Usf Microbiology Lab Midterm, Vandenbergh

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Simple Microscope

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uses only a single lens (magnifying glass)

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Light microscope

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uses light to view specimens

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4 types of lightmicroscopes

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Bright field, dark field, phase-contrast, and fluorescent microscopes.

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Bright field microscopes....

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light up background while the specimens appear dark

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Electron microscopes

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use a beam of electrons and magnets to allow higher degrees of magnification.

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Compound microscopes

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employ multiple lenses for better magnification

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Ocular lens

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lens you are physically looking through

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Objective lens

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lens directly above your specimen

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Condenser

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made up of two lenses that focus the light rays coming from the light source onto the specimen

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Magnification

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enlargement of a specimen

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Total magnification

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magnification of ocular lens * magnification of the objective lens being used

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Parafocal

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the ability of the microscope to stay in focus when adjusting the objective lens to a higher power

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Parcentric

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the ability of the microscope to keep the specimen you have in focus centered each time you increase the magnification

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Field of view

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area you are viewing through the microscope

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Resolution

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the smallest distance two closely spaced objects can be separated and still be differentiated as two individual objects

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Empty resolution

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an increase in magnification without the required increase in resolution

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Depth of field

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"3D resolution", as magnification increases there is a decrease in the 3D power of the microscope.

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Culture media

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solutions that contain nutritional requirements of microorganisms; can be broth, solid, or semisolid

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Agar

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a solidifying agent, derived from seaweed, which is a complex carbohydrate and has no nutritional value

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Solid mediums require an agar concentration of __% and semisolid mediums require an agar concentration of __%.

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Solid: 1.5-1.8%. Semisolid: <1.0%.

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Broth media can be used to grow large amounts of bacteria, but...

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cannot determine the different types of cells in the mixed culture because the bacteria are in suspension

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Agar deeps

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agar poured into test tubes

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Agar slant

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agar poured into test tubes and cooled on an angle

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Agar plates

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Agar poured into plates, the most common.

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Chemically defined media

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used when an organism's exact nutritional needs are known and its chemical makeup is known. The precise nutritional requirement of and organism must be known.

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Complex media

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made up of complex substances such as peptones, beef and yeast extracts and its exact composition is unknown. These media are used in lab because they are cheap and easy to prepare.

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Enriched media

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are a complex medium that has an additional additive(s) added to it to allow growth of fastidious organisms.

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Fastidious

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organisms that do not grow well on basic, complex media and require additional growth factors or nutritional needs in order to be grown

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Sterilization

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destroys all microorganisms and spores in a medium

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Autoclaving

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a popular method of sterilization; process includes pressure, high temperature, and hot steam in a chamber.

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Staining

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technique used to color the specimens of a slide

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Morphologies

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shapes of bacteria

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Cocci

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spheres

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Bacilli

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rod-shaped

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Vibrios

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slightly curved rods, comma shaped

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Coccobacilli

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short rods

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Spirilla

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spiral shaped

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Pleomorphic

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multiple shapes

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Diplo-

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cells arrange as pairs

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Staphylo-

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cells arrange as grape like clusters

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Strepto-

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cells arrange in chains

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Tetrad-

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a quartet of cells

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Sarcina

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two quartets of cells that form a 3D cube

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Smear

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a sample of bacteria applied to a slide and allowed to dry so staining techniques can be performed

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Fixation

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"fixing" or "gluing" cells to the slide

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Direct stain

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staining of the specimen, but not the background. Light passes through the background and the colored cells are easily visible

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Negative stain

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stains the background, but not the specimen. The background will be colored and the light will pass through the cells

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Simple stain

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stains the bacterial smear with a single dye. Cells are colored against an unstained background

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Differential stain

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a stain that used at least three chemical reagents on a smear.

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Differential primary stain

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initially gives color to all cells

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Differential decolorizer

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used to give a color contrast based on the chemical makeup of the cell. Will usually decolorize one type of cell while the other cell type maintains the primary stain color

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Differential counterstain

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gives a contrasting color from the primary stain to cells that were decolorized

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Gram stain

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the most famous differential stain. Developed by Hans Christian Gram (1884) and used to differentiate bacteria based on their cell wall composition. Differentiates into gram positive and gram negative bacteria

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Gram positive bacteria

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have cell walls with thick layers of peptidoglycan. Appear purple. (primary stain)

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Gram negative bacteria

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have cell walls with thin layers of peptidoglycan. Appear pink. (counterstain)

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Pure culture

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contains only one type of microorganism, is important in the identification and classification of bacteria

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Subculturing

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transfer of microorganisms from one medium to another

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Aseptic transfer

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requires that subculturing be performed with sterile instruments and under sterile conditions to prevent contamination from outside sources

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Streak plate technique

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reduces the number of organisms of the surface on an agar plate. This allows the different species of bacteria in the culture to be spread far enough apart on the agar that the individual cells can multiply without touching.

