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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?


Endospores in gram stain
Endospores in Gram stain


Endospore stain malachite green safranin
Endospore stain:Malachite green + safranin




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


Micro labs review

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


Micro labs review

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?