Gram staining helps to identify bacterial pathogens in specimens and cultures by their gram reaction grampositive and gramnegative and morphology coccirod. Pdf a gramstaining technique combining staining with two fluorescent stains, oregon greenconjugated wheat germ agglutinin wga and hexidium iodide. It is almost always the first test performed for the identification of bacteria. However, within tissue sections, adaptations of this technique e. An optimized staining technique for the detection of gram. The process of identifying bacteria in the sample involves using different agents, such as crystal violet dye, iodine mordant, decolorant and safranin. Some laboratories may choose to use a cytospin slide centrifuge to concentrate body fluids for s mear preparation. Laboratory procedure manual centers for disease control. In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear figure 1. The gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria. Stains and dyes are frequently used in histology the study of tissue under the microscope and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses disease at a microscopic level. Gram staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by danish bacteriologist hans christian gram in 1884. The primary stain of the grams method is crystal violet. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram.
Gram stain is a very important differential staining technique used in the initial characterization and classification of bacteria in microbiology. Stains may be used to define biological tissues highlighting. Gram stains from blood cultures and direct specimen. Reagents of gram stain because the gram stain reaction is more widely used and studied than any other stain, it is also the most open to variation in protocol. The original formulation comprised of aniline gentian violet, lugols iodine, absolute alcohol and bismarck brown. 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. Overheating of smears when heat fixation is used d. The gram stain the gram stain, originally developed in 1884 by christian gram, is probably the most important procedure in all of microbiology. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Clinically, gram stain results allow for rapid intervention with appropriate antibiotics. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms.
The modified gram stain technique was performed in the same fashion as described in the gram stain above, with the only addition of a counterstain after the first dehydration step of 100 % alcohol fig. Gramstaining is a specific staining technique invented in 1844 by the. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. Crystal violet dye, iodine, alcohol 95% ethyl alcohol, saffranin dye principle. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Grams iodine acts as a mordant helps to fix the primary dye to the cell wall. 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.
The gram stain dallas county community college district. Csf and other body fluids requiring centrifugation. Gram stain workshop for the laboratory generalist aphl. A primary stain originally anilinegentian violet was used, but this has been replaced by the more stable crystal violet. Explain the principle of the gram stain procedure, including. It is very difficult to observe bacteria without adding color to them. It is a primary test which quickly and cost effectively divides bacteria into one of two types. Add the primary stain crystal violet and incubate 1 minute. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. 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. Get a printable copy pdf file of the complete article 582k, or click on a page. Recorded on august 19, 2011 using a flip video camera. Gram stains quickly tell not only if a bacterium is grampositive or gramnegative, but also staining the cells reveals the shape of the bacterium its cell morphology. Gramstain is one of the commonly used differential stains.
The structure of the flagellar basal body differs between. This method has been recommended to increase gram stain sensitivity and to decrease centrifugation and examination time for more rapid results. Types of staining techniques used in microbiology and. Gram staining requires a sample, which can be blood, sputum or mucus, states healthline. Gram staining is still the cornerstone of bacterial identification and taxonomic division. Gram positive bacteria dont lyse in 3% koh so dna is released. Bacteria are transparent and microscopic clear, like glass. A staining technique is often applied to the cells to color them. The word gram is always spelled with a capital, referring to hans christian gram, the inventor of gram staining gram staining principles gram staining is used to determine gram status to classify bacteria broadly. Flood slide with crystal or gentian violet 60 seconds. The gram stain, the most widely used staining procedure in bacteriology. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. Modification in gram staining methods since the original procedure of gram, many variations of the gram staining technique have been published. Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle.
Gram staining is most widely staining technique used in mo examination. 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. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. The gramstaining process discovered in 1882 published 1884 by hans christian gram, a danish bacteriologist and plays an important role in the classifying the bacteria. 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. The name comes from its inventor hans christian gram who developed the technique in 1882 published 1884. Carefully decolorize with 95% ethanol until thinnest parts of the smear are colorless. Gently flood the smear with gram s iodine and let stand for 1 minute. Crystal violet may also be used as a simple stain because it dyes the cell wall of any bacteria. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. Gram staining is an empirical method of differentiating bacterial species into two large groups gram positive and gram negative based on the chemical and physical properties of their cell walls. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Some of them have improved the method, others include some minor technical variants of no value.
