How to identify Streptococcus pneumoniae?

By Acharya Tankeshwar •  Updated: 06/17/21 •  6 min read

Streptococcus pneumoniae (pneumococci) is a part of the normal nasopharyngeal and oropharyngeal flora. It is an important etiological agent of upper and lower respiratory tract infections (URTI and LRTI), bacteremia, and septicemia. Streptococcus pneumoniae is also associated with otitis media, sinusitis, meningitis, and endocarditis.

Lanceolate diplococci. Image source:
Lanceolate diplococci. Image source:

Pneumococci are Gram positive lancet shaped diplococci (intracellularly or extracellularly), non-motile, and encapsulated. They occur in pairs with the broader end opposed, hence called Gram positive diplococci. S. pneumoniae is a fastidious bacterium, which grows best at  at 35-37°C with ~5% CO2 (or in a candle-jar).

Laboratory diagnosis

Laboratory diagnosis of Streptococcus pneumoniae infection is based on finding characteristics shape of the organism in the sample, characteristic colony morphology, biochemical reactions, susceptibility to certain diagnostic discs, and latex agglutination test.


Specimens used for the laboratory diagnosis of Streptococcus pneumoniae of may be

Sample Collection

  1. Sputum:
    Collect > 1.0 ml expectorated sputum in a sterile screw-capped container.
  2. Lung aspirate/ pleural fluid
    Collect > 1.0 ml by percutaneous needle aspiration in a sterile screw-capped tube.
  3. Blood:
    Clean the venipuncture site with 70% alcohol and iodine, allow it to evaporate and collect blood aseptically in a culture broth with an anticoagulant. In case of adults, collect 5-10 ml blood in culture bottle, for children < 12 year old, collect 1.5-2.0 ml blood. Mix the blood and broth by rotating gently to avoid clotting.
  4. Cerebrospinal fluid (CSF):
    Clean the skin over L3-L4 inter-space with 70% alcohol and iodine. Collect > 1.0 ml CSF in a sterile screw-capped tube. Keep the CSF in an incubator at 35-37 degree centigrade, if it is not processed immediately.
  5. Exudates from joints/middle ear
    Collect > 1.0 ml by aspiration in a sterile screw-capped tube or add directly in a culture broth used for blood culture.


Sample Transport

Streptococcus pneumoniae is a fastidious bacteria. Care must be taken during transport of specimen. Specimens must be transported promptly to the laboratory preferably within 1-2 hours.

Blood can only be transported after collecting in a culture broth containing appropriate anticoagulant. The inoculated medium can be held at room temperature (20°C– 25 °C) for 4 – 6 hours before incubation at 37 °C. The samples during transportation should be protected from extremes of temperature (less than 18 °C, more than 30 °C) and direct sunlight.

Culture and  identification during suspected Streptococcus pneumoniae infection

Flow chart for identification and characterization of a S. pneumoniae isolate
Flow chart for identification and characterization of a S. pneumoniae isolate
  1. Microscopy and Staining 

gram positive cocci streptococcus
Gram positive diplocci: Streptococcus spp

2. Culture and Sensitivity 

Colony morphology

Draughtsman colonies of S. pneumoniae

Alpha-hemolytic colonies are further identified by the following confirmatory tests.

Identification of Streptococcus pneumoniae by biochemical reactions.

Mnemonics: “Streptococcus pneumoniae is a BOSS” i.e. Bile Soluble, Optochin Sensitive

      A. Optochin test (6 mm disc with 5µg).

Alpha hemolysis in Blood Agar by Streptococcus pneumoniae, Observe the zone of inhibition around Optochin disk.

Observe the zone of growth inhibition around the disc and interpret as:

B.  Bile solubility test.

Bile solubility test: Streptococcus pneumonie colonies are lysed by bile
Bile solubility test: Streptococcus pneumoniae colonies are lysed by bile


Note:  This test can also be done directly onto the colony and the colony is lysed by the addition of bile solution.

Interpretation of Optochin and Bile solubility test

Antimicrobial susceptibility

Reporting of results: Streptococcus pneumoniae isolated and resistance patterns with tested antibiotics

3. Detection of the antigen

C-carbohydrate antigen of the Streptococcus pneumoniae can be detected in the urine (Read:Pneumococcal Urinary Antigen Testing (UAT): Principle, Procedure and Results ) for the diagnosis of pneumonia and in CSF for the diagnosis of pneumococcal meningitis.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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