Sputum DigestionDecontamination for Mycobacteriology Culture

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Sputum DigestionDecontamination for Mycobacteriology Culture


    Johns Hopkins University

    Baltimore, MD USA

    Sputum Digestion/Decontamination for Mycobacteriology Culture- Guidelines

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

    Charache, MD; Effective (or Post) Date: 10 Feb 09

    Review History Date of last review: 11 May 2010

    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.

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 also 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.

    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 SMILE Document 8 August 2008


    Johns Hopkins University

    Baltimore, MD USA

    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 Methods:

    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.

NaOH Method

    Higher concentrations of sodium hydroxide can both liquefy sputum, and concurrently kill the contaminating normal flora however, it is more toxic to mycobacteria. Sodium hydroxide should be used at the lowest concentration that will effectively digest and decontaminate the specimen. If excessive contamination occurs with the use of 2% NaOH, increase the concentration to 3 or 4% rather than increasing the time of exposure. Harsh decontamination can kill 20 to 90% of the mycobacteria in a clinical specimen.


    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

    5. Weitzman, I. (2007) p In H.D. Isenberg (ed.) Clinical Microbiology

    Procedures Handbook American Society for Microbiology, Washington, D.C.

SMILE Document 8 August 2008


    Johns Hopkins University

    Baltimore, MD USA

     Sputum Digestion/Decontamination for Mycobacteriology Culture - SOP

    Peggy Coulter MDE, MT (HEW) Document Effective

    International QA/QC Coordinator Number Date Author(s), Name &

    Title Patricia Charache, MD; Pro6.7--15 30 Sep 08


    Approved By Name, Title Signature Date

    SOP Annual Name, Title Signature Date Review

     Version # [0.0] Revision Date Description (notes)

    Revision [dd/mm/yy]


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

Associated Forms:

SMILE Document 8 August 2008


    Johns Hopkins University

    Baltimore, MD USA


    There are several approved methods for the digestion-decontamination procedure. The two most useful for processing sputum are outlined below. Both can be used for liquid culture media as well as solid media, while most other methods kill the mycobacteria when used for liquid systems.

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

    4. Mycobacteriology Culture

Abbreviations Used:

    ; AFB = Acid Fast Bacilli

    ; BSC = Bio-Safety Cabinet

    ; SOP = Standard Operating Procedure

Analytic Procedure

    Specimen Information:

    Specimen collection- Refer to Mycobacteriology Laboratory Specimen Collection/Receiving


    Specimen types