TECHNIQUES FOR MICROSCOPIC STUDY & BACTERIAL STAINING

• The condenser control knob is located beneath and behind the condenser and is used to adjust the height of the condenser.
• Fine and course adjustment knobs are located on the arm of the microscope near the base and are used to focus the objective lenses.
• The arm supports the binocular body and the revolving nosepiece, and is held with one hand when carrying the microscope, with the other hand beneath the base to support the weight of the microscope.
• The binocular body holds the ocular lenses in their proper positions.
• Total magnification achieved when the objective is used in conjunction with a x10 objective ocular lens is x4.
• Simple staining procedure involves the use of methylene blue to stain the cells so that their size, shape, and morphologic arrangement can be determined.
• Structural staining procedures include capsule stains, flagella stains, and endospore stain.
• Capsule stains are used to determine whether the organism is encapsulated.
• Flagella stains are used to determine whether the organism possesses flagella and, if so, their number and location on the cell.
• Endospore stain is used to determine whether the organism is a spore-former and, if so, to determine whether the spores are terminal or subterminal spores.
• Differential staining procedures include Gram stain and Acid-fast stain.
• Gram stain is used to differentiate between Gram-positive and Gram-negative bacteria.
• Acid-fast stain is used to differentiate between acid-fast and non-acid-fast bacteria.
• Total magnification achieved when the objective is used in conjunction with a x100 objective ocular lens is x10.
• Total magnification achieved when the objective is used in conjunction with a x1000 objective ocular lens is x100.
• Techniques for the microscopic study of bacteria include using stains, which are salts composed of a positive and a negative ion, one of which is colored (i.e., the chromophore), and basic dyes, where the chromophore is in the positive ion (i.e., dye + Cl-).
• Living bacteria are difficult to see with the average light microscope because they appear almost colorless when viewed individually, even though the culture as a whole may be highly colored.
• Stains are used to color bacteria for easier viewing and study.
• Staining is the process of coloring a microorganism with a dye to make the structures more discernible.
• A smear is a thin film of material used for microscopic examination.
• Fixing uses air and heat to attach microorganisms on a slide, and also kills the organisms and preserves the morphology.
• Simple staining is the coloration of bacteria by applying a single solution of stain to a fixed smear.
• The method is sufficient to observe bacterial shape and morphologic arrangement.
• For simple staining, a dye such as methylene blue is applied to a fixed smear, rinsed, dried, and examined using the oil immersion lens of the microscope.
• Gram staining, developed by Christian Gram in 1884, is a method that distinguishes between bacteria based on the thickness of their peptidoglycan.
• The principle of Gram staining is that certain bacteria have thicker peptidoglycan such that when they are stained with primary dye they are not easily decolorized and thus retain the primary stain (Gram positive) while bacteria with thinner peptidoglycan are easily decolorized but retain the secondary stain (Gram negative).
• The procedure for Gram staining involves applying Crystal violet (Primary stain) for 1 minute, Lugol’s iodine (Mordant) for 1 minute, Acetone alcohol (Decorolizer) for 10 - 30 seconds, and Safranin (Secondary stain) for 30 seconds.
• Acid Fast staining, developed by Paul Ehrlich, is a method that distinguishes between bacteria based on their ability to resist decolorization by acid alcohol.
• The principle of Acid Fast staining is that some bacteria are able to resist decolorization by acid alcohol due to the presence of lipids that bind tenaciously with the primary stain.
• The procedure for Acid Fast staining involves applying Carbol fuchsin (Primary stain) for 5 minutes (heat), Acid alcohol (Wash) for 15 seconds, Methylene blue (Secondary stain) for 1 minute, and Acid fast - take up the primary stain (pink) Non acid fast - take up the secondary stain (blue).
• Endospore stain is a stain that demonstrates spore structure in bacteria as well as free spores.
• Capsule stain is a stain that demonstrates the presence of capsules surrounding cells, also known as negative-staining, for example, nigrosin or India ink.
• Flagella stain is a stain that demonstrates the presence and arrangement of flagella.
• Cytoplasmic inclusion stains are stains that identify intracellular deposits of starch, glycogen, polyphosphates, hydroxybutyrate and other substances.