512 Journal of Food Protection, Vol. 47, No. 7, Pages 512-519 (July 1984) Copyright*', International Association of Milk, Food, and Environmental Sanitarians Comparison of Aspergillus Differential Medium and Aspergillus flavuslparasiticus Agar for Enumerating Total Yeasts and Molds and Potentially Aflatoxigenic Aspergilli in Peanuts, Corn Meal and Cowpeas L. R. BEUCHAT Department of Food Science, University of Georgia Agricultural Experiment Station, Experiment, Georgia 212 (Received for publication November 28, 1983) ABSTRACT Aspergillus differential () and Aspergillus flavus/parasiticus agar () were compared for enumerating total yeasts and molds as well as the A. flavus group in raw peanuts, corn meal and cowpeas. Colony counts were made after plates were incubated at and C; incubation times of and h were evaluated. at C enhanced recovery of yeasts and molds compared to incubation at C. for h at C on or was optimum for detecting A. flavus in corn meal. Use of for detecting A.flavus/parasiticusmay have some advantage over, since orange-yellow color development of reverse colonies is quicker and more intense on. Ideally, culture media for isolating and enumerating yeasts and molds in foods should support recovery of all viable propagules. In addition, media should restrict growth of spreading molds, inhibit development of bacterial colonies, and aid in identification of fungi to at least the genus level. Formulation of such media is not easily achieved. Adjustment of media to acid ph may well inhibit bacterial growth, but is also likely to be lethal to stressed cells of yeasts and molds (2,72). Antibiotics such as chloramphenicol, chlortetracycline, gentamycin and oxytetracycline, when incorporated into media at ph near neutrality, serve to more effectively reduce bacterial contaminants in most foods without interfering with rescusitation of fungi (70). Rose bengal (9) and dichloran (2,6-dichloro-4-nitroanaline) (5,6) are among chemical reagents which are effective in retarding the rate of spread of mold colonies. Rose bengal is often incorporated into colonies of fungi, thus interfering with identification, and has been shown to inhibit growth of some yeasts (8). Dichloran inhibits mold colony spreading without affecting recovery when used in antibiotic-supplement potato dextrose agar (7). King et al. (77) formulated a containing both rose bengal and dichloran, which restricted spreading molds and recovered a wide range of species from foods. A combination of rose bengal, dichloran and streptomycin in a enabled isolation of Aspergillus flavus from peanuts (7). Bothast and Fennell (4) described a (Aspergillus differential, ) which facilitated enumeration of A. flavus propagules in grains by providing a rapid method for differentiating between members of the A. flavus group and other common storage fungi. The was subsequently proven valuable for use in medical mycology (14). Amendment of with dichloran aided in enumerating A. flavus in cottonseeds (5) but not in a variety of other dry foods (3). Pitt et al. (73) recently reported an effective selective for enumeration of A. flavus and A parasiticus, which was developed by modifying. Colonies can be enumerated on the new, Aspergillus flavus and parasiticus agar (), after 42 h of incubation at C, compared to 72 h at 28 C for. The study reported here was designed to compare the suitability of chloramphenicol-supplemented and for enumerating total yeast and mold propagules as well as the A. flavus group in raw peanuts, corn meal and cowpeas. The influence of incubation ( and C) and time ( h and h) on detection of viable populations was determined. MATERIALS AND METHODS Samples analyzed Ten samples (lots) each of raw peanuts, cowpeas and corn meal were obtained from commercial sources. Peanuts and cowpeas had not been subjected to further processing after harvesting, whereas corn meal was obtained in processed form from local grocers. Both peanuts and corn meal are not uncommonly contaminated with A. flavuslparasiticus,
MEDIA FOR YEASTS AND MOLDS 513 whereas the extent of contamination of cowpeas with this group of fungi has not been determined. Four subsamples (20 g) of each lot were separately combined with 80 ml of 0.1 M potassium phosphate buffer (ph 7.0) containing 0.01% Tween 80 and homogenized for 2 min using a Colworth Stomacher. Test materials were allowed to settle to the bottom of the Stomacher bag for 2-3 min before the wash liquid was serially diluted in phosphate buffer in preparation for surface-plating on recovery media. Media evaluated Two media were evaluated for their suitability to support growth of yeasts and molds and, in particular, to detect potentially aflatoxigenic A. flavus and A. parasiticus. Aspergillus Differential Medium () (4) consists of 15 g of tryptone, 10 g of yeast extract, 0.5 g of ferric citrate and 15 g of agar per liter of distilled water; the was adjusted to ph 6.5 and sterilized by heating at 121 C for 15 min. This formula was modified by adding 0.1 g of chloramphenicol/l before sterilizing. The second evaluated was Aspergillus flavus and parasiticus Agar () (13). The formulation of this was 20 g of yeast extract, 10 g of peptone, 0.5 g of ferric ammonium citrate, 0.1 g of chloramphenical, 0.002 g of dichloran (2,6-dichloro-4-nitroaniline; 1.0 ml of a 2% ethanolic solution) and 15 g of agar per liter of distilled water. The ph of the was adjusted to 4.5 before