533563106
Document type: SOP
Document code: TB 03-02
SPECIMEN DECONTAMINATION
Confidentiality: none
TABLE OF CONTENTS
1.
PURPOSE............................................................................................................................................ 2
2.
SCOPE................................................................................................................................................. 2
3.
RESPONSIBILITIES ............................................................................................................................ 2
4.
CROSS-REFERENCES ...................................................................................................................... 2
5.
PROCEDURES .................................................................................................................................... 2
5.1. Equipment................................................................................................................................................ 2
5.2. Supplies ................................................................................................................................................... 3
5.3. Reagents .................................................................................................................................................. 3
5.4. Preparation of solutions ............................................................................................................................ 3
5.4.1. NaOH - sodium citrate working solution ....................................................................................................... 3
5.4.2 0.5% NALC-2% NaOH-1.45% sodium citrate solution ................................................................................. 4
5.4.3. 4% NaOH solution (for pre-processing only) ................................................................................................. 4
5.4.4. 0.067 M Phosphate Buffer, pH 6.8 ................................................................................................................ 4
5.5. Specimen processing ................................................................................................................................. 5
5.6. Re-processing of specimens ....................................................................................................................... 6
5.6.1 Processed specimens ................................................................................................................................... 6
5.6.2 Growth on solid media ................................................................................................................................. 7
5.6.3 Liquid culture ............................................................................................................................................... 7
5.7. Quality control ......................................................................................................................................... 7
6.
REFERENCE ........................................................................................................................................ 7
7.
CHANGE HISTORY ............................................................................................................................. 8
This SOP template has been developed by FIND for adaption and use in TB laboratories
Release date: ddMMMyy
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533563106
1.
PURPOSE
This SOP describes the procedure for digestion and decontamination of specimens from non-sterile
sources for growth and detection of mycobacteria in the _________________TB Laboratory.
Mycobacteria are recovered optimally from clinical specimens when methods both to release them
from body fluids and cells (digestion) and to remove or sufficiently reduce competing organisms
(decontamination) are used. N-Acetyl-L-cysteine (NALC) is a mucolytic agent that at concentrations of
0.5 to 2.0% can rapidly digest even tenacious sputa from cystic fibrosis patients within 2 minutes.
Decontamination is achieved by the addition of sodium hydroxide. One advantage of using NALCNaOH method is that a very good mucolytic agent can be used with reduced concentrations of a
decontaminating agent (the final NaOH concentration in sputum is 1%).
2.
SCOPE
This SOP covers all procedures involving handling of non-sterile specimens in the
_________________TB Laboratory.
3.
RESPONSIBILITIES
All staff members working in the _________________TB Laboratory are responsible for the
implementation of this SOP. 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:
Refer to SOPs listed under TB 01 (General Procedures), TB 02 (Specimen Handling), TB 04 (Culture
and Drug Susceptibility Testing), TB 05 (Molecular Methods) and TB 06 (Equipment Use and
Maintenance)
5.
PROCEDURES
5.1. Equipment
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Class II biological safety cabinet (BSC)
Refrigerated centrifuge with aerosol-free sealed centrifuge cups
Vortex mixer
Timer
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5.2. Supplies
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Splash-proof container
Disinfectant
Disinfectant soaked towel
Sterile 50-ml conical polypropylene screw-cap tubes (graduated)
Sterile 15 ml conical polypropylene screw-cap tubes (graduated)
Sterile disposable 10μl loops
Sterile filtered tips
Automatic pipettes
Sterile graduated transfer pipettes, 3ml
Sharps containers
Test tube racks
Disposable spatula
5.3. Reagents
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5.4.
NALC powder
Sodium hydroxide (NaOH)
Sodium citrate x 2 H2O or anhydrous sodium citrate.
N-acetyl-L-cysteine (NALC)
Na2HPO4
KH2PO4
Preparation of solutions
5.4.1. NaOH - sodium citrate working solution
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Prepare an appropriate volume of NaOH-sodium citrate working solution according to Table 1.
Table 1. Preparation of 2% NaOH-1.45% Sodium Citrate Working Solution
Volume needed (ml)
NaOH (grams)
Sodium Citrate x 2 H2O (grams)
250
5
3.125
500
10
7.25
1000
20
14.5
2000
40
29.0
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Add the correct amount of NaOH and sodium citrate together to two thirds of the required
volume of distilled water in a conical flask and dissolve completely.
Once dissolved completely, make up to the appropriate volume with distilled water.
Dispense 250ml volumes into screw cap Duran bottles. Put autoclave tape on each bottle,
label and date and autoclave for 15 minutes at 121°C, 15 psi. Allow to cool.
Store at room temperature for up to 4 weeks.
