390 The Difco Manual
Limitations of the Procedure
1. The addition of some carbohydrates to the basal medium may cause
an acid reaction. To restore the original pH (and color of the
medium), add 0.1 N sodium hydroxide on a drop-by-drop basis. Take
care not to make the medium too alkaline, which would prevent
fermentation from occurring within the usual incubation period.
2. When inoculating tubes, stab gently and do not use a loop. Rough
stabbing or using a loop to stab may give the false appearance of
gas production when mechanical splitting of the medium is what
actually occurred.
References
1. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
Scott’s diagnostic microbiology, 9th edition. Mosby-Year Book,
Inc., St. Louis, MO.
2. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
R. H. Yolken. (ed.). 1995. Manual of clinical microbiology,
6th edition. American Society for Microbiology, Washington, D.C.
3. Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T.
Williams. 1994. Bergey’s manual of determinative bacteriology,
9th edition. Williams & Wilkins, Baltimore, MD.
4. Ewing, W. H. 1986. Edwards and Ewing’s identification of
Enterobacteriaceae, 4th edition. Elsevier Science Publishing Co.,
Inc., New York, NY.
5. Bacteriological Analytical Manual, 8th edition. 1995. AOAC
International, Gaithersburg, MD.
6. MacFaddin, J. F. 1985. Media for isolation-cultivation-
identification-maintenance of medical bacteria. Williams &
Wilkins, Baltimore, MD.
Packaging
Phenol Red Agar Base 500 g 0098-17
Phenol Red Lactose Agar 500 g 0100-17
Phenol Red Mannitol Agar 500 g 0103-17
Phenol Red Carbohydrate Media Section II
Phenol Red Carbohydrate Media
Bacto
®
Phenol Red Broth Base
.
Bacto Phenol Red Dextrose
Broth
.
Bacto Phenol Red Lactose Broth
.
Bacto Phenol Red
Mannitol Broth
.
Bacto Phenol Red Saccharose Broth
Intended Use
Phenol Red Carbohydrate Media are basal media used with added
carbohydrates in differentiating pure cultures of bacteria based on
fermentation reactions.
Summary and Explanation
The fermentative properties of bacteria are valuable criteria in their
identification.
1,2,3,4
A basal medium for determining the fermentation
reactions of microorganisms must be capable of supporting growth of
test organisms and be free from fermentable carbohydrates. Vera
5
used
a fermentation test medium employing the pH indicator phenol red
and obtained highly accurate results.
Phenol Red Broth Base is recommended for use to determine the ability
of organisms to ferment various carbohydrates.
6,7,8,9
Different fermentable
substances may be added in any desired concentration. The concentration
of carbohydrate generally employed for testing fermentation reactions
of bacteria is 0.5 to 1%. Some investigators prefer to use 1% rather
than 0.5% to ensure against reversion of the reaction due to depletion
of the carbohydrate.
Phenol Red Broth Base is an excellent substrate for streptococci, as
well as for other less fastidious bacteria, the growth promotion of the
medium can be greatly improved for fastidious, microaerophilic, and
obligately anaerobic strains by the addition of a small amount of Bacto
Agar (0.1-0.2%). A medium containing this small quantity of agar may
be heated it to the boiling point to drive out the dissolved air. The tubes
are then cooled to below 40°C, without excessive agitation, just prior
to inoculation. The fermentation reaction of gonococci may be determined
by using 0.8% Bacto Agar and adding 5% sterile fresh rabbit serum to
the sterile Phenol Red Broth Base containing the selected carbohydrate.
Coagulase Plasma EDTA can be added to Phenol Red Mannitol Broth
to prepare Coagulase Mannitol Broth. This medium is useful in determining
the ability of Staphylococcus aureus to ferment mannitol and to
coagulate plasma.
10
Principles of the Procedure
Proteose Peptone No. 3 and Beef Extract provide the carbon and nitrogen
sources required for good growth of a wide variety of organisms. Sodium
Chloride maintains the osmotic balance of the medium. Phenol Red
serves as an indicator, turning from red-orange to yellow when acid is
produced during fermentation of the added carbohydrates.
Formula
Phenol Red Broth Base
Formula Per Liter
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 10 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.018 g
Final pH 7.4 ± 0.2 at 25°C