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13MEDICAL SCIENCES
CHANGES REVEALED BY TRANSMISSION ELECTRON MICROSCOPY (TEM) IN CANDIDA ALBICANS CULTURES INOCULATED WITH OIL OF OREGANO
Moroianu O.,
Doctoral School, University "Ovidius" of Constanta, Romania
Popescu N.,
Central Medical Iowemed from Constanta, Romania
Stefanov C.,
Faculty of Medicine, Universitatea "Ovidius" Constanta, Romani
Dobrin N.,
Faculty of Medicine, Universitatea "Ovidius" Constanta, Romani
Rosoiu N.
Academy of Romanian Scientists, Bucharest, Romania Professor Emeritus, Faculty of Medicine, University "Ovidius" of Constanta, Romania
https://doi.org/10.5281/zenodo.7079215
Abstract
The study focused on section imaging and electron microscopic structure analysis of Candida albicans culture treated with oregano essential oil. In the study we used cultures of Candida albicans ATCC pre-treated with essential oil of oregano which were initially left for 72 h at the thermostat, in order to be inhibited by the essential oil of oregano. We took samples from this environment and treated them properly so that they could be viewed and analyzed with the transmission electron microscope.
Keywords: Candida albicans, osmium tetraoxide, Epon 812, specimen
Abbreviations: h = hour, mm = millimeter, 1 M -molar concentration (expressed in mol/liter), ATCC = standard culture of Candida albicans, ATCA = trichlo-roacetic acid (phosphotungtic acid)
GA = glutaral - dehyde, DDSA = dodecenyl suc-cinic anhydride, NMA = methyl nadic anhydride, DMP30 = 2, 4, 6 - dimethylaminomethyl phenol, TEM = Transmission electron microscope/ transmission electron microscopy Introduction
Transmission Electron Microscopy (TEM) Transmission electron microscopy (TEM) is used to produce images of a sample by illuminating the sample with electrons (eg electron beam) in vacuum and detecting the electrons transmitted through the sample. Finally, using TEM we can see the columns of atoms present in crystalline samples.
A typical transmission electron microscope consists of the following components (Figure 1):
Figure 1. Schematic representation of typical transmission electron microscope components(1)
• Electron Cannon: Generates the electron beam. This is usually positioned at the top of the microscope column. The electron emitter is placed in a conical Wehnelt cylinder and the beam exits through the small central hole in the top of the cone.
• Electron column: consists of the electron gun assembly at the top, a column filled with a set of electromagnetic lenses, the sample introduction port and pres-surization chamber, and a set of apertures that can be moved in and out of the beam path. The column contents are under vacuum.
• The electromagnetic lens system: they give the shape of the electron beam, which circulates in a spiral trajectory. Each lens is constructed from a coil of copper wire through which electrical current is passed. There is a hole in the center through which the beam travels.
• Sample introduction port/pressurization chamber: is where the sample is introduced into the electron column, into the path of the high voltage electron beam. Before reaching the path of the beam, the sample must pass through the pressurization chamber, which has the role of sealing hermetically to protect the vacuum inside the electronic column.
• Main control panel and operational controls: the instrument can be controlled via external panels using buttons, switches and joysticks.
• Image Capture: At the base of the column is a viewing chamber with a window and adjustable binoculars. The image is projected on the screen in the viewing room. Binoculars are available to focus the image. The screen in the viewing room is only for producing a temporary image. To collect a permanent image, a CCD camera is inserted into the beam path. This allows the image to be collected in digital form. The exposure time can be adjusted to suit the beam parameters and control the desired image quality.
Current 3D localization microscopy approaches are fundamentally limited in their ability to image thick, densely labeled specimens (1).
Due to the increasing popularity of electron cryo-microscopy (cryoEM) in the structural analysis of large biological molecules and macro-molecular complexes and the need for simple, rapid and efficient readout, there is a persuasive need for improved detectors (2).
