3 Microscopic Techniques and Staining Methods

Transcript

Hello!! Welcome to the third topic of the week that is Microscopic techniques and sustaining methods. Microscopy is an important element for plant diagnosis because we can see directly the micro organisms associated with disease plants and it help us to establish a initial etablishment of the relationship between the observe microorganism along with the symptoms it is produced in the host plants. So for microscopy we need certain training procedure as well and we will be looking into those aspects that are used for common plant pathological work. So, broadly Microscopy can be categorized in 2 broad groups: one is Optical microscopy and the other one is Electron microscopy. In Optical microscopy the conventional light microscopy, Flurescence microscopy and confocal microscopy comes into existence. Otherwise Optical microscopy is also known as light microscopy and it involves the usage of visible light and one or lens to produce an enlarged image of an object that is placed in the focal plane of the lens. This can either branch off into transmission, where the beam of light passes through the sample. There are many applications of Optical microscopy such as in microbilogy, nanophysics and biotechnology but in plant pathology it is mostly known for using diagnostics of infected plant tissues. So, Light microscopes normally have 3 types of magnifications:

The low power (10X)

High Power (40X)

Oil immersion (100X)

And they have the capacity to magnify 10 times, 40times and 100 times accordingly. So, Light Microscopy or Optical Microscopy can be of two types: Normal Binocular Microscope and Inverted Binocular Microscope. In Normal Binocular Microscope the light passes from the bottom through the specimen whereas, in case of Inverted Binocular Microscope the light resource is present on the top and the specimen is present in the base were the light passes through from the top to bottom.

Incase of Electron microscopy: The most common electron microscopes are Scanning electron microscopy (SEM) and Transmission electron miscroscopy (TEM). This is a form of microscopy that uses electron beams to create an image of the object being used. They have a much higher magnification than light microscopes. This allows us to see smaller specimens with greater detail. The resolution is abled to be increased because as the electrons travel faster their wavelength becomes shorter so there is a direct correlation between reducing wavelength and increasing resolution. So, this is the reason because the electrons move much faster, so the wavelength of electrons are very small and because of that it is able to have better resolution from the electron microscopy.

There are two types of electron microscopes that are used one is Transmission and the other one is Scanning electron microscopes. TEM involves shooting a high voltage beam through a thin layer of specimen and gathering information about the structure whereas, SEM in contrast produces images by detecting secondary electrons that have been emitted of the surface due to excitation by the primary electron beam. So, the basic principles it is of two distinct types – in case of transmission Electron moves through the thin layer of specimen whereas in case of SEM the image is gathered from the detecting secondary electrons that have been emitted from the surface of the specimen. Here is an example of comparative microscopy of light of optical microscope SEM and TEM. Here you can see that rust fungal pathogen spore that is germinating and this is on the surface of leaf under light or optical microscopy. This is the image generated under scanning electron microscopy and whereas, this is the image generated on transmission electron microscopy where this is the fungal spore, storium and this is the hastorium. This structure can be detected through three sectioning of the fungal infecting sight and this is how the images are created by optical. Light microscopy, scanning microscopy and transmission electron microscope.

Staining of fungal structures is very important to observe. As we have seen in the previous slide how fungal spores we can be visualize under light microscopy. So, Lactophenol Cotton Blue Stain is the common stain that is used for staining the fungal structures. So, it is formulated with lactophenol, which serves as a mounting fluid and cotton blue serves as a stain. The organisms suspended in the stain are killed due to the presence of phenol and cotton blue is an acid dye that stains the chitin present in the cell walls of fungi. That’s how we are able to see the fungal structures under light microscopes in a very clear cut manner. So in totality Phenols that is used it kills all the living organisms like fungal spores, mycelium then the Lactic acid that is used as a Lacta phenol – preserves fungal structures and Cotton blue – it is the stain that stains the chitin of the fungal cell walls. So, that’s how we are able to visualize the fungal structures with the use of Lacto phenol cotton blue.

So how to prepare a Lacto phenol cotton blue solution (LPCB)- so these are the ingredients : Cotton Blue, Phenols, Lactic acid and Distilled water. An the stain is prepared over two days. On the first day one has to dissolve cotton blue in distilled water and leave it overnight to eliminate the insoluble dye. On the second wearing gloves add the phenol crystals to the lactic acid in a glass beaker and place on magnetic stirrer until the phenol is dissolved. Then finally add glycerol. Filter the Cotton blue and distilled water solution into the phenol/glycerol/lactic acid solution. Mix and store at the room temperature.

So in brief, this is the procedure how lactophenol cotton blue (LPCB) is prepared over two days time and this is the most common staining agent that is used for staining fungal pathogens. So, normally observing fungal spore on glass slides is done at low power and high power and Hyaline spores are basically stain with lacto phenol cotton blue and we can see the spores very clearly in comparison to the same spore without staining . Coloured spores like in case of bicolories or Altrneria normally no staining agent is used and they are still observed under low and high power because of their own inheriting colours the staining is not required for visualizing of such fungus under microscope. Then there is another technique for observation of spores on intact leaf surface and what we need to do here is that we need to clear the chlorophyll content of the leaves on which the spores are present.

So, the leaf simple Leaf clearing technique involves: Ethanol and acetic acid solution in the ratio of 3:1 and it is to help in removal of chlorophyll from the leaves. After clearing the leaves staining was done by Coomassie blue for visualization of pathogens like Erysiphe pisi that causes powdery mildew on different pea leaves. After staining, leaves are kept on glass slide and observed under compound light microscope for conidial presence and their germination. So leaf clearing technique is used mostly to remove the chlorophyll content of the leaves followed by staining of the fungal structures like spores or germinating spores so that we can see the association of fungal spores with the particular host plant and its development on host plants.

Then Gram staining of bacteria – it’s an important staining procedure for differentiating gram positive bacteria from gram negative bacteria. Any isolated bacteria from infected plant samples can be first differentiated with the help of this gram staining procedure. So the procedure comprises of preparation of a bacterial smear on a glass slide, then we should go for air drying of the bacterial smear. Then it is stained with crystal violet and it is done for approximately 30 seconds, then bacteria gets stained with bluish purple colour then it is washed with water followed by adding iodine staining solution, it works like a mordant and it forms a complex with the crystal violet and gives a crystal violet iodine complex. The, this is subjected to alcohol treatment for few seconds to a minute and this alcohol treatment can differentiate between the gram positive bacteria gram negative bacteria. In case of gram positive bacteria they will retain the crystal violet colour and they can be confirmed as gram positive bacteria. The appearance will be like this, it will be kind of bluish purple. Then, some of the bacteria will be destained after the alcohol treatment and they can be done counter stain with another stain that is Safranin and then the colours of the bacteria will turn into pink and this will confirmed at those bacterial cells are gram negative in nature and their appearance will look this under normally oily immersion. So this is how we can differentiate the bacterial pathogen that is isolated from an infected plant and they can be differentiated on the basis of gram staining whether the pathogen is gram positive bacteria or gram negative bacteria and then, visualizing the structures and other things.

So, in short we have seen the common microscopic techniques that are used for diagnosis of plant pathogens that is associate with plant diseases and then simple and staining methods that are most commonly used for staining of fungal pathogens and bacterial pathogens for differentiating them and correlating them with the associated disease symptoms. So, with this we have come to an end of the topic microscopic techniques and staining methods. In the next topic we will be discussing about diagnosis seed borne pathogens. Till then..

Thank you very much.