Media Components:

                                 40 ml          Tomato Juice
                                 10 g.           Peptonized Milk
                                 10 g.           Pancreatic Digest of Casein
                                 12 g.           Agar
                                 excess         dH₂0
                                 1                 5 L Flask
                                 20               Sterile Petri Dishes
                                 20               T-soy Agar Dishes

Source of S. lactis:

                                 Fresh whole milk

Isolation and Confirmation Assay Materials:

                                 Crystal Violet
                                 Gram's Iodine
                                 95% Ethyl Alcohol
                                 Safranin 
                                 Congo Red
                                 Maneval's solution
                                 Malachite Green
                                 Bunsen burner
                                 Inoculating loop
                                 Glass microscope slides
                                 Compound light microscope
                                  Immersion oil
                                 Motility test medium (solidified in test tubes)
                                 Hydrogen peroxide reagent
                                 Cytochrome oxidase reducing agent
                                 T-soy broth
                                 MacConkey's agar
                                 Sheep's blood agar
                                 Anaerobic chamber device
                                 Carbohydrate utilization tubes (lactose and glucose)
                                 4^o Celsius incubator
                                 37^o Celsius incubator
                                 55^o Celsius incubator

1.  Preparation of media:

                   i)    To 5 L flask add tomato juice, peptonized milk, pancreatic digest of casein, and                          agar. 

                   ii)   Fill 5 L flask containing ingredients with dH₂O to 1 Liter.

                   iii)   Dissolve media components.

                   iv)  Autoclave media mixture.

                   v)   Pour media in petri dishes and allow to ample time to solidify.

2.  Isolation of Streptococcus lactis:

                   i)   Inoculate tomato juice agar plates with fresh whole milk using streak plate                                   technique.

                   ii)  Incubate at 37^oC for 48 hours in an anaerobic chamber device.

                   iii) Isolate and restreak colonies with characteristic morphologies on T-Soy agar.

3.  Confirmatory Assays (optional):

                   i)  Perform the following confirmatory assays for organism identification:
                                - Gram stain 
                                - Capsule stain 
                                - Endospore stain
                                - Motility stab agar incubated for 48 hours at 37 ^oC
                                - Oxidase/catalase assay
                                - Incubation for 48 hours at 37 ^o C in T-Soy Broth
                                - Incubation for 48 hours at 37 ^o C on MacConkey’s medium
                                - Incubation for 48 hours at 37 ^o C on sheep's blood agar medium
                                - Incubation for 48 hours at 37 ^o C in anaerobe chamber
                                - Carbohydrate Utilization (Glucose and Lactose) 
                                - 48 hour incubation in adverse temperature conditions (4^oC and 55 ^oC)