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?
Endospores in Gram stain
Endospore stain:
Malachite green + safranin
Flagella stain
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 mannitol fermenters from non-fermenters
• MacConkey agar
– SELECTS for Gram-negative rods
– DIFFERENTIATES lactose fermenters fron non-fermenters
• LES Endo agar
– SELECTS for Gram negative rods
– DIFFERENTIATES coliform bacteria (Enterobacteriaceae) from Salmonella
Mannitol salt agar: mannitol
fermenters 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:
A. 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
B. 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 106 organisms/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?
UV light
Antimicrobial drugs
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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?
KB test
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 nonpathogenic 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 nonpathogenic, 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
Hemolysis
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
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What are the “indicator organisms”?
MPN test for lactose fermentation
MPN index = ?
+ BGLB tubes / LES endo plates – why?
What sugar fermentation profile
indicates a fecal coliform?