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Johns Hopkins University

Baltimore, MD USA

Specimen Digestion/Decontamination for Mycobacteriology Culture-

Guidelines

Author: Peggy Coulter Document Number: Pro67-C-15-G

Effective (or Post) Date: 10 Feb 09
Review History Version V 1.1
Revised Date: 13 May 2013
Date of last review 14 May 2013 Reviewed by: Heidi Hanes
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s
specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering
their use in other applications. If you have any questions contact SMILE.

CAP Accreditation Checklist:

Question pertaining to Mycobacteriology Culture can be found under Mycobacteriology –
Concentration, Inoculation, Incubation Section of the CAP Accreditation Laboratory
Microbiology Checklist.
Background Information:
Specimens submitted for Mycobacteriology culture are most frequently obtained from body sites
where other pathogens and/or normal flora reside. Because mycobacteria are slow growing and
require long incubation times these contaminating organisms can overgrow in cultures, blocking
the ability to detect the presence of the mycobacteria. Digestion and decontamination procedures
are used in processing sputum for examination and culture of sputum specimens. This step
performs two major functions: 1) sputum is liquefied (digestion), permitting the mycobacteria to
be released from the thick sputum so that they can be subsequently concentrated by
centrifugation, and 2) contaminating normal flora is preferentially killed (decontamination).
Unfortunately, it must be recognized that the strong alkaline reagent usually used for the
decontamination step is also toxic to mycobacteria. The degree to which the mycobacteria in the
specimen are killed is a function of the concentration of the alkali used, the length of time over
which the organisms are exposed, and the temperature at which the exposure occurs.
Appropriate digestion and decontamination procedures, culture media, and conditions of
incubation must be selected to facilitate optimal recovery of the mycobacteria. These
considerations are especially important for paucibacillary disease, i.e. sputum from patients who
present with non-cavitary disease, who excrete only small numbers of organisms in their sputum.
These populations include patient with early infections, and those who are HIV-infected.
The success of digestion/decontamination depends upon the following considerations:
 The concentration of organisms present in a given specimen;
 The resistance of tubercle bacilli to the concentration of the strongly alkaline or acidic
digesting solutions used;
 The length of time the mycobacteria are exposed to the digestant;
 The temperature of the room, in which the exposure is carried out
 The amount of heat generated by the specimen centrifugation step; and
 The efficiency of the centrifuge used in the sedimentation step.

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Several methods are available for digestion and decontamination of sputum specimens for
culturing of Mycobacteria, but not all are acceptable for all specimens or for all systems used to
culture for mycobacteria subsequently. The choice must be based upon technical capability, and
upon the quality and type of equipment, supplies and reagents available. Optimally for quality
control reasons, only a single method should be used in a given laboratory. Whichever method is
used, meticulous care must be taken to prevent laboratory cross-contamination of specimens
during processing. Risk of contamination occurs especially when digestant and/or buffer are
added subsequently to a batch of tubes. A single positive culture for M. tuberculosis can lead to
diagnosis of tuberculosis and a false-positive culture affects not only clinical management of the
patient, but also epidemiological investigations and public health controls.

Major Liquefaction/Decontamination Processing Method:

N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH) Method
NALC (a mucolytic agent) combined with sodium hydroxide is the preferred method for the
digestion step because it is the least toxic to the mycobacteria, and therefore provides the highest
yield of positives. NALC perform the liquefaction step, and permits the use of a lower, (1%)
concentration of NaOH than that required when NALC is omitted. Sodium citrate is also
included to bind heavy metal ions, which, if present in the specimen, can inactivate the acetyl-
cysteine. The digestant must be made fresh daily due to the rapid loss of acetyl-cysteine activity
when the compound is in solution.

A variety of specimen types may be processed using this method, making it particularly useful
for processing specimens from patients with few organisms, as seen in non cavitary disease
which includes patients with concurrent HIV infections. This is the manufacturer approved
method for processing specimens using the BD BBL™ MGIT™ Mycobacteria Growth Indicator
Tube.

