Hi everyone! Sorry for the delay in my post there was a bit mixed up in my remembering of the weeks.

I am working in the food and water microbiology lab now. In the lab, the main testing that we were instructed to focus on is on the testing of food samples. Ying Ying has roughly described of routine testing of food and the basics of food tesing principles. Now I am going to touch on the testing of specific pathogen, Salmonella. The most common testing for Salmonella in our lab is the testing of chocolate samples from overseas companies.

For Salmonella testing of chocolate sample:

25 grams of chocolate sample is being weighed and 225ml of skim milk is being added to the chocolate sample. For normal Salmonella testing, buffered peptone broth is being added instead of skim milk. However, as this is a request from the company overseas, we have to follow their instruction as that is their way of testing there. After the addition of skim milk, the food is being homogenized by the stomacher. The stomacher has been described in Ying Ying’s blog posting so I shall not talk about it anymore. After stomaching at 230rpm for 30s, phosphate buffer is then added to the homogenate. After that, the chocolate homogenate is then taken to the incubator for incubation at 37◦C.

After incubation for 24 hours, 1ml of the chocolate homogenate is being pipetted into Tetrathionate Brilliant-green Bile Enrichment Broth (TTB) and 0.1ml is pipetted into Rappaport and Vassiliadis (RV Broth). This is a selective enrichment step to improve the growth of Salmonella. For TTB, 0.2ml of iodine and 0.01ml of brilliant green is added before the chocolate sample is added.

For TTB (incubate at 35◦C):
- Only organisms which possess the enzyme tetrathionate reductase can grow (Only Proteus and Salmonella)
- Proteus can be inhibited by adjusting the pH to about 6.5 or the addition of novobiocin.
- Bile salts promote the growth of enteric bacteria and inhibit the growth of bacteria that do not normally live in the intestine.
- Brilliant green inhibits gram-positive bacterial flora.
For RV Broth (incubate at 42◦C):
- Allows only Salmonella growth by exploiting the following characteristics of Salmonella:
o Ability to survive at high osmostic pressure
o Multiply at low pH values (pH: 5.5)
o More resistant to malachite green (increase amounts)
o Less demanding nutritional requirements

After incubation for 1 day at their respective incubators, the samples from both the TTB and RV Broth is streaked onto Hektoen Enteric (HE agar), Bismuth Sulfite (BS agar) and Xylose Lysine Desoxycholate (XLD agar). 3 different types of agar plates are used as each of them supports the growth of different Salmonella species so any form of Salmonella can be detected.

Mode of action:
Hektoen Enteric agar
- Contains bromothymol blue and acidic fuchsin indicators. Differentiates lactose-positive colonies from lactose-negative colonies.
- Additional carbohydrates (sucrose and salicin) helps early detection of slow lactose-fermenting organisms to prevent false-postive results.
- Thiosulphate and ferric citrate causes H2S positive colonies to become black in colour.
- Blue green colonies with black centres would indicate presence of Salmonella..

Bismuth Sulfite agar
- Selective media for isolation of salmonella species.
- Bismuth Sulfite and Brilliant Green act together as a selective agent by suppressing the growth of coliforms.
- H2S production by salmonella due to presence of sulphur.
- Reduction of bismuth ions forms metallic lusture around the colonies.
- Black centre, light edges surrounded by a black precipitate with metallic luster would indicate presence of Salmonella.

Xylose Lysine Desoxycholate agar
- Degradation of xylose, lactose and sucrose to acid causes phenol red indicator to change to yellow.
- Decarboxylation of lysine by salmonella species alters pH to alkaline causes purple colouration around the colonies.
- H2S production indicated by thiosulfate and iron(III) salt forms a precipitate of black iron sulfide in the colonies.
- Red colonies, translucent, sometimes with a black centre would indicate presence of Salmonella.


If there are any suspicious colonies, biochemical tests should be carried out.
The following descriptions below are the biochemical tests for the confirmation of Salmonella.

