Schematic showing the modified gram stain procedure. The traditional gram stain procedure is completed in either case top box. Jun 10, 2006 such staining methods are called differential staining methods, these include gram staining and acid fast staining. Gram stain protocols american society microbiology. Heat fix the bacteria to the slide by passing it through the flame of. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. To a gram positive cell the counter stain stains the cell, but the cells are already so heavily stained by crystal violet that the addition of pink does not change the dark purple color of the cells.
Flood the gram s iodine for 1 minute and wash with water. Oct 24, 2019 the gram stain is a differential method of staining used to assign bacteria to one of two groups gram positive and gramnegative based on the properties of their cell walls. Microbiology 101 the gram stain outbreak news today. Variations in gram staining results learn microbiology. How to perform a gram staining procedure wonderhowto.
Huckers modification method gram staining procedure is used to microscopically identify gram positive and gram negative bacteria. The gram stain is a complex and differential staining procedure that remains a. The purpose of the gram stain is to show whether the bacteria are gram positive purplecolored, gram negative pinkcolored, or both. Sep 05, 2014 this general microbiology practical lecture explains the gram staining procedure and the principle behind it. Different bacteria stain differently to a common staining procedure. Gramnegative and gram positive are more than just the results of tests to identify sickness. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents.
Apr 04, 2016 gram variable observations in gram staining the gram staining procedure does not always give clearcut results. Huckers modification method gram staining procedure. If the control slides do not prove satisfactory, the staining procedure is not accepted. Gram stain principle, reagents, procedure, steps, results.
An infected appendix, or any tissue containing both negative and positive gram rods. Bacteria species can be separated into two large groups, which are the gram positive and gramnegative groups through the gramstaining sridhar rao, n. The cell wall can stain either positive or negative, depending on its chemistry. 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. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. Wash off with dh2o, blot dry and examine under microscope. This is applied and stains the gram negatives pink.
Gram staining is a method commonly used to determine the chemical make up of the cell wall of bacteria. 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 crystal violet stain is the primary stain, which stains everything in the smear blue. Briefly, the average number of lactobacilliary morphotypes per oil immersion field will be quantified. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. Staining flood the entire slide when crystal violet, iodine and safranin are applied. Apr 12, 2016 gram stain is the most common staining technique used diagnostically within both the clinical setting and in research laboratories to differentiate between gram positive and gram negative microorganisms in various types of tissues 17, 26.
This video tutorial show you medical doctors how exactly to perform a gram staining procedure. For this project, youll need a heatfixed bacteria slide, gram s lodine, crystal violet, safranin, decolorizer, and water. Decolorization the critical step of the gram staining procedure is the decolorization step. Materials and methods aseptic technique was used throughout the experiment. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Gram staining or grams method is a way of classifying bacteria into two large groups. Air dry and heat fix crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram.
It is one of the differential stains that are used to characterize bacteria in one of two groups. Gramstaining is usually the first step in identification bacteria and can be used in characterizing bacteria. Combining the objective lens and eyepiece allows for higher magnification than using a single lens alone. The reagents you will need to successfully perform this operation are. The specimen should be mounted and fixed on a slide before you procede to stain it. Gram staining procedure questions and study guide quizlet. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. This will ensure that all areas are stained evenly. The name comes from its inventor, hans christian gram.
Responsibility microbiologist or above of microbiology laboratory. Grams stain does not cross the bacterial cytoplasmic membrane. Place slide with heat fixed smear on staining tray. Add the primary stain crystal violet and incubate 1 minute. Evaluation of a new technique for the demonstration of gonococci. Gram staining is a four part procedure which uses certain dyes to make a bacterial cell stand out against against its background. Hold the slide in a tilted downward position and allow the decolorizer to flow over the smear. 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. A gramstaining technique combining staining with two fluorescent stains, oregon. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. With these types of organisms, gram positive and gramnegative cells may be present within the same preparation 40.
I will also at some point add information on the use of fluorescence microscopy to reduce gstaining to a onestep procedure. The name comes from the danish bacteriologist hans christian gram, who developed the technique. We developed a rapid and sensitive method for the differentiation of gram positive and gramnegative. If the morphology is uncertain, the gram stain reaction and morphology from a fourhour broth culture should be evaluated. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Steps place a small drop of bacterial sample on a slide. Occurrence of a phosphoric ester in certain bacteria. Scope this sop is applicable for gram staining in microbiology laboratory of pharmaceutical company name. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. Hopps method, utilizes a slide basket and staining dishes for.
The developed magnetic gram stain method was then used to. Use a dropper to apply the primary stain crystal violet to the slide and allow it to sit for 1 minute. Gram negative bacteria will be pink and gram positive bacteria will be purple. 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. If the slide is heat fixed, allow it to cool to the touch before applying the stain. Make a slide of tissue or body fluid that is to be stained. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. The standard staining protocol used to differentiate between gram positive and gram negative bacteria was adapted from moyes, reynolds and breakwell 2005. Prepare the smear of suspension on the clean slide with a loopful of sample. Grams method stains bacteria according to the chemical and physical properties of their cell walls. The procedure involves staining bacterial samples with crystal violet, which.
Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. The gram stain is the most widely used staining procedure in bacteriology. Positive and negative control slides should be used every time the staining procedure is performed except for gram staining where positive controls may be enough unless a new batch of stain is made. The counter stain used in the gram stain is safranin pink. The gram stain, the most widely used staining procedure in bacteriology. Gently flood the smear with grams iodine and let stand for 1 minute. Overheating of smears when heat fixation is used d. Gram staining is a differential staining technique that differentiates bacteria into two groups. Batch staining for demonstration of gram positive and gram. Sop on gram staining objective to lay down the procedure for gram staining. Bet you didnt know gram stains are performed on body fluid or biopsy when infection is presumed. Endospores are difficult to stain with standard staining techniques because they. 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 gramnegative, based on the chemical and physical properties of their cell.
Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1. Request pdf grams stain does not cross the bacterial. 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 stain is probably one of the most commonly used staining procedures used in the field of microbiology. Grampositive organisms in tissues were easily seen even at low.
An optimized staining technique for the detection of gram. Gram staining or grams method is an emprical method of differentiating bacterial species into two large groups gram positive and gramnegative based on the chemical and physical properties of their cell wall. Scribd is the worlds largest social reading and publishing site. Gently flood smear with crystal violet and let stand for 1 minute. These organisms are usually filamentous, gram positive rods of varying length that often form chains, but occasionally, they may stain. Experiment to perform gram staining of bacteria with figure. The primary dye is crystal v iolet and the secondary dye is usually either safranin o or basic fuchsin. Gram stain permits the separation of all bacteria into two large groups, those which retain the primary dye gram positive and those that take the color of the counterstain gram negative. Gram staining free download as powerpoint presentation. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. The bv score for gram staining will be calculated by nugents method 1991. Some organisms are gramvariable and may appear either gram negative or gram positive according to the conditions.