OXIDATIVE-FERMENTATIVE MEDIUM (OF BASAL MEDIUM)

Cat. no. Y51 OF Base Medium, 13x100mm Tube, 3ml 20 or 100 tubes/box
Cat. no. Y57 OF Dextrose, 13x100mm Tube, 3ml 20 or 100 tubes/box
Cat. no. Y58 OF Fructose, 13x100mm Tube, 3ml 20 or 100 tubes/box
Cat. no. Y54 OF Lactose, 13x100mm Tube, 3ml 20 or 100 tubes/box
Cat. no. Y52 OF Maltose, 13x100mm Tube, 3ml 20 or 100 tubes/box
Cat. no. Y53 OF Mannitol, 13x100mm Tube, 3ml 20 or 100 tubes/box
Cat. no. Y55 OF Sucrose, 13x100mm Tube, 3ml 20 or 100 tubes/box
Cat. no. Y56 OF Xylose, 13x100mm Tube, 3ml 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics OF Basal Medium is recommended for the detection of oxidation or fermentation of carbohydrates by bacteria.

SUMMARY

OF Basal Medium was developed by Hugh and Leifson to aid in the identification of gram-negative bacteria on the basis of their ability to oxidize or ferment a specific carbohydrate. (6)

As compared to other OF Media, Hugh and Leifson's formula employs a low peptone/carbohydrate ratio and a minimal amount of agar. The decreased amount of peptone reduces the formation of alkaline amines which can ultimately mask the small quantities of acid that may be produced from oxidative metabolism. (5) The increased carbohydrate results in an increase in the amount of acid that may be formed. The small amount of agar added to the medium provides a semi-solid structure which concentrates the acid at the point of reaction, thereby facilitating visual interpretation of the pH shift.

Proper performance of the OF test requires an organism to be inoculated to two tubes of each OF Medium. Once inoculated, one tube is overlaid with mineral oil or melted paraffin. The other tube is left open to the air.

Oxidative utilization of the carbohydrate will result in acid production (yellow) in the open tube only. Fermentative utilization of the carbohydrate will result in acid production (yellow) in both the open and closed tubes. Acidic changes in the overlaid tubes are considered to be a result of true fermentation, while acidic development in the open tubes are due to oxidative utilization of the carbohydrate present. Asaccharolytic organisms will not produce acid in either tube.

FORMULA

Ingredients per liter of deionized water:*

Sodium Chloride 5.0gm
Pancreatic Digest of Casein 2.0gm
Dipotassium Phosphate 0.3gm
Bromothymol Blue 0.08gm
Agar 2.0gm

Additionally, OF Media with carbohydrate contains 10.0gm/L of specific carbohydrate.

Final pH 6.8 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt, store at 2-8ºC. (except Cat. no. Y51, store at 2-30ºC.) away from direct light. Media should not be used if there are any signs of contamination, deterioration or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Specimen collection is not applicable since this medium is not intended for primary isolation from clinical specimens. As a general rule, infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Consult listed references for information on specimen collection. (1-5)

Method of Use:

1. Allow media to equilibrate to room temperature.

2. Obtain pure, isolated colonies from an 18-24 hour culture.

3. For each test organism, inoculate tubes in duplicate. Inoculate by stabbing the agar to approximately 1/4 inch from the bottom.

4. Apply sterile mineral oil, sterile melted paraffin, or sterile melted petrolatum to one of each duplicate tubes. Tighten the cap of the overlaid tube, and loosen the cap of the non-overlaid tube.

5. Incubate both tubes aerobically at 35ºC. for up to 14 days.

6. Examine tubes daily.

7. A control tube of OF Base Medium (Cat. no. Y51) should be inoculated and incubated in parallel with the OF tests.

INTERPRETATION OF RESULTS

A positive carbohydrate utilization test is indicated by the development of a yellow color in the medium.

A negative carbohydrate utilization test is indicated by the absence of a yellow color (media remains green or turns blue).

The method of metabolism is determined as follows:

Carbohydrate Utilization Open Tube
(No overlay)
Closed Tube
(Oil overlay)
Oxidation Positive (Yellow) Negative (Green)
Fermentation Positive (Yellow) Positive (Yellow)
Non-oxidizer/Non-fermenter Negative (Green or Blue) Negative (Green)
Both oxidation and fermentation Positive (Yellow) Positive (Yellow)

Note: Color reactions should be compared to the OF Base Medium (Cat. no. Y51) control tube.

LIMITATIONS

Organisms that only oxidize dextrose will not ferment any other carbohydrate. Other carbohydrates will only be oxidized. The overlaid (closed) tube, therefore, may be omitted when determining other carbohydrate utilization of such organisms.

Some microorganisms do not grow in OF Basal Medium. It may be necessary to use another basal medium containing dextrose to confirm the negative reaction.

Some mineral oils are acidic and may result in erroneous results.

