Bacterial Cell Staining and Preparation for Immunological Studies

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

1. Perform all bacterial work in a Biosafety Cabinet, Biosafety Level 2 laboratory.
2. 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.
3. The next day, use a 1:100 dilution of the overnight culture to inoculate a subculture and grow them in TSB (1 mL) for 3 h to the exponential phase, mea....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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