Biochemical Tests Flashcards
is used to identify bacterial species based
on the differences in the biochemical reactions of each of
the different types of bacteria
Biochemical test
The is used to identify bacteria that
produce cytochrome c oxidase, an enzyme of the
bacterial electron transport chain (ETC)
oxidase test
t is based on the principle that
certain bacteria produce indophenol blue from the
oxidation of dimethyl-p-phenylenediamine and anaphtho
Principle of Oxidase tes
Oxidase POSITIVE bacteria:
o Pseudomonas
o Vibro cholerae
o Neisseriae
o Campylobacter
o Helicobacter / Haemophilus
o Aeromonas
o Alcaligene
Precaution while performing oxidase test:
o Do not use nickel-base alloy wires containing
chromium and iron wire (nichrome wire).
o Interpret test within 10 seconds.
o Perform test using 5% SBA or on a medium without fermentable sugar
maintains osmotic pressure. in tsi
Nacl
are the fermentable
carbohydrates
Lactose, Sucrose, and Dextrose
S make H2S (Hydrogen sulfide) indicator system
sodium thiosulfate and ferrous sulfate
is reduced to H2S by several species of bacteria
and H2S combines and form insoluble black precipitates.
FeSO4 present in the medium
Thiosulfate
how long is the incubation for tsi
Incubation is for 18 to 24 hours in order to detect the
presence of sugar fermentation, gas production, and H2S
production
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 or sodium thiosulfate
TSI
agar slants contain a 1% concentration of
sucrose and lactose, and 0.1% glucose
TSI
IN TSI, it is incorporated into
the medium to detect acid production from
carbohydrate fermentation.
phenol red
tsi results color
The indicator is pink at alkaline pH and yellow in acidic pH, at neutral pH it remains red
Yellow butt (A) and red slant (K)
- due to fermentation of glucose (phenol indicator turns yellow due to persisting acid formation in the butt)
- The slant remains red (alkaline) (K) because of
limited glucose in the medium. Therefore, limited acid formation, which does not persist.
A yellow butt (A) and slant (A)
due to fermentation of lactose and/or sucrose.
- Yellow slant and butt due to high concentration of these sugars leading to excessive acid formation in the entire medium.
o noted by splitting agar.
Gas formation
o seen by blackening of agar.
Gas formation (H2S)
red butt (K) and slant (K)
introduces that none of the sugars were fermented and neither gas nor H2S produced.
Slant Butt Gas H2S
of Escherihia,
Acid (A) Acid (A) Pos (+) Neg (-)
Klebsiella,
Slant Butt Gas H2S
Acid (A) Acid (A) Pos (+) Neg (-)
Enterobacte
Slant Butt Gas H2S
Acid (A) Acid (A) Pos (+) Neg (-)
Slant Butt Gas H2S
shigella
Shigella,
Alkaline (K) Acid (A) Neg (-) Neg (-)
Slant Butt Gas H2S
Serratia
Serratia
Alkaline (K) Acid (A) Neg (-) Neg (-)
Slant Butt Gas H2S
salmonella
Salmonella,
Alkaline (K) Acid (A) Pos (+) Pos (+)
Slant Butt Gas H2S
proteus
Proteus
Alkaline (K) Acid (A) Pos (+) Pos (+)
Slant Butt Gas H2S
Pseudomonas
Pseudomonas
Alkaline (K) Alkaline (K) Neg (-) Neg (-)
If an Enterobacteriaceae contains amino acid
decarboxylase, amines produces by decarboxylae action
cause an ________ pH, and _________
Amino acid decarboxylation
alkaline ph
bromocresol purple
Amino acid decarboxylation
result
alkaline = purple = positive/amino acid is decarboxylated
acidic = yellow/glucose fermentation acidifie s broth
Decarboxylation patterns are essential for the genus
identification of
Klebsiella, Enterobacter, Eschericia, and
Salmonella
Enterobacter aerogenes, E. cloacae,
Proteus mirabilis, and Shigella sonnei
Aim: To differentiate gram-negative bacilli based on decarboxylation or deamination of the lysine and the formation of hydrogen sulfide (H2S).
Lysine Iron Agar
Lysine decarboxylation is indicated by an increase in
alkaline (purple)
medium
Lysine deamination is detected by a
red slant.
Dextrose fermentation is indicated by a
purple slant and a
yellow butt
lia
deaminate lysine which
results in a distinctive red slant over an acid (yellow) butt. give example
Proteus and Providencia spp.
To determine the ability of microbe to
degrade amino acid tryptophan.
Indole Production Test
indole production test result
: Development of cherry red colour at the
interface of the reagent and the broth, within seconds after adding the Kovacs’ reagent indicates the presence of indole and the test is positive.
If no color change is observed, then the test is negative and so organisms are not
capable of producing tryptophanase
To differentiate E. coli and E. aerogene
mr
e. coli +
e. aerogenes -
determine the ability of microbes to oxidize glucose with
production and stabilization of high content of acid end
product.
mr
differentiate the E.coli and E. aerogenes by the
production of 2,3-butanediol and
acetoin via glucose fermentation
VP
e. coli -
e. aerogenes +
RESULT IN VP
nterpretation: Development of crimson red color indicates positive test for E. aerogenes; and no color indicates negative test
Principle: This test determines the capability of some organisms to produce non-acidic or neutral end products, such as acetyl methyl corbinol (acetoin), from the organic acid that results from glucose metabolism
vp
Aim: To determine the ability of the microbes to ferment
citrate as sole carbon source
Citrate Test
cARBON AND NITROGEN SOURCE IN CITRATE TEST
Sodium citrate as the carbon source, Ammonium
(NH4
+) as a nitrogen source
CITRATE TEST RESULT
+ = BLUE
- = NO COLOR
UREASE TEST RESULT
+ = REDDISH PINK TO RED
- = YELLOW
The hydrolysis of urea is catalysed by specific enzyme urease to yield
2 moles of ammonia, water, CO2.
o determine the ability of
microbes to ferment sugars with the
production of an acid and/or gas
Sugar Fermentation Test
If fermenting bacteria are grown in a liquid culture
medium containing the carbohydrate, they may
produce ______as by-products of the
fermentation.
If fermenting bacteria are grown in a liquid culture
medium containing the carbohydrate, they may
produce organic acids as by-products of the
fermentation.
Gases produced during the fermentation process can
detect by using a small, inverted tube (Durham tube)
within the liquid culture medium
Sugar Fermentation TesT
Sugar Fermentation TesT RESULT
Interpretation:
o If the medium changes from colorless to yellow and
gas bubble is found in Durham’s tube then it indicates
acid and gas production.
o If no change observed in the color of medium then
sugar is not degraded by the organism
To determine the ability of some microbes to reduce
nitrate (NO3
-
) to nitrites (NO2
-
) or beyond the nitrite stage
NITRATE REDUCTION
RESULT OF NITRATE REDUCTION
+ = RED
