The Gram stain and simple stain Smear
Preparation of smear and application of simple stain to observe size,shape, arrangement, color of the microorganisms using oil immersion objective.And application of differential staining method and classify microorganism as Gram positive, Gram negative.
• Crystal violet
• Distilled Water
• Bibulous paper
• E.coli in Nutrient broth culture
• Sterile Glass slide
• İnoculating loop
• İmmersion oil
• Bunsen burner
• Methylene blue
To preparation of smear, firstly in aseptic conditions a sterile microscopic slide was taken.It was flamed through a bunsen burner.After that, a few loops of E.coli broth culture was taken. The inoculum was putted on the slide.Then was waited for air drying.Next, heat fixation was applied and smear was prepared.
In Gram staining process, firstly the ready smear was placed to staining pool.Then, a few drops of Crytal violet was added and was waited for 1 minute.After waiting, it was washed with water. Next, a few drops of Iodine in order to form CV-I complex was added and was waited for 1 minute.Then,it was washed with water again. After the this step, the smear was decolorized by using alcohol and it was washed with water.After that, 1 drop of Safranine was added and was waited for 1 minute.Then, it was washed with water and was dried with a paper.Finally, before it was examined under microscope at 100x, a few drops of oil added on the slide.And was decided the bacterial smear is G(+) or G(-).
Bacteria are too small to see without the aid of a microscobe. Even with a microscope, bacteria cannot be seen easily. Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope.There are many different ways to stain bacteria so that they can be more easily visualized under the microscope. Some stains can also be used to identify and classify bacteria. The Gram stain is a differential stain that allows you to classify bacteria as either Gram-positive or Gram-negative.
Using a single stain to color a bacterial cell is commonly referred to as a simple stain. The most common dyes for this type of stain are methylene blue, fuchsin, and crystal violet. Staining times for most simple stains are relatively short, usually from 30 seconds to 2 minutes, depending on the affinity of the dye. After a smear has been stained for the required time, it is washed off gently, blotted dry, and examined directly under oil immersion. This type of slide is useful in determining basic morphology and the presence or absence of certain kinds of granules.
Bacteria that decolorize easily are called Gram-negative (red) and those that retain the primary stain are called Gram-positive (purple). Bacteria stain differently because of differences in their cell walls. Gram-positive cell walls consist of many layers of peptidoglycan. The crystal violet-iodine complex is larger than either the crystal violet or iodine molecules that entered the cell and the complex cannot pass through this thick cell wall. Gram-negative bacteria have a thin layer of peptidoglycan and an outer lipopolysaccharide layer. The alcohol dissolves the lipopolysaccharides so that the crystal violet-iodine complex can wash out of the cell.
Before bacteria can be stained, a smear of bacteria must be made on a slide and heat fixed. A smear is made by spreading a bacterial suspension on a clean slide and letting it air dry. The dry smear is heated on a hot plate or passed through a flame several times to heat fix it. Heat fixing denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell to break, a process called autolysis. The heat also enhances the adherence of bacterial cells to the slide. Finally, it is very important to prepare thin smears. If a smear is too thick one will not be able to see individual cells, their arrangement, or the details of their microstructures. Thick smears with large clumps of cells can entrap the stain preventing it from being washed. The first step in preparing a smear depends on whether the organism has been growing in liquid or solid media. Two or more loopfuls of liquid media containing the organism can be placed on the slide. One can use an inoculating loop to disperse organisms from solid media into a drop of water on the slide.