1.0 OBJECTIVE:
Standard operating procedure for detection of E. coli and Salmonella spp. in animal urine samples
2.0 SCOPE:
This SOP is applicable to Animal House Facility of Lab Name
3.0 RESPONSIBILITY:
In-charge Animal house shall collect the urine sample and will maintain the record.
In-charge Microbiology is responsible for overall compliance of this procedure.
4.0 INTRODUCTION:
Salmonoella enterica serovar typhimurium is a Bio-safety level (BSL) 2 bacterial pathogen, indicating the potential to cause disease in humans. With the safety and standard operating protocols described below, the risk of exposure and disease is minimal. For transmission to humans, exposure of bodily fluids with contaminated materials must occur. Transmission to humans by the aerosol route with these pathogens does not occur, however, since centrifugation has the potential to create aerosols, all centrifuges in the lab are fitted with aerosol-tight rotor lids and/or sample buckets. E. coli can cause urinary tract infections. With proper procedures used, no specific infection risk attributed. Antibiotics can be used as treatment (Ampicillin and clavulanic acid, for example).
5.0 PROCEDURE:
CULTURE AND CENTRIFUGATION OF BACTERIA
Small frozen aliquots (~ 10 microliters) of each strain will be put onto a bacterial growth plate, and re-streaked for isolation of single colonies. These plates will be incubated overnight at 37ºC in a lab specific incubator, and then stored at 4ºC. On the day prior to a planned infection, a individual colony for each strain of bacteria to be used will be picked from the bacterial growth plate, and inoculated into liquid broth (2-5 ml) using snap cap tubes (disposable test tubes), and allowed to incubate overnight (300 rpm on an orbital shaker). The concentration of log-phase cultures will be verified by determining the optical density of the culture (1 OD 600 = 1.0 x 10e8 bacteria per ml). The log-phase culture will then the aliquoted into eppendorf tubes and centrifuged (14K rpm for 2 minutes at room temperature). Although these bacteria are not known to be transmitted by the aerosol route only certified sealed (aerosol tight) rotors and chambers with (O-rings) will be used for centrifugation. Bacterial pellets will be washed with sterile PBS, and adjusted to the desired concentration. All these procedures will be done under standard BSL-2 laboratory conditions.
INFECTION OF MICE
Stocks of bacteria will be transported to the animal suite (WMBB 1-514A) in screw-cap polystyrene containers containing diluted bacteria enclosed in an outer sealable plastic (Ziplock) type bag. Prior to leaving the main laboratory, the outside of these sealable plastic Ziplock bags will be sprayed with disinfectant (10% bleach). Within room 1-514A, all animal work will be done in a BSL-2/ABSL-2 certified biosafety hood under RAR standard operating procedures. All laboratory staff will have been oriented by RAR staff to ABSL-2 procedures prior to beginning work with mice. For infections, mice will be restrained in dedicated holders (intravenous inoculation), or by hand (for oral-gastric inoculations). All mouse holders (for IV infection), hood counters, and shield surfaces will be sprayed and wiped with disinfectant before and after each use.
Salmonella infection: 5-8 week old mice housed in the BSL2 facility will be inoculated once with 1x104 live Salmonella bacteria, either i.v. or by oral gavage. Do not recap needle, dispose of directly into sharps container. Mice will be euthanized 1, 2, or 3 days later.
TISSUE HARVEST
At the indicated endpoints, we will euthanize the mice (using CO2) and recover spleen, liver, bone marrow, and thymus for FACS analysis. Dissection of mice infected with Salmonella, or E. coli, will occur exclusively in biosafety cabinets located either in MBB 1-514A. Dissected organs will be transported to the main lab on ice in closed screw cap tubes on ice, and sprayed with 10% bleach as a disinfectant prior to leaving the animal suite.
Mice expressing severe signs of distress prior to the endpoints (prostration, hunched posture) will be euthanized within 12 hours. Carcasses will be double bagged, labeled with BIOHAZARD sticker as “SALMONELLA TYPHIMURIUM” or “E. COLI” and placed in the BSL2 Safety Facility Freezer.
6.0 PRECAUTIONS:
Protective laboratory coats, gowns, smocks, gloves or uniforms designated for lab use will be worn while in the laboratory.
All infectious agent cultures, stocks, animal carcasses and/or tissues, sharps used to inoculate animals, etc. and bedding from infected animals will be disposed of as hazardous waste (i.e. put in an appropriately labeled red biohazard bag, and decontaminated By autoclaving.
7.0 REFERENCES:
Cowie, R. A., & Makkar, H. P. (2013). Quality assurance for microbiology in feed analysis laboratories FAO.
Balthrop, J., Brand, B., Cowie, R. A., Danier, J., de Boever, J. L., de Jonge, L. H. & Piotrowski, C. (2011). Quality assurance for animal feed analysis laboratories (No. 14). FAO.
8.0 ABBREVATIONS:
SOP: Standard Operating Procedure
cfu: colony forming unit
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