sterilizing at 121 C for 15 min. Plating procedure Sterilized media were cooled to 45-55 C and poured to a depth of 5-7 mm into Petri plates. The plates were allowed to remain at ambient for 18-26 h before serial dilutions (0.1 ml) of test samples were spread over the surface with sterile glass rods. Quadruplicate plates of each dilution were prepared. Two plates of each dilution were incubated (non-inverted) at C and at C. The total number of colonies of yeasts and molds as well as the number of colonies showing orange-yellow reverse coloration indicative of potentially aflatoxigenic aspergilli were recorded after 42-44 h of incubation. A second set of experiments identical to that described above was conducted, except that plates were incubated for h before colonies were counted. Statistical analysis Data presented represent means of four subsamples (replicates) analyzed in quadruplicate for each lot. Statistically significant differences (P=s0.05) were determined using information derived from analysis of variance and Duncan's multiple range test (75). RESULTS AND DISCUSSION Data from experiments comparing and for enumerating total yeasts and molds in ten lots of peanuts are listed in Table 1. In three lots, had a significant effect on counts obtained after h of incubation; after h, significant differences resulting from incubation were noted in only two lots. Significant increases in counts were obtained in three lots by extending the incubation time to h. One incubation was not clearly superior to the other for supporting colony development. Overall, and performed equally with regard to supporting colony development by yeasts and molds. Results from experiments using corn meal are shown in Table 2. Populations of yeasts and molds detected on 5 of 10 lots incubated for h were significantly influenced by incubation ; populations recovered from six lots incubated for h were significantly affected by. In these lots, incubation at C appeared to enhance recovery compared to C. Significant decreases in counts were obtained by extending the incubation time from h to h. This was due to overgrowth of plates, making enumeration of colonies extremely difficult after h. The type of ( or ) did not substantially affect colony development. Total populations of yeasts and molds recovered from 9 of 10 lots of cowpeas incubated at h and h were significantly influenced by and/or recovery (Table 3). An incubation of C was clearly superior to C, regardless of the recovery. Neither nor was judged to be better in supporting colony development. Overall, an extension of incubation time was not beneficial to increase the number of fungal propagules detected. Populations of A. flavuslparasiticus recovered from peanuts, corn meal and cowpeas are summarized in Table 4. One lot each of peanuts (No. 5) and cowpeas (No. 7) was positive for A. flavuslparasiticus, whereas eight lots of corn meal contained the mold. Detection of A. flavuslparasiticus in the peanut sample was not influenced by time, or recovery. In corn meal, 6 of 8 lots in which A. flavuslparasiticus was detected showed significantly higher counts if incubated at versus C when plates were examined after h of incubation; differences between s and recovery media were essentially eliminated by extending the incubation time to h. However, within and, an extension of incubation time resulted in significant increases in populations of A. flavuslparasiticus in 5 of 8 lots. The optimum conditions for detection of the mold group in corn meal, then, are judged to be h of incubation at C using either or. Observations on populations of A. flavuslparasiticus in the one positive lot of cowpeas are contrary to those noted for corn meal. An incubation of C was better than C for detecting the mold. This was due to overgrowth of plates at C, thus probably retarding growth and pigment production by A. flavusl parasiticus. Data listed in Table 5 represent composites of total yeast and mold populations within food types. For peanuts, incubation at C on for h gave significantly higher counts than incubation at C on for h. With corn meal and cowpeas, incubation of plates at C was clearly superior to incubation at C, but the incubation time and plating were essentially without affect. The observation that colony development was enhanced at C confirms the general view that growth of field and storage fungi is favored by s in the 20- C range. Composite data for A. flavuslparasiticus are presented in Table 6. Data for peanuts and cowpeas were derived from one lot each, and thus have limited value with respect to establishing the best combination of, incubation and time for detecting A. flavusl parasiticus. A large number of contaminated lots would
514 BEUCHAT TABLE 1. Total yeast and mold populations in peanuts detected on and incubated at and 3TC for h and h. Lot No. 10 Population per g a h h 42 74 38 71 11 b 51 a 12 b 49 a 2173 2445 26 2150 139 128 151 160 5212a 72 c 2448 b 96 c 28 b 55 ab 28 b 80 a 169 209 199 256 162 186 168 174 706 841 1040 801 491 398 312 434 89 b 82 58 74 21 80 114 90 2194 3461 2283 609 201 129 221 170 2511 ab 82 b 3209 a 78 b 75_ab 89 a 39 b 56 ab 8 311 331 329 199 162 252 235 692 720 679 804 510 431 456 381 "Mean values within the same lot and incubation time which are not followed by the same letter are significantly different (P= 0.05). b Mean value is significantly greater (P= 0.05) than value for other incubation time.
