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Procedure and Form 6 Staining Procedures

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Procedure and Form 6 Staining Procedures

Procedure and Form 6: Staining Procedures

    CLIA Level of Complexity

    Moderate for Acid-Fast Stain, High for Albert’s Stain (Zaias’s Modification) and Anagen Dye (1% DACA), High for Wright’s Stain and Paragon Multiple Stain (PMS) except when

    used for Tzanck smears which are Moderate, High for Gram Stain of all samples except

    endocervical and urethral which are Moderate.

    Laboratory

    Review Policy

    This procedure manual is reviewed by the Laboratory Director annually and at other

    times as required by major changes in procedure or other circumstances affecting

    laboratory performance of the test.

    Record of Changes

    All changes should be made in the space provided or on inserted or attached sheets.

    Each change or notation should be referenced to the appropriate paragraph number and

    signed by the Laboratory Director.

REVIEW BY LABORATORY DIRECTOR

    Date Signature

Date of first use of these procedures: _________________________

    Date of last use of these procedures: _________________________

    (Copies of all procedures must be retained for two years after last date of use.)

Procedure and Form 6: Staining Procedures (continued)

    Principle of Tests

    Various stains are used to identify skin and other tissue pathology. Staining procedures

    are described below.

    ? The Gram stain is used for identification of most bacteria and Candida albicans.

    ? Wright’s Stain is a polychromatic acid and basic dye that may be used for

    examination of cytologic specimens obtained from the skin as well as the blood and

    other tissues.

    ? Paragon Multiple Stain (PMS) is a polychromatic acid and basic dye that may be

    used for examination of cytologic specimens obtained from the skin as well as the

    blood and other tissues.

    ? Acid-Fast Stain is used for the identification of “acid-fast” mycobacteria such as

    Mycobacterium leprae, M. tuberculosis and atypical mycobacteria. The acid-fast

    stain method described here is a modification of the Ziehl-Neelsen technique. ? Albert’s Stain (Zaias’s modification) was originally developed for staining diphtheria

    bacilli; it is also useful for staining the true fungi that cause mycetoma.

    ? Anagen Dye is used to stain the amino acid citrulline a bright-red color. Plucked

    anagen hairs regularly contain an internal root sheath rich in citrulline, which stains

    positively. Plucked telogen hairs do not contain the inner root sheath and are

    therefore easily distinguished.

    Diagnostic Value

    Stained skin and tissue specimens are used to diagnose and identify a variety of

    pathologies and causal agents.

    Clinical Indications

    Staining of patient samples may be indicated for a large number of symptoms including

    those listed in the following sections of the Laboratory Procedure Manual: III KOH

    Examination Skin Samples, Vaginal Wet Preparation / KOH Examination, Tzanck (Cytodiagnostic)

    Smears, Cytodiagnosis of Molluscum Contagiosum, and Microscopic Hair Shaft

    Evaluation.

    MODIFICATIONS AND UPDATES

    Paragraph Changes Date Director's Signature

Procedure 6: Staining Procedures (continued)

    Patient Preparation

    No special patient preparation is required.

    Specimen Collection

    ? Specimens may include smears, blood, tissue or body fluids.

    ? Specimens should be collected using standard techniques appropriate for the site and diagnosis.

    Slide/Specimen Rejection

    ? Presence of foreign material that obscures the diagnostic finding. ? Cracked slide.

    ? Inappropriately labeled slide.

    ? Other (specify) _______________________________________________________ . Specimen Handling, Storage, Transport, Preservation and Identification

    ? Specimen collection supplies are located ____________________________________

     ____________________________________________________________________ .

    ? Specimen is stored ____________________________________________________ . ? Specimen is transported ________________________________________________ . ? Specimen is preserved __________________________________________________

     ___________________________________________________________________ .

    ? Slides are uniquely identified ______________________________________________

     ___________________________________________________________________ .

    ? Record the patient’s name, date and test requested, and specimen source (if

    applicable) before performing the analysis.

    ? Specimens will be disposed of according to federal, state, and local laws. MODIFICATIONS AND UPDATES

    Paragraph Changes Date Director's Signature

Procedure and Form 6: Staining Procedures (continued)

    Referral of Specimen for Processing (Check applicable) ? No materials, equipment, reagents, or test procedures are used. The sample is sent

    to the following CLIA-certified laboratory for processing. (Indicate laboratory name,

    address, phone number, contact person, and append or attach documentation of

    laboratory’s Quality Control procedures.)

