Infection of Epithelial Cells With Salmonella enterica - Bacterial Pathogenesis: Methods and Protocols - page 192

Internalized bacteria can be assayed by recovery of colony-forming units,
in a classical gentamicin assay or by microscopical analysis after fixation and
staining using Salmonella -specific antibodies. For both assays, epithelial cells
are grown in monolayers and infected with Salmonella for various lengths of
time. Because of the time lag that occurs before the onset of intracellular repli-
cation, invasion is usually monitored at 1-1.5 h post-infection and replication
at 4.5-8 h or later. Both invasion and replication can be affected by numerous
factors—for example, host cell type, viability, confluency, and growth condi-
tions. Adhering to a standardized routine can minimize most experimental
variability. The gentamicin assay, which involves solubilization of the cells and
plating the released bacteria, yields information on the total number of colony-
forming units (i.e., viable bacteria) in a host cell monolayer. In contrast, the
microscopical assay determines the number of bacteria inside individual cells
but cannot differentiate between live and dead bacteria. Together these assays
are indispensable tools for any lab studying Salmonella -host cell interactions.
2. Materials
1. Glycerol stocks of S. enterica . This protocol has been optimized for serovar
Typhimurium SL1344 (11) ( see Note 1 ).
2. Luria-Bertani (LB) Broth, Miller: 10 g/L Bacto tryptone and 5 g/L Bacto
yeast extract 10 g/L NaCl. For plates add 15 g/L of Bacto-agar. Sterilize by
autoclaving.
3. Antibiotic stocks (stored at -20 °C): streptomycin, 100 mg/mL in H 2 O and
gentamicin sulfate, 50 mg/mL in H 2 O.
4. Minimal essential medium (MEM) with Earle's salts without l-glutamine (Invit-
rogen, Carlsbad, CA).
5. Sodium pyruvate, 100 m M in H 2 O.
6. l-glutamine, 200 m M in H 2 O.
7. Trypsin EDTA, 1×: solution of 0.25% Trypsin and 2.21 m M EDTA in HBSS
without sodium bicarbonate, calcium, and magnesium. Available pre-made from
suppliers of cell-culture materials.
8. Fetal bovine serum (FBS): Performance tested, mycoplasma tested, virus tested,
bacteriophage tested, and endotoxin tested. To heat inactivate, thaw the serum
slowly at 37 °C and mix the contents of the bottle thoroughly. Place the thawed
bottle of serum into a 56 °C water bath containing enough water to immerse the
bottle to just above the level of the serum. Swirl the serum every 5-10 min to
ensure uniform heating and to prevent protein coagulation at the bottom of the
bottle. After 30 min at 56 °C, cool the serum immediately. Store at -20 °C.
9. Growth medium (GM): MEM supplemented with 2 m M l-glutamine( see Note 2 ),
1m M sodium pyruvate, and 10% heat-inactivated FBS.