For use in the gram s staining method for the initial differentiation of gram positive and gram negative bacteria. Jun 10, 2006 such staining methods are called differential staining methods, these include gram staining and acid fast staining. Some laboratories may choose to use a cytospin slide centrifuge to concentrate body fluids for s mear preparation. 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. Laboratory perspective of gram staining and its significance in. 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. Summary and explanation the gram stain was originally devised by christian gram in 1884. Learn vocabulary, terms, and more with flashcards, games, and other study tools. To learn the techniques of gram staining, nigrosin staining and koh test. This was developed in 1884 by a danish bacteriologist, hans christian gram. Gram stain synonyms, gram stain pronunciation, gram stain translation, english dictionary definition of gram stain.
Fourteen samples containing bacteria were tested one time each and once for each bacterial type. Csf and other body fluids requiring centrifugation. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. 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 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. The aim of this study was to validate a simplified grading of gram stained vaginal smears for the diagnosis of bv in gum clinics. Gram staining of bacteria from environmental sources. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Actinomyces israelii gram stain grampositive bacteria that form long. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. In recent reports, the impact of gram staining results on patient mortality has been documented. We developed a rapid and sensitive method for the differentiation of gram positive and gram negative bacteria, utilizing a polycarbonate membrane filter, crystal violet, iodine, 95% ethanol, and 6% carbol fuchsin, that can be completed in 60 to 90 s. A rapid method for distinction between gram negative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 gram positive and 55 gram. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents.
This protocol is taken from bensons microbiological applications. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. The contrast between a cell, which is primarily water, and the background, which is water, is poor. Get a printable copy pdf file of the complete article 3. The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria. It is used for initial assessment of specimens prior to culture and is an important step in the workup and identification of pathogens isolated from clinical specimens.
Google scholar mittwer t, bartholomew jw, kallman bj. Laboratory manual in general microbiology eleventh edition 1. The gram stain is one of the most commonly performed tests in the clinical microbiology laboratory, yet it is poorly controlled and lacks standardization. These online assessment tools will help you strengthen your understanding of the gram stain method. Spore stain procedure schaefferfulton method youtube. The traditional gram stain procedure is completed in either case top box. 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. Gram stain definition of gram stain by the free dictionary. Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. February 2, 2015 acharya tankeshwar bacteriology, staining techniques in microbiology 45. As a result, bacteria are classified as either gram positive or gram negative. To visualize these cells, the contemporary gram stain procedure applies the counterstain safranin to the smear causing cells to appear red or pink. The primary stain of this method is crystal violet, which can be sometimes substituted with equally effective methylene blue. Gram staining technique is the widely used differential staining technique in bacteriology.
An optimized staining technique for the detection of gram. Rapid method for detection of grampositive and negative. Pdf gram staining is an empirical method of differentiating bacterial. Gram staining is an empirical method of differentiating bacterial species into two large groups gram positive and gram negative based on the chemical and. Introduction to staining live specimens are difficult to see with the bright field microscope. Staining and streaking protocols for simple stain, gram stain, streak plate technique and culture maintenance lab 2a. Apr 12, 2016 schematic showing the modified gram stain procedure. Notes on the gram stain with description of a new method. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Gram staining of bacteria from environmental sources protocol. In spite of his defective method, his name lives on jay hardy, cls, sm ascp hile working with infected lung tissue.
The results of testing the bacterial stock cultures diluted to 1. 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. 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. The patients were a highly selected group and no prevalence data were obtained. 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. It is important to note that the expected results of the modern gram stain are best generated from young, actively growing 1824 hourold cultures. Gram stain article about gram stain by the free dictionary. At lower concentrations, the gram stain of a clinical specimen seldom reveals organisms even if the culture is positive. Pdf gram staining procedure find, read and cite all the research you need on researchgate. The gram stain procedure is a differential staining procedure that involves multiple steps. Objectives to learn the technique of smear preparation. 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. You will be asked about differential staining, gram positive and.
It is also known as gram staining or gram s method. In addition this stain also allows determination of cell morphology, size, and arrangement. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology. The gram stain is a classical and important staining technique that remains widely used by environmental microbiologists. Occurrence of a phosphoric ester in certain bacteria. Available literature does not mention them, but they are so simple that acetone must have been used in this manner before. Rapid method for the differentiation of grampositive and. This method has been recommended to increase gram stain sensitivity and to decrease centrifugation and examination time for more rapid results. Different bacteria stain differently to a common staining procedure. A staining technique used to classify bacteria in which a bacterial specimen is first stained with crystal violet, then treated with an. It is one of the differential stains that are used to characterize bacteria in one of two groups.
The staining procedure differentiates organisms of the domain. It is a primary test which quickly and cost effectively divides bacteria into one of two types. Protocols for using the gram stains in the microbiology laboratory. The standard gram s staining method can be used to differentiate intact, morphologically similar bacteria into two groups.
The composite criteria described by amsel et al 3 were used as the reference method as this is still regarded as the gold standard. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. 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. Validation of a simplified grading of gram stained vaginal. Although technologic advances, such as malditofbased identification, have allowed for dramatic reductions in the time required for blood pathogen identification, 8, 10, 11 they have yet to replace the gram stain as the most economical and efficient method of acquiring actionable data from a positive blood culture result. I hope this site and the others are helpful to you as free online atlases to. 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. With the increasing cost of alcohol and the difficulty of obtaining the pure substance, the following uses of acetone for alcohol may prove of interest. Beyer individual supplies microscope slide clothes pin inoculating loop or wood splint paper towels shared supplies crystal violet stain safranin red stain grams iodine ethyl alcohol denatured water bottle rinsing basin alcohol burner wax pencil matches microscope create a staining area on your microscope slide obtain a clean microscope slide draw a.
It was once the best rapid test in microbiology, but it is no longer trusted by many clinicians. 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. Oct 16, 2012 this video demonstrates the gram staining method for identifying unknown bacter. The name comes from the danish bacteriologist hans christian gram, who developed the technique. 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.