BIOCHEMICAL TESTS
1.Phenol-Red Fermentation Assays
a. What are two common by-products of sugar fermentation? How is phenol-red broth designed
to measure both?
Two common byproducts of sugar fermentation are acid and gas (mainly hydrogen and carbon
dioxide).
Acid production - Acid production is indicated by the color changes of medium from normal red
to yellow and will remain red for no acid production.
Gas production - During fermentation, any gas produced by the microbes gets trapped in the
inverted Durham tube and obvious bubbles appear in the inverted Durham tube.
b. What does a pink-red color after incubation indicate about the bacterium of interest? Explain
your conclusion.
It indicates the production of nitrogenous products (pH > 8.2)
c. What does a yellow color after incubation indicate about the bacterium of interest? Explain
your conclusion.
Phenol red is yellow at the pH < 6.8 indicating the production of acid by the fermentation of
carbohydrate. If the organism is able to utilize the carbohydrate, an acid by-product is created,
which turns the media yellow.
2.Triple Sugar Iron Agar
a. What happens when fermenting bacteria uses up all the glucose present in TSI media? Explain
your conclusion.
The glucose fermenting bacteria ferment the glucose present in the media and produces acidic
byproducts leading to the drop of pH in the media. This pH drops results in the phenol red to
change its color from deep red to yellow. During the initial hours, the full media will become
yellow due to the fermentation of the whole glucose present in the media, but after that the color
of the slant will become red again. This is because the amount of glucose present in the slant is
less as compared to butt where more media is present. The low acid production in the slant
, oxidizes rapidly causing an increase in the pH to alkaline condition. Thus, the color of the slant
will become red. Hence, after depletion of the entire glucose the TSI media will see as yellow at
the bottom and red color at the slant. But some glucose fermenting bacteria are capable of
fermenting lactose or sucrose also, in such cases after depletion of the glucose; they will start
fermenting lactose or glucose.
b. Why is hydrogen sulfide H2S production observed primality in the butt of the tube? Explain
your conclusion.
The H2S produced during the reaction and reacts with the ferrous sulfate present in the medium.
H2S is a colorless gas and when it reacts with ferrous Sulphate - produces ferrous sulfide.
Ferrous sulfide is black and is insoluble in the media leading to form as a black precipitate at the
bottom of the tube.
c. How is sulfur reduction and glucose fermentation observed in TSI media? Explain your
conclusion.
Some bacteria use thiosulphate anion present in the medium as a terminal electron acceptor
leading to reducing it to sulfide. The H2S gas produces reacts with the ferrous sulphate and
produces ferrous sulfide, which will be seen as a black precipitate at the bottom of the tube.
Some examples of H2S producing bacteria are Salmonella, Citrobacter etc. The glucose
fermenting bacteria, ferment the glucose in the media and produces acidic byproducts causing a
pH drop in the media. In acidic condition the color of phenol red will become yellow. During the
initial hours of incubation, the full media will become yellow (both slant and butt) due to the
fermentation of the glucose, but after that the color of the slant will become red again. This is
because the amount of glucose present in the slant is less as compared to butt where more media
is present. The low acid production in the slant oxidizes rapidly causing an increase in the pH to
alkaline condition. The color of the slant will become red and the observation of TSI slant for
glucose fermenting bacteria will be yellow but and red slant.
3. Catalase Test
a. What benefit is derived from the actions of catalase for a bacteria?
1.Phenol-Red Fermentation Assays
a. What are two common by-products of sugar fermentation? How is phenol-red broth designed
to measure both?
Two common byproducts of sugar fermentation are acid and gas (mainly hydrogen and carbon
dioxide).
Acid production - Acid production is indicated by the color changes of medium from normal red
to yellow and will remain red for no acid production.
Gas production - During fermentation, any gas produced by the microbes gets trapped in the
inverted Durham tube and obvious bubbles appear in the inverted Durham tube.
b. What does a pink-red color after incubation indicate about the bacterium of interest? Explain
your conclusion.
It indicates the production of nitrogenous products (pH > 8.2)
c. What does a yellow color after incubation indicate about the bacterium of interest? Explain
your conclusion.
Phenol red is yellow at the pH < 6.8 indicating the production of acid by the fermentation of
carbohydrate. If the organism is able to utilize the carbohydrate, an acid by-product is created,
which turns the media yellow.
2.Triple Sugar Iron Agar
a. What happens when fermenting bacteria uses up all the glucose present in TSI media? Explain
your conclusion.
The glucose fermenting bacteria ferment the glucose present in the media and produces acidic
byproducts leading to the drop of pH in the media. This pH drops results in the phenol red to
change its color from deep red to yellow. During the initial hours, the full media will become
yellow due to the fermentation of the whole glucose present in the media, but after that the color
of the slant will become red again. This is because the amount of glucose present in the slant is
less as compared to butt where more media is present. The low acid production in the slant
, oxidizes rapidly causing an increase in the pH to alkaline condition. Thus, the color of the slant
will become red. Hence, after depletion of the entire glucose the TSI media will see as yellow at
the bottom and red color at the slant. But some glucose fermenting bacteria are capable of
fermenting lactose or sucrose also, in such cases after depletion of the glucose; they will start
fermenting lactose or glucose.
b. Why is hydrogen sulfide H2S production observed primality in the butt of the tube? Explain
your conclusion.
The H2S produced during the reaction and reacts with the ferrous sulfate present in the medium.
H2S is a colorless gas and when it reacts with ferrous Sulphate - produces ferrous sulfide.
Ferrous sulfide is black and is insoluble in the media leading to form as a black precipitate at the
bottom of the tube.
c. How is sulfur reduction and glucose fermentation observed in TSI media? Explain your
conclusion.
Some bacteria use thiosulphate anion present in the medium as a terminal electron acceptor
leading to reducing it to sulfide. The H2S gas produces reacts with the ferrous sulphate and
produces ferrous sulfide, which will be seen as a black precipitate at the bottom of the tube.
Some examples of H2S producing bacteria are Salmonella, Citrobacter etc. The glucose
fermenting bacteria, ferment the glucose in the media and produces acidic byproducts causing a
pH drop in the media. In acidic condition the color of phenol red will become yellow. During the
initial hours of incubation, the full media will become yellow (both slant and butt) due to the
fermentation of the glucose, but after that the color of the slant will become red again. This is
because the amount of glucose present in the slant is less as compared to butt where more media
is present. The low acid production in the slant oxidizes rapidly causing an increase in the pH to
alkaline condition. The color of the slant will become red and the observation of TSI slant for
glucose fermenting bacteria will be yellow but and red slant.
3. Catalase Test
a. What benefit is derived from the actions of catalase for a bacteria?
