The gram stain differentiates bacteria into two fundamental varieties of cells. The steps required to do a gram stain microbiology. Gently flood smear with crystal violet and let stand for 1 minute. Gram stain article about gram stain by the free dictionary. The preliminary analysis of bacterial clinical samples is gram stain differentiation. I would like to give an elaborate explanation for the reagents being used in this gram staining and why it is used. Knowing the chemical make up makes it easier to manipulate the bacteria for various purposes. Gram staining is a well established method of staining bacteria. The gram stain is the most frequently used stain in a clinical microbiology laboratory and is usually the first step in identifying bacteria. The smear will appear as a purple circle on the slide. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Grampositive cells will retain the stain even after being washed in the solvent, but the stain will be removed from the gramnegative cells. Microbiology lecture 2 gram positive and gram negative.
The first reagent crystal violet is the primary stain. Crystal violet dye is added for one minute to stain the cell wall. If this was really a gram stain, then the nucleus of the cells would be decolorized and would end up being pink, not purple. Here i would like to give a brief overview over the basics of the gram stain. Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011 novaultra special stain kits principle. Use of the gram stain in microbiology pubmed ncbi nih. Gently flood the smear with gram s iodine and let stand for 1 minute. The end result is easiertoread and accurate staining. Christian grams to differentiate between gram positive and negative bacteria. Gram positive bacteria retain the crystal violetiodine complex after decolorization, are not counterstained, and appear purple gram negative bacteria have a different cell wall structure, dont retain the crystal violetiodine complex after decolorization, and so are counterstained by safraninfuchsin and appear pinkred paraffin sections. The gram stain virtual interactive bacteriology laboratory. Gram negative gently stirred into a drop of 3% koh potassium hydroxide, the bacteria lyse break open, releasing their dna making the liquid viscous in 4560 secs. Abstractthe gram stain for the demonstration of bacteria is a popular.
Groups for inspiration and financial assistance options for photographers. Gram afb stain kits reduce the presence of artifacts through a unique bd manufacturing process. Mohamed eladawy demonstrator of microbiology and immunology under supervision of. For more help on other staining techniques check out our other staining demonstrations.
Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Place slide with heat fixed smear on staining tray. Crystal violet primary stain, grams iodine mordant, 95% alcohol decolorizing agent, safranin counter stain prepare smears of organisms by spreading cell mass from a colony or by taking a loopful from the broth as a thin film on the slide. The gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. When the bacteria is stained with primary stain crystal violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. Step by step instruction on how to prepare a gram stain. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. As a result, bacteria are classified as either grampositive or gramnegative. Touch a colony with a loop and mix the bacteria with the water until there is a uniform, thin film of microorganisms on the slide. If flash is not working for you we have an alternate video version. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884.
This test can be used to confirm and determine gram stain reaction when if gram stain reagents arent available. It also explains the differences in characteristics of gram positive and. And again, because the stain is only on the external layers of the bacteria, pay attention to just the external layer. Gram staining is generally the first step in identifying an unknown.
I also have videos on the gram stain, flagella stain, endospore stain, capsule stain and more if you want to check them out. The primary stain crystal violet binds to peptidoglycan, coloring cells purple. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Thank you for watching microbiology lecture 2 gram positive and gram negative bacteria gram staining principle. Grampositive bacteria produces a bluepurple color with this staining procedure while gramnegative ones are red in color 5.
Below is a procedure that works well in the teaching laboratories. Wash slide in a gentle and indirect stream of tap water for 2 seconds. The gram stain should almost always be done as the first step in diagnosis of a bacteria infection the gram stain is named after the danish scientist hans christian. Gram staining is a method commonly used to determine the chemical make up of the cell wall of bacteria. Yomna hagag demonstrator of microbiology and immunology recorded by. Cover the slide with crystal violet stain and wait one minute. Gram staining is a quick procedure used to look for the presence of bacteria in tissue samples and to characterise bacteria as grampositive or gramnegative, based on the chemical and physical properties of their cell walls. Gram positive bacteria dont lyse in 3% koh so dna is released. This site is not hosting any videos and is only linking. This is a picture of a wright stain a stain meant to stain wbcs, not bacteria. Gram stain procedure step by step instruction on how to prepare a gram stain. Bacterial characteristics gram staining video khan academy. The cationic dye crystal violet is used to stain the nucleic acids of the microorganisms and background tissues.
