Here is a table which elaborates different types of staining techniques with their purpose and examples. In the past, conventional identi fi cation of fungi relied on the combination of morphological and physiological properties. Using theses bacterial culture techniques students discover and isolate the bacteria present around us. The most useful staining procedure in medical microbiology. Kits are available commercially, or formulas for reagents are listed in appendix b. 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. It is almost always the first test performed for the identification of bacteria. In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. Version 4202400003 lab report assistant this document is not meant to be a substitute for a formal laboratory report. An important laboratory technique for the amplification and detection of specific dna sequences as, for. Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol. A counterstain, safranin, is then used to give color to the nonsporeforming bacteria.
The name comes from the danish bacteriologist hans christian gram, who developed the technique gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. Gram stain pure strain cultures to determine whether gram negative or gram positive. It is another important differential staining procedure. It is most commonly used to identify mycobacterium spp. Flagella stain principle, procedure and result interpretation. Gram staining is a differential staining technique that differentiates bacteria into two groups. Gram stain developed in 1884 by hans gram, a danish microbiologist. Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. Introduction bacteria are too small to see without the aid of a microscope.
General microbiology morphology of bacterial cell, staining procedures for bacteria, culture media for bacteria, cultivation and isolation techniques for bacteria, preparation of pure cultures. Alternative staining techniques kinyoun or acid fast stain are therefore used that take advantage of the resistance to destaining after lengthier initial staining. With proper staining technique gram positive bacteria will stain purple. Bacterial morphology and staining techniques handson labs, inc. Pdf staining techniques and biochemical methods for the. Gramnegative bacteria are those bacteria that do not retain crystal violet dye in the gram staining protocol. Observe the slide under the microscope, using proper microscope technique. In recent years, morphological studies, supplemented with staining techniques and biochemical methods, still play an. Types of staining techniques used in microbiology and. Flagella are too thin to be visualized using a bright field microscope with ordinary stains, such as the gram stain, or a simple stain. Following isolation of bacteria, students characterize the bacteria with household disinfectant products and. At the end of the procedure, spores stain green and other cells stain red. Different bacteria stain differently to a common staining procedure. For demonstrating gramnegative and grampositive in tissue.
Observation of bacteria with conventional bright field microscopy yields relatively little useful information. Grampositive bacteria gramnegative bacteria provides useful information for disease treatment. Classic gram staining techniques involve the following steps. In a similar way as in acidfast, in the gram stain, a bacterial smear is dried and then heatfixed to denature the cell proteins and to cause bacteria to adhere to the glass slide. Staining, therefore, is of primary importance for the. The gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure. The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. Similar to a simple stain, it allows for assessment of bacterial cell morphology e. Failure to follow these directions may cause staining artifacts and disrupt the normal morphology of bacteria and cells. Gram negative bacteria give pink colour gram positive. Bacteria vary in certain physiological and biological properties. Such staining methods are called differential staining methods, these include gram staining and acid fast staining. Bacterial smears provide scientists with the means to look at microorganisms preparation of bacterial smears is required for bacterial staining procedures. Gram staining detects a fundamental difference in the cell wall composition of bacteria.
Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. 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. Methods of culturing microorganisms 3 five basic techniques 4 fig. However, while gram staining is a valuable tool for the identification of a bacterial organism, not all bacteria can be definitively classified by this technique. Some staining techniques involve the application of only one dye to the. 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. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet.
Introduction as bacteria consist of clear protoplasmic matter, differing but slightly in refractive index from the medium in which they are growing, it is difficult with the ordinary microscope, except when special methods of illumination are used, to set them in the unstained condition. Gram stain is a very important differential staining techniques used in the initial characterization and classification of bacteria in microbiology. Bacterial taxonomy today depends upon the extent of dna sequence homology. Some staining techniques, such as the gram and ziel neelsen stains, although of great diagnostic. The primary stain of the grams method is crystal violet. These bacteria have cell wall with high lipid content such as mycolic acid a group of branched chain hydroxy lipids, which prevent dyes from readily binding to cells. Staining techniques staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Microscopic slides endospores a stained preparation of bacillus subtilis showing endospores as uncoloured and the vegetative cell as. The staining procedure differentiates organisms of the domain. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. The staining characteristics of bacteria in the gram stain are very useful in classification.
This staining technique divides bacteria into two groups namely acidfast and non acidfast. Malachite green is used with heat to force the stain into the cells and give them color. Both bacteria s, positive and negative, cell wall is composed of peptidoglycan,the grampositve has a thicker wall and both will take up the crystal violet. Some staining techniques, such as the gram and ziel neelsen stains, although of great diagnostic value because of their differential staining properties for specific bacteria, reveal little internal structure. Some bacteria such as mycobacteria are not reliably stained due to the large lipid content of the peptidoglycan. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis tb along with some others of this genus. Bacillus spp and clostridium spp are main endospore producing bacterial genera.
This procedure is extensively used in the diagnosis of tuberculosis and leprosy. Perform bacterial gram staining with remainder of the culture from step 1. The microscope is a very important tool in microbiology, but there are limitations when it comes to. The smear is then stained with crystal violet dye, which is rinsed off and replaced with an iodine solution. 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. To learn the techniques of smear preparation, gram staining, nigrosin staining and correlating the results of gram staining with koh test. The gram stain is a classical and important staining technique that remains widely used by environmental microbiologists. Gramstaining is a differential staining technique that uses a primary stain and a. 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.
There are three kinds of staining techniques as mentioned below. The steps of the gram stain procedure are listed below and illustrated in table 1. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology. Classification of bacteria basic microbiology online. Capsule staining principle, reagents, procedure and result. Preparing smears disperses bacterial cells on a slide so that individual cells can be easily visualized under the microscope. 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. Types, techniques, preparations and procedures microscopy refers to the practice that involves the use of a microscope for the purposes of observing small scale structures that cannot be viewed using the naked eye and often cell staining is necessary as s tructures are. Types of different staining techniques of microorganisms. The gram stain was first used in 1884 by hans christian gram.
Distinguishes bacteria of two large and medically important groups. While some eucaryotes, such as protozoa, algae and yeast, can be seen at magnifications of 200x400x, most bacteria can only be seen with. Gram staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by danish bacteriologist hans. Some staining techniques utilize these differences to stain the bacteria differently. Gram positives are violet, while gram negatives are red. Gram staining of bacteria from environmental sources. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram.410 1245 642 423 680 236 19 1513 725 763 897 1141 423 332 100 158 1000 1465 686 228 315 622 9 1079 1165 498 1076 1134 667 774 241 799 359 507 1414 1109 104 87 1465 743 19 968 309 51 370 10 419 1496 675