The gram stain is a type of differential stain that allows a microbiologist to. If you continue browsing the site, you agree to the use of cookies on this website. The staining procedure as originally presented by gram used. Gram staining is initially established by the physician hans christian gram, which was from denmark. Here are the following main principles of gram staining techniques. Distinguishes bacteria of two large and medically important groups. Often the first test conducted on an unknown species. Occurrence of a phosphoric ester in certain bacteria. A wet mount technique is used for staining bacterial flagella, and it is simple and useful when the number and arrangement of flagella are critical to the identification of species of motile bacteria. Place slide on staining tray or hold with forceps above the sink. To learn the techniques of gram staining, nigrosin staining and koh test. These are called gramnegative bacteria and they appear red after staining.
What are the factors that affect the gram staining procedure. Record the qc results on the gram stain qc sheet provided in annex 1. 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. Also, because depending on whether its gram positive or gram negative, thats going to determine which kind of drugs will be effective, and which will not be effective. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. This was developed in 1884 by a danish bacteriologist, hans christian gram. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they dont rinse out during the staining procedure. The gram stain procedure is a differential staining procedure that involves multiple steps. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. Ankur choudhary print question forum no comments 1. 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. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent.
During the early days of bacteriology the detection of. It is also a key procedure in the identification of. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology. Add the primary stain crystal violet and incubate 1 minute. Bacteria that stain purple with the gram staining procedure are termed. It is also known as gram staining or gram s method. Gram positive bacteria gram negative bacteria provides useful information for disease treatment. It is one of the differential stains that are used to characterize bacteria in one of two groups. Gram stain or gram staining, also called grams method, is a method of staining used to. Jan 17, 2016 this video provides instructions on how conduct a gram stain.
Scope this sop is applicable for gram staining in microbiology laboratory of pharmaceutical company name. The gram stain is the most widely used staining procedure in bacteriology. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the. 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. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than. Gram stain principle, reagents, procedure, steps, results. Dieter claus information centre for european culture collections, mascheroder weg 1b, d3300 braunschweig, germany introduction when bacteria are stained with certain basic dyes and treated with iodine some. Before ethanol treatment, the purple colour in gram negative bacteria is mainly due to the staining of outer lipid layer and thin peptidoglycan wall with crystal violet. The procedure steps of gram stain prepare and fix the specimen to the microscope slide before staining. Planted digital learning library gram stain procedure youtube. This standard operating procedure is applicable to all technical staff in the microbiology section that have been trained in performing this procedure and are competent and authorized to perform this procedure.
Add crystal violet liquid a onto the fixed smear slide. The gram staining procedure is one of the most basic and important tests performed in the clinical microbiology laboratory. Histology special stain methods, techniques, protocols. The book contains more than three hundred color images, the majority of which come from direct smear examination, such as gram stain and other staining procedures. Sop for microbial staining procedures standard operating procedure to stain the bacterial cells, bacterial spores and fungus. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Hold the slide in a tilted downward position and allow the. Google scholar mittwer t, bartholomew jw, kallman bj. June 12, 2018 by sagar aryal 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. Fields stain is a histological method for staining of blood smears. The filter gram stain was approximately 100fold more sensitive than the slide gram stain.
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. Asm journals are the most prominent publications in the field, delivering uptodate and authoritative coverage of both basic and clinical microbiology. The gram staining technique is the most important and widely used microbiological differential staining technique. Gram variable observations in gram staining the gram staining procedure does not always give clearcut results. Sign in to view the content sakshat amrita virtual lab. The gram stain procedure click start lab from a liquid culture, take a loopful of bacteria emulsify it in a small drop of water or saline on the slide. Laboratory perspective of gram staining and its significance in. Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle. The name comes from the danish bacteriologist hans christian gram, who developed the technique. Grams method involves staining the sample cells dark blue, decolorizing those. Acidfast stain principle, procedure, interpretation and examples. Bacteria are often classified into grampositive and gramnegative. The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Gram staining is a differential staining technique that differentiates bacteria into two groups.
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. Responsibility microbiologist or above of microbiology laboratory. Ppt gram stain procedure powerpoint presentation free. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope.
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. 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. Gram stain developed in 1884 by hans gram, a danish microbiologist. There are many factors that can affect gram staining procedures, some of them are. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram. How to do a grams stain test grams stain is one of the most frequently used processes in identifying bacteria and is used daily in hospitals.
On ethanol treatment, lipids are dissolved in ethanol and peptidoglycan wall is very thin to hold the stain so gram. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. The procedure involves staining bacterial samples with crystal violet. Decolorization the critical step of the gram staining procedure is the decolorization step. Huckers modification method gram staining procedure. These organisms are usually filamentous, gram positive rods of varying length that often form chains, but occasionally, they may stain gram negative. Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1. In microbiology, gram staining is an important test used because it can determine the presence of bacteria in a sample, as well as differentiate between the two distinct bacteria species, which are gram positive and gram negative bacteria. It has to be one of the most repeated procedures done in any lab. The mold phase and the microscopic structure of the identified fungal species relating to the initial findings of the direct smear have been linked to avoid bias.
Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. Flood slide with crystal or gentian violet 60 seconds. If the staining procedure is performed correctly, gram positive bacteria will be purple, while gram negative bacteria will be pink. 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 a complex and differential staining procedure that remains a useful test performed in microbiology laboratories. Flagella are too thin to be visualized using a bright field microscope with ordinary stains, such as the gram stain, or a simple stain. For gram positive staphylococcus aureus is used, for gram negative e. The gram stain is a very important preliminary step in the initial characterization and classification of bacteria.
It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. For confirmation of bacterial presence and identity, we used standard staining including gram and acidfast staining 33, 34. Oct 24, 2019 both gram positive and gram negative bacteria pick up the pink stain, but it is not visible over the darker purple of the gram positive bacteria. The crystal violet stain is the primary stain, which stains everything in the smear blue. 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. Nov 20, 20 gram stains just a few to remember slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Flagella stain principle, procedure and result interpretation. Oil red o staining protocol ellis papanicolaou staining protocol. 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. 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.
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. An infected appendix, or any tissue containing both negative and positive gram rods. 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. The difference in result is due to the differences in the cell wall structure or composition of bacteria that causes the different in the reaction with the series of reagents in gram staining talaro, 2007. Gram positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red. Flood the surface of the slide with crystal violet stain and let sit for one minute. What is the importance of gram staining in microbiology.
Nissl staining protocol for frozenvibratome sections. In thin films the red blood cells are fixed and parasitized cells can be identified by their morphology. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. The gram stain and differential staining journal of. Corynebacterium diptheria seen in gram staining 35. This theory relies on the fact that the ppg is found in layers and the stain molecules are trapped within the many layers of the gp cw when they form the complex with the mordant iodine molecules. It was developed by danish microbiologist hans christian gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. This will ensure that all areas are stained evenly. Gram positive rods, gram negative rods, gram postive cocci and gram negative cocci see images below. Remel gram stain kit, 4 x 250 ml bottles related applications. Jan 14, 2017 9 advantages and disadvantages of gram staining jan 14, 2017 oct 26, 2016 by editor in chief gram staining, which is also referred to as the grams method, is a scientific technique of staining that is used to differentiate the species of bacteria into 2 main groups, namely the grampositive and the gramnegative bacteria. A magnetic gram stain for bacterial detection ncbi nih.
The most accepted theory about the rationale for the gram staining process is the one proposed by salton. Acidfast stain principle, procedure, interpretation and. The gram stain the gram stain, originally developed in 1884 by christian gram, is probably the most important procedure in all of microbiology. Currently, the only control method for determining the validity of this.
The definitive diagnosis of malaria infection and other parasites is still based on finding such organisms in blood films. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. 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 relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. Bacteria that stain purple with the gram staining procedure are termed gram positive. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition.
Thermo scientific remel gram stain kit is for qualitative procedures to differentiate gram positive from gram negative microorganisms. As noted above, the decolorization step is critical to the success of the procedure. Widely used method for the identification of unknown bacteria. It is a primary test which quickly and cost effectively divides bacteria into one of two types. Gram stain principle, laboratory continuing education. Gram staining technique is the widely used differential staining technique in bacteriology. The most useful staining procedure in medical microbiology. It is feasible to combine the essential features of the 2 most useful bacteriologic staining procedures, i. Overheating of smears when heat fixation is used d. He help to distinguish klebsiella pneumonia to pneumococci. 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.
This should be a thin, not milky, suspension or it will not stain. Make a slide of tissue or body fluid that is to be stained. 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. Cover the smear with crystal violet, the primary stain, for 20 seconds. 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. Flood the slide with grams iodine and time for one. The gram s iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram. Introduction observation of bacteria with conventional bright field microscopy yields relatively little useful information. To prepare the bacteria culture slides, a volume of 1 ml of bacterial. Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology.
Principle, procedure, interpretation, examples and animation last updated. Staining flood the entire slide when crystal violet, iodine and safranin are applied. In addition this stain also allows determination of cell morphology, size, and arrangement. Laboratory perspective of gram staining and its significance. Huckers modification method gram staining procedure is used to microscopically identify gram positive and gram negative bacteria. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes.
Gram positive bacteria that include most of the firmicutes, and gram negative bacteria that include the rest. Module the gram stain procedure michigan state university. After 5 s, add liquid b and gently shake the slide to mix liquids a and b for 30 s. 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 stain reagents are evaluated by staining the following recommended bacterial strains. Unless it is a gram stain of a pure culture, there normally should be material staining. Protocols for using the gram stains in the microbiology laboratory. Objectives to learn the technique of smear preparation. This should be a thin, not milky, suspension or it will not sta. The staining procedure differentiates organisms of the domain. It is called a differential stain since it differentiates between gram positive and gram negative bacteria. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. Sop on gram staining objective to lay down the procedure for gram staining. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope.
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