Jun 10, 2006 gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. 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. Start studying micro lab 3preparation of bacterial smearssimple stainingnegative staininggram stain. Dissolve 2 g crystal violet in 20 ml of 95% ethyl alcohol.
The original formulation comprised of aniline gentian violet, lugols iodine, absolute alcohol and bismarck brown. Gram stain principle, reagents, procedure, steps, results. Flood the smear with gram s iodine solution and leave for 1 minute. Field stain a which is methylene blue and azure dissolved in a phosphate buffer solution. Audience this basic level course is designed for new or existing public health and clinical laboratory professionals, individuals with a science background who are entering or reentering the. Make a smear of the suspected specimen on a clean glass slide. Transfer a drop of the suspended culture to be examined on a slide with an inoculation loop.
Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. The direct microscopic examination of specimens and cultures can provide a rapid presumptive diagnosis meanwhile gram staining is a differential stain that provide morphological information regarding the shape of cell,the type of cell arrangement that is single. Drain off the iodine wash the slide for the again in a gentle stream of tap water. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. The filtergram stain detected 2 to 100 bacteria, whereas the slidegram stain failed to detect less than.
The gram stain is the most common differential stain used in microbiology. Most bacteria have no colour, so they generate little contrast in the microscope field. Sg5155 our gramstain reagents are intended to be used as a differential stain for the microscopic examination of bacterial cultures and laboratory specimens. Pdf lab report of microbiology 3 akbar haqi academia. Microscopic slides endospores a stained preparation of bacillus subtilis showing endospores as uncoloured and the vegetative cell as violet gram endospore stain a stained preparation of bacillus subtilis showing endospores as green and the vegetative cell as red. The unique cellular components of the bacteria will determine how they will react to the different dyes. The staining spectrum includes almost all bacteria, many fungi, and parasites such as. All cells, grampositive and gramnegative, are stained with crystal violet appear purple. A common procedure, the gram stain, differentiates. Thermo scientific remel gram stain kit is for qualitative procedures to differentiate grampositive from gramnegative microorganisms. 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 or gram staining, also called grams method, is a method of staining used to distinguish and classify bacterial species into two large groups. 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. Pdf mlt 415 lab report smear preparation and gram stain. Gram stain reagents for in vitro use only catalogue no. The two major groups of bacteria can be divided into gram positive and gram negative. The name comes from the danish bacteriologist hans christian gram, who developed the technique. The gram stain procedure has been basically unchanged since it was first developed in 1884. This is applied and stains the gram negatives pink. The gram stain is the first test performed on specimens and often leads to clinical decision making and antibiotic treatment. This is in contrast to gramnegative bacteria, which cannot retain the.
He help to distinguish klebsiella pneumonia to pneumococci. Gram positive cells form a crystal violetiodine complex. The counter stain used in the gram stain is safranin pink. The technician decides to make a gram stain of the specimen. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. After smear is rinsed with water, an iodine mordant solution is applied. In addition this stain also allows determination of cell morphology, size, and arrangement. This technique is commonly used as an early step in identifying pathogenic bacteria.
Gram stain is one of the most common staining procedures. This can result in areas that are under decolorized, thus organisms that are truly gramnegative will appear grampositive. Both gram positive and gram negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet. Safranin o, an indicator of cell chondrogenesis, is a cationic dye that stains acidic proteoglycan present in cartilage tissues.
Gram stain, grampositive, gramnegative, heat fixation, methanol fixation. The gram stain ranks as one of the most important stains for bacteria. To obtain useful information from gram stained preparations, you should. In 1883, karl friedlander investigated differential staining of bacterial cells in tissue. It is one of the differential stains that are used to characterize bacteria in one of two groups. The direct microscopic examination of specimens and cultures can provide a rapid presumptive diagnosis meanwhile gram staining is a differential stain that provide morphological information regarding the shape of cell,the type of. Gloves, laboratory safety glasses, and a lab coat are recommended. The name comes from the danish bacteriologist hans christian gram, who developed the technique gram staining differentiates bacteria by the chemical and physical. Staining of bacterial cells objective to learn the techniques of smear preparation, gram staining, nigrosin staining and correlating the results of gram staining with koh test. The objective of gram stain this test differentiates the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiation of microorganisms. Details vary from one gram stain protocol to another, mainly in the timing and the composition of the decolorizing agent.
Oct 05, 2018 gram stain is one of the most common staining technique used for examining specimens suspected to contain bacteriologic agents. Thermo scientific remel gram stain kit is for qualitative procedures to differentiate grampositive from gram. Giemsa staining technique principle, preparation, procedure. Giemsa stain is commonly used when there is need to examine the blood smear for the parasites but is a good stain for routine examination of blood smear and used to differentiate nuclear and cytoplasmic morphology of the various cells of the blood like. Gram staining is the single most useful test in the microbiology laboratory given its simplicity and.
