Measuring the Migration and Biofilm Formation of Various Bacteria

Summary

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

Abstract

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923 and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Introduction

The mammalian host represented by the human body contains a large number of microorganisms. These microorganisms are widely distributed in the mouth, digestive tract, intestine, and blood of the host and have different effects on the host's health. The oral cavity is host to a plethora of microbes that can modulate the host's susceptibility to infections. Microbes such as Streptococci (e.g., S. mitis/oralis, S. pseudopneumoniae, and S. infantis) and Prevotella spp. colonize the oral cavity, forming a multispecies biofilm on the tongue surface causing bad breath and functioning as a microbial reservoir for microbial infection. These pathogens can infect the jawbone by infiltrating the periodontal ligaments that hold the tooth root in the jawbone¹. The characterization of these microbes isolated from the host body is often a tedious process, particularly when the microbes exhibit individual traits requiring specific treatment and growth conditions. Microbes, such as the pathogenic Helicobacter pylori, Clostridium difficile, and Fusobacterium nucleatum, thrive in the gut's harsh environment, with specific oxygen, nutrient, and growth requirements, presenting a challenge in the characterization processes, particularly in investigating the pathogenicity of these microbes. Therefore, a standardized method of isolating and investigating these microorganisms is needed for scientists and medical practitioners to develop new medical treatments.

This protocol uses microbes that thrive in the bone matrix of rats. Traditionally, with the exception of the osseous system represented by the jaw, where the presence of teeth makes the bone matrix more susceptible to infection than other bones¹, it is generally believed that the host's healthy bone is a sterile environment. However, studies have found that microorganisms enter the systemic circulation through the intestinal wall, ultimately affecting bone mineralization². As a proof-of-concept, we use the described protocol to characterize the biochemical properties of microbial isolates from the femur and tibia of healthy SD rats (Corynebacterium stationis, Staphylococcus cohnii subsp., and Enterococcus faecalis). These microbial isolates were selected as the bone is a closed and hypoxic environment, and characterizing these microbes from bone can be a challenging task. Various articles have detailed the processes used in studying these microbes; however, there are few that provide a complete protocol to identify host-isolated microorganisms.

In establishing the proper culture conditions, the oxygen requirements of the microbe need to be understood via the use of Fluid Thioglycollate Medium (FTM). Microbes with different oxygen requirements form stratified layers in the clear FTM liquid³. Based on the stratification profile, the oxygen requirement of the microbe is then used to investigate the growth of the cells. Microbes that thrive on the surface of the FTM liquid are obligate aerobes, whereas microbes that grow at the bottom are obligate anaerobes. Microbes that grow as a suspension in the FTM liquid are either facultative or aerotolerant anaerobes. The microbial growth rate is established by observing the exponential growth phase of the microbial cells. The growth profile is then compared to the biofilm formation of the microbe. Biofilms are largely composed of multiple species that directly and indirectly affect each other's health. During this process, beneficial interactions among microbial communities select for attachment, providing a spatial structure that favors the evolution of active reciprocal interactions. For example, the co-culture of Paenibacillus amylolyticus and Xanthomonas retroflexus exhibit facultative symbiotic interactions in a static environment, promoting rapid biofilm growth¹³. Microbes adapt to the host tissues via biofilm formation for sustained localization, protecting themselves against harsh environments and evading the host's immune system^4,5,6,7. Biofilms are usually dense and multilayered structures that help microorganisms resist external subcritical stimuli; for example, E. faecalis in dental pulp enhances its resistance to antibiotics by increasing biofilm formation when faced with subconcentrations of tetracycline and vancomycin⁸.

