This protocol shows the use of contact mode atomic force microscopy to observe changes in cell morphology due to a specific disease or treatment. It is highly recommended to use sealed containers for this procedure to avoid contamination and analyze the fixed sample as soon as possible to avoid aging. Begin by fixing the samples on the slide by gently passing the slide over a flame several times until the sample is dry.
Place the fixed samples in a Petri dish and transport them to the atomic force microscope equipment for observation. Next, turn on the computer and the atomic force microscope. Then select the contact mode, ContAI-G probes, and auto slope options in the software.
Set the default values for the Z controller by setting the set point at 20 nanometers, P gain at 10, 000, I gain at 1, 000, D gain at zero, and tip voltage to zero millivolts. Using the 70 micrometer scan range head, place the samples in the AFM contact mode. Obtain a quick view of the surface of the area under the probe by using a camera mounted on two lenses integrated into the scan head.
Now, choose the desired area by manually displacing it in the XY plane. Allow the probe to electronically approach the sample surface until contact is reached. Electronically adjust the XY measurement plane of the scanner and sample surface by reducing the XY slope direction.
Set the standard parameters of the software to one line per second and 256 points per line. Then perform a full scan range of a 70 by 70 micrometer area. Select the smaller area using the zoom tool.
Select a new area from the sample by retracting the cantilever electronically and moving the sample along the XY plane. Then perform a new scan as demonstrated before. Next, use the line by line leveling in the tools menu of the image in the filter selection to perform data processing.
Optimize the maximum and minimum height in the color bar to obtain the best image resolution. Finally, perform the image analysis using the analysis menu. Select the initial and final points with the desired tool to determine the length and distance.
Atomic force microscopic analysis of the bacterial cultures showed that the Staphylococcus aureus cultures were distributed by zones with cocci aggregates. Increased scales showed that there was a greater appreciation of population distribution and cocci morphology. The AFM contact mode images allowed the determination of the size of the cocci which showed an average width of 1.25 micrometers.
Pseudomonas hunanensis showed a homogenous distribution on the entire glass support surface forming an adherent monolayer. The cultures were rod-shaped with a length of 1.9 micrometers and a width of 0.9 micrometers. Micro dilution exposure of magnesium oxide nanoparticles of Staphylococcus aureus resulted in the loss of a smooth surface and homogenous contours due to severe cellular structure deterioration.
Vesicle formation and cytosolic material release were observed in cultures at higher concentrations than the MIC. Similar results were obtained in E.coli cultures with the controls showing smooth surfaces highlighting its homogenous rod-like shape. The structure completely disappeared at 1, 000 PPM nanoparticle exposure with only distinguishable silhouettes.
It is important to perform sample fixing correctly as it is essential for further analysis. The image filter helps provide good image resolution and sample details. Using the fixing method, AFM non-contact modes could be applied which can be helpful in the study of mechanical properties.
This methodology could be an inexpensive and useful tool for medical and microbiology research focused on bacterial cell damage analysis.
