Section III

T Triple Sugar Iron Agar

Triple Sugar Iron Agar • TSI Agar

Intended Use Principles of the Procedure
This medium conforms with specifications of The United States TSI Agar contains three sugars (dextrose, lactose and sucrose),
Pharmacopeia (USP). phenol red for detecting carbohydrate fermentation and
ferrous ammonium sulfate for detection of hydrogen sulfide
Triple Sugar Iron Agar (TSI Agar) is used for the differentia-
production (indicated by blackening in the butt of the tube).
tion of gram-negative enteric bacilli based on carbohydrate
fermentation and the production of hydrogen sulfide. Carbohydrate fermentation is indicated by the production of
gas and a change in the color of the pH indicator from red to
Summary and Explanation yellow. To facilitate the detection of organisms that only
TSI Agar is used for the determination of carbohydrate ferment dextrose, the dextrose concentration is one-tenth the
fermentation and hydrogen sulfide production in the identifi- concentration of lactose or sucrose. The small amount of acid
cation of gram-negative bacilli.1,2 It is recommended in the produced in the slant of the tube during dextrose fermentation
USP for use in performing Microbial Limit Tests.3 oxidizes rapidly, causing the medium to remain red or revert
Hajna developed the formulation for TSI Agar by adding sucrose to an alkaline pH. In contrast, the acid reaction (yellow) is
to the double sugar (dextrose and lactose) formulation of Kligler maintained in the butt of the tube because it is under lower
Iron Agar.4 The addition of sucrose increased the sensitivity of oxygen tension.
the medium by facilitating the detection of sucrose-fermenting After depletion of the limited dextrose, organisms able to do
bacilli, as well as lactose and/or dextrose fermenters. so will begin to utilize the lactose or sucrose.2
Carbohydrate fermentation is detected by the presence of To enhance the alkaline condition of the slant, free exchange
gas and a visible color change (from red to yellow) of the pH of air must be permitted by closing the tube cap loosely. If the
indicator, phenol red. The production of hydrogen sulfide is tube is tightly closed, an acid reaction (caused solely by
indicated by the presence of a precipitate that blackens the dextrose fermentation) will also involve the slant.
medium in the butt of the tube.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences
in the development and testing of media for industrial and clinical applications, per the referenced publications.

Identity Specifications Identity Specifications

Difco™ Triple Sugar Iron Agar BBL™ TSI Agar
Dehydrated Appearance: Pink, free-flowing, homogeneous. Dehydrated Appearance: Fine, homogeneous, free of extrane-
Solution: 6.5% solution, soluble in purified ous material, may contain many
water upon boiling. Solution is red, minute to very small tan specks.
slightly opalescent. Solution: 5.94% solution, soluble in purified
Prepared Appearance: Red, slightly opalescent. water upon boiling. Solution is medium
to dark, red-orange to orange-red,
Reaction of 6.5% clear to slightly hazy.
Solution at 25°C: pH 7.4 ± 0.2
Prepared Appearance: Medium to dark, red-orange to orange-
red, clear to slightly hazy.
Cultural Response
Reaction of 5.94%
Difco™ Triple Sugar Iron Agar
Solution at 25°C: pH 7.3 ± 0.2
Prepare the medium per label directions. Inoculate with fresh cultures
by the stab and streak method and incubate at 35 ± 2°C for 18-24
hours.
Cultural Response
BBL™ TSI Agar
ORGANISM ATCC™ RECOVERY SLANT BUTT GAS H 2S
Prepare the medium per label directions. Inoculate with fresh cultures
Escherichia coli 25922 Good A A + – by the stab and streak method and incubate at 35 ± 2°C for 24 hours.
Pseudomonas aeruginosa 9027 Good K K – –
ORGANISM ATCC™ RECOVERY SLANT BUTT GAS H 2S
Salmonella choleraesuis
subsp. choleraesuis Escherichia coli 25922 Good A A + –
serotype Enteritidis 13076 Good K A + + Pseudomonas aeruginosa 27853 Good K K – –
Shigella flexneri 12022 Good K A – – Salmonella choleraesuis
A = Acid K = Alkaline subsp. choleraesuis
serotype Typhimurium 14028 Good K A +/– +
Shigella flexneri 12022 Good K A – –
A = Acid K = Alkaline