MEDIA FOR YEASTS AND MOLDS 515 TABLE 1. Total yeast and mold populations in corn meal detected on and incubated at and C for and h. Lot No. 10 Population per g a h h 20 18 12 25 69125 a 39625 b 71000 a 495 b 5528 4359 4921 4586 23100 a 14575 b 24038 a 21288 a 12875 a 675 b 12400 a 2775 b 134750 b 571 c 156875 a 545 c 29000 ab 24125 b 325 a 27875 ab 21350 15275 22463 15638 35875 625 40750 35000 14313 a 10775 b 15125 a 10275 b a Mean values within the same lot and incubation time which are not followed by the same letter are b Mean value is significantly greater (P^O.05) than value for other incubation time. 31 34 32_ b 28 665 63875 60500 585 1228 ab 950 b 14 a 1019 ab 18238 a 13350 b 180 a 16413 a 10600 a 693 b 11000 a 1173 b 121625 a 525 b 1175 a 525 b 29875 a 19950 b 21863 ab 24750 ab 12788 14875 17950 150 36500 34875 385 625 20225 a 9988 b 21213 a 10138 b significantly different (P=S0.05).
516 BEUCHAT TABLE 3. Total yeast and mold populations in cowpeas detected on and incubated at and C for and h. Lot No. 10 * Population per g a h h 296 a 16 b 226 a 10 b 15 a 1 b 16 a 1 b 509/ 54 b 441 a 61 b 22 b 19 b 64 ab 228 a 14 45 12 126 86 a 5b 92 a 9b 190 b 166 b 6 a 160 b 550 a 48 b 458 a 13b 1500 a 112b 1562 a 112b 285 a 20 b 298 a 16 b 215 a 16 b 246 a 42 b 35 a 4b 19 ab 5b 272 b 15 c 2775 a 12 c j45a 41 a 292 b 251 b 41 a 28 ab 0c 6 be 65 a 6b 65 a 6b 245 170 244 200 722 a 38 c 320 b 16 c 1525 a 38 b 1450 a 25 b 325 a 1 b 295 a 2b "Mean values within the same lot and incubation time which are not followed by the same letter are significantly different (Pss0.05) b Mean value is significantly greater (Ps 0.05) than value for other incubation time.
MEDIA FOR YEASTS AND MOLDS 517 TABLE 4. Aspergillus flavus populations in peanuts, corn meal and cowpeas detected on and incubated at and ITCfor and h. Food Peanuts Corn meal Cowpeas Lot No." 5 2 4 5 6 7 8 9 10 7 Population per g b h h 16 16 15 12 344 449 345 426 38 b 91 a 19 b 72 a 20 b 59 a 29 b 32 b 202 b 489 a 218 b 460 a 46 45 50 38 15 b 39 a 36 ab 29 ab 25 c 59 ab 41 be 76 a 0b 48 a 0b 36 a a 9b 31a 11 b 19 21 21 20 441 ab 452 a 349 b 441 ab 64 61 73_ 84 60_ 56 49 59 348 b 450 a 470 a 466 a 52 50 36 49 40 36 39 II 66 54 51 42 35 36_ 45 18 a 2 b 10 ab 2b a Lot numbers for peanuts, corn meal and cowpeas correspond to those listed in Tables 1, 2 and 3, respectively. All other lots were negative for A. flavus. b Mean values within the same lot and incubation time which are not followed by the same letter are significantly different (P«0.05). c Mean value is significantly greater (Pss0.05) than value for other incubation time.
