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.
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.
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).
- 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
- 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
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:
- 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 then the organism is probably a Haemophilus species that requires only factor V, such as H. parainfluenzae (v factors are supplied by the colonies of S. aureus)
- 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.
Haemophilus influenzae is the first free-living organism to have its genome (complete genetic code) sequenced.
References and further readings
- Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology.
- Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
Acharya TankeshwarHello, 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.
Bordetella pertussis: Properties, Pathogenesis, Lab Diagnosis
Agent of whooping cough (pertussis), Bordetella pertussis is a small gram-negative coccobacilli.
Acinetobacter: Disease, Properties, Resistance
Acinetobacter are gram-negative coccobacilli, among them Acinetobacter baumannii accounts for most infections in humans in hospital settings.