Orange Serum Agar

Intended Use

Orange Serum Agar is used for cultivating aciduric microorganisms, particularly those associated with spoilage of citrus products.

Summary and Explanation

he low pH of fruit juices makes citrus fruit products susceptible to spoilage by yeasts, molds and the bacteria Lactobacillus and Leuconostoc.1 In the 1950s, Hays investigated spoilage in frozen concentrated orange juice. He found that an agar medium containing orange serum (juice) was superior to Lindegren Agar in isolating the microorganisms responsible for spoilage causing a buttermilk off-odor.2 In a later comparative study, Murdock, Folinazzo and Troy found Orange Serum Agar, pH 5.4 to be a suitable medium for growing Leuconostoc,
Lactobacillus and yeasts.3 Stevens described preparation of dehydrated agar media containing orange serum.4 The BBL formula for Orange Serum Agar differs only in a lightly increased orange serum content and in the incorporation of less agar.
Orange Serum Agar is included in recommended methods for examining fruit beverages.1

Principles of the Procedure

Orange Serum Agar and Orange Serum Broth Concentrate 10× contain peptone as a source of carbon and nitrogen for general growth requirements. Orange serum provides the acid
environment favorable to recovering acid-tolerant microorganisms. Yeast extract supplies B-complex vitamins which stimulate growth. Dextrose is the carbohydrate. Agar is the solidifying agent in Orange Serum Agar.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.
Identity Specifications
Orange Serum Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material,
                                    may contain dark tan particles.
Solution:                       4.5% solution, soluble in purified water upon
                                    boiling. Solution is light to medium, yellow to
                                    tan; clear to slightly hazy.
Prepared Appearance:     Light to medium, yellow to tan; clear to slightly hazy.
Reaction of 4.5%
Solution at 25°C:            pH 5.5 ± 0.2
Cultural Response
Orange Serum Agar
Prepare the medium per label directions. Inoculate streak plates with fresh cultures and incubate for 66-72 hours at 30-32°C; for Penicillium roquefortii, incubate at 23-27°C. Inoculate pour plates with Lactobacillus plantarum and incubate for 66-72 hours at 30-32°C.

Directions for Preparation from Dehydrated Product

1. Suspend 45 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Dispense in quantities under 50 mL and autoclave at 121°C for 10 minutes. For larger quantities, increase the autoclave time. Avoid overheating with consequent darkening and poor
solidification.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

1. For the plate count method, prepare serial 10-fold dilutions of the test material.
2. Add 1 mL of test sample to a sterile Petri dish.
3. Add 18-20 mL of molten agar (cooled to 45-50°C) and swirl plate gently to mix well.
4. Allow to solidify before incubatin

Expected Results

Record colony morphology for each type of growth.

Limitations of the Procedure

1. Orange Serum Agar is not a differential medium. Perform microscopic examination and biochemical tests to identify isolates to genus and species if necessary.
2. If Orange Serum Agar is divided into aliquots and allowed to solidify, remelt only once. Repeated heating may produce a softer medium.

*Store at 2-8°C.

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