Record prepared solution in the NaOH-Sodium Citrate Solution Preparation Logbook.
Use:
NaOH-Sodium Citrate Solution Preparation Logbook_form.doc
Location:
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5.4.2. 0.5% NALC-2% NaOH-1.45% sodium citrate solution
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Pre-weigh 0.25g amounts of N-acetyl-L-cysteine into labeled sterile screw cap bottles and
store in the refrigerator. Sufficient bottles for approximately 1 month can be prepared at one
time.
Use:
NALC Logbook_form.xls
Location:
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Immediately prior to use aseptically transfer 50ml of NaOH-sodium citrate solution into a
sterile graduated conical tube.
Aseptically add 0.25g NALC into the solution and allow to dissolve completely before use.
The solution should be prepared just before use and used on the day of preparation.
Record prepared solution in the NALC-NaOH-Sodium Citrate Solution Preparation Logbook.
Use:
NALC-NaOH-Sodium Citrate Solution Preparation Logbook_form.doc
Location:
5.4.3. 4% NaOH solution (for pre-processing only)
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Prepare 4% sodium hydroxide solution (for use in re-processing only) according to Table 2.
Table 2. Preparation of 4% NaOH
Total Volume Needed for (ml)
Sodium hydroxide (grams)
500
20
1000
40
2000
80
3000
120
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Dissolving of sodium hydroxide in water is highly exothermic reaction. Add sodium hydroxide
pellets to water slowly, allowing the solution to cool down before addition of the next portion.
Dissolve the pellets in approximately two thirds the total volume of water required in a conical
flask and make up to the final volume with distilled water once the pellets are fully dissolved.
Dispense 250ml volumes into screw cap Duran bottles. Put autoclave tape on each bottle,
label and date and autoclave for 15 minutes at 121°C, 15 psi. Allow to cool.
Store at room temperature for up to 4 weeks.
Record prepared solution in the 4% NaOH Solution Preparation Logbook.
Use:
4% NaOH Solution Preparation Logbook_form.doc
Location:
5.4.4. 0.067 M Phosphate Buffer, pH 6.8
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Dissolve Na2HPO4 and KH2PO4 in distilled water (Table 3). Check pH on meter or with pH
strips.
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Table 3. Preparation of 0.067 M phosphate buffer, pH 6.8
Total Volume Needed for (ml)
Disodium phosphate (Na2HPO4)
Monopotassium phosphate
ahydrous (grams)
(KH2PO4) (grams)
1000
4.74
4.54
2000
9.47
9.07
3000
14.20
13.60
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If final buffer requires pH adjustment, add Na2HPO4 powder to raise the pH of the solution or
KH2PO4 to lower it.
Distribute the buffer into 500ml volumes in screw capped Duran bottles. Label and date and
put autoclave tape over the lid.
Autoclave at 121°C, 15 psi. for 15 minutes. Allow to cool. Store at room temperature
unopened for up to 4 weeks.
Record prepared solution in the Phosphate Buffer pH 6.8 Preparation Logbook.
Use:
Phosphate Buffer Preparation Logbook_form.xls
Location:
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Check solutions immediately before use for decontamination. Do not use if turbid or a
precipitate is present
5.5. Specimen processing
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The procedure should be performed over disinfectant-soaked paper towels in the biological
safety cabinet.
Process no more than twelve samples per batch (ten specimens plus 2 controls).
Record specimen details on the Specimen Processing Worksheet.
Use:
Specimen Processing Worksheet_form.doc
Location:
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Label centrifuge tubes with specimen numbers and place in a rack in the BSC.
Opening only one specimen and one tube at a time transfer up to 10 ml of specimen into
50 ml sterile disposable conical tube. For very small or viscous specimens, add a small volume
of phosphate buffer to the specimen container and mix to loosen the specimen to facilitate
sputum transfer.
Add equal amount of fresh NALC-NaOH-sodium citrate working solution to each tube, opening
only one tube at a time. Addition of equal amount of NALC-NaOH-sodium citrate working
solution is critical. Lesser amount will lead to under-decontamination and will cause
contamination of culture. More than equal amount will lead to higher concentration of NaOH
and will decrease number of viable bacilli.
Recap the tube tightly and agitate on a vortex mixer for no more than 30 seconds. Avoid
excessive agitation, as it may inactivate NALC and cause the specimen to gel.
Let the tube stand for 15 min at room temperature to decontaminate the specimen.
Make sure the specimen is completely liquefied. If still mucoid add small amount of NALC
powder (30 – 35 mg) directly to the specimen tube. Mix well by inverting the tube several
times.
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Processing time can be extended for up to 20 – 25 min but no longer because prolonged incubation in
the presence of sodium hydroxide greatly affects the recovery and time-to-detection of mycobacteria.
