Modified Field's Stain Kit

Cat. no. Z15 Modified Field's Stain Kit 2 bottles/kit
Each kit contains:
Z15A - Modified Field's Stain Solution A, 15ml
Z15B - Modified Field's Stain Solution B, 15ml

1 bottle
1 bottle

INTENDED USE

Hardy Diagnostics Modified Field's Stain Kit is recommended for the rapid staining of protozoans, such as Acanthamoeba and Trichomonas species.

SUMMARY

Depending on the specimen source, many stains are available for the identification of protozoans, including the original Field's stain, modified Ziehl-Neelson stains, Giesma stain, Wright's stain, and Wheatley's trichrome stain. For certain clinical specimens, modified Field's stain has been shown to be a superior method, being simpler and faster to perform, and providing sharper contrast, than traditional protozoan stains. (1,2) This method was developed by Field in 1941, and later modified by Field, et al. in 1963; the modification being the use of methanol (instead of aqueous buffer) as the solvent for solution B. (1)

Modified Field's Stain has been used for the detection of microfilaria, Leishmania , Plasmodium (malaria), Babesia , Trypanosoma , Trichomonas , and Acanthamoeba species. (2) Staining can facilitate early detection of protozoans directly from certain patient specimens. (1) Eye, CSF, and urogenital (including urine sediments) specimens generally do not tend to stain well using the Wheatley modification (used for intestinal tract specimens) or Gomori's trichrome (used for histology specimens). Hardy Diagnostics Modified Field's Stain Kit is a simple, rapid, and superior method for detecting protozoans, as well as visualizing their nucleolus, nucleus, vacuoles, and flagella, if present. (1,2)

REAGENT FORMULA

Modified Field's Stain Solution A (Cat. no. Z15A):
Sodium Phosphate 2.6gm
Potassium Phosphate 2.6gm
Methylene Blue, Certified 1.6gm
Azure I (Azure B) 1.0gm

Ingredients per liter of methanol:*

Modified Field's Stain Solution B (Cat. no. Z15B):
Eosin 2.0gm

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC., in the dark. Product should not be used if there are any signs of deterioration or if the expiration date has passed. Do not expose to excessive heat or moisture. Product is light sensitive; protect from light.

The expiration date applies to the product in its intact packaging when stored as directed.

PRECAUTIONS

Warning! Staining solutions are hazardous in nature. Wear protective gloves and wash thoroughly after use. Avoid contact with eyes, skin, or clothing. Avoid breathing the fumes or vapors. Use only in an adequately ventilated room.

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(3-8)

Refer to the keyword "Specimen Collection" on the Hardy Diagnostics Technical Document website for a description of specimen collection methods.

Specimen Preparation:

If patient specimen is liquid (such as urine), the sample should be mixed and an aliquot should be transferred to a sterile screw-capped tube or microcentrifuge tube. Centrifuge this tube for 3 to 5 minutes at 500xG, decant supernatant, and smear sediment on a clean glass slide. Allow to air dry at room temperature. Do not heat fix.

If patient specimen is blood, prepare routine thick and thin blood films. Allow to air dry at room temperature. Do not heat fix.

If patient specimen is solid (such as corneal scrapings or a vaginal swab), the sample may be directly smeared on a clean glass slide. Allow to air dry at room temperature. Do not heat fix.

Preparation of smears from Culture:

Broths (such as Modified Diamonds Medium, Cat. no. K02 or Acanthamoeba Broth, Cat. no. K225) should be mixed and an aliquot should be transferred to a sterile screw-capped tube or microcentrifuge tube. Centrifuge this tube for 3 to 5 minutes at 500xG, decant supernatant, and smear sediment on a clean glass slide. Allow to air dry at room temperature. Do not heat fix.

Plates (such as Non-Nutrient Agar, Cat. no. G225) should be scraped where feeding tracks are visible and directly smeared on a clean glass slide. Allow to air dry at room temperature. Do not heat fix.

Staining Procedure:

1. Fix smears with 5 to10 drops of solution B, then immediately add equal drops of solution A.

2. Allow to stain for approximately 20 seconds.

3. Carefully and gently rinse with tap water. Once the stain is washed off, gently tilt the slide to allow excess water to run off.

4. Air dry slide at room temperature.

5. Examine the smear at 1,000X, using the oil immersion objective.

INTERPRETATION OF RESULTS

When examined under oil immersion, protozoans and their cytoplasm should stain purplish blue; the nucleolus and flagella should stain dark blue and dark reddish purple, respectively. Nuclei, brain cells, and bacteria should stain pink. Consult appropriate references for descriptions, drawings or pictures for specific information. (1,2,4,6)

A. castellanii #1

A. castellanii (ATCC ® 30010) trophozoite stained with Modified Field's Stain Kit. Nucleus is visible. 1,000x.

A. castellanii #2

A. castellanii (ATCC ® 30010) trophozoite stained with Modified Field's Stain Kit. Nucleus and acanthapodia are visible. 1,000x.

LIMITATIONS

Staining must be done within 12 hours of smear preparation, as the quality of the stain contrast will decrease after 12 hours.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, glass slides, immersion oil, culture media, pipets, microscopes, and incinerators are not provided.

QUALITY CONTROL

Test Organisms* Results
Blood Parasite Control Slide Visualization of protozoa and organelles

* If a protozoan is not available to perform QC with, labs may order and stain blood parasite control slides (Cat. no. SL9010)

USER QUALITY CONTROL

It is recommended that each new lot of stain be tested with known positive controls and retested each week of use thereafter. (3,5)

The microscope should be calibrated (within the last 12 months), and the objectives and oculars used for the calibration procedure should be in place on the microscope when objects are measured. (3,5)

It is recommended that positive controls be run in parallel with patient specimens and that results from this staining procedure be reported only if positive control smears are acceptable ( Acanthamoeba , Trichomonas ). If blood parasites are a possibility, the WBCs contained in the specimen will serve as the internal control; if the WBCs look good on the thick and thin films, any parasites present will also look good.

Physical Appearance

Modified Field's Stain Solution A should appear clear, and blue in color.
Modified Field's Stain Solution B should appear clear, and red to orange in color.

REFERENCES

1. Pirehma, M., et al. 1999. Field's stain - a rapid staining method for Acanthamoeba spp. Parasitol. Res.; 85:791-793.

2. Afzan, M.Y., et al. 2010. Modified Field's staining - a rapid stain for Trichomonas vaginalis. Diagn. Microbiol. Infect. Dis.; 68:159-162.

3. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

4. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

5. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

7. Lillie, R.D. 1991. H. J. Conn's Biological Stains, 9th ed. Williams & Wilkins Company, Baltimore, MD., 1977. Reprint by Sigma Chemical Company.

8. Garcia, L.S., 2007. Diagnostic Medical Parasitology, 5th ed. American Society for Microbiology, Washington, D.C.


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