To learn the techniques of gram staining, nigrosin staining and koh test. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. Multiple choice questions on gram staining mcq biology. This will ensure that all areas are stained evenly. Staining of the bacteria forms the foremost and the most important step in the identification of bacteria. Currently, the only control method for determining the validity of this. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. This standard operating procedure is applicable to all technical staff in the microbiology section that have been trained in performing this procedure and are competent and authorized to perform this procedure.
Gently flood smear with crystal violet and let stand for 1 minute. The direct microscopic examination of specimens and cultures can provide a rapid presumptive diagnosis meanwhile gram staining is a differential stain that provide morphological information regarding the shape of cell,the type of cell arrangement that is. To use and relate the gram stain to the study of bacterial cell morphology, and as an important step in the identification of a bacterial species. Gram staining of bacterial cells is neither an accurate nor elaborate technique, but nevertheless it is practically useful to distinguish two big domains of eubacteria species. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. The gram staining procedure is one of the most basic and important tests performed in the clinical microbiology laboratory.
The procedure of giemsa staining varies as per the purpose of staining that means whether the staining is done for the examination of blood cells or to find the parasites in the blood smear and accordingly the blood smears are prepared as. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. An infected appendix, or any tissue containing both negative and positive gram rods. Pdf gram staining procedure find, read and cite all the research you need on researchgate. The gram stain is a classical and important staining technique that remains widely used by. The gram stain differentiates bacteria into two fundamental varieties of cells. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Gram stain is one of the most common staining procedures.
Staining flood the entire slide when crystal violet, iodine and safranin are applied. Cover the smear with gram s iodine, the mordant, for 1 minute. Gram staining technique is the widely used differential staining technique in bacteriology. It was developed by danish microbiologist hans christian gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques. Modification in gram staining methods since the original procedure of gram, many variations of the gram staining technique have been published. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. Gram positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red. Gram staining is a quick procedure used to look for the presence of bacteria in tissue samples and to characterise bacteria as gram positive or gram negative, based on the chemical and physical properties of their cell. In brief, the procedure involves the application of a solution of iodine potassium iodide to cells previously stained with crystal violet or gentian violet. The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria.
Gram staining is a microbiologic procedure used to differentiate gram negative from gram positive bacteria. Biology multiple choice questions and answers for different competitive exams. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized. Gram staining of bacteria from environmental sources.
This staining procedure differentiates microbes into two basic groups. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. Thermo scientific remel gram stain kit is for qualitative procedures to differentiate gram positive from gram negative microorganisms. The gram stain, the most widely used staining procedure in bacteriology. Gram staining is a differential staining technique that differentiates bacteria into two groups. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram.
The gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. Place slide with heat fixed smear on staining tray. Gram stain is one of the most common staining technique used for examining specimens suspected to contain bacteriologic agents. Method of staining used to distinguish and classify bacterial species into two large groups. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Some of them have improved the method, others include some minor technical variants of no value. The document covers the methods for the staining procedures commonly used in clinical microbiology laboratories for the identification of pathogens and dyes stains used for the differentiation of blood cells, for example, methylene blue stain used for. Gram stain or gram staining, also called gram s method, is a method of staining used to distinguish and classify bacterial species into two large groups. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. The gram stain reaction and koh test results were compared with the. In spite of his defective method, his name lives on jay hardy, cls, sm ascp hile working with infected lung tissue. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. It is called a differential stain since it differentiates between gram positive and gram negative bacteria.
Gram stain purpose, procedure and preparation microscopemaster. Gently flood the smear with gram s iodine and let stand for 1 minute. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram. The most widely used staining procedure in microbiology is the gram stain, discovered by the danish scientist and physician hans christian joachim gram in 1884. Remel gram stain kit, 4 x 250 ml bottles related applications. Unless it is a gram stain of a pure culture, there normally should be material staining. The gram stain is the most widely used staining procedure in bacteriology. The gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure.
The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Gram staining or gram s method is an emprical method of differentiating bacterial species into two large groups gram positive and gram negative based on the chemical and physical properties of their cell wall. Bacteria that stain purple with the gram staining procedure are termed gram positive. Cover the smear with crystal violet, the primary stain, for 20 seconds. Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
Prepare and fix the specimen to the microscope slide before staining. The name comes from the danish bacteriologist hans christian gram, who developed the technique. Flood the gram s iodine for 1 minute and wash with water. Hold the slide in a tilted downward position and allow the. In 1883 hans christian gram discovered an important differential staining method that is used extensively today. Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1. This was developed in 1884 by a danish bacteriologist, hans christian gram. Occurrence of a phosphoric ester in certain bacteria. The primary stain of this method is crystal violet, which can be sometimes substituted with equally effective methylene blue. Objectives to learn the technique of smear preparation. The cells are colored red or violet so as to distinguish its group. The gram stain is a type of differential stain that allows a microbiologist to. Prepare the smear of suspension on the clean slide with a loopful of sample. The primary stain crystal violet binds to peptidoglycan, coloring cells purple.
The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Gram positive bacteria that include most of the firmicutes, and gram negative bacteria that include the rest. Gram stain principle, reagents, procedure, steps, results. Bacteria that retain the initial crystal violet stain purple are said to be gram positive, whereas those that are decolorized and stain red with carbol fuchsin or safranin are said to be gram negative. The gram stain procedure was originally developed by the danish physician hans christian gram to differentiate pneumococci from klebsiella pneumonia. The isolated bacteria are stained by various methods depending upon the bacteria in focus. What is gram stain test staining procedure, principle. The procedure steps of gram stain prepare and fix the specimen to the microscope slide before staining. The gram stain procedure is a differential staining procedure that involves multiple steps. Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. If the slide is heat fixed, allow it to cool to the touch before applying the stain. Decolorization the critical step of the gram staining procedure is the decolorization step. The gram stain is a complex and differential staining procedure that remains a useful test performed in microbiology laboratories.
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