The filter gram stain was approximately 100fold more sensitive than the slide gram stain. You will be asked about differential staining, grampositive and. The gram stain technique basic protocol is a differential staining technique since it. 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. 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. Gram staining is a differential staining technique that differentiates bacteria into two groups. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. Gram stain hans christian gram history and principles of gram. Nov 04, 2016 i would like to give an elaborate explanation for the reagents being used in this gram staining and why it is used.
They are transparent, and their detection is difficult under living and unstained conditions. Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. Gram stain principle bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as gram positive bacteria. The ziehlneelsen acidfast stain is a specialized stain used to target mycobacteria, nocardia, and some parasites. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized. 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. Principle, procedure, interpretation, examples and animation. The lipoid capsule of the mycobacteria is of such high molecular weight that it is waxy at room temperature and successful penetration by the aqueousbased staining solutions such as grams is prevented. Gram positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red. The gram stain is a staining procedure used to not only make. Get a printable copy pdf file of the complete article 3. Most bacteria can be differentiated by the gram reactions due to difference in their cell wall structure. Acidic dye stains the basic components of the cell and basic dye stain the acidic components of the cell. Calcofluor white staining technique was used as a comparative stain specifically for the identification of onychomycosis fungal infection.
The most important and widely used differential stain for bacteria is the gram stain. It oxidizes the carbon to carbon bonds between two adjacent hydroxyl groups. The different response of the two groups to the gram stain is based on fundamental differences in cell wall structure and composition. Principle giemsa stain is used to differentiate nuclear andor cytoplasmic morphology of platelets, rbcs, wbcs, and parasites 1,2. The chitin containing structures that fluoresce bright white under ultraviolet light in a fluorescent microscope. Gram staining is initially established by the physician hans christian gram, which was from denmark. The stain is retained because the cell wall of a grampositive bacteria is not as permeable as the cell wall of a gramnegative bacteria.
The gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. Corynebacterium diptheria seen in gram staining 35. Christian grams to differentiate between gram positive and negative bacteria. Gram stain principle, reagents, procedure, steps, results. I would like to give an elaborate explanation for the reagents being used in this gram staining and why it is used. Gramnegative pseudomonas aeruginosa bacteria pinkrods. The filtergram stain was approximately 100fold more sensitive than the slidegram stain. There are three major types of staining techniques. The cell walls for grampositive microorganisms have a higher peptidoglycan and lower lipid content than gramnegative bacteria.
The most dependable stain for blood parasites, particularly in thick films, is giemsa stain containing azure b. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. A monograph of gram stained preparations of clinical specimens marler ms mtascpsm, linda m. Name the 3 solutions and times involved in the ziehlneelsen stain 1 primary stain carbolfuchsin 5 mins. Calcofluor white staining online microbiology notes. So the gram stain, when you first apply it to a bacteria, it just stains the whole thing purple, and then youll wash it off. Pdf gram staining procedure find, read and cite all the research you need on researchgate.
Google scholar mittwer t, bartholomew jw, kallman bj. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. And because the gram negative bacteria has this very thin peptidoglycan layer in their cell wall, it washes right off, and later theyll restain it with something called safranin, which isnt important, but they come in. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Gram staining is still the cornerstone of bacterial identification and taxonomic division. In spite of his defective method, his name lives on jay hardy, cls, sm ascp hile working with infected lung tissue. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. The next most commonly used diagnostic technique is acidfast staining that is used primarily to detect mycobacterium tuberculosis and other severe infections. Pas stain principle initial reaction the first reaction makes use of a periodic acid that acts as an oxidizing agent.
Bacterial characteristics gram staining video khan. The gram stain is a complex and differential staining procedure that remains a. The structure of the organisms cell wall determines whether the organism is gram psitive or negative. In gram s stain, the section was stained with crystal violet and gram iodine, followed by decolorization. The gram stain the gram stain, originally developed in 1884 by christian gram, is probably the most important procedure in all of microbiology. Gram stain principle, laboratory continuing education. 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. In grams stain, the section was stained with crystal violet and gram iodine, followed by decolorization. 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. Gram stain principles, method and value flashcards quizlet. What is the principle and procedure of gram staining.
