Characteristic gram negative bacteria gram positive bacteria wall structure they have a thin lipopolysaccharide exterior cell wall. A modified gram stain was developed and tested as an alternative to gram stain that improves the contrast between gram positive bacteria, gram negative bacteria and host tissue. Bacterial classification, structure and function columbia university. After heatfixing the smear, crystal violet is applied. They are transparent, and their detection is difficult under living and unstained conditions. Misinterpretation of the gram stain has led to misdiagnosis or delayed. Gram staining is an essential procedure used in the identification of bacteria and is frequently the only method required for studying their morphology. National public health laboratory world health organization. Grampositive bacteria thick layer of peptidoglycan90% of cell wall stains purple. The gram stain technique basic protocol is a differential staining technique since it reacts differently with different types of bacteria based on their cell wall. In a similar way as in acidfast, in the gram stain, a bacterial smear is dried and then heatfixed to denature the cell proteins and to cause bacteria to adhere to the glass slide. Gram staining lab introduction gram staining is a very important technique used in biology labs all over the world. It has to be one of the most repeated procedures done in any lab.
The acid fast stain was discovered by ehrlich, who found that after staining with. Bacteria viability was evaluated by two different tests. Then, the cells are treated with a mordant, gram s iodine, which enters into the cells and binds to the primary stain forming an insoluble dyemordant complex crystal violetiodine complex, thus. 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. The goal of the current study was to develop a modification of the gram stain that enhances the contrast between bacteria and host tissue. Lab 34 bacterial vaginosis centers for disease control. Slides are sequentially stained with crystal violet, iodine, then destained with alcohol and counterstained with safranin. Method of staining used to distinguish and classify bacterial species into two large groups. Thus, various staining methods are developed to facilitate bacterial detection.
The primary stain of this method is crystal violet, which. Key difference gram stain vs acid fast bacteria are very small microorganisms. The gram stain and differential staining journal of. Stain dark purple due to retaining the primary dye. The staining procedure differentiates organisms of the domain. Gram stain, a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria. Gram stain developed in 1884 by hans gram, a danish microbiologist. Asm journals are the most prominent publications in the field, delivering uptodate and authoritative coverage of both basic and clinical microbiology. To stain bacteria, a uniform smear is applied to a glass side and allowed to dry.
An optimized staining technique for the detection of gram positive. A gram stain is a lab test used to detect bacteria or fungi in a sample taken from the site of a suspected infection. Excess water left on the slide will dilute reagents, particularly grams iodine. The gram stain the gram stain, originally developed in 1884 by christian gram, is probably the most important procedure in all of microbiology. Older cultures contain more ruptured and dead cells. This cell is easily lysed and it is metabolically active but unable to reproduce. Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. All other cases of bacterial meningitis are reportable when a culture result becomes available. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Some species of bacteria are described as gram variable and may stain blue or red or show both colors e. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. Full text full text is available as a scanned copy of the original print version. Used staining liquid should be discarded in the liquid waste chemical containers 2. Some staining techniques, such as the gram and ziel neelsen stains, although of great diagnostic value because of their differential staining properties for specific bacteria, reveal little internal structure.
The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology. By exploiting this phenomenon, an alternative gram staining technique has been developed. Gram stain hans christian gram history and principles of gram. Introduction bacteria are too small to see without the aid of a microscope. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. Using a solvent to remove the stain from some types of bacteria. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. Experiment to perform gram staining of bacteria with figure. The gram stain differentiates bacteria into two fundamental varieties of cells. Brown and hopps method was the most satisfactory for distinguishing gram positive and gram negative bacteria.
The gram stain procedure was originally developed by the danish physician hans christian gram. Gram staining technique is the widely used differential staining technique in bacteriology. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. The 100x oil immersion lens is essential for viewing individual bacteria. This was developed in 1884 by a danish bacteriologist, hans christian gram. Gardnella has an unusual gram positive cell wall structure that causes bacteria of this genus to stain gram negative or gram variable sadhu et al 1989. Gram positive bacteria stain bluepurple and gram negative bacteria stain red. 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.
Get a printable copy pdf file of the complete article 582k, or click. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than 1,000 bacteria. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Objectives to learn the technique of smear preparation. Pdf gram staining is an empirical method of differentiating bacterial. In gram staining, the bacteria cells are first stained with a primary stain, crystal violet that enters into the cells and stain them purpleblue. Gram positive rods, gram negative rods, gram postive cocci and gram negative cocci see images below.