Chemotaxis enables microorganisms to move according to chemical gradients, and signaling pathways are widely distributed in a variety of pathogenic bacteria. Some pathogenic microorganisms migrate to specific sites, under the guidance of chemical signals, to cause infectious diseases¹⁴. For example, Xanthomonas spp. express 19 chemoreceptor and 11 flagellin proteins in the host, triggering bacterial binding and, ultimately, ulceration¹⁵. There are also specific proteins in the bacteria (pectin-binding proteins) that guide the specific migration of bacteria to nutrients, which can lead to better growth¹⁶. This is also critical for bacteria that may exist in nutrient-poor environments. Microbial cells often rely on chemotactic motility to draw themselves to a conducive growth environment while evading other predatory cells and toxins that harm cellular viability. Building on previously established soft agar approaches to determine the chemotaxis, we develop a diffusible method to generate a chemoattractant gradient for testing the microbial chemotaxis.

This paper describes the methods for determining the growth conditions, biofilm formation, and chemical tropism of bacteria, using Corynebacterium stationis, Staphylacoccus cohnii, Enterococcus faecalis, Staphylacoccus aureus ATCC 25923, and Enterococcus faecalis V583 as examples (see Figure 1). The optimization of microbial growth conditions uses FTM to determine the oxygen requirements of the microbe, while biofilm formation uses glass surfaces as a solid backing, and the biofilm mass is counterstained with crystal violet. The microbe's chemical tropism relies on its chemotactic behavior, where through 3D printing (Supplemental Figure S1), a standardized method is used to generate a chemical reservoir for the chemoattractant in a soft agar matrix (Supplemental Figure S2).

Protocol

NOTE: See the Table of Materials for details about all the materials and equipment used in this protocol. Use aseptic techniques to avoid contamination.

1. Bacterial recovery to get a single colony

1. Prepare solid agar plates.
   1. Prepare Terrific Broth (TB) medium containing agar, with each liter of liquid containing 11.8 g of tryptone, 23.6 g of yeast extract, 9.4 g of K₂HPO₄, 2.2 g of KH₂PO₄, and 15 g of agar, pH = 7.
   2. Autoclave the solution at 121 °C for 20 min using an air-permeable cap or bottle-sealing film with an air vent.
   3. Before the liquid cools and solidifies, pour the TB broth containing agar into 10 cm plastic Petri dishes at a volume of 25 mL per plate.
      NOTE: The broth should be poured into the plate before it solidifies at temperatures lower than 60 °C. Use aseptic techniques to prevent contamination.
2. Bacterial inoculation to the plate
   1. Take out the target strains stored at −80 °C and thaw them at room temperature in a biological safety cabinet.
   2. Use a 10 µL pipette to take up 10 µL of bacterial suspension and spread it on the TB plate by streaking.
   3. Seal the plate to prevent contamination. Place the plate in a biochemical incubator at 37 °C. Check colony growth visually after ~24 h. Pick single colonies and amplify them by PCR to confirm that they contain only one type of bacteria.

2. Bacterial liquid culture to logarithmic growth phase

1. Prepare liquid culture medium.
   1. Refer to the preparation of solid agar plates in Step 1.1.1. to prepare TB liquid medium, 1 L of which contains 11.8 g of tryptone, 23.6 g of yeast extract, 9.4 g of K₂HPO₄, and 2.2 g of KH₂PO₄, pH ~7.
      NOTE: Do not add agar. Bacteria grow faster in a liquid nutrient environment.
   2. Autoclave the solution at 121 °C for 20 min using an air-permeable cap or bottle-sealing film with an air vent.
   3. Store the liquid culture medium at 4 °C.
2. Bacterial inoculation and cultivation
   1. In the biological safety cabinet, pick a single colony with an inoculating loop or a 10 µL pipette and inoculate the bacteria in the prepared TB liquid medium in an Erlenmeyer flask.
   2. Seal the Erlenmeyer flask with gauze and place it in a shaker (200 rpm) at 37 °C to cultivate the bacteria.
      ​NOTE: The liquid medium becomes gradually turbid over 5 h; the time taken to become turbid varies with different bacteria.

