1 - FIND



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

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 NALC-NaOH 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

• Class II biological safety cabinet (BSC)

• Refrigerated centrifuge with aerosol-free sealed centrifuge cups

• Vortex mixer

• Timer

5.2. Supplies

• 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

• NALC powder

• Sodium hydroxide (NaOH)

• Sodium citrate x 2 H2O or anhydrous sodium citrate.

• N-acetyl-L-cysteine (NALC)

• Na2HPO4

• KH2PO4

5.4. Preparation of solutions

5.4.1. NaOH - sodium citrate working solution

• 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 |

• 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: | |

5.4.2. 0.5% NALC-2% NaOH-1.45% sodium citrate solution

• 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: | |

• 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)

• 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 |

• 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

• Dissolve Na2HPO4 and KH2PO4 in distilled water (Table 3). Check pH on meter or with pH strips.

Table 3. Preparation of 0.067 M phosphate buffer, pH 6.8

|Total Volume Needed for (ml) |Disodium phosphate (Na2HPO4) ahydrous (grams)|Monopotassium phosphate (KH2PO4) (grams) |

|1000 |4.74 |4.54 |

|2000 |9.47 |9.07 |

|3000 |14.20 |13.60 |

• 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: | |

• Check solutions immediately before use for decontamination. Do not use if turbid or a precipitate is present

5.5. Specimen processing

• 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: | |

• 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.

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).

• 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: | |

• 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

• 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.

• 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

• 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:

• 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.

7. CHANGE HISTORY

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

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