An In Vitro Technique to Study Antimicrobial Treatments on Bacterial Aggregates

Published: September 29, 2023

Abstract

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.

Protocol

1. Prepare synthetic cystic fibrosis medium (SCFM2) NOTE: Preparation of SCFM2 comprises three main stages outlined below (Figure 1). Sterilization of porcine mucin Prepare sterile mucin at a final concentration of 5 mg/mL in SCFM2. For example, for a 5 mL volume of SCFM2, weigh 25 mg of Type II mucin in a sterile Petri dish, and place it into an ultraviolet (UV) sterilizer for 4 h, gently agitating every hour. <…

Representative Results

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…

Declarações

The authors have nothing to disclose.

Materials

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

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An In Vitro Technique to Study Antimicrobial Treatments on Bacterial Aggregates. J. Vis. Exp. (Pending Publication), e21646, doi: (2023).

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