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