FLAGELLA STAIN

Cat. no. Z87 Flagella Stain, 0.5oz. Polyethylene Dropper Bottle, 15ml 1 bottle

INTENDED USE

Hardy Diagnostics Flagella Stain is recommended for use in detecting the presence and arrangement of flagella on the bacterial cell.

SUMMARY

Bacterial flagella, due to their narrow diameter, cannot be seen with a light microscope. The Flagella Stain provides a method for viewing bacterial flagella by employing crystal violet in an alcoholic solution as the primary stain. During the staining procedure, the alcoholic solution evaporates and leaves a precipitate around the flagella, increasing its apparent size to the human eye. The Flagella Stain also contains tannic acid and aluminum potassium phosphate as mordants, and phenol as an antifungal agent.

Hardy Diagnostics Flagella Stain is a simple, rapid, qualitative method for detecting bacterial flagella and their shape, length, curvature, arrangement and number on the cell. (2,5) This method was developed by Ryu in 1937, and also later described by Kodaka, et al. in 1982. (4,7) This test has been found especially useful in providing taxonomic and identifying information about motile bacteria, and more recently, anaerobic bacteria. (2,4,5)

REAGENT FORMULA

Ingredients per liter of deionized water:*

Aluminum Potassium Sulfate 57.0gm
Phenol 25.0gm
Tannic Acid 20.0gm
Crystal Violet, Certified 6.0gm

* 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.

PRECAUTIONS

Warning! Contains Phenol! Harmful if swallowed, inhaled, or absorbed through skin. Causes irritation. Avoid contact with eyes, skin mucous membranes, and clothing. If contact occurs, flush immediately with large amounts of water for at least 15 minutes, remove contaminated clothing, and call a physician. Avoid breathing gas, vapor, dust or mist. Wash clothing before reuse. Keep container tightly closed at all times.

Warning! Staining solutions are hazardous in nature. Wear appropriate safety apparel when working with staining solutions. It is recommended that staining procedures be performed under a hood and with adequate ventilation.

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product used with other biochemical tests to identify cultures of isolated organism. Consult listed references for information on specimen collection. (1-3)

Organism Preparation:

For best results it is recommended that an 18-48 hour old, well isolated colony taken from a Blood Agar plate be used for the Flagella Stain.

Streak the organism to be examined for isolation on a Blood Agar plate (Cat. no. A10). Incubate in an appropriate environment at 35ºC. Some non-fermentative organisms grow better at 25ºC., and are more likely to be flagellated at this growth temperature.

Smear Preparation:

1. With a wax pencil, draw a border around the clear portion of a microscope slide.

2. Approximately one centimeter from the frosted edge, place a drop of deionized (or distilled) water.

3. With a sterile inoculating loop, gently touch a colony of the culture to be examined and then lightly touch the drop of water without touching the slide. Do not mix.

4. Tilt the slide so the drop will flow to the opposite end of the slide.

5. Allow the slide to air dry, at room temperature. Do not heat fix.

Staining Procedure:

1. Flood the slide with Flagella Stain. Let the slide sit for approximately four minutes. However, staining time may need to be adjusted to between two to eight minutes, depending on the age of the stain, air currents and room temperature, and staining solution depth over the smear. (5,6)

2. Carefully rinse stain by leaving slide on staining rack and allowing water to flow over the surface of the slide. Do not tilt slide while rinsing.

3. 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 slide microscopically using the oil immersion objective. Begin the examination at thinner areas of the smear and work towards the center. Look for fields with several isolated cells rather than clumps of bacteria.

INTERPRETATION OF RESULTS

When examined under oil immersion, bacteria and their flagella should stain purple. Consult appropriate references for descriptions, drawings or pictures for specific information. (2,4,5,7)

LIMITATIONS

The presence of fermentable substances in the growth medium, age of the culture, and the temperature of incubation have all been shown to affect the degree of flagellation.(5) For optimal results, use cultures of log phase of organism from Blood Agar plates (Cat. no. A10).

Care is needed in transferring organisms from the culture to the water droplet on the slide, as flagella are easily dislodged from the bacterial cells.

The length of staining time may affect staining quality. A short staining time may result in poorly stained flagella. If this occurs, increase staining time. Prolonged staining may result in noticeable precipitation of the crystal violet on the slide. In this case, decrease the staining time.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, glass slides, wax pencils, immersion oil, culture media, pipets, microscopes, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Results
Klebsiella pneumoniae
ATCC® 13883
No flagella seen
Proteus vulgaris
ATCC® 8427
Peritrichous flagella seen
Pseudomonas aeruginosa
ATCC® 27853
Monotrichous flagella seen

USER QUALITY CONTROL

It is recommended that each new lot of stain be tested with known positive and negative controls and retested each week of use thereafter. (1,3)

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. (1,3)

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.

Physical Appearance

Flagella Stain should appear opaque, and blue-violet in color.

REFERENCES

1. 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.

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

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

4. Kodaka, H., et al. 1982. Practical procedure for demonstrating bacterial flagella. J. Clin. Microbiol.; 16:948-952.

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

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

7. Ryu, E. 1937. A simple methos of staining bacterial flagella. Kitasato Arch. Exp. Med.; 14:218-219.


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