Huckers modification method gram staining procedure. To learn the techniques of gram staining, nigrosin staining and koh test. Below are all common mistakes made during gram staining. The gram stain, the most widely used staining procedure in bacteriology. Gram staining a useful laboratory technique sciencedirect. College of medicine alimam mohammad bin saud islamic university first year 20 2014 1 by. Gram staining is a rapid, easily learned laboratory technique. Start studying lab exam 2 pore stain, gram stain, acid fast stain.
Gramstaining is a four part procedure which uses certain dyes to make a bacterial cell stand out against against its background. The name comes from the danish bacteriologist hans christian gram, who developed the technique. Cover the smear with crystal violet stain and leave for 1 minute. A counterstain, such as the weakly water soluble safranin, is added to the sample. Gently flood smear with crystal violet and let stand for 1 minute. Acidfast stain principle, procedure, interpretation and. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes.
Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. Hold the slide at a 45 degree angle and rinse throughly with water 6. His method for identifying these two groups became the first step in any bacterial identification process. Student procedures illinois institute of technology. The gram stain the gram stain, originally developed in 1884 by christian gram, is probably the most important procedure in all of microbiology. An optimized staining technique for the detection of gram. Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. Hold the slide at a 45 degree angle and rinse throughly with water 8.
An optimized staining technique for the detection of gram positive and gram negative bacteria within tissue. Sop on gram staining objective to lay down the procedure for gram staining. Gram staining gram staining is the single most important and universally used staining technique in the laboratory. Bios 318 staining and interpretation rice university. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. 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 is a quick procedure used to look for the presence of bacteria in tissue samples and to characterise bacteria as grampositive or gramnegative, based on the chemical and physical properties of their cell. Gram stain protocols american society of microbiology.
Ankur choudhary print question forum no comments 1. Heat fix the bacteria to the slide by passing it through the flame of a bunsen burner three. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The automated gramstaining characterisation of bacterial cells using colour and cell wall properties by extracting geometric features has been carried out by. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Although commonly used in other clinical areas, gram staining has not been in standard nursemidwifery practice. Wash slide in a gentle and indirect stream of tap water for 2 seconds. A decolorizer such as ethyl alcohol or acetone is added to the sample. Performing the staining without the decolorization process would result in labeling both grampositive and negative species, since the gramnegative species would also retain the cvtco. It is called a differential stain since it differentiates between grampositive and gramnegative bacteria. These organisms are usually filamentous, gram positive rods of varying length that often form chains, but occasionally, they may stain gram negative. The gram stain is the most widely used staining procedure in bacteriology.
Next, a grams iodine solution iodine and potassium iodide. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Gram positive bacteria will be purple and gram negative bacteria will be colorless. Staining procedures scope of document this test procedure contains the methods for the staining procedures commonly used in clinical microbiology laboratories for the identification of pathogens. A rapid method for distinction between gramnegative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71. This gram stain kit allows you to differentiate bacteria using the gram staining method. The name comes from its inventor hans christian gram who developed the technique in 1882 published 1884. Gram staining tips page 2 of 2 some specimens on preparation will have areas on the slide that are thicker than others. The staining involves 3 major stepsprocesses that include.
Gram stain is one of the most common staining procedures. Module common staining technique microbiology 24 notes indeterminate groups as well. Materials and methods aseptic technique was used throughout the experiment. Scope this sop is applicable for gram staining in microbiology laboratory of pharmaceutical company name. Postlab reflectio n for use with either the virtual or handson labpracticum 1. Gram staining is a differential staining technique that differentiates bacteria into two groups. Most bacteria can be differentiated by the gram reactions due to difference in their cell wall structure. 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 word gram is always spelled with a capital, referring to hans christian gram, the inventor of gram staining gram staining principles gram staining is used to determine gram status to classify bacteria broadly. The advantages of such a procedure, particularly as augmentation to wet mounts, suggest that adoption of gram staining may be of great use in the clinical area. Prepare the smear of suspension on the clean slide with a loopful of sample. Record the qc results on the gram stain qc sheet provided in annex 1. Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011 novaultra special stain kits principle. It is one of the differential stains that are used to characterize bacteria in one of two groups.
