Microbiology Laboratory


Written by MicroDok

Sputum culture is often recommended in the diagnoses of lower respiratory tract infection (e.g. pneumonia and pulmonary tuberculosis). Lower respiratory tract infections (LRTIs) are infections/diseases that occur below the voice box (larynx) i.e. in the bronchi and trachea. In this case, an induced or expectorated sputum specimen and not saliva is requested and obtained from the patient. The patient is asked to take a deep breath and then cough deeply to discharge what he or she coughed up into a sputum specimen collection container.

AIM: To isolate pathogenic bacteria from sputum specimens as an aid in the diagnosis of lower respiratory tract infections (e.g. pneumonia).

MATERIAL/APPARATUS: Sputum specimen, incubator, anaerobic jar, blood agar (BA), chocolate agar (CA), inoculating loop, grease pencil, face mask.


  1. Cover face with face mask to avoid splashing of aerosols that might emanate while working on the specimen.
  2. Label the BA and CA plates with the patients name and laboratory number using the grease pencil.
  3. Using a sterilized inoculating loop, collect a loopful of the sputum specimen.
  4. Inoculate it on both the CA and BA plates.
  5. Incubate the BA plate aerobically in the incubator at 37oC overnight.
  6. Incubate the CA plate anaerobically in an anaerobic jar at 37oC overnight.
  7. After incubation, examine both plates and look out for a significant growth of bacteria.
  8. Perform biochemical tests (oxidase test, catalase test, coagulase test) to identify the isolated bacteria.
  9. Perform antimicrobial susceptibility test only when a significant pathogen have been isolated


Look especially for a significant growth of Streptococcus pneumonia, Haemophilus influenza, and Staphylococcus aureus and report.

NOTE: Sputum specimens should always be collected in leak-proof specimen containers and, such specimens should be treated with caution in order to avoid cross-contamination due to infected aerosol production which may occur during handling and processing of the specimen.

The culture and antimicrobial sensitivity testing of sputum specimens for Mycobacterium tuberculosis (the causative agent of tuberculosis – TB) are usually undertaken in a Tuberculosis reference center/laboratory that is normally outside the hospital environment. This is because aerosols from sputum specimens carrying M. tuberculosis can easily become airborne and cause infection. Though some hospitals have facilities for carrying out the microscopy, culture, and sensitivity of sputum specimens suspected to contain M. tuberculosis. It is also advisable to work under a laminar flow biological safety cabinet when handling and processing sputum specimens suspected to contain M. tuberculosis in order to prevent aerosols from the specimen from coming into contact with the microbiologist.

Anaerobic jar is used for the incubation of cultures in chocolate agar (CA) plates because the organisms for which this media (CA) is meant to isolate anaerobes/obligate aerobes and thus require a CO2 atmosphere for growth. The anaerobic jar increased CO2 tension which enhances the growth of the bacteria.

Illustration of an Anaerobic jar

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