Google scholar mittwer t, bartholomew jw, kallman bj. Smears that are too thick will be difficult to decolorize and imposable to read. Pdf a flowcytometric gramstaining technique for milk. Flood the gram s iodine for 1 minute and wash with water. At this point, slides were immersed in alcoholic saffron american master tech scientific, inc. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Staining techniques in microbiology linkedin slideshare. 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. Materials and methods aseptic technique was used throughout the experiment. Five techniques for gram staining bacteria in paraffin sections were compared. It was devised by a danish physician, hans christian gram, in 1884.
Bacteria that stain purple with the gram staining procedure are termed. Make a slide of tissue or body fluid that is to be stained. Because their methodshowedmorestained bacteria and greater homogeneity and contrast, brown and brenn achieved the highest score. Pdf replacing a century old technique modern spectroscopy. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram. A gram staining technique combining staining with two fluorescent stains, oregon. Humberstones methoddidnotstain thecorynebacteriaandstained other grampositive bacteria pink. After staining, bacteria differentiation is possible because grampositive bacteria have a purple color. A new technique for gram staining paraffinembedded tissue. The purpose of the gram stain is to show whether the bacteria are gram positive purplecolored, gram negative pinkcolored, or both. Basic microbiologymlsbmic224 staining methods l 16, 17. In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. A flowcytometric gramstaining technique for milkassociated. In todays lab exercise, you will be preparing a bacterial smear for gram.
A gramstaining technique combining staining with two fluorescent stains, oregon. How to do a grams stain test grams stain is one of the most frequently used processes in identifying bacteria and is used daily in hospitals. This test can be used to confirm and determine gram stain reaction when if gram stain reagents arent available. Staining is the process by which color is added to bacteria so they can be viewed under the light microscopes in our classroom. Gram stain protocols american society of microbiology. Evaluation of a new technique for the demonstration of gonococci. Grampositive bacteria that include most of the firmicutes, and gramnegative bacteria that include the rest. The crystal violet stain is the primary stain, which stains everything in the smear blue. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the isolates were gramnegative bacteria. Anewtechniquefor gramstainingparaffinembeddedtissue 189 acceptable staining of grampositive bacteria. Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
Alternatively, the gram stain is able to differentiate between gram positive and gram negative bacteria, and is the most used technique for classifying bacterial smears in vitro. Stains combine chemicallywith the bacterial protoplasm. Bartholomew 1962 has pointed out that each variation in the gram staining procedure has a definite. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. Gram staining is a differential staining technique that differentiates bacteria into two groups. Glass slides, bunsan burner, cotton, sample, microscope. In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air. Huckers modification method gram staining procedure is used to microscopically identify gram positive and gram negative bacteria. Prepare the smear of suspension on the clean slide with a loopful of sample.
February 2, 2015 acharya tankeshwar bacteriology, staining techniques in microbiology 45. 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. These organisms are usually filamentous, gram positive rods of varying length that often. Place slide with heat fixed smear on staining tray. A video showing the gram stain technique for visualising bacteria. Huckers modification method gram staining procedure. Gramstaining is usually the first step in identification bacteria and can be used in characterizing bacteria.
Module common staining technique microbiology 24 notes indeterminate groups as well. It has to be one of the most repeated procedures done in any lab. Gram stain, a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Gently flood smear with crystal violet and let stand for 1 minute. What is the importance of gram staining in microbiology. Gram staining gram staining is the single most important and universally used staining technique in the laboratory. Slide preparation purulent material should be selected whenever possible.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Occurrence of a phosphoric ester in certain bacteria. Replacing a century old technique modern spectroscopy can supplant gram staining. Gram negative wall structure gives rise to differences in staining, toxicity, and effects of drugs and disinfectants.
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