Triple Sugar Iron (TSI) differentiates gram-negative enteric bacilli based on carbohydrate fermentation and production of hydrogen sulphide. TSI contains dextrose, sucrose, lactose, phenol red (detection of carbohydrate fermentation) and ferrous ammounium sulphate (detection of hydrogen sulphide). The gas produced during carbohydrate fermentation will change the phenol red indicator from red to yellow. Formation of hydrogen sulphide will cause blackening in the butt of the test tube. To facilitate the detection of organism that only ferments dextrose/glucose, the dextrose/glucose concentration is 1/10 the concentration of lactose or sucrose. The acid produced at the slant oxidizes rapidly, causing the medium to remain red or revert to an alkaline pH. However, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension. Loosely capping will allow the exchange of gases.

Lysine Iron Agar (LIA) aids the differentiation of enteric bacilli based on the ability to decarboxylase lysine and to produce hydrogen sulphide. LIA contains bromcresol purple indicator (detection of lysine decarboxylase production), lysine (detection of enzymes lysine decarboxylase and lysine deaminase), ferric ammonium citrate and sodium thiosulfate (both are indicators of hydrogen sulphide). The gas produced during carbohydrate fermentation will cause the bromcresol purple indicator to remain purple. Formation of hydrogen sulphide will cause blackening of the medium due to production of ferrous sulfides. Production of lysine decarboxylase would cause an alkaline reaction (purple). Deamination of lysine would cause an acid slant (red).

Urease Broth is used in parallel with TSI and LIA. Used for the detection of Proteus organism as it causes hydrolysis of urea (colour change to pink).

Indole test is used in the differentiation of genera and species. Tryptone water is used due to its high content of tryptophan. Any microorganism with the enzyme Tryptophanase will cleave tryptophan to produce indole and other products. Kovac’s reagent will then react with indole to form a red ring. False negative results will be produced if the pH falls below (7.4-7.8).

Methyl Red test is used to test the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Organisms that form large amounts of acid from glucose fermentation causes the pH to fall below 4.4 (colour becomes red).

Voges Proskauer (VP) test is used to detect the production of acetylmethylcarbinol (acetoin) from the fermentation of glucose/dextrose. If positive, acetoin is oxidized in the presence of oxygen and potassium hydroxide to diacetyl which reacts with peptone water to give off a red colour.

Decarboxylase media are used in differentiation of gram-negative enteric bacilli based on the production of arginine dihydrolase and lysine and ornithine decarboxylase. When dextrose is being fermented, the bromocresol purple indicator changes from purple to yellow. The acidic condition also stimulates decarboxylase activity. When the amino acid is degraded, a corresponding amine is being produced. The amine then elevates the pH of the medium, causing a colour change of the indicator from yellow to purple.

Simmon’s Citrate agar is used for the differentiation of gram-negative enteric bacteria on the basis of critrate utilization. Organisms that are able to utilize ammonium dihydrogen phosphate and sodium citrate as the sole source of nitrogen and carbon will produce an alkaline reaction by changing the bromthymol blue indicator from green to blue (alkaline).


After incubation, if the biochemical tests results are as follow, the chocolate sample can be presumed to contain Salmonella:

TSI (incubate at 35◦C for 24hours): Acid butt (yellow), alkaline slant (red) with H2S production (black)
LIA (incubate at 35◦C for 24hours): Alkaline reaction (purple) with H2S production (black)
Urease test: Negative (no colour change)
Indole test (Tryptone water + Kovac’s reagent): Negative
Methyl Red test: Positive (distinct red colour)
VP test: Negative (no colour change)
Base decarboxylase: Negative (yellow)
Glucose/Xylose decarboxylase: Negative (yellow)
Lysine decarbosylase: Positive (purple)
Ornithine decarboxylase: Positive (purple)
Simmon’s Citrate: Blue
Gram staining: Gram-negative bacilli

After that, serological test is being carried out. Each presumptive Salmonella isolate (using TSI agar slant capture) is being tested against Polyvalent O (somatic) and Polyvalent H (flagella, phase I and II) antisera. For both Polyvalents, there is agglutination. This shows that the presumptive Salmonella can be confirmed as Salmonella.

Jeremy Lee
TG01
0503168G