**5.3.5 Imvic test**

144 Salmonella – A Dangerous Foodborne Pathogen

Colonies selected are purified. If time is restricted, this purifying process may be ignored.

a. Every colony selected is planted as to allocate single colony to a separate Mac Conkey

c. *Salmonella* colonies appear as transparent and colorless in Mac Conkey agar. Sometimes the centre appears as pink. If there are many organisms that fermantates the lactose, the precipitated area around *Salmonella* colonies which are next to them becomes

d. Planting is performed from typical colonies to normal slant agars. Cultures are

e. Preparation is prepared from slant agar cultures and stained with gram method. If

A passage specified in the following is performed from 24 hours, purified slant agars to

a. It is immersed to the bottom by a loop and then it is planted into slanted part. It is

b. Urease reaction, glucose and mannitol fermentation and gas formation are recorded. Cultures with positive urease reaction converts the medium to a dark purple color. Mannitol fermentation is characterized by bottom part turning into yellow; and gas formation is characterized by appearance of gas bubbles in the agar. *Salmonellas* are urease negative. On the other hand, they fermantate glucose and mannitol with or

c. Urease postitive cultures that do not fermentate glucose or mannitol are assessed as

a. The tube is hold vertically and planting is performed by immersing to 2 cm depth with

b. Tubes are incubated at 35 to 37 °C for 24 hours vertically. If reactions are not significant,

c. Sucrose and salicine fermentation, motility, H2S and indole formation are recorded. Color change in the agar, conversion from original blue-green color to yellow shos that sucrose or salicine or both are fermentated. Darkening of the lead acetate paper indicates H2S formation and indol paper becoming red indicates indole formation.

a. Suspicious single colony is taken from purified culture or selective agar medium and inoculation is performed by immersing to the bottom with a loop of triple sugar iron

c. Cultures that do not give reactions specific to *Salmonellas* are not taken into the assessment. Typical reaction in TSI agar is red color on the slant part, "alkaline reaction"

Typical *Salmonellas* are motile, indole positive, Sucrose and Salicine negative.

**5.3 Biochemical tests for identification of** *Salmonellas* 

The following procedure is applied for purification process.

b. Reversed plates are incubated at 35 to 37 °C for 24 hours.

cultures are pure, the following mediums are used for passages.

**5.3.1 Purificitaion of colonies selected** 

incubates at 35 to 37°C for 24 hours.

incubated at 35 to 37°C for 24 hours.

**5.3.2 Planting to Gillies medium 1 and assesment** 

**5.3.3 Planting to Gillies medium II and assessment** 

they are waited for another 24 hours.

**5.3.4 Planting to TSI AGAR and assessment** 

agar (TSI) or by drawing to the slant part.

b. Cultures are incubates at 35 to 37 °C for 18 to 24 hours.

Gillies medium 1 and Gillies medium 2.

without forming gas.

a loop.

negative in terms of *Salmonella*.

agar plate.

transparent.

(I=indole, M=methyl red, V=voges pros cover, C=citrate)

INDOL= Planting is performed into tryptophan medium. After incubation at 37°C for 24 hours, 0,2 to 0,5 cc of Kovac indicator is dripped into indole medium. Red circle formation is positive (*Figure 11)*.

Fig. 11. Indole Test

Isolation and Identification of Salmonellas from Different Samples 147

CITRATE=Simmons citrate; line style planting is performed to citrate medium. If the bacteria used citrate as a carbon source, the color of the medium will turn from green into

**Reaction** *Salmonella* 

Indole negative

Citrate positive

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

Methyl Red positive

Voges Pros Kover negative

blue. The test is positive (*Figure 14)*.

Fig. 14. Citrate test

Table 2. IMVIC Test Results

**5.3.6 Triple tube method** 

IMVIC test results for *Salmonella* were given in Table 2.

and 1st tubes. It is left for incubation at 37°C for 18-22 hours.

1. Matters that we may observe in the tube:

METHYL RED= Planting is performed into BGB (buffered glucose bouillon) or peptone medium. This test shows pH change in 0,5% buffered glucose medium. 5-6 drops of methyl red indicator is dripped on 1 cc. of medium. If pH drops under 4,2, red color occurs and the result is positive. If there is no color change, it is negative (*Figure 12)*.

Fig. 12. Methyl RED Test

VOGES PROS COVER= 0,2 cc 40% KOH is dripped into 1 cc BGB medium. Then, 0,6 cc of alpha naphtol indicator is added. Test results 20-30 minutes after.

 If red circle appears, the result is positive. In positive cases, acethyl-methyl-carbinol, final catabolism product of glucose occurs. If there is no color change, it is negative (*Figure 13)*.

Fig. 13. Voges Pros Cover test

CITRATE=Simmons citrate; line style planting is performed to citrate medium. If the bacteria used citrate as a carbon source, the color of the medium will turn from green into blue. The test is positive (*Figure 14)*.

Fig. 14. Citrate test

146 Salmonella – A Dangerous Foodborne Pathogen

METHYL RED= Planting is performed into BGB (buffered glucose bouillon) or peptone medium. This test shows pH change in 0,5% buffered glucose medium. 5-6 drops of methyl red indicator is dripped on 1 cc. of medium. If pH drops under 4,2, red color occurs and the

VOGES PROS COVER= 0,2 cc 40% KOH is dripped into 1 cc BGB medium. Then, 0,6 cc of

 If red circle appears, the result is positive. In positive cases, acethyl-methyl-carbinol, final catabolism product of glucose occurs. If there is no color change, it is negative (*Figure 13)*.

alpha naphtol indicator is added. Test results 20-30 minutes after.

result is positive. If there is no color change, it is negative (*Figure 12)*.

Fig. 12. Methyl RED Test

Fig. 13. Voges Pros Cover test

IMVIC test results for *Salmonella* were given in Table 2.


Table 2. IMVIC Test Results
