This article details the growth and analysis of Neisseria gonorrhoeae, a human-specific pathogen. The study focuses on the bacterium's ability to extract essential metals from the host environment, which is critical for its survival.
Begin with a culture of Neisseria gonorrhoeae, a human-specific pathogen grown on a metal-supplemented nutrient medium.
During human infection, this bacterium expresses outer membrane receptors that bind to host proteins carrying metal ions.
This enables the pathogen to extract essential metals and survive in the host environment.
Streak single colonies onto a fresh culture plate and incubate to obtain actively growing colonies.
Inoculate a single healthy colony into a metal-depleted medium in a baffled flask. Then, incubate with shaking to promote bacterial growth.
Once the culture has doubled, dilute it with fresh metal-depleted medium to lower the cell density.
As the bacteria grow without access to external metals, their internal metal reserves are gradually depleted.
This metal scarcity triggers the transcription of metal-responsive genes that encode outer membrane receptors exhibiting a high affinity for human metal-binding proteins.
The bacteria are now ready for downstream analysis.
Two days before beginning the experiment, streak gonococci from freezer stocks onto GC medium plates for a no more than 24 hour incubation at 37 degrees Celsius.
14 to 16 hours before the growth experiment, streak single colonies onto fresh GC medium plates. On the day of the experiment, add 5 to 10 milliliters CDM to an acid-washed 125 milliliter baffled side-arm flask and use this medium to blank a Klett colorimeter. Use a sterile cotton-tipped swab to inoculate 20 Klett units of CDM from healthy single colonies.
Generation of a healthy Neisseria gonorrhoeae inoculum for subculture into the 96-well plates for liquid growth assays is critical to the success of all downstream experiments. Incubate the cultures at 37 degrees Celsius in 250 revolutions per minute for one to two hours until approximately one mass doubling before diluting the cultures with a sufficient volume of CDM to reach half of the initial culture density. Then, return the culture to the shaking incubator.
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