Therefore, to see bacteria with the microscope, it is necessary to apply colour by using a staining reagent. The gram stain was developed by christian gram in 1884 and modified by hucker in 1921. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. Objectives to learn the technique of smear preparation. Sg5155 our gram stain reagents are intended to be used as a differential stain for the microscopic examination of bacterial cultures and laboratory specimens. After completing the gram stain procedure, the technician views the slide under the brightfield microscope and sees purple, grapelike clusters of spherical cells figure 4. The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria.
Cover a heat fixed smear with a basic dye crystal violet. The gram stain was first used in 1884 by hans christian gram. Sign in to view the content sakshat amrita virtual lab. In 1883 hans christian gram discovered an important differential staining method that is used extensively today. Finally, a counter stain safranin, is applied to stain those cells gram negative that have lost the primary stain as a result of decolorization gram reaction grampositive bacteria are those that are stained dark blue or violet by gram staining. This staining procedure differentiates microbes into two basic groups. Finally, a counter stain safranin, is applied to stain those cells gram negative that have lost the primary stain as a result of decolorization gram reaction gram positive bacteria are those that are stained dark blue or violet by gram staining. A fundamental laboratory technique that is introduced in general biology and microbiology courses is staining of bacterial cells on glass slides for visualization and characterization purposes. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. Saturate the smear with crystal violet for 1 minute. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the 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 gram positive or gram negative, based on the chemical and physical properties of their cell. It is the basic component of the stain and field stain b made up of eosin y in a buffer solution which is the acidic component of the stain. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. Perform a bacterial smear, as discussed in figure 352 on page 150 of your lab manual. To learn the techniques of gram staining, nigrosin staining and koh test. 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.
Gram staining technique is the widely used differential staining technique in bacteriology. Csf and other body fluids requiring centrifugation. The two major groups of bacteria can be divided into grampositive and gramnegative. Make a thin film of the material on a clean glass slide, using a sterile loop or swab for viscous specimens. This method has been recommended to increase gram stain sensitivity and to decrease centrifugation and examination time for more rapid results. Author hopes the book will save the time and efforts of research workers, students. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. Oct 24, 2019 the primary stain crystal violet binds to peptidoglycan, coloring cells purple. Gramstained smears are due to errors in the preparation of the slide, such as making the smear too thick, excessive heatfixing, and under or overdecolorization. Gram stained smears are due to errors in the preparation of the slide, such as making the smear too thick, excessive heatfixing, and under or overdecolorization. Gram staining is initially established by the physician hans christian gram, which was from denmark. The gram staining method is named after hans christian gram, the danish. Gram smear preparation in an automated microbiology laboratory.
The objective of gram stain this test differentiates the bacteria into grampositive and gramnegative bacteria, which helps in the classification and differentiation of microorganisms. Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology. The purpose of handbook of media, stains and reagents in microbiology is to collect all necessary information in one book. Hardy diagnostics gram stain kits and reagents are used to stain microorganisms from cultures or specimens by the differential gram method. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. Gram stain is one of the most common staining technique used for examining specimens suspected to contain bacteriologic agents. Gram stain, gram positive, gram negative, heat fixation, methanol fixation. Cover the smear with crystal violet stain and leave for 1 minute. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Some laboratories may choose to use a cytospin slide centrifuge to concentrate body fluids for s mear preparation. Distribute iml in each tube, steam sterilize at 121c for 20 min. Stain protocols biol 2420 smith 2010 page 2 of 4 gram stain 1. Cover the slide with crystal violet solution and allow to act for about 30 seconds.
The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. Positive test, green to blue colour of medium after inoculation and incubation. Staining involves the sample preparation onto slides, fixation which aims to. To a gram positive cell the counter stain stains the cell, but the cells are already so heavily stained by crystal violet that the addition of pink does not change the dark purple color of the cells. Heatfix the smear by passing the slide over flame 23 times quickly. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram. A comparison of heat versus methanol fixation for gram. Stains microbiology resource center truckee meadows. In short, the process of gram staining comprises the use of a solution of gram iodine or potassium iodide to the cells which are use to stain before with crystal violet or gentian violet. Gram staining protocol for bacterial differentiation. Purpose, procedure and preparation gram stain is probably one of the most commonly used staining procedures used in the field of microbiology. It is one of the differential stains used to characterize bacteria as either gram positive bacteria or. Mix the crystal violet and ammonium oxalate monohydrate solutions to make the crystal violet stain.
This was developed in 1884 by a danish bacteriologist, hans christian gram. 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. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. Gram s iodine iodine and potassium iodide is applied as a mordant or fixative. Stain protocols 2010 austin community college district. The gram stain remains one of the oldest, trusted, simplest and most reliable testing techniques in the microbiology laboratory. Mix 45 ml glacial acetic acid and 55 ml distilled water and heat to boiling in a conical flask with cotton plugging. This is in contrast to gram negative bacteria, which cannot retain the. Gram staining or gram s method is an emprical method of differentiating bacterial species into two large groups gram positive and gram negative based on the chemical and physical properties of their cell wall. The gram stain technique is based on the differential structure of the. The examiner may consider the smear to be adequately decolorized if gram positive purple bacteria.
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