The gram stain the gram stain, originally developed in 1884 by christian gram, is probably the most important procedure in all of microbiology. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. The gram stain test was first described more than a century ago, and is well established for identifying and characterizing more common bacteria, especially in the laboratory research setting. Gram positive cells form a crystal violetiodine complex. To differentiate between the two major categories of bacteria. Gram positive bacteria have a plasma membrane then peptidoglycan on top while gram negative bacteria have a plasma membrane, then peptidoglycan and another outer membrane on top. Stain dark purple due to retaining the primary dye called crystal violet in the cell wall. Gram s iodine iodine and potassium iodide is applied as a mordant or fixative. Oral microbiology bacteria causing inflammations and abscesses within the oral cavity. 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.
Flood slide with crystal or gentian violet 60 seconds. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Problems interpreting gram stain slide biology stack exchange. Basic microbiologymlsbmic224 staining methods l 16, 17.
Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology. The following chart represents a classification based on bergerys manual of determinative bacteriology. Counterstain staining with safranin turns gram negative bacteria red. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. The following are some common causes of poor gram stain results a. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. The proper sequence of reagents in the gram stain procedure is.
An alternative qc source is material scraped from between teeth with a wooden applicator stick. In 1883 hans christian gram discovered an important differential staining method that is used extensively today. Slide safety precautions gram stain materials will stain skin and other materials. Grampositive bacteria become gramnegative when their cell wall is ruptured or removed. Dotson md, jeanine wienerkronish md, in critical care secrets fourth edition, 2007. 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 filtergram stain detected 2 to 100 bacteria, whereas the slidegram stain failed to detect less than. Decolorizer removes the stain from gramnegative bacteria becoming colorless 4. The filtergram stain was approximately 100fold more sensitive than the slidegram stain. A chemical bond between the crystal violet and the bacteria 3. Gram staining is a differential staining technique that differentiates bacteria into two groups.
Counterstain staining with safranin turns gramnegative bacteria red. What is the importance of gram staining in microbiology. The objective of this study was to assess the utility of semiquantitative scores. 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. Evaluation of semiquantitative scoring of gram staining. A new technique for gram staining paraffinembedded tissue. In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air. 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 process of identifying bacteria in the sample involves using different agents, such as crystal violet dye, iodine mordant, decolorant and safranin.
Gram negative organisms lose the crystal violet stain and contain only the counterstain safranin and thus appear red. This is commonly performed by using a standing technique called the gram stain, which is based is a type of phenotypic identification method and it undertaken so that the microbiologist can understand the general profile for microorganisms. Bacteria that retain the initial crystal violet stain purple are said to. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. 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 differential method of staining used to assign bacteria to one of two groups gram positive and gram negative based on the properties of their cell walls.
The traditional gram stain procedure is completed in either case top box. Culture is necessary for definitive identification and susceptibility testing. Although gram stain is useful in staining bacteria, certain fungi such as candida. Apr 12, 2016 schematic showing the modified gram stain procedure. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. Using spectroscopy, researchers upended the belief that bacteria completely absorb the dye used to identify different bacterial types.
Evaluation of semiquantitative scoring of gram staining or. Pdf 2 overview biol 552 has a challenging goal, which is to give students practical. With the current theory behind gram staining, it is thought that in grampositive bacteria, the crystal violet and iodine combine to form a larger. Before staining, the position of the smear on the slide should be confirmed by making a scratch with the help of a needle. Counter stain with loeffers methylene blue for 3 min.
The filter gram stain was approximately 100fold more sensitive than the slide gram stain. Schematic showing the modified gram stain procedure. Gram staining is the most sensitive method by which to visualize and recognize nocardiae in clinical specimens. Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. Gram staining is still the cornerstone of bacterial identification and taxonomic division. The cviodine complexes were then counterstained with safranin 35 and used for the acquisition of gramstain images.
Gram staining in diagnostic microbiology slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. Gram stain results, including organism morphology, can. This is in contrast to gram negative bacteria, which cannot retain the. Table 2 use of bacteriocins on gramnegative bacteria in foods. Sputum gram s stain and culture can be performed noninvasively and are inexpensive diagnostic tests. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. In the gram stain procedure see table the slide is flooded with crystal violet the primary stain, followed by grams iodine the mordant, which chemically bonds the alkaline crystal violet to the cell wall. A monograph of gramstained preparations of clinical specimens marler ms mtascpsm, linda m. After staining, bacteria differentiation is possible because gram positive bacteria have a purple color. The slide is then decolorized with acetone, absolute alcohol, or a mixture of the two.
Pdf strategies for the use of bacteriocins in gramnegative. Common pitfalls in this method are that heat fixation may cause grampositive cells to stain gramnegative and older cultures may give. It is also known as gram staining or gram s method. Use results of gram stain in conjunction with other clinical and laboratory findings. The most important and widely used differential stain for bacteria is the gram stain. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. The gram stain was first used in 1884 by hans christian gram. The method is named after its inventor, the danish scientist hans christian gram 18531938, who developed the technique in 1884 gram 1884.
