LAB 3 Morphological Characteristics of Bacteria Protocols for Endospore Stain Capsule Stain Motility Stab and Wet Mount INTRODUCTION Bacteria are characterized by the presence or absence of a number of different structures Endospores capsules and flagella are three such examples Each of these structures is visible with light microscopy if the correct staining procedure is employed ENDOSPORES are survival structures In poor growth conditions some genera may sporulate Rather than dying endospores survive in a dormant state Endospores are unique to Bacteria and are formed by a limited number of bacterial genera The soil bacteria within the genera Bacillus and Clostridium are the most familiar The stepwise process of sporulation is triggered by poor growth conditions see the discussion of the process of sporulation in your text The transition from vegetative cell to endopsore requires an environmental signal and then a series of steps The The endospore forms within the vegetative cell A wall forms around a copy of the bacterial chromosome capturing some ribosomes proteints and DNA The endospore forming within the cell can be visualized using the light microscope As the sporulation process continues layers form within the spore making it very dense Exterior to the spore the vegetative cell dies At the completion of sporulation oval spores are visible using light microscopy Endospores cannot replicate However they allow survival in lean times In fact they are resistant to extreme environmental conditions such as high temperatures dryness toxic chemicals and UV radiation The dormant structure allows cell survival until conditions favorable to cell growth returns Favorable growth conditions signal the process of endospore germination Germination of a spore results in a break in the spore wall and the outgrowing of a new vegetative cell The newly formed vegetative cell is capable of growth and reproduction The dense endospore is impenetrable by basic dyes In the Gram stain the spore is not stained In early stages of sporulation it can be seen as a clear oval in the stained vegetative cell A special differential stain can be used to color the endospore and differentiate it from the vegetative cell In the Endospore Staining Procedure heat is needed to drive the stain Malachite Green into the spore Once stained spores retain the green dye whereas washing with water removes the malachite green from the vegetative cell The counterstain safranin is used to color the vegetative cell The size of the endospore and its position within the vegetative cell is characteristic for a given species CAPSULES are slippery structures found exterior to the cell wall of many bacteria Capsules may be involved in bacterial virulence cells with capsules are less likely to be phagocytized or in bacterial adherence such as the adherence of Streptococcus mutans to teeth Capsules are normally composed of polymers of sugars and or proteins They have no net charge and will not bind charged dye particles Negative staining where the background is stained revealing an unstained structure of interest is useful for demonstrating capsules In Maneval s method cells are mixed on a slide with a drop of the pH indicator congo red pH 3 or below the color is blue and at pH 5 and above the color is red Congo red does not penetrate the capsule and provides a colored background The sample is e then allowed to air dry Note that heat fixing is not used for the capsule stain Protein capsules are destroyed by heat Heating samples will destroy protein capsules Heating can also dehydrate cells As the cells dehydrate they will shrink and false capsule like areas will form around the cell After air drying the slide is flooded with Maneval s solution a combination of acetic acid and acid fuchsin The acetic acid lowers the pH in the sample and causes the Congo red to change from red to blue The acid fuchsin penetrates through the capsule and stains the cell a bright red The unstained capsule is clearly seen using the light microscope as white in this red white and blue preparation FLAGELLA are structures that mediate bacterial motility Motile bacteria may move toward a source of nutrition or away from a toxin by the process of chemotaxis The number and mode of attachment of flagella as well as their location on the cell surface are characteristic of a species Flagella arrangements include monotrichous single flagellum located at one end of a cell amphitrochous single flagellum located at both ends of a cell lophotrichous a group of flagella at one cell end and peritrichous where flagella are found around the cellular perimeter Flagella are too thin to be seen in the light microscope without the use of special staining procedures These technically difficult procedures are used to load molecules onto the flagella to increase their width Figures 3 33 3 34 3 35 3 36 The presence of flagella indicate that the bacterium is capable of motility But motility can also be detected by direct visualization of bacteria in a wet mount Motility is characterized by directional movement of organisms It must be distinguished from Brownian motion or streaming In Brownian motion a consequence of molecular vibration cells wiggle in place In streaming a current carries the bacteria en masse from one area of the slide to another An indirect method of testing for motility is through the use of a motility stab A motility stab contains growth media and low percentage of agar in a test tube To inoculate the test organism the tube of semi solid media is stabbed with a needle inoculated with bacteria The tube is incubated As the bacteria grow nonmotile bacteria will grow only along the stab line Motile bacteria will have the capacity to move around in the medium A motility stab that is positive for motility will show a fuzzy stab line or no stab line bacteria have moved throughout the tube A motility stab that is negative for motility will show growth only along the stab line bacteria cannot move away from the site of inoculation Figure 5 25 Additional mechanisms of bacterial locomotion include gliding and motion by axial filament contraction Gliding is movement of bacteria along solid surfaces by an unknown mechanism Axial filament contraction occurs in spirochetes These unusual spiral shaped bacteria have a filament that stretches from end to end of the bacterial cell When the filament contracts the organism moves OBJECTIVES To learn the role of the bacterial