Part II: Microscopy
specimen collection

In tuberculosis diagnosis attention tends to be focussed on the problems of microscopy, while an often overlooked problem is that of obtaining adequate specimens. Correct collection and transportation of specimens to the laboratory are important to ensure that results are accurate and reliable.

Containers
An essential prerequisite for the safe collection of satisfactory specimens is a robust, leak-proof 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. Specimens should be collected in clean containers that are free from paraffin and other waxes or oils. These materials may appear in smears as acid-fast artefacts or may react with other bacteria and cause them to appear to be acid-fast.

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 a water-tight seal 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. This container is reusable after thorough cleaning and sterilisation in boiling water for at least 30 minutes.

Collection procedures
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 if there are respiratory symptoms.

Collecting a good sputum specimen requires that the patient be given clear instructions. Aerosols containing tuberculosis bacteria may be formed when the patient coughs to produce 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 a sputum specimen early in the morning before the patient has eaten since food particles in smears make them difficult to examine.

Because tuberculosis lesions in the lungs may drain intermittently, it is possible for a specimen to be negative on one day and positive the next. For this reason, three specimens should be collected for diagnosis as follows:

• one spot specimen when the patient first presents to the health  service
• one early morning specimen (preferably the next day)
• one spot specimen when the early morning specimen is submitted for  examination

These should not be pooled but should be sent to the laboratory as single specimens. For follow-up of treatment at regular intervals and to determine outcome at the end of treatment, one specimen should be collected.

If a patient has a productive cough, obtaining a sputum specimen is a fairly straight-forward procedure. However, if a patient finds it difficult to produce sputum, other methods may be used to obtain pulmonary secretions, such as sputum induction. 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.

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