Download Property/Enzyme Testing For

Week 8 W
New:
2nd
•Exp 24: IMViC
•Part A: Indole Test
•Part B: Citrate Test
•Exp 22: Carbohydrate
Fermentation
Lab Two pg 158
•Exp 25: Hydrogen
Sulfide Test
•Exp 26: Urease Test
•Exp 23: Triple Sugar
Iron Agar Test
Lab Two pg 162
•Exp 30: Oxidase Test
1st
•Charts
10/10
Lactose Fermentation—which one
is positive?
Ex 22: Carbohydrate Fermentation
(by facultative anerobes)
• Fermentation w/ Phenol Red (pH indicator)
• - Red at neutral ph (7)= no Fermentaion occured
• A Yellow at acidic ph (<7)= Fermentation
• G
microorganisms that produce gas (CO2) will
show as a bubble in the inverted Durham tube
• Some bacteria metabolize proteins (peptones) for energy
instead of the carbohydrate, which cause the broth to
turn basic (alkaline).
Results To ID Gram Negative Bacteria
MICROORGANISM
E.coli (EC)
P.
aeruginosa
(PA)
P.vulgaris
(PV)
E. aerogenes
(EA)
Serratia
marcescens
(SM)
shape/
arrangement
rods
rods
rods
rods
rods
color, size, form,
margin,
elevation,
texture
white,
small,
circular,
entire,
convex ,
wet
white/green,
small, circular,
entire, convex,
mucoid
white, small,
circular,
undulate,
wet
white, small,
circular, entire,
convex, wet
red centers,
small, circular,
entire, convex,
wet
Notation
Morphology
TSA
(cultural
characteristics)
PEA
+/-
growth
MSA
+/-
growth
Starch Hydrolysis
+/-
amylase
Gelatin Liquefac.
+/-
Lactose Ferm.
A (G) /- ferm.
Dextrose Ferm.
(glucose)
A (G) /- ferm.
Sucrose Ferm.
A (G) /- ferm.
gelatinase
■
■
TSI Slant
Alk or A
TSI Butt
Alk or A (G)
TSI H2S
+/- H2S prod.
SIM Indole Prod.
tryptophanase
■
SIM H2S
+/- H2S prod.
■
SIM Motility
+/- motility
■
+/-
TSI Results
Exp. 23:TRIPLE SUGAR IRON AGAR
COLOR/CONDITION
INTERPRETATION
RED SLANT/ RED BUTT
(Alk/Alk)
No carbohydrate fermentation.
Instead, peptones are catabolized
aerobically and/or anaerobically.
RED SLANT/
YELLOW BUTT
(Alk/A)
Only glucose fermentation has occurred.
Peptones are catabolized aerobically.
YELLOW SLANT/
YELLOW BUTT
(A/A)
Lactose and/or sucrose fermentation has
occurred.
GAS FORMED (G)
Seen as cracks in the agar, bubbles, or
the entire slant may be pushed out of the
tube.
BLACK PRECIPITATE
(+/-)
H2S has been produced.
How to Identify or Differentiate an Organism
(based on what we have learned to date)
isolate pure culture (using SPD)
determine cultural characteristics
determine microscopic morphology
determine Gram + or Gram -
use Special Media and biochemical tests to differentiate
Then use reference sources:
Bergey’s Manual, Lab Manual & Journal
Ex 24: IMViC Part A: Indole Test
• Medium: SIM Deep
• Chemical Components:
Tryptophan and Kovac’s Reagent (indicator)
• Property/Enzyme Testing For:
Tryptohanase degrades tryptophan to prod indole
• Results:
+ = cherry red layer
- = no color change
• Interpretation of Results: Kovac’s Reagent tests for
indole- a byproduct of tryptophan degradation.
Absence of red coloration demonstrates that the
substrate tryptophan was not hydrolyzed and indicates
an indole- neg reaction due to lack of tryptophanase.
