This study outlines a method for quantifying group B Streptococcus (GBS) in a mouse model. The technique involves vaginal swabbing and bacterial culture on differential agar to visualize GBS colonies.
Take a female mouse colonized vaginally with group B Streptococcus or GBS, an opportunistic bacterial pathogen of the vaginal tract.
Restrain the mouse and insert a buffer-moistened swab into the vaginal lumen.
Gently rotate the swab while applying slight pressure to the vaginal wall to dislodge surface-bound bacteria.
Immerse the swab in a buffer and agitate to suspend the bacteria.
Serially dilute the suspension in the buffer and plate it on a chromogenic differential agar containing a substrate linked to a chromogen.
Incubate to allow bacterial growth.
During incubation, GBS expresses a specific enzyme that hydrolyzes the chromogenic substrate.
The released chromogen undergoes spontaneous oxidation and dimerization, forming a pink or mauve-colored compound.
This results in pink or mauve GBS colonies, visually distinct from other bacterial colonies.
Count the colonies to quantify the GBS load and assess its persistence in the mouse model.
Just prior to swabbing, pre-wet the cotton in PBS, and restrain one of the mice as just demonstrated.
Insert the swab 10 millimeters into the vaginal lumen, and gently rotate four times clockwise, and four times counterclockwise, while applying slight pressure to the vaginal wall.
Place the swab into a 1.5 milliliter microcentrifuge tube containing 100 microliters of PBS, and vortex the tube and swab for about 15 seconds to release the bacteria from the cotton fibers. Then, serially dilute each sample in PBS, and plate 20 microliters of 1 to 10 through 1 to 10,000 dilutions on differential medium agar plate, prepared per the manufacturer's instructions, for incubation at 37 degrees Celsius for 24 hours. GBS colonies will appear as either bright pink or mauve in color.
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