Final Enrichment Protocol

Materials

Ingredients for Lactobacillus Selection Oxgall Agar:

• Sodium Acetate * 3H2O 25.0g
• Dextrose 20g
• Agar 15g
• Pancreatic Digest of Casein 10g
• KH2PO4 6g
• Yeast Extract 5g
• Ammonium Citrate 2g
• Oxgall 1.5g
• Polysorbate 80 1g
• MgSO4 .575g
• FeSO4 .034g
• MnSO4 .12g
• Acetic Acid, glacial 1.32mL

 Other materials:

• Orange juice concentrate
• 1000ml glass jars
• One pack of sterilized plates
• 5000ml flask
• parafilm
• 95% alcohol
• safranin
• crystal violet dye
• iodine
• water
• clean slides
• microscope
• staining tray

Method

Media Preparation

• Add all media ingredients except for Acetic Acid to distilled/ deionized H2O bring volume to 998.7 mL, mix thoroughly, bring to boil and then add acetic acid, mix heat while stirring bring to 90-100 degrees Celsius for 2-3min. Do not autoclave, pour into sterile Petri dishes. Keep pH between 5.5 +/- .2 at 25 degrees Celsius.

Preparation of Orange Juice

• Take concentrated orange juice and dilute it with tap water.

                1)    100mL concentrate, 700mL water

• Leave the sample at room temperature left uncovered. After 6-8 days Lactobacillus “slime” should be present. Slime should be extracted from jars and streaked onto Lactobacillus Selection Oxgall Agar.

• When extracting slime make sure to take the top yellowish looking slime from dilution in jar.

Streak Plate Procedure

• Label bottom of plat with date
• Divide the plate into four different quadrants
• Streak the initial inoculums over quadrant 1
• Flame the loop and allow it to cool
• Streak area 2 by starting on a streak of area 1
• Flame loop again and cool on agar
• Streak area 3 by going over one streak in area 2
• Flame the loop and allow to cool
• Streak are 4 by starting on area 3 and make sure to end in middle of plate
• Parafilm plate and invert and let stay in room temperature
• Keep Plates at room temp. pH 5.6-6.2, seal plates with parafilm. Observe characteristics of colony.
• Look for smooth, glistening, opaque, color will vary from no pigment to yellow and red. No odor.

Gram Stain

• Place slide on staining tray making sure to have put wax pencil circle mark on slide
• Flame loop and cool with distilled water
• Take a drop of distilled water after cooled on loop an place on slide
• Flame loop and inoculate with organism
• Dip loop in water drop on slide
• Allow slide to dry
• Heat fix slide after complete drying
• Flood slide with crystal violet and let stay for 30 seconds
• Gently rinse dye with distilled water
• Flood slide with iodine for 10 seconds
• Rinse with distilled water
• Flood slide with 95% alcohol until stain no longer washes off
• Quickly rinse with distilled water
• Flood slide with safranin for 30 seconds
• Rinse slide with distilled water
• Blot slide with blotting paper making sure not to rub slide
• After slide is completely dry observe bacterial cells at 1000x magnification (use oil)

After you have determined organism is the isolate continue to keep culture growing for later use either on LSO plate or TSA plate.

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