Transcript yellow
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