Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. 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. What is the importance of gram staining in microbiology. Gram staining requires a sample, which can be blood, sputum or mucus, states healthline. Oct 24, 2019 the primary stain crystal violet binds to peptidoglycan, coloring cells purple. An alternative qc source is material scraped from between teeth with a wooden applicator stick. Evaluation of semiquantitative scoring of gram staining. Before staining, the position of the smear on the slide should be confirmed by making a scratch with the help of a needle. 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. 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. 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. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than. Choice of culture media for inoculation can be made empirically based on grams stain report.
Mordant gram s iodine causes a stain to become more tightly bound to the cell. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Culture is necessary for definitive identification and susceptibility testing. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. 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. Counterstain staining with safranin turns gramnegative bacteria red. Gram stains have limited application in the diagnosis of. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. The method is named after its inventor, the danish scientist hans christian gram 18531938, who developed the technique in 1884 gram 1884. Decolorizer removes the stain from gramnegative bacteria becoming colorless 4. The gram stain is used to differentiate between different types of bacteria based on the biochemical properties of their cell walls. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions.
Counter stain with loeffers methylene blue for 3 min. The following chart represents a classification based on bergerys manual of determinative bacteriology. Schematic showing the modified gram stain procedure. In 1883 hans christian gram discovered an important differential staining method that is used extensively today. Empirical choice of antibiotics can be made on the basis of gram stains report. Gram negative organisms lose the crystal violet stain and contain only the counterstain safranin and thus appear red. Sputum gram s stain and culture can be performed noninvasively and are inexpensive diagnostic tests. A new technique for gram staining paraffinembedded tissue. Gram iodine, 510 ml bunsen burner gram safranin, 510 ml compound microscope materials included in kit. 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.
The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. The different response of the two groups to the gram stain is based on fundamental differences in cell wall structure and composition. 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. Pdf 2 overview biol 552 has a challenging goal, which is to give students practical.
Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. The gram stain is a type of differential stain that allows a microbiologist to. To understand how the gram stain reaction affects gram positive and gram negative bacteria based on the biochemical and structural di. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. The filtergram stain detected 2 to 100 bacteria, whereas the slidegram stain failed to detect less than.
Basic microbiologymlsbmic224 staining methods l 16, 17. The slide is then decolorized with acetone, absolute alcohol, or a mixture of the two. Mordant grams iodine causes a stain to become more tightly bound to the cell. 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. In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air.
To differentiate between the two major categories of bacteria. Decoloring will remove the stain on the outer membrane of gram negative bacteria. Stain dark purple due to retaining the primary dye called crystal violet in the cell wall. The most important and widely used differential stain for bacteria is the gram stain. 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. Pdf strategies for the use of bacteriocins in gramnegative. Grampositive bacteria become gramnegative when their cell wall is ruptured or removed.
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. This staining procedure differentiates microbes into two basic groups. Gram stain image analysis of the sizes, shapes and morphologies of the. Module common staining technique microbiology 24 notes indeterminate groups as well. A monograph of gram stained preparations of clinical specimens marler ms mtascpsm, linda m. The gram stain dallas county community college district. Gram positive cells form a crystal violetiodine complex. Gram s iodine iodine and potassium iodide is applied as a mordant or fixative. Sputum grams stain and culture can be performed noninvasively and. Gram stain results, including organism morphology, can be affected. Oct 23, 20 semiquantitative gram stain and culture methods are still commonly used for diagnosing ventilatorassociated pneumonia vap, due to its convenience. Both gram positive and gram negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet.
Using spectroscopy, researchers upended the belief that bacteria completely absorb the dye used to identify different bacterial types. 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. After staining, bacteria differentiation is possible because gram positive bacteria have a purple color. Wear chemical splash goggles, chemicalresistant gloves, and a chemicalresistant apron. The gram stain was first used in 1884 by hans christian gram. 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. Apr 12, 2016 schematic showing the modified gram stain procedure. 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.
Problems interpreting gram stain slide biology stack exchange. 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. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Table 2 use of bacteriocins on gramnegative bacteria in foods. The objective of this study was to assess the utility of semiquantitative. The original formulation comprised of aniline gentian violet, lugols iodine, absolute alcohol and bismarck brown. The gram stain was first used in 1884 by hans christian gram gram,1884. These staining procedures are used to distinguish organisms based on staining properties. Gram stain results, including organism morphology, can. Gram staining is the most sensitive method by which to visualize and recognize nocardiae in clinical specimens. Gram staining is a differential staining technique that differentiates bacteria into two groups.
This is in contrast to gram negative bacteria, which cannot retain the. The proper sequence of reagents in the gram stain procedure is. Decolorizer removes the stain from gram negative bacteria becoming colorless 4. The following are some common causes of poor gram stain results a. Semiquantitative gram stain and culture methods are still commonly used for diagnosing ventilatorassociated pneumonia vap, due to its convenience. The cviodine complexes were then counterstained with safranin 35 and used for the acquisition of gram stain images. The following points highlight the top two staining techniques for recognition of bacteria. This sensitization is important in food microbiology, because most pathogens that cause. Although gram stain is useful in staining bacteria, certain fungi such as candida. The filtergram stain was approximately 100fold more sensitive than the slidegram stain. 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. A monograph of gramstained preparations of clinical specimens marler ms mtascpsm, linda m. Oral microbiology bacteria causing inflammations and abscesses within the oral cavity. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology.
Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. 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. The filter gram stain was approximately 100fold more sensitive than the slide gram stain. 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. Modification of the brown and brenn gram stain for the differential staining of grampositive and gramnegative bacteria in tissue sections. Gramstain image analysis of the sizes, shapes and morphologies of the. Crystal violet sticks primarily to the peptidoglycan. With the current theory behind gram staining, it is thought that in grampositive bacteria, the crystal violet and iodine combine to form a larger. Slide safety precautions gram stain materials will stain skin and other materials. Bacteria that retain the initial crystal violet stain purple are said to.
The cviodine complexes were then counterstained with safranin 35 and used for the acquisition of gramstain images. At this point, slides were immersed in alcoholic saffron american master tech scientific, inc. The basic principle of gram staining is the properties of certain bacteria cell walls to retain the crystal violet dye. The traditional gram stain procedure is completed in either case top box. Pdf laboratory exercises for microbiology biology 552. An optimized staining technique for the detection of gram. It is also known as gram staining or gram s method. The process of identifying bacteria in the sample involves using different agents, such as crystal violet dye, iodine mordant, decolorant and safranin. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Counterstain staining with safranin turns gram negative bacteria red. Although gram stain is useful in staining bacteria, certain fungi such as candida and cryptococcus are observed as gram positive yeasts.