ZN Microscopy_TB 04-01_V1.0.doc
`Document type: SOP
Document code: TB 04-01
ZIEHL NEELSEN MICROSCOPY
Confidentiality: none
Table of Contents
1.
PURPOSE...................................................................................................................... 2
2.
SCOPE .......................................................................................................................... 2
3.
RESPONSIBILITIES ...................................................................................................... 2
4.
CROSS-REFERENCES ................................................................................................. 2
5.
PROCEDURES .............................................................................................................. 2
5.1 Materials required ............................................................................................................................. 2
5.1.2 Smear preparation ...................................................................................................................... 2
5.1.3 Staining ....................................................................................................................................... 3
5.1.4 Microscopy .................................................................................................................................. 3
5.2 Preparation of reagents and solutions .............................................................................................. 3
5.2.1 Staining solution (Carbol fuchsin, 1%; Phenol, 5%; 1 L) .............................................................. 3
5.2.2 Decolorizing solutions ................................................................................................................. 4
5.2.3 Quality control of freshly prepared staining reagents ................................................................. 4
5.3 Preparation of smears ....................................................................................................................... 5
5.4 Staining of slides ................................................................................................................................ 6
5.5 Reading of smears ............................................................................................................................. 6
5.6 Reporting of results ........................................................................................................................... 7
5.7 Storage of slides ................................................................................................................................ 7
5.8 Troubleshooting ................................................................................................................................ 7
6.
REFERENCES ............................................................................................................... 8
7.
CHANGE HISTORY ....................................................................................................... 8
This SOP template has been developed by FIND for adaptation and use in TB laboratories
Release date: ddMMMyy
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ZN Microscopy_ TB 04-01_V1.0.doc
1.
PURPOSE
This SOP describes the Ziehl-Neelsen method for acid-fast bacilli (AFB) microscopy. The property of
acid-fastness of mycobacteria is based on the presence of mycolic acid in their cell walls. Primary
stain (fuchsin) binds cell wall mycolic acids. Intense decolourization (strong acid) does not release
primary stain from the cell wall easily so AFBs keep the red colour of fuchsin following acid treatment.
Counterstain (methylene blue) provides contrasting background to improve visualization.
2.
SCOPE
This SOP covers the performance of smear microscopy in the ___________ TB Laboratory.
3.
RESPONSIBILITIES
All staff members working in the ___________ TB Laboratory are 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
5.1 Materials required
5.1.1. Reagent preparation
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Balance with a sensitivity of 0.1 g
Brushes to clean bottles before reuse
Containers for newly prepared stains (dark amber glass bottles or plastic bottles)
Distilled or purified water
Filter paper, large, adapted to funnels
Flasks (conical or flat-bottomed balloons), capacity at least one litre
Measuring cylinders
Funnels, large ones to fill bottles
Labels for bottles
Magnetic stirrers
Carbol fuchsin, > 85% of purity
Phenol crystals
Alcohol (95% ethanol)
Sulphuric (>95%) or hydrochloric (37%, fuming) acid
Distilled water
Methylene blue
5.1.2 Smear preparation
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BSC
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ZN Microscopy_ TB 04-01_V1.0.doc
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Sharps container
Biohazard bag for waste disposal
New slides
Pencil for frosted and diamond pencil for unfrosted slides
Slide holder
Wooden sticks or disposable loops
5.1.3 Staining
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Bunsen burner or spirit lamp
Forceps
Small funnel
Filter paper to fit small funnel
Slide staining rack
Timer
5.1.4 Microscopy
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LED microscope (light mode)
Immersion oil
Lens cleaning solution
Lens cleaning paper
Tissue for drying slides/blotting off immersion oil
5.2 Preparation of reagents and solutions
5.2.1 Staining solution (Carbol fuchsin, 1%; Phenol, 5%; 1 L)
The following procedure for staining solution preparation will be followed:
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Weigh 10 g of Basic fuchsin and 50 g of phenol crystals separately. Utilize correction factor when
purity is <85% as follows:
Correction factor =
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weight required (gm)
purity (as a decimal)
Measure 100 ml of alcohol and 850 ml of distilled water separately.
Combine alcohol and phenol crystals in a conical flask and stir until phenol dissolves.
Add Carbol Fuchsin powder and stir until dissolved.
