Part III: Culture
specimen collection

The presence of acid-fast bacilli in a clinical specimen may be confirmed either by microscopy or by culture. However, since individual mycobacterial species cannot be identified by smear examination, the definitive diagnosis of tuberculosis can only be made if M. tuberculosis is isolated from the clinical specimen.

In tuberculosis bacteriology attention tends to be focused on the problems of microscopy, culture and identification systems, while an often overlooked problem is that of obtaining adequate specimens. The advantages of subtle decontamination techniques, sensitive culture media and simple identification schemes will not be fully realised unless specimens are collected with the utmost care and promptly transported to the laboratory.

Containers
An essential prerequisite for the safe collection of a satisfactory specimen is a robust, leakproof and clean container. Containers must be rigid to avoid crushing in transit and must possess a water-tight wide-mouthed screw top to prevent leakage and contamination.

To facilitate the choice of a container the following specifications are recommended:

• Wide-mouthed (at least 35mm in diameter) so that the patient can expectorate easily inside the container without contaminating the  outside
• Volume capacity of 50ml
• Made of translucent material in order to observe specimen volume and quality without opening the container
• Made of single-use combustible material  to facilitate disposal
• Screw-capped to obtain an airtight seal  and to reduce the risk of leakage during transport
• Easily-labelled walls that will allow  permanent identification

An alternative container is the 28ml Universal bottle, which is a heavy glass, screw-capped bottle with a wide neck. This container is reusable after thorough cleaning and sterilisation in boiling water for at least 30 minutes.

Collection procedures
Sputum specimens
Although M. tuberculosis is capable of causing disease in almost any organ of the body, more than 85% of tuberculosis disease in high prevalence countries is pulmonary. Therefore, sputum is the specimen of choice in the investigation of tuberculosis and should always be collected. If extra-pulmonary disease is suspected, sputum should be collected in addition to any extra-pulmonary specimens.

A good sputum specimen consists of recently-discharged material from the bronchial tree, with minimum amounts of oral or nasal material. Satisfactory quality implies the presence of mucoid or mucopurulent material and is of greater significance than volume. Ideally, a sputum specimen should have a volume of 3-5ml, although smaller quantities are acceptable if the quality is satisfactory.

Collecting a good sputum specimen requires that the patient be given clear instructions. Aerosols containing tubercle bacilli may be formed when the patient produces a sputum specimen. Patients should, therefore, produce specimens either outside in the open air or away from other people and not in confined spaces such as toilets.

In some countries, patients may present first to the laboratory for diagnosis. It is therefore appropriate that laboratory staff know the correct way of collecting sputum specimens. This procedure is described in Annex 2. It is best to obtain sputum early in the morning before the patient has eaten or taken medication (which may interfere with the growth of tubercle bacilli). If sputum specimens are collected for diagnostic purposes, tuberculosis chemotherapy should not be started until the specimens have been collected.

Because of the increased sensitivity of culture, a single good-quality sputum specimen may suffice. Some patients shed mycobacteria irregularly and in small numbers; for these patients the chance of obtaining a positive culture result will be improved if more specimens are cultured.

Specimens should be transported to the laboratory as soon as possible after collection. If a delay is unavoidable the specimens should be refrigerated to inhibit the growth of unwanted micro-organisms.

Other specimens
If a patient has a productive cough, obtaining a sputum specimen is a fairly straightforward procedure. However, if a patient finds it difficult to produce sputum, other methods may be used to obtain pulmonary secretions for diagnosis. Collection techniques fall outside the scope of this document and will not be discussed. However, induced sputum resemble saliva and it is important that these specimens be marked ?induced? in order not to be discarded as unsuitable.

Because M. tuberculosis may infect almost any organ in the body, the laboratory should expect to receive a variety of extra-pulmonary specimens, eg. body fluids, tissues, pus and urine. These specimens may be divided into two groups, namely:

• aseptically collected specimens, usually free from other micro-organisms
• specimens known to contain contaminating normal flora or specimens not collected aseptically

Aseptically collected fluids
Body fluids (spinal, pleural, pericardial, synovial, ascitic, blood, pus, bone-marrow) should be aseptically collected in a sterile container by the physician using aspiration techniques or surgical procedures. For fluids that may clot, sterile potassium oxalate (0.01-0.02ml of 10% neutral oxalate per ml fluid) or heparin (0.2mg per ml) should be added. Specimens should be transported to the laboratory as quickly as possible.

Aseptically collected tissues
Aseptically collected tissue specimens should be placed in sterile containers withoutfixatives or preservatives. If the specimen is to be sent by mail it should be protected from drying by adding sterile saline and packing the container in dry ice or maintaining a temperature of 4-15EC. Specimens should be transported to the laboratory as quickly as possible.

Specimens expected to be contaminated
Urine is the most commonly encountered extra-pulmonary specimen that requires processing before culture. To minimise excessive contamination of urine specimens the external genitalia should be washed before the specimens are collected and the urine should be immediately processed or refrigerated. Three early morning, voided midstream specimen should be collected.