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Micro labs - review. BIOL260 Winter 2012. Ubiquity. What organisms grow best at room temperature? ___°C? At body temperature? = ___°C? What kind of medium is TSA? . Appearance of growth on plates. Bacteria vs fungi (molds)?. Aseptic technique. What does it mean?
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Micro labs - review BIOL260 Winter 2012
Ubiquity • What organisms grow best at room temperature? ___°C? • At body temperature? = ___°C? • What kind of medium is TSA?
Appearance of growth on plates • Bacteria vs fungi (molds)?
Aseptic technique • What does it mean? • Why do we need to use it? • What does sterile broth look like?
Inoculation of media • Streaking for isolation – how and why? • Can you tell if growth on a slant is pure culture? • How do you inoculate a broth? • A slant? • A deep?
Microscope • What type of microscope did we use for our labs?
Simple and differential stains • What is the difference? • How do you prepare a slide for staining?
The Gram stain! • Know the steps including names of all reagents & what their function is • Know what the staining properties of bacteria tell you about the organisms • Know what can go wrong
Misc other stains • Endospores • How do they appear in Gram stain • What is one species of bacteria that forms endospores • Flagella • How do they appear in the Gram stain? • What result would you expect for a motility test? • Acid fast bacteria • How do they appear in Gram stain? • What is the primary genus of acid fast bacteria?
Chemically defined media • Glucose mineral agar = glucose salts agar (GSA)
Complex/undefined media • Trypticase soy agar (TSA) • Tripticase soy yeast agar (TSY) • Mueller Hinton agar
Selective media • Antibiotics, toxic dyes, bile and other selective inhibitors of bacterial growth
Differential media • pH indicators: • Brom cresol purple • Phenol red
Selective & differential media • Eosin methylene blue (EMB) • SELECTS for Gram negative rods: eosin is toxic to Gram positive bacteria • DIFFERENTIATES lactose fermentors from non-lactose fermenters: acid produced during lactose fermentation causes the colonies to appear dark pink or purple • Mannitol salt agar • SELECTS for salt-tolerant Gram positive cocci = Staphylococcus species • DIFFERENTIATES mannitolfermenters from non-fermenters • MacConkey agar • SELECTS for Gram-negative rods • DIFFERENTIATES lactose fermentersfron non-fermenters • LES Endo agar • SELECTS for Gram negative rods • DIFFERENTIATES coliform bacteria (Enterobacteriaceae) from Salmonella
Mannitol salt agar: mannitolfermenters turn the agar yellow MacConkey agar: lactose fermenters turn the agar yellow
Viable cell count • Determine the cells/ml in the original stock solution based on a bacterial count plate, given the following: • 1 ml of a 1:100 dilution (10 -2 dilution) of the stock culture was added to the count plate and the count plate contains 72 bacterial colonies • 0.1 ml of a 1:1000 dilution (10 -3 dilution) of the stock culture was added to the count plate and the count plate contains 115 bacterial colonies
Count plate ex. 1 Number of colonies divided by dilution factor times volume, in ml, of diluted medium that was plated: = 72 x 1/10-2 x 1ml = 72 x 102 = 7.2 x 103 organisms per ml in stock culture
Count plate ex. 2 Number of colonies divided by dilution factor times volume, in ml, of diluted medium that was plated: = 115 x 1/10-3/0.1ml = 115 x 103/ 0.1ml = 115 x 104= 1.15 x 106organisms/ml in stock culture
Aerobic/anaerobic lab: oxygen requirements • Aerobes: require oxygen • Obligate anaerobies: require that there NOT be oxygen • Facultative anaerobes: can grow in either aerobic or anaerobic conditions • The shake agar/deep tube: what did we use this for? Do you remember how to read it?
UV light • What is the effect of UV light on bacteria? • What factors will influence how much damage is done by UV light? • Why did we incubate the plates in the dark?
Antimicrobial drugs • What is the Kirby-Bauer test? • What does it tell you about the organism? • What is a “zone of inhibition”? • What do you need to know in order to interpret the results of a KB test? What about a disinfectant?
Transformation lab • What is transformation? • What was the positive control for this lab? • What was the negative control? Why did we use controls? • What was the function of the streptomycin in the TSY plate used in step 2 of this exercise?
Normal skin microbiota • What types of organisms normally inhabit our skin? • Which of the normal skin inhabitants can grow in both anaerobic and aerobic conditions? What are these types of organisms called? • TSY + glucose + brom cresol – what does this medium allow us to determine?
Skin microbiota • Staphylococcus epidermidis: Gram positive, facultative, coag negative • Staphylococcus aureus: Gram positive, facultative; coag positive • Micrococcus luteus: Gram positive coccus, aerobe • Propionibacterium acnes, P. granulosum: Gram positive, anaerobic coryneform (diptheroid) rod • Bacillus subtilis, B. cereus: Gram positive rods, facultative
Coagulase test • Used to differentiate coagulase-producing species of staphyloccus (coagulase-postive staph) from non-coagulase producing species (coagulase-negative staph) – important in differentiating pathogenic from non-pathogenic isolates
Throat culture lab • What types of organisms are normal inhabitants of your throat? • What type of plates do you use to observe hemolysis? • What does the type of hemolysis tell us about an organism? • What organism causes strep throat? Is it hemolytic? If yes, what type of hemolysis?
Sore throat: is it viral or bacterial? • If the only bacteria isolated are non-pathogenic, this SUPPORTS it being a viral infection • It doesn’t CONFIRM it being a viral infection because you have not actually isolated a virus • Viruses DO NOT grow on bacterial culture plates
Identification of gram negative rods • What is phenol red used for? How do you interpret a test that uses this dye as an indicator? • What is a durham tube?
Differential media: fermentation broths 1: No acid, no gas 2A. Weak acid, no gas 2B. Strong acid, no gas 3A & 3B: Strong acid + gas
Negative urea Positive urea
Water lab • What are the “indicator organisms”? • MPN test for lactose fermentation • MPN index = ? • + BGLB tubes / LES endo plates – why?