Resources
1. Cernoch, P.L. et al. (1994). Cumitech 16A: Laboratory diagnosis of the Mycobacterioses.
ASM Press. Washington, DC.
2. Forbes, B.A., et al (2007). Laboratory detection and identification of mycobacteria; Proposed
guideline. CLSI document M48-P. Clinical and Laboratory Standards Institute, Wayne, PA.
3. Kent , P.T. and Kubica, G. P. (1985). Public Health Mycobacteriology. A Guide for the Level
III Laboratory. U.S. Dept. Health and Human Services. Center for Disease Control.
4. SA Healthinfo, Tuberculosis Part III: Culture homogenization and decontamination. Obtained
from the World Wide Web on 14 March 2007 at HREF="http://www.sahealthinfo.org/tb"
5. Weitzman, I. (2007) p 7.1.2.1-7.1.2.9. In H.D. Isenberg (ed.) Clinical Microbiology
Procedures Handbook American Society for Microbiology, Washington, D.C.

Digestion Decontamination for Mycobacterial Cultures V1.1 SMILE Document

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Specimen Digestion/Decontamination for Mycobacteriology Culture - SOP

Peggy Coulter MDE, MT (HEW) Document Effective
Author(s), Name & International QA/QC Coordinator Number Date
Title Pro67-C-15 13 May 2013

Approved By Name, Title Signature Date

SOP Annual Name, Title Signature Date

Review

Version # [0.0] Revision Date Description (notes)

Revision [dd/mm/yy]
History

Distributed Name (or location) # of copies Name (or location) # of copies

Copies to

Associated Forms:

Digestion Decontamination for Mycobacterial Cultures V1.1 SMILE Document

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Baltimore, MD USA

Purpose
There are different approved methods for processing specimens from various body sites. The one
used for processing sputum outlined below can be used for the BD BBL MGIT (Mycobacteria
Growth Indicator Tube) liquid and solid culture media.

Pre-analytic Procedure
Refer to the following procedures:
1. Mycobacteriology Laboratory Specimen Collection/Receiving
2. Safety in the BSL-3 Laboratory
3. Use of the Bio-Safety Hood in the Mycobacteriology Laboratory

Abbreviations Used:
 AFB = Acid Fast Bacilli
 BSC = Bio-Safety Cabinet
 SOP = Standard Operating Procedure

Specimen Information:
Specimen collection- Refer to Mycobacteriology Laboratory Specimen Collection/Receiving
SOP.

Analytic Procedure
Specimen types
 Specimens that require decontamination include sputum, bronchial secretions, washings, or
biopsies, skin, soft tissue, gastric lavage, stool specimens, urines and all other specimens
from sites contaminated with normal microbial flora.
o Gastric aspirates
 If the volume is greater than 10 ml, concentrate by centrifugation.
 Re-suspend sediment in about 5ml of sterile water and then decontaminate.
 Add a small amount of NALC powder if the specimen is thick or mucoid.
o Stool
 Suspend 1 g of feces in 5ml of Middlebrook broth.
 Agitate the suspension on a vortex mixer for 5 s.
 Precede to the procedure steps below.
 Sterile sites not requiring decontamination include CSF, bone marrow, blood, pleural fluid,
and sterile biopsy sites. These should be processed following specimen handling in the
Mycobacteriology Culture SOP.
 When a specimen is determined to be unacceptable, a repeat specimen must be requested.
o Unacceptable specimens include unlabeled or inadequately identified specimens, dry
swabs, and those received in previously used containers, containers that are non-
sterile or cannot be tightly sealed.
o Specimens held unrefrigerated prior to processing for greater than one hour may lead
to bacterial overgrowth at levels that prevent detection of mycobacteria
o Sputum specimens collected at different times must never be pooled as this greatly
increases the contamination rates.

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Specimen storage
 Specimens should be delivered to the laboratory as soon as possible to avoid overgrowth by
contaminants and normal respiratory flora.
 Specimens not processed within one hour of collection must be refrigerated at 2 – 8° C.
 If gastric lavage specimens are to take longer than 4 hours prior to processing, 100 mg. of
sodium carbonate must be added to the container to neutralize the high acidity of the
specimen.