It has to be one of the most repeated procedures done in any lab. The gram stain was first used in 1884 by hans christian gram gram,1884. Because the purple stain imparts its color to all cells, it is referred to as a primary stain step 2 of gram stain after a short time, the purple dye is washed off, and the smear is covered with iodine, a mordant. A monograph of gram stained preparations of clinical specimens marler ms mtascpsm, linda m. After staining, bacteria differentiation is possible because grampositive bacteria have a purple color. Mordant gram s iodine causes a stain to become more tightly bound to the cell. Pdf laboratory exercises for microbiology biology 552.
The gram stain differentiates bacteria into two fundamental varieties of cells. Gram stains have limited application in the diagnosis of. Both gram positive and gram negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet. The gram stain procedure enables bacteria to retain color of the stains, based on the differences in the chemical and physical properties of the cell wall. Wear chemical splash goggles, chemicalresistant gloves, and a chemicalresistant apron.
A gram stain and culture of the material from an infected site are the most commonly performed microbiology tests used to identify the cause of a bacterial infection. Oct 23, 20 semiquantitative gram stain and culture methods are still commonly used for diagnosing ventilatorassociated pneumonia vap, due to its convenience. Apr 03, 2011 gram staining in diagnostic microbiology slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. The objective of this study was to assess the utility of semiquantitative. To understand how the gram stain reaction affects gram positive and gram negative bacteria based on the biochemical and structural di. Decoloring will remove the stain on the outer membrane of gram negative bacteria. The cviodine complexes were then counterstained with safranin 35 and used for the acquisition of gram stain images. This staining procedure differentiates microbes into two basic groups. This sensitization is important in food microbiology, because most pathogens that cause.
Gram iodine, 510 ml bunsen burner gram safranin, 510 ml compound microscope materials included in kit. The gram stain is used to differentiate between different types of bacteria based on the biochemical properties of their cell walls. Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. 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. Wash the smear and air dry the smear and observe under microscope. 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.
Choice of culture media for inoculation can be made empirically based on grams stain report. 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. If you continue browsing the site, you agree to the use of cookies on this website. At this point, slides were immersed in alcoholic saffron american master tech scientific, inc. Gram stain image analysis of the sizes, shapes and morphologies of the.
In a smear that has been stained using the gram stain protocol, the. Gram staining requires a sample, which can be blood, sputum or mucus, states healthline. Only a few studies, however, have assessed the reliability of these methods when compared with quantitative cultures, a current standard for the diagnosis of vap. Modification of the brown and brenn gram stain for the differential staining of grampositive and gramnegative bacteria in tissue sections. In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear figure 1.
The following points highlight the top two staining techniques for recognition of bacteria. Done correctly, grampositive organisms retain the crystal violet stain and appear blue. The major difference in the modified gram stain procedure occurs during the dehydration process and is the application of alcoholic saffron lower right box, highlighted in red. The basic principle of gram staining is the properties of certain bacteria cell walls to retain the crystal violet dye. Apr 12, 2016 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. The different response of the two groups to the gram stain is based on fundamental differences in cell wall structure and composition.
These staining procedures are used to distinguish organisms based on staining properties. Additional procedures, such as culture, must be used to confirm findings suggested by gram stain. The gram stain is a type of differential stain that allows a microbiologist to. Although gram stain is useful in staining bacteria, certain fungi such as candida and cryptococcus are observed as gram positive yeasts. Decolorizer removes the stain from gram negative bacteria becoming colorless 4. Oct 24, 2019 the primary stain crystal violet binds to peptidoglycan, coloring cells purple. An optimized staining technique for the detection of gram. On the basis of their reaction to the gram stain, bacteria can be divided into two large groups.
Crystal violet sticks primarily to the peptidoglycan. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming gram variable and gram. If gram positive diplococci flattened at the outguidelines for the interpretation of gram stains normal flora in respiratory secretions normal flora in the female genital tract presumptive identification of microorganisms from gram stain correlation of findings i1a staphylococci. The original formulation comprised of aniline gentian violet, lugols iodine, absolute alcohol and bismarck brown. The gram stain dallas county community college district.
Gram stain results, including organism morphology, can be affected. May 01, 2015 a gram stain and culture of the material from an infected site are the most commonly performed microbiology tests used to identify the cause of a bacterial infection. Module common staining technique microbiology 24 notes indeterminate groups as well. The method is named after danish scientist hans christian gram 1853 to 1938, who developed the technique in order to distinguish between two different bacterial causes of pneumonia streptococcus pneumoniae and. If grampositive diplococci flattened at the outguidelines for the interpretation of gram stains normal flora in respiratory secretions normal flora in the female genital tract presumptive identification of microorganisms from gram stain correlation of findings i1a staphylococci. Empirical choice of antibiotics can be made on the basis of gram stains report.
The cell walls for gramr positive microorganisms have a higher peptidoglycan and. Mordant grams iodine causes a stain to become more tightly bound to the cell. From developing new therapies that treat and prevent disease to helping people in need, we are committed to improving health and wellbeing around the world. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. The yellow contrast in the connective tissue collagen within skin tissue is. Acidic dyes combine more strongly with cytoplasmic components of bacteria.