Bacterial Cell Staining and Preparation for Immunological Studies

Published: November 30, 2023

Abstract

Source: Yang, J. et al., Automated, High-Throughput Detection of Bacterial Adherence to Host Cells. J. Vis. Exp. (2021)

This video demonstrates the staining and preparation of bacterial cells for immunological studies using red fluorescent dye. The stained bacteria are diluted and resuspended in a growth medium to achieve the desired bacterial concentration for host cell infection, representing the multiplicity of infection.

Protocol

1. Bacterial growth and staining Perform all bacterial work in a Biosafety Cabinet, Biosafety Level 2 laboratory. Inoculate all bacterial cultures, including Pseudomonas aeruginosa (PAO1), Escherichia coli, Listeria monocytogenes,  Bacillus subtilis, etc., from the frozen glycerol stock and grow them in Tryptic Soy Broth (TSB, 3 mL) in a shaking incubator at 37 °C overnight maintained at 250 rpm. T…

Divulgaciones

The authors have nothing to disclose.

Materials

10x PBS VWR 45001-130
BactoView Live Red Biotium 40101 Bacteria staning dye
Centrifuge Eppendorf 5810R
E. coli Laboratory stock 
F-12k medium ATCC  302004 A549 cell culture medium
Fetal bovine serum Corning 35-016-CV
L. monocytogenes NIST collections
OD600 DiluPhotometer IMPLEN
P. aeruginosa Dr. Lori Burrows laboratory stock
P. aeruginosa ΔpilA Dr. Lori Burrows laboratory stock
S. agalactiae NIST collections
S. aureus BEI NR-46543
S. aureus ΔsaeR BEI NR-48164
S. rubidaea NIST collections
Typical soy broth Growcells MBPE-4040

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Citar este artículo
Bacterial Cell Staining and Preparation for Immunological Studies. J. Vis. Exp. (Pending Publication), e21805, doi: (2023).

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