Ex 25: Hydrogen Sulfide Test (& motility)
• Medium: SIM Deep
• Chemical Components:
Cysteine and Thiosulfate
Ferrous sulfate (indicator)
• Property/Enzyme Testing For:
Ability to ferment sulfur-containing amino acids or inorganic
sulfur compounds
• Results:
+ = black precipitate
- = no black precipitate
• Interpretation of Results: Organisms capable of H2S (colorless)
production, will show blackening of the butt b/c of precipitation of
insoluble ferrous sulfide. [Pathway 1: Some bacteria can degrade
sulfur-containing peptones to amino acids (such as cysteinesource of H2S). Pathway 2: Inorgainc sodium thiosulfate is the
substrate for H2S. ]
• Medium: SIM Deep
• Chemical Components:
not applicable
• Property/Enzyme Testing For:
flagellated organisms
• Results:
+ = motile
- = nonmotile
• Interpretation of Results: When culture growth
(turbidity) of flagellated organisms is not restricted to
the line of inoculation.
Ex 24: IMViC Part D: Citrate Test
• Medium: Citrate Agar Slant
• Chemical Components:
Bromthymol blue (indicator)
• Property/Enzyme Testing For:
Capability of using citrate as a carbon source.
• Results:
+ = blue
- = green
• Interpretation of Results: In absence of fermentable
glucose or lactose, some bacteria are capable of
fermenting citrate. Byproducts of this reaction are
alkaline (emission of CO2 combines with salt to form
Na2CO3) which changes bromthymol blue from green to
blue.
Ex 26: Urease Test
• Medium: Urea broth
• Chemical Components:
Phenol red (indicator)
• Property/Enzyme Testing For:
Urease degrades amide compounds.
• Results:
+ = bright pink
- = salmon color (no change)
• Interpretation of Results: Especially helpful to identify
Proteus vulgaris. As organisms splits urea, presence of
alkaline ammonia (NH3) creates alkaline environment
turning phenol red deep pink.
Exp. 30: Oxidase Test
• Medium: TSA plate
• Chemical Components:
Oxalate
• Property/Enzyme Testing For:
Cytochrome oxidase aids aerobic respiration
• Results:
+ = purple/black
- = no color change or light pink
• Interpretation of Results: Pseudomonas auruginosa, all aerobic,
some facultative anerobes, and microaerophiles are oxidative
positive. Whereas Enterobacteriaceae (E. coli and Enterobacter
aurugenes) are oxidative negative. Oxidase is important in the
electron transfer chain during aerobic respiration. Oxalate reagent
serves as an e- donor becoming oxidized to a blackish color in the
presence of oxidase and free O2.
WEEK 8w TESTS
(per table)
CULTURE
MEDIA
PROCEDURE
Ex 24: IMViC
Part A: Indole Test
EC, PA, PV,
EA
4 SIM
deeps
- Ex 24 pt A: Ref. pages: 166-167.
- Ex 25: Ref. pages:178.
- Stab SIM in center to butt w/ needle.
- Incubation: 37oC 24 Hrs.
- Next week: add Kovac’s reagent for
indole test.
Ex 24: IMViC
Part D: Citrate Test
EC, PA, PV,
EA
4 Citrate
slants
- Ref. pages: 169-170.
- Streak surface w/ needle.
- Incubation: 37oC 24 Hrs.
Ex 26: Urease Test
EC, PA, PV,
EA
4 Urea
broths
- Ref. pages:181-182.
- Heavily inoculate: use 2 loopfulls of org.
- Incubation: 37oC 24 Hrs.
Ex 30: Oxidase Test
EC, PA, PV,
EA
2 TSA
plates
- Ref. pages:199-200.
- Divide plates in half, single line steak org.
- Incubation: 37oC 24 Hrs.
- Next week: add oxalate.
Ex 25: Hydrogen
Sulfide Test
(& motility)
(2organism
/plate)