Standard Operating Procedures
Working with Streptococcus pneumoniae
Last Updated: July 16, 2011
I. Purpose
The purpose of this standard operating procedure is to outline the proper procedures for handling Streptococcus pneumoniae in the facilities of Laboratory Animal Resources at the University of Montana-Missoula. This procedure describes safety precautions required for infecting mice with S. pneumoniae and for working in animal rooms where S. pneumoniae is present, cleaning dirty cages, and disposing of S. pneumoniae-contaminated waste.
II. Accident, Injuries, and Potential Exposures
- In the event of a percutaneous or mucous membrane exposure to S. pneumoniae
- Initiate first aid at the worksite:
- Wash contaminated skin with soap and water
- Irrigate contaminated eyes and mucous membranes for 10 minutes, using normal saline or water
- The disease is usually self-limiting and should not cause a serious illness
- In the absence of clinical signs following accidental exposure to S. pneumoniae, a physician may be seen
- Individuals exhibiting clinical signs associated with S. pneumoniae infection after accidental exposure must see a physician
- Symptoms include:
- Tiredness, fever, headache, body aches, joint pain, shaking chills, coughing with sputum, chest pain, shortness of breath
- Self-treatment includes:
- Drink plenty of nonalcoholic fluids to stay hydrated, anti-fever medications (acetaminophen or ibuprofen), avoid cigarette or tobacco smoke, and avoid cough suppressants
- See a physician if:
- Individual has shortness of breath, chest pain, confusion, or is coughing up yellow, green or brown sputum.
- Treatment will most likely include the use of antibiotics for a typical duration of 5-14 days
- In the event of a spill in the biosafety cabinet
- Wipe spill area immediately with MB-10
- Place all clean-up materials into a biohazard bag for autoclaving, spray bag with MB-10
- Autoclave all contaminated materials before final disposal
III. Personal Protective Equipment (PPE)
- Conduct all manipulations with S. pneumoniae that involve animals in the Biological Safety Cabinet (BSL2) in HSB 004 or SB117.
- House all animals treated with S. pneumoniae in HSB 004 in specifically denoted cubicle
- Wear personal protective equipment whenever you are working with S. pneumoniae or S. pneumoniae-treated animals and their wastes, or when entering HSB004 animal cubicle during the 24 h following inoculations
- Inoculating animals:
- Wear 2 pairs of disposable lab gloves, a disposable lab coat (used only for this purpose), shoe covers, and a mask covering the nose and mouth
- Waste receptacle containing 10% bleach
- MP-10 spray bottle
- Work only in biosafety cabinet
- When entering HS 004 animal cubicle up to 24 hr after inoculation:
- Wear 1 pair of disposable lab gloves, shoe covers, a disposable lab coat (used only for this purpose), mask covering the nose and mouth
- You may hang the disposable lab coat on a hook in the cubicle for use the following day only
- Harvesting infected mouse tissue:
- Wear 2 pairs of disposable lab gloves, a disposable lab coat (used only for this purpose), shoe covers, and mask covering the nose and mouth
- Waste receptacle containing 10% bleach in the hood
- MP-10 spray bottle
- Work only in BSL2 cabinet
- When observing or entering HSB 004 when S. pneumoniae work is being conducted in biosafety cabinet:
- Wear disposable lab gloves, a lab coat, shoe covers and mask covering the nose and mouth
- Remove protective clothing.
- When moving from biosafety cabinet to another area, remove outer gloves in biosafety cabinet and place them in a biohazard bag kept inside cabinet area.
- When finished for the day, place outer gloves in biohazard bag kept inside biosafety cabinet area, remove inner gloves, mask and lab coat and place in biohazard bag (disposable lab coat may be hung in the cubicle following inoculations and used the following day for harvesting tissue). Dispose of shoe covers when exiting HSB 004.
- LAR staff will autoclave the bagged biohazardous waste before final disposal.
IV. Administration of S. pneumoniae To Mice
- Conduct all S. pneumoniae experimentation involving animals in HSB 004.
- Procedure
- Conduct all procedures in a biosafety cabinet (BSC2) using PPE
- Inoculate mice with 107 CFU of S. pneumoniae (50 µl maximum volume) by Intra-Tracheal/Oropharyngeal Aspiration
- Anesthetize mice with isoflurane
- An anesthetized mouse will be suspended on an angled board by hooking the front teeth on a suspended rubber band, the tongue gently extended out of the oral cavity, and the inoculum will be instilled in the trachea using a blunt-ended 25-ga needle.
- Return inoculated mice to their cages and observe for adverse effects of anesthesia or inoculation
- Monitor mice a minimum of twice daily after inoculation
- Clean biosafety cabinet: spray contents with MB-10, remove all biosafety bags, dispose of 10% bleach, spray with MB-10 and wipe down
- House inoculated and control mice for 24 hours in HSB 004 specified cubicle
- Euthanize mice 24 hours after S. pneumoniae inoculation using Euthasol followed by cervical dislocation in the BSL2 cabinet
- Place empty, dirty cages into a biohazard bag in the biosafety cabinet
- Harvest lungs in the biosafety cabinet and freeze tissue in liquid nitrogen for shipment to MSU
- Place mouse carcass into a dated and labeled bag and place inside red biohazard bag in HSB 015A biohazard freezer
- Cleaning and disinfecting the biosafety cabinet and area
- At the end of the experimental day, spray the biosafety cabinet and contents with MB-10. Close all biohazard bags containing contaminated materials, remove from BSC2 and place into a larger bag for autoclaving. Dispose of 10% bleach solutions down drain
- Place all cages, with lids on, into a biosafety bag for autoclaving. Mark bag as cages, with your name and date.
- Spray inside of BSC2 with MB-10 and wipe down area
- Place PPE into biosafety bag for autoclaving
- Mark all autoclave bags with your name and date, set aside for autoclaving by LAR staff
- Make sure area is clean and secure before you leave
V. Animal Housing
- Place animals into clean caging immediately prior to S. pneumoniae inoculations
- House S. pneumoniae-treated animals in HSB 004 in a specified cubicle
- When S. pneumoniae is present, place a sign on cubicle door and outer door with the following information
- Biohazard symbol
- ABSL-2
- Contact name and phone number(s)
- S. pneumoniae-treated animals are present
- Entrance is restricted to authorized personnel
- Gloves, shoe covers, lab coat and face mask must be worn
- Label the mouse cages to indicate that hazardous material is present (S. pneumoniae).
- 24 hr after inoculation
- Animals will be euthanized and tissues collected
- Put cages, with lids on, into autoclave bags and LAR staff will autoclave
- Place mouse carcass into a dated and labeled bag and place inside red biohazard bag in HSB 015A biohazard freezer
- Final cage cleaning.
- After the cages and contents have been autoclaved, the LAR staff will empty and wash cages using normal procedures
- Final cubicle cleaning by LAR staff.
- Wear PPE including gloves, regular lab coat and face mask
- ASC room thoroughly
