Satellitism Test for the Identification of Haemophilus influenzae: Introduction, Principle, Procedure and Result Interpretation

Satellitism test for the identification of Haemophilus influenzae

Satellitism Test for the Identification of Haemophilus influenzae

Satellitism test or satellite test for  Haemophilus influenzae is positive as shown above picture. This test uses to identify Haemophilus influenzae. It is a fastidious organism and thus needs extra ingredients like hemin (factor X) and  NAD (factor V) to grow. It uses the X factor to produce essential respiratory enzymes such as cytochromes, catalases, and peroxidase where as the V factor is an electron carrier in the organism’s oxidation-reduction system.

Principle of Satellitism Test

H. influenaze requires both X and V factors for growth . Most strains of Haemophilus species do not grow on 5% sheep blood agar, which contains X factor but lacks V factor. Staphylococcus aureus produces the V factor as a metabolic by-product when growing in a culture media containing blood. Thus,  Haemophilus species  may grow on sheep blood agar very close to the colonies of Staphylococcus aureus (as V factor); this phenomenon is known as satelliting and the test to detect it called satellitism test.

Test Requirements for Satellitism Test

  • 5% sheep blood agar plate
  • Physiological saline/ peptone water
  • Nutrient agar or  tryptic soy agar
  • Suspected growth of H. influenzae ( any organism growing on chocolate agar  and not blood agar that shows in Gram stain , gram negative coccobacilli or shor rods)
  • Bunsen burner
  • Inoculating loop
  • Sterile swab sticks
  • S. aureus (ATCC 25923)
  • Incubator ( CO2)
  • Control strains ( H. influenaze  ATCC 43065 and S. aureus ATCC 25923 )

Test Procedure of Satellitism Test 

  1. First wear gloves
  2. Mix a loopful of suspected colonies of Haemophilus colonies in about 2 ml of sterile physiological saline or sterile peptone water.
  3. Make sure none of the chocolate agar media is transferred.
  4. Using a sterile swab stick, inoculate the organism suspension on a plate of nutrient agar or tryptic soy agar and a plate of blood agar
  5. Streak a pure culture of S. aureus across each of the inoculated plates.
  6. Incubate both plates in a CO2 enriched atmosphere at 35 to 37°C for 18-24 hours.
  7. Examine the culture plates for growth and satellite colonies.


Observe the growth of organisms on inoculated agar plates like nutrient agar or tryptic soy agar and blood agar.

Result Interpretations

  1. 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.
  2. The colonies near the column of Staphylococcus aureus growth are larger than those furthest from it.
  3. 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 Haemophilus parainfluenzae.


  1. This test is performed on blood agar and blood agar contains only X factor and V factor is produced by growing S. aureus.
  2. Satellitism is also shown by variant streptococci like Abiotrophia defectiva and Granulicatella species. They do not grow on blood agar, since it lacks the nutritional requirements, but will grow in the medium surrounding staphylococci, which supplies the needed nutrients. In Gram stain, these organisms are cocci, filaments, and bulbous forms in smear.
  3. H.  influenzae is the first free-living organism whose complete genome has been sequenced.
  4. H.  influenzae strains have been classified into 6 capsular types (a-f) based on polysaccharide capsules.
  5.  Among them, only type b is responsible for 95% of the disease.
  6. Some microorganisms grow only on chocolate agar and will no grow on blood agar even with a staphylococcus dot. These organisms include Haemophilus ducreyi, Francisella tularensis, and some Methylobacterium species.

# Chocolate agar with bacitracin for screening  Haemophilus ||Use of 10U bacitracin: Bacitracin is a polypeptide antibiotic derived from Bacillus subtilis that functions to block cell wall formation by interfering with the dephosphorylation of the lipid compound that carries peptidoglycans to the growing microbial cell wall. Haemophilus is resistant to bacitracin (10U) where as most common bacteria are sensitive. It makes easier to screen Haemophilus influenzae in sputum growing around the bacitracin disc as shown below-

#Haemophilus influnezae on Gram stain as shown below-

#Haemophilus influnezae satellite test: Positive  as shown below-

# Variety of Haemophilus species identification on basis of X, V,XV, factors, blood agar and Xylose test as shown below-


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