A Research Note

A Selective Medium for the Isolation and Differentiation of Gluconobacter and Acetobacter
M. C. ClRlGLlANO

ABSTRACT
A new medium permits selective isolation of acetic acid bacteria

within 3 days and differentiation between Gluconobacter and Acetobacter within 48 hr. Dextrose Sorbitol Mannitol (DSM) Agar was devised to provide differentiation of Gluconobacter and Acetobacter based on the preferential oxidation of carbon sources. Selectivity is achieved by acidification and the incorporation of cycloheximide to inhibit yeast and mold growth. To minimize interference by other acid tolerant gram positive bacteria, best results were obtained with the addition of 29.5 micrograms of brilliant green or 0.lg of sodium desoxycholate per liter of medium. D.S.M. has been used in our laboratory as both a selective and differential medium in beverage and fermentation quality control procedures.

INTRODUCTION
YEAST, and acetic and lactic acid bacteria are the microorganisms most frequently cited for spoilage in the beverage industry. The types of spoilage produced include off-flavor development, slime-like growth, and gas formation (Lott and Carr, 1964; Frazier, 1967). With an increase in the production volume of still beverages packaged in plastic containers, the significance of the non-fermentative acetic acid bacteria has grown considerably in recent years (Sand, 1977). Several explanations have been given for this increasing importance including t h e resistance of Gluconobacter and related bacteria t o bottling plant sanitizers; the ability to grow in the presence of benzoic and/or sorbic acid; and high free oxygen levels often characterizing still drinks in plastic containers (Sand, 1977). It is therefore desirable that one has a means by which the acetic acid bacteria can be singled out so that appropriate measures might be taken t o control them. Traditionally, the acetic acid bacteria have been isolated on media used for the isolation of yeasts. Selectivity is normally achieved by acidification t o a pH that favors acetic acid bacteria growth, and excludes the growth of Pseudornonas (Shimwell e t al., 1960), while differentiation of the two genera is facilitated b y subsequent biochemical characterization. The ability of A c e t o b a c t e r t o oxidize lactate and acetate (Buchanan and Gibbons, 1974), as well as ethanol (Gibbs and Shapton, 1968) are metabolic attributes that have been exploited in the past. DeLey (1961) also implies that preferential carbohydrate utilization might be used t o differentiate. We now wish to report the development of a new medium, Dextrose Sorbitol Mannitol Agar (D.S.M.) which incorporates specific carbohydrate substrates, at a pH of less than 4.5 or between 5.0 and 5.5, and which allows the rapid isolation and differentiation of A cetobacter and Gluconobacter.

industrius (ATCC 23776), G. oxydans subsp. melanogenes (ATCC 15163), and G. oxydans subsp. oxydans (ATCC 23773); and three subspecies of Acetobacter, i.e. A . pasteurianus subsp. pasteurianus (ATCC 23765), A . aceti subsp. aceti (ATCC 15973), and A . aceti subsp. xylinus (ATCC 10245 and 23770); were obtained from the American Type Culture Collection, Rockville, MD 20852. Thirty-three additional acetic acid bacteria isolates were obtained from laboratory still beverage platings. All cultures were maintained on mannitol agar (ATCC, 1978) and cultured weekly in mannitol broth. The incubation temperature for test and maintenance procedures was 32C. Cultures were prepared 72 hr in advance of each test plating. Appropriate dilutions were then made in Butterfield Buffer with 0.1% peptone (Scott Laboratories) to deliver from 100-300 colony forming units (C.F.U.s). Plates were poured, spread plated in duplicate, and incubated for from 3-5 days. The growth response of each organism on Dextrose Sorbitol Mannitol (DSM) agar, Potato Dextrose Agar (PDA), and Rogosa SL (RSL) agar (Difco) was recorded as a function of the range of colony diameters with time. DSM differential streak plates, slants, and semi-solid tubes were also prepared directly from test plate isolates. DSM agar when used as a primary isolation media contained the following ingredients (per liter): log of (Difco) Proteose Peptone $3; 3g of (Difco) yeast extract; 15g calcium lactate (FisherC104); lg dextrose; lg d-sorbitol; 2g d-mannitol; lg monopotassium phosphate N.F.; 0.02g manganese sulfate monohydrate; 0.03g bromo cresol purple (Difco); 0.004g cycloheximide (Upjohn); 0.lg sodium desoxycholate (Difco) or 29.5 micrograms of brilliant green (Difco); and 15g of agar, (3g for semi-solid). The calcium lactate was added before autoclaving or, to prevent clouding of the medium, after by dissolving, with mild heating, 15g in 100 ml of distilled water and filter sterilizing using a 0.45 millimicron membrane filter. Ten ml of solution were then added to 100 ml of sterile basal agar. Cycloheximide was dissolved in 95% ethanol and added to the medium before autoclaving. Aqueous stock solutions of brilliant green and sodium desoxycholate were prepared in advance, filter sterilized, and also added prior to autoclaving. Sterilization was accomplished by autoclaving at 121C for 15 min. The medium was acidified after autoclaving to a pH of 4.2-4.4 by adding 1.5 ml of hydrochloric acid (10%). The agar was used as is (pH 4.8-5.0) when DSM was used as primary isolation medium for beverage systems with a pH of less than 4.5, or when employed in differential techniques.

