dark, return slides to the warm silver solution.  Recheck at 2-3 minute intervals until desired intensity is achieved.
    1. Staining at room temperature will require an overall longer incubation time.
  3. Microwave Modification:
    1. Incubate slides in a plastic Coplin jar containing Silver-Methenamine Working Solution and microwave for 1 minute at 70°C. 
    2. Check microscopically for adequate development. If additional incubation is required, return slides to the warm Silver-Methenamine Working Solution.  Recheck at 2-3 minute intervals.
  4. Rinse in three to four changes of distilled water.
    1. Never use tap water at this step.
  5. Tone in Solution E: Gold Chloride 0.1%, Aqueous until sections turn gray; 20 seconds to 1 minute.
  6. Rinse well in distilled water.
  7. Remove unreduced silver in Solution F: Sodium Thiosulfate 2%, Aqueous for 2 minutes.
  8. Wash in running tap water for 5 minutes; rinse in distilled water.
  9. Counterstain in Solution G: Light Green SF Yellowish 0.02%, Aqueous for 2 minutes.
    1. Over counterstaining could mask organisms. 
  10. Dehydrate quickly in two changes each of 95% and 100% ethyl alcohol. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

 

RESULTS:

Candida sp. Sharply outlined in black
Background Green
Negative lung Negative for fungus

 

PROCEDURE NOTES:

  1. Acid clean all glassware/plasticware (Part 12086) and rinse thoroughly in several changes of distilled water.  Cleaning glassware with bleach is not equivalent to acid washing.
  2. Plastic (Parts 5500, 5501), plastic-tipped or paraffin coated metal forceps must be used with any silver solution to prevent precipitation of silver salts.  No metals of any kind should be in contact with any silver solution. Only glass thermometers should be used.
  3. Drain staining rack/slides after each step to prevent solution carry over.
  4. Do not allow sections to dry out at any point during staining procedure.
  5. The suggested microwave procedure has been tested at Newcomer Supply using an “EB Sciences”, 850 watt microwave oven with temperature probe and agitation tubes.  This procedure is reproducible in our laboratory.  It is nonetheless a guideline and techniques should be developed for your laboratory which meet the requirements of your situation. Microwave devices should be placed in a fume hood or vented into a fume hood, according to manufacturer’s instructions, to prevent exposure to chemical vapors.
  6. Preheating Silver-Methenamine Working Solution to 45°C-60°C prior to incubation is suggested for timely silver development. A water bath can be used for preheating if a microwave is unavailable. Begin preheating the silver solution approximately 20-30 minutes before use.
  7. Staining slides at higher temperatures will cause the development reaction to happen faster, but may also cause precipitate to form in the working silver solution and deposit on the slides.  Maintaining the silver solution between 45°C-60°C will help to minimize precipitate.
  8. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

 

REFERENCES:

  1. Carson, Freida L., and Christa Hladik. Histotechnology: A Self-Instructional Text. 3rd ed. Chicago, Ill.: American Society of Clinical Pathologists, 2009. 239-243.
  2. Grocott, R G, “A Stain for Fungi in Tissue Sections and Smears using Gomori Methenamine Silver Nitrate Technic”. American Journal of Clinical Pathology 25 (1955): 975-979.
  3. Koski, John. "Silver Methenamine Borate (SMB): Cost Reduction with Technical Improvement in Silver Nitrate-Gold Chloride Impregnations." The Journal of Histotechnology 4.3 (1981): 115-119.
  4. Sheehan, Dezna C., and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 245-246.
  5. Modifications developed by Newcomer Supply Laboratory.