Rate and Extent of Helicobacter pylori Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 143

3.3. Cultivation of Helicobacter pylori
Because H. pylori is a biosafety level 2 pathogen, all manipulations of
live bacteria should be performed in a tissue-culture hood. Streak bacteria
from a frozen glycerol stock onto a blood agar plate using a sterile inocu-
lation loop. A heated and humidified microaerophilic environment (5%
O 2 , 10% CO 2 , 85% N 2 ; 37 °C; 98% humidity) is required for optimal
H. pylori growth. These conditions can be achieved using a special incubator.
Alternatively, a microaerophilic atmosphere can be generated using CampyPak
pouches that can then be placed in a 37 °C tissue-culture incubator. Depending
on the H. pylori strain employed, growth will be apparent after 1 or 2 days.
Harvest bacteria from agar plates by scraping into 1 mL of sterile PBS
supplemented with 10 mM glucose. Pellet bacteria in a microfuge (7000 × g ,
2 min, 4 °C). Discard the supernatant and resuspend the pellet in 1 mL of
PBS/glucose (pipette gently, do not vortex). Pellet the bacteria once again and
resuspend organisms in PBS/glucose. Repeat for a total of three or four washes.
If desired, bacteria can be opsonized with specific antibody or complement ( see
Note 6 ). Determine bacterial concentration by measuring the absorbance of a
1:10 dilution of the stock at 600 nm. Store washed H. pylori on ice until use.
We routinely check all H. pylori preparations for motility and viability before
infection of macrophages. To assess motility, place a drop of washed bacteria
onto a microscope slide. Overlay bacteria with a coverslip and view using a
phase-contrast microscope. Molecular Probes BacLight Live/Dead reagents are
used to quantify viability of washed bacteria. Dilute 0.1 mL of H. pylori in 1
mL of PBS containing 3 μL of a 1:1 mixture of the live-dead stains (3.34 m M
Styo9 and 20 m M propidium iodide ) . After 15 min at room temperature, place
an aliquot of stained bacteria onto a microscope slide, overlay with a large
coverslip, and examine the sample using an immunofluorescence microscope
equipped with a ×40 dry objective and filters for FITC and rhodamine fluores-
cence. By this assay, live bacteria fluoresce bright green, dead bacteria are
bright red, and damaged organisms may appear orange. Typically, more than
95% of H. pylori are viable, motile, and spiral shaped. Overgrown or spent
cultures will accumulate clumps of non-viable cocci. Discard samples if fewer
than 90% of bacteria are viable.
3.4. Synchronized Phagocytosis
Centrifugation of H. pylori onto macrophages at low temperature allows
particle binding but not phagocytosis, and rapid transfer of samples to 37 °C
supports synchronized ingestion. Aspirate medium from dishes of macrophages.
Place 1 mL of fresh 37 °C medium into each dish and return dishes to the