Voges
Proskauer Test:
This
test determines the ability of microorganism to ferment glucose. The end
products
of glucose metabolism,pyruvic acid, is further metabolized by using
Butylene glucol pathway to produce neutral end such as acetoin and 2,3 butanediol.
When Barrit's reagent A ( 40% KOH) and Barrit's reagent B (5% solution of alpha
naphthol) is added it will detect the presence of acetoin, the precursor in the
2,3- butanediol synthesis. Acetoin in the presence of Oxygen and Barrit's
reagent is oxidized to diacetyl, where alpha naphthol act as a catalyst.
Diacetyl then reacts with guanidine components of peptone to produce a cherry
red colour
Citrate
Utilization Test:
This
test determines the ability of microorganism to utilize Citrate. Some bacteria
have the capability to convert the salts of organic acids, for example, Sodium
citrate to alkaline carbonates. Sodium citrate is one of the important
metabolite of Kreb's cycle. Certain bacteria use citrate as the sole carbon
source. Citrate utilization requires a specific membrane transporter and
citrate lyase activity. Citrate is converted to Oxalo acetic acid by citrate
lyase and oxaloacetate decarboxylase activity will convert oxaloacetate to
pyruvate with the release of carbondioxide. The other products of the reaction
are acetate, Lactic acid, formic acid etc. The carbondioxide reacts with sodium
and water to form sodium carbonat.
TThe
triple sugar- iron agar test is designed to differentiate among the different
groups or genera of the Enterobacteriaceae, which are all gram negative bacilli
capable of fermenting glucose with the production of acid, and to distinguish
them from other gram negative intestinal bacilli. This differentiation is based
on the differences in carbohydrate fermentation patterns and hydrogen sulfide
production by the various groups of intestinal organisms. Carbohydrate
fermentation is detected by the presence of gas and a visible colour change
(from red to yellow) of the pH indicator, phenol red. The production of
hydrogen sulfide is indicated by the presence of a precipitate that blackens
the medium in the butt of the tube. TSI Agar contains three fermentative
sugars, lactose and sucrose in 1% concentrations and glucose in a concentration
of 0.1%. Due to the building of acid during fermentation, the pH falls. The
acid base indicator Phenol red is incorporated for detecting carbohydrate
fermentation that is indicated by the change in colour of the medium from
orange red to yellow in the presence of acids. In case of oxidative decarboxylation
of peptone, alkaline products are built and the pH rises. This is indicated by
the change in colour of the medium from orange red to deep red. Sodium
thiosulfate and ferrous ammonium sulfate present in the medium detects the
production of hydrogen sulfide (indicated by blackening in the butt of the
tube). To facilitate the detection of organisms that only ferment glucose, the
glucose concentration is one-tenth the concentration of lactose or sucrose. The
small amount of acid produced in the slant of the tube during glucose
fermentation oxidizes rapidly, causing the medium to remain orange red or
revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained
in the butt of the tube since it is under lower oxygen tension. After depletion
of the limited glucose, organisms able to do so will begin to utilize the
lactose or sucrose. To enhance the alkaline condition of the slant, free
exchange of air must be permitted by closing the tube cap loosely. If the tube
is tightly closed, an acid reaction (caused solely by glucose fermentation)
will also involve the slant.
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