Source: Gannon, A. D., et al. Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model. J. Vis. Exp. (2021).
This video demonstrates an in vitro technique to study the impact of antimicrobial treatment on Pseudomonas aeruginosa (Pa) aggregates. Fluorescently-labeled Pa cells are grown as aggregates, mimicking the growth pattern during infection. Upon challenging the aggregates with a sublethal concentration of the antibiotic colistin, the real-time assessment of the bacteria-antibiotic interaction is performed via fluorescence microscopy.
1. Prepare synthetic cystic fibrosis medium (SCFM2)
NOTE: Preparation of SCFM2 comprises three main stages outlined below (Figure 1).
2. Real-time assessment of antimicrobial tolerance in bacterial aggregates
3. Visualizing aggregates during antibiotic treatment with confocal laser scanning microscopy (CLSM)
NOTE: This section describes the use of confocal laser scanning microscope and image capture software for the imaging of Pa aggregates in SCFM2. The goal is to observe and characterize the remaining (tolerant) bacterial biomass after treatment with antibiotics. The steps outlined can be performed with success on other confocal microscopes, although the instrument operating manual should be referenced for specific guidance.
4. Propidium iodide staining of Pa aggregates
NOTE: Propidium iodide (PI) is commonly utilized as a staining reagent to identify non-viable (dead) bacterial cells in culture. Here, it is used to identify aggregates sensitive to antibiotic treatment applied in section 3. Throughout this protocol, the expression and detection of GFP in Pa cells is used as the main proxy for cell viability. This final step allows confocal imaging to be used once more to identify the spatial positioning of live/dead aggregates in relation to each other. Additionally, aggregates are identified as live/dead for further downstream cell sorting in section 3.
5. Isolating live cells from aggregates using a Fluorescence-activated cell sorting (FACS) approach
NOTE: FACS presents a powerful platform to sort and isolate groups of cells according to a fluorescently tagged phenotype. Here, FACS is used to isolate live (antibiotic-tolerant) aggregates from non-viable aggregates.
Table 1: Preparation of salt, amino acid, DNA, and mucin stocks required for buffered base of synthetic cystic fibrosis sputum medium, SCFM2.
Chemical | Stock Concentration (M) | Final concentration (mM) | Notes |
NaH2PO4 | 0.2 | 1.3 | |
Na2HPO4 | 0.2 | 1.25 | |
KNO3 | 1 | 0.35 | |
K2SO4 | 0.25 | 0.27 | |
NH4Cl | 2.28 | Add solid directly to buffered base | |
KCl | 14.94 | Add solid directly to buffered base | |
NaCl | 51.85 | Add solid directly to buffered base | |
MOPS | 10 | Add solid directly to buffered base | |
Ser | 0.1 | 1.45 | |
Glu HCl | 0.1 | 1.55 | |
Pro | 0.1 | 1.66 | |
Gly | 0.1 | 1.2 | |
Ala | 0.1 | 1.78 | |
Val | 0.1 | 1.12 | |
Met | 0.1 | 0.63 | |
Ile | 0.1 | 1.12 | |
Leu | 0.1 | 1.61 | |
Orn HCl | 0.1 | 0.68 | |
Lys HCl | 0.1 | 2.13 | |
Arg HCl | 0.1 | 0.31 | |
Trp | 0.1 | 0.01 | Prepared in 0.2 M NaOH |
Asp | 0.1 | 0.83 | Prepared in 0.5 M NaOH |
Tyr | 0.1 | 0.8 | Prepared in 1.0 M NaOH |
Thr | 0.1 | 1.07 | |
Cys HCl | 0.1 | 0.16 | |
Phe | 0.1 | 0.53 | |
His HCl H2O | 0.1 | 0.52 | |
Salmon Sperm DNA | 0.6 mg/mL | ||
Porcine Mucin | 5 mg/nL |
Table 2: Preparation of additional stocks required for buffered base of synthetic cystic fibrosis sputum medium, SCFM2.
Chemical | Stock concentration (M) | Final concentration (mM) | Notes |
Dextrose (D-glu2cose) | 1 | 3 | |
L-lactic acid | 1 | 9.3 | Adjust to pH 7.0 with NaOH |
CaCl2*H2O | 1 | 1.75 | |
MgCl2*6H2O | 1 | 0.61 | |
FeSO4*7H2O | 1 mg/mL | 0.0036 | Make fresh daily |
N-acetylglucosamine | 0.25 | 0.3 | |
1,2-dioleoyl-sn-glycero-3-phosphocoline (DOPC) | 25 mg/mL | 100 µg/mL | Incubate for at least 20 min at 37 °C after addition |
Figure 1: Preparation and inoculation of SCFM2 medium. (A) Buffered base is prepared using salts and amino acids listed in Table 1 and Table 2. Buffered base can be stored at 4 °C for up to 30 days, but must be protected from light exposure. (B) Mucin and DNA are added to an aliquot of buffered base and dissolved into solution overnight at 4 °C. (C) Lipid and additional stocks are added to the overnight solution and incubated at 37 °C with agitation at 250 rpm for 20 min. SFCM2 is then inoculated with washed, log phase cells at an OD600 = 0.05. Abbreviations: SCFM2 = synthetic cystic fibrosis sputum medium.