Overlay the slide with grams iodine and let stand for one minute 7. Huckers modification method gram staining procedure is used to microscopically identify gram positive and gram negative bacteria. 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. Each uk smi method has an individual record of amendments. Introduction observation of bacteria with conventional bright field microscopy yields relatively little. This can result in areas that are under decolorized, thus organisms that are truly gram negative will appear gram positive. Differentiate into gram positive and gram negative bacteria.
In this staining, method bacteria are classified into two groups that are. The cell wall can stain either positive or negative, depending on its chemistry. Automated gramstaining characterisation of bacterial. Hearfix the smears, gram stain them with the above procedure, and examine them. National public health laboratory world health organization. It is a primary test which quickly and cost effectively divides bacteria into one of two types. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Overheating of smears when heat fixation is used d.
The grams iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram. It has to be one of the most repeated procedures done in any lab. At lower concentrations, the gram stain of a clinical specimen seldom reveals organisms even if the culture is positive. 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. Gently flood the smear with grams iodine and let stand for 1 minute. Use a dropper to apply the primary stain crystal violet to the slide and allow it to sit for 1 minute. By modifying the above procedure, the detection strategy can be applied to detect both grampositive and gramnegative bacteria.
Staining and interpretation of smears preparing a smear gram stain procedure and examination negative staining spore staining observation of living bacteria important information such as shape and degree of motility can be obtained by observation of living bacteria with the phase contrast or dark field microscope. 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. Principle, reagents, procedure, step, result interpretation of gram stain. Gram staining is still the cornerstone of bacterial identification and taxonomic division. 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. 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. Compare the appearance of the cells in the two smears. 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. Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology.
Gram staining is a universal staining technique used for identification and classification of organisms. The specimen should be mounted and fixed on a slide before you procede to stain it. Pdf gram staining procedure find, read and cite all the research you need on researchgate. 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. Flood the smear with gram s iodine solution and leave for 1 minute. Drain off the iodine wash the slide for the again in a gentle. Objectives to learn the technique of smear preparation. Pour off the stain and gently rinse the excess stain with a stream of water from a. Sop for microbial staining procedures standard operating procedure to stain the bacterial cells, bacterial spores and fungus. Responsibility microbiologist or above of microbiology laboratory. Knowing the chemical make up makes it easier to manipulate the bacteria for. Every student and professional who works with bacteria has to, at some point, learn how to do a gram stain. The primary stain of this method is crystal violet, which can be sometimes substituted with equally effective methylene blue. Place slide on a staining rack and overly the smear with crystal violet solution.
Use this kit to teach students how to do a gram stain for a fun and informative biology lesson. Acidfast stain principle, procedure, interpretation and examples. Place slide with heat fixed smear on staining tray. Gram stain principle, reagents, procedure, steps, results. The cationic dye crystal violet is used to stain the nucleic acids of the microorganisms and background tissues. For gram positive staphylococcus aureus is used, for gram negative e. The primary dye is crystal v iolet and the secondary dye is usually either safranin o or basic fuchsin. Gram staining is a method commonly used to determine the chemical make up of the cell wall of bacteria. The reagents you will need to successfully perform this operation are. The crystal violet stain is the primary stain, which stains everything in the smear blue.
The gram stain is a differential method of staining used to assign bacteria to one of two groups grampositive and gramnegative based on the properties of their cell walls. Introduction quality assurance many of the stains that are described in this national standard method nsm are commercially available. The purpose of the gram stain is to show whether the bacteria are gram positive purplecolored, gram negative pinkcolored, or both. Gram staining procedure was discovered by hans christian gram in 1884.
When focusing the broth smear use the technique suggested for thin smears. Bacteria which stain purple with the gram staining procedu re are termed grampositive. Flood the gram s iodine for 1 minute and wash with water. This 4 min incubation represents a short addition to the staining process already in clinical use. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Learn vocabulary, terms, and more with flashcards, games, and other study tools. 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. To be visible on a slide, organisms that stain by the gram method must be present in concentrations of a minimum of 104 to 105 organismsml of unconcentrated staining fluid. The gram stain is a complex and differential staining procedure that remains a useful. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment.
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