Cat. no. G91 Orange Serum Agar, 15x60mm Plate, 11ml 10 plates/bag
Cat. no. G181 Orange Serum Agar, 15x100mm Plate, 18ml 10 plates/bag


Hardy Diagnostics Orange Serum Agar is used for the cultivation and enumeration of aciduric, putrefactive microorganisms, such as Lactobacillus spp., Bacillus spp., Leuconostoc spp., Clostridium spp., yeasts and molds in citrus fruit juices and juice concentrates. (5)

This product is not intended to be used for the diagnosis of human disease.


The microbial population of citrus foods and juices varies greatly depending on how foods are processed and preserved. A small percentage of contaminated fruit can "seed" the operating equipment with spoilage organisms. Equipment that is used for fruit juice preparations is often found to be a significant source of contamination. There are many specific operations, or areas, where microbial buildup can occur such as presses, extractors, finishers, mills, pipelines and conveyors. Geotrichum candidum has been labeled the "machinery mold" because of its tendency to accumulate on fruit processing equipment. Aerobic plate counts can provide an index for assessing the sanitation of citrus fruit and juice processing equipment. (8)

In the production of citrus concentrates, juice may be held in stainless steel tanks for 30 to 120 minutes before high-temperature evaporation. It is during this holding period that the product is most susceptible to microbial spoilage. The spoilage of unpasteurized fruit juices is most often due to aciduric organisms such as lactic acid bacteria and yeast with Saccharomyces , Candida and Torulopsis being the most commonly isolated spoilage organisms. (8)

Commercially prepared and packaged citrus fruit juices and products have a pH range of 2.9 to 4.0. For example, the pH of orange juice is usually 3.0 to 4.0 and other acidic foods like tomato juice have pH that ranges from of 3.9 to 4.4. (7) Because of the low pH of fruits and fruit juices, aciduric molds and yeast are the microbes that are most often encountered in contaminated citrus products. Of the aciduric bacteria, the lactic acid group, primarily Lactobacillus and Leuconostoc spp., is most often observed. (8) On occasion, C. pasteurianum is responsible for the spoilage of products with low pH such as tomatoes, pears, figs and pineapples. (7) While potential contaminants of food products, pathogenic bacteria are not usually encountered in citrus fruit products due to the low pH and the pasteurization process during manufacturing. However, non-pasteurized apple cider has been reported to be responsible to over 200 cases of salmonellosis. Studies have shown that Salmonella enterica is able to survive up to 30 days in apple juice with a pH of 3.6. (8)

Foodborne yeasts and molds include several hundred species. These organisms, due to their wide range of pH and temperature tolerances, as well as their assortment of hydrolytic enzymes, have the ability to grow in most low pH food. Contamination of foods by yeast and molds result in substantial losses to the producer, processor and the consumer. Several of the foodborne molds, and possibly yeast, may be hazardous to human and animal health due to their ability to produce mycotoxins. Yeast and molds may also elicit allergic reactions or infections in  those individuals who are aged or those receiving chemotherapy or antibiotics. (7)

Orange Serum Agar is specially formulated for the isolation, cultivation and enumeration of the aciduric bacteria, mold and yeasts seen in citrus foods, juices and other foods with low pH values. Orange Serum Agar contains: casein peptone as a nitrogen source, yeast extract to provide B-complex vitamins to stimulate growth, and dextrose as a carbohydrate source. In order to create an acidic media that favors the recovery of aciduric organisms, orange powder is added to the media, while potassium phosphate serves as a buffer.


Ingredients per liter of deionized water:*

Casein Peptone 15.5gm
Dextrose 4.0gm
Orange Powder 3.5gm
Yeast Extract 3.0gm
Dipotassium Phosphate 2.5gm
Agar 17.0gm

Final pH 5.5 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



1. Filter juice sample through a sterile bacteriological membrane filter, pore size 0.45um. (8)

2. Place the membrane filter on the surface of prepared Orange Serum Agar plates. Avoid trapping bubbles between the agar and filter.

3. Invert plates and incubate for 1-5 days at 35ºC.

Consult listed reference for additional information regarding the Orange Serum Agar membrane filtration. (8)


Determine the microbial colony count and note the morphology of each specific colony type. However further tests must be performed on all growth in order to differentiate and identify organisms. (5) Microscopic examination must be performed on all molds for complete identification.



Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Lactobacillus acidophilus
ATCC ® 4356
J 1-5 days 35°C Aerobic Growth; 10-100 colonies
Weisella paramesenteroides
ATCC ® 33313
J 1-5 days 35°C Aerobic Growth; 10-100 colonies
Aspergillus brasiliensis
formerly A. niger
ATCC ® 16404

J 1-5 days 35°C Aerobic Growth; 10-100 colonies

User Quality Control

Physical Appearance

Orange Serum Agar should appear slightly opalescent, and light to medium amber in color.


1. Anderson, N.L, et al. Cumitech 3B; Quality Control and Quality Assurance Practices in Clinical Microbiology , Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology , American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology , C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook , Vol. I & II. American Society for Microbiology, Washington, D.C.

5. MacFaddin, J.F. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria , Vol. I. Williams & Wilkins, Baltimore, MD.

6. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22-A2. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

7. Landry, W.L., Schwab, M.J., Lancette, G.A. 1995. Bacteriology Analytical Manual , 8th ed. AOAC International, Gaithersburg, MD.

8. Vanderzant, C. and Splittstoesser, D.F. 1992. Compendium of Methods for the Microbiological Examination of Foods . American Public Health Association, Washington, D.C.

ATCC is a registered trademark of the American Type Culture Collection.