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

Introduction

Satellitism test or satellite test is applied to identify Haemophilus influenzae. It is a fastidious bacterium and thus it 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 whereas the V factor is an electron carrier in the organism’s oxidation-reduction system.

Principle of Satellitism Test

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

Test Requirements for Satellitism Test

• 5% sheep blood agar (BAP) 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 short rods)
• Bunsen burner
• Inoculating loop
• Sterile swab sticks
• S. aureus (ATCC 25923)
• CO₂ Incubator
• Control strains (H. influenaze ATCC 43065 and S. aureus ATCC 25923 )

Test Procedure

1. First put on gloves
2. Mix a loopful of suspected colonies of Haemophilus colonies ( test organism) 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 test organism suspension on a plate of nutrient agar or tryptic soy agar and a plate of blood agar and spread uniformly.
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. Observe the culture plates for growth and satellite colonies.

Observation

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

Result- Interpretations of Satellitism Test 

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.

Keynotes on Satellitism Test 

1. The satellitism assay 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, as it lacks 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. Haemophilus  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 not grow on blood agar even with a staphylococcus dot. These organisms include Haemophilus ducreyi, Francisella tularensis, and some Methylobacterium species.

Further Readings

1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
2. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
3. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
4. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
7. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
8. https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958