Acid-Fast Culture Health Article

Specimen collection

Specimens to be processed for the recovery of mycobacteria are obtained and handled using specific guidelines to ensure successful growth, isolation, and identification of the causative organism. Containers must be sterile, leak-proof, and labeled properly. After collection, if the specimen cannot be processed within one hour, refrigeration is required but no longer than overnight. However, blood samples must be placed in the proper media and incubated immediately at 35-37°C.

The most often requested specimens are pulmonary specimens (secretions) which must be obtained before any treatment (antibiotic therapy) is given. Pulmonary specimens may be obtained in several ways: spontaneously produced (expectorated) sputum; aerosol-induced sputum; bronchioscopic aspirations, washings and brushings; gastric aspirates, and lavages (washings) from patients who have swallowed sputum through the night. Saliva is not acceptable as a specimen for the recovery of mycobacteria and is usually rejected as a contaminated specimen. A series of early morning sputum specimens are recommended over a three-day period. The ideal amount of sputum specimen for processing and recovery of mycobacteria is 5-10 mL of sputum. Upon rising in the morning, the patient is instructed to cough deeply to produce sputum (expectorated sputum). A patient who is unable to bring up any sputum is given an aerosol treatment (aerosol-induced sputum) by a respiratory therapist in order to recover a sufficient amount of sputum for culture.

Other specimens requested for culture and recovery of mycobacteria are early morning, voided urine specimens; fecal specimens; tissue and body fluids (pleural, pericardial and peritoneal fluids), cerebrospinal fluid (CSF), bone marrow aspirates, and blood. Blood and stool specimens are usually cultured from AIDS patients. These specimens reveal numerous mycobacteria when infection is present in these patients. Wound or skin lesions (abscesses) require a technique using aspiration of the specimen into a syringe rather than the use of a swab to obtain the specimen.

Specimens not suitable for culture and usually rejected are 24-hour urine specimens, pooled sputum, saliva, and swabs containing pulmonary secretions. The high rate of contamination as well as the reduced rate of mycobacteria recovery in these specimens renders them unsuitable.

Specimen processing

Decontamination and digestion of sputum specimens is necessary to recover mycobacteria for culture and identification. The process of decontamination (removing unwanted bacteria) and digestion (breaking down mucous and protein) of sputum specimens is necessary to release the mycobacteria that may be present but are trapped in the mucous, and also to kill the unwanted bacteria (normal flora). Specimens from sterile body sites (blood, tissue, and body fluids, etc.) do not need the process of decontamination and digestion as do sputum samples. If the process of decontamination and digestion is not done or done improperly, recovery of mycobacteria from sputum samples is inhibited causing a false-negative report. Mucous, cells, and normal bacterial flora (from the oral cavity) entrap and enmesh the mycobacteria in sputum. A common decontaminant is sodium hydroxide (4%) which is also used as a mucolytic agent (for liquifaction or digestion of mucous). A combination is often used which consists of N-acetyl-L-cysteine (NALC) and a lower concentration (2%) of sodium hydroxide. This combination gives a better recovery rate when used together as a mucolytic-decontaminant. Liquifaction of the thick mucous in sputum is necessary to free the mycobateria trapped in it without harming the mycobacteria, and decontamination kills the normal flora (bacteria from the mouth, throat and oral cavity) which interfere with the recovery of mycobacteria. The final product is reduced (concentrated) from the original 5-10 mL volume, and a portion of the resulting specimen is transferred by sterile technique to either sterile solid, tube or plate media, and liquid media, while another portion is used to make several smears on glass slides for staining.