MicroLab Exam 1 Study Guide
MicroLab Exam 1 Study Guide MIC301
Popular in Microbiology Lab
Popular in Microbiology
verified elite notetaker
verified elite notetaker
verified elite notetaker
verified elite notetaker
verified elite notetaker
verified elite notetaker
This 5 page Study Guide was uploaded by Alison Kohn on Saturday January 31, 2015. The Study Guide belongs to MIC301 at University of Miami taught by Multiple professors in Fall. Since its upload, it has received 175 views. For similar materials see Microbiology Lab in Microbiology at University of Miami.
Reviews for MicroLab Exam 1 Study Guide
So much better than office hours. Needed something I could understand, and I got it. Will be turning back to StudySoup in the future
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 01/31/15
Alison Kohn Microbiology Lab Exam 1 Study Guide Exercise 0 Microscopes See lab manual for microscope parts ocularseyepieces two of them we use a 15X eyepiece objective lens the rotating lens that capture rays from the specimen 4X 10X 40X lOOX total magni cation found by multiplying the eyepiece by the objective stage platform that specimen is placed on condenser concentratesfocuses light on specimen diaphragm lets in more or less light under specimen f1ne focus knob fine adjustments course adjustment knob large adjustments revolving turret allows for rotation of objectives Never remove any part of microscope or use a microscope that looks damaged If you don t know if you re seeing the specimen or debris rotate the eyepiece move the stage or clean your lens parfocal stays in focus when magnification is changed 4OX high dry objective specimen will not be clearly visible because of the refractive quality of the glass and the air differences in the refractive properties cause light to bend as it passes from light to air lOOX oil immersion don t go back to 40X with oil immersion Exercise 1 Slants Stabs Streaks Kingdom Monera made of all prokaryotic organisms including bacteria and cyanobacteria They grow in diverse habitats and are identified by studying individual species cyanobacteria photosynthetic prokaryotes gram negative Microbial culture along with diagnostic methods can be used to identify the cause of a disease Microorganisms need to be isolated to be studied They thrive at different optimal conditions pH oxygen level pressure temperature etc 2 mediums liquid nutrient broth the turbidity measured by optical density spectrophotometer is a growth estimate solid medium you add agar galactan while melts at around 100 C and solidifies at around 42 C pour plate procedure inoculating prior to agar hardening aseptic technique inoculation that maintains purity through sterilization heat filtration and chemicals but we will use Bunsen burner bacterial growth lag turning on off of certain genes bc of environment exponential constant rate of binary fission stationary due to growth of limiting factors death Procedure Inoculated soil E coli and M luteus in a broth medium and streaked them too Did slants and stabs with E coli and M Luteus Exercise 2 Motili Han in Dro Wet Mount Bacteria are locomotive Flagella long ne threads that probably originate in the cytoplasm monotrichous one lophotrichous a bunch on one side amphitricous bunches on both sides peritrichous all over true motility actual position of cell is changed brownian movement result of molecular bombardment and characterized by to and fro cell movement gliding motility secretion of slime that moves bacterium types of motility swarming movement in a group swimming use of agella twitching pili retraction gliding slime secretions with focal adhesion points sliding spreading by growth detection of motility used a stab in TTC agar triphenyltetrazolium chloride which turns red when metabolized Horizontal spread and diffuse growth indicate motility intracellular motion shigella and listeria Hanging drop method used when it s necessary to observe viable organisms for a long period of time Organisms are suspended in a hanging drop on a concave slide 4 drops of oil and 2 loopfuls in center then slide is placed on top Procedure wet mount with E coli and M Luteus brownian Hanging drop with euglena amoeba and paramecium Exercise 3 Simple staining smears and Gram staining Simple stains increases contrast between background and bacteria one color used Differential stains increases contrast and distinguishes between types of bacteria All bacterial dyes are synthetic products An acid dye is the salt of a color acid and a base dye is the salt of a color base They owe their colors to their anions and cations respectively The salts are used because free acidbase dyes are insoluble in water or don t stick to bacteria well Bacterial Cells have a slight negative charge on their surface near neutral pH Basic dyes are most often used because of the inherent positive charge of the dye s salt which sticks to the negative charge of the bacterial cell envelope Smears water and loop of bacteria then dry it over Bunsen burner Don t overheat it because it could shatter or the bake the stain onto the slide Evaporation after heating is slow simple staining prepare smear stain for amount of time rinse with DI water then blot with bibulous paper gram staining stain with a primary dye crystal violet then a mordant iodine then a decolorizing agent ethyl alcohol then a counterstain safranin The mordant forms a complex with the primary dye that forms an insoluble precipitate Because gram positive have a thick peptidoglycan layer the complex can t be removed and the color is of the primary dye gram positive are purple gram negative are red to determine if an organism is gram negative a drop of 3 KOH can be placed above a loopful of the organism If the loop is lifted and strings come up it is gram negative The strings are from the release of genetic material of the gram negative bacteria Exercise 4 Morphology ShapesArrangements cocci circle streptococciin lines staphylococcigrapelike clusters bacilli rods spirilla corkscrew spiral pleomorphic different shapes spores typically made by bacillus bacteria They are highly resistant to environmental stresses