Culturing Non-typeable Haemophilus influenzae

Begin with a non-typeable Haemophilus influenzae strain, a human respiratory tract pathogen.

Streak the bacterial culture onto nutrient-rich selective media and incubate it under CO₂-enriched conditions to mimic the respiratory tract environment.

During incubation, the bacteria utilize nutrients and multiply, forming colonies.

Transfer a colony into selective liquid media and incubate to promote bacterial growth in liquid culture.

Transfer an aliquot into a microcentrifuge tube.

Centrifuge to separate the bacteria and discard the supernatant.

Resuspend the bacteria in a buffer, centrifuge again to wash and separate the bacteria, and discard the residual media-containing supernatant.

Resuspend the bacteria in the buffer and dilute.

Using a spectrophotometer, measure the optical density at 600 nm to estimate the bacterial concentration.

Plate the diluted bacterial culture onto selective media and incubate to form colonies.

Count the colonies to calculate colony-forming units for preparing the inoculum for future use.

Plate the NTHI on a chocolate agar plate. Incubate the plate upside down overnight at 37 degrees Celsius with 5% carbon dioxide. The following day, culture the bacteria in a brain-heart infusion broth.

Then, in a microcentrifuge tube, add one milliliter of the broth. Spin the mix down at 4,000 g for five minutes at room temperature. Then, discard the supernatant and resuspend the pellet in one milliliter of PBS. Repeat this wash step one more time.

Next, dilute 100 microliters of resuspended solution in 900 microliters of PBS and measure the culture's absorbance at 600 nanometers. Use this information to adjust the inoculum to 20 million CFUs per milliliter.

Next, determine the viability and CFUs of the inoculum. Culture one to ten serial dilutions of the broth on chocolate agar plates, and incubate the plates overnight.