Contact Mode Atomic Force Microscopy as a Rapid Technique for Morphological Observation and Bacterial Cell Damage Analysis

Summary

Here, we present the application of atomic force microscopy (AFM) as a simple and fast method for bacterial characterization and analyze details such as the bacterial size and shape, bacterial culture biofilms, and the activity of nanoparticles as bactericides.

Abstract

Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the sample preparation for observation. Atomic force microscopy (AFM) is a very useful characterization technique due to its high resolution in three dimensions and because of the absence of any requirement for vacuum and sample conductivity. AFM can image a wide variety of samples with different topographies and different types of materials.

AFM provides high-resolution 3D topography information from the angstrom level to the micron scale. Unlike traditional microscopy, AFM uses a probe to generate an image of the surface topography of a sample. In this protocol, the use of this type of microscopy is suggested for the morphological and cell damage characterization of bacteria fixed on a support. Strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas hunanensis (isolated from garlic bulb samples) were used. In this work, bacterial cells were grown in specific culture media. To observe cell damage, Staphylococcus aureus and Escherichia coli were incubated with different concentrations of nanoparticles (NPs).

A drop of bacterial suspension was fixed on a glass support, and images were taken with AFM at different scales. The images obtained showed the morphological characteristics of the bacteria. Further, employing AFM, it was possible to observe the damage to the cellular structure caused by the effect of NPs. Based on the images obtained, contact AFM can be used to characterize the morphology of bacterial cells fixed on a support. AFM is also a suitable tool for the investigation of the effects of NPs on bacteria. Compared to electron microscopy, AFM is an inexpensive and easy-to-use technique.

Introduction

Different bacterial shapes were first noted by Antony van Leeuwenhoek in the 17th century¹. Bacteria have existed in a great diversity of shapes since ancient times, ranging from spheres to branching cells². Cell shape is a fundamental condition for bacterial taxonomists to describe and classify each bacterial species, mainly for the morphological separation of gram-positive and gram-negative phyla³. Several elements are known to determine bacterial cell forms, all of which are involved in the cell covers and support as components of the cell wall and membrane, as well as in the cytoskeleton. In this way, scientists are still elucidating the chemical, biochemical, and physical mechanisms and processes implicated in determining bacterial cell forms, all of which are defined by clusters of genes that define bacterial shapes^2,4.

Additionally, scientists have shown that the rod shape is likely the ancestral form of bacterial cells, since this cell shape appears optimal in cell-significant parameters. Thus, cocci, spiral, vibrio, filamentous, and other forms are regarded as adaptations to various environments; indeed, particular morphologies have evolved independently multiple times, suggesting that the shapes of bacteria could be adaptations to particular environments^3,5. However, throughout the bacterial cell life cycle, the cell shape changes, and this also occurs as a genetic response to damaging environmental conditions³. The bacterial cell shape and size strongly determine the stiffness, robustness, and surface-to-volume ratio of the bacteria, and this characteristic can be exploited for biotechnological processes⁶.

Electronic microscopy is used to study biological samples due to the high magnification that can be reached beyond light-based microscopes. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are the most commonly used techniques for this purpose; however, samples require some treatments before they are placed into the chamber of the microscope in order to obtain appropriate images. A gold cover on the samples is required, and the time used for total image acquisition should not be too long. In contrast, atomic force microscopy (AFM) is a technique widely used in the analysis of surfaces but is also employed in the study of biological samples.

There are several types of AFM modes used in surface analysis, such as contact mode, non-contact mode or tapping, magnetic force microscopy (MFM), conductive AFM, piezoelectric force microscopy (PFM), peak force tapping (PFT), contact resonance, and force volume. Each mode is used in the analysis of materials and provides different information about the surface of the materials and their mechanical and physical properties. However, some AFM modes are used for the analysis of biological samples in vitro, such as PFT, because PFT allows for obtaining topographical and mechanical data on cells in a liquid medium⁷.

In this work, we used the most basic mode included in every old and simple AFM model-the contact mode. AFM uses a sharp probe (around <50 nm in diameter) to scan areas less than 100 µm. The probe is aligned to the sample in order to interact with the force fields associated with the sample. The surface is scanned with the probe to keep the force constant. Then, an image of the surface is generated by monitoring the motion of the cantilever as it moves across the surface. The gathered information provides the nano-mechanical properties of the surface, such as the adhesion, elasticity, viscosity, and shear.

