**5.3.6 Triple tube method**

Single colony that identification is desired is made suspension in the bouillon or a 3rd tube. Incubation is performed at 37°C for 3 to 4 hours if required. Plantings are performed to 2nd and 1st tubes. It is left for incubation at 37°C for 18-22 hours.

1. Matters that we may observe in the tube:

Isolation and Identification of Salmonellas from Different Samples 149

The mixture is distributed into screw tubes as 7 to 8 ml after boiling. It is sterilized in the

The mixture is distributed into screw tubes as 5 to 6 ml after boiling. It is sterilized in the

L-Tryptophane 0,3 g Potassium dihidrogen phosphate (KH2PO4) 0,1 g Dipotassium hydrogen phosphate (K2HPO4) 0,1 g NaCl 0,5 g Urea 2 g Ethanole (95%) 1ml Phenol red(0,2 %) 1.25 ml Distilled water 1,000 ml Ph 6,5

The mixture is sterilized by the filter after dissolving. It is distributed into sterile tubes. If

Peptone (casein) 5 g Neopeptone 5 g Mannitol 2 g Potassium nitrate 1,7 g Phenol red(0,2 %) 20 ml Distilled water 1,000ml Agar 2,5 g

autoclave at 121°C for 15 minutes. Tubes are frozen as oblique.

autoclave at 121°C for 15 minutes. Ph is adjusted to 7.

there is no filter, it is sterilized by tyndalisation method (*Table 3)*.

Table 3. Identification Schedule of *Salmonellas* According to triple Tube Method

2. TUBE:

3. TUBE:


Phennyaline deaminase Oxidase

	- a. Mannitol fermentation: It is understood by conversion of the color from red into yellow.
	- b. Motility: It is smeared through the middle and it is reproduced to right and left alon the planting line.
	- c. Nitrate reduction: 4 drops each from indicators A and B are dripped.
	- a. UREASE formation is observed by conversion o the medium into red color.
	- b. Indole: 0,5 ml of Kovacs indicator is added from the side of the tube slowly. Red color indicates that the test is positive.
	- c. Tryptophane deaminase: 5 drops of medium is transferred into a sterile agglutination tube via a pipette before addition of Kovacs indicator to the medium. 1 drop of 10% FeCl3 is added on it. If the color turns into red tile color within 3 to 5 minutes, test is positive.

Mediumd used in triple tube method

1. TUBE: klikler I A, or TSI agar are used.


The mixture is distributed into screw tubes as 7 to 8 ml after boiling. It is sterilized in the autoclave at 121°C for 15 minutes. Tubes are frozen as oblique.

2. TUBE:

148 Salmonella – A Dangerous Foodborne Pathogen

yellow color.

red into yellow.

of gas bubbles.

yellow.

positive.

Mediumd used in triple tube method

g. Other tests: Beta galactosidase

2. Matters that we may observe in the tube:

3. Matters that we may observe in the tube:

color indicates that the test is positive.

1. TUBE: klikler I A, or TSI agar are used.

alon the planting line.

Oxidase

a. Glucose fermentation: It is understood by turning of the bottom of the tube to

b. Lactose fermentation: The color of sloped surface of the medium turns from orange

d. Lyzine decarboxylase: 4 ml of 4N NaOH and 2 ml of Chloroform are added on the culture. It is kept at room temperature for 15 minutes, 1 ml from chloroform layer is taken by Pasteur pipette. Equal quantities of ninhydrin (from 0,1% solution in chloroform) is added and kept at room temperature for 10 minutes. Formation of

e. Gas formation: It is understood by biodegradation of the medium and occurrence

f. ONPG Test: Loop full culture which was taken from the surface of the medium is dispersed with 0,25 ml of physiological saline. 0,25 ml ONPG solution is added on this and it is kept in the drying oven at 37°C for 30 minutes. Formation of fixed

a. Mannitol fermentation: It is understood by conversion of the color from red into

b. Motility: It is smeared through the middle and it is reproduced to right and left

b. Indole: 0,5 ml of Kovacs indicator is added from the side of the tube slowly. Red

c. Tryptophane deaminase: 5 drops of medium is transferred into a sterile agglutination tube via a pipette before addition of Kovacs indicator to the medium. 1 drop of 10% FeCl3 is added on it. If the color turns into red tile color within 3 to 5 minutes, test is

c. H2S formation: It is understood by formation of black color in the medium.

violet color at the end of this period shows that the test is positive.

yellow color at the end of this period was evaluated as positive.

c. Nitrate reduction: 4 drops each from indicators A and B are dripped.

a. UREASE formation is observed by conversion o the medium into red color.

Peptone 20 g Lactose 10 g Glucose 1 g Sodyum thiosulphate 0,2 g Ferroammonium sulphate 0,3 g NaCl 6 g Agar 17 g Phenol red(0,2 %) 12.5 ml Distilled water 1,000 ml

Ph 7

Phennyaline deaminase


The mixture is distributed into screw tubes as 5 to 6 ml after boiling. It is sterilized in the autoclave at 121°C for 15 minutes. Ph is adjusted to 7.

3. TUBE:


The mixture is sterilized by the filter after dissolving. It is distributed into sterile tubes. If there is no filter, it is sterilized by tyndalisation method (*Table 3)*.

Table 3. Identification Schedule of *Salmonellas* According to triple Tube Method

Isolation and Identification of Salmonellas from Different Samples 151

media.

Fig. 15. API Test Result for *Salmonella* 

to Phoenix ID broth.

(BD Sparks-USA).

**5.3.8 Full automatic bacteria identification device** 

Some amount of material is taken and transferred into selenite F bouillon (bio-Merieux SA-France). It is incubated at 37°C for 16 to 24 hours in the drying oven. Single colony planting is performed to *Salmonella* -Shigella agar (bio-Merieux SA-France) after the period has passed. It is incubated at 37°C for 16 to 24 hours in the drying oven. Bacteria is made suspension to provide 0,40-0,60 McFarlantd turbidity from reproduced suspicious colonies

1 drop of Phoenix AST indicator (Phonex AST Indicator solution, BD Sparks, Benex Limited, Shannon, Ireland) to Phoenix AST broth (Phonex AST borth, BD Sparks, Benex Limited, Shannon, Ireland). 25 µl ID broth is taken and pipetted into AST Broth (BD Phoenix NMIC/ID-82 Sparks- USA). ID and AST broths are transferred to ID and AST pplate. Identification and antibiogram process are performed as full automatically in Phoenix-100

which is probably typical of *Edwardsiella tarda* Biogroup 1. Clinical laboratories usually run this test in Triple Sugar Iron Agar in which the organism's fermentation of sucrose (with consequent high acid production) tends to negate the H2S reaction, and – as a result – the organism is mis-characterized throughout the literature as H2S negative even though it shows a positive reaction in KIA and other H2S-detecting
