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TSI test (triple
sugar iron agar)
Lab 5
This test use to
1- detect organisims that ferment different types of sugars
2- detect the bacteria that degrade amino acid and
produce H₂S gas (H₂S gas will react with Fe and produce
black precipitate between the slant and butt).
FeSO4 + H2S
FeS↓ + H2SO4
(Black ppt.)
The media used in this test contain
1- three sugars (glucose, lactose .sucrose)
2- pepton (protein has amino acid and sulfur)
3 –phenol red (as indicator sugar fermentation)
4- FeSO4 ) as source of iron and sulfur)
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 .
Result s :
1-If there is no fermentation = red color
2-If bacteria ferment lactose and sucrose =
3-If bacteria ferment glucose = yellow butt
4-If bacteria produce gas from fermentation = cracks
in the butt.
5- Also If bacteria produce H₂S =black preciptate
Pseudomonas E.Coli
Sugar fermentation
To determine if a bacterium can ferment a
particular carbohydrate and determine the end
products of that fermentation .
Media ingredients:
0.5%-1.0% of the carbohyrate to be tested ( use:
lactose, glucose , sucrose, dulcitol, saccharose,
sorbitol, and mannitol), nutrient broth, and the pH
indicator phenol red(red at pH 7.4 and yellow below pH 6.8).
The test organism is inoculated into a broth
containing the test sugar and incubated for 3 days
at 37C°.
Result s :
1-If there is no fermentation = remain Red color(-ve)
2-If there is fermentation = yellow color (+ve)
3-If there is fermentation and gas prduction =
yellow color and displacement of medium from
the Durham tube (+ve)with gas .
Carbohydrate Fermentation tubes
(+) with
Starch Hydrolysis
Some bacteria are capable of using starch as a source of
carbohydrate, but in order to do this, they must first
hydrolyze or break down the starch so it may enter the cell.
The bacterium secretes an exoenzyme, which hydrolyzes the
starch by breaking the bonds between the glucose molecules.
Make a single streak line, with the organism on the starch agar
Incubate at 37°C until the next lab period.
Iodine will be added to the plate. Iodine reacts with starch to produce a dark
brown or blue/black color.
+ ve =If starch has been hydrolyzed, there will be a clear zone around the
bacterial growth.(Left side)
- ve = If starch has not been hydrolyzed, the agar will remain a dark
brown or blue/black color .(Right side)
Nitrate Reduction Test
It is used to determine if an organism is capable of
reducing nitrate (NO3-) to nitrite (NO2-) or other
nitrogenous compounds via the action of the enzyme
nitratase (also called nitrate reductase).
All enterobacteriacae give(+ve) result , so by this test
can differentiate enterobacteriacae from G-ve nonenteric bacteria .
The test organism is inoculated into nitrate tubes
containing the nitrate broth and incubated at 37ºC
for 24 hour .
After addition of specific reagents.
1-Tube 1 is (-) it did not turn red and has no gas
2- tube 2 is (+) because it is red indicating presence of nitrite,
3- tube 3 is also (+) while there is no red color to indicate the
presence of nitrite there is nitrogen gas
Oxidase Test
This test is used to identify microorganisms containing the enzyme
cytochrome oxidase (important in the electron transport chain).
It is commonly used to
1- distinguish between oxidase negative (-ve) Enterobacteriaceae and
oxidase positive(+ve) Pseudomonas and Neisserria.
2- differentiate Neisserria and G⁺ve cocci
Transfer loopful of test organism to the filter paper saturated with
(tetramethyl-p-phenylene diamine dihydrochloride) reagent .
Result s :
When the electron donor(T.P.d.d) is oxidized by
cytochrome oxidase it turns a dark purple. This is
considered a positive result. In the picture below the
organism on the right (Pseudomonas aeruginosa) is
oxidase positive.
Gelatin Hydrolysis Test
The ability of microorganisms to hydrolyze gelatin Some bacteria
produce a proteolytic enzyme called gelatinase, that
hydrolyzes gelatin .
Most of the Enterobacteriaceae are gelatin-hydrolysis-test –negative.
Bacteria like Vibrio, Bacillus, and Pseudomonas are gelatin-positive.
Inoculated nutrient gelatin media(first tube) with test organisms
and let the second tube without inoculation as a control.
Tubes are incubated for at least 48 hrs and then placed into the
refrigerator for approximately 30 minutes.
+ = refrigerated gelatin remains liquid (at least partially)
- = the gelatin is still intact , refrigerated gelatin is solid.