In the AFM contact mode, the cantilever is scanned across the sample at a fixed deflection. This allows one to determine the height of the samples (Z), and this represents an advantage over the other electronic microscope techniques. The AFM software allows the generation of a 3D image scan by the interaction between the tip and sample surface, and the tip deflection is correlated to the height of the sample through a laser and a detector.

In static mode (contact mode) with constant force, the output presents two different images: the height (z topography) and the deflection or error signal. Static mode is a valuable, simple imaging mode, especially for robust samples in air that can handle the high loads and torsional forces exerted by static mode. The deflection or error mode is operated in constant force mode. However, the topography image is further enhanced by adding the deflection signal to the surface structure. In this mode, the deflection signal is also referred to as the error signal as the deflection is the feedback parameter; any features or morphology that appear in this channel are due to the "error" in the feedback loop or, rather, due to the feedback loop required to maintain a constant deflection setpoint.

AFM's unique design makes it compact - small enough to fit on a tabletop - while also having high enough resolution to resolve atomic steps. The AFM equipment has a lower cost than the equipment for other electronic microscopes, and the maintenance costs are minimal. The microscope does not require a lab with special conditions such as a clean room or an isolated space; it only needs a vibration-free desk. For AFM, the samples do not need to undergo elaborate preparation like for other techniques (gold cover, slimming); only a dry sample has to be attached to the sample holder.

We use AFM contact mode to observe bacterial morphologies and the effects of NPs. The population and cellular morphology of bacteria fixed on a support can be observed, as well as the cellular damage produced by nanoparticles on the bacterial species. The images obtained by AFM contact mode confirm that it is a powerful tool and is not limited by reagents and complicated procedures, making it a simple, fast, and economical method for bacterial characterization.

Protocol

1. Bacterial isolation and identification

1. Isolation of an endophytic strain from garlic bulb meristems:
   1. Place 2 mm fragments of meristems from previously peeled, disinfected, and cut garlic bulbs on trypticase soy agar (TSA) as a rich growth medium, and incubate at 25 °C for 1 day.
   2. Based on the morphological differences of the bacterial colonies-shape, size, color, edge, transmitted light, reflected light, texture, consistency, and pigment production-describe the different observed morphotypes, and purify by cross-streaking a representative colony of each morphotype.
   3. Preserve all the purified bacterial strains in 30% glycerol at −80 °C.
   4. Identify a randomly selected strain (9AP) by sequencing the 16S rDNA. Extract the DNA following the method of Hoffman and Winston⁸.
   5. Amplify the 16S rRNA by polymerase chain reaction (PCR) with 100 ng of DNA, 1x polymerase buffer, 4 mM MgCl₂, 0.4 mM dNTPs, 10 pmol each of 27F/1492r universal oligonucleotides, and 1 U of DNA polymerase⁹. Use the following thermal cycler conditions: 95 °C for 5 min for the initial denaturalization; followed by 35 cycles of 1 min at 94 °C as the denaturalization temperature, 1 min at 56 °C as the hybridization temperature, and 1 min at 72 °C as the extension temperature; and then 10 min at 72 °C as a final extension.
   6. Capillary-sequence the PCR product using the same oligonucleotides; use the dideoxy-terminal method with the matrix installation kit for capillary sequencing¹⁰, and perform electrophoresis in an automated multicapillary system¹¹.
   7. Manually edit the sequences, compare the sequence with the GenBank library using a BLAST search, and tentatively identify the strain with the gold standard of at least 98.7% identity with the closest species^12,13.
      NOTE: The strain 9AP was identified as belonging to the species of Pseudomonas hunanensis, with an identity of 99.86% with the sequence for the strain P. hunanensis LV (JX545210).

2. Bacterial sample preparation for morphological observation by AFM

1. Under sterile conditions, place a drop of the bacterial suspension (Pseudomonas hunanensis, Staphylococcus aureus) on a glass slide.
2. Fix the samples on the slide by dry heating. Pass the slide gently over a flame several times until the sample is dry.
   NOTE: The fixation of samples is a routine technique in microbiology to observe fixed prokaryotic organisms and to simulate their structure as living cells as closely as possible.
3. Place the fixed samples in a Petri dish, and take them to the AFM equipment for observation.
   ​NOTE: As the samples can be altered by weather conditions over time, set aside a maximum time of 24 h for analysis in the AFM equipment. Keep the samples free of dust.

3. Antibacterial effect of MgO nanoparticles against bacteria

NOTE: The synthesis and characterization of MgO NPs have been published previously¹⁴. In this work, the antibacterial activity of the nanomaterials was estimated based on the Clinical and Laboratory Standards Institute (CLSI) manual using macrodilution and microdilution methods for inhibition^15,16.