RESULTS & DISCUSSION

A c e t o b a c t e r grown o n DSM effect a change in color from yellow t o purple while producing a white precipitate of calcium carbonate. Precipitated calcium carbonate contiguous with cell growth often clears to form a translucent halo encircling well isolated colonies. Gluconobacter, unable t o oxidize lactate, will preferentially oxidize the minor carbohydrate constituents producing acetic acid and maintaining the yellow color of the medium. Gluconobacter, and several species o f d c e t o b a c t e r , will also produce a scattered crystalline precipitation of calcium-5-ketogluconate after extended periods of incubation. Among the selective gram positive bacterial inhibitors screened, i.e. crystal violet, brilliant green, and sodium desoxycholate, brilliant green was found t o be the least inhibitory to the acetic acid bacteria. Sodium desoxycholate reduced the growth response of all A c e t o b a c t e r tested, while crystal violet completely inhibited the A . aceti subspecies.

MATERIALS & METHODS THREE SUBSPECIES of Gluconobacter, i.e. G. oxydans subsp.

Author Cirigliano is affiliated with the Microbiological Services Dept., T.J. Lipton, Inc. 800 Sylvan Ave., Englewood Cliffs, NJ
07632.

1038-JOURNAL OF FOOD SCIENCE- Volume 47 (1982)

In our evaluation of acidifying agents, hydrochloric acid was chosen over acetic acid due to the reduced growth response of Gluconobacter to the latter. In our comparative performance evaluations of PDA, RSL, and DSM the response of Acetobacter and Gluconobacter strains on DSM was far superior t o that observed on
Table I-Comparative growrh response of A TCC type cultures on DSM, RSL, and PDA as a function of the range of colony diameters after 72 hr a t 3 2 C

Organism

DSM

RSL

PDA

Gluconobacreroxydans 0.8-1.1 mm subsp. oxydans

0.1-1.0 mm 0.1-1.0 mm

(ATCC 23773)
Gluconobacter oxydans 0.5-1.3 mm subsp. melanogenes

0.1-0.6 mm 0.1-0.4 mm

species tested, seemed t o be greatly improved at pH 5.0 with a shift from acetic acid to hydrochloric acid as an acidulant, and the incorporation of mannitol and sorbitol. The growth response of the ATCC type cultures, on each of the media evaluated, is described in Table 1 as a function of the range of colony diameters after 72 hr. When employed as a differential medium, DSM was used with no selective additions at a pH 5.0. In our laboratory the streak plate technique, the agar slant, and/or the tubed semi-solid vehicle were successfully applied t o this purpose, and correctly genotyped all 33 isolates obtained from laboratory still beverage platings. These were subsequently speciated (Buchanan et al., 1974; Gibbs and Shapton, 1968) and identified as being predominately Acetobacter aceti, i.e. subspecies xylinus

REFERENCES
0.1 -0.5 mm 0.1 mm
American Type Culture Collection. 1978. Catalogue of Strains I, 13th ed. Am. Type Culture Collection, MD. Buchanan, R.E. and Gibbons, N.E. 1974. Bergeys Manual of Determinative Bacteriology. 8th ed. p. 2 5 1 ; 276. The Williams and Wilkins Co., Baltimore. MD. DeLey, J. 1961. Comparative carbohydrate metabolism and a proposal for a phylogenetic relationship of the acetic acid bacteria. J. Gen. Microbiol. 2 4 : 31. Frazier. W.C. 1967. Food Microbiology, 2nd ed. McGraw-Hill Book Company. New York. Gibbs, B.M. and Shapton, D.A. 1968. Identification Methods for Microbiologists, part B. p. 1. Academic Press, New York. Lott. A.F. and Carr. J.G. 1964. Characteristics of an organism causing spoilage i n an orange juice beverage. J. Appl. Bact. 2 7 ( 3 ) : 379. Sand, F.E.M.J. 1977. Gluconobacter, still drinks and plastic containers. Soft Drinks Trade J. Oct: 371. Shimwell. J.L., Can, J.G.. and Rhodes, M.E. 1960. Differentiation of Acetomonas and Pseudomonas. J. Gen. Microbiol. 2 3 : 283. Ms received 1 2 / 2 6 / 8 1 ;accepted 1 2 / 2 8 / 8 1 .
~~ ~ ~~

(ATCC 15163)
Gluconobacter oxydans 0.2-0.9 mm su bsp. indusrrius

(ATCC 23776)
Acetobacrer aceri subsp. xylinus

1.1-2.2 mm

1 .O-2.0 mm

0.1 mm

(ATCC 23 770)
Acerobacrer aceri s bsp aceri u

0.6-3.0 mm

0.2-1.0 mm 0.1-0.6 mm

(ATCC 15973)
Acerobacrer pasteurianus 0.8-2.2 mm s bsp. pasteurianus u

0.8-1.4 mm 0.1 -0.6 mm

(ATCC 23765)
Acerobacrer aceti subsp. xylinus

0.8-3.8 mm

0.1 -1.2 mm

No growth

(ATCC 10245)

PDA or RSL when compared with or without selective additions. In particular the growth of the Gluconobacter

The authors thanks Dr. J. Flickenger and Dr. S. Glassner for their support during this study.

Volume 47 (1982)-JOURNAL OF FOOD SCIENCE-7039

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