Alternate gram staining technique using a fluorescent. Add the primary stain crystal violet and incubate 1 minute. Gram staining is a differential technique that provides an easy differentiation ppt. Microbiology lecture 2 gram positive and gram negative bacteria gram staining principle this second microbiology lecture will explain the properties of gram positive and gram negative bacteria. Stains, or dyes, contain salts made up of a positive ion and a negative ion. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure.
The gram stain is a type of differential stain that allows a microbiologist to identify the differences between organisms andor differences within the same organism. Grams stain is a laboratory staining technique that distinguishes between two groups of bacteria b y the identification of differences in the structure of their cell walls. Gram staining is a basic technique used for identification and classification of the cell. The microscopicanalysis using gram staining technique. Slide preparation purulent material should be selected whenever possible. In addition to fixation, staining is almost always applied to color certain features of a specimen before examining it under a light microscope. A gramstaining technique combining staining with two fluorescent stains, oregon greenconjugated wheat germ agglutinin wga and hexidium iodide hi followed by flowcytometric detection is described. Find powerpoint presentations and slides using the power of, find free presentations research about staining techniques ppt.
Grampositive bacteria that include most of the firmicutes, and gramnegative bacteria that include the rest. The advantages of such a procedure, particularly as augmentation to wet mounts, suggest that adoption of gram staining may be of great use in the clinical area. Microbiology lecture 2 gram positive and gram negative. It is very difficult to observe bacteria without adding color to them. Overheating of smears when heat fixation is used d. The first page of the pdf of this article appears above.
Bacteria are transparent and microscopic clear, like glass. 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. Gramstaining is a specific staining technique invented in 1844 by the. A fundamental laboratory technique that is introduced in general biology and microbiology courses is staining of bacterial cells on glass slides.
In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. To learn the techniques of gram staining, nigrosin staining and koh test. Google scholar mittwer t, bartholomew jw, kallman bj. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. A comparison of heat versus methanol fixation for gram. The peptidoglycan layer of gramnegative bacteria is covered by a membrane and is not labeled by the lectin. Occurrence of a phosphoric ester in certain bacteria. Gram stain, a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria. Objectives to learn the technique of smear preparation. Since the original procedure of gram, many variations of.
Although gram staining diagnostics can be highly specific, its sensitivity to detect the presence of bacteria is relatively low, requires additional reagents, is time consuming, and can depend on. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. To stain rickettsia by using gimenez staining method. Smears that are too thick will be difficult to decolorize and imposable to read. Gram staining technique free download as powerpoint presentation. Gram staining of bacteria from environmental sources. The gram stain and differential staining journal of. Staining is the process by which color is added to bacteria so they can be viewed under the light microscopes in our classroom. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. The gram stain is a differential staining technique that allows scientists to detect differences in bacteria based on the variable structure of the bacterial cell wall. View and download powerpoint presentations on staining techniques ppt.
The best way for students to print out the powerpoint show is to download the pdf version. Staining technique an overview sciencedirect topics. Three introductory chapters cover gramstain procedures, quality control, evaluation, and more. 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. The gram stain is a classical and important staining technique that remains widely used by environmental microbiologists. 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 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. Provide student with opportunity to perform a gram stain, with controls and an unknown practice evaluating gram stain results perform an isolation streak plate technique aseptically. Module common staining technique microbiology 24 notes indeterminate groups as well.
To undertake a gram stain, heat fix the bacterial sample as described earlier and make sure you have grams crystal violet stain, grams safranin or another counterstain, grams iodine, either 95% alcohol or acetone alcohol, water, blotting paper, gloves and a stop digital clock or a. Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Grampositive organisms contain a highly crosslinked layer of peptidoglycan that retains the primary dye, crystal violet cv, following the application of the mordant, iodine i. At this point, slides were immersed in alcoholic saffron american master tech scientific, inc. Similar to a simple stain, it allows for assessment of bacterial cell morphology e. Gram staining is a rapid, easily learned laboratory technique. It includes introduction, history,principle, method of gram staining procedure slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Make a slide of tissue or body fluid that is to be stained. 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.
It is pleomorphic because it shows different shapes of bacteria that is cocci, rod and spiral shape. 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 method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. Stains may be used to define biological tissues highlighting. Gram stain, grampositive, gramnegative, heat fixation, methanol fixation. Replacing a century old technique modern spectroscopy. Depending on the type of dye, the positive or the negative ion may be the chromophore the colored ion. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. How to prepare an isolation streak plate powerpoint show or pdf printout printing. An optimized staining technique for the detection of gram. The primary staining technique used to differentiate bacteria is the gram stain. 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. For this reason it is strongly recommended to use very young cultures for the staining procedure, after growth has become just visible. The gram stain technique basic protocol is a differential staining technique since it.
Fluorescencelabeled wheat germ agglutinin binds specifically to nacetylglucosamine in the outer peptidoglycan layer of grampositive bacteria. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. It is also helpful in studying morphological characters of cells. It was devised by a danish physician, hans christian gram, in 1884. Although commonly used in other clinical areas, gram staining has not been in standard nurse. 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. Gram staining is still the cornerstone of bacterial identification and taxonomic division. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. The original formulation comprised of aniline gentian violet, lugols iodine, absolute alcohol and bismarck brown. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. By exploiting this phenomenon, an alternative gram staining technique has been developed. Gram stain principle, reagents, procedure, steps, results.
Bisen prakash s microbes in practice, ik international, new delhi, 2014 pp 9155. The structure of the flagellar basal body differs between. Some bacteria, after staining with the gram stain, yield a gramvariable pattern. It is a useful technique in the diagnosis of the causative agent of a clinical infection. Rickettsia is a nonmotile, gramnegative,nonspore forming, pleomorphic bacteria. 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. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. Flood slide with crystal or gentian violet 60 seconds.