Jun 29, 1988 - Ivan Sorvall, Inc., Newtown, Conn.) at ca. 2,200 x g for 20 ... of a sterile, molten 2% Noble agar solution (Noble agar,2.0 g; deionized water, 100 ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1988,
p.
3185-3186
Vol. 54, No. 12
0099-2240/88/123185-02$02.00/0 Copyright © 1988, American Society for Microbiology
Disk Inoculum-Solid Medium Method To Test Carbon and Nitrogen Assimilation by Yeast Isolates KERRY J. MOORE,* MICHAEL G. JOHNSON, AND SHANE P. McCLARY Department of Food Science, University of Arkansas, Route 11, Fayetteville, Arkansas 72703 Received 29 June 1988/Accepted 27 September 1988
Carbon and nitrogen assimilation for 50 yeasts isolated from White Riesling fermentation were tested by using a disk inoculum-solid medium method. This method was quicker and gave results comparable to the conventional liquid medium methods. Yeast characteristics (growth response, pigment production, morphology) could also be compared with this method.
Carbon and nitrogen assimilation abilities of yeast isolates are among the first properties tested (2-4) in identifying and classifying yeasts. The recommended tests are conventionally done in liquid medium or by an auxanographic method whereby a solid medium containing all the nutrients and growth factors required by a yeast, except a carbon source, is seeded with the test yeast and then different carbon compounds are added at isolated sites on the surface of the hardened agar medium. An enhanced area of yeast growth around a particular carbon source indicates that compound can be assimilated and used for growth by that yeast (5). Previous research done in our laboratory on identification of yeasts from wine musts (K. J. Moore, M. G. Johnson, and J. R. Morris, J. Food Sci., in press) indicates that the liquid medium test methods are too time-consuming and laborintensive, while the conventional solid medium test methods often give equivocal and nonreproducible results. The laborsaving multipoint inoculator method (1) was considered but was discarded because of the cost of fabricating this device as well as the potential for colony crowding and overlap on a 100-mm-diameter plate if 20 or more yeasts are inoculated per plate. In an effort to reduce the time and labor and minimize the expense required to obtain unequivocal results, a study was undertaken to (i) determine whether a disk inoculum-solid medium (DISM) method could be used to test carbon and nitrogen assimilation by yeast isolates from wine musts and (ii) compare the rapidity and reproducibility of this DISM method with conventional static liquid medium methods. Fifty yeast isolates were randomly selected from one lot of yeasts recovered from must fermentations of White Riesling (Vitis vinifera) grapes. Six known pure yeast cultures included as controls in the assimilation assays were Debaryomyces hansenii, Kloeckera apiculata, Kluyveromyces fragilis NRRL Y-2415, Pichia membranaefaciens, Rhodotorula mucilaginosa, and Saccharomyces cerevisiae ATCC 4132. Yeast isolates were maintained on yeast malt agar slants at 4°C. Yeast strains were brought to a state of active growth by loop transferring them from yeas.t malt slants into tubes containing 3 ml of yeast malt broth, followed by incubation at 25°C for 36 to 48 h. Cells were then harvested by centrifugation, using a superspeed centrifuge (Model SS-3; Ivan Sorvall, Inc., Newtown, Conn.) at ca. 2,200 x g for 20 min at room temperature (ca. 25°C). The isolates were washed twice with sterile deionized water before suspending the cells in sterile deionized water to approximate a McFar*
Corresponding author.
land barium sulfate standard number 3 turbidity value (Difco manual, 10th ed., Difco Laboratories, Inc., Detroit, Mich., 1984), which was equivalent to an A640 of ca. 0.30 and a yeast count of 106 cells per ml. Sterile, 6-mm-diameter, blank antibiotic sensitivity disks (Difco Laboratories, Inc., Detroit, Mich.) were placed into empty sterile disposable petri dishes. Approximately 3 ml from each washed yeast suspension was aseptically poured into individual petri dishes containing the sterile blank disks. The yeast-saturated disks (each containing a volume of ca. 30 ,ul) were then aseptically transferred, in duplicate, to the surfaces of appropriate prepoured plates of agar medium for assay studies. Blank disks, saturated only with sterile deionized water, were also planted on the plating medium as control disks. Although it may be possible to plant 8 to 10 disks (i.e., for studying four to five yeasts in duplicate) plus a single uninoculated control disk per plate, we chose to plant only four disks (for studying two yeasts in duplicate) plus a control disk per plate. This was done to eliminate any potential for colony crowding in the event that some of these wine must yeasts were highly filamentous or rapid spreaders. Carbon assimilation tests. A 2.2-ml amount of a lOx strength filter-sterilized solution (yeast nitrogen base, 6.7 g; carbon source, 5.0 g [exception was raffinose, 10.0 g]; deionized water, 100 ml) was added to tubes containing 20 ml of a sterile, molten 2% Noble agar solution (Noble agar, 2.0 g; deionized water, 100 ml). After the two solutions were carefully mixed together, plates were poured and allowed to dry for 1 to 2 days at room temperature before inoculation with the disks. The carbon compounds studied included glucose (as a positive control), galactose, maltose, sucrose, cellobiose, trehalose, lactose, melibiose, raffinose, melezitose, D-xylose, L-arabinose, D-ribose, L-rhamnose, erythritol, ribitol, D-mannitol, myo-inositol, succinic acid, and citric acid. Plates were incubated lid-side up at 25°C and examined after 2, 5, 7, 14, and 21 days. Macroscopically visible growth around the yeast-impregnated disks during incubation on any carbohydrate-containing plate was considered positive for the ability of that yeast to assimilate that particular carbon compound. Nitrogen assimilation tests. A 2.2-ml amount of a 1OX strength filter-sterilized solution (yeast carbon base, 11.7 g; nitrogen source was potassium nitrate [NO3], 0.78 g, sodium nitrite [NO2], 0.26 g, or L-lysine, 0.56 g; deionized water, 100 ml) was added to tubes containing 20 ml of a sterile, molten 2% Noble agar solution. After the two solutions were carefully mixed together, plates were prepared and disk inoculated as described above. Plates were incubated lid3185
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APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Carbon and nitrogen assimilation results for control yeast isolates, using the DISM method Control yeast isolatesa Compound
Carbonb Glucose Galactose Maltose Sucrose Cellobiose Trehalose Lactose Melibiose Raffinose Melezitose
D-Xylose L-Arabinose D-Ribose L-Rhamnose Erythritol Ribitol D-MannitOl myo-inositol Succinic acid Citric acid None Nitrogenc NO3
NO2 L-Lysine
D. hansenii
Kloeckera apiculata
Kluyveromyces fragilis NRRL Y-2415
P. membranaefaciens
R. mucilaginosa
S. cerevisiae ATCC 4132
+(10)CD +(10)CD +(10)CD +(11)CD +(8)CD + (8)CD + (7)CD +(8)CD +(9)CD +(9)CD +(9)CD +(8)CD +(7)CD
+(8)CD +(7)CD
+(10)CD +(9)CD
+(9)PD _
+(9)CD +(7)CD +(9)CD +(9)CD
+(9)PD +(8)CD +(9)PD +(9)CD
-
+(8)PD +(11)CD +(7)CD +(9)CD +(10)CD +(7)CD +(8)CD --
+(9)CD
_
+(7)CD
-
+(8)CD +(7)CD
-
+(7)CD +(7)CD
_
-
-
+(8)CD +(8)CD +(9)CD +(7)G +(7)G
+(7)G
+(8)CD -
-
+(9)CD + (8)CD
-
+(8)CD
-
+(8)G +(7)CD
--___ --___ +(7)G +(9)PD +(9)CD +(8)CD
None a +, Positive for growth; value in parentheses, growth diameter, in millimeters, of colony (includes disk diameter of 6 mm for carbon assimilation tests); colony appearance code: CD, creamy, dull; PD, pasty, dull; G, glisteny. -, Negative for growth. b Results given are those obtained after 7 days of incubation. ' Results given are those obtained after 7 days of incubation of replica plates.
side up at 25°C and examined after 2 and 7 days. On day 7 of incubation, the isolates were replica plated with sterile velveteen onto fresh plates of the same medium (to avoid possible false-positive results due to excretion of soluble nitrogenous compounds by the yeasts [2]) and onto plates of potato dextrose agar (to monitor culture viability) and examined 2 and 7 days later. A moderate amount of growth on the nitrogen-containing replica plates after incubation for 7 days was considered positive for the ability of that yeast to assimilate that particular nitrogen compound. Liquid medium assimilation tests were performed simultaneously by the methods of van der Walt and Yarrow (5). Using the DISM method, test results for carbon and nitrogen assimilation for six selected known yeast cultures were obtained (Table 1). The DISM method gave carbon and nitrogen assimilation patterns comparable to those obtained with conventional static liquid medium (2, 3) methods (data not shown). The DISM method routinely gave final carbon assimilation results within the first 7 days of incubation, with any test result which was negative after 7 days remaining so at 21 days. Thus, the DISM method is more rapid than the conventional static liquid medium method, particularly for yeasts showing slow rates of carbon assimilation. It is possible to obtain liquid assimilation test results in less than 21 days by agitating the cultures in tubes in a special near-horizontal rotating rack device (5), but this extra expense can be avoided by using the DISM method. Using several selected biochemical and physiological tests (2, 3), the predominant yeasts among the 50 wine must isolates tested were identified by the DISM method and
confirmed by the liquid method to be species of Cryptococcus, Hanseniaspora, Pichia, Saccharomycopsis, Trichosporon, Zygosaccharomyces, Rhodotorula, Candida, Metschnikowia, and Saccharomyces (data not shown). In summary, the DISM method allows one, with minimum labor, to inexpensively compare several yeast cultures on one plate for relative growth responses, pigment production, and colony morphologies. This method also has been successfully adapted in our laboratory to test wine must yeast isolates for production of hyphae-pseudohyphae and extracellular amyloid compounds. This research was supported in part by funds from the Arkansas State Board of Higher Education 10/10 grant program. The authors thank Pat Stout for typing the manuscript. LITERATURE CITED 1. Beech, F. W., and J. G. Carr. 1955. A multipoint inoculator for plating bacteria or yeasts. J. Gen. Microbiol. 13:408-410. 2. Kreger-van Rij, N. J. W. (ed.). 1984. The yeasts: a taxonomic study, 3rd ed. Elsevier Science Publishing, Inc., Amsterdam, The Netherlands. 3. Lodder, J. (ed.). 1970. The yeasts: a taxonomic study, 2nd ed.
North-Holland Publishing Co., Amsterdam. 4. Rose, A. H. 1987. Responses to the chemical environment, p. 540. In A. H. Rose and J. S. Harrison (ed.), The yeasts, 2nd ed., vol. 2. Academic Press, Inc., Orlando, Fla. 5. van der Walt, J. P., and D. Yarrow. 1984. Methods for the isolation, maintenance, classification and identification of yeasts, p. 45-104. In N. J. W. Kreger-van Rij (ed.), The yeasts: a taxonomic study, 3rd ed. Elsevier Science Publishing, Inc., Amsterdam.
