Protocol

1. Obtain sample of yogurt
2. Prepare HYA Agar media
3. Dilute yogurt and streak onto plates
4. Isolate colonies on the HYA agar
5. Perform various tests to identify the isolated organism

Method

1. Obtain a commercial brand yogurt for use in the isolation of Streptococcus thermophilus
 

2. Prepare HYA Agar Medium
Per Liter (makes 40 plates)
Agar: 15g
Proteose peptone #3: 10g
Beef extract: 1 g
Lactose solution: 10 ml
Galactose solution: 10 ml
Glucose solution: 10 ml
pH 6.8 plus or minus .2 @ 25 degrees Celsius

Lactose solution (per 10 ml):
lactose: 5g, add to distilled water to make 10 ml

Galactose Solution (per 10 ml):
galactose: 2.5g, add to distilled water to make 10 ml

Glucose Solution (10 ml)
glusoce: 2.5g, add to distilled water to make 10 ml

Preparation:
Add components (except the 3 solutions) to distilled water and bring volume to 970 ml.  Mix. Gently heat and bring to boiling. Adjust pH to 6.8, autoclave for 15 minutes at 15 psi, 121 degrees Celsius.  Cool to 45-50 degrees Celsius.  Add solutions.  Mix and pour into dishes.
 

3. Streak one HYA Agar plate with a small yogurt sample straight from the container.  Streak a second HYA Agar plate with a small amount  yogurt diluted with saline solution.  Incubate the plates at 37 degrees Celcius for at least 24 hours.
 

4. Observe growth on HYA Agar plates.   S. thermophilus colonies should be very small and white.
 

5. Characteristics of S.thermophilus include, we have found that it is gram positive, mesophile, catalase negative and it will ferment glucose and lactose, but not mannitol. Perform a Gram stain test, a catalase test, and innoculate glucose and mannitol sugar tubes to check for fermentation.
    Gram Stain:  shows whether organism is gram positive or gram negative; also shows shape of organism
    Catalase Test:  indicates presence of catalase enzyme (bubbles indicate organism is aerobic)
    Sugar Tubes:  tube is originally red; change in color to yellow indicates fermentation of sugar