X and V factor test

X and V factor test

Avibacterium paragallinarum, cultivated on bovine blood agar together with a streak of Staphylococcus aureus. A. paragallinarum has increased growth with bigger colonies close to the streak of Staphylococcus aureus, which cause hemolysis and release of the  V factor from the erythrocytes.

Image: Ingrid Hansson & Karl-Erik Johansson (BVF, SLU). - Click on the image to enlarge it.


Some bacteria produce substances that other bacteria need for growth. Examples of such substances are the X factor and the V factor. The X-Factor, which is heat stable, can be protoporphyrin IX, hemin or other iron-containing porphyrins, and are found in red blood cells as well as extracellularly in blood. The V-factor, which is a heat sensitive, may be nicotinamide adenine dinukleotide (NAD) eller nicotinamide adenine dinukleotide phosphate (NADP), and is predominantly intracellular in red blood cells. X and V factor dependence can be detected either by the X and V factor test or by a disc test and the result can provide valuable information when identifying bacteria.

X and V factor test

The X and V factor test is used to determine how a certain bacterium grows on blood agar and on a minimal medium (for instance TSA) near a streak of a bacterium that produces the V factor. First, the two agar plates are inoculated with the bacterium to be examined, and then a streak of Staphylococcus aureus is done on both plates. S. aureus produces the V-factor during growth. The plates are then incubated in 5-10% CO2 at 37°C for 2-3 days. Bacteria, which only require V factor, will form larger colonies (called satellite colonies) around the S. aureus streak on both plates, while bacteria that require both V and X factors will only grow on the blood agar plate and give larger satellite colonies around the S. aureus streak.

Disc test

With a disc test, one can also investigate whether a bacterium requires the V factor or both the X and V factors. This test is carried out with three filter paper discs impregnated with V factor, X factor and a mixture of X and V factors, respectively. The discs should then be applied onto an agar plate, where the bacterium to be examined has been inoculated. This agar plate (e.g. TSA) must not contain X or V factor. The bacteria will then grow out as satellite colonies near the respective disc with the growth factor(s) that the bacterium in question needs.

Hematin agar

Hematin agar is used to cultivate fastidious bacteria and it contains blood that has been hemolysed by heating. The X-factor, which is heat stable, has thus been released from the red blood cells. The V-factor, which is a heat sensitive, has been partially destroyed, although the NAD+ nucleosidase (NADase), which is present in blood, has been inactivated by the heat.

Why do bacteria need X and V factors?

The X factor (hemin) is needed for synthesis of cytochrome C and other iron-containing respiratory enzymes. The V factor (NAD and NADP) is required for reduction-oxidation (redox) processes in the cell metabolism.