1.11 LAS MEDIDAS CAUTELARES PERSONALES
1.11.2 FUNDAMENTO CAUTELAR
1.11.1.1 DETENCIÓN PREVENTIVA
Stock concentration Final concentration
(mg/ml) (!Ag/ml)
20 30
50 50
40 25
Salmonella isolates were routinely grown on blood, XLD and MacConkey agars at 37°C for 1 8-24 h under aerobic conditions. The freeze-dried cultures of Salmonella and other bacterial isolates were re suspended in 0.5 ml of nutrient broth, and a few drops of suspension were placed on an agar plate and incubated as above. From frozen glycerol stocks, a few microliters were inoculated and grown on agar plates. For Salmonella biochemical tests, colonies were grown on TSI agar, urea agar and lysine decarboxylase broth at 3 7°C for 24-48 h. E. coli strains, including recombinants, were grown on L B agar and/or in L B broth containing appropriate antibiotics and/or supplements at 3 7°C under aerobic conditions. Liquid cultures were grown in tubes or flasks with a volume of at least 4 times that of the culture volume, and were shaken at approximately 225 rpm on an Innova 4000 incubator shaker (New Brunswick Scientific
2.2.3
Measurement of cell density of culturesCell densities of recombinant E. coli liquid cultures were calculated from the OD values of cultures at 600 nm (OD6oo). The double beam spectrophotometer (Helios a, Unicam, UK) was calibrated to zero at 600 nm using 2 blank polystyrene cuvettes (Global Science & Technology Ltd., Auckland, NZ) each containing 1 ml of sterile L B broth. Routinely, 1 00 f-ll o f culture and 900 f-ll o f sterile LB broth were mixed carefully to avoid bubbles in a cuvette. One of the blank cuvettes was replaced with the one containing 1 : 1 0 diluted culture, and the OD6oo reading was recorded. All samples were analysed in duplicate.
2.2.4
Storage of culturesB acterial cultures were kept for short periods (up to 1 week) at 4°C. For longer-term storage, cultures of recombinant E. coli, Salmonella and other bacteria were stored as glycerol stocks at -70°C. B acteria were streaked onto an appropriate agar plate and incubated overnight at 3 7°C. For the storage of Salmonella and other bacterial isolates, several loopfulls of bacterial cells from overnight agar plates were mixed in 1 ml of 1 5% glycerol in trypticase soy broth in cryovials (Nalgene Nunc I nternational, Roskilde, Denmark). For recombinant E. coli, a single colony was picked from the agar plate either with a sterile toothpick or a pipette tip, and inoculated into 5 ml of L B broth. When the recombinant E. coli cultures reached 0.6-0.8 at OD6oo, 800 f-l l o f liquid culture was mixed with 200 f-ll of sterile glycerol (20% v/v) in a cryovial by inversion or a brief vortex. The cryovials were then stored at -70°C.
2 .3
D NA extractions
2.3.1
Extraction of genomic DNA2. 3. 1 . 1 Boiling method
For most PCR reactions, genomic DNA was obtained by boiling a suspension of bacteria. Each strain was grown on an agar plate overnight at 3 7°C. A single colony
was resuspended in 20 "",1 of distilled water, and heated at 1 00°C for 1 0 min. It was immediately cooled on ice for 5 min, and centrifuged at 1 3 ,000 x g at 4°C for 1 0 min. The supernatant containing DNA was stored at -20°C until used.
2. 3. 1. 2 Phenol-chloroform method
F or preparation of the S. Brandenburg genomic library, genomic DNA was extracted using a phenol-chloroform method. A loopful of Salmonella culture from a frozen glycerol stock was streaked onto a blood agar plate. After an overnight incubation at 3 7°C, a single colony was grown in 6 ml of LB broth. The overnight culture was centrifuged at 2,000 rpm for 1 0 min, and the cell pellet was resuspended in 2 ml of TE buffer pH 8.0 ( 1 0 mm Tris.CI, 5 mm ethylenediamine tetraacetic acid [EDTAJ). The cell suspension was transferred into a microcentrifuge tube, and centrifuged at 1 2,000 x g for 1 0 min. The pellet was re suspended in 1 ml of TE buffer pH 8.0 containing 1 0 "",1 of 50 mg/ml lysozyme stock (Cat. No. 1 -243-004, Roche, Mannheim, Germany) and incubated at 3 7°C for 1 h. Fifty microlitres of 1 0% sodium dodecyl sulfate (SDS) was added to the tube and the mixture was vortexed. After addition of 1 0 "",1 of 20 mg/ml proteinase K (Cat. No. 745-723, Roche), the samples were vortexed again and incubated at 55°C for 2 h. Genomic DNA was isolated with two phenol-chloroform isoamyl alcohol (25 :24 : 1 , v/v; Cat. No. 1 5593-03 1 , Life Technologies) extractions to remove contaminating proteins. Briefly, 1 ml of phenol-chloroform-isoamyl alcohol was added to the suspension, vortexed and centrifuged at 1 3 ,000 x g for 1 0 min. The upper aqueous phase was removed to a c lean m icrocentrifuge tube and phenol chloroform extraction was repeated one more time. The upper aqueous phase (approximately 1 m l ) was then aliquoted i nto two microcentrifuge tubes. Subsequently, ethano l precipitation was performed to remove salts from the preparation. One hundred microlitres (0. 1 volume) of 3 M sodium acetate pH 5 .0, and 1 ml (2 volumes) of 1 00% ice-cold ethanol were added to each tube, vortexed and incubated at -20°C for at least 30 min. The tubes were centrifuged at 1 3 ,000 x g for 30 min at 4°C. After removing the supernatant, the pellet was washed by mixing with 1
ml (2 volumes) of 70% ice-cold ethanol and centrifuging at 1 3,000 x g for 5 min at 4°C. The resulting DNA pellet was vacuum dried using an SC 1 00 Speed Vac (Savant Instruments Inc., Holbrook, NY, U SA) and resuspended in 1 00 "",1 of sterile distilled
water. Where RNA removal was desired, 90 !ll of isolated genomic DNA was incubated with 1 0 !ll of 1 0 mg/ml RNase (Cat. No. 1 09- 1 42, Roche) at 3 7°C for 1 h. Subsequently, DNA was isolated with two phenol-chloroform-isoamyl alcohol extractions and ethanol precipitation as above. The DNA pellets were dried, resuspended in sterile water and stored at -20°C.