     _______________________________________________________________________

     _______________________________________________________________________

    Materials and Equipment Used

    Maintenance logs will be kept up-to-date for all equipment and daily temperature logs

    will be completed for equipment that must operate in a specific temperature range (e.g.,

    heat source if slides must be heat fixed at a certain temperature). (See Section III

    Laboratory Procedure Manual for Log Sheet templates.)

    (Check applicable)

    ? Microscope.

    ? Microscope slides and coverslips.

    ? Microscope immersion oil.

    ? Heat source for heat fixation of slide (open flames are prohibited near flammable

    liquids).

    ? Slide washing / staining jar.

    ? Blotting paper.

    ? Other (specify)

    _______________________________________________________________

    ? Other (specify)

    ________________________________________________________________

    MODIFICATIONS AND UPDATES

    Paragraph Changes Date Director's Signature

Procedure and Form 6: Staining Procedures (continued)

    Gram Stain Reagents

    ? Crystal Violet Reagent. 1 gm Crystal violet, 5 gm Sodium bicarbonate (NaHCO3),

    100 ml Distilled water

    ? Gram’s Iodine Solution. 1 gm Iodine, 2 gm Potassium Iodide (KI), 200 ml Distilled

    water

    ? Decolorizing Solution. 3 volumes Acetone, 7 volumes Alcohol ? Counter Stain. 0.5 gm Safranine O, 100 ml Distilled water

    Wright’s Stain Reagents

    ? Wright’s Stain. 0.3 gm Wright’s dye, 3 ml Glycerin, 100 ml Methyl alcohol

    ? Wright’s Buffer. 6.63 gm Potassium phosphate, 2.56 gm Dibasic sodium phosphate,

    1 liter Distilled water

    PMS Stain Reagents

    ? PMS Stain. 0.356 gm Toluidine blue, 0.135 gm Basic fuchsin, 50 ml Ethyl alcohol

    (30%)

    ? Isopropyl alcohol (70%)

    Acid-Fast Stain Reagents

    ? Carbol Fuchsin. 0.3 gm Basic fuchsin, 10.0 ml Ethyl alcohol, 5.0 ml Phenol, 95 ml

    Distilled water, 2-3 drops Tergitol No. 7

    ? Acid-alcohol. 97.0 ml Ethyl alcohol (95%), 3.0 ml Hydrochloric acid (HCl)

    ? Methylene Blue. 0.3 gm Methylene blue, 30.0 ml Ethyl alcohol (95%), 100.0 ml KOH

    (0.1% w/v)

    ? Malachite Green. 220 ml Malachite green (2% aqueous solution), 30 ml Glacial

    acetic acid, 50 ml Glycerol

    MODIFICATIONS AND UPDATES

    Paragraph Changes Date Director's Signature

Procedure and Form 6: Staining Procedures (continued)

    Albert’s Stain Reagents

    ? Albert’s solution No. 1: 0.15 gm Toluidine blue, 0.2 gm Malachite green, 1.0 ml

    Glacial acetic acid, 2.0 ml Alcohol (95%), 100 ml Distilled water (Let stand 24 hours

    and filter; keep as a “stock” solution) ? Distilled water

    ? Glycerin

    Anagen Dye Reagent

    Dimethylaminocinnamaldehyde (1%) in hydrochloric acid

    Reagent Preparation

    Gram Stain, Wright’s Stain, Paragon Multiple Stain, Acid-Fast Stain, and Albert’s Stain

    are commercially available. Receipt of each new batch, lot, or shipment of reagent will

    be recorded on a Receipt Log (See Section IV Quality Assessment Manual for Log

    Sheet templates).

