Lab Final Exam Flashcards
T/F:
Tiny earth searches for antibiotic-resistant bacteria in the soil because as soil erodes, antibiotic-resistant bacteria can end up in our waterways.
FALSE
Antibiotic-producing bacteria
T/F:
TSA with cyclohexamide is a differential medium for antibiotic producers.
FALSE
Selective medium (against fungi)
T/F:
To make a ten-fold dilution, you transfer 100uL of stock to 900 uL of water.
TRUE
T/F:
The serial dilution plate grew ALL bacteria in your soil sample.
FALSE
Not all bacteria can be grown in culture (only ~30% can)
T/F:
Antibiotic producing bacteria are resistant to all antibiotics.
FALSE
Only resistant to the antibiotic it produces.
T/F:
After you quadrant streak a plate, you should put it in the fridge.
FALSE
Grow in incubator
Good/Bad Choice:
After observing the growth of your patch plate, you want to keep the plate to use it again next week, so you put it in the incubator.
BAD CHOICE
Put in refridgerator
Convert 1.5 cm to mm
Convert 0.7 cm to mm
15 mm
7 mm
Draw a ten-fold serial dilution through 10^-5.
How much is transferred each time?
How many tubes?
How much diluent (water) is in each tube?
1 mL transferred each time (100 uL)
5 tubes (not including stock solution)
9 mL in each tube (900 uL)
See drawn picture on iPad
Design a protocol to test whether bacteria isolated from your garden produce any antibiotic compounds capable of inhibiting the growth of E. coli.
How would you interpret the results?
Why would you want to sample soil from a vibrant garden?
Protocol:
1. Harvest soil sample (aseptic technique)
2. Culture soil sample (serial dilutions)
3. Pick diverse colonies (patch plate)
4. Screen colonies for ability to inhibit E. coli growth (patch plate with E. coli lawn; observe clearing)
5. Isolate pure cultures
Results:
Clearing = zone of inhibition
- Indicates inhibition of E. coli growth
- Potential antibiotic-producer (antimicrobial activity)
More vegetation = more microbial diversity
Interpret chart about Kirby Bauer results.
Which drug should be used for treatment?
What can you conclude about Chloramphenicol?
Kanamycin should be used for treatment (S - sensitive)
Chloramphenicol must be used in combination with another antibiotic to clear infection (I - intermediate)
See iPad drawing.
Biochemical Tests
MSA
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Selective agent = 7.5% NaCl
- Selects for Staph
- Inhibits G-
Indicator = phenol red
- Differentiation of S. aureus from other Staphylococcus species
Results:
- Growth = halotolerant
- Yellow = mannitol fermentation
- No color change (red) = no fermentation
Mannitol Salt Agar
Selective & differential
G+ media
Substrate = mannitol
Good growth + yellow = possible S. aureus
Good growth + red = Staph other than S. aureus
Biochemical Tests
BEA
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Selective agent = oxgall (bile)
- Separates Group D Streptococci from other Strep spp.