If powder is not dissolving, add 100 ml of water and continue stirring.
Check for remaining powder or crystals on the bottom.
If these are seen, continue stirring with occasional heating.
It may be preferable to leave the mixture stirring with slight heating overnight.
Only after Carbol Fuchsin is completely dissolved add the remaining water to make 1 L total and
mix.
Perform Quality Control (see Section 5.2.3).
If results of QC are satisfactory, label appropriate clean container for freshly prepared stain with:
“1% Carbol Fuchsin”, batch number, date prepared, expiry date (shelf life of the stain 6 months)
and initials. Date first opened should be recorded on bottle.
Filter prepared stain into the container.
Record batch preparation in ZN Stains Preparation Logbook.
Use:
ZN Stains Prep Logbook_form.doc
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5.2.2 Decolorizing solutions
5.2.2.1 Hydrochloric acid in alcohol, 3%; 1 L
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Add 970 ml of 95% alcohol to a conical flask.
Measure 30 ml of concentrated hydrochloric acid in a cylinder.
Pour HCl slowly 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 5.2.3).
If results of QC are satisfactory, label appropriate clean container for freshly prepared stain with:
“3% acid alcohol”, batch number, date prepared, expiry date (shelf life of the stain 6 months) and
initials. Date first opened should be recorded on bottle.
Record batch preparation in ZN Stains Preparation Logbook (see above for location).
5.2.2.2 Sulphuric acid 25%, 1 L
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Add 750 ml of cold distilled water into a conical flask.
Measure 250 ml of concentrated Sulphuric acid into a cylinder.
Pour Sulphuric acid very slowly into the flask containing water, directing the flow of acid along the
inner side of the flask with constant swirling.
As soon as dissolving of acid generates a lot of heat it is necessary to stop a few times to cool it of
a bit, or even holding it under the tap with cold water.
Mix well by swirling the flask.
Perform Quality Control (see Section 5.2.3).
If results of QC are satisfactory label appropriate clean container for freshly prepared stain with:
“25% Sulphuric acid”, batch number, date prepared, expiry date (shelf life of the stain 6 months)
and initials. Date first opened should be recorded on bottle.
Record batch preparation in ZN Stains Preparation Logbook (see above for location).
5.2.2.3 Counterstaining solution (methylene blue, 0.1%; 1 L)
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Weigh 1 g of methylene blue powder.
Add the powder to 1 L of water.
Swirl and stir until dissolved.
Perform Quality Control (see Section 4.5).
If results of QC satisfactory, label appropriate clean container for freshly prepared stain with:
“0.1% Methylene blue”, batch number, date prepared, expiry date (shelf life of the stain 6 months)
and initials. Date first opened should be recorded on bottle.
Record batch preparation in ZN Stains Preparation Logbook (see above for location).
5.2.3 Quality 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:
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Stain positive control slides one time and negative control slides three times sequentially (without
reading) in order to check for the presence of environmental mycobacteria in water used for
preparation of stains.
Read all slides once.
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ZN Microscopy_ TB 04-01_V1.0.doc
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Record results of quality control in Quality Control of ZN Stains.
Use:
QC ZN Stains_form.doc
Location:
Unacceptable results of quality control are as follow:
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AFBs in positive control slides are not strongly red
Negative control remains red after decolorization
Background is not properly decolorized
Stain deposit is present on the QC slides
If there are unacceptable results:
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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 error found in stain preparation and staining procedure, prepare new staining solutions from a
new batch of stains and reagents:
 Carbol Fuchsin: if AFBs are not strongly red.
 De-colorization solution: if negative control remains red after decolorization
and background of the slides is not properly de-colorized.
 Methylene blue: if one or both of negative control slides have AFB clumps.
The solution could be contaminated.
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5.3 Preparation of smears
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Label the slide using a pencil or diamond pen using the laboratory number.
Record laboratory numbers on the ZN Microscopy Worksheet.
Use:
ZN Microscopy Worksheet_form.doc
Location:
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Place the slide on the slide holder.
Proceed for smear preparation:
 For a direct sputum smear select a small potion of purulent/mucopurulent
material and transfer it to the slide with a wooden applicator or inoculation
loop.
 If the smear is performed 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.
 If smear is from culture on solid media, put one drop of sterile saline on the
slide. Use a sterile loop to transfer a very small growth onto the drop and
gently rub it onto the slide.
