Detection of chlamydia trachomatis inclusions in mccoy and. Biologist hans christian gram devised one of the most important staining techniques used in microbiology to identify bacteria under a. Detection of fungi with various stains is quick and simple, and can be optimized through the use of readytouse depending on the consistency of the specimen material, it may be necessary to perform a simple pretreatment step with alkali prior to staining. The interaction force will depend on the nature of the sample, the probe tip and the distance between them. The vegetative bacterial cells are stainable with aqueous dyes, but endospores possess. Can be changed by staining, which increases contrast between specimen and surrounding medium.
A laboratory manual pdf using our direct download link mentioned at the end of this article. The previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. The cell walls of fungi will bind the stain calcofluor white, which greatly enhances visibility of fungal element in tissue or other specimens. Jun 10, 2006 bacteria vary in certain physiological and biological properties. Negative staining principle, procedure and result interpretation objectives of negative staining to perform a negative staining procedure. Such staining methods are called differential staining methods, these include gram staining and acid fast staining. By exploiting this phenomenon, an alternative gram staining technique has been developed. Special staining endospore capsule flagella metachromatic granules spore staining if spore bearing organisms are stained with ordinary dyes, or by grams stain, the body of the bacillus is deeply coloured, whereas the spore remains unstained.
Module context this module introduces microbiological laboratory techniques to be used for analysis of coliforms bacteria can be used as indicators of pollution. Testing sensitivity to antimicrobial substances 21 2. Principle of negative staining negative staining requires the use of an acidic stain such as india ink or nigrosin. Screening techniques and its details general microscience. The flagella can be visualized by the special staining techniques in which their thickness is increased by mordanting, or. Microbiology is a popular option for practical work in schools. Gram stain is a very important differential staining techniques used in the initial characterization and classification of bacteria in microbiology. The stain must not be allowed to evaporate and dry on the slide. Gram staining of bacteria from environmental sources protocol. Techniques for controlling variability in gram staining of. Hot zn stain is the usual method in which we heat the smear to enhance the dye penetration. Introduction bacteria are too small to see without the aid of a microscope. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism. Gram staining primary stain step 1 crystal violet is the primary stain used in the gram staining process it is not specific for gram positive cells and it stains everything the same color purple.
Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. He did not share his techniques with other scientists. Staining can be performed with basic dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial cytoplasm. Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. Microbiology module common staining technique microbiology 20 notes 2 common staining technique 2. Bacterial flagella are tiny hairlike organelles of locomotion. Cover the slide with filtered carbol fuchsin and heat until steam rises. Laboratory safety rules observations and study of structure of microbes microbial physiology. Alternate gram staining technique using a fluorescent. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. 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. Learn staining techniques microbiology with free interactive flashcards.
Classification of bacteria basic microbiology online. 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. Different bacteria stain differently to a common staining procedure. Toluidine blue staining was used to detect chlamydia trachomatis inclusions in both mccoy and hela229 cells from clinical specimens. Allow the preparation to stain for 5 min, heat gently. An introduction to microbiology, aseptic technique and safety page number preparation safety guidelines 1 risk assessment 1 good microbiological laboratory practice gmlp 3.
Flowchart of capsule staining by manevals method mechanism. Scribd is the worlds largest social reading and publishing site. Stains and dyes are frequently used in histology the study of tissue under the microscope and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses disease at a microscopic level. It is commonly used to directly detect fungal element and to observe the subtle characteristics of fungi grown in culture. Stains and dyes are used to color bacterial cells and increase their contrast can be seen more easily with a microscope. In microbiology the concept of staining is very important because it highlights the structures of microorganisms allowing them to be seen. Do not allow the pipette to touch any other surface. This method was more sensitive than iodine staining for detecting c trachomatis inclusions in both mccoy and hela229 cells and also more sensitive than giemsa staining for detecting chlamydial inclusions in hela229 cells. The microbiology society, in association with the microbiology in schools advisory committee misac. Staining, therefore, is of primary importance for the. Manual of histologic staining methods of the armed forces institute of pathology armed forces institute of pathology u. This chapter of the manual is concerned mainly with risks from infectious agents, and also with physical and chemical injuries in microbiology laboratories. Staining will increase the contrast between a cell and its background. Staining is a technique used to enhance contrast in samples, generally at the microscopic level.