The authors have nothing to disclose.
Amino acids | |||
Alanine | Acrs Organics | 56-41-7 | |
Arginine HCl | MP | 1119-34-2 | |
Asparagine | Acrs Organics | 56-84-8 | Prepared in 0.5 M NaOH |
Cystine HCl | Alfa Aesar | L06328 | |
Glutamic acid HCl | Acrs Organics | 138-15-8 | |
Glycine | Acrs Organics | 56-40-6 | |
Histidine HCl H2O | Alfa Aesar | A17627 | |
Isoleucine | Acrs Organics | 73-32-5 | |
Leucine | Alfa Aesar | A12311 | |
Lysine HCl | Alfa Aesar | J62099 | |
Methionine | Acrs Organics | 63-68-3 | |
Ornithine HCl | Alfa Aesar | A12111 | |
Phenylalanine | Acrs Organics | 63-91-2 | |
Proline | Alfa Aesar | A10199 | |
Serine | Alfa Aesar | A11179 | |
Threonine | Acrs Organics | 72-19-5 | |
Tryptophan | Acrs Organics | 73-22-3 | Prepared in 0.2 M NaOH |
Tyrosine | Alfa Aesar | A11141 | Prepared in 1.0 M NaOH |
Valine | Acrs Organics | 72-18-4 | |
Antibiotic | |||
Carbenicillin | Alfa Aesar | J6194903 | |
Day-of Stocks | |||
CaCl2* 6H2O | Fisher Chemical | C79-500 | |
Dextrose (D-glucose) | Fisher Chemical | 50-99-7 | |
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) | Fisher (Avanti Polar Lipids) | 4235-95-4 | Shake 15-20 min at 37 °C to evaporate chloroform |
FeSO4 * 7H2O | Acrs Organics | 7782-63-0 | This stock equals 1 mg/mL, MUST make fresh |
L-lactic acid | Alfa Aesar | L13242 | pH stock to 7 with NaOH |
MgCl2 * 6H2O | Acrs Organics | 7791-18-6 | |
N-acetylglucosamine | TCI | A0092 | |
Prepared solids | |||
Porcine mucin | Sigma | M1778-100G | UV-sterilize |
Salmon sperm DNA | Invitrogen | 15632-011 | |
Stain | |||
Propidium iodide | Alfa Aesar | J66764MC | |
Salts | |||
K2SO4 | Alfa Aesar | A13975 | |
KCl | Alfa Aesar | J64189 | Add solid directly to buffered base |
KNO3 | Acrs Organics | 7757-79-1 | |
MOPS | Alfa Aesar | A12914 | Add solid directly to buffered base |
NaCl | Fisher Chemical | S271-500 | Add solid directly to buffered base |
Na2HPO4 | RPI | S23100-500.0 | |
NaH2PO4 | RPI | S23120-500.0 | |
NH4Cl | Acrs Organics | 12125-02-9 | Add solid directly to buffered base |
Consumables | |||
Conical tubes (15 mL) | Olympus plastics | 28-101 | |
Conical tubes (50 mL) | Olympus plastics | 28-106 | |
Culture tubes w/air flow cap | Olympus plastics | 21-129 | |
35 mm four chamber glass-bottom dish | CellVis | NC0600518 | |
Luria-Bertani (LB) broth | Genessee Scientific | 11-118 | |
Phosphate-buffered saline (PBS) | Fisher Bioreagents | BP2944100 | |
Pipet tips (p200) | Olympus plastics | 23-150RL | |
Pipet tips (p1000) | Olympus plastics | 23-165RL | |
Serological pipets (5 mL) | Olympus plastics | 12-102 | |
Serological pipets (25 mL) | Olympus plastics | 12-106 | |
Serological pipets (50 mL) | Olympus plastics | 12-107 | |
Ultrapure water (RNAse/DNAse free); nanopure water | Genessee Scientific | 18-194 | Nanopure water used for preparation of solutions in Table 1 |
Syringes (10 mL) | BD | 794412 | |
Syringes (50 mL) | BD | 309653 | |
0.22 mm PES syringe filter | Olympus plastics | 25-244 | |
PS cuvette semi-mico | Olympus plastics | 91-408 | |
Software | |||
Biorender | To prepare the figures | ||
FacsDiva6.1.3 | Becton Dickinson, San Jose, CA | ||
Imaris | Bitplane | version 9.6 | |
Zen Black | |||
Equipment | |||
FacsAriallu | Becton Dickinson, San Jose, CA | ||
LSM 880 confocal laser-scanning microscope | Zeiss |