Most strains of Haemophilus spp does 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.
Why 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.
Procedure of Satellitism test to identify Haemophilus influenzae
- 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.
- Using a sterile swab, inoculate the organism suspension on a
plate of nutrient agar or tryptic soy agar
b. a plate of blood agar
- Streak a pure culture of S. aureus across each of the inoculated plates
- Incubate both plates in a carbon dioxide enriched atmosphere at 35 to 37°C for 18-24 hours.
- Examine the culture plates for growth and satellite colonies
Observation and interpretations
The suspected colonies are of Haemophilus influenzae if:
- Growth is seen in the blood agar but not in the nutrient agar (or tryptic soy agar) plate
- The colonies near the column of S. aureus growth are larger than those furthest from it
If satellite colonies are present on both blood and nutrient agar plates than the organism is probably a Haemophilus species that requires only factor V, such as H. parainfluenzae.
Note: Very occasionally satellitism is shown by strains of Neisseria, Streptococcus species, and diphtheroids.
Haemophilus influenzae is the first free-living organism to have its genome (complete genetic code) sequenced