TRIPLE SUGAR IRON (TSI)
Triple Sugar Iron may also be used to determine H2S production as well as carbohydrate fermentation.
L to R The first tube is an un-inoculated control. The second tube is A/A, G (fermentation of glucose and lactose or sucrose with gas). The third tube is alk/A (glucose fermentation only & peptone catabolized). The last tube is alk/A, H2S (glucose fermentation only H2S produced)
INTRODUCTION:
Triple Sugar Iron medium is a differential medium that can distinguish between a number of Gram-negative enteric bacteria based on their physiological ability (or lack thereof) to:
a. metabolize lactose and/or sucroseb. conduct fermentation to produce acidc. produce gas during fermentationd. generate H2S.
The medium contains 1.0% each of sucrose and lactose and 0.1% glucose. If only glucose is fermented, acid produced in the butt will turn it yellow, but insufficient acid products areformed to affect the methyl red in the slant. However, if either sucrose or lactose are fermented, sufficient fermentation products will be formed to turn both the butt and the slant yellow. If gas is formed during the fermentation, it will show in the butt either as bubbles or as cracking of the agar. If no fermentation occurs (as for an obligate aerobe), the slant and butt will remain red.
The medium also contains ferrous sulfate. If the bacterium forms H2S, this chemical will react with the iron to form ferrous sulfide, which is seen as a black precipitate in the butt (a black butt).
PREPARATION OF MEDIA: (for 40 tubes)(NOTE: prepare at beginning of class.)
Weigh out medium, dissolve: 17.8 g dry medium, add to 300 mL water, heat to boiling to dissolve.
Deliver aliquots to tubes: 6 mL to16 x 150 mm tubes with a repipet. Cover with closures.
Autoclave capped aliquoted tubes 13 lb, 15 min.
Creating the slant: Using a ringstand and clamp, clamp the rack so that the tubes (with liquid medium in them) have a 3 cm slant with a 2-3 cm butt. Let cool until solid. Incubate 48 hr at37ºC to assure sterility.
INOCULATION:
Inoculate the slants with a pure culture by streaking over the entire surface of the slant (zig-zag to cover surface)and then stabbing deep into the butt.
Incubate at 37ºC for 24 hours (48 hr may be necessary to show all H2S reactions).
TRIPLE SUGAR IRON AGAR: INTERPRETATION OF RESULTS
SCORING THE SLANTS: Examine the slant and butt, and record data using the following criteria:
INTRODUCTION:
Triple Sugar Iron medium is a differential medium that can distinguish between a number of Gram-negative enteric bacteria based on their physiological ability (or lack thereof) to:
a. metabolize lactose and/or sucroseb. conduct fermentation to produce acidc. produce gas during fermentationd. generate H2S.
The medium contains 1.0% each of sucrose and lactose and 0.1% glucose. If only glucose is fermented, acid produced in the butt will turn it yellow, but insufficient acid products areformed to affect the methyl red in the slant. However, if either sucrose or lactose are fermented, sufficient fermentation products will be formed to turn both the butt and the slant yellow. If gas is formed during the fermentation, it will show in the butt either as bubbles or as cracking of the agar. If no fermentation occurs (as for an obligate aerobe), the slant and butt will remain red.
The medium also contains ferrous sulfate. If the bacterium forms H2S, this chemical will react with the iron to form ferrous sulfide, which is seen as a black precipitate in the butt (a black butt).
PREPARATION OF MEDIA: (for 40 tubes)(NOTE: prepare at beginning of class.)
Weigh out medium, dissolve: 17.8 g dry medium, add to 300 mL water, heat to boiling to dissolve.
Deliver aliquots to tubes: 6 mL to16 x 150 mm tubes with a repipet. Cover with closures.
Autoclave capped aliquoted tubes 13 lb, 15 min.
Creating the slant: Using a ringstand and clamp, clamp the rack so that the tubes (with liquid medium in them) have a 3 cm slant with a 2-3 cm butt. Let cool until solid. Incubate 48 hr at37ºC to assure sterility.
INOCULATION:
Inoculate the slants with a pure culture by streaking over the entire surface of the slant (zig-zag to cover surface)and then stabbing deep into the butt.
Incubate at 37ºC for 24 hours (48 hr may be necessary to show all H2S reactions).
TRIPLE SUGAR IRON AGAR: INTERPRETATION OF RESULTS
SCORING THE SLANTS: Examine the slant and butt, and record data using the following criteria:
