Fluorescence Microscopy_TB 04-02_V1.0.doc

TABLE OF CONTENTS

1.PURPOSE

2.SCOPE

3.RESPONSIBILITIES

4.CROSS-REFERENCES

5.PROCEDURES

5.1Materials required......

5.1.1. Reagent preparation......

5.1.2. Smear preparation......

5.1.3. Staining......

5.1.4. Microscopy......

5.2Preparation of reagents......

5.2.1Staining solution (Auramine 0.1%, Phenol, 3%; 1 L)......

5.2.2Decolorizing solution: Hydrochloric acid in alcohol, 0.5%; 1 L......

5.2.3Counterstaining solution (potassium permanganate, 0.5%; 1 L)......

5.3Quality control of freshly prepared staining reagents......

5.4Preparation of smears......

5.5Staining of slides......

5.6Reading of smears......

5.7Reporting of results......

5.8Recording results......

5.9Storage of slides......

5.10Troubleshooting......

6.REFERENCES

7.CHANGE HISTORY

1.PURPOSE

This SOP describes fluorescence acid-fast bacilli (AFB) microscopy in the ______TB Laboratory. The property of acid-fastness of mycobacteria is based on the presence of mycolic acid in their cell wall. Primary stain (Auramine) binds cell wall mycolic acids. Intense decolourization (strong acid) does not release primary stain from the cell wall easily and AFBs keep the fluorescent bright colour of Auramine. Counterstain (potassium permanganate) provides contrasting background. Fluorescent stains are usually organic substances which absorb ultraviolet light and remit part of the energy as light of longer wavelength which can be observed through the eyepiece as fluorescence. When exposed to ultraviolet light, the fluorescent bacilli are perceived as brightly coloured organisms against a dark background.

2.SCOPE

This SOP covers microscopy procedures performed at the ______TB Laboratory.

3.RESPONSIBILITIES

All staff members working in the ______TB Laboratoryare responsible for the implementation of this operating procedure.All users of this procedure who do not understand it or are unable to carry it out as described are responsible for seeking advice from their supervisor.

4.CROSS-REFERENCES

See: / Document Matrix_TB 01_01_V1.0.doc
Location:

5.PROCEDURES

5.1Materials required

5.1.1. Reagent preparation

  • Balance, with a sensitivity of 0.1 g
  • Brushes to clean bottles before reuse
  • Containers for the newly prepared stains (dark amber glass bottles or plastic bottles)
  • Distilled or purified water
  • Flasks (conical or flat-bottomed balloons), capacity at least one litre
  • Measuring cylinders
  • Labels for bottles
  • Magnetic stirrer
  • Auramine, certified grade
  • Phenol crystals
  • Alcohol (70% ethanol)
  • Hydrochloric (37%, fuming) acid
  • Distilled water
  • Potassium Permanganate, certified grade

5.1.2. Smear preparation

  • BSC
  • Sharps container
  • Biohazard bag for waste disposal
  • New slides
  • Pencil for frosted and diamond pencil for unfrosted slides
  • Slides holder
  • Wooden sticks or disposable loops

5.1.3. Staining

  • Forceps
  • Slides staining rack
  • Timer

5.1.4. Microscopy

  • Fluorescent microscope
  • Len cleaning solution
  • Lens cleaning paper

5.2Preparation of reagents

Stains used in the ______TB Laboratorywill generally be purchased as pre-prepared reagents, where possible. However, in some circumstances (such as unavailability of commercial supplies), staining solutions will be prepared in the ______TB Laboratory. The following procedure for staining solution preparation will be followed:

5.2.1Staining solution(Auramine 0.1%, Phenol, 3%; 1 L)

  • Weigh 1.0 g of Auramine powder and 30 g of phenol crystals separately.
  • Measure 100 ml of alcohol and 870 ml of distilled water separately.
  • Combine alcohol and Auramine powder in a conical flask and stir until Auramine dissolved.
  • Add phenol crystals to water and stir until dissolved.
  • Only after Auramine is completely dissolved combine the two solutions.
  • Top off with distilled water to fill to a total volume of 1 L.
  • Perform Quality Control (see Section 5.5).
  • If results of QC satisfactory label appropriate clean container for freshly prepared stain with: “0.1% Auramine”, batch number, date prepared, expiry date (shelf life of the stain 6 months) and initials. Date of first opening should be mentioned.
  • Record prepared batch inthe Fluorescence StainsPreparation Logbook.

Use: / Fluorescence Stains Prep Logbook_form.doc
Location:
  • Store in the dark

5.2.2Decolorizing solution: Hydrochloric acid in alcohol,0.5%; 1 L

  • Add 995 ml of 70% alcohol to a conical flask.
  • Carefully add 5 ml of concentrated hydrochloric acid into the flask containing alcohol, directing the flow of acid along the inner side of the flask with constant swirling.
  • Mix well by swirling the flask.
  • Perform Quality Control (see Section 5.5).
  • If results of QC are satisfactory label appropriate clean container for freshly prepared stain with: “0.5% acid alcohol”, batch number, date prepared, expiry date (shelf life of the stain 6 months) and initials. Date of first opened should be mentioned.
  • Record prepared batch in Fluorescence Stains Preparation Logbook (see above for location).

5.2.3Counterstaining solution(potassium permanganate, 0.5%; 1 L)

  • Weigh 5 g of potassium permanganate powder.
  • Add the powder to 1 L of water.
  • Swirl and stir until dissolved.
  • Perform Quality Control (see Section 5.5).
  • If results of QC are satisfactory, label appropriate clean container for freshly prepared stain with: “0.5% potassium permanganate”, batch number, date prepared, expiry date (shelf life of the stain 6 months) and initials. Date of first opened should be mentioned.
  • Record prepared batch in Fluorescence Stains Preparation Logbook (see above for location).
  • Store in the dark.

5.3Quality control of freshly prepared staining reagents

  • Quality control must to be performed for each batch of staining, decolorizing and counterstaining solutions before using them routinely.
  • Control slides include two known positive (1+) and two known negative slides.
  • Stain positive control slides one time and negative control slides three times in raw (without reading) in order to check for the presence of environmental mycobacteria in water used for preparation of stains.
  • Read all slides once.
  • Record results of quality control in Quality control of Auramine Stains.

Use: / QC Auramine Stains_form.doc
Location:

Unacceptable results of quality control are as follows:

  • AFBs in positive control slides are not bright yellow
  • Negative control remains bright yellow after de-colorization
  • Background is not properly de-colorized

If there are unacceptable results:

  • Check for the method of preparation of reagents.
  • If preparation of staining solutions seems to be correct repeat a few more slides and ensure the staining procedure is correct.
  • If no errors are found in stain preparation and staining procedure prepare new staining solutions from a new batch of stains and reagents:
  • Auramine in case AFBs are not bright yellow
  • De-colorization solution if negative control remains bright yellow after de-colorization and background of the slides not properly de-colorized.

5.4Preparation of smears

Label the slide using a pencil or diamond pen using the laboratory number.Record laboratory numbers on the Auramine Microscopy Worksheet.

Use: / Auramine Microscopy Worksheet_form.doc
Location:
  • Place the slide on the slide holder
  • Procedure for smear preparation:
  • For a direct sputum smear select a small potion of purulent or mucopurulent material to the slide with a stick /loop and transfer it to the slide.
  • If the smear is to be done after specimen decontamination or liquid culture, transfer one drop of the material to the slide with a sterile pipette or tip used for media inoculation. Do not splash.
  • Albumin can be used to help specimen stick to slide.
  • For smear to bedone from culture on solid media put one drop of sterile saline on the slide and transfer a very small growth with sterile loop into the drop and gently rub it.
  • Spreadthe material carefully over the area equal to about 1-2cm,do not touch the border and take care to avoid placing too much on the slide, the thickness of smear should be such that a newspaper can be read through the smear if held under the slide
  • The slide bearing the smear should be left to dry in air at room temperature.
  • As soon as they are dry, hold the slide with forceps and fix them by passing them through a Bunsen burner or spirit flame three times in a quick succession.

