The pathogenic Haemophilus influenzae can be differentiated from its non-pathogenic relative, Haemophilus parainfluenzae, by its dependence on an external supply of two growth factors, known as 'X' (haem) and 'V' (NAD). Haemophilus influenzae requires both, while Haemophilus parainfluenzae requires only the 'V' factor, since it can make its own haem. This facility may be tested by supplying both growth factors, alone and in combination, to a lawn of bacteria to be tested. In the image above, the bacteria can only grow around the disc containing both 'X' and 'V' factors, indicating that it is the pathogen, Haemophilus influenzae.
X and V dependence may also be demonstrated by the phenomenon of 'satellitism'. The second image above illustrates this. A lawn of test bacteria is plated onto a fresh blood agar plate. This provides a supply of haem. An inoculum of Staphylococcus aureus, which can provide NAD, is placed on the plate and the culture is incubated. Haemophili can be seen growing larger near to the staphylococcal colony, where the supply of NAD is greatest. Colonies of haemophili growing further from the staphylococcal colony are proportionately smaller, as the supply of NAD from the staphylococcus diminishes. This test is good for detecting haemophili, but cannot distinguish the pathogenic Haemophilus influenzae from Haemophilus parainfluenzae.