Gram stain reagents are checked weekly and also when a new lot of stain is to be put into use, as per the clia quality control regulatory notice published july 18, 1997. Links to pubmed are also available for selected references. In the gramnegative cell wall, the amount of lipids is very high, when the cell is dissolved in alcohol, it leads to the formation of large pores in the cell. On the basis of their reaction to the gram stain, bacteria can be divided into two large groups. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. 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. The polychromatic pap stain involves five dyes in three solutions. Principle, procedure, interpretation, examples and. Atcc 25923, staphylococcus aureus and attc 25922, escherichia coli. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. The csf concentration of bacteria does alter the likelihood of visualizing an organism. Natural dye hematoxylin is the nuclear stain which stains cell nuclei. The gram stain should almost always be done as the first step in diagnosis of a bacteria infection the gram stain is named after the danish scientist hans christian. Pas stain periodic acidschiff stain procedure and uses.
The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. 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. Flood slide with crystal or gentian violet 60 seconds. The gram stain learning the basics richard tom thomson, jr. 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. The gram stain dallas county community college district. Iodine forms complexes with crystal violet to fix the stain.
The filtergram stain detected 2 to 100 bacteria, whereas the slidegram stain failed to detect less than. The name comes from the danish bacteriologist hans christian gram, who developed the technique. Differentiate into gram positive and gram negative bacteria. The stain is retained because the cell wall of a gram positive bacteria is not as permeable as the cell wall of a gram negative bacteria. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on the slide. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. It has to be one of the most repeated procedures done in any lab. You will be asked about differential staining, gram positive and. The gram stain procedure distinguishes between gram. This test differentiates the bacteria into grampositive and gramnegative bacteria, which helps in the classification and differentiation of microorganisms. The cell walls for gram positive microorganisms have a higher peptidoglycan and lower lipid content than gram negative bacteria. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the. The lipoid capsule of the acidfast organism takes up carbolfuchsin and resists decolorization with a dilute acid rinse.
The gram stain was first used in 1884 by hans christian gram gram,1884. Papanicolaou stain includes both acidic and basic dyes. Every student and professional who works with bacteria has to, at some point, learn how to do a gram stain. 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. When the bacteria is stained with primary stain crystal violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol.
Cover the fixed smear with crystal violet stain for 30 seconds and then wash off with water. Gram stain reagents are evaluated by staining the following recommended bacterial strains. Jul 01, 2019 microbiology lecture 2 gram positive and gram negative bacteria gram staining principle this second microbiology lecture will explain the properties of gram positive and gram negative bacteria. Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as grampositive bacteria. This test differentiates the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiation of microorganisms. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Grampositive bacteria stain deep violet to blue and gramnegative bacteria stain pink to red.
Microbiology lecture 2 gram positive and gram negative. Jul 28, 2017 bacteria are very small microorganisms. A monograph of gramstained preparations of clinical specimens marler ms mtascpsm, linda m. The gram stain is a type of differential stain that allows a microbiologist to. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. As a result, bacteria are classified as either grampositive or gramnegative. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on. Occurrence of a phosphoric ester in certain bacteria. The gram stain was developed by christian gram in 1884 and modified by hucker in 1921.
Microbiology module common staining technique microbiology 22 notes 2. Microbiology lecture 2 gram positive and gram negative bacteria gram staining principle this second microbiology lecture will explain the properties of gram positive and gram negative bacteria. It is recommended that, in order to determine the tendency of an organism with regard to the gram stain, more than one staining procedure be used, and that preparations of the culture be prepared at various stages of growth from 12 hours to several days in age. Bacterial characteristics gram staining video khan academy. The lipoid capsule of the mycobacteria is of such high molecular weight that it is waxy at room temperature and successful penetration by the aqueousbased staining solutions such as gram s is prevented. These online assessment tools will help you strengthen your understanding of the gram stain method. In addition this stain also allows determination of cell morphology, size, and arrangement.
When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting declorization while others get decolorized by a decolorizer. Stain dark purple due to retaining the primary dye called crystal violet in the cell wall. Full text full text is available as a scanned copy of the original print version. The gram stain, the most widely used staining procedure in bacteriology. The gramreaction rely upon the chemical nature of the bacterial cell wall, especially the lipids which comprise 1122% in gramnegative cell wall and 14% in the grampositive cell wall. Principle, reagents, procedure, step, result interpretation of gram stain.
First of all, the gram staining was named after dr. Thus, various staining methods are developed to facilitate bacterial detection. In the gram negative cell wall, the amount of lipids is very high, when the cell is dissolved in alcohol, it leads to the formation of large pores in the cell. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. The gram reaction rely upon the chemical nature of the bacterial cell wall, especially the lipids which comprise 1122% in gram negative cell wall and 14% in the gram positive cell wall.
506 390 958 684 219 1390 211 561 373 252 1390 305 197 950 164 352 1534 1062 1373 3 207 432 812 1145 1593 758 66 543 849 174 1039 1125 168 1454 1001 95 1223