Module the gram stain procedure michigan state university. Grampositive organisms will appear blueblack or purple while gramnegative organisms will appear. This is followed by a second reagent grams iodine, which acts as a mordant and helps the color to stick to the cell wall. Overheating of smears when heat fixation is used d. Microbiology 101 the gram stain outbreak news today. Gram did not actually use safranin as a counter stain in the original procedure gram negative cells would be colorless. The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. Occurrence of a phosphoric ester in certain bacteria.
Click to download this application note on gram staining in medical microbiology. What is the principle and procedure of gram staining. Developed in the late 1800s, this staining procedure has become crucial in medical laboratories and. This is gram stain by aimei nadrah on vimeo, the home for high quality videos and the people who love them. This is because the cell walls of gramnegative cells contain more lipids fatty substances. This protocol is taken from bensons microbiological applications. This video provides instructions on how conduct a gram stain. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Alternate gram staining technique using a fluorescent lectin. Learners view video clips demonstrating grams staining procedure. Grams method stains bacteria according to the chemical and physical properties of their cell.
Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. Gram staining or grams method is a way of classifying bacteria into two large groups. This should be a thin, not milky, suspension or it will not stain properly. Gram staining procedure animation microbiology principle, procedure, interpretation duration. The gram stain is a differential method of staining used to assign bacteria to one of two groups grampositive and gramnegative based on the properties of their cell walls. The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria. The name comes from the danish bacteriologist hans christian gram, who developed the technique gram staining differentiates bacteria by the chemical and physical properties of their cell walls. A microscopy staining method for staining and identifying bacteria, based on the differential retention of a crystal violetiodine complex within the cell membrane. The method for mounting bacteria on a microscope slide using aseptic technique is shown. Gram stain procedure 1 heatfix the glass slide containing the specimen. Gram stain set instructions introduction one of the most frequently cited criteria by which bacteria are classified involves the reaction of individual species to the gram staining procedure.
The gramstain is a differential stain that requires the use of at least three chemical reagents applied sequentially to a heatfixed smear. Bacteria that retain the initial crystal violet stain purple are said to. The gram stain procedure click start lab from a liquid culture, take a loopful of bacteria emulsify it in a small drop of water or saline on the slide. Gram staining and clinical applications linkedin slideshare.
He instead recommended using bismarck brown as a counter stain to enable tissue cell nuclei to be visualized. The cell wall can stain either positive or negative, depending on its chemistry. Bd bbl gram qc slides and bd bbl afb qc slides eliminate the need for the laboratory staff to maintain stock qc cultures and prepare qc slides. Flood slide with crystal or gentian violet 60 seconds. Staind its been awhile official music video by staind. The name comes from its inventor, hans christian gram. Bhanu prakash usmle, fmge and neet pg 18,162 views. Bhanu prakash usmle, fmge and neet pg 16,787 views. Student procedures illinois institute of technology. Replacing a century old technique modern spectroscopy can. Gram stain or gram staining, also called grams method, is a method of staining used to distinguish and classify bacterial species into two large groups. Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle.
Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. So even though right here it says capsule, cell wall, and plasma membrane, youll see in a minute that these layers are really different between gram positive or gram negative bacteria, which is. In histology, depending upon the procedure used, staining would require from four to 17 steps and almost as many solutions. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. Based on differences in cell wall components, bacteria are categorized as either grampositive stains dark purple or gramnegative stains pink. First of all, the gram staining was named after dr. Bacterial characteristics gram staining video khan. Planted digital learning library gram stain procedure youtube. Imagecdc the staining procedure for bacteria was discovered over 100 years ago by hans christian gram. Gram staining is still the cornerstone of bacterial identification and taxonomic division.
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