Gram staining requires a sample, which can be blood, sputum or mucus, states healthline. As a result, bacteria are classified as either grampositive or gramnegative. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. What is the importance of gram staining in microbiology. This is the most important staining technique in bacteriology. The staining technique, developed in the late 1700s by christian gram classifies the rigid cell walled bacteria into one of two groups based on whether they are able to resist the decolorizing action of an alcoholic. Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as grampositive 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. During the early days of bacteriology the detection of bacterial cells. In regressive staining differentiation is the removal of washing out of the excess stain until the colour is retained only in the tissue components to be studies. Gram iodine mordant forms crystal violet iodine complexes.
Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. 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. Make a slide of tissue or body fluid that is to be stained. Gram staining helps to identify bacterial pathogens in specimens and cultures by their gram reaction grampositive and gramnegative and morphology coccirod. It is a useful technique in the diagnosis of the causative agent of a clinical infection. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Iodine, but not the dye, can be extracted from gramstained s. Similar to a simple stain, it allows for assessment of bacterial cell morphology e. Stains may be used to define biological tissues highlighting. Mycobacterium species resist the effect of the acid. 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. The distinctiveness of gramstaining depends on numerous factors, including the staining technique, the reagent condition, the composition of the growth medium, and the age of the culture 3, 4. 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 primary stain of this method is crystal violet, which can be sometimes substituted with equally effective methylene blue. Modification in gram staining methods since the original procedure of gram, many variations of the gram staining technique have been published. Csf and other body fluids requiring centrifugation. Bartholomew 1962 has pointed out that each variation in the gram staining procedure has a. It is also helpful in studying morphological characters of cells. Except for humberstones method, all methods gave table comparison ofsix methodsfor staining grampositive andgramnegative bacteria in paraffin sections method staphylo strepto coryne clostri escheri proteus klebsiella pseudo haemococcus coccus bacterium dium chia monas philus a number 4 4. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. In order to perfect our gram staining laboratory technique and use those skills to address the issue of possible contamination in our dairy processing. After staining, bacteria differentiation is possible because grampositive bacteria have a purple color. A rapid method for distinction between gram negative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 gram positive and 55 gram negative bacterial strains. Gram staining of bacteria from environmental sources.
To differentiate between the two major categories of bacteria. Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups. Five techniques for gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Types of staining techniques used in microbiology and. Staining is technique used in microscopy to enhance contrast in the microscopic image. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. At this point, slides were immersed in alcoholic saffron american master tech scientific, inc. Gram staining is still the cornerstone of bacterial identification and taxonomic division. The identification, morphology, some extracellular and intracellular components of microorganisms can be determined and detected through the staining. Laboratory procedure manual centers for disease control. The process of identifying bacteria in the sample involves using different agents, such as crystal violet dye, iodine mordant, decolorant and safranin. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. A flowcytometric gramstaining technique for milkassociated bacteria article pdf available in applied and environmental microbiology 695.
Gram staining was given by christian gram in 1884 and it is also called differential staining because it differentiates bacterial in different groups. Some laboratories may choose to use a cytospin slide centrifuge to concentrate body fluids for s mear preparation. Pdf a flowcytometric gramstaining technique for milk. 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.
While this method of estimating probabilities directly from counts works fine in. 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 bv score for gram staining will be calculated by nugents method 1991. Another differential stain technique is the acidfast technique.
Therefore, when the decolorizer is applied to the smear. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. Gram staining is a basic technique used for identification and classification of the cell. Place slide with heat fixed smear on staining tray. Principle, procedure, interpretation, examples and. The gram staining technique is the most important and widely used microbiological differential staining technique. 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.
Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Then the cells are washed with a dilute acidalcohol solution. Gram stain is a very important differential staining technique used in the initial characterization and classification of bacteria in microbiology.
Crystal violet primary stain violet colored, stains all microorg 2. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary while others get decolorized by a decolorizer. This technique differentiates species of mycobacterium from other bacteria. 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. Gram staining or gram stain, also called grams method, is a method of staining used to differentiate bacterial species into two large groups gram. The stain is retained because the cell wall of a grampositive bacteria is not as permeable as the cell wall of a gramnegative bacteria. Stains and dyes are frequently used in biological tissues for viewing, often with the aid of different microscopes. 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. Manual of methods for pure culture study of bacteria. Overheating of smears when heat fixation is used d. Module common staining technique microbiology 24 notes indeterminate groups as well. Gram staining and its molecular mechanism sciencedirect. 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. Handbook of histology methods for bone and cartilage springerlink.
Get a printable copy pdf file of the complete article 3. Make a thin film of the material on a clean glass slide, using a. To understand how the gram stain reaction affects gram positive and gram negative bacteria based on the biochemical and structural di. Pdf basic staining techniques for cartilage sections.
Laboratory perspective of gram staining and its significance in investigations of infectious diseases yunusa thairu 1, idris abdullahi nasir 1, yahaya usman 2 1 department of medical microbiology, university of abuja teaching hospital, gwagwalada, abuja, nigeria 2 department of medical laboratory science, faculty of medicine, ahmadu bello university, zaria, kaduna, nigeria. Comparison of gramstaining and alternate methods, koh. The gram stain is a classical and important staining technique that remains widely used by environmental microbiologists. Impregnation it is the deposition of salts of heavy metals on or around cells, tissue constituents etc. 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 scientific method includes all of the following except. In addition this stain also allows determination of cell morphology, size, and arrangement. Some of them have improved the method, others include some minor technical variants of no value. Briefly, the average number of lactobacilliary morphotypes per oil immersion field will be quantified.
Notes on the gram stain with description of a new method. Gently flood the smear with gram s iodine and let stand for 1 minute. Gram staining technique free download as powerpoint presentation. 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. Flood slide with crystal or gentian violet 60 seconds. Gently flood smear with crystal violet and let stand for 1 minute. Brown and hopps method was the most satisfactory for distinguishing grampositive and gramnegative bacteria. A new technique for gram staining paraffinembedded tissue.
Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. In spite of his defective method, his name lives on jay hardy, cls, sm ascp. Staining is a technique used to enhance contrast in samples, generally at the microscopic level. This method has been recommended to increase gram stain sensitivity and to decrease centrifugation and examination time for more rapid results.
Gram staining 2 free download as powerpoint presentation. Staining is an important step to observe microorganisms more clearly, to differentiate between microorganisms as well as to differentiate parts in microorganism bagyaraj et al, 2005. Stains may be used to define and examine bulk tissues highlighting, for example, muscle fibers or connective. The staining procedure is followed by mordanting and differentiation. These organisms are usually filamentous, gram positive rods of varying length that often form chains, but occasionally, they may stain. Add the primary stain crystal violet and incubate 1 minute. 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. An optimized staining technique for the detection of gram. Gram staining is a simple procedure if done with precision and accuracy can guide in diagnosis of several bacterial infections. Gram staining is a differential staining technique that differentiates bacteria into two groups. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Bacteria and cells are visualised without enabling differentiation of bacteria.
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