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Pure stock culture

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a single bacterial colony aseptically transferred to a new medium

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Colony morphology

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macroscopic observation of the colony's growth patterns on an agar plate

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Cell morphology

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microscopic observation of the form and structure of the cells when stained

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Capsule stain

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Culture is grown in a milk broth. Crystal violet is used to stain the background, and decolorizer (copper sulfate) will decolorize the capsule leaving it clear forming a "halo". Heat is not used because it would denature proteins on the capsule.

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Endospore stain (Malachite Green method)

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heat is used to help drive the primary stain, malachite green, into the spore. After steaming both the spores and the vegetative cells are green. Vegetative cells are decolorized with water. Unstained vegetative cells are counterstained with safranin. Spores are green, cells are pink.

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Germination

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when the spore becomes a metabolically active vegetative cell because conditions have become favorable again

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Acid-fast stain

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Organisms that are able to resist decolorization with acid-alcohol are called acid-fast. Primary stain is Carbol Fuchsin (Red), decolorizer is acid-alcohol, and counterstain is methylene blue. Non-acid-fast organism will be blue, while acid-fast organism will be red.

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Hydrolytic Enzymes

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use water to split complex molecules in a process called hydrolysis

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Starch hydrolysis

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Starch is a carbohydrate made up of glucose subunits. Amylase breaks down starch to maltose and glucose. When testing for starch, iodine addition will cause agar to turn dark. If no starch is present no color change will occur and a clear halo will form.

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Casein hydrolysis

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Casein is a milk protein that gives milk its white color. Its broken down into amino acids by caseinase. When testing for casein milk agar is used for bacterial inoculation. If caseinase is produced it will hydrolyze casein in medium and produce a clear halo around inoculation. If it doesn't produce caseinase, no clear halo will form.

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Gelatin hydrolysis

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Gelatin is produced from collagen, a protein that makes up animal connective tissue. Gelatinase breaks down gelatin into amino acids. When testing for gelatinase; if positive, medium will turn to liquid; if negative, medium will stay solid.

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Lipid hydrolysis

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Lipids a generally fats and are composed of a glycerol molecule with 3 fatty acid chains attached. Lipases hydrolyze lipids. Tween 80 is a detergent also known as Polysorbate 80. When testing for lipase activity: yellow/straw color to pink color indicates a positive result. No color change indicates negative result for lipase production.

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Food poisoning

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refers to the pathogens that can cause disease present in food

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Food spoilage

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refers to microorganisms present on food that cause it to become inedible

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Serial dilution

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diluting a fixed amount of the original (stock) solution a series of times, usually diluting it a ten fold each time.

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Blanks

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stock is diluted into these, which contain a specified amount of buffer or water

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Dilution factor

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refers to the degree of the original sample has been diluted in each tube

df = (vol. sample added)/(total volume)

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CFU

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Colony Forming Unit.

CFU/mL = (CFU)/(volume plated)(df from tube plated)

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Bacteriophage

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viruses that infect bacteria

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Lytic cycle/lysogeny

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the phage's genome is immediately replicated and new phage particles are synthesized. The phage will actually digest the bacterial cell's own DNA in an effort to focus the cell's energy to make its own DNA and phage particles. Once the phage particles are complete the host cell will lyse, releasing the newly synthesized phage, which will then infect neighboring cells.

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Prophage

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During lysogeny the phages DNA becomes integrated into the host's genome

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Pour plate technique

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inoculated top agar is poured on top of agar plate. Top agar is allowed to solidify and then incubated. Bacteria added to top layer grow as a uniform lawn over the agar plate. When phage is added along with the bacterial cells, the phage will infect a bacterial cell and start the cycle of replication, lyse, and infection of neighboring cells. This leads to plaques being observes in the lawn of bacterial growth where the phage has killed bacterial cells.