1. Estimation of the minimum inhibitory activity (MIC) and bactericides (CMB)
   1. Pregrowth of the strains
      1. Inoculate an appropriate amount of Escherichia coli or Staphylococcus aureus into nutritious broth to obtain a final concentration of 1 × 10⁸ CFU/mL.
      2. Incubate the suspension at 37 °C (± 2 °C) for 18-24 h.
   2. Postgrowth of acclimatized strains
      1. Immerse a sterile bacteriological loop in the suspension, touching the bottom of the tube.
      2. Perform streaking with the loop on eosin and methylene blue agar and nutrient agar.
      3. Incubate the agar plates at 37 °C (± 2 °C) for 24 h.
   3. Selection of colonies for the preparation of the standardized inoculum
      1. Take three to five colonies with the same morphological type by touching the top of the colony in the Petri dish with a bacteriological loop.
      2. Transfer and immerse the selection in 3-5 mL of Müller-Hinton broth.
      3. Incubate at 37 °C (± 2 °C) to achieve a turbidity of 1 × 10⁸ CFU/mL or 2 × 10⁸ CFU/mL.
         NOTE: In this work, McFarland turbidity standards were used as a reference for the bacteriological suspensions; specifically, the 0.5 standard corresponds approximately to a homogeneous E. coli suspension of 1.5 × 10⁸ cells/mL. A UV-Vis spectrophotometer was used to measure the turbidity.
      4. Take 1 mL, and add it to 9 mL of sterile broth. Take 200 µL of this dilution, and add it to 19.8 mL of sterile broth to obtain a final concentration of 5 × 10⁵ CFU/mL.
   4. Required concentrations of MgO nanoparticles
      1. Use an ultrasonicator for the preparation of the solutions.
      2. Suspend the nanomaterial in sterile water at twice the concentration required to obtain serial solutions.
      3. Add these suspensions to sterile tubes containing Müller-Hinton broth to achieve the new range of concentrations required for the experiment.
         NOTE: The following MgO NPs concentrations were applied for microdilution: 30 ppm, 60 ppm, 120 ppm, 250 ppm, 500 ppm, 1,000 ppm, 2,000 ppm, 3,000 ppm, 4,000 ppm, and 8,000 ppm.
   5. Preparation of the microdilution¹⁵
      1. Prepare a new and sterile 96-well microtiter plate (microplate). Label column No. 12 of the microplate as a sterility column; label column No. 11 as a growth control column.
      2. Add 120 µL of the nanoparticle suspensions with the different concentrations to columns No. 1-10.
      3. Inoculate 120 µL of bacteria (5 × 10⁵ CFU/mL) into the wells in columns No.1-10.
         NOTE: For this study, row G and row H were controls with ceftriaxone at concentrations suggested by CLSI standards: 8 ppm, 4 ppm, 2 ppm, 1 ppm, 0.5 ppm, 0.25 ppm, 0.125 ppm, 0.06 ppm, and 0.03 ppm.
      4. Incubate the 96-well microplate for 24 h at 37 °C (35 °C ± 2 °C). Finally, analyze the wells.
2. Observation of the MgO nanoparticle-induced morpho-structural changes in the bacterial strains by AFM
   1. Take 250 µL from the wells of the microdilution tests, mix with 750 µL of sterile water, and centrifuge at 2,000 × g from 2 min to 5 min at 5 °C.
   2. Wash the precipitates three times with 1 mL of sterile water each time, and at the end of the washes, add 500 µL of water to them.
   3. To obtain the AFM images, take 10-20 µL of the final suspension of each starting well, perform a smear on a slide previously cleaned by ultrasound (30 min), and dry at room temperature for 1 h.

4. AFM measurements

NOTE: Here, the atomic force microscope in contact mode was mounted on an anti-vibration workstation that allowed the isolation of the microscope from any mechanical vibrational sources and kept the system leveled. Electrical interference is diminished with line filters and surge protection. The AFM used here auto-aligns the laser beam to the photodetector.

