Lab Practical Exam 1 - Study Guide

Lab Practical Exam 1 Week one Brightf ield Microscopy Brightfield microscope: allows light rays to pass directly to the eye without being deflected by an intervening opaque plate Be able to label the microscope The limit for most light microscope is 1000x due to resolving power, its ability to completely separate two objects in a microscopic field Bacteria Colony: mass of cells that arose from a single cell Growth media/medium: nutrient-rich mixture on which organisms grow TSA= tryptic soy agar (solid) TSB= tryptic soy broth (liquid) Inoculation: introducing bacteria to media Culture: bacteria that are growing in their media Week Two Asepti c technique Aseptic technique: transfer bacteria from one medium to another without introducing contaminants 1. 2. 3. 4. 5. 6. 7. Gloves and goggles Disinfect work area Sterilize loop or needle Flame mouth of tube Plate cover is barely opened Sterilize loop or needle Disinfect work area Smear Prep Good smears are critical for discerning: 1. The morphology of cells 2. Arrangement of cells 3. Internal structures such as endospores and inclusions Goals of a smear: 1. To cause the cells to adhere to the microscope slide so that they are not washed off during subsequent staining and washing procedures 2. No cell shrinkage 3. Prepare thin smears Gram stain Hans Christian Gram came up with gram stain Differential stain for 2 types of bacteria based on cell wall composition Gram+ is blue/purple and has more peptidoglycan in the cell wall Gram- is pink/red and has less peptidoglycan in the cell wall Steps and outcomes: 1. None-heat fixed cells a. + has no color b. – has no color 2. Crystal violet (primary stain) a. + is purple b. – is purple 3. Gram’s Iodine (mordant) a. + is purple (causes insoluble complex in g+ cells) b. – is purple 4. Ethyl Alcohol (decolorization) a. + is purple b. – has no color 5. Safranin a. + is purple b. – is pink Moti lity Motility is independent movement Brownian motion: bombardment od molecules with cells, uniform drifting Bacillus cereus: rod-shaped, motile, gram positive Chemotaxis: move toward nutrients in the environment or away from harmful substances Week Three Pure Culture Techniques Pure culture contains only a single kind of an organism, whereas a mixed culture contains more than one kind Streak-plate method: rubbing culture in quadrants Pour plate: Dipping and pouring, needs aseptic technique Spore stain Endospore resistance is in part due to their protein coat/exosporium Schaeffer-Fulton Method: utilizes malachite green to stain an endospore and safranin to stain the vegetative portion of the cell Green endospore and pink sporangium 1. Malachite green 2. Safranin Dorner Method: produces a red spore with colorless sporangium; nigrosine is used as dark background for contrast. Both are stained by sporangium is decolorized by the diffusion of carbolfuchsin into nigrosine Acid Fast stain Mycobacterium and nocardia have high lipid content (mycolic acid) Need to use methods that make walls more permeable to stains Ziehl-Neelsen method: the primary stain, carbolfuchsin contains phenol and heat (mordant) which facilitate the penetration of the carbolfuchsin into the cell fuchsin stain acid alcohol methylene blue Kinyun acid-fast method: modification in which the concentrations of primary stain, basic fuchsin, and phenol are increased, making it unnecessary to heat the cells during the staining procedure Acid fast are stained pink M. smegmatis (rod) Non-acid fast are stained blue S. epiermidis (cocci) We grow bacteria in agar plates Ubiquity of bacteria: they are very common/found everywhere E.coli is white and found in fecal matter S. Aureus is yellow and found on the skin P. Aeruginosa is green and found in the environment