Microbiology lecture 2 gram positive and gram negative. Gram stain principle, reagents, procedure, steps, results. Feb 04, 2016 christian gram, a danish physician in 1884 developed a staining technique to distinguish two types of bacteria. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than 1,000 bacteria. Therefore, to see bacteria with the microscope, it is necessary to apply colour by using a staining reagent. The gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria. Gram staining is based on the ability of bacteria cell wall to retaining. Pdf laboratory perspective of gram staining and its significance in. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram negative bacteria. Subsaharan african journal of medicine vol 1issue 4octdec 2014 168. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell.
To learn the techniques of gram staining, nigrosin staining and koh test. An optimized staining technique for the detection of gram positive. Smears or films of bacterial cultures and clinical specimens are usually fixed by heat, the slide being first throughly dried in air and then heated gently in a flame. Antibacterial activity of ethanol and water extracts of leaves of lagerstroemia speciosa l pers. It was devised by a danish physician, hans christian gram, in 1884. Simple stains use basic dyes which are positively charged. Bacterial characteristics gram staining video khan academy. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Bacteria are generally studied when fixed and stained. Special stains have been developed over time for identifying bacteria species, differentiating them morphologically, and even characterizing there very special features.
The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of. Alternatively, the gram stain is able to differentiate between gram positive and gram negative bacteria, and is the most used technique for classifying bacterial smears in vitro. The name comes from the danish bacteriologist hans christian gram, who developed the technique. The gram stain differentiates bacteria into two fundamental varieties of cells. The acidfast stain is a differential stain used to identify acidfast organisms such as mycobacterium from nonacid fast organisms. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. Gram stain, a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria. In particular, we were interested in evaluating the number of cells adhering to the slides following the gram staining procedure. This crosssectional study of gram negative staining bacterial contamination of milk meant for human consumption was carried out from october 2010 to may 2011 in gondar town, ethiopia.
If bacterial cells are present, they usually appear either purple or reddish brown, depending on whether or not the crystal violet is retained after the alcohol rinse. Gram positive bacteria stain bluepurple and gram negative bacteria stain red. And if it stains well, it stains purple, and if it gets washed off and stained with something else, it turns. Bacteria are first stained with crystal violet or gentian violet. Difference between gram stain and acid fast compare the. Others that are not stained by crystal violet are referred to as gram negative, and appear red. 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 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 first page of the pdf of this article appears above. Our objective was to examine gram staining results following fixation of both gram positive staphylococcus aureus and gram negative escherichia coli bacteria using heat versus methanol as means of fixation. This video demonstrates the gram staining method for identifying unknown bacter. Gram stains quickly tell not only if a bacterium is gram positive or gram negative, but also staining the cells reveals the shape of the bacterium its cell morphology.
Occurrence of a phosphoric ester in certain bacteria. To stain bacteria, a uniform smear is applied to a glass side and allowed to dry. Gram stain or gram staining, also called gram s method, is a method of staining used to distinguish and classify bacterial species into two large groups. Jun 12, 2018 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. 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. 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. Clinically, gram stain results allow for rapid intervention. The gram stain and differential staining journal of. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram.
Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Cell wall structure identifies either cell is gram positive or negative in nature during the procedure when we stained by primary stain and secure it by a mordant. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. The acid fast stain was discovered by ehrlich, who found that after staining. Gram staining of bacteria from environmental sources. Gram stain of sputum or examination of a bronchoscopically obtained specimen from a patient with anaerobic pneumonia reveals numerous polymorphonuclear leukocytes with an abundance of intracellular and extracellular bacteria. We will be monitoring our bioreactors for bacterial contamination. Bacterial classification, structure and function columbia university. Rapid method for detection of grampositive and negative.
The red dye safranin stains the decolorized gram negative cells redpink. In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. The links on this page provide the virtual microbiology classroom documents, web pages and videos useful in preparing for this lab. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram.
The presence of fungi should not be overlooked during the direct examination of the clinical specimens for bacteria. Different bacteria stain differently to a common staining procedure. Difference between gram positive and gram negative bacteria. May 19, 20 gram staining or gram s method is a method of differentiating bacterial species into two large groups gram positive and gram negative. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. Gram staining technique is the widely used differential staining technique in bacteriology. They include mycobacteria, mycoplasma, chlamydia and the rickettsial complex. The gram negative bacteria are decolorized, and are then ounterstained light pink by the subsequent application of safranin, neutral red or dilute carbol fuschin. Pdf difference between gram positive and gram negative bacteria. Subsaharan african journal of medicine vol 1issue 4 octdec 2014 168. A comparison of heat versus methanol fixation for gram. Objectives to learn the technique of smear preparation. Gram staining allows for quick visualization of bacterial morphology and broad cellular distinction from a wide range of environmental samples.
This was developed in 1884 by a danish bacteriologist, hans christian gram. The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Gram positive bacteria that include most of the firmicutes, and gram negative bacteria that include the rest. Three introductory chapters cover gram stain procedures, quality control, evaluation, and more. The two categories of bacteria based on gram staining are gram positive bacteria and gram negative bacteria. Here are the following main principles of gram staining techniques. It is also known as gram staining or gram s method.
