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 t....

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 bacteria....

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 microsc.......

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

List of materials used in this article

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