Cover the smear with gram s iodine, the mordant, for 1 minute. Gramnegative bacteria are stained with greater homogeneity, the contrast between grampositive and gramnegativebacteriais increased,andthestaining ofthe tissue is such that the tissue can be identified. A new modification and application of the gram stain. Cover the smear with crystal violet, the primary stain, for 20 seconds. The gram stain the gram stain, originally developed in 1884 by christian gram, is probably the most important procedure in all of microbiology. Full text full text is available as a scanned copy of the original print version. To understand how the gram stain reaction affects gram positive and gram negative bacteria based on the biochemical and structural di. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition.
You will be asked about differential staining, grampositive and. The century old gram stain remains the mainstay of rapid diagnosis. Pdf gram staining procedure find, read and cite all the research you need on researchgate. Sterilize the inoculating loop on a flame of a bunsen burner. 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. Planted digital learning library gram stain procedure. Gram staining tips page 2 of 2 some specimens on preparation will have areas on the slide that are thicker than others. 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 gram stain is the most important staining procedure in microbiology. Gramstaining is a four part procedure which uses certain dyes to make a bacterial cell stand out against against its background. Gram staining is generally the first step in identifying an unknown. The gram staining procedure is one of the most basic and important tests performed in the clinical microbiology laboratory. Introduction this slide set is an introduction to the use of grams stain as a rapid tool in the laboratory diagnosis of infectious disease.
The book contains more than three hundred color images, the majority of which come from direct smear examination, such as gram stain and other staining procedures. The gram staining method is named after hans christian gram, the. Grease or oil free slides are essential for the preparation of microbial smears. Carefully decolorize with 95% ethanol until thinnest parts of the smear are colorless.
This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. Get a printable copy pdf file of the complete article 260k, or click on a page image below to browse page by page. Record the qc results on the gram stain qc sheet provided in. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. The gram stain is a type of differential stain that allows a microbiologist to. 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.
Gram stain purpose, procedure and preparation microscopemaster. Gram stain, grampositive, gramnegative, heat fixation, methanol fixation. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. Principle, procedure, interpretation, examples and animation last updated.
Get a printable copy pdf file of the complete article 3. To prepare the bacteria culture slides, a volume of 1 ml of bacterial. A comparison of heat versus methanol fixation for gram. With these types of organisms, grampositive and gramnegative cells may be present within the same preparation.
Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Gently flood smear with crystal violet and let stand for 1 minute. A fundamental laboratory technique that is introduced in general biology and microbiology courses is staining of bacterial cells on glass slides. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. An optimized staining technique for the detection of gram. Gram variable observations in gram staining the gram staining procedure does not always give clearcut results. 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. Ankur choudhary print question forum no comments 1. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Gram negative wall structure gives rise to differences in staining, toxicity, and effects of drugs and disinfectants.
Make a slide of tissue or body fluid that is to be stained. In summary, the present modification stains larger numbers of gramnegative bacteria. Free microbiology photos of gram stained slides viewed through compound microscope and with naked eye. Acetone alcohol is one reagent used in this process to provide the color differentiation. 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.
Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. 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. Bet you didnt know gram stains are performed on body fluid or biopsy when infection is presumed. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the. Gram stain principle, reagents, procedure, steps, results. It is one of the differential stains used to characterize bacteria as either gram positive bacteria or. The difference in result is due to the differences in the cell wall structure or composition of bacteria that causes the different in the reaction with the series of reagents in gram staining talaro, 2007. If the slide is heat fixed, allow it to cool to the touch before applying the stain. 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. Gram staining is a simple procedure if done with precision and accuracy can guide in diagnosis of several bacterial infections. Principle, procedure and results february 2, 2015 acharya tankeshwar bacteriology, staining techniques in microbiology 45 gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. Grampositive bacteria have a thick peptidoglycan layer and stain purple, while.
These online assessment tools will help you strengthen your understanding of the gram stain method. The mold phase and the microscopic structure of the identified fungal species relating to the initial findings of the direct smear have been linked to avoid bias. Protocols for using the gram stains in the microbiology laboratory. These organisms are usually filamentous, gram positive rods of varying length that often. The gram stain is a complex and differential staining procedure that remains a useful test performed in microbiology laboratories. Add the primary stain crystal violet and incubate 1 minute.
Sop for microbial staining procedures standard operating procedure to stain the bacterial cells, bacterial spores and fungus. Links to pubmed are also available for selected references. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. Overheating of smears when heat fixation is used d. Module the gram stain procedure michigan state university.
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. At this point, slides were immersed in alcoholic saffron american master tech scientific, inc. To differentiate between the two major categories of bacteria. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. The gram stain is a differential staining procedure that shows which bacteria are grampositive or gramnegative based on their stain color. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. Prepare and fix the specimen to the microscope slide before staining. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Different bacteria stain differently to a common staining procedure. For confirmation of bacterial presence and identity, we used standard staining including gram and acidfast staining 33, 34. Currently, the only control method for determining the validity of this. Transfer a loopful of culture or the specimen by sterile loop and make a smear at the center. 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.
Gram staining 2 free download as powerpoint presentation. Gently flood the smear with gram s iodine and let stand for 1 minute. Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle. For this project, youll need a heatfixed bacteria slide, grams lodine, crystal violet, safranin, decolorizer, and water. June 12, 2018 by sagar aryal 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. It has to be one of the most repeated procedures done in any lab. Sign in to view the content sakshat amrita virtual lab. The stain is retained because the cell wall of a grampositive bacteria is not as permeable as the cell wall of a gramnegative bacteria. Flood slide with crystal or gentian violet 60 seconds. This video provides instructions on how conduct a gram stain. Such staining methods are called differential staining methods, these include gram staining and acid fast staining. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as grampositive bacteria. Some organisms are gramvariable and may appear either gram negative or grampositive according to the conditions.
This can result in areas that are under decolorized, thus organisms that are truly gramnegative will appear grampositive. As a result, bacteria are classified as either grampositive or gramnegative. How to perform a gram staining procedure wonderhowto. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. It is used to differentiate between gram positive organisms and gram negative organisms. For gram positive staphylococcus aureus is used, for gram negative e. The staining procedure differentiates organisms of the domain. A new technique for gram staining paraffinembedded tissue. Gram stain is one of the most common staining procedures. Simple and rapid, requiring only minutes as opposed to hours to even a day for sophisticated immunological techniques. Explain the mechanism of the gram stain why gram negative cells lose the primary dye during decolorization. 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. The primary stain crystal violet binds to peptidoglycan, coloring cells purple.