Lecture 8: Bacterial Stains - PDF

LECTURE 8 Bacterial Stains Ms. Peters  Bacteria Staining The lack of pigment in most cells make structural details of organisms difficult to visualize under a light microscope. As such cells are subjected to dyes and stains that binds to cellular components and make...

LECTURE 8 Bacterial Stains Ms. Peters  Bacteria Staining The lack of pigment in most cells make structural details of organisms difficult to visualize under a light microscope. As such cells are subjected to dyes and stains that binds to cellular components and make them more visible. Specific staining techniques can be used to determine the cells’ biochemical or structural properties, such as type of cell wall and presence or absence of endospores Information ascertained from staining can help laboratory scientists in identifying and classifying infection causing microorganisms and thus assist with a medical diagnosis.  Bacteria Staining Based on the types and number of dyes used, staining can be categorized into different types.  Bacteria Staining Simple Stains Are basic dyes that produce color contrast to bacteria components The same color is given to all the bacteria in the smear Used to determine a bacteria size, morphology and arrangement but does not provide any additional information. Basic stains have positive charges that bind strongly to negatively charged cell components such as bacterial nucleic acids and cell walls.  Bacteria Staining Simple Stains Cont. E.g., methylene blue or basic fuchsin Bacillus (rod) shaped bacteria Staphylococcus sp. (simple stain, simple stained with crystal violet. methylene blue)  Bacteria Staining Differential Stains Uses two stains to impart different colors to differentiate between different types of microorganisms on a slide E.g., Gram’s Staining, Acid Fast Staining, Endospore Staining  Bacteria Staining Differential Stains Cont. – Gram staining Used in the initial characterization and classification of bacteria. Helps to identify bacterial pathogens by their Gram reaction (Gram-positive and Gram-negative) and morphology It uses crystal violet as the primary stain, iodine as mordant and safranin as counterstain. Crystal violet is applied, then the mordent. This is followed by adding the decolorizer (acetone). Finally, the counterstain, safranin is added. The gram-positive bacteria retain the primary stain even after acetone treatment while gram-negative bacteria lose the primary stain and take up the counterstain  Bacteria Staining Differential Stains Cont. Gram staining – Analysis of the stained slide shows gram-positive bacteria in blue-purple color and the gram-negative bacteria in red color.  Bacteria Staining Differential Stains Cont. Acid Fast Staining (Ziehl-Neelsen Technique) – A differential stain used to detect bacteria with mycolic acid cel walls (differentiate acid fast from non - acid fast bacteria) – Carbol fuchsin, the primary stain, is applied. It is a highly lipid soluble containing phenol. – This property helps the stain penetrate the cell wall. This penetrations is assisted by heat. – The carbol fuchsin binds to the mycolic acid in the mycobacteri cell wall. – After the primary stain, an acid decolorizer is applied. – The decolorizer strips the stain from cells but are unable to penetrate acid fast organisms  Bacteria Staining Differential Stains Cont. Acid Fast Staining (Ziehl-Neelsen Technique) – Finally, the smear is counterstained with malachite green, or methylene blue which stains the background material, providing a contrast color against which the red AFB can be seen. – Analysis of the stained slide, reveals red acid - fast bacteria against a blue or green background while non - acid fast bacteria will take up the counter stain and appear blue or green  Bacteria Staining Differential Stains Cont. Endospore Staining – Endospore staining methods are important in bacterial identification. – The primary stain, malachite green floods the slide which is then heated for 5 minutes. (observing for continuous steam) – The slide is then gently washed with water and counterstained with safranin for 2 minutes. – Analysis of the slide will reveal endospores stained green from the primary stain while the parent cells will stain red from the counterstain  Bacteria Staining Differential Stains Cont. Negative Staining (Capsule Stain) – Capsules in yeast and bacteria can be detected using a negative staining procedure. – The background of the slide is stained by acidic stains, the capsule remains unstained and the cells within the capsule are stained with basic stains – Negative stain is applied to a slide followed by the bacterial isolate under investigation. A smear is made and left to air dry – The smear is then flooded with crystal violet for about 1 minute, drained and left to air dry.  Bacteria Staining Differential Stains Cont. Negative Staining Cont. – Analysis of the slide reveals encapsulated cells as indicated by clear zones surrounding the cells – E.g., of negative stains used are India ink, Nigrosin, Congo red.  Bacteria Staining Differential Stains Cont. Negative Staining Cont. India Ink Stain – particularly used for the identification of C. neoformans  Bacteria Staining Impregnation Staining Bacterial cells and structures too thin to be seen under the light microscope are thickened using silver salts on their surface to make them visible E.g., bacterial flagella and spirochetes. – Flagella stain » Silver nitrate makes flagella appear larger than they are » Can be used to determine arrangement of flagella for identification » Two techniques for staining flagella are in use: A wet-mount procedure (Ryu method) Dried-smear preparation (Leifson staining technique)  Bacteria Staining Other Staining Methods – Acridine orange Stain » This staining method is used to confirm the presence of bacteria in blood cultures. » The detection of mycoplasmas (absent cell wall ) » Binds to and stains the nucleic acid – Cytoplasmic inclusion stains » Identifies intracellular deposits such as starch and glycogen » E.g., Albert staining is used to stain the metachromatic granules of C. diphtheriae. – Auramine-Rhodamine stain » Fluorochrome staining method used to enhance the detection of mycobacteria directly in patient specimens  Fungal Staining Other Staining Methods – Calcofluor White Staining » Used to directly detect fungal elements in fungi grown in culture. » The cell walls of fungi will bind the stain enhancing the visibility of fungal elements in tissue or other specimens. – Lactophenol cotton blue (LPCB) wet mount » Most widely used method of staining and observing fungi.