3. FTM experiment and growth curve determination

1. Fluid Thioglycollate Medium (FTM) preparation
   1. Prepare Thiolglycollate Medium (TM), each liter of which contains 15 g of tryptone, 5 g of yeast extract, 5 g of glucose, 0.5 g of L-cysteine, 0.5 g of sodium thioglycolate, 2.5 g of sodium chloride, 0.001 g of resazurin, and 0.75 g of agar.
      NOTE: Commercially available solid medium can easily be reconstituted with water and sterilized (Step 3.1.2.).
   2. Weigh 29.3 g of solid Thioglycollate Medium, add 1 L of distilled water, heat and stir until completely dissolved and until the solution turns pink.
   3. Add TM (19 mL) to a test tube with a sealing plug. After sealing, autoclave the solution at 121 °C for 20 min using an air-permeable cap or bottle-sealing film with an air vent.
      NOTE: After heating, the solution becomes light yellow.
2. Inoculation, cultivation, and observation of bacteria
   1. Prepare the bacterial suspension to grow to OD 0.6-0.8 in liquid TB medium (see Step 2.2.).
   2. Open the test tube sealing plug under aseptic conditions in the biological safety cabinet, add 1 mL of the bacterial culture to the test tube containing FTM as soon as possible, and immediately seal the test tube again.
   3. Shake the tube gently to make sure the bacteria are homogeneously distributed in the liquid and place the test tube at 37 °C for static culture.
   4. After 48 h of incubation, observe the growth of different bacteria in FTM in various test tubes and take photographs.
3. Determination of growth curve under aerobic conditions
   1. Prepare the initial bacterial culture with a suitable OD value (see Step 2.2.), and culture the bacteria until they reach the logarithmic growth phase.
      NOTE: Corynebacterium stationis, Staphylacoccus cohnii, Enterococcus faecalis, Staphylacoccus aureus ATCC 25923, and Enterococcus faecalis V583 used in this protocol took ~5 h to reach the log phase.
   2. Add 200 µL of the bacterial culture (from Step 3.3.1) in TB liquid medium to each well of a 96-well plate in triplicate. Use a microplate reader to determine the OD value at 600 nm, and dilute the different bacterial cultures so that the OD difference between the different bacteria-containing wells and TB liquid medium (blank) is between 0.01 and 0.02.
   3. Take a new 96-well plate, add TB medium, transfer the different diluted bacterial cultures from Step 3.3.2., in triplicate, to the plate, and place it on an ultra-clean workbench.
   4. Seal the 96-well plate with sealing tape, put it in the microplate reader, and set the program as follows:
      1. Set the temperature to 37 °C ± 0.5.
      2. Set the Kinetic Loop to a total of 60 cycles, each for 30 min.
      3. Set the absorbance to 600 nm.
      4. Set Shaking to 1,600 s in a cycle.
      5. Run the program and stop it when the growth curve plateaus.
4. Determination of growth curve under anaerobic conditions
   1. Follow Step 3.3. but keep the 96-well plate with the TB medium and bacterial suspensions in an anaerobic incubator before sealing the plate with sealing tape. Pump and fill the anaerobic box with nitrogen to reduce the oxygen concentration to <3%. Seal the 96-well plate with sealing tape and leave it in the anaerobic box.

4. Biofilm-formation ability test

1. Culture the bacteria to logarithmic growth phase in liquid medium.
   1. Refer to Section 2 to inoculate the bacteria in the TB liquid medium.
   2. Cultivate the bacteria overnight in a shaker at 37 °C, ensuring that the OD₆₀₀ of the bacterial culture medium is between 0.6 and 0.8.
2. Divide the bacterial culture and cultivate under different conditions.
   1. When the OD₆₀₀ of the bacterial culture is between 0.6 and 0.8, add 10 mL of this bacterial suspension to a glass test tube.
   2. Divide each bacterial culture into two groups: aerobic and anaerobic conditions, and perform all operations in triplicate.
      1. For the aerobic group, wrap the test tube containing the bacterial suspension with tin foil and incubate it in a 37 °C water bath at a constant temperature.
      2. For the anaerobic group, wrap the tube containing the bacterial suspension with tin foil and transfer it to an anaerobic incubator. Remove the tin foil in the anaerobic incubator and reduce the oxygen concentration in the incubator to <3% by pumping and filling it with nitrogen. Close the test tube with a frosted glass stopper, take it out after sealing, and incubate it in a water bath at 37 °C.
   3. Every 6 h, take three test tubes from each of the two groups (aerobic/anaerobic) to observe the growth of the biofilm at the bottom, and stain following Step 4.3.
3. Discard the bacterial suspension and dry the biofilm.
   1. Take out the test tubes at different time points and carefully aspirate the upper layer of the bacterial suspension. Observe the white biofilm adhering to the bottom of the test tube.
      NOTE: Take care not to disturb the biofilm when aspirating the bacterial suspension.
   2. Carefully add 2 mL of phosphate-buffered saline (PBS) along the wall of the test tube, shake it gently to wash away the planktonic bacteria adsorbed in the biofilm, and carefully aspirate the PBS buffer.
   3. Put the test tube with the biofilm adsorbed on the bottom into the oven to dry for 30 min.
4. Staining
   1. Take out the test tubes, add 2 mL of 0.1% crystal violet staining solution to each test tube, and stain the biofilms for 30 min at room temperature.
   2. Use a pipette to carefully aspirate the crystal violet dye solution. Carefully add distilled water along the wall of the test tube to wash away the remaining crystal violet dye solution, repeating 3x-5x until the added distilled water became almost colorless. Observe the purple biofilm at the bottom of the test tube.
   3. Dry the test tube again.
5. Dissolve the biofilm with 95% ethanol and measure the OD₆₀₀ of the biofilm solution.
   1. Take out the dried test tube, add 10 mL of 95% ethanol to each test tube, and shake thoroughly to make sure that the crystal violet was fully dissolved in the ethanol.
   2. With a volume of 200 µL per well, add crystal violet in ethanol to a 96-well plate, and measure its absorbance at a wavelength of 600 nm.
      ​NOTE: According to Beer-Lambert's law, the absorbance is linearly related to the concentration of the solution, so the value of OD₆₀₀ can reflect the concentration of crystal violet, which reflects the amount of biofilm in the bacterial suspension at different times.

