Table of Contents
- Introduction to the Satellitism Test
- Objectives of Satellitism Test
- Principle of Satellitism Test
- Requirements for Satellitism Test
- Procedure of Satellitism Test
- Result and Interpretation of Satellitism Test
- Precautions
- Applications of Satellitism Test
- Limitations of Satellitism Test
Introduction to the Satellitism Test:
- Bacteria belonging to the genus Haemophilus have specific nutritional requirements for growth and survival.
- Two essential factors play a crucial role in this process: the X-factor and the V-factor.
- The X-factor, also known as hemin or protoporphyrin IX, is involved in essential cellular functions.
- The V-factor, nicotinamide adenine dinucleotide (NAD), acts as a coenzyme in various metabolic reactions.
- The Satellitism Test capitalizes on these differing needs for X and V factors among Haemophilus species. This test allows us to differentiate, particularly between Haemophilus influenzae and other pathogenic Haemophilus species.
- Here's the key distinction: H. influenzae requires both the X and V factors for growth. Conversely, most other pathogenic Haemophilus species can thrive with just one of these factors. By analyzing the growth pattern in the Satellitism Test, we can identify the specific Haemophilus species present in a sample.
Objectives of Satellitism Test
- To definitively identify the presence of Haemophilus influenzae in a bacterial sample.
Principle of Satellitism Test
- Blood agar medium provides only the X-factor, but H. influenzae requires both X and V factors to grow. Since H. influenzae can't hemolyze blood or grow without the V-factor, it can't grow alone in blood agar medium.
- However, Staphylococcus aureus is hemolytic and releases the V-factor (NAD) into the medium, allowing H. influenzae to grow in its vicinity. This phenomenon, where H. influenzae grows in close proximity to S. aureus colonies, is known as "satellitism".
Requirements for Satellitism Test
1. Culture Media
Blood Agar Medium is used for the Satellitism test.
Composition of Blood Agar Base per 1000 mL
- Beef Heart Peptone/Meat Extracts: 10g
- Peptone/Tryptose: 10g
- Sodium Chloride: 5g
- Agar: 15g
- pH: 7.3 ± 0.2 at 25°C)
Preparation of Blood Agar Plate
- Mix Blood Agar Base powder with water according to the manufacturer's instructions.
- Heat to boiling to dissolve all components, then autoclave at 121°C and 15 lbs pressure for 15 minutes.
- Cool to 40-45°C, then add 5% (5-10% v/v) sterile defibrinated Sheep Blood with constant stirring.
- Pour 25 mL of the mixture into a sterile Petri plate and let it solidify at room temperature.
2. Reagents
- None required.
3. Equipment
- Petri Plates
- Micropipette
- Cotton Swab/Inoculating Loop
4. Test Organisms
- Sample Bacteria (Haemophilus spp.)
- Staphylococcus aureus ATCC 25923
5. Control Organisms
- Haemophilus influenzae ATCC 35056
- Haemophilus haemolyticus ATCC 33390
Procedure of Satellitism Test
- Prepare a suspension of identified Haemophilus spp. colonies (or suspected Haemophilus influenzae).
- Prepare a suspension of S. aureus.
- Using a sterile inoculating loop, streak the Haemophilus spp. suspension in a blood agar medium plate in a single direction (e.g., horizontally).
- Using another sterile loop, streak a line of S. aureus suspension perpendicularly across the Haemophilus spp. streak (e.g., vertically).
- Incubate the plate at 35±2°C for approximately 24 hours in a 5% CO2 environment.
Result and Interpretation of Satellitism Test
- Growth of small colonies only in the close vicinity of the S. aureus colonies indicates H. influenzae.
- H. haemolyticus and H. parahaemolyticus are hemolytic in nature; hence, can grow without S. aureus.
- Growth of bacteria across the plate – near and away from S. aureus colonies – indicates Haemophilus spp. that either:
- Need X-factor only
- Are hemolytic
Precautions
- Ensure the blood agar plate is sterile and not haemolyzed before inoculation.
- When transferring Haemophilus colonies for suspension, avoid transferring any chocolate agar medium.
- Incubate in the presence of 5% CO2.
Applications of Satellitism Test
- Identify isolated Haemophilus spp. as Haemophilus influenzae.
- Differentiate Brucella spp. from Haemophilus spp.
Limitations of Satellitism Test
- Unable to separate H. influenzae and H. aegyptius.
- H. ducreyi does not grow in Blood agar even with S. aureus.
- Requires prior identification of bacteria as Haemophilus spp. using Gram staining or other biochemical tests.
- May yield false positives with:
- Nutritionally variant Streptococci (NVS) like Abiotrophia defectiva and Granulicatella species.
- Neisseria spp.
- Diphtheroid.