    Reagent Labeling

    Commercially prepared reagents are labeled by the manufacturer. Reagents prepared

    by the laboratory or transferred from manufacturer’s container to smaller containers must

    be labeled. Labels must include:

    ? Name of reagent

    ? Expiration date

    ? Preparation date

    ? Warning if reagent is hazardous

    ? Strength or concentration of reagent

    ? Storage requirements

MODIFICATIONS AND UPDATES

    Paragraph Changes Date Director's Signature

Procedure and Form 6: Staining Procedures (continued)

    Reagent Storage, Use and Handling (Check applicable)

    ? Reagents are stored ____________________________________________________

     _____________________________________________________________________

     _____________________________________________________________________

     _____________________________________________________________________

     _____________________________________________________________________

     _____________________________________________________________________

     _____________________________________________________________________

    ? To ensure that each new batch, lot, or shipment of reagent provides the expected

    positive and negative results in this procedure, at the time of its first use, each new

    batch, lot, or shipment of each reagent will be tested as described below under Test

    Procedure and Quality Control Procedures and the results of these analyses will be

    recorded on a Reagent Quality Assessment Log Sheet (See Section IV Quality

    Assessment Manual for Log Sheet templates).

    ? Do not use reagents after expiration date.

    ? Reagents are disposed of according to federal, state and local laws.

    ? Reagents are stored according to the manufacturer’s instructions and temperature

    logs of storage sites are maintained as appropriate.

    ? Material safety data sheets are located ______________________________________

     ____________________________________________________________________ .

    Calibration

    Not applicable.

MODIFICATIONS AND UPDATES

    Paragraph Changes Date Director's Signature

    Procedure and Form 6: Staining Procedures (continued) Gram Stain Procedure

    1. Gently heat-fix the specimen.

    2. Flood the slide with crystal violet and allow to stand for one minute.

    3. Rinse the slide with water.

    4. Cover with Gram’s iodine and let stand for one minute. 5. Rinse with water.

    6. Decolorize with acetone:alcohol solution. Let the decolorizing solution drip off the

    slide until no further color comes off in the drippings.

    7. Rinse with water.

    8. Counterstain with safranine for 30 seconds.

    9. Rinse with water.

    10. Dry without blotting and examine with a microscope.

    MODIFICATIONS AND UPDATES Paragraph Changes Date Director's Signature

    Procedure and Form 6: Staining Procedures (continued) Wright’s Stain Procedure

    1. Heat fixation is not needed with Wright’s stain. Heating will distort the cells. 2. Apply the Wright’s stain and let the slide stand for two minutes. 3. Apply the buffer and blow gently on the slide to provide even distribution of the

    reagents.

    4. Let stand three to four minutes until a green-colored metallic sheen forms.

    5. Flood the slide with tap water to remove the reagents.

    6. Blot the edges and allow the slide to air-dry before examination with a microscope.

    PMS Standard Procedure

    1. Fix the specimen immediately after obtaining it by placing the slide in 70% isopropyl

    alcohol for three minutes.

    2. Dip the slide five times in 70% isopropyl alcohol diluted 1:4 with water.

    3. Dip the slide five times in water.

    4. Cover the slide with PMS for about five seconds.

    5. Rinse with water.

    6. Blot the edges of the slide.

    7. Place a drop of water on the specimen. Place a coverslip over the water and

    examine with a microscope.

    MODIFICATIONS AND UPDATES Paragraph Changes Date Director's Signature

Procedure and Form 6: Staining Procedures (continued)

    PMS Rapid Procedure

    1. If the specimen is dry, such as scales, place a drop of water over the specimen.

    2. If the specimen is wet, such as from a blister or a pustule, or after it has been

    prepared as in the step above, heat gently to evaporate water and fix the specimen. 3. Apply 1 to 2 drops of PMS for 5 to 50 seconds.

    4. Rinse with water. Blot excess water from the edges of the slide.

    5. Place a drop of water over the stained specimen. Place a coverslip over the water

    and examine with a microscope.

    Acid-Fast Stain Procedure

    1. Fix the specimen with heat or absolute alcohol.

    2. Apply carbol fuchsin.

    3. Wash with water.

    4. Decolorize with acid-alcohol. Let the acid-alcohol drip over the slide until no further

    color comes off with the drippings.

    5. Counterstain for one to five minutes with methylene blue or malachite green.

    6. Blot and allow to air-dry before microscopic examination. Oil immersion and 1,000-

    fold magnification will be needed to see the acid-fast organisms.

MODIFICATIONS AND UPDATES

    Paragraph Changes Date Director's Signature

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