Exam 1 Notes

Exam 1 Notes - Exam 1 Microbiology Lab SAFETY...

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Exam 1 Microbiology Lab SAFETY INFORMATION: -Spills and injuries- Notify TA -Autoclave wastes after use -Broken glass bucket -Slides & slide covers -> Sharps container -Fire Safety -Label tubes/plate correctly (name, date, section, what is it?) HANDLING MICROSCOPES: -Oil is ONLY for the 100X Objective lens and must be wiped off after use with LENS PAPER 4X = 40X Magnification 10X = 1000X Magnification -Eyepiece (10) X Objective Lens ASEPTIC TECHNIQUE: -Microorganisms are everywhere -Sterilization and disinfection are vital to the study of MO’s -Possible means of contamination must be a constant thought -Disinfect benches before and after -Keep lids closed as much as possible -Never touch the “insides” (tube/plate/orifices) with anything that is not sterile -Flame loops/needles and other tools before and after EXPERIMENT 1: SMEAR PREP: 1) Clean a slide (Bon Ami) and dry it 2) Flame Loop 3) Remove cap, flame tube 4) Remove one loop full 5) Flame tube, replace plug 6) Smear loop onto center of slide 7) Flame loop 8) Allow slide to dry 9) Heat fix the slide (briefly) EXPERIMENT 2: SIMPLE STAIN PROCEDURE: 1) Place slide on staining rack 2) Cover smear with crystal violet, wait for 30-40 sec. 3) Gently wash off the dye, blot dry lightly 4) Observe under increasing magnification EXPERIMENT 3: SEPARATION AND ISOLATION OF BACTERIA FROM A MIXED CULTURE: 1) Obtain and label one petri dish of nutrient agar
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2) Use aseptic technique as described in Ex. 1 (flaming loop and tube) to obtain one loop of bacterial culture 3) Lift lid of dish and make primary, secondary, tertiary, and quaternary streaks being sure to flame in between 4) Flame loop. Invert dish, and incubate at 30C until next class 1) Nutrient agar (NA) Petri dish should be labeled, exposed to the air for at least 15 min, and incubated 30C until next class= Air Plate = Start of unknown 2) Continue Ex. 3- Gram stain each type pf colony you see The Gram Stain: DIFFERENTIAL STAIN: Technique to distinguish two or more types of MO based on their physical and/or chemical interactions with the reagents used -Hans Christian Gram- Danish (1884) -Takes advantage of the differences in cell walls peptidoglycan layers -Two major types: Gram (+), Gram (-) GRAM (+): Thick layer of peptidoglycan and Teichoic Acid GRAM (-): Periplasmic space- Outside cytoplasm -Contains very thin layer of peptidoglycan -Contains LPS (Lipo-Poly-Saccharides) HOW THE STAIN WORKS: 1) Crystal violet is used as a 1 stain (1 min rinse) 2) Lugol’s iodine is the mordanting reagent which complexes the crystal violet and peptidoglycan (1 min rinse) 3) Alcohol is the decolorizing agent which: (A) Disrupts the outer membrane and washes away the crystal violet OR (B) dehydrates the thick PG layer effectively trapping crystal violet (10 sec rinse) -Alcohol dissolves crystal violet and dehydrates the PG layer trapping crystals 4) Gram (+) cells are now violet, Gram (-) cells are void of color
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Exam 1 Notes - Exam 1 Microbiology Lab SAFETY...

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