MATERIALS REQUIRED BUT NOT PROVIDED

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

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
OF Base Medium:
Escherichia coli
ATCC® 25922
D 18-48hr 35°C Aerobic Growth; no color change (top may turn blue at 48 hours) in open tube and in oil overlaid tube
OF Dextrose:
Escherichia coli
ATCC® 25922
D 18-24hr 35°C Aerobic Growth; acid (yellow) reaction and gas in open tube and in oil overlaid tube
Pseudomonas aeruginosa
ATCC® 27853
D 18-24hr 35°C Aerobic Growth; acid (yellow) reaction in open tube; no change in oil overlaid tube
OF Fructose:
Klebsiella pneumoniae
ATCC® 13883
D 18-24hr 35°C Aerobic Growth; acid (yellow) reaction in open and oil overlaid tube
Branhamella (Moraxella) catarrhalis
ATCC® 25240
D 18-24hr 35°C Aerobic Growth, negative fermentation and oxidation - green
OF Lactose:
Enterobacter cloacae
ATCC® 23355
D 18-24hr 35°C Aerobic Growth; acid (yellow) reaction in open and oil overlaid tube
Branhamella (Moraxella) catarrhalis
ATCC® 25240
D 18-24hr 35°C Aerobic Growth; negative oxidation and fermentation-green
OF Maltose:
Klebsiella pneumoniae
ATCC® 13883
D 18-24hr 35°C Aerobic Growth; acid (yellow) reaction in open and oil overlaid tube
Branhamella (Moraxella) catarrhalis
ATCC® 25240
D 18-24hr 35°C Aerobic Growth; no color change in either tube
OF Mannitol:
Enterococcus faecalis
ATCC® 29212
D 18-24hr 35°C Aerobic Growth: acid (yellow) reaction in open and oil overlaid tube
Escherichia coli
ATCC® 25922
D 18-24hr 35°C Aerobic Growth; acid (yellow) reaction in open and oil overlaid tube
Branhamella (Moraxella) catarrhalis
ATCC® 25240
D 18-24hr 35°C Aerobic Growth, negative oxidation and fermentation reactions - green
OF Sucrose:
Enterobacter aerogenes
ATCC® 13048
D 18-24hr 35°C Aerobic Growth; acid (yellow) reaction in open and oil overlaid tube
Branhamella (Moraxella) catarrhalis
ATCC® 25240
D 18-24hr 35°C Aerobic Growth; negative oxidation and fermentation - green
OF Xylose:
Enterobacter aerogenes
ATCC® 13048
D 18-24hr 35°C Aerobic Growth; acid (yellow) reaction in open and oil overlaid tube
Branhamella (Moraxella) catarrhalis
ATCC® 25240
D 18-24hr 35°C Aerobic Growth; negative oxidation and fermentation - green

User Quality Control

PHYSICAL APPEARANCE

OF Basal Media should appear slightly opalescent, and green in color.

OF Dextrose, E. coli

Escherichia coli (ATCC® 25922) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Dextrose (Cat. no. Y57) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.

OF Dextrose, S. flexneri

Shigella flexneri (ATCC ® 12022) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Dextrose (Cat. no. Y57) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.

OF Dextrose, P. aeruginosa

Pseudomonas aeruginosa (ATCC ® 27853) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Dextrose (Cat. no. Y57) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.


OF Fructose, K. pneumoniae

Klebsiella pneumoniae (ATCC ® 13883) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Fructose (Cat. no. Y58) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.

OF Fructose, B. catarrhalis

Branhamella (Moraxella) catarrhalis (ATCC ® 25240) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Fructose (Cat. no. Y58) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.


OF Lactose, E. cloacae

Enterobacter cloacae (ATCC ® 23355) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Lactose (Cat. no. Y54) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.

OF Lactose, B. catarrhalis

Branhamella (Moraxella) catarrhalis (ATCC ® 25240) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Lactose (Cat. no. Y54) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.


OF Mannitol, E. faecalis

Enterococcus faecalis (ATCC ® 29212) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Mannitol (Cat. no. Y53) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.

OF Mannitol, E. coli

Escherichia coli (ATCC ® 25922) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Mannitol (Cat. no. Y53) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.



OF Mannitol, B. catarrhalis

Branhamella (Moraxella) catarrhalis (ATCC ® 25240) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Mannitol (Cat. no. Y53) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.




OF Maltose, K. pneumoniae

Klebsiella pneumoniae (ATCC ® 13883) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Maltose (Cat. no. Y52) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.

OF Maltose, B. catarrhalis

Branhamella (Moraxella) catarrhalis (ATCC ® 25240) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Maltose (Cat. no. Y52) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.


OF Sucrose, B. catarrhalis

Branhamella (Moraxella) catarrhalis (ATCC ® 25240) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Sucrose (Cat. no. Y55) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.

OF Sucrose, E. aerogenes

Enterobacter aerogenes (ATCC ® 13048) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Sucrose (Cat. no. Y55) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC


OF Xylose, E. aerogenes

Enterobacter aerogenes (ATCC ® 13048) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Xylose (Cat. no. Y56) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.

OF Xylose, B. catarrhalis

Branhamella (Moraxella) catarrhalis (ATCC ® 25240) growing in Oxidative-Fermentative Base (Cat. no. Y51) and OF Xylose (Cat. no. Y56) media. Inoculated in duplicate, with Mineral Oil (Cat. no. Z79) overlay applied to one of each duplicate. Incubated aerobically (overlay tubes had tightened caps) for 24 hours at 35ºC.


REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, 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 & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Hugh, R. and Leifson, E.J. 1953. Bacteriol.; 66:24-26.


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