X-Factor Requirement
(porphyrin synthesis test)
DESCRIPTION
The most reliable test for the determination of  X-factor bacterial need is the test for porphyrin synthesis. Method is used mainly for
Haemophilus species.
Hemin-independent bacteria produce porphyrins and porphobilinogen, substances involved in the hemin biosyntetic pathway in the
presence of delta-aminolevulinic acid.
Hemin-dependent bacteria lack the enzymes for producing these substances.
MEDIUM PREPARATION
Composition
Delta-aminolevulinic acid ..................................................2 mM
MgSO
4 ..............................................................................0.08 mM
in 0.1 M phosphate buffer,  pH = 6.9

PROCEDURE
Distribute in test tubes and inoculate bacteria (rich inoculum), then incubate 4 hours
at 37 °C.
RESULTS
Read results under UV light (360 nm). If a red fluorescence appears then the strain
produced porphyrins and is X-factor independent.
Alternatively Kovac's reagent may be used instead of UV light. Add the reagent, shake the tube, then allow phases to separate. A red
color of the water phase indicates a X-factor independent strains (porphobilinogen produced). But false positive results may be
obtained with indole-producing strains.
REFERENCES:
1. H. Raducanescu, V.Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. J. G.Holt et al., 1994.Begey’s manual of Determinative Bacteriology, 9
th-edition, Williams & Wilkins.
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