1. Turn on the computer and the AFM, then select the Contact mode, the contAI-G probes, and the Auto Slope options in the software tools. The default values of the Z-controller are Setpoint = 20 nM, P-Gain = 10,000, I-Gain = 1,000, D-Gain = 0, and Tip voltage = 0 mV.
2. Place the samples in the AFM contact mode using a 70 µm scan range head. We used ContAI-G silicon cantilevers with a spring constant of 0.13 N/m.
3. Use a camera device mounted on two lenses integrated into the scan head to obtain a quick view of the surface of the area under the probe.
4. Manually perform a displacement in the XY plane in order to choose the desired area. The probe is made to electronically approach the surface sample until the contact conditions are reached. Electronically adjust the XY measurement plane of the scanner and sample surface by reducing the slopes in the XY directions.
5. Use the standard parameters of the software: 1 line per second at 256 points per line.
6. First, perform a full scan range of a 70 µm x 70 µm area; then, select a smaller area using the zoom tool. The AFM optimizes the chart range in Z automatically.
   NOTE: By decreasing the lines per second time, the total scan time increases, the quality of the scan is more defined, and some noise from the measurement is also cleared.
7. In order to select a new area from the sample, retract the cantilever electronically, and move the sample along the XY plane. Realize a new scan using the procedure described in step 4.5 and step 4.6.
   NOTE: The scans obtained are shown in a single-color bar scale, in which the light colors are associated with higher altitudes, and the dark colors are associated with deeper altitudes. The AFM software generates a 3D view of the scan that allows the appreciation of the details of the measured surface.
8. Perform the data processing using the line by line leveling in the Tools menu of the image in the filter selection, which is the most used and simplest leveling method.
   NOTE: This leveling method takes every horizontal or vertical line produced in the AFM image and fits it to a polynomial equation.
9. Optimize the maximum and minimum height in the color bar on the right in order to obtain the best image resolution.
10. Perform the image analysis by using the Analysis menu in the tools bar. Determine the heights and distances by selecting the initial and final points once the desired tool is selected.
    NOTE: Users must try the different filters of the Tools menu to avoid tip-image artifacts due to the flattening used in the leveling process. Image artifacts appear as streak lines and are shown in some of the AFM images.

Results

Images of the morphology and size of S. aureus and P. hunanensis strains, as well as the population organization of both strains, were taken by atomic force microscopy in contact mode. The S. aureus images showed that its population was distributed by zones with aggregates of cocci (Figure 1A). With an increase in scale, there was a greater appreciation of the population distribution and morphology of the cocci (Figure 1B). The mi...

Discussion

Microscopy is a technique commonly used in biological laboratories that allows for the investigation of the structure, size, morphology, and cellular arrangement of biological samples. To improve this technique, several types of microscopes can be used that differ from each other in terms of their optical or electronic characteristics, which determine the resolution power of the instrument.

In scientific research, the use of microscopy is required for the characterization of bacterial cells; f...

Materials

Name	Company	Catalog Number	Comments
AFM EasyScan 2	NanoSurf	discontinued	Measurement Media
bacteriological loop	No aplica	not applicable	instrument for bacterial inoculation
BigDye Terminator v3.1	ThermoFisher Scientific	4337455	Matrix installation kit
Bioedit	not applicable	version 7.2.5	Sequence alignment editor
Cary 60 spectrometer	Agilent Technologies	not applicable
ceftriazone	Merck	not applicable	antibiotic
centrifuge	eppendorf	not applicable	to remove particles that interfere with AFM
ContAI-G Silicon cantilever	BudgetSensors	ContAl-G-10	Measurement Media
eosin and methylene blue agar	Merck	not applicable	bacterial culture medium
Escherichia coli	American Type Culture Collection	ATCC 25922	bacterial strain
GoTaq Flexi DNA Polymerase	Promega	M8295	PCR of 16S rRNA gene
microplate	Thermo Scientific	10558295	for microdilution analysis
Müller-Hinton broth	Merck	not applicable	bacterial culture medium
nutrient agar	Merck	not applicable	bacterial culture medium
nutritious broth	Merck	not applicable	bacterial culture medium
Petri dishes	not applicable	not applicable	growth of bacteria
Pseudomonas hunanensis 9AP	not applicable	not applicable	isolated from the garlic bulb by CNRG
Sanger sequencing	Macrogen	not applicable	sequencing service
ScienceDesk Anti-Vibration workstation	ThorLabs
slides	not applicable	not applicable	glass holder for bacterial sample analysis
Staphylococcus aureus	American Type Culture Collection	ATCC 25923	bacterial strain
Thermalcycler	Applied Biosystems	Veriti-4375786	PCR amplification
Trypticasein soy agar	BD	BA-256665	growth media
ultrasonicator	Cole-Parmer Ultrasonic Processor, 220 VAC	not applicable	for mixing the nanoparticle dilutions