23- Incubate for 15 min at 37 °C with rapid aeration (e.g.,
250 rpm). Use this time to prechill a centrifuge to 4 °C and
place 20 mM sodium citrate on ice. - Add 1 mL ice-cold 20 mM sodium citrate to the mixture and
incubate on ice for 5 min. - Centrifuge the mixture at 4 °C for 10 min at approximately
~ 4000 × g). - Discard the supernatant (see Note 12).
- Resuspend the pellet in 1 mL ice-cold 20 mM sodium citrate
and incubate on ice for between 45 min and 1.5 h. - Spread 100 μL aliquots onto phage agar plates containing the
appropriate antibiotic for selection of transductants (see Note 13 ). - Incubate for 24–48 h at 37 °C until healthy bacterial colonies
appear, then restreak and incubate again overnight to ensure
complete loss of residual phage particles. - Grow 5 mL overnight culture of each strain in BHI at 37 °C
with rapid aeration (e.g., 250 rpm). - Subculture in 50 mL to OD 600 = 0.01 and grow as above until
the culture reaches an appropriate growth phase for your
experiment. - Stop growth by incubating on ice for 15 min. Use this time to
prechill a centrifuge to 4 °C. - Vortex the samples well and precisely measure the OD 600
immediately prior to the next step. - Centrifuge the mixture at 4 °C for 10 min at approximately
~ 4000 × g). Use this time to prechill 15 mL tubes on dry ice. - Discard the supernatant, then resuspend the pellet in sterile,
ice-cold PBS to approximately 1 × 1010 CFU/mL (see
Note 14 ). Vortex well. - Pipette 100 μL aliquots into 15 mL tubes on dry ice. Allow to
freeze. - Transfer to − 80 °C for long term storage and at least overnight
prior to first concentration test. Samples are viable for several
years (periodically retest the concentration as described below). - Defrost three aliquots of each strain on ice. Add sterile, ice-
cold PBS to each tube so that its final concentration is
1 × 108 CFU/mL (see Note 14). - Vortex well, then make a decimal serial dilution series of each
aliquot. Plate 100 μL of 10−^4 , 10−^5 , and 10−^6 dilutions onto
BHI agar without antibiotics. - Incubate plates overnight at 37 °C. This will enable you to detect
any nonstaphylococcal contamination and precisely calculate the
average final concentration of each strain’s aliquots (see Note 15).
3.4 In Vivo Studies:
Preparation
of Bacteria for Mouse
Infection
Construction and Use of Staphylococcus aureus Strains to Study Within-Host...