and staining In this lab we force spores to take up stain by steaming for a long time capsules mucilaginous materials composed of polysaccharides that form a matrix around the bacteria When gram stained they appear as a halo Contributes to an organisms virulence by increasing its resistance to the body s defenses granules presence of metachromatic granules can diagnostically determine the presence of diphtheria bacilli agella the diameter is below the light microscope resolving power so they must be stained with mordant to layer the agella surface and increase the diameter bacterial pigments some are lipoid and insoluble and some diffuse into the medium These pigment producing bacteria are called chromogenic Negative staining stains the background so that the organism stands out Put a loop into NigrosineIndia Ink then spread it throughout slide and let it dry Acid fast stain ZiehlNeelsen differential stain which measures a bacteria s resistance to decolorization by acids Acid fast bacteria have a mycolic layer and are not easily decolorized by acids We make a smear then we stain and steam we steam to allow the dye to penetrate the lipid layer with carbol fuschin red the decolorize with acid alcohol then counterstain with methylene blue The acid fast keep the red and the non acid fast retain the blue Spore stain Schaeffer Fulton We make a smear then steam with malachite green for 10 minutes the counterstain with safranin then rinse The spores will be green and the vegetative cells will be red Exercise 5 Part 1 Enzymatic Reactions Enzymes are used to facilitate cell activities They utilize chemicals as a source of energy We can use enzymatic tests to find a pattern of activity to help identify bacteria Extracellular enzymes enzymes produced by the bacteria found in the supernatant culture medium Intracellular enzymes enzymes found only after deathlysis of the cell constituitive enzymes those produced at all times by a particular cell adaptive enzymes those produced only in response to the presence of their speci c substrate Carbohydrate Utilization The most common products of carb breakdown is C02 and hydrogen acid and gas Each of our test tubes will have an inverted vial Durham Tube and pH indicator phenol red Air bubbles indicate gas production and yellow coloring indicate acid production Starch Hydrolysis Some bacteria can break down complex starches into simple sugars dextrins maltose glucose When starch is exposed to iodine it turns a blue color When starch has been hydrolyzed its cleavage products do not turn blue A clear zone around the area in which the organisms have grown indicates hydrolysis of the starch in the agar Casein hydrolysis Casein is the principal protein of milk which exists as a colloidal suspension and gives milk its opaque appearance This experiment determines if peptonization protein breakdown occurs Peptonization occurred if there is a clear zone apparent in the agar Gelatin Hydrolysis The protein gelatin is secured by the hydrolysis of collagen a component of connective tissue and tendons Gelatinase hydrolyzes collagen If the gelatin is liquid it has been hydrolyzed by proteolytic enzymes Pathogenic are usually gelatinase positive If we agitate the solution there could be a false negative from solid mixing with liquid Blood Agar we will use TSA trypticase soy agar with sheep blood Beta hemolysis no red color present because hemolysin was released and completely broke down the red blood cells Gamma hemolysis no effect on red blood cells Alpha hemolysis green tint due to the presence of biliverdin which partially breaks down red blood cells Exercise 5 Part 2 Metabolic Reactions Catalase Activity catalase breaks down hydrogen peroxide to release oxygen gas Bubbles means it is catalase positive Catalos positive organisms are usually aerobic Catalase negative organisms include Enterococcus Leuconostoc Lactobacillus and Clostridium Procedure was to add 3 H202 and watch for bubbles AMC and Methyl Red Test characterize gram negative nonsporeforming rods Bacteria convert acid products arising from carbohydrate metabolism to neutral products and carbon dioxide They test the ability to convert acid products to neutral products For both tests we inoculated MRVP mediums and incubated Voges Proskaueh VP Test tests for presence of neutral products acetylmethylcarbinol AMC Identification of bacillus and gram negative rods After incubation we agitated the mixture and added alphanaphthol and KOH solution If it turned red AMC acetoin is present If it is yellow or copper there was no AMC Methy Red Test After incubation add 12 alcohol solution drops of methyl red If it turns red acidic it is MR positive If it stays yellow neutral pH it is MR negative Indole Test useful because only some bacteria form indole Indole is a nitrogen containing compound formed by break down of tryptophan we use tryptone as a substrate for this test protein sparing action of carbs When glucose is added to tryptonecontaining medium the fermentation of the carbohydrate interferes with the breakdown of trytophan into indole as the acid produced interferes with enzyme activity we use tryptone and tryptoneglucose broth then add kovacs solution if top layer is red it is positive for indole If green it s negative Citrate Test differentiates on basis of citrate utilization metabolized citrate will produce an alkaline reaction and basic environment high pH Broth contains bromthymol blue indicator which is green at neutral ph negative result and blue and basic pH positive result Hydrogen Sul de Production Activity of some bacteria on sulfurcontaining amino acids frequently results in the liberation of H28 Slanted on a thiosulfateiron medium that contains lactose and dextrose 101 Phenol Red indicator is used which is yellow at acidic pH and red at neutral pH Black line across stab means H282 was produced A yellow butt and red slant mean dextrose was produced A yellow butt and slant means lactose was utilized An elevatedcracked butt means gas was produced A red butt means the organism could not ferment lactose or dextrose
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'