But, the vast majority of individual bacterial cells are nearly completely colorless. Laboratory Exercises in Microbiology 5 th. Definition safranin Term What is the primary stain of a gram stain? Additionally, this species has not been successfully cultured in the laboratory on an artificial medium; therefore, diagnosis depends upon successful identification using microscopic techniques and serology analysis of body fluids, often looking for antibodies to a pathogen. These results are often helpful in identifying the microbe. Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Begin with a drop of acidic stain at one end of a clean slide 2.
Acid-Fast Stains Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. However, the decolorized Gram negative cells are stained red. Once the smear is sufficiently washed, it is placed on a piece of blotting paper. We have already alluded to certain techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for sample preparation in greater detail. Those that retain the crystal violet are Gram-positive and those that do not retain it are Gram-negative. A more reliable method to identify cell morphology would be to use special stains to identify specific parts of a microbe like endospores, which is usually present in gram positive bacteria. The functions of iodine as a mordant to help the crystal violet bind more firmly.
If the chromophore is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an acidic dye. Gram-negative cells stain pink Acid-fast stain After staining with basic fuchsin, acid-fast bacteria resist decolonization by acid-alcohol. Alternatively, flood smear with methanol for 1 minute. Basic Dyes: Methylene Blue, basic fuchsin, crystal violet, safranin and malachite green have positively charged groups salts. These ionisable dyes are divided into two classes based on their nature of their charged group: 1. Human cheek cells stained by simple staining with methylene blue.
Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-negative cells have other implications with clinical relevance. These enzymes break the starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. Crystal violet is first applied, followed by the mordant iodine, which fixes the stain Figure. If too much culture is added to the water, you will not see clearly stained individual bacteria. An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged dyes. In some cases, specific stains can be used to visualize certain structures flagella, capsules, endospores, etc. Observe under oil immersion Negative Stain Preparation 1.
The India ink has colored the background brown, leaving the cells their natural color. Using the distilled water wash bottle, gently wash off the excess methylene blue from the slide by directing a gentle stream of water over the surface of the slide. Thus acidic dyes stain best under acidic conditions when proteins and many other molecules carry a positive charge; basic dyes are most effective at higher pHs. Air dry and observe under the microscope Why negative staining? Change of objective to get closer and closer to the sample. Staining Techniques Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope. Also, where determining the accurate size is crucial, a negative stain can be used because it produces minimal cell shrinkage Exercise: 1. Chemical fixative penetrate cells and react with cellular components, usually proteins and lipids and render them inactive, insoluble and immobile.
In preparation for staining, a small sample of microorganisms is placed on a slide and permitted to air dry. In a negative staining technique, a negatively charged stain colors the background, leaving the cells light colored and unstained. The smear is next decolourized by washing with ethanol or acetone. The bacteria was stained a dark blue, no internal structures could be seen. Continue until the image comes into focus. Simple Stain The simple stain can be used to determine cell shape, size, and arrangement.
The longer you leave the dye on, in general, the darker the stain. Simply washing off the dye would result in removal of the cells along with the excess dye. As a result, the dye-iodine complex is not retained within the cell and permeates out of it during the process of decolourisation. All of these dyes work well in bacteria because they have positively charged cationic colored ions chromophores. Unfortunately, if you were to try staining bacterial cells in this manner you would find that there was too much background unbound dye to allow for visualization of the cells. Acidic dyes, because of the negative charge, bind to positively charged cell structures.
Let's take a quick look at a few of the more common categories of staining techniques. The slide is now ready to be viewed under a microscope. This kills the bacteria, attaches the cells to the slide, and enhances the stain uptake. This suggests damage to the individual cells or that decolorizer was left on for too long; the cells should still be classified as gram-positive if they are all the same species rather than a mixed culture. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. Allow the stain to sit for 60 seconds, then rinse the slide with water to remove excess stain.
Care must be taken not to flood the slide, which would be a wastage of staining solution. There are many different staining techniques. Used to distinguish acid-fast bacteria such as M. It is subsequently focused using fine adjustment until a clear image appears. Saline may also be transferred using a bacteriological loop.
Finally smear is counter-stained with a simple basic dye different in color from Crystal violet. The slide should feel very warm but not too hot to hold. Center the area to be studied, apply immersion oil directly to the smear, and focus the smear under oil with the 100X objective. The function of fixation is to kills bacteria rendering safe handling and prevents autolysis by inactivating the autolytic enzymes. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. At the end of the procedure, spores stain green and other cells stain red. Bacteria are slightly negatively charged at pH 7.