Available in colors other dyes that preferentially stain gram positive bacteria include membrite fix and calcofluor white surface stains, nucspot live dna stains. For meningococcal meningitismeningococcemia any the following presents as a suspect case and is reportable. Gram staining is a differential staining technique that differentiates bacteria into two groups. Cells from old cultures may stain gram negative even if the bacteria are gram positive. Difference between gram stain and acid fast compare the. In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air dry. An optimized staining technique for the detection of gram. A fundamental laboratory technique that is introduced in general biology and microbiology courses is staining of bacterial cells on glass slides. Gram positive bacteria are bacteria that give a positive result in the gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their cell wall gram positive bacteria take up the crystal violet stain used in the test, and then appear to be purplecoloured when seen through an optical microscope.
Grampositive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. In the gram stain, an insoluble crystal violetiodine complex is formed inside the cell, and this complex is extracted by alcohol from gramnegative but not from grampositive bacteria. Gram stain, gram positive, gram negative, heat fixation, methanol fixation. A bacterium is referred as a protoplast when it is without cell wall. It is also known as gram staining or gram s method. The peptidoglycan layer of gram negative bacteria is covered by a membrane and is not labeled by the lectin. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram negative bacteria. It gives relatively quick, preliminary results as to whether microbes are present and, if so, the general types causing an infection. Pdf laboratory perspective of gram staining and its significance in. The acidfast stain is a differential stain used to identify acidfast organisms such as mycobacterium from nonacid fast organisms. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram.
Introduction observation of bacteria with conventional bright field microscopy yields relatively little useful information. 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. This will allow you to focus quickly and to look for areas of purulence. While some eucaryotes, such as protozoa, algae and yeast, can be seen at magnifications of 200x400x, most bacteria can only be seen with. Cytoplasm of gram positive bacteria is more acidic, hence can retain the basic dye crystal violet for longer.
Get a printable copy pdf file of the complete article. Gram positive bacteria gram negative bacteria provides useful information for disease treatment. Gram stain primary test where gram positive and gram negative terms come from. A comparison of heat versus methanol fixation for gram. Gram staining is still the cornerstone of bacterial identification and taxonomic division. The peptidoglycan layer is thick effect of dye do not retain the crystal violet dye, and react only with a counter stain, generally stain pink. Fluorescent gram stains wheat germ agglutinin wga is a carbohydratebinding lectin that preferentially binds to gram positive bacteria fig. It is almost always the first test performed for the identification of bacteria. Gram positive pathogenic bacteria are staphylococci, streptococci, pneumococci, etc gram negative.
Gram staining allows for quick visualization of bacterial morphology and broad cellular distinction from a wide range of environmental samples. Gram stain is reliable only on cells from cultures that are in the exponential phase of growth. It was devised by a danish physician, hans christian gram, in 1884. The gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions.
Gramnegative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out. A differential stain like that invented by hans christian gram in 1882 will give you more information and allow you to group the stainable bacteria into more groupings. The gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria. Gram stain and culture, using the vortex elution method according to the clsi 40a document. Alternate gram staining technique using a fluorescent lectin. A new technique for gram staining paraffinembedded tissue. The name comes from the danish bacteriologist hans christian gram, who developed the technique. It is a technique used to differentiate types of bacteria using certain physical and chemical characteristics of their cell walls. Gram smears should be discarded in the autoclave bin 12. Five techniques for gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies.
In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear figure 1. 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. Basic fuchsin binds to lipids in the cell wall and turns mycobacteria red while other bacteria stain blue. Gram staining of bacteria from environmental sources.
It allows a large proportion of clinically important bacteria to be classified as either gram positive or negative based on their morphology and differential staining. The cell walls of grampositive organisms retain this complex after treatment with. Evaluation of the eswab transport system to collect. To learn the techniques of gram staining, nigrosin staining and koh test. 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. Gram staining is a differential staining technique, which separates bacteria into two groups known as gram positive bacteria and gram negative bacteria.
413 1230 828 387 730 1188 1410 787 163 1604 1395 1229 578 1087 1410 981 490 204 954 474 373 867 600 1169 974 96 1330 638 664 32 24 457 710 651 413 917 246 1396 853 742 1016