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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

Micro labs - review

BIOL260

Winter 2012

ubiquity
Ubiquity
  • What organisms grow best at room temperature? ___°C?
  • At body temperature? = ___°C?
  • What kind of medium is TSA?
appearance of growth on plates
Appearance of growth on plates
  • Bacteria vs fungi (molds)?
aseptic technique
Aseptic technique
  • What does it mean?
  • Why do we need to use it?
  • What does sterile broth look like?
inoculation of media
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
Microscope
  • What type of microscope did we use for our labs?
simple and d ifferential stains
Simple and differential stains
  • What is the difference?
  • How do you prepare a slide for staining?
the gram stain
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
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
Chemically defined media
  • Glucose mineral agar = glucose salts agar (GSA)
complex undefined media
Complex/undefined media
  • Trypticase soy agar (TSA)
  • Tripticase soy yeast agar (TSY)
  • Mueller Hinton agar
selective media
Selective media
  • Antibiotics, toxic dyes, bile and other selective inhibitors of bacterial growth
differential media
Differential media
  • pH indicators:
    • Brom cresol purple
    • Phenol red
selective differential media
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
slide20

Mannitol salt agar: mannitolfermenters turn the agar yellow

MacConkey agar: lactose fermenters turn the agar yellow

viable cell count
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
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
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
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
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
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
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
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
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
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
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
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
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
Differential media: fermentation broths

1: No acid, no gas

2A. Weak acid, no gas

2B. Strong acid, no gas

3A & 3B: Strong acid + gas

slide38

Negative urea

Positive urea

water lab
Water lab
  • What are the “indicator organisms”?
  • MPN test for lactose fermentation
  • MPN index = ?
  • + BGLB tubes / LES endo plates – why?
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