Biochemical Test Reaction Interpretation of Gram Negative Bacterium: TSI, SIM, Citrate Utilization and Urea Hydrolization Test
Biochemical Test Reaction for Gram-Negative Bacilli or Rods
The most common routine tests used for biochemical test reaction for gram-negative rods are Triple Sugar Iron(TSI) agar test, Sulphide Indole Motility(SIM) test, Citrate Utilization test, and Urea hydrolyzation test.
Triple Sugar Iron(TSI) agar-R/Y, no gas production, No hydrogen sulfide production
Sulfide Indole Motility(SIM) medium-Indole-Positive, Motile but no hydrogen sulfide production
Citrate Utilization test-Positive
Urea hydrolyzation test-Positive
Result- Suggestive for Providencia rettgerii
Observation and Interpretation TSI test
Examine the reaction in the slant and the butt also observe for gas and hydrogen sulfide production.
Yellow: Acid reaction
Red: Alkaline reaction
Blackening of the medium: H2S production
Bubbles, cracks, or displacement of the medium: Gas production
Interpretation Of Carbohydrate
A/A: Glucose and lactose or sucrose fermented
K/A: Only glucose fermented or non-lactose fermenter
K/K: No carbohydrate fermented or non-glucose fermenter
Result Interpretation of SIM Test
Positive H2S test: blackening of the medium
A negative H2S test: absence of blackening
Positive motility test: a diffuse zone of growth flaring from the line of inoculation
Negative motility test: restricted growth along the stab line
Indole positive test: a pink to red color ring is formed at the top of the medium after the addition of Kovac’s reagent
Indole negative test: A yellow color denotes a negative indole test after the addition of Kovac’s reagent
Escherichia coli ATCC 25922: Growth; Motility: positive, H2S: negative, and Indole: positive (It turns pink after addition of Kovac’s reagent)
Salmonella enterica ATCC 14028: Growth; Motility: positive, H2S: positive, and Indole: negative
Result And Interpretation In Citrate Utilization Test
Citrate utilization test positive: The growth will be visible on the slant surface and the medium will be an intense blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue.
Citrate utilization test negative: Trace or no growth will be visible. No color change will occur; the medium will remain the green color of the uninoculated agar. Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium, thus a citrate utilization negative test culture will be virtually indistinguishable from an uninoculated slant.
Uninoculated (UN): No growth and no color change; slant remains green
Negative control: No growth and no color change; slant remains green
Positive control: Growth with color change from green to intense blue along the slant
Result Interpretation of Urea Hydrolyzation Test
Organisms that hydrolyze urea rapidly (e.g. Proteus species) may produce positive reactions within 1 or 2 hours; less active species (e.g. Klebsiella species) may require up to 4 days. In routine diagnostic laboratories, the urease test result is read within 24 hours.
If an organism produces a urease enzyme, the color of the slant changes from light orange to magenta/ bright pink/ red.
If an organism does not produce urease, the agar slant and butt remain light orange (medium retains original color).
References
- Jean F. Mac Fadden Biochemical tests for Identification of Medical bacteria
- Bailey’s and Scott’s Diagnostic Microbiology-13th Edition
- Mackie and McCartney Practical Medical Microbiology-14th Edition
- ASM Clinical Microbiology Procedures Handbook-2nd Edition
- Monica Cheesbrough Distinct Laboratory Practice in Tropical Countries…2nd Edition