Electronic pneumatic injection (EPI) is a technique for dermal drug delivery, which is increasingly being used in clinical practice. Immediate cutaneous distribution was visualized using ex vivo confocal microscopy (EVCM) (3).
To study the immunological features of the secretion of the prostate by electron microscopy in patients with chronic recurrent bacterial prostatitis (4).
Conventional two-photon microscopes use photo-multiplier tubes, which enable high sensitivity but can detect relatively few photons per second, forcing longer pixel integration times and limiting maximum imaging rates (5).
Understanding the effect of external conditions, temperature in particular, on novel nanomaterials is of great significance. The powerful ability of scanning
tunneling microscopy (STM) to characterize topography and electronic levels on a single molecule scale is ut (6).
Scanning probe microscopy (SPM) is considered one of the most powerful tools for nanoscale studies that are becoming increasingly important, and SPM has shown rapid development. Atomic force microscopy (AFM), in particular, is the widely used SPM system (7).
Super-resolution microscopy can reveal the subtle biological processes hidden behind the optical diffraction barrier (8).
Material and methods
The study was conducted between July 1, 2019 and May 20, 2020 at the Faculty of Medicine of the "Ovidius" University in Constanta.
For transmission electron microscopy (TEM) we took 2 samples from the Sabouroud medium seeded with Candida albicans samples, from a calibrated assortment, called ATCC; the medium was initially treated with oregano essential oil; I inserted the plate into the thermostat, at the standard temperature of 37°C; this was kept for 72 h at the thermostat (10), (11), (12).
After these 72 h in which Candida albicans was also inhibited by oregano oil, oil containing carvacrol (13) and thymol (14), we sectioned the necessary TEM material.
The purpose of the study was to obtain images and analyze them.
The samples from the experimental variants were processed by the JASTROW method and analyzed from an ultrastructural point of view by transmission electron microscopy through the following work steps:
• prefixing,
• fixation,
• dehydration,
• inclusion in epoxy resins.
a. Prefixation in 1M cacodylate buffer with 2.7% glutaraldehyde (GA)
The sample is immersed in 2.5% buffered GA + 2% paraformaldehyde in 0.1 M Sorensen buffer, pH 7.4. The resulting sediment is resuspended in prefixing medium consisting of 1M cacodylate buffer, 1M sucrose and 2.7% GA, at a temperature of + 5°C. It is homogenized and kept at a temperature of +4°C for 2 hours. The prefixed homogenate is centrifuged at 800g, which constitutes the second centrifugation for 15 minutes, and another 10 minutes at 1300g. The operation is performed with a centrifuge with cooling. The supernatant is removed by decantation, and the sediment is sufficiently condensed, attached to the bottom of the centrifuge tube. 1M cacodylate buffer and 1M sucrose, without GA, at +4°C is placed in a watch bottle, in which the firmly detached sediment from the tube is placed. It is portioned into small specimens with a side of 0.5 mm. The washing liquid is removed and the second wash is carried out using the same procedures, which has the role of removing excess fixative.
It is very important that after pre-fixation any trace of glutaraldehyde is washed away, the residues of which will prevent the binding of osmium in the post-fixing solution.If traces of aldehydes are not removed
from the tissue, upon postfixation membrane lipids will not fix well with OsO4 (9).
b. Fixation in 2% osmium tetraoxide.
Specimens are placed in small glass tubes containing 2 ml of cold fixative medium consisting of 2% (w/v) osmium tetraoxide (OsO4) dissolved in bidistilled water. The fixing itself is done for 1 hour at +4°C. During this interval the specimens turn black due to their oxidation by the osmium tetraoxide, this shows that the fixation has been well done. The fixative is slowly removed by decantation and replaced with cac-odylate buffer and cold sucrose to wash and remove excess osmium. The washing operation is done 2 times with cold cacodylate buffer. Each wash takes 5 minutes.
