Satellitism Test for Haemophilus influenzae

By Acharya Tankeshwar •  Updated: 05/03/22 •  3 min read

Most strains of Haemophilus spp do not grow on 5% sheep blood agar, which contains hemin (factor X) but lacks NAD (factor V).

Staphylococcus aureus produces NAD as a metabolic byproduct when growing in a culture media containing blood. Therefore, Haemophilus spp may grow on sheep blood agar very close to the colonies of Staphylococcus aureus (as it produces NAD-factor V); this phenomenon is known as satelliting.

Satellitism test for the identification of Haemophilus influenzae (Image source:

Why does Haemophilus need X and V Factor? 

Haemophilus influenzae uses factor X to produce essential respiratory enzymes such as cytochromes, catalases, and peroxidase. Factor V is used as an electron carrier in the organism’s oxidation-reduction system.

Test organism

Any organism growing only on chocolate agar and not on blood agar plate that is suggestive of Haemophilus or Francisella by Gram stain (Gram-negative coccobacilli or short rods).

Quality Control

Procedure of Satellitism test

  1. Mix a loopful of suspected colonies of Haemophilus colonies in about 2 ml of sterile physiological saline (or sterile peptone water). Make sure none of the chocolate agar media is transferred.
  2. Using a sterile swab, inoculate the organism suspension on a plate of nutrient agar or tryptic soy agar
    b. a plate of blood agar
  3. Streak a pure culture of S. aureus across each of the inoculated plates
  4. Incubate both plates in a carbon dioxide-enriched atmosphere at 35 to 37°C for 18-24 hours.
  5. Examine the culture plates for growth and satellite colonies

Observation and interpretations

A positive result for a tiny Gram-negative rod or coccobacilli indicates the organism is in the genus Haemophilus.

The suspected colonies can be presumptively identified as Haemophilus influenzae if:

  1. Growth is seen in the blood agar but not in the nutrient agar (or tryptic soy agar) plate
  2. The colonies near the column of S. aureus growth are larger than those furthest from it

Note: Very occasionally satellitism is shown by strains of Neisseria, Streptococcus species, and diphtheroids.

Interesting Fact:
Haemophilus influenzae is the first free-living organism to have its genome (complete genetic code) sequenced.

References and further readings

  1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology.
  2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition

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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