OS 214: Digestive System Micro Preceptors

Biochemical Activity of Microorganisms Micro Exam

Outline **complete permanent acidification of both slant and butt if the

bacteria are also lactose-fermenting
I.
Action on Proteins and
Nitrogenous Compounds C. Production of Indole
A. Hydrolysis of Gelatin
B. Production of Hydrogen Sulfide The indole test is a biochemical test performed on bacterial
C. Production of Indole species to determine the ability of the organism to split indole
D. Reduction of Nitrate to Nitrites from the amino acid tryptophan through the action of
E. Decomposition of Urea tryptophanase.
II. Action on Carbohydrates
A. Carbohydrate Fermentation The Ehrlich's reagent (aldehyde indicator) added after the
B. Production of Acetyl-Methyl- ether, is used when performing the test on non-fermenters and
Carbinol and other acids from anaerobes. Indole reacts with an aldehyde to produce a red-
Glucose colored product.
III. Other Biochemical Activities
Interpretation of Results
A. Citrate Utilization
B. Motility Test Color Interpretation
IV. Results Red/red-violet Positive for indole
Disclaimer: This trans is just a supplement of the manual Yellow Negative for indole
already provided by the Microbiology Dept. This will deal
mostly on the principles behind the biochemical tests. Please **Indole production is a key test for the identification of
refer to the manual for the steps on how to perform each test. Escherichia coli. (yields a red ring)
God bless sa exam! 
D. Reduction of Nitrate to Nitrites
I. Action on Proteins and Nitrogenous Compounds
The nitrate reduction test permits differentiation between these
A. Hydrolysis of Gelatin
species which are nitrate positive and N. gonorrhoeae (nitrate-
negative). Nitrate reductase reduces nitrate to nitrite.
Gelatin is hydrolyzed by the enzyme gelatinase. Hydrolyzed
gelatin will not solidify even if placed in ice or refrigerated.
Organisms that have gelatinase include Serratia,
Xenorhabdus, S. aureus, S. marcescens, and Proteus species.

B. Production of Hydrogen Sulfide

Triple Sugar Iron (TSI) agar is a differential medium that can

distinguish between a number of Gram-negative enteric bac-
teria based on their physiological ability (or lack thereof) to:

a. metabolize lactose and/or sucrose

b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S.

The medium contains 1.0% each of sucrose and lactose and

0.1% glucose. The medium also contains ferrous sulfate. Figure 1. Detection of nitrite in medium

• If the bacterium forms H2S, this chemical will react Interpretation of Results
with the iron to form ferrous sulfide, which is seen as
a black precipitate in the butt (a black butt). Color Interpretation
Purple/red/maroon Positive for nitrite
Interpretation of Results Absence of color Nitrate not reduced to nitrite
(absence of nitrate reductase) OR
Slant color Interpretation nitrate reduction occurred but
Light orange Non-fermenting organism; original converted rapidly to ammonia or
color of TSI gaseous nitrogen
Red/pink Does not ferment either lactose or
sucrose; alkalinization (by- E. Decomposition of Urea
products of glucose fermentation
further used for amine production) urease
Yellow Ferments lactose and/or sucrose; Urea -----> ammonia + CO2
acidification (glucose fermenta-
tion) Urease is an enzyme that breaks the carbon-nitrogen bond of
amides to form carbon dioxide, ammonia, water.
Butt color/condition Interpretation
Members of genus Proteus are known to produce urease.
Red/pink No fermentation; bacteri-
um is an obligate aerobe
Urease can be detected by plating bacteria onto an amide
Yellow Some fermentation; fac- containing medium, specifically urea. When urea is broken
ultative anaerobe down, ammonia is released and the pH of the medium
With gas Seen as bubbles or cracks increases (becomes more basic). This pH change is detected
in the agar by a pH indicator that turns pink in a basic environment. A pink
Black Positive for H2S medium indicates a positive test for urease.
November 11, 2008 | Tuesday Page 1 of 2
Noems, Vic, Jow, Kitts
 OS 214: Digestive System Micro Preceptors

Biochemical Activity of Microorganisms Micro Exam

B. Motility Test
II. Action on Carbohydrates
Another important differential test among Enterobacteriaceae
A. Carbohydrate Fermentation and many other bacteria is motility. It can be done by growing
bacteria in semisolid media. The media are prepared as butt
Many tests used to differentiate among members of the tubes. The organism is inoculated by stabbing a straight wire
Enterobacteriaceae determine the organism’s ability to utilize a carrying the inoculums once vertically into the center of the
carbohydrate with the production of acid metabolic end agar butt to a depth of approximately 2 cm. After incubation,
products. In addition to detecting acid, carbohydrate motility is evident as a haze of growth extending into the
fermentation can be modified to detect the formation of gas by agar from the stab line.
placing a very small, inverted glass tube (Durham tube) into the
broth. If gas is produced during fermentation of the IV. Results
carbohydrate, it is collected in the inverted tube and is visible
as a bubble. Biochemical Escherichia Salmonella Shigella
Test coli typhi flexneri
PLEASE REMEMBER!!! TSI A/Ag+ K/A; +H2S K/A; no
Color Sugar H2S
Black Sucrose Motility +/- + -
Brown Maltose Indole + - +/-
Red Lactose Lysine +/- +
Green Glucose decarboxylase
Orange Mannitol Lysine +/- -
deaminase
Examine the tubes for production of acid and/or gas. Methyl Red Test + + +
Interpretation of Results Voges- - - -
Proskauer
Color Interpretation Citrate - - -
yellow Fermented the sugar Utilization
pink Did not ferment the sugar
Urea - - -
Mannitol + + +
B. Production of Acetyl-Methyl-Carbinol and other
Maltose + + -
acids from Glucose
Lactose + - -
Methyl Red Test Glucose + - +/-
-test for the formation of acids from glucose Sucrose + - -
-positive test: red color (indicates a decrease in pH of Gel - - -
the solution) Nitrate + + +
-negative test: yellow color

Vogues-Proskauer Test GREETINGS!!!

-test for the formation of a compound called acetyl-
methyl carbinol (also known as acetoin) from glucose Noems: Ka-stress ang trans na to! waah! Sana makatulong pa
-pink color indicates formation of acetyl-methyl carbinol rin.  Hello sa lahat ng mga kaibigan ko! Thank you sa lahat
ng nakaalala ng bday ko!  Di na ko pedia! waah! Get well
III. Other Biochemical Activities soon kitts! At ikaw, help me understand, let me understand. 
A. Citrate Utilization
Jowel: Bading ako. 
The citrate test is used to determine the ability of a bacterium
Dinno: Good luck sa atin sa TRP! Go raphy! Animo! 
to utilize citrate as its only source of carbon. The ability to
metabolize citrate for example is useful for differentiating
Victor: I love you.
among enterobacteriaceae.

Simmons Citrate Agar is a medium containing citrate as the
sole carbon source and ammonium salts as the sole nitrogen
source. Organisms that metabolize citrate utilize ammonium
salts releasing ammonia and increasing the pH of the medium.
Bromthymol blue is present in the medium as the indicator dye.
It is green at neutral pH and deep blue above pH 7.6.
Bacteria can break the conjugate base salt of citrate into
organic acids and carbon dioxide. The carbon dioxide can
combine with the sodium from the conjugate base salt to form
a basic compound, sodium carbonate. A pH indicator in the
medium detects the presence of this compound by turning blue
(a positive test).
Interpretation of Results
Organism Color
Salmonella typhi Royal blue (positive test)
Escherichia coli Green (negative)
Ada: Text lang ng text!  I love 2012! 
Jenni: Haha, sa sariling trans walang greeting pero dito meron
;P Hi 2012! Sana mamemorize natin lahat ang mga positive at
negative na results at madifferentiate natin kung ano ang red
sa orange kahit di katanggap tanggap. Greet ko raw si Ada. Hi
Ada! Go for Renal! Hi SL-mates! We’re growing in number! Hi
researchmates! Hi PSF! Hanapin mo yung fire tree. :p God
bless sa micro exam :D
Leann: toxic naman ng nutrition module. Tsaka, kanina pa
kaming kain nang kain ng junkfood. So much for studying all
the recommended energy intake tables. Haha good luck sa atin
sa exam bukas.  I just want so much to get everything over
with—the 6 weeks, the easter egg hunting in the middle of
November, the surveys, the presentations—and go home. Nor,
mira, okay ba ang nilipatan natin? Hi sa mga seatmates. Ada,
till the next module. hehehe God bless everyone.

November 11, 2008 | Tuesday Page 2 of 2

Noems, Vic, Jow, Kitts