5.5Staining of slides

  • Include QC slides with each days reading
  • Place the slide on the staining rack over a sink. Keep distance in between slides otherwise there is a possibility that acid-fast bacilli might float off one slide and become attached to the next slide.
  • Pour Auramine solution over the slide to that the smear is completely covered.
  • Do not heat.
  • Leave for 20 minutes.
  • Thoroughly rinse the slide with distilled water. Chlorine present in tap water can interfere with fluorescence.
  • Pour the acid solution over the slides.
  • Allow for to act 3minutes.
  • Gently rinse again with distilled until all macroscopically visible stain has been washed off.
  • Flood smear with potassium permanganate solution for 1minute (proper timing is critical because counterstaining for longer time may quench the fluorescence).
  • Wash off with distilled water.
  • Stand the slide on edge to drain.
  • Air dry on slide rack.

5.6Reading of smears

  • Start reading with QC slides. The positive control ensures the staining capability of the solutions and of the staining procedure. The negative control confirms that acid-fast contaminants are not present in the stains and in other solutions.
  • Use 25x or 40x objective together with 10x.
  • Systematically examine the smear by scanning it in the horizontal direction and move back (at least 100 fields are to be examined before smear reported AFB negative).
  • Stop and observe each field before moving onto the next field.
  • Keep slide boxes containing Auramine-stained smears closed when not in use to minimize fading.
  • Examine fluorochrome stained smears within 24 hours after staining as the fluorochrome may fade with time. Smears that cannot be examined immediately should be kept in the dark.
  • If only a few organisms are seen the result is doubtful and is to be confirmed by Ziehl Neelsen staining.
  • Analyze results on a weekly and monthly basis for percentage of positive results. Investigate any sharp differences from the norm.

5.7Reporting of results

Follow the WHO and IUATLD reporting scale:

Reporting scale / AFB seen
200 - 250x / 400x
Negative / 0 / 0
Scanty / 1-29 AFB per 1 length (30 fields) / 1-19 AFB per 1 length (40 fields)
1+ / 30-299 AFB per 1 length (30 fields) / 20-199 AFB per 1 length (40 fields)
2+ / 10-100 AFB per 1 field on average / 5-50 AFB per 1 field on average
3+ / >100 AFB per field on average / >50 AFB per field on average

5.8Recording results

Use: / Auramine Microscopy Worksheet_form.doc
Location:
  • Record results directly on the worksheets; do not use other pieces of paper for initial recording. If errors are made on the worksheets, put one line through the writing (so the original text is visible) and make the correction. Sign and date the correction in a footnote.

5.9Storage of slides

  • Store all slides in slide boxes in the chronological order of the laboratory number.
  • Slides will be retrieved later for the purposes of quality assurance.

See: / Microscopy QA_TB 04-03_V1.0.doc
Location:

5.10Troubleshooting

  • Possible causes of false positive results
  • Unfiltered Carbol Fuchsin
  • Using a scratched slide
  • Re-use of old slides
  • AFB floated off one slide and became attached to another during staining procedure because of insufficient distance between slides.
  • Inadequate decolourization
  • Drying of Auramine on the slide (crystal arise)
  • Bulk staining
  • Possible causes of false negative results
  • Poor quality of specimen
  • Taking improper portion of specimen for smear preparation
  • Excessive decolonization
  • Use of poorly prepared staining solutions
  • Over staining with potassium permanganate
  • Under staining with Auramine solution
  • Auramine solution kept in light
  • Reading fewer microscopic fields than recommended

6.REFERENCES

World Health Organisation. Laboratory Services in Tuberculosis Control. WHO, 1998

Use of fluorochrome staining for detecting acid-fast mycobacteria. Centers for Disease Control, 2000

7.CHANGE HISTORY

New version # / date / Old version # / date / No. of changes / Description of changes / Source of change request

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