Visualization of Bacterial Motility Using Redox Dye–Supplemented Semisolid Medium

Abstract

Source: Chu, W., and Zhuang, X. Visualizing Bacterial Motility Based on a Color Reaction. J. Vis. Exp. (2022)

This video demonstrates a redox dye-based semisolid agar assay for assessing bacterial motility by visually distinguishing motile and non-motile strains.

Protocol

Bacterial strains

NOTE: Eighty strains were isolated from the aquatic environment and identified using an automated bacteria identification instrument (see the Table of Materials), including Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Vibrio spp., Klebsiella pneumoniae, and Aeromonas hydrophila (Table 1). Staphylococcus aureus (see the Table of Materials) was used as a negative nonmotile control; Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium (see the Table of Materials) were used as positive control strains.

1. Identify test bacterial strains to be used for motility analysis.
2. Include negative nonmotile controls and motile positive control strains.

TTC-enhanced bacterial motility observation

1. Pick single colonies of test bacteria from agar plates and inoculate them into the semisolid media with TTC (2,3,5-triphenyltetrazolium chloride ) by puncturing them with inoculating needles.
2. Culture the tubes at 37 °C in the incubator for 24-48 h to observe the results.
3. Observe the growth state: characterize the bacteria as nonmotile (-) if only the puncture line is red. Characterize the bacteria as motile (+) if the red color spreads lightly outward along the puncture line.

Table 1: Comparison of bacterial motility.

Strain		Semisolid medium with 0.4% agar		Semisolid medium with 0.4% agar and 0.005% TTC
		24 h	48 h	24 h	48 h
P. aeruginosa ATCC27853		+	+	+	+
E. coli ATCC25922		+	+	+	+
S. typhimurium ATCC14028		+	+	+	+
S. aureus ATCC25923		-	-	-	-
E. coli (15)		12	14	13	14
Salmonella spp. (8)		7	8	8	8
A. hydrophila (20)		18	20	20	20
Vibrio spp. (8)		7	8	8	8
P. aeruginosa (24)		18	20	22	23
K. pneumoniae (5)		-5	-5	-5	-5
Numbers indicate the numbers of positive strains (+) and negative stains (-).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacto Agar	Difco
Escherichia coli	ATCC	ATCC25922	Positive control
Pseudomonas aeruginosa	ATCC	ATCC27853	Positive control
Staphylococcus aureus	ATCC	ATCC25923	Negative nonmotile control
Tryptose	OXOID
TTC (2,3,5-Triphenyl-tetrazolium chloride)	Sigma	298-96-4
VITEK 2 automated microbial identification system	Bio Mérieux