Reagents/Media:
Reagents used for digestion/decontamination are dependent on precise adherence to the required
procedures.
1. Fresh working NALC-NaOH Solution - Directions for preparing NALC-NaOH Solution or
alternatively BD BBL™ MycoPrep™ Specimen Digestion/Decontamination Kit are included
in Appendix A
2. Phosphate buffer solution (pH 6.8)
3. BBL™ MGIT™ Mycobacteria Growth Indicator Tube
4. BACTEC™ MGIT™ 960 Supplement Kit including:
a. BACTEC MGIT Growth Supplement
b. BBL MGIT PANTA Antibiotic Mixture vial

Required Supplies:
1. 50 ml sterile conical centrifuge tubes
2. Vortex mixer
3. Centrifuge- capable of speed 3,000–3,500 x g, fixed angle rotor with aerosol-free safety
centrifuge cups
4. Funnel and waste container filled 1/3 full with an approved disinfectant solution
5. Disposable sterile pipettes
6. Slide warmer set between 65 and 75°C
7. Glass microscope slides (a frosted end for labeling with a pencil is useful)

Quality Control:
Negative - Process a negative water or buffer control with each run of specimens.
Positive - Sputum spiked with a mycobacterial species should be processed as a positive control
once per week.

Each new lot or shipment of MGIT tubes may be parallel tested using the following strains and
dilutions. The test culture should not be more than 15 days old. Prepare a suspension from
growth on solid medium, which is well dispersed, free of large clumps, and with a turbidity of
0.5 McFarland standard. When 0.5 mL of diluted suspension is inoculated into an MGIT tube
supplemented with enrichment, it should be detected as instrument positive within the time frame
shown below:

M. tuberculosis (ATCC 27294) 1:500 within 6-10 days

M. kansasii (ATCC 12478) 1:50,000 within 6-11 days
M. fortuitum (ATCC 6841) 1:5,000 within 1-3 days

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Record Quality Control growth results on a weekly basis.

Digestion, Decontamination and Concentration Procedure Steps:

Follow laboratory bio-safety practices for all procedure steps.

1. Prepare fresh working digestant/decontamination solution or BD BBL™ MycoPrep™

Specimen Digestion/Decontamination Kit as described in Appendix A.

2. Prepare the BSC for use following the Use of the Bio-Safety Hood in the Mycobacteriology
Laboratory SOP.

3. Reconstitute a lyophilized vial of BBL MGIT PANTA Antibiotic Mixture with 15 mL of

BACTEC MGIT Growth Supplement.
*Once reconstituted, the PANTA mixture must be stored at 2 – 8°C and used within 5 days.

4. Label the MGIT tubes with the specimen number.

5. Unscrew the cap and aseptically add 0.8 mL of Growth Supplement/MGIT PANTA
Antibiotic Mixture to each labeled MGIT tube.
*For best results, the addition of Growth Supplement/MGIT PANTA Antibiotic Mixture
should be made just prior to specimen inoculation.

6. If the specimen was not collected in a sterile, labeled 50 ml disposable centrifuge tube,
transfer the entire specimen to a labeled 50 ml conical tube.
*No more than 10 ml of specimen may be processed per conical tube. The remaining sample
should be transferred to second tube and processed. Repeat for all patient specimens.

7. Stagger the tubes in the rack to prevent cross contamination.

*Do not process more specimens in a batch than the centrifuge will hold.

8. Opening only one tube at a time, to the first specimen tube add an equal amount of fresh
working NACL-NaOH solution, rotate and invert the tube, ensuring the mixture coats the
entire interior surface. Vortex the mixture for 10-15 seconds. Repeat this step for each
specimen in the batch.
*Start the timer for 15 minutes after adding the solution to the first tube in the batch.

9. Allow the specimens to stand the entire 15 minutes. During the incubation time check each
specimen by slightly tilting the tube and observing for liquefaction.
*If a specimen is very mucoid with no change during these checks, add a small amount of
NALC directly to the tube, vortex and allow to stand until the end of this incubation time.

10. After the digestant has remained in the first tube for 15 minutes, begin with the first
specimen and fill the first tube to the 50 ml mark with phosphate buffer by slowly pouring
the buffer down the side of the tube avoiding splashing or contamination. Tighten the cap and
wipe the outside of each tube with the disinfectant soaked towel, then invert the tube several

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times to mix thoroughly. Repeat this step on each of the remaining specimens in the batch,
mixing well after each addition.

11. Load tubes in aerosol-free safety centrifuge cups. Centrifuge tubes for 15 minutes at 3,000 -
3,500 x g. Allow aerosols to settle a few minutes before removing tubes from the centrifuge
cups.

12. Opening one tube at a time, pour off the supernatant into a waste container filled 1/3 full with
approved disinfectant solution. Wipe the lip of the conical tube with a disinfectant soaked
towel and then recap.
*The use of a funnel is preferred. (Pour slowly so as not to disturb the pellet and be sure to
not touch the funnel while pouring.

