Step 3. Imaging
Channel: Bad Idea
Date: 2021-02-21

All living organisms consist of cells. There are two main types of cells: prokaryotic and eukaryotic. The division to prokaryotes and eukaryotes reflects levels of cellular organization. Cells in prokaryotes lack a membrane-bound nucleus. Prokaryotes do not have mitochondria, or any other membrane-bound organelles. All their intracellular components are enclosed by the cell membrane, rather than separated in different cellular compartments.
One of the methods to study cells and cellular structures is microscopy. Light microscopy allows to magnify (enlarge) cells up to 1000 times and resolve (ability to distinguish two adjacent objects) details as small as 0.2μm. Light microscopy does not allow observe many details. However, cell size, shape and some internal structures can be easily determined with the help of light microscopy.
The principal of light microscopy is to shine light through a specimen and examine it under magnification. It is important that the specimen is not very thick so that light can pass through it. The major optical parts of a microscope are the objective lens, the eyepiece (oculars), the condenser and the light source. The objective lenses function to magnify the object. The eyepiece functions to deliver the image to the eye or to the camera. Eyepieces also magnify the image, but do not increase the ability to see fine details. The condenser functions to focus the light source on the specimen and to provide a uniform illumination of the sample
The use of different staining techniques helps to identify and observe different components of the cells.
Cell staining is a technique that can be used to better visualize cells and cell components under a microscope. In staining, a dye solution is used to give the color to cells and its components. Cell components, such as a nucleus or a cell wall, or the entire cell can be stained by different dyes. Staining can be performed on both, fixed (non-living cells) and on living cells.
Staining is possible because stain can bind to specific macromolecular cellular components (protein, nucleic acids). Stains are chemicals that contain chromophores, chemical groups that impact color. There are two types of dyes: basic stains and acidic stains. A basic dye is cationic (positively charged) and will therefore react with material that is negatively charged. The macromolecular composition of bacterial cell gives the cell a negative charge which attracts and binds with basic dyes. Some examples of basic dyes are crystal violet, safranin, and methylene blue. Acid dyes have negatively charged chromophores and are repelled by the bacterial surface. Some dyes require the use of a mordant, which is a chemical compound that reacts with the stain to form an insoluble, colored precipitate. The mordant stain will remain in the sample when the excess dye solution is washed away.
There are two types of staining of cells: simple and differential. In simple staining, just one dye is used. Simple staining is used to visualize morphological shape and arrangement of microorganisms. The Gram stain is a differential stain because it uses two contrasting stains that allow distinguishing between different types of bacteria.

Bacteria are divided into two groups on the basis of the Gram staining. By this technique, Gram positive bacteria stain purple and Gramnegative bacteria stain red. In Gram staining, the bacteria are first stained with crystal violet. Crystal violet stains both gram-positive and gram-negative cells purple because the dye enters the cytoplasm of both types of cells. Then cells are treated with mordant which is Gram's iodine. Iodine forms large crystals with the dye that are too large to escape through the cell wall. The bacteria are then rinsed with ethanol which dehydrates the peptidoglycan of gram-positive cells to make it more impermeable to the crystal violet-iodine complexes. In gram-negative cells, alcohol dissolves the outer membrane and leaves small holes in the thin peptidoglycan layer through which crystal violet-iodine complexes diffuse. The addition of safranin (the counterstain) turns the cells pink. Because gram-negative bacteria are colorless after the alcohol wash, safranin provides a contrasting color to the primary stain (crystal violet).
Gram staining procedure involves heat fixing of cells. This procedure is very important because it allows cells to get attached to the glass slide and not to get washed off the slide during the staining procedure. In addition, heat kills bacteria and alters cells so they readily accept dyes. It is important to not overheat the sample to avoid destroying cells on the slide. However, at low temperatures, cells will not stick to the glass slide. Specimens are required to be air-dried before heating. Heat fixing of wet samples will boil cells and vapor pressure inside the cells will burst them destroying their structure