Understanding Light Microscopy: Types, Techniques, and Structures in Microbial Visualization
This guide explores the different types of structures observable under a light microscope, including various bacterial shapes like filaments, spirals, and cocci. It highlights the importance of ocular and objective lenses in enlarging images, as well as the role of light wavelength and angle in discrimination properties. Techniques such as darkfield and interference microscopy improve visualization of microorganisms. Additionally, it covers staining methods like Gram and negative staining for better contrast, alongside the capabilities of advanced electron microscopy for high-resolution imaging of viruses and cellular structures.
Understanding Light Microscopy: Types, Techniques, and Structures in Microbial Visualization
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Presentation Transcript
Types of structures seen in light microscope Branche filaments, spirals, vibrio, thic rod, tetraede – 4 cocci, staphylococci– grapes of cocci, streptococci – chain of cocci, diplococci – double of cocci ( lancets shape – flame and candle, coffee beans), different spirals. Leptospira interrogans ?
Microscope - light Enlargement: 2 systems of lenses to make the picture enlarge – ocular lenses nad objective lenses Objective lenses: –power x10: general overview – x 40: big microbes (parasites and their cysts and eggs, filamentous fungi) – x 100 with immersion oil : bacteria, yeast, morphological details Ocular lenses: usually x 10, x 25 The power of microscope equals the multiplication of the objective and occcular lenses power Discirmination properties: are given by the wave lenghth of the light beam and by the angle in wich the light beam enters the lens of the objective – numeric aperture: 2 /um –
Light microscope Index of light of the background and of the bacteriu, is almost the same = the discrimination is bad – native smear – living bacteria (movement, budding - stained, colored smears (the contrast between microbe and bacgound is greate) – better discrimination + identification of some subceluluar structures Darkfield microscopy – the sample is visualised by the light beam entering from the perifery – large angle (0,1/um – 0,2/um) – Treponema, Borrelia, Leptospira Microscopy with interference – filtration system visualise the phase differences of the light beam after its passing through objects with different density The system anable 3 dimensional picture
Improoving the discrimination properties of light microscope Vibrio, rod with spores, sprial filamentous rods, spirochets, staphylococci, streptobacili – rods in chain, streptococci, tetrades, diplococci
Negative staining amelioration of the contrast against the dark background- Burri method – capsule is uncolored, - Gram staining – negative staining of staphylococci or spores that are not stained
Fluorescein microscope Mercury vacuum lamp emits the light of shorter wavelenght The smear is prepared with fluorochromes – compositions that can absorbe the short lenght´s ultraviolet or ultrablue light and can emit energy of higher wavelenght Fluorochromes that stain the smear – fluorescence staining – that after the beam touch it will emit the green fluorescein light Highly sensitive
Electron microscop Uses the magnet – not lenses – to direct the beam of electrons throught speciment to the screen. This process uses the shorter wavelenghts of light.Higher discrimination Visualisation of viruses and subcellular structures 2 types – transmission – beams enter directly throught the sample – scanning – beams enter the sample in the angle 3 dimmensional picture
Possibilities in visualisation of bacterial sporesnegative staining, WirtzConcklin, transmission electron microscopy, scanning electron microscopy
