Culturing Non-typeable Haemophilus influenzae

Abstract

Source: Venuprasad, K. et. al., Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation. J. Vis. Exp. (2016)

This video demonstrates the culturing of non-typeable Haemophilus influenzae and the preparation of a dose-standardized inoculum for downstream colonization or infection studies.

Protocol

1. Culturing non-typeable Haemophilus influenzae (NTHi) and preparing the inoculum

1. Plate NTHi on a chocolate agar plate and keep the plate upside down in a humidified CO₂ incubator overnight at 37 °C and 5% CO₂. The following day, culture the bacteria in brain heart infusion broth. Then, add 1 ml of the broth into a sterile 1.5 ml tube.
2. Centrifuge at 4,000 x g for 5 min at room temperature. Then, discard the supernatant and resuspend the pellet in 1 ml of sterile 1x phosphate-buffered saline (PBS). Repeat this step once.
3. Transfer 100 µl of the re-suspended solution into 900 µl of 1x PBS and measure the absorbance of the diluted culture at 600 nm wavelength in a spectrophotometer.
4. Determine the viability and colony-forming units (CFUs) of the inoculum by culturing 1:10 serial dilutions on chocolate agar plates and incubating overnight at 37 °C and 5% CO₂.
5. Based on the viability and CFUs determined from the previous step, adjust the inoculum to a final concentration of 20 x 10⁶ CFU/ml.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chocolate agar plate	Fisher Scientific	CAS50-99-7
1.5 ml microcentrifuge tube	Light Labs	A-7001-R
1X PBS	Gibco	10010-023