Haemophilus influenzae is a fastidious organism which grows best at 35-37°C with ~5% CO₂ (or in a candle-jar) and requires hemin (X factor) and nicotinamide-adenine-dinucleotide (NAD, also known as V factor) for growth. The standard medium used for growth of H. influenzae is a chocolate agar plate (CAP), which can be prepared with heat-lysed horse blood, a good source of both hemin and NAD, although sheep blood can also be used. Growth occurs on a CAP because NAD is released from the blood during the heating process of chocolate agar preparation (the heating process also inactivates growth inhibitors) and hemin is available from non-hemolyzed as well as hemolyzed blood cells. Alternatively, NAD can be included as a component of liquid H. influenzae growth media supplements, (available commercially or prepared in the laboratory), which are incorporated into the chocolate agar. H. influenzae appear as large, round, smooth, convex, colorless-to-grey, opaque colonies on a CAP. Encapsulated strains appear more mucoidal than non-encapsulated strains, which appear as smaller, compact grey colonies. No hemolysis or discoloration of the CAP is apparent. While H. influenzae produce a pungent indol smell, plates should not be opened in order to smell the cultures. H. influenzae cannot grow on an unsupplemented BAP (The Center for Disease Control).

The studies reported that 38% of H. influenzae genes are critical for growth or viability of colonies on rich medium. Putatively essential genes were no more likely to have been previously characterized than genes with nonessential functions. In several cases, putatively essential genes that had been identified are nonessential in other organisms (Mohd-Zain et al., 2004). Studies on H. influenzae can potentially detect essential cellular pathways that have several alternate mechanisms in organisms with larger genomes. Alternatively, H. influenzae may contain additional factors that become lethal when the essential gene is depleted. Moreover, organisms with larger genome sizes compared with those with smaller coding capabilities may have an increase in gene functions that seem redundant under a given growth condition (Karlin et al., 1996).