Mic 224 Lab 9

TSI and Carbohydrate Tests

Triple Sugar Iron Test (TSI)

• Triple sugar iron agar (TSI) is a differential medium that contains 1 % lactose, 1% sucrose, and 0.1% glucose (dextrose), ferrous sulfate, and the pH indicator phenol red.

• This test is normally performed on Gram negative bacteria

• It is used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates.

TSI Test• As with the phenol red fermentation broths, if an organism can

ferment any of the three sugars present in the medium, the medium will turn yellow as acid is produced (A). 

• If an organism can only ferment glucose, the small amount of glucose in the medium is used by the organism within the first ten hours of incubation. After that time, the reaction that produced acid reverts in the aerobic areas of the slant, and the medium in those areas turns red, indicating alkaline conditions (K).

• If an organism is incapable of fermentation, the conditions will be alkaline and produce a red color

• If the butt of the slant is obscured by the precipitate, look at the top of the slant to determine if the organism could ferment only dextrose (red), or if it could ferment either lactose and/or sucrose (yellow). 

TSI Test

• If an organism can reduce sulfur, the hydrogen sulfide gas which is produced will react with the iron to form iron sulfide, which appears as a black precipitate. If the precipitate is formed, it can mask any acid/alkaline results. Sulfur reduction requires an acidic environment, so if the black precipitate is present, some fermentation took place.

• Organisms that produce hydrogen sulfide gas contain the enzyme cysteine desulfurase and the co-enzyme pyridoxyl phosphate

• This reaction occurs only in the butt of the tube

TSI Test

• If the fermentation produced gas, you may see fissures (cracks) in the medium, or the entire slant may be raised above the bottom of the test tube.

TSI Test Procedure

• You will need 4 TSI slants. Inoculate a TSI slant for each of the following organisms:– E.coli, Citrobacter freundii, Proteus rettgeri, and

Pseudomonas aeruginosa

• Make sure to label each test tube with the appropriate organism name

• With a sterile stab transfer the organism from the stock culture to the TSI slant. Stab to the bottom of the tube to inoculate the butt portion and when exiting inoculate the slant portion of the tube

Possible TSI Results

Results (slant/butt) Symbol Interpretation

Red/yellow K/A Glucose fermentation only; Peptone catabolized

Yellow/yellow A/A Glucose and lactose and/or sucrose fermentation

Red/red K/K No fermentation; Peptone catabolized

Red/no color change K/NC No fermentation; Peptone used aerobically

Yellow/yellow with bubbles A/A,G Glucose and lactose and/or sucrose fermentation; Gas produced

Red/yellow with bubbles K/A,G Glucose fermentation only; Gas produced

Red/yellow with bubbles and black precipitate K/A,G, H2S Glucose fermentation only; Gas produced; H2S produced

Red/yellow with black precipitate K/A, H2S Glucose fermentation only; H2S produced

Yellow/yellow with black precipitate A/A, H2S Glucose and lactose and/or sucrose fermentation; H2S produced

No change/no change NC/NC No fermentation

 

A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction

TSI ResultsK/K K/A A/A +g A/A +g,+H2S A/K +g, +H2S

Carbohydrate Fermentation Test

• Carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate.

• Fermentation patterns can be used to differentiate among bacterial groups or species. For example, all members of the Enterobacteriaceae family are classified as glucose fermenters because they can metabolize glucose anaerobically. Within this family however, maltose fermentation differentiates Proteus vulgaris (positive) from Proteus mirabilis (negative).

• The media contains a single carbohydrate (0.5-1%), beef extract, peptone and the pH indicator phenol red– Yellow: acid production indicative of fermentation pH 6.8 or below– Red: alkaline conditions no fermentation pH 8.4 or above

• The tube also contains a durham tube– If gas is produced as a by-product of fermentation the durham tube will

trap a bubble

Carbohydrate Fermentation• During the fermentation process, an organic substrate serves as the

final electron acceptor. The end-product of carbohydrate fermentation is an acid or acid with gas production. Various end-products of carbohydrate fermentation can be produced. The end-product depends on the organisms involved in the fermentation reaction, the substrate being fermented, the enzymes involved, and environmental factors such as pH and temperature. Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide, and hydrogen.

• Fermentation reactions are detected by the color change of a pH indicator when acid products are formed. This is accomplished by adding a single carbohydrate to a basal medium containing a pH indicator. Because bacteria can also utilize peptones in the medium resulting in alkaline by-products, the pH changes only when excess acid is produced as a result of carbohydrate fermentation.

Carbohydrate Test Procedure

• You will need 4 tubes of each carbohydrate type: 4 glucose, 4 sucrose, 4 mannitol, and 4 lactose tubes

• Inoculate 1 organism into each carbohydrate type. – Ex: inoculate E.coli into 1 glucose tube, 1 sucrose

tube, 1 mannitol tube, and 1 lactose tube– Repeat this for the other three organisms; C. freundii,

P.rettgeri, and P.aeruginosa

• Be sure to label all tubes!!

Carbohydrate Test Results