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. In simple staining, a single dye is used to emphasize particular structures in the specimen. Some staining techniques, such as the gram and ziel neelsen stains, although of great diagnostic value because of their. 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 difficult to discern due to insufficient contrast.
Stains and dyes are frequently used in biological tissues for viewing, often with the aid of different microscopes. Biochemical tests the sole purpose of biochemical test in microbiology is to differentiate between various classes of bacteria by studying their enzymatic activities. One covers the fixed smear with stain for specific period, after which this solution is washed off with water and slide blotted dry. Has a safety section and frequent safety cautions throughout. Principle, procedure, interpretation, examples and animation last updated. Basic practical microbiology a manual compiled by john grainger. Staining techniques in microbiology a presentation by g. The effect of the staining technique on morphological and.
View lab report lab report 2 staining technique from hmm 1011 at multimedia university, bukit beruang. So in this method first we use congo red stain as it is acidic stain it stains the background in red colour. The sample of soil used is serially diluted in sterile distil water. Histologic staining options available include, but are not limited to. Objectives to learn the technique of smear preparation. 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. Gram staining of bacteria from environmental sources. The most important lesson from 83,000 brain scans daniel amen tedxorangecoast duration. Originating in the cytoplasm beneath the cell wall, they extend beyond the cell, usually equaling or exceeding it in length. The gram stain is a classical and important staining technique that remains widely used by environmental microbiologists. Alright, now in this part of the article, you will be able to access the free pdf download of microbiology.
Module common staining technique microbiology 22 notes 2. To use and relate the gram stain to the study of bacterial cell morphology, and as an. Alternative staining techniques kinyoun or acid fast stain are therefore used that take advantage of the resistance to destaining after lengthier initial staining. Fluorescencelabeled wheat germ agglutinin binds specifically to nacetylglucosamine in the outer peptidoglycan layer of grampositive bacteria. An introduction to microbiology, aseptic technique and safety preparation. As we know capsules are nonionic in nature so they do not get stain easily. Alcohols used for wiping the skin may cause hemolysis and artifacts. The staining method was found to significantly affect the dimensions of the boar sperm head. Staining is a valuable technique used in microscopy to enhance contrast in the microscopic image. Choose from 500 different sets of staining techniques microbiology flashcards on quizlet. Differential staining techniques viewing bacterial cells.
Observe the slide under the microscope, using proper microscope technique. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one. Then apart from all this some other staining techniques are there for identification of fungus like lactophenol cotton blue staining, and many other techniques. Gram staining differentiates bacteria based on the. The gram stain is a differential stain commonly used in the microbiology laboratory that. His daughter donated 100 of his microscopes to the. By using the negative staining method demonstration of capsule becomes easy. These staining procedures are used to distinguish organisms based on staining properties. Staining simply means coloring of the micro organisms with the dye that emphasizes and elucidate different important structures of microorganisms including bacteria, virus, protozoa and etc. It is used to distinguish between grampositive and gramnegative bacteria, which have distinct and consistent differences in their cell walls. The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Staining methods histology and cytology 152 notes sources of errors irregular distribution of the blood smear on a glass slide may result in an erroneous cell counts.
The following points highlight the top two staining techniques for recognition of bacteria. Holding the bottle of broth in your left hand, insert the pipette into the bottle and withdraw 10 ml of broth. To learn the techniques of gram staining, nigrosin staining and koh test. Also, because negative staining involves deposition of heavy atom stains. They were so difficult to focus, he built a new one for each specimen, a total of 419. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. The media comes to temperature, meaning it cools down. Gram staining is a differential staining technique that differentiates bacteria into two groups. Here source used for screening of microorganism is generally soil. Hans christian gram, is one of the most important staining techniques in microbiology. Bsci 424 pathogenic microbiology fall 2000 the gram stain the gram stain is the most important and universally used staining technique in the bacteriology laboratory.
