Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Inoculation of Neisseria musculi
• Procedure: Quantification of Neisseria musculi
• Procedure: Verification of Neisseria musculi in oral-swab suspensions
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Mice tested for endogenous Neisseria (all negative)	Incubator	-
2	Mice inoculated with Neisseria musculi (oral cavity)	Pipettor	-
3	Weekly or biweekly oral swabs and quantification of Neisseria musculi	Incubator	Number of colony-forming units
4	Verification of Neisseria musculi	PCR machine	-

Equipment and supplies

• Incubator
• Petri dishes (agar plates)
• BD BBL CultureSwab Plus Transport System (Fisher Scientific)
• Pipettor
• PCR machine

Reagents and solutions

• Neisseria musculi strain AP2365 (rifampin-resistant)
• GCB Medium Base (Becton Dickinson)
• Agar
• Kellogg's supplements I and II
• Vancomycin
• Trimethoprim
• Rifampin
• Phosphate-buffered saline
• Internal transcribed spacer (ITS) primers specific for AP2031 (used to isolate AP2356)

Procedure: Inoculation of Neisseria musculi

1. Mice are allowed to rest in the animal facility after shipping for 2 wks before inoculation.
2. Oral cavities are swabbed to test for endogenous Neisseria (all mice were found to be negative).
   • Oral cavities are swabbed using the culture swab transport system.
   • Swabs are suspended in GCB medium base plus Kellogg's supplements I and II.
   • Dilutions of the suspensions are plated on GCB agar containing vancomycin (2mg/L) and trimethoprim (3 mg/L).
   • Bacteria are counted after incubation for 48h at 37°C, 5% CO₂.
3. On the day of inoculation, AP2365 is swabbed from an agar plate and resuspended in phosphate-buffered saline at an OD₆₀₀ of 2.0.
4. Mice are manually restrained and 50 µL of the bacterial suspension is pipetted into the oral cavity of each mouse.

Procedure: Quantification of Neisseria musculi

1. Oral cavities are swabbed weekly or biweekly.
2. Swab suspensions in GCB medium base are plated on GCB agar containing rifampin (40mg/L).
3. Plates are incubated for 48h at 37°C, 5% CO₂.
4. Colony-forming units are counted.

Procedure: Verification of Neisseria musculi in oral-swab suspensions

1. Samples from each colony growing on GCB-rifampin agar are used for verification of N. musculi.
2. Internal transcribed spacer (ITS) primers specific to sequences that are highly conserved among Neisseria species are used for colony PCR (ITS sequences were found to be identical to strain AP2031 (used to isolate AP2356)).

Data collected by investigator

• Number of colony-forming units (weekly or biweekly)