574
 Triple Sugar Iron Agar, cont.

Formulae Procedure
Difco™ Triple Sugar Iron Agar To inoculate, carefully touch only the center of an isolated
Approximate Formula* Per Liter colony on an enteric plated medium with a cool, sterile needle,
Beef Extract ................................................................ 3.0 g
Yeast Extract .............................................................. 3.0 g
stab into the medium in the butt of the tube, and then streak
Pancreatic Digest of Casein ...................................... 15.0 g back and forth along the surface of the slant. Several colonies
Proteose Peptone No. 3 ............................................. 5.0 g from each primary plate should be studied separately, since
Dextrose ..................................................................... 1.0 g mixed infections may occur.
Lactose ..................................................................... 10.0 g
Sucrose .................................................................... 10.0 g Incubate with caps loosened at 35°C and examine after 18-24
Ferrous Sulfate ........................................................... 0.2 g
Sodium Chloride ........................................................ 5.0 g hours for carbohydrate fermentation, gas production and
Sodium Thiosulfate .................................................... 0.3 g hydrogen sulfide production. Any combination of these reactions
Agar ......................................................................... 12.0 g may be observed. Do not incubate longer than 24 hours
Phenol Red ............................................................... 24.0 mg
because the acid reaction in the slant of lactose and sucrose
BBL™ TSI Agar fermenters may revert to an alkaline reaction.
Approximate Formula* Per Liter
Pancreatic Digest of Casein ...................................... 10.0 g
Peptic Digest of Animal Tissue ................................. 10.0 g Expected Results
Dextrose ..................................................................... 1.0 g Compare reactions produced by the unknown isolate with those
Lactose ..................................................................... 10.0 g produced by the known control organisms.
Sucrose .................................................................... 10.0 g
Ferrous Ammonium Sulfate ........................................ 0.2 g Carbohydrate fermentation is indicated by a yellow coloration
Sodium Chloride ........................................................ 5.0 g
Sodium Thiosulfate .................................................... 0.2 g of the medium. If the medium in the butt of the tube becomes
Agar ......................................................................... 13.0 g yellow (acidic), but the medium in the slant becomes red
Phenol Red ............................................................... 25.0 mg (alkaline), the organism being tested only ferments dextrose
*Adjusted and/or supplemented as required to meet performance criteria.
(glucose).
Directions for Preparation from A yellow (acidic) color in the slant and butt indicates that
Dehydrated Product the organism being tested ferments dextrose, lactose and/or
1. Suspend the powder in 1 L of purified water: sucrose.
Difco™ Triple Sugar Iron Agar – 65 g;
A red (alkaline) color in the slant and butt indicates that the
BBL™ TSI Agar – 59.4 g.
Mix thoroughly.
organism being tested is a nonfermenter. T
2. Heat with frequent agitation and boil for 1 minute to Hydrogen sulfide production results in a black precipitate in
completely dissolve the powder. the butt of the tube.
3. Dispense into tubes and autoclave at 118-121°C (per label Gas production is indicated by splitting and cracking of the
directions) for 15 minutes. medium.
4. Cool in a slanted position so that deep butts are formed.
5. Test samples of the finished product for performance using For final identification, perform biochemical tests and other
stable, typical control cultures. identification procedures with a pure culture of the organism.
Consult appropriate references for further information.5-7

Limitations of the Procedure

1. Hydrogen sulfide production may be evident on Kligler Iron
Agar but negative on Triple Sugar Iron Agar. Studies by
Bulmash and Fulton8 showed that the utilization of sucrose
could suppress the enzymatic mechanisms responsible for
H2S production. Padron and Dockstader9 found that not
all H2S-positive Salmonella are positive on TSI.
2. Sucrose is added to TSI to eliminate some sucrose-fermenting
lactose-nonfermenters such as Proteus and Citrobacter spp.1
3. Further biochemical tests and serological typing must be
performed for definite identification and confirmation of
organisms.
4. Do not use an inoculating loop to inoculate a tube of Triple
Uninoculated Escherichia coli Salmonella Shigella flexneri Sugar Iron Agar. While stabbing the butt, mechanical
Tube ATCC™ 25922 enteritidis ATCC™ 12022
ATCC™ 13076 splitting of the medium occurs, causing a false positive
result for gas production.1