While some eucaryotes, such as protozoa, algae and yeast, can be seen at magnifications of 200x400x, most bacteria can only be seen with. The gram staining method, named after the danish bacteriologist who. The tip may be dragged across the surface, or may vibrate as it moves. Types of staining techniques used in microbiology and. They are slightly more elaborate than simple staining techniques that the cells may be exposed to more than one dye or stain, for instance use of gram staining which divides bacteria into two classesgram negative and gram positive. 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. Colouration of microorganisms by applying single dye to a fixed smear is termed simple staining. Staining microscopic specimens microbiology openstax.
Microorganisms not only very small, they are also transparent. Some bacteria such as mycobacteria are not reliably stained due to the large lipid content of the peptidoglycan. What this does is it kills the cell so it is dead and therefore cannot move around on your slide when lookin. Mar 15, 2017 to fix a sample of bacteria to a slide, you have to first heat it over a bunson burner called heat fixing or something similar to one. Choose from 500 different sets of microbiology staining techniques flashcards on quizlet. Introduction quality assurance many of the stains that are described in this national standard method nsm are commercially available. Do not let the specimens dry at any stage of the staining procedure. Two of the most important concerns are resolution and contrast.
In microbiology the concept of staining is very important because it highlights the structures of microorganisms allowing them to be seen under a microscope simple and. To ensure safer downloads, we have uploaded this pdf ebook file to our online file repository. Special stains differential stains basic techniques of. This manual, which explains the basic techniques necessary to carry out microbiology experiments safely and effectively, is intended as a guide for teachers and technicians. Check out the quiz on gram staining technique and enhance your knowledge about the concept. As light passes through a glass slide, air, and the objective lens, it bends each time, causing loss of light. Their fine protein structure requires special staining techniques for demonstrating them with the light microscope. Stains are used to highlight structures in clinical specimens, often when viewed with the aid of different microscopes. Feb 03, 2015 flagella staining flagella is the organ of locomotion, which is one or more unbrached, long filament. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology. Under anaerobic conditions, grampositive staining occurred in all test organisms with quick fixing techniques with both absolute methanol and formalin.
Has a convenient, portable 6 x 9 trim size, a spiral binding and soft cover, making it ideal for use on the lab bench surface. 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. Leeuwenhoeckssimple microscopes allowed him to magnify images from 100 to 300 x. Cell staining in microscopy types, techniques, preparations and procedures. An alternative is to use a dye such as nigrosin or congo red, acidic, negatively charged dyes. Bacteria that retain the initial crystal violet stain purple are said to be grampositive, whereas those that are decolorized and stain red with carbol fuchsin or safranin are said to be gramnegative. Some staining techniques utilize these differences to stain the bacteria differently. Dispense 3 ml of broth into the glass culture tube. The purple, crystalviolet stained cells are referred to as grampositive cells, while the red, safranindyed cells are gramnegative figure 2. Whereas in cold zn staining, instead of heating, we increase the concentration of the basic dye and phenol and incorporate a wetting agent chemical. Types of different staining techniques of microorganisms.
This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. This fluorochrome staining method is used to enhance the detection of mycobacteria directly in patient specimens and initial characterization of cells grown in culture. The lactophenol cotton blue stain methods for clinical specimens and colonies clarified in separate titles. To understand the benefit obtained from visualizing unstained microorganisms. In the screening of antibioticproducing microorganism, a crowded plate technique is used. Modified bismarck brown staining for demonstration of soft tissue mast cells pdf. Hmm1011 basic microbiology lab report 2 staining technique name. Risks from infection are reduced by good laboratory techniques and secure facilities, which aid in the containment of pathogens. The gram stain is a type of differential stain that allows a microbiologist to identify the differences between organisms andor differences within the same organism. Demonstrates those techniques that will be used frequently for studying microbes in the laboratory. The presence or absence of flagella and their number and arrangement are characteristic of different genera of bacteria. Overheating of smears when heat fixation is used d. 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. Protocols for using the gram stains in the microbiology laboratory.
Accurate gram staining of american type culture collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber. Test and improve your knowledge of microbiology laboratory techniques with fun multiple choice exams you can take online with. Microscopy using the microscope 22 stained preparations 23 making a smear 23 a simple stain 24 a differential stain. An optimized staining technique for the detection of gram positive. Some staining techniques involve the application of only one dye to the sample. When two substances have a different index of refraction, the light will bend as it passes from one material to another.