Growth and hemolysin production by Haemophilus pleuropneumoniae cultivated in a chemically defined medium

A chemically defined medium (CDM) has been developed which supports both growth and hemolysin production by Haemophilus pleuropneumoniae. Although the growth rate in stationary cultures was substantially slower in CDM than in trypticase soy broth plus 0.6% yeast extract (TSBYE) and slightly slower than in heart infusion broth (HIB), extracellular hemolysin activity in CDM was slightly higher than in HIB and 16-fold greater than in TSBYE. Maximum hemolytic activity was produced in CDM in early to mid log phase of growth. Hemolytic activity in sterile, cell-free culture supernatant fluids persisted for over 10 days at 4 °C and 3–5 days at 37 °C, but was completely destroyed at 56 °C after 30 min. Total hemolysin inactivation was also achieved in the presence of trypsin or pronase (10 units/mL), but no decrease in hemolytic activity was noted in the presence of DNase or RNase. Iron had little effect on the hemolytic activity in the early stages of growth. However, in the later stages of growth, iron had a pronounced effect with hemolyic activity decreasing as the iron concentration increased from 1 to 500 μM. None of these iron concentrations had any effect on the hemolytic activity when added directly to prepared cell-free culture supernatant fluids. The extracellur hemolysin produced by H. pleuropneumoniae in CDM appears to be a heat-labile protein the activity of which is influenced by iron at certain phases of growth.