tests or polymerase chain reaction tests on clinical samples obtained from a sterile body site. In this respect, H. influenzae cultured from the nasopharyngeal cavity or throat would not indicate H. influenzae disease, because these sites are colonized in disease-free individuals. However, H. influenzae isolated from cerebrospinal fluid or blood or joint fluid would indicate H. influenzae infection. Culture Bacterial culture of H. influenzae is performed on agar plates, the preferable one being chocolate agar, with added X (hemin) and V (nicotinamide adenine dinucleotide) factors at 37 °C in a CO2-enriched incubator. The ideal CO2 concentration for the culture is ~5%. Blood agar growth is only achieved as a satellite phenomenon around other bacteria. Colonies of H. influenzae appear as convex, smooth, pale, grey, or transparent colonies. Gram stained and microscopic observation of a specimen of H. influenzae will show Gram-negative coccobacillus. The cultured organism can be further characterized using catalase and oxidase tests, both of which should be positive. Further serological testing is necessary to distinguish the capsular polysaccharide and differentiate between H. influenzae b and nonencapsulated species. Although highly specific, bacterial culture of H. influenzae lacks sensitivity. Use of antibiotics prior to sample collection greatly reduces the isolation rate by killing the bacteria before identification is possible. Recent work has shown that H.influenzae uses a highly specialized spectrum of nutrients where lactate is a preferred carbon source. H. influenzae will grow in the hemolytic zone of Staphylococcus aureus on blood agar plates; the hemolysis of cells by S. aureus releases factor V which is needed for its growth. H. influenzae will not grow outside the hemolytic zone of S. aureus due to the lack of nutrients such as factor V in these areas. Latex particle agglutination The latex particle agglutination test (LAT) is a more sensitive method to detect H. influenzae than is culture. Because the method relies on antigen rather than viable bacteria, the results are not disrupted by prior antibiotic use. It also has the added benefit of being much quicker than culture methods. However, antibiotic sensitivity testing is not possible with LAT alone, so a parallel culture is necessary. Molecular methods Polymerase chain reaction (PCR) assays have been proven to be more sensitive than either LAT or culture tests, and are highly specific. These PCR tests can be used for capsular typing of encapsulated H. influenzae strains. Pathogenicity H. influenzae is generally found within and