13. Using a sterile disposable transfer pipette, re-suspend the pellet by adding 1-2 mL of
phosphate buffer. Gently mix the tube contents.

14. Add 0.5 mL of the concentrated specimen suspension to the prepared BBL™ MGIT™
Mycobacteria Growth Indicator Tube. Also add a drop (0.1 - .25 mL) of specimen to a
Lowenstein-Jensen agar slant or other conventional solid medium.
*Refer to the Mycobacteriology Culture SOP.

15. With the same pipette, place a drop of the suspension onto a clean, labeled glass microscope
slide. Prepare a smear over an area of 1 by 2 cm of the slide.
*Refer to the AFB smear SOP.

16. Tightly recap the MGIT tube and mix well. Leave the inoculated tubes at room temperature
for 30 minutes before loading in to the MGIT system.
*Refer to the Mycobacteriology Culture SOP.

17. Load inoculated BBL™ MGIT™ Mycobacteria Growth Indicator Tube into the instrument
following manufacturers’ instructions for the duration of the recommended 42 day testing
protocol.

18. For specimens in which mycobacteria with different incubation requirements are suspected, a
duplicate MGIT tube can be set up and incubated at the appropriate temperature; e.g., 30 or
42°C. Inoculate and incubate at the required temperature. These tubes must be manually read
(refer to the BACTEC MGIT Instrument User’s Manual).

Post-analytic Procedure
Specimen Retention:
Store processed specimens in AFB refrigerator for as long as space allows (approximately two
weeks).

Calculations:
Quality monitors are compiled monthly to monitor the digestion process of the lab.

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Expected Values:
 Contamination rates of 8 - 10% are considered to be acceptable for solid culture
media.
 Liquid culture media will have a higher contamination rate which will be monitored
for acceptability.
 A contamination rate of less than 5% suggests overly harsh decontamination.
 A contamination rate of greater than 10% growth suggests inadequate
decontamination, incomplete digestion, reagent and/or media contamination or
environmental contamination.

Interpretation of Results:
Contamination rates higher than 10% on solid media will be investigated to determine whether
equipment, reagent or personnel are causing the high rates. Data is included in the monthly
quality assurance report.

Method Limitations:
 The procedure is dependant on strict adherence to recommended techniques, timing,
temperature, and biochemical requirements. Any deviation from the SOP will not provide
appropriate clinical care.
 The NaOH procedure is very robust and may kill up to 60% of tubercle bacilli in clinical
specimens, and may give a false negative result, especially in cases of paucibacillary disease
as seen in early disease, or in many HIV positive patients.
 Additional contributory factors such as heat build-up in the centrifuge step may also kill
tubercle bacilli.

Appendix
Appendix A -NALC-NaOH Method Reagent

References:
1. Cernoch, P.L. et al. (1994). Cumitech 16A: Laboratory diagnosis of the Mycobacterioses.
ASM Press. Washington, DC.
2. Clinical Laboratory Standards Institute (CLSI). Clinical Laboratory Technical Procedure
Manuals; Fourth Edition. CLSI Document GP2-A4 (ISBN 1-56238-458-9). Clinical and
Laboratory Standards Institute, Wayne, PA
3. Forbes, B.A., et al (2007). Laboratory detection and identification of mycobacteria; Proposed
guideline. CLSI document M48-P. Clinical and Laboratory Standards Institute, Wayne, PA.
4. Kent , P.T. and Kubica, G. P. (1985). Public Health Mycobacteriology. A Guide for the Level
III Laboratory. U.S. Dept. Health and Human Services. Center for Disease Control.
5. SA Healthinfo, Tuberculosis Part III: Culture homogenization and decontamination. Obtained
from the World Wide Web on 14 March 2007 at HREF="http://www.sahealthinfo.org/tb"

Digestion Decontamination for Mycobacterial Cultures V1.1 SMILE Document

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Johns Hopkins University
Baltimore, MD USA

6. Weitzman, I. (2007) p 7.1.2.1-7.1.2.9. In H.D. Isenberg (ed.) Clinical Microbiology

Procedures Handbook American Society for Microbiology, Washington, D.C.
7. BBL™ MGIT™ Mycobacteria Growth Indicator Tube with BACTEC™ MGIT™ 960
Supplement Kit package insert, Becton, Dickinson and Company, 2011.

Digestion Decontamination for Mycobacterial Cultures V1.1 SMILE Document