c. Dehydration of specimens
For dehydration, I use serial baths of cold ethyl alcohol, in concentration of 30%, 50% and 70%. In each alcohol bath, the specimens are kept for 10 minutes. In this dehydration phase, the specimens are contrasted in the second bath of 70% ethyl alcohol, which contains 0.5% uranyl acetate and 1% phosphotungtic acid or tri-chloroacetic acid (ATCA). Contrasting is done at room
temperature for 14 h, after which the specimens are dehydrated at room temperature with ethyl alcohol in a concentration of 90%, 95% followed by 2 baths in 100% ethyl alcohol and 2 baths of propylene oxide. Each bath lasts 10 minutes.
d. Inclusion in epoxy resins Specimens with Candida Albicans culture cells were placed in Epon812 - (consisting of DDSA, NMA and DMP30 as a polymerization agent) mixture 1:1 with propylene oxide. Impregnation is done at room temperature for 16 hours, after which they are taken and placed in transparent capsules. The capsules are filled with pure EPON 812 (epoxy resin), kept for 3h at room temperature and 72h in a thermostat heated to a temperature of 67 °C. At the end of polymerization, the black specimens are at the top of the capsule. The specimens thus prepared are subject to evaluation from the point of view of morphological qualities.
Fine sections were double stained with uranyl acetate and lead acetate, after which they were examined with a Tecnai T12 Microscope produced by FEI, which is located at the Faculty of Medicine of the "Ovidius" University of Constanta.
(a) (b) (c) (d)
Figure 2 (a, b, c, d). Epoxy resin blocks with Candida albicans sample and oregano essential oil analyzed
from different angles
Discussion and results Evaluation of sections
Overall characterization was performed using photomicrographs taken at x2900- x30000. From these, I selected some representative images and later interpreted what I obtained.
Caracterizarea in ansamblu s-a efectuat utilizand microfotografii efectuate la marimi de x2900- x30000. Dintre acestea, am selectat cateva imagini representative si ulterior am interpretat ceea ce am obtinut.
Figure 4. Photomicrograph size x2900, Candida albi-cans culture inoculated with oregano essential oil. The presence of numerous vacuoles and the absence of cell organelles and nucleus can be observed
Figure 5. Photomicrograph size x4800, Candida albicans culture inoculated with oregano essential oil. It can be seen how the number of cells was drastically reduced following the action of oregano essential oil.
Figure 6. Photomicrograph size x6800, Candida albi-cans culture inoculated with oregano essential oil. Absence of cell organelles and nucleus can be observed.
Figure 7. Photomicrograph size x9300, Candida albi-cans culture inoculated with oregano essential oil. Cell wall and cell membrane damage can be seen.
Figure 8. Photomicrograph size x9300, Candida albi-cans culture inoculated with oregano essential oil. Cell wall and cell membrane damage can be seen.
Figure 9. Photomicrograph size x13000, Candida al-bicans culture inoculated with oregano essential oil. Cell wall and cell membrane damage can be seen.
Figure 12. Photomicrograph size x13000, Candida al-bicans culture inoculated with oregano essential oil. Cell wall and cell membrane damage can be seen.
Figure 13. Photomicrograph size x18500, Candida albicans culture inoculated with oregano essential oil. Cell wall and cell membrane lesions can be observed.
Figure 14. Photomicrograph size x18500, Candida al- Figure 15. Photomicrograph size x23000, Candida albicans culture inoculated with oregano essential oil. bicans culture inoculated with oregano essential oil. Damage to the cell wall and cell membranes can be Dissociation between the cell wall and the membrane seen. can be seen.
Figure 16. Photomicrograph size x23000, Candida albicans culture inoculated with oregano essential oil. Dissociation between the cell wall and the membrane can be observed.
Figure 17. Photomicrograph size x30000, Candida albicans culture inoculated with oregano essential oil. Dissociation between the cell wall and the membrane can be seen.
Conclusions
Oregano essential oil can be used successfully as an antifungal for conditions caused by Candida Albi-cans. This antifungal works by dissociating the cell wall and the candida membrane due to the thymol and carvacrol in the composition. The antifungal action of oregano essential oil is irreversible and has a residual effect.
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