An optimized staining technique for the detection of gram. Gram negative bacteria tend to exhibit lower fluorescence intensity than gram positive bacteria, because of their cell structure outer membrane impedes penetration of cfda. Most bacteria have no colour, so they generate little contrast in the microscope field. Laboratory perspective of gram staining and its significance in. Gram staining simple english wikipedia, the free encyclopedia. Khan academy is a nonprofit with the mission of providing a free, worldclass education for anyone, anywhere. The gram negative bacteria appear colorless and gram positive bacteria remain blue. 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. Acinetobacter baumannii and brucella species are gramnegative organisms that are vulnerable to misinterpretation as gram positive or gram. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. 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. Bacterial characteristics gram staining video khan.
Apr 12, 2016 an optimized staining technique for the detection of gram positive and gram negative bacteria within tissue. Oct 24, 2019 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. Introduction observation of bacteria with conventional bright field microscopy yields relatively little useful information. This suggests gramnegative bacteria are more leaky, causing these thinwalled lipidrich cells to lose. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. The difference between the two groups is believed to be due to a much larger.
The viabilitycytotoxicity assay kit for bacteria features dual staining. The bacteria concentrated on the surface of the membrane are stained with a cationic dye toluidine blue that can be visualized by the naked eye. Available in colors other dyes that preferentially stain gram positive bacteria include membrite fix and calcofluor white surface stains, nucspot live dna stains. We sometimes are culturing li as the organism of choice and in this case we want to make sure it is n. Gram staining principles gram staining is used to determine gram status to classify bacteria broadly. Typically, a mixture of gram stain reactions and morphologies are seen, including pale staining gram negative rods with. After heatfixing the smear, crystal violet is applied. Principle, reagents, procedure, step, result interpretation of 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.
For example, gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. To visualize decolorized gram negative bacteria, a red counter stain such as safranin is used after decolorization treatment appearance of the gram positive coccus and gram negative bacillus at different stages of the gram staining procedure are illustrated below. In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air dry. The gram stain is a type of differential stain that allows a microbiologist to. Milk is highly prone to contamination and can serve as an efficient vehicle for human transmission of foodborne pathogens, especially gram negative bacteria, as these are widely distributed in the environment. Gram staining is an essential procedure used in the identification of bacteria and is frequently the only method required for studying their morphology. Gram staining or gram s method is a way of classifying bacteria into two large groups. Dmao green for live cells, and ethdiii red for dead cells fig. After staining, the membrane is treated with ethanolacetic acid ph 2. The partially negatively charged bacterial cell walls forms an interaction with the dyes utilized in gram staining thus prompting the colors of the dye to adhere to the bacteria s cell wall. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. Gram s method stains bacteria according to the chemical and physical properties of their cell walls. During such times, the gram stain is the only technique available in the clinical microbiology laboratory for direct detection of fungi from these specimens.
Bacteria that retain the initial crystal violet stain purple are said to be gram positive, whereas those that a. Apr 04, 2016 a staining technique used to classify bacteria. Differentiate into gram positive and gram negative bacteria. Gram staining of bacterial cells is neither an accurate nor elaborate technique, but nevertheless it is practically useful to distinguish two big domains of eubacteria species. Gram staining is the differential staining that is used to differentiate the bacteria majorly into two groups i. The identification of an unknown organism typically begins with a gram stain. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Nov 25, 2019 prokaryotes bacteria and gram staining gram positive and gram negative btec101, day 5 why are we learning this. Two basic methods provide foundations for differential staining and detection of bacteria. The gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure.
Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. 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 chained, clustered of gram negative and gram positive bacteria. Method of staining used to distinguish and classify bacterial species into two large groups. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent.
The name comes from its inventor, hans christian gram. Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear figure 1. So now we know about the three main shapes that bacteria can take, but we havent talked too much about the color yet, and this color comes from a special stain called the gram stain, and it basically stains the outside of the bacteria. Pdf gram staining is an empirical method of differentiating bacterial species into two large groups grampositive. Gram negative bacteria tend to be more resistant to certain antibiotics than gram positive bacteria. Eyram ganu gram staining introduction in this lab experiment, we were introduced to the technique of gram staining. Gram positive bacteria stain blue while gram negative stain red. The gram stain easily divides bacteria into two groups, grampositive and gramnegative, on the basis of. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Gram staining of bacteria from environmental sources protocol.
Gram staining for differentiating bacterial species youtube. Gram staining is a differential staining technique, which separates bacteria into two groups known as gram positive bacteria and gram negative bacteria. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. The gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial. Gram positive bacteria has only the inner plasma membrane and no outer membrane. The most common stain being gram staining, acidfast staining, endospore staining. The gram positive bacteria resist decoloration and remain stained a dark purple colour. The gram stain is a classical and important staining technique that remains widely used by environmental microbiologists.
A comparison of this new method with well known gram stain methods demonstrates its superiority in differentially staining gram positive and gramnegative bacteria in control experimental tissue lesions when types of fixatives, duration of tissue storage prior to paraffin embedment, types of embedding agents, and section thickness are varied. The acid fast stain was discovered by ehrlich, who found that after staining with. Each of these stains aims at identifying and characterizing bacteria based on their morphologies. Jun 10, 2006 such staining methods are called differential staining methods, these include gram staining and acid fast staining.
However, within tissue sections, adaptations of this technique e. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology. 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. Find information and process for the preparation of gram staining regent. Fluorescent gram stains wheat germ agglutinin wga is a carbohydratebinding lectin that preferentially binds to gram positive bacteria fig. Gram positive and gram negative on the basis of cellwall difference and by the sequential application of crystal violet, iodine, alcohol and safranin. We will discuss this and other applications of gram staining in more detail in later chapters. Learn for free about math, art, computer programming, economics, physics, chemistry, biology, medicine, finance, history, and more. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement.
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