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University of Tabuk
Faculty of Applied Medical Science
Department of Medical Laboratory Technology
Mr.AYMAN.S.YOUSIF
M.SC IN Microbiology
&IMMUNOLOGY
Academic Year: 1434-1435 (2013-2014)
General Procedure of Bacteriological Diagnosis
specimens
Cultivation in suitable types of media
Morphologic Identification
Microscopy & Staining
Biochemical tests ( Identification and Isolation )
Sub culture in the special types of media for confirmation
Serological Test
Susceptibility Testing ( to select the suitable antibiotics for
treatment the pathogenic isolated bacteria from the specimen ) 2
Urea Hydrolysis (urea test)
 Urea can be broken down with the help of the enzyme
urease, producing the alkaline product of ammonia
plus carbon dioxide. That causes the pH indicator
phenol red to turn a beautiful shade of hot pink
(pink-red) .
OBJECTIVES:
 Understand the reactions of bacteria in urea broth.
THE PROCEDURE:
1. Inoculate the tube of urea broth with your unknown
bacterium.
2. Incubate over night at 37 degrees C.
INTERPRETATION:
 The alkaline reaction turns the pH
indicator to hot pink or red colour .
 A yellowish color is still a negative
reaction, although acidic.
 Some bacteria will produce a WEAK
reaction, with a bit of pink in the
tube.
 This should be recorded as weak +.
 It is a good idea to compare your tube
with an uninoculated to make sure
that you do not have a weak + result.
Triple sugar iron agar (TSI)
OBJECTIVE:
It is used to
Differentiate Enterobacteriaceae
based on the ability to
 Reduce Sulfur
 Ferment Carbohydrates.
Triple Sugar Iron (TSI) Agar
Is a Differential medium that contains .
 Yeast extract
0.3% (% = grams/100 mL)
 Beef extract
0.3%
 Peptone
1.5%
 Proteose peptone 0.5%
Total Protein = 2.6%
 Lactose
1.0%
 Sucrose 1
1.0%
 Glucose
0.1%
Carbohydrate = 2.1%
1Absent
in Kligler Iron Agar
Triple Sugar Iron (TSI) Agar
 Ferrous sulfate
 Sodium thiosulfate
 Sodium chloride
 Agar (1.2%)
 Phenol red
 pH = 7.4
Triple sugar iron agar (TSI)
THE PROCEDURE:
1. Inoculate the tube of TSI media with your unknown
bacterium (stabbing and zig zag on the surface ).
2. Incubate over night at 37 degrees C.
 If an organism can ferment any of the three sugars
present in the medium, the medium will turn yellow.
 If an organism can only ferment dextrose (Glucose) ,
the small amount of dextrose in the medium is used by
the organism within the first ten hours of incubation.
 If an organism can reduce sulfur, the hydrogen sulfide
(H2S) which is produced will react with the iron to form
iron sulfide, which appears as a black precipitate.
Results (slant/butt)
Symbol
Interpretation
Red/yellow
K/A
Glucose fermentation only; Peptone
catabolized
Yellow/yellow
A/A
Glucose and lactose and/or sucrose
fermentation
Red/red
K/K
No fermentation; Peptone catabolized
Red/no color change
K/NC
No fermentation; Peptone used
aerobically
Yellow/yellow with bubbles
A/A,G
Glucose and lactose and/or sucrose
fermentation; Gas produced
Red/yellow with bubbles
K/A,G
Glucose fermentation only; Gas
produced
K/A,G, H2S
Glucose fermentation only; Gas
produced; H2S produced
Red/yellow with black precipitate
K/A, H2S
Glucose fermentation only; H2S
produced
Yellow/yellow with black precipitate
A/A, H2S
Glucose and lactose and/or sucrose
fermentation; H2S produced
Red/yellow with bubbles and black
precipitate
A=acid production; K=alkaline reaction; G=gas
production; H2S=sulfur reduction
Results (slant/butt)
Symbol
Interpretation
Red/yellow
K/A
Shigella , Providencia
Yellow/yellow
A/A
Serratia marcescens 2
Yersinia enterocolitica 2
Red/red
K/K
Nonfermenters such as Pseudomonas
Yellow/yellow with bubbles
A/A,G
Escherichia coli , Klebsiella pneumoniae ,
Klebsiella oxytoca , Enterobacter aerogenes
Enterobacter cloacae , Serratia marcescens 1, 2
Red/yellow with bubbles
K/A,G
Salmonella serotype Paratyphi A
Red/yellow with bubbles and
black precipitate
Yellow/yellow with black
precipitate
2
Non-lactose, sucrose fermenter
K/A,G, H2S
A/A, H2S
Salmonella (most serotypes) .
Proteus mirabilis.
Edwardsiella tarda .
Citrobacter freundii
Proteus vulgaris1
1Non-lactose, sucrose fermenter
OXIDASE TEST
 The oxidase test is a key test to differentiate between the
families of Pseudomonadaceae (ox +) and Enterobacteriaceae
(ox -)
 Is useful for identification of many other bacteria, those that
have to use oxygen as the final electron acceptor in aerobic
respiration, and produce cytochrome oxidase enzyme.
OBJECTIVE:
 Test for the enzyme oxidase on your unknown isolates.
Materials Needed:
 Oxidase Reagent. (Tetramethyl-p-phenylenediamine dihydrochloride)
 Wooden Rods.
 Filter Paper .
OXIDASE TEST
THE PROCEDURE:
 A piece of filter paper in a clean Petri dish is soaked with
a few drops of oxidase reagent.
 Using a piece of stick or glass rod (not an oxidized wire
loop) remove a colony of the test organism and smear it
on the filter paper.
 Look for the development of a blue-purple colour within
a few seconds
 When the organism is oxidase-producing, the
phenylenediamine in the reagent will be oxidized to a
deep purple colour.
 Alternatively an oxidase reagent strip can be used.
OXIDASE TEST
Result
 Blue-purple colour - Positive oxidase test (within 10 seconds)
Pseudomonas aeruginosa , N. gonorrhoeae , Vibrio cholerae
 No blue-purple colour - Negative oxidase test (within10seconds)
Escherichia coli