5. Bacterial chemotaxis test

1. Prepare differential eutrophic agar plates.
   1. Refer to Step 1 to prepare a half-dose MH medium containing 0.5% agar, each liter of which contains 11.8 g of tryptone, 23.6 g of yeast extract, 9.4 g of K₂HPO₄, 2.2 g of KH₂PO₄, and 5 g of agar, pH ~7.
   2. Refer to Step 1 to prepare 5x TB medium containing 0.5% agar, each liter of which contains 59 g of tryptone, 118 g of yeast extract, 47 g of K₂HPO₄, 11 g of KH₂PO₄, and 5 g of agar, pH ~7.
   3. Autoclave the solution at 121 °C for 20 min using an air-permeable cap or bottle-sealing film with an air vent.
   4. Before the liquid is cooled and solidified, pour 25 mL of half-dose MH broth (containing 0.5% agar) per plate, cover the plate with a 3D printed lid, and place it in a biological safety cabinet. After cooling, remove the cover to observe the cylindrical well (1.4 cm diameter) in the semisolid medium.
      ​NOTE: The 3D printed lid with resin material cannot be sterilized using high-temperature steam. To kill the surface bacteria, it can be sprayed with 95% ethanol and then irradiated with a UV lamp for 30 min in a biological safety cabinet. When removing the lid, apply force in the vertical direction and lift it carefully to prevent damage to the semisolid agar.
   5. Before the 5x TB broth (containing 0.5% Agar) solidifies, use a pipette to add the 5x TB broth to the well in the semisolid plate until the broth is flush with the surface of the medium.
   6. Cool the plate in a biological safety cabinet to obtain a semisolid agar plate containing eutrophic bodies. Take care to avoid plate contamination by environmental microorganisms.
      NOTE: The semisolid medium is still somewhat fluid and, hence, must be fully cooled before being stored upside down.
2. Bacterial inoculation, cultivation, and photography
   1. Place the agar plate with the semisolid culture medium in a biological safety cabinet and cool it for ~30 min under UV irradiation to prevent bacterial contamination.
   2. Use a 10 µL pipette to take up 4 µL of the bacterial suspension cultured to an OD of ~0.6-0.8, insert the pipette tip into the center of the plastic Petri dishes, and pipet out the bacterial suspension.
   3. Wait for the bacterial suspension to be absorbed into the medium for 1 min. When the bacterial suspension has been fully absorbed, divide the plate into two groups (aerobic/anaerobic) in triplicate.
   4. Seal the aerobic group with sealing tape and place those plates upside down in a 37 °C biochemical incubator for static culture. Seal the anaerobic group in an anaerobic box as described in Step 4.2, inverting the plates at 37 °C for growth.
   5. Check the spread and migration of bacteria every 12 h (at 0 h, 12 h, 24 h, 36 h, and 48 h), and use the imaging system to take images of the plate.
3. Use ImageJ software to calculate bacterial migration distances.
   1. Import the image file acquired using the imaging system.
   2. Set the scale bar and apply it to all the images.
      1. When taking images, add a ruler, create a line segment, select a straight-line segment (e.g., 1 cm, and then set the length of this line segment to 1 cm), and mark the length of the board.
      2. Open the Set Scale window; click on Analyze | Set Scale.
      3. Type the actual length in Known distance and Unit of length.
      4. Check Global.
      5. Enter the folder address of the original image and the output file address.
   3. Use the straight line tool to select the communities one by one and adjust the tolerance until the straight tool line fits the boundary of the bacterial colony extending to both sides.
   4. Measure the migration distance of the colony from the center to both ends separately and derive the straight-line length by clicking on Analyze | Measure.
   5. Repeat Steps 5.3.3-5.3.4 until all the communities are measured and save the results to a .csv file for further analysis.

Results

This work describes the approaches taken to characterize the isolated microbes from the host microbiome (Figure 1). As a proof-of-concept, three microbes were isolated from the SD rat host (C. stationis, S. cohnii, and E. faecalis), and two commercially acquired microorganisms (S. aureus ATCC 25923 and E. faecalis V583) were tested using this protocol. To establish the oxygen requirements of individual microbes using FTM, we added two control orga...

Discussion

We isolated and identified five species of bacteria by sequential methods. The growth of bacteria has minimal nutrient requirements: the minimal medium-a medium containing only inorganic salts, a carbon source, and water. Although the bacteria in the experimental group were found on MH solid plates, we used half-dose MH medium to verify the chemotaxis of the bacteria and achieved good results. However, we also performed control experiments using minimal medium. M9 basic medium was used in the experiment (see Tabl...

Materials

Name	Company	Catalog Number	Comments
Chemical/Solution
1% crystal violet dye solution	Solarbio	G1062	100 mL
Agar	Sigma-Aldrich	V900500	Used to obtain semi-solid plates, 20 g
Centrifuge tube	Corning	430790	15 mL
Fluid thioglycollate medium	Kinghunt	K0001	29.3 g
Mueller Hinton II Broth medium	Solarbio	NO.11865	100 g
Petri dishes	Bkman	B-SLPYM90-15	Plastic Petri dishes, circular, 90 mm x 15 mm
Potassium Chloride	VETEC	WXBC4493V	0.2 g
Potassium Dihydrogen Phosphate	aladdin	04-11-7758	0.24 g
Sodium chloride	Macklin	S805275	8.0 g
Sodium phosphate dibasic	aladdin	7558-79-4	1.44 g
Terrific Broth medium	Solarbio	LA2520	200 g
Kits/ Equipment
Anaerobic incubator	Longyue
Biochemical incubator	Blue pard	LRH-70
Microplate reader	Spark
Tanon 5200multi imaging system	Tanon	5200CE
Thermostatic water bath	Jinghong	DK-S28