Staining techniques 1. Slide is flooded with a basic dye such as Crystal violet or Methylene blue for a minute or so. In1921, this technique was further developed by Hucker. Immunohistochemical Staining Techniques. II. In this staining technique a longer heat treatment and prolonged staining technique. STAINING AND BACTERIAL CELL MORPHOLOGY I. Observing dead cells by making use of chemical nature of their unicellular, body. Gram Staining. Cell wall structure of Gram+ and Gram- 22 GRAM STAIN Cell Color Calcofluor White Staining: It is commonly used to directly detect fungal elements and to observe the subtle characteristics of fungi grown in culture. 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. Differentiation of cells: Differences in the staining reaction such as that between acidophilic and basophilic cells help greatly in the identification of certain cell types found in smears. This staining was named as gram staining to honor the contribution of Hans Christian Gram. Actin (skeletal muscle) CD3 (T cell lymphocytes) CD20 (B cell lymphocytes) CD117 (c-kit expression) Cytokeratin AE1/AEC (epithelial cells) Desmin (muscle, Cytoplasm of smooth, striated and cardiac muscle) Factor VIII (endothelial cells) 3. In a previous section, we described the detection of immune cell expansion by quantitating antigen-specific T cells using tetramer staining. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. The positive charged dye is attached to the Bacterial cell which has negative charge and thus staining takes place. OBJECTIVES • To learn the technique of smear preparation. This type of stain allows us to observe the shape, size and arrangement of bacteria. . The Procedure of Leishman staining may vary as per the purpose of staining that means whether the staining is done for the examination of Blood cells Morphology, Toxic Granules in Leucocytes, Type of Anemia etc. 2. Acid Fast Stain. White blood cells comprise a diverse collection of leukocytes mediating a variety of immunologically related functions. and also, the protocol may vary as per the Standard Operating Procedures (SOPs) of the Laboratory. Morphological Unknown I. These antibody staining methods often require the use of frozen section histology. Almost incidentally he noticed that white blood cells were also brilliantly colored by the solution. Bacterial Staining Techniques I. Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. Mycobacterium species due to its special cell wall resist the effect of the decolorizer acid-alcohol and retain the color of the primary stain carbolfuchsin stain and stains the acid fast cells in bright red color. An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged dyes. Mycobacterium species contains large amount of mycolic acid in its cell wall. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. The specimen does not need to be heat-fixed prior to negative staining. In cell biology and biology in general, cytochemistry is the study of the (bio-chemical) chemical constituents of the cell. Principles: Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of the microscopic image. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Purpose The purpose of this lab is to learn how to prepare a wet mound, to learn proper staining techniques and to examine human cheek cells and onion skin cells. This technique is effective for vegetative cells. In 1891, Romanowsky successfully used methylene blue solution to detect malarial parasites in blood. . negative … Endospore gets stained due to longer heat treatment, prolonged staining and heavy concentration of stain. Here we pass he slide from flame for about 25 times in addition we use concentrated stain that is 7.6 % Malachite green for about 10 minutes. Other advanced techniques include in situ hybridization to identify specific DNA or RNA molecules. Differential Staining Techniques. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. Staining can be performed with basic dyes such as crystal violet or methylene blue,... simple stain procedure. Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is typically used for capsule staining. • To learn the techniques of Gram staining, nigrosin staining and KOH test. Staining is of different types depending on their purpose; simple staining using a single stain to provide a contrast between the sample and the background; differential staining using more than one dyes in order to differentiate between cells or between organelles of a cell. This technique involves culturing immune cells in the presence of Golgi block to inhibit intracellular protein transportation. The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell. The primary objective of pathological techniques is the diagnostic classification of pathologically altered tissue and the assessment of cell morphology ().In addition to post-mortem examination, histological and cytological evaluation of tissue is the main task in pathology. The Gram stain procedure is a differential staining procedure that involves multiple steps. This staining technique is used to demonstrate the presence of acid and alcohol fast bacilli (AAFB) which have waxy envelopes that make them difficult to stain and decolorize. An acid-fast stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not. PROCEDURE OF LEISHMAN STAINING. This is The Most Important staining technique in Bacteriology. Due to high lipid content, most dyes cannot enter easily through the cell wall, so Mycobacterium species cannot be stained by Gram staining. In 1882, Hans Christian gram invented the staining technique and published in 1884. 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. Romanowsky found that best staining was obtained when methylene blue reagent was aged and formed a surface film. Hypothesis It is predicted that all cells, no matter if it be plant or animal, will be amalgamated in large groups. Bacterial Staining Techniques II I. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results. Another differential staining technique is acid-fast technique which differentiates species of Mycobacterium from other bacteria. Both progressive and regressive nuclear staining techniques can be used in Papanicolaou stain. Gram Staining Procedure/Protocol: Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. The cell wall of most bacteria has an overall net negative charge and thus can be stained directly with a single basic (positively charged) stain or dye. Gram-staining was firstly introduced by Cristian Gram in 1883 which used to differentiate the Gram-positive microorganisms and Gram-negative microorganisms. Differential Staining Techniques Viewing Bacterial Cells. 4. Acid fast staining is a differential staining technique which differentiate acid fast and non-acid fast bacteria. INTRODUCTION Also that most of… Differential Stains: Gram Stain and Acid-fast Stain II. This is achieved by staining. Called immunohistochemistry, this technique has greatly increased the ability to specifically identify categories of cells under a microscope. Individual cell types can be microscopically distinguished by gross morphology and by staining with cytochemical dyes. Common Cell Biology Techniques The following list covers some of the more commonly used cell biology techniques – it is by no means exhaustive. The Gram Staining Steps Include: In this method, heat is used to help drive the primary stain into the waxy cell walls of these difficult-to-stain cells. Staining Techniques Simple stain techniques. 1. Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of the microscopic image.Stains and dyes are widely used in the scientific field to highlight the structure of the biological specimens, cells, tissues etc. 21 Common Staining Technique MICROBIOLOGY MODULE Microbiology Notes 2.2 TERMS RELATED TO STAINING Stain A stain is a substance that adheres to a cell, giving the cell color. STAINING TECHNIQUES 2. Which fixes the cells of slide, kill most of the micro-organism and prepare them for staining. The cell bodies vary greatly in diameter (15–60 μm), have several long or short dendritic processes and adrenergic vesicles can be demonstrated in the substance of the cell body by specific fixation and staining techniques. Auramine-Rhodamine technique: This fluorochrome staining method is used to enhance the detection of mycobacteria directly in patient specimens and initial characterization of cells grown in culture. Using cytochemistry, it has also become possible for scientists and technicians to localize various chemical components or elements, which may be enzymatic or non-enzymatic in nature. Summary. • 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. Cytochemistry Methods, Specific Techniques and Stains in Microscopy What is Cytochemistry? Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents.