Lab final 2
Triple Sugar Iron (TSI) Test:
Purpose: Detects carbohydrate fermentation, gas production, and hydrogen sulfide (H₂S) production.
Carbohydrates in TSI:
Glucose (0.1%) – limited quantity.
Lactose (1%) and Sucrose (1%) – abundant quantities.
Results:
Slant/Butt (K/A, A/A, etc.):
K (Alkaline, red) = No fermentation.
A (Acid, yellow) = Fermentation occurred.
Gas Production: Cracks or bubbles in the agar.
H₂S Production: Black precipitate in the butt.
Result
Interpretation
K/A
Glucose fermentation only.
A/A
Glucose + lactose and/or sucrose fermentation.
K/K
No carbohydrate fermentation (non-fermenter).
Black butt
H₂S production (positive).
Gas present
Fermentation with gas production.
B. Urea Test:
Purpose: Tests for the production of urease enzyme, which hydrolyzes urea into ammonia and CO₂.
Results:
Positive: Bright pink (alkaline reaction due to ammonia production).
Negative: Yellow or no color change (no ammonia production).
C. Citrate Test:
Purpose: Tests for the ability to use citrate as the sole carbon source via the enzyme citrate permease.
Results:
Positive: Blue color (alkaline reaction due to citrate metabolism).
Negative: Green (no growth or citrate utilization).
2. Enzymes Being Tested
Test
Enzyme(s) Involved
Reaction
TSI Test
Various fermentative enzymes
Ferments glucose, lactose, and/or sucrose. Produces H₂S if sulfur-reducing enzymes are present.
Urea Test
Urease
Hydrolyzes urea to ammonia and CO₂.
Citrate Test
Citrate Permease
Transports citrate into the cell for metabolism.
3. Procedure Summary
A. TSI Test:
Stab the butt of the TSI agar with a sterile needle.
Streak the slant with the same needle.
Incubate at 35°C for 18-24 hours.
Observe and record slant/butt color, gas production, and H₂S production.
B. Urea Test:
Inoculate urea broth or slant with a loopful of bacteria.
Incubate at 35°C for 18-24 hours.
Observe the color change (yellow to pink indicates positive).
C. Citrate Test:
Streak the slant of Simmons’ citrate agar with a loopful of bacteria.
Incubate at 35°C for 18-24 hours.
Observe for color change (green to blue indicates positive).
4. Sources of Error
Inoculation Issues:
Over-inoculating can overwhelm the media and produce false results.
Improper stabbing or streaking for TSI may miss key reactions.
Incubation Errors:
Insufficient incubation time may lead to false negatives.
Prolonged incubation may alter true results (e.g., reversion of TSI slant to red due to ammonia accumulation).