 Spread the material carefully over an 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 held under the slide can
be read through the smear.
 The newly smeared slide should be left to air dry at room temperature.
 As soon as they are dry, hold the slides with forceps and fix them by passing
them through a Bunsen burner or spirit flame three times in quick succession.
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ZN Microscopy_ TB 04-01_V1.0.doc
5.4 Staining of slides
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Include QC slides with each day’s reading.
Place the slides 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 freshly filtered Carbol Fuchsin solution over the slides so that the smears are completely
covered.
Heat from below with spirit flame until steam rises. Repeat twice during 5 minutes.
Leave for 10 additional minutes.
Thoroughly rinse slides with distilled or running water.
Pour the acid solution over the slides.
Allow to act for 3 minutes.
Gently rinse again with distilled or tap water until all macroscopically visible stain has been washed
off.
Flood smears with methylene blue solution for 1 minute.
Wash off with distilled or running water.
Stand the slides on edge to drain.
Air dry on slide rack.
5.5 Reading of smears
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Start reading with QC slides.
Plug the microscope into the electrical source and switch on the light.
Place the slide on the stage with smear-side up.
Focus the smear using the 10x objective by turning coarse adjustment knob.
Adjust the distance between the ocular lenses until both right and left images become one.
Fine focus the image with right eye while looking into the right eyepiece by turning the fine
adjustment knob.
Focus the image with left eye looking into the left eyepiece by turning the dioptre ring.
Put one drop of immersion oil on the smear. Let it fall freely on to the slide. Avoid touching smear
with applicator as that may cause false positive results.
Change to the 100x objective and be sure the condenser is raised as high as possible to maintain
the intensity of the light. Open the condenser iris to 70-80% of the aperture diameter. Focus the
smear by turning only the fine focus adjustment knob.
To avoid pushing the objective through the slide, never adjust the stage upward while looking
through the eyepiece.
To view the next slide, the entire procedure does not have to be repeated. Turn the 100x objective
away from the field and clean it with lens paper and lens cleaning solution. Add a drop of
immersion oil on a new stained smear and insert it on the stage, then carefully turn the 100x
objective.
Systematically examine the smear by scanning it in the horizontal direction. One sweep through
2 cm of smear would be approximately 100 high power fields.
Stop and observe each field before moving onto the next field.
Read at least 100 high power fields before reporting a negative result.
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5.6 Reporting of results
Follow the WHO/UAILTD reporting scale
Reporting scale
AFB seen
Negative
No AFB seen in at least 100 fields
Actual number
1-9 AFB per 100 fields
1+
10-99 AFB per 100 fields
2+
1-10 AFB per field at least in 50 fields
3+
More than 10 AFB per field in at least 20 fields
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Record results on the ZN Microscopy Worksheet when reading smears prepared from specimens
(see above for location).
Record results on the Positive MGIT Culture Work up when reading smears prepared from
positive MGIT cultures.
Use:
Positive MGIT Culture Work-Up_form.doc
Location:
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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.
5.7 Storage of slides
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Remove oil from smears by placing the slides face down on a paper towel and leaving them in this
position overnight.
Store all slides in slide boxes in the chronological order of the laboratory number.
Slides will be later retrieved for the purposes of quality assurance.
See:
Microscopy QA_ TB 04-03_V1.0.doc
Location:
5.8 Troubleshooting
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Possible causes of false positive results
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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 decolorization
Contaminated lens or immersion oil
Drying or Carbol Fuchsin on the slide leading to crystal formation.
Bulk staining
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ZN Microscopy_ TB 04-01_V1.0.doc
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Possible causes of false negative results
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Poor quality of specimen.
Taking improper portion of specimen for smear preparation.
Excessive decolorization.
Use of poorly prepared staining solutions.
Overstaining with methylene blue.
Overheating during fixing.
Reading fewer than 100 microscopic fields.
REFERENCES
World Health Organisation. Laboratory Services in Tuberculosis Control. WHO, 1998
The Public Health Service National Tuberculosis Reference Laboratory and the National Laboratory
Network. IUATLD, 1998.
7.
CHANGE HISTORY
New version #
/ date
Old version #
/ date
No. of
changes
Description of changes
Source of
change request
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