3)  Triple Sugar Iron(TSI) Agar Test:  
                     The TSI agar test  is used to differentiate among the members
of the family Enterobacteriaceae and between Enterobacteriaceae
and other intestinal bacteria. The authors have chosen
three common bacteria in the family Enterobacteriaceae:
Escherichia coli, Proteus vulgaris, and Shigella flexneri.  All
three are facultatively anaerobic gram-negative rods. In a
TSI tube, E. coli produces an acid butt, an acid or alkaline
slant, and no H2S, but does produce gas. P. vulgaris produces
an acid butt, an acid or alkaline slant, H2S, and gas. S.
flexneri produces an acid butt, an alkaline slant, no H2S, and
no gas. For the other intestinal bacteria, the authors have
chosen Alcaligenes faecalis and Pseudomonas aeruginosa.
Both of these intestinal bacteria are gram-negative aerobic
rods. In a TSI tube, A. faecalis produces an alkaline butt, alkaline
slant, H2S, and gas; P. aeruginosa, an acid butt, alkaline
slant, H2S, and gas.
                                    As originally described in 1911 by F. F. Russell, the
triple sugar iron (TSI) agar test is generally used for
the identification of enteric bacteria (Enterobacteriaceae).
It is also used to distinguish the Enterobacteriaceae
from other gram-negative intestinal bacilli by
their ability to catabolize glucose, lactose, or sucrose,
and to liberate sulfides from ferrous ammonium sulfate. TSI agar slants contain a 1%
concentration of lactose and sucrose, and a 0.1% glucose
concentration. The pH indicator, phenol red, is also incorporated into the medium to detect acid production 
from carbohydrate fermentation. 
   Often Kligler Iron Agar (named after I. J. Kligler
in 1917), a differential medium similar to TSI, is used
to obtain approximately the same information.
TSI slants are inoculated by streaking the slant
surface using a zig-zag streak pattern and then stabbing
the agar deep with a straight inoculating needle. Incubation is for 18 to 24 hours in
order to detect the presence of sugar fermentation, gas
production, and H2S production. The following reactions
may occur in the TSI tube;
1. Yellow butt (A) and red slant (A) due to the
fermentation of glucose (phenol red indicator turns
yellow due to the persisting acid formation in the
butt). The slant remains red (alkaline) (K) because
of the limited glucose in the medium and, therefore,

limited acid formation, which does not persist.
2. A yellow butt (A) and slant (A) due to the
fermentation of lactose and/or sucrose (yellow slant
and butt due to the high concentration of these
sugars) leading to excessive acid formation in the

entire medium. 
3. Gas formation noted by splitting of the agar.
4. Gas formation (H2S) seen by blackening of the
5. Red butt (K) and slant (K) indicates that none of
the sugars were fermented and neither gas nor H2S were produced.

Please visit these links for references:
Cited By Kamal Singh Khadka
Assistant Professor Pokhara University(PU), Regional College Of Science & Technology(RE-COST), Prithvi Narayan Campus(PNC), Novel Academy(NA). 


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