Sara Hamilton                                     11/17/03

 

 

 

Growth of Listeria monocytogenes for injection

 

Hazards: Listeria is a biosafety level 2 organism and poses a potential hazard to laboratory personnel. Although symptomatic infection is rare in healthy individuals, severe disease can result in immunocompromised persons. This includes pregnant women who can suffer abortion, preterm labor, or stillbirth in extreme cases. Ingestion is the most likely route of exposure, but skin and eye infections are also possible.

 

Protection: Gloves and eye protection

 

Waste: Dispose of bacterial plates into biohazard waste containers. Liquid cultures can be bleached with a 20% bleach solution for 30 minutes and poured down the sink. Reusable materials can be sprayed with 70% ethanol and wiped down.

 

Spill Clean-Up: Wipe up spill with paper towels and place in biohazard waste containers. Spray effected area with 70% ethanol and wipe down.

 

Procedure:

Note: This procedure should be performed in the BL2 pathogen room (6-231C).

1. Obtain a 1mL aliquot of bacteria from the –80⁰C freezer and thaw in 37⁰C water bath.
2. Also thaw stock of streptomycin sulfate (Sigma S6501) (50 mg/mL) from the –20⁰C freezer (note: All of our Listeria strains are resistant to streptomycin. Some strains carry additional antibiotic resistance markers.)
3. After flaming bottle over a bunsen burner, pour or pipette ~ 9 mL sterile tryptic soy broth (BD 211825) (30g/L) (liquid found at 4⁰C) into a 50 mL conical tube. Also pour/pipette ~5mL into a second tube (this will serve as a blank). Reflame bottle, cap, and put away. Make sure to examine for signs of contamination before use (ie. cloudy liquid).
4. Add streptomycin to both tubes of broth at a 1:1000 dilution (50 mg/mL final concentration).
5. Vortex
6. Add bacteria to the 9mL tube
7. Loosely cap both tubes and place securely in bacterial shaker (37⁰C at 250 RPM) (Note: Listeria will grow at lower temperatures, but it will take significantly longer to get to log phase.)
8. Grow to an O.D.₆₀₀ of ~0.1 (this should take about 2 hours)
9. Measure the optical density of the solution and calculate how many bacteria you have. An O.D.₆₀₀ of 0.1 is estimated to be 10⁸ CFU/mL and a typical primary infection is in the range of 1-3 x 10³ CFU/mouse for BALB/c and 5 x 10³ for B6.
10. Dilute bacteria into sterile saline for injection (it is not necessary to spin down bacteria unless you are in injecting very high numbers).
11. Make sure to plate an aliquot of the injected solution onto tryptic soy broth agar plates (containing streptomycin). Then grow the plates for ~24 hours and count the colonies to determine the actual number of organisms you injected. (The optical density reading is only an estimate!) 100 colonies is a good number to shoot for (being 2-fold off is not unusual).
12. Bleach remaining LM for 30 minutes and dispose of down the sink. Dispose of plates in biohazard waste containers.