Visualizing Bacterial Motility Based on a Color Reaction

Summary

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

Abstract

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Introduction

Bacterial motility plays a critical role in bacterial pathogenicity, biofilm formation, and drug resistance¹. Bacterial motility is closely associated with pathogenicity and is necessary for bacterial colonization during early infection of host cells². Biofilm formation is closely related to bacterial motility, where bacteria adhere to the surface of solid media through motility. Bacterial motility has long been considered to be positively correlated with biofilm formation. A high degree of bacterial drug resistance due to biofilm can lead to persistent infections that are a threat to human health^3,4,5. Therefore, it is important to detect bacterial motility. The bacterial motility test is mainly used to examine the motility of different forms of bacteria in the living state, which can indirectly determine the presence or absence of flagella and, thus, has an important role in the identification of bacteria.

There are direct and indirect methods to detect bacterial motility⁶. As bacteria with flagella show motility, it is possible to detect whether bacteria are motile indirectly by detecting the presence or absence of flagella. For example, it is possible to detect motility indirectly by electron microscopy and flagellar staining to indicate that bacteria are motile. It is also possible to detect by direct methods, such as suspension drop and semisolid puncture methods.

The semisolid puncture method commonly used in undergraduate microbiology laboratories to detect bacterial motility inoculates the bacteria into the puncture in the semisolid agar medium containing 0.4-0.8% agar, according to the direction of bacterial growth. If the bacteria grow along the puncture line to spread around, cloud-like (brush-like) growth traces appear, indicating the presence of flagella and, therefore, motility. If there are no puncture-line growth traces, the bacterium is neither flagellated nor motile.

However, this method has its drawbacks: the bacteria are colorless and transparent, the flagellar activity is affected by the physiological characteristics of the living bacteria and other factors, and the concentration of agar and the small diameter of the test tube. Moreover, aerobic bacteria are only suitable for growth on the agar surface, affecting the observation of bacterial motility. Hence, to improve this experiment, 2,3,5-triphenyltetrazolium chloride (TTC) (colorless) was added to the medium to establish a more reliable and intuitive method of determining bacterial motility than the current direct-puncture method using intracellular dehydrogenases to catalyze the formation of a red product of TTC^7,8,9,10.

Protocol

1. Preparation of semisolid medium

1. Traditional semisolid agar
   1. Prepare the traditional semisolid agar according to the bacterial motility test medium recipe using the basic ingredients¹¹. Dissolve 10 g of Tryptose, 15 g of NaCl, 4 g of agar in enough distilled water, adjust the pH to 7.2 ± 0.2, and make up the final volume to 1,000 mL.
   2. Autoclave the agar at 121 °C for 20 min, and dispense it into 10 mL test tubes as a 3-cm high semisolid medium.
2. Traditional semisolid agar with TTC
   1. After autoclaving the conventional semisolid medium, cool it to 50 °C, add 5 mL of sterile 1% TTC solution to 100 mL of medium, mix, and dispense it into 10 mL test tubes to form a 3-cm-high semisolid medium.

2. 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.

3. TTC-enhanced bacterial motility observation

1. Pick single colonies of test bacteria from agar plates and inoculate them into the above two semisolid media (steps 1.1.2 and 1.2.1) by puncture using 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 line¹².

4. Effect of different agar concentrations on bacterial motility

1. Prepare semisolid media containing 0.3%, 0.5%, and 0.8% agar and inoculate them by puncture, as described above. Observe the results after 24-48 h of incubation.

Results

Both standard strains and isolated strains were compared for motility detection, and the results are shown in Table 1. Due to the absence of flagella, Staphylococcus aureus and Klebsiella pneumoniae only grew along the inoculated line on both traditional and TTC semisolid media. In contrast, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium showed growth in all directions around the inoculated line after culturing for 24 h on TTC semisolid m...

Discussion

The detection of bacterial motility by the semisolid medium method is affected by many factors^13,14. Bacterial growth conditions, such as oxygen (aerobic on agar surface, nonaerobic at the bottom of the tube with the semisolid medium), pH, and temperature, can affect the viability of bacterial flagella, which can lead to reduced motility or even loss of motility¹⁵. In addition, some mucus-type bacteria as their motility can be affected by ...

Materials

Name	Company	Catalog Number	Comments
Bacto Agar	Difco
Escherichia coli	ATCC	ATCC25922	Positive control
Pseudomonas aeruginosa	ATCC	ATCC27853	Positive control
Salmonella typhimurium	ATCC	ATCC14028	Positive control
Staphylococcus aureus	ATCC	ATCC25923	Negative nonmotile control
Tryptose	OXOID
TTC	Sigma	298-96-4
VITEK 2 automated microbial identification system	Bio Mérieux