575
Section III
T Triple Sugar Iron Agar, cont.

5. A pure culture is essential when inoculating Triple Sugar Availability

Iron Agar. If inoculated with a mixed culture, irregular Difco™ Triple Sugar Iron Agar
observations may occur. AOAC BAM BS10 CCAM CMPH COMPF EP MCM7 SMD
6. Tubes should be incubated with caps loosened. This allows SMWW USDA USP
Cat. No. 226540 Dehydrated – 500 g
a free exchange of air, which is necessary to enhance the
alkaline condition on the slant.1 BBL TSI Agar
™

AOAC BAM BS10 CCAM CMPH COMPF EP MCM7 SMD

References SMWW USDA USP

1. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, Cat. No. 211749 Dehydrated – 500 g
vol. 1. Williams & Wilkins, Baltimore, Md. 221038 Prepared Slants – Pkg. of 10*
2. Forbes, Sahm and Weissfeld. 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby, 221039 Prepared Slants – Ctn. of 100*
Inc., St. Louis, Mo.
3. United States Pharmacopeial Convention, Inc. 2001. The United States pharmacopeia 25/The *Store at 2-8°C.
national formulary 20 – 2002. United States Pharmacopeial Convention, Inc., Rockville, Md.
4. Hajna. 1945. J. Bacteriol. 49:516.
5. Ewing. 1985. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
Publishing Co., Inc., New York, N.Y.
6. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed.
American Society for Microbiology, Washington, D.C.
7. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriol-
ogy, 9th ed. Williams & Wilkins, Baltimore, Md.
8. Bulmash and Fulton. 1964. J. Bacteriol. 88:1813.
9. Padron and Dockstader. 1972. Appl. Microbiol. 23:1107.

Tryptic Nitrate Medium

Intended Use detected by various test methods and is used in differentiating
Tryptic Nitrate Medium is used for differentiating microor- organisms from clinical samples, foods and dairy products.1-5
ganisms based on nitrate reduction.
Principles of the Procedure
Summary and Explanation Peptone is a source of nitrogen, amino acids and vitamins.
Tryptic Nitrate Medium is a differential, semi-solid, general Dextrose provides carbohydrates. Potassium nitrate provides
purpose medium that supports growth of aerobes as well as the basis for nitrate reduction. Disodium phosphate is a buff-
facultative and obligate anaerobes.1 The formulation includes ering agent. The low agar content, which allows varying
potassium nitrate which can be reduced by certain organisms degrees of anaerobiosis in the medium, supports growth of
to either nitrite or nitrogen gas. Nitrate reduction can be organisms with various oxygen requirements.

User Quality Control

Identity Specifications
Difco™ Tryptic Nitrate Medium
Dehydrated Appearance: Beige, free flowing, homogeneous.
Solution: 2.5% solution, soluble in purified
water upon boiling. Solution is light
amber, clear to slightly opalescent.
Prepared Appearance: Light amber, slightly opalescent.
Reaction of 2.5%
Solution at 25°C: pH 7.2 ± 0.2

Cultural Response
Difco™ Tryptic Nitrate Medium
Prepare the medium per label directions. Inoculate and incubate at 35 ±
2°C for 18-24 hours; incubate Clostridium sporogenes anaerobically. Test
for nitrate reduction using Difco™/BBL™ Nitrate A, B and C Reagents.

INOCULUM NITRATE
ORGANISM ATCC™ CFU RECOVERY REDUCTION
Clostridium sporogenes 11437 102-103 Good – Uninoculated Escherichia coli Clostridium
Tube ATCC™ 25922 sporogenes
Escherichia coli 25922 102-103 Good + ATCC™ 11437
Staphylococcus aureus 25923 102-103 Good +

576