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