10. Fix the confluent epithelial substratum by adding 0.5 mL of 4%
paraformaldehyde (PFA) solution per well for 1 h at room
temperature.
11. Aspirate the 4% PFA solution and wash the cell substratum
three times with 1 mL PBS per well to remove any residual PFA
solution. Use immediately or briefly store in refrigerator after
adding 0.5 mL PBS per well.
12. Streak a S. pneumoniae strain from glycerol stock onto a blood
agar plate and grow overnight.
13. Inoculate the streak from above step into 3–5 mL of THY
media in a 10-mL glass tube or a 15-mL conical tube and
grow statically at 37
C to an OD 600nm of approximately 0.6.
Bacteria from this step can be harvested for challenge models as
the planktonic phenotype.
14. Dilute S. pneumoniae 1:10 in CDM to an OD 600nm of approximately 0.06 and seed 0.5 mL S. pneumoniae suspension in
each well containing fixed epithelial cells.
15. Incubate at 34
C for bacterial biofilm formation for 48 h,
carefully exchanging the supernatant with 0.5 mL fresh CDM
media approximately every 12 h. Avoid disturbing the biofilm
as much as possible.
16. To disperse biofilms with heat, wash the biofilms with fresh
CDM medium and expose the S. pneumoniae to 38.5
C for
4 h.
17. Collect and pellet biofilm-released bacteria by centrifugation at
9000 Â g for 2 min at 4
C in a microcentrifuge.
18. Resuspend pellets in 0.5 mL 0.9% NaCl and incubate for 5 min
at room temperature.
19. Pellet the biofilm-dispersed pneumococci by centrifugation at
9000 Â g for 2 min in a microcentrifuge. Remove and discard
supernatant by pipetting without disturbing the pellet. The
pellet can be stored at À80
C at this time (Fig. 2).
3.10 Pneumococcal
Challenge Model
1. After allowing for 14 days to pass following the secondary
immunization described above, challenge mice with 1 Â 10
4
(sepsis model) or 1 Â 10
6 (pneumonia model) CFU of pneumococci strains through intraperitoneal or intranasal (with
isoflurane) administration, respectively. Bacteria utilized in
these studies can be prepared using either planktonic pneumococci (see step 13 in Subheading 3.9) or using biofilmdispersed pneumococci (step 19 in Subheading 3.9; see Fig. 3).
2. Monitor mice monitored every 4 h for signs of morbidity
(huddling, ruffled fur, lethargy, and abdominal surface temperature). Mice found to be moribund should be euthanized via
CO 2 asphyxiation and cervical dislocation (see Note 10).
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Ruiquan Qi et al.
paraformaldehyde (PFA) solution per well for 1 h at room
temperature.
11. Aspirate the 4% PFA solution and wash the cell substratum
three times with 1 mL PBS per well to remove any residual PFA
solution. Use immediately or briefly store in refrigerator after
adding 0.5 mL PBS per well.
12. Streak a S. pneumoniae strain from glycerol stock onto a blood
agar plate and grow overnight.
13. Inoculate the streak from above step into 3–5 mL of THY
media in a 10-mL glass tube or a 15-mL conical tube and
grow statically at 37
C to an OD 600nm of approximately 0.6.
Bacteria from this step can be harvested for challenge models as
the planktonic phenotype.
14. Dilute S. pneumoniae 1:10 in CDM to an OD 600nm of approximately 0.06 and seed 0.5 mL S. pneumoniae suspension in
each well containing fixed epithelial cells.
15. Incubate at 34
C for bacterial biofilm formation for 48 h,
carefully exchanging the supernatant with 0.5 mL fresh CDM
media approximately every 12 h. Avoid disturbing the biofilm
as much as possible.
16. To disperse biofilms with heat, wash the biofilms with fresh
CDM medium and expose the S. pneumoniae to 38.5
C for
4 h.
17. Collect and pellet biofilm-released bacteria by centrifugation at
9000 Â g for 2 min at 4
C in a microcentrifuge.
18. Resuspend pellets in 0.5 mL 0.9% NaCl and incubate for 5 min
at room temperature.
19. Pellet the biofilm-dispersed pneumococci by centrifugation at
9000 Â g for 2 min in a microcentrifuge. Remove and discard
supernatant by pipetting without disturbing the pellet. The
pellet can be stored at À80
C at this time (Fig. 2).
3.10 Pneumococcal
Challenge Model
1. After allowing for 14 days to pass following the secondary
immunization described above, challenge mice with 1 Â 10
4
(sepsis model) or 1 Â 10
6 (pneumonia model) CFU of pneumococci strains through intraperitoneal or intranasal (with
isoflurane) administration, respectively. Bacteria utilized in
these studies can be prepared using either planktonic pneumococci (see step 13 in Subheading 3.9) or using biofilmdispersed pneumococci (step 19 in Subheading 3.9; see Fig. 3).
2. Monitor mice monitored every 4 h for signs of morbidity
(huddling, ruffled fur, lethargy, and abdominal surface temperature). Mice found to be moribund should be euthanized via
CO 2 asphyxiation and cervical dislocation (see Note 10).
472
Ruiquan Qi et al.