Satellitism Test for Haemophilus influenzae

Most species of Haemophilus require both X and V factors for growth. As V factors are released erythrocytes into the medium, it is available for bacteria to utilize. In contrast, hemin is found inside intact RBCs and is unavailable until erythrocytes are lyzed. So H. influenaze fails to grow in blood agar.

Staphylococcus aureus produces NAD as a metabolic byproduct when growing in a culture media containing blood and also releases hemin while lyzing RBCs. Therefore, Haemophilus spp may grow on sheep blood agar very close to the colonies of Staphylococcus aureus; this phenomenon is known as satelliting.

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 an electron carrier in the organism’s oxidation-reduction system.

Test organism

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

Quality Control

• Demonstrate satelliting on each lot of blood agar plate using staphylococcus and H. influenzae ATCC 43065
• Periodically verify that the strain has not become contaminated.

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

• If satellite colonies are present on both blood and nutrient agar plates, then the organism is probably a Haemophilus species that requires only factor V, such as H. parainfluenzae (the colonies of S. aureus supply v factors)
• Some microorganisms only grow on chocolate agar and will not grow on blood agar, even with Staphylococcus dot. These include Francisella tularensis, some Methylobacterium, and Haemophilus ducreyi.
• Haemophilus haemolyticus and H. parahaemolyticus may not demonstrate the satellite phenomenon, since they are hemolytic.

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
3. Musher DM. Haemophilus Species. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 30. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8458/