518 BEUCHAT TABLE 5. Total yeast and mold populations for composites of ten lots each of peanuts, corn meal and cowpeas detected on and incubated at and C for and h. Food Peanuts Corn meal Cowpeas time (h) a Mean values within food types which are not followed by the same letter are significantly different (P=s0.05). Population perg a 913 a 685 ab 446 ab 555 ab 677 ab 764 ab 427 ab 283 b 34594 a 31748 a 14062 b 15911 b 996 a 791 a 16738 b 15609 b 347 b 349 b 49 c 36 c 348 b 571 a 74 c 57 c TABLE 6. Aspergillus flavus populations for composites of lots of peanuts, corn meal and cowpeas detected on and incubated at and C for and h. Food Peanuts Corn meal Cowpeas time (h) Population perg a 16 a 19 a 16 a 21 a 15 a 21 a 12 a 20 a 86 c 140 abc 160 a 150 abc 92 be 138 abc 146 abc 154 ab a 18 ab 9 be 2c 34 a 10 be 11 be 2c a Mean values within food types which are not followed by the same letter are significantly different (P«0.05). Values were determined only from those lots in which A. flavus was detected. JOURNAL OF FOOD PROTECTION, VOL, 47, JULY 1984
MEDIA FOR YEASTS AND MOLDS 519 serve as a better base from which to easily draw conclusions. Such was true for corn meal, eight lots of which contained A. flavuslparasiticus. at C for h retarded development of the mold group compared to incubation at C for h on ; incubation time and did not influence counts obtained on. Pitt et al. (14) reported that colonies of A. flavusl parasiticus recovered on developed better reverse orange-yellow color than did colonies on. This observation was also made in the present study, particularly when comparing colonies on and after 42-44 h of incubation. In summary, and performed similarly as media for enumerating total fungal propagules on peanuts, corn meal and cowpeas. Any differences in performance can be largely attributed to variability in mycoflora present in various lots. The use of for detecting the A. flavuslparasiticus group may have some advantage over for inexperienced workers, since color development of reverse colonies is quicker and more intense on. Observations from previous studies in our labortory indicate that compares favorably with chloramphenicol-supplemented plate count agar (PCA) for enumerating total yeasts and mold in legumes. However, statistical analysis of results was not made. It cannot be advised at this time, then, that or be used as media to enumerate total yeasts and molds concurrently with the A. flavus group on grains and legumes. Further studies comparing antibiotic-supplemented PCA, and for their suitability to recover yeasts and molds from seeds and meals will be necessary before one or both of the latter media can be recommended for use when enumerating total fungal population and A. flavuslparasiticus concurrently. ACKNOWLEDGMENT This work was supported in part by the Bean/Cowpea Collaborative Research Support Program, U.S. Agency for International Development. REFERENCES 1. Bell, D. K., and J. L. Crawford. 1967. A Botran-amended for isolating Aspergillus flavus from peanuts and soil. Phytopathology 57:939-941. 2. Beuchat, L. R. 1979. Comparison of acidified and antibiotic-supplemented potato dextrose agar from three manufacturers for its capacity to recover fungi from foods. J. Food Prot. 42:427-428. 3. Beuchat, L. R. 1979. Survival of conidia of Aspergillus flavus in dried foods. J. Stored Prod. Res. 15:25-31. 4. Bothast, R. J., and D. I. Fennell. 1974. A for rapid identification and enumeration of Aspergillus flavus and related organisms. Mycologia 66:365-369. 5. Hamsa, T. A. P., and J. C. Ayres. 1977. A differential for the isolation of Aspergillus flavus from cottonseed. J. Food Sci. 42:449-453. 6. Henson, O. E. 1981. Dichloran as an inhibitor of mold spreading in fungal plating media: Effects on colony diameter and enumeration. Appl. Environ. Microbiol. 42:656-660. 7. Henson, O. E., P. A. Hall, R. E. Arends, E. A. Arnold, R. M. Knecht, C. A. Johnson, D. J. Pusch, and M. G. Johnson. 1982. Comparison of four media for the enumeration of fungi in dairy products - A collaborative study. J. Food Sci. 47:9-932. 8. Janes, L., and R. H. Tilbury. 1981. A comparison of media and methods for the enumeration of yeasts and moulds in refined sugar products. J. Appl. Bacterid. 51:xiv. (Abstr.) 9. Jarvis, B. 1973. Comparison of an improved rose bengal chlortetracycline agar with other media for the selective isolation and enumeration of moulds and yeasts in foods. J. Appl. Bacteriol. 36:723-727. 10. Jarvis, B. 1978. Methods for detecting fungi in foods and beverages. pp. 471-504. In L. R. Beuchat (ed), Food and beverage mycology. AVI Publishing Co., Inc., Westport, CT. 11. King, D. A., Jr., A. D. Hocking, and J. I. Pitt. 1979. Dichloranrose bengal for enumeration and isolation of molds from foods. Appl. Environ. Microbiol. :959-964. 12. Nelson, F. E. 1972. Plating ph as a factor in apparent survival of sublethally stressed yeasts. Appl. Microbiol. 24:236-239. 13. Pitt, J. I., A. D. Hocking, and D. R. Glenn. 1983. An improved for the detection of Aspergillus flavus and A. parasiticus. J. Appl. Bacteriol. 54:109-114. 14. Salkin, I. F., and M. A. Gordon. 1975. Evaluation of Aspergillus differential. J. Clin. Microbiol. 2:74-75. 15. Statistical Analysis System. 1979