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 produce NAD as a metabolic byproduct when grow 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 needs 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.
Haemophilus influenzae will grow in the hemolytic zone of Staphylococcus aureus on blood agar plates.
The hemolysis of erythrocytes by S. aureus releases nutrients vital to the growth of H. influenzae (NAD, factor V).
The NAD diffuses into the surrounding medium and stimulates the growth of H. influenzae in the vicinity of the Staphylococcus aureus.
H. influenzae will not grow outside the hemolytic zone of S. aureus.
This is known as satelliting. For Haemophilus spp. the satellite test substutes for V factor test.
Procedure of satellitism test to identify Haemophilus influenzae
1. Mix a loopful of suspected colonies of Haemophilus colonies in about 2 ml of sterile saline.
2. Using a sterile swab, inoculate the organism suspension on a plate of blood agar
3. Streak a pure culture of S. aureus across of the inoculated blood agar plate.
4. Incubate the plate in a carbondioxide enriched atmosphere at 35-37ºC for 18-24 hours.
5. Examine the culture plate for growth and satellite colonies.
Observation and interpretation
The suspected colonies are of Haemophilus influenzae if:
1) Growth is seen in the blood agar plate.
2) The colonies near the column of S. aureus growth are larger than those furthest from it.
Abiotrophia defectiva and Granulicatella adiacens
A. defectiva and G. adiacens colonies can grow as satellite colonies around other bacteria, such as Staphylococcus aureus.
However most cinical microbiologists have overlooked the fact that A. defectiva and G. adiacens isolates can show discrepant satellite testing results based on the different kinds of nutrients contained in blood agar plates produced by different manufactures.