An In Vitro Technique to Study Antimicrobial Treatments on Bacterial Aggregates

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).

1. Sterilization of porcine mucin
   1. 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.
   2. After 4 h, transfer the UV-treated mucin into autoclaved 1.7 mL tubes under sterile conditions, and store at -20 °C.
   3. To confirm com....

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) .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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