- Inhibits G-
Indicator = ferric citrate
- Oxidized iron
Results:
- Black precipitate = + (esculin hydrolysed in presence of bile)
- No blackening = -
Bile Esculin Agar
Selective, & differential
Undefined, G+ medium
Other ingredients:
- Beef extract (nutrients & energy)
- Digest of gelatin (nutrients & energy)
- Esculin (horse chestnut tree bark extract)
Biochemical Tests
EMB
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Selective agent = eosin & methylene blue dyes
- Inhibits G+
Indicators = eosin & methylene blue dyes
- Isolation of fecal coliforms (lactose fermenters)
Results:
- Dark purple/black = lactose fermentation
- Pink/mucoid growth = lactose fermentation
- Metallic = LOTS of fermentation
- Colorless = no fermentation
Eosin-Methylene Blue Agar
Selective & differential
G- media
Substrate = lactose
E. coli = metallic green
Biochemical Tests
HEA
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Selective agent = bile salts
- Inhibits G+
Indicators:
1. Sodium thiosulfate (reduces sulfur to H2S)
2. Bromothymol blue & acid fuschin dyes (pH change)
Results:
- Blue/green = no sugar fermentation (Shigella)
- Blue/green + black precipitate = sugar fermentation, sulfur reduction (Salmonella)
- Pink/orange grow = sugar fermentation
Hektoen Enteric Agar
Selective & differential
Gram- media
Substrates:
- Lactose
- Sucrose
- Salicin
Used to isolate/differentiate Salmonella & Shigella from other enterics in pt stool samples or dairy/poultry
Biochemical Tests
MAC
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Selective agents = bile salts & crystal violet
- Inhibit G+
Indicator = neutral red dye
- Isolate & differentiate Enterobacteriaceae based on ability to ferment lactose
Results:
- Colorless = no fermentation (pH > 6.8)
- Pink/red = lactose fermentation (pH < 6.8)
MacConkey Agar
Selective & differential
G-media
Substrate = lactose
Biochemical Tests
CNA
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Selective agents:
1. Colistin (antibiotic): G- membrane integrity
2. Nalidixic Acid (antibiotic): DNA replication
Indicator: sheep blood
- Differentiation based on hemolysis
- Isolates Staphylococci, Streptococci, & Enterococci from clinical specimens
Results:
- Alpha hemolysis = partial
- Beta hemolysis = complete
- Gamma hemolysis = none
Columbia CNA Agar
Selective & differential
G+ media
Yeast extract provides B vitamins
Biochemical Tests
TSIA
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Indicators:
- Phenol red (pH)
- Ferrous sulfate (H2S)
Results:
A/A = yellow slant & butt
- Fermentation
K/A = red slant & yellow butt
- Glucose fermentation
- Proteins catabolized aerobically with alkaline products (reversion)
K/K = red slant & butt
- NO fermentation
- Peptone catabolized aerobically & anaerobically
- NOT from Enterobacteriaceae
K/NC = red slant & no change in butt
- NO fermentation
- Peptone catabolized aerobically
- NOT from Enterobacteriaceae
NC/NC = no change in slant/butt
- Organism growing slowly or not at all
- NOT Enterobacteriaceae
H2S = black precipitate in agar
- Sulfur reduction
- Acid condition (from fermentation of lactose)
G = cracks/lifting in agar
- Gas production
Triple Sugar Iron Agar or Kliger Iron Agar (KIA)
Differential ONLY
- Differentiate Enterobacteriaceae from other G- rods
Sugars:
- Glucose (0.1%)
- Lactose (1%)
- Sucrose (1%)
Biochemical Tests
SIM
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Indicators:
1. Ferrous ammonium sulfate
2. Kovac’s reagent (DMAB)
Results:
- Black precipitate = sulfur reduction
- Red/pink = indole production
- Turbidity away from stab = motile
Sulfur reduction, Indole production, Motility
Differential ONLY
- ID of Enteric bacteria species
Reduced agar concentration (0.5%) makes motility possible
Indole produced if bacteria has tryptophanase
Black precipitate = ferric sulfide (H2S combied with indicator)
Biochemical Tests
PRSBs
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Indicator = phenol red (pH)
- Differentiates Enterobacteriaceae from other G- rods
- Differentiates G+ fermenters
Results:
A/G = yellow broth & bubbles
- Sugar fermented with acid & gas products
A/- = yellow broth & no bubbles
- Sugar fermented with acid products ONLY
-/- = red broth & no bubbles
- NO fermentation
K = pink broth (no bubbles)
- NO sugar fermentation
- Peptone degraded with alkaline end products
Phenol Red Sugar Broths
Differential ONLY
Durham tube - collects gas from fermentation
4 carbohydrate versions:
1. Glucose (green cap)
2. Sucrose (yellow cap)
3. Lactose (red cap)
4. Mannitol (white cap)
Biochemical Tests
Citrate
- Selective (how/what organisms)?
- Differential (how/what organisms)?
- Result interpretation (color/meaning)
Indicator = bromothymol blue (pH)
- Differentiates based on which organism can use citrate as sole carbon source
Results:
- Blue = citrate utilization + (alkaline byproduct)
Differential ONLY
- Citrate permease allows citrate into cell for citrate catabolism/fermentation
Defined media
