17. Special Stains: Endospore Stain, Capsule Stain, and Acid-fast Stain Introduction A few genera of bacteria such as the Gram positive rods of the genus Bacilli have the ability to produce resistant survival forms called endospores. They should not be confused with the spores of fungi and plants because they are not used for reproduction. Endospores are resistant to heat, dryness, radiation, and many disinfectants. Endospore formation (sporulation) occurs through a complex series of events. One structure is produced within each vegetative or dividing cell. Once the endospore is formed, the vegetative portion of the bacterium is degraded and the dormant endospore is released. The green round structures are the endospores which trap malachite green. Vegetative cells stain red. The endospore is able to survive for long periods of time until environmental conditions again become favorable for growth. The endospore germinates producing a single vegetative bacterium. Some bacterial strains synthesize a capsule, a layer of polysaccharides or proteins that coats cells. The presence of a capsule is linked to virulence because it interferes with phagocytosis. Capsules are visualized by negative staining using dyes which are either too large to penetrate the cell or are negatively charged and repelled by the cell. Sample preparation for negative staining are not heat fixed because the heat would destroy the morphology of the capsule. Acid-fast stain was developed to stain Mycobacteria, Gram positive bacteria that do not stain well in a Gram stain because of the waxes present in the outside cell wall. The waxy coats are an important virulence factor. The procedure can be used to detect the organisms that cause Hansen’s disease (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis). Mycobacterium smegmatis is a commensal organism of the human body and serves as model for mycobacteria. Cords: because of their waxy outer membranes, Mycobacterium cells do not always separate after replication and form cords or rope-like aggregates. They appear as red clumps. Relationship to Class Instruction: Chapters 3 and 10, and 16: stains, identification, and virulence factors
Lab worksheet due __________________________
Purpose of Laboratory Perform endospore stain using the Schaeffer-Fulton method Perform negative stain of Klebsiella pneumoniae Demonstration of acid-fast stain of Mycobacterium smegmatis and observation of prepared slides
Material and Methods Organisms: Bacillus subtilis Klebsiella pneumoniae Mycobacterium smegmatis Staphylococcus epidermidis