(If longer decontamination time is used this must be recorded in the daily worksheet).
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Add sterile phosphate buffer, pH 6.8 up to the 45 ml mark. This will reduce the continued
action of the NaOH and lower the viscosity of the mixture. Recap the tubes tightly and mix well
by inverting several times.
Using aerosol-free sealed centrifuge cups centrifuge the specimen tubes at 3,000g for 20
minutes. Open the aerosol-free sealed centrifuge cups inside the biological safety
cabinet ONLY. Remove the centrifuge tubes.
Opening one tube at a time carefully with one uninterrupted movement decant the
supernatant into the splash-proof discard container containing approximately 5cm depth of
suitable disinfectant making sure that the sediment is not lost during decanting. Recap the
tube.
Opening one tube at a time draw up an appropriate volume of phosphate buffer, pH 6.8 into
sterile disposable Pasteur pipette (usually 1-2ml, depending on the number of tests being
performed). Add the buffer along the wall of the tube holding the end of the pipette close to the
sediment to prevent aerosolization. Mix thoroughly with the pipette. Discard the pipette into
sharp container.
Recap the tube tightly. Proceed to next tube.
Swab the tube with disinfectant soaked paper towel in the event of the outer surface of
the tube becoming contaminated during decanting.
Proceed for inoculation of solid and MGIT media.
Use:
MGIT Culture and Drug Susceptibility Testing_TB 05-02_V1.0.doc
Solid Mycobacterial Culture_TB 05-01_V1.0.doc
Location:
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Mix suspension by pipetting up and down several times prior to inoculation of media. A single
sterile transfer pipette can be used per specimen for inoculating several tubes of medium,
provided it does not become contaminated during the procedure.
Keep processed specimens one week in refrigerator in order to re-process specimens that
become contaminated.
5.6. Re-processing of specimens
Re-process specimens that grow contaminates in both tubes/plates.
5.6.1. Processed specimens
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Select the processed specimens that grew contaminated within one week and were kept in the
refrigerator.
Add sterile phosphate buffer, pH 6.8 up to 5 ml in each centrifuge tube.
Perform specimen processing procedure (Section 5.5), but using an equal volume of 4%
sodium hydroxide solution (in place of NALC-sodium hydroxide-sodium citrate solution)
Inoculate media tubes/bottles labeled “RS” (reprocessed) in addition to other identification
information
Record contamination in the laboratory register against the specimen number as “Both
contaminated” and date followed by “Reprocessed” and date of re-processing.
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Complete a new Specimen Processing Worksheet for the batch of reprocessed specimens,
indicating that they are reprocessed using RS followed by original laboratory number.
Use:
Specimen Processing Worksheet_form.doc
Location:
5.6.2. Growth on solid media
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Transfer generous amount of contaminated AFB positive (confirmed by ZN microscopy) growth
from solid media with loop into 5 ml of sterile phosphate buffer, pH 6.8 in 15 ml Falcon tube
with 8-10 glass beads (3 mm)
Vortex to mix. Transfer the suspension to a 50 ml sterile disposable centrifuge tube.
Process with 4% NaOH as in Section 5.5. “Specimen processing”
Inoculate media tubes/bottles labeled “RC” (reprocessed culture) in addition to other
identification information
Record contamination in the laboratory register against the specimen number as “Both
contaminated” and date followed by Reprocessed and date of re-processing.
Complete a new Specimen Processing Worksheet (see above for location) for the batch of
reprocessed cultures, indicating that they are reprocessed using “R” followed by original
laboratory number.
5.6.3. Liquid culture
Transfer 5 ml of contaminated AFB positive (confirmed by ZN microscopy) culture into 50 ml sterile
disposable centrifuge tube using sterile transfer pipette. Proceed as in 5.6.2.
5.7. Quality control
Process positive and negative controls together with every batch of processed specimens:
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Negative control: 5 ml of phosphate buffer
Positive control: 5 ml of M. tuberculosis H37Rv suspension (0.5 McFarland standard) in
phosphate buffer (TB 05-05 Mycobacterial Suspensions & Viable Counts). Positive control
culture must not be older than two weeks after growth appearance.
Record QC samples the same way as specimens. Note the date the QC strain was sub-cultured on
the Specimen Processing Worksheet.
6.
REFERENCES
Kent P.T., and G.P. Kubica. 1985. Public Health Mycobacteriology. A Guide for the Level III
Laboratory. U.S. Depatment of Health and Human Services, Centers for Disease Control, Altlanta, GA.
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7.
CHANGE HISTORY
New version #
/ date
Old version #
/ date
No. of
changes
Description of changes
Source of
change request
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