University of Tabuk
Faculty of Applied Medical Science
Department of Medical Laboratory Technology
Mr.AYMAN.S.YOUSIFM.SC IN Microbiology
&IMMUNOLOGY
Academic Year: 1434-1435 (2013-2014)
2
specimens
General Procedure of Bacteriological Diagnosis
Morphologic Identification
Sub culture in the special types of media for confirmation
Biochemical tests ( Identification and Isolation )
Susceptibility Testing ( to select the suitable antibiotics for treatment the pathogenic isolated bacteria from the specimen )
Serological Test
Microscopy & Staining
Cultivation in suitable types of media
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) AgarIs 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) AgarFerrous sulfateSodium thiosulfateSodium chlorideAgar (1.2%)Phenol redpH = 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
Red/yellow with bubbles and black precipitate
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
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 2Yersinia 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
K/A,G, H2S Salmonella (most serotypes) .Proteus mirabilis.Edwardsiella tarda .
Yellow/yellow with black precipitate
A/A, H2S Citrobacter freundiiProteus vulgaris11Non-lactose, sucrose fermenter
2
Non-lactose, sucrose fermenter
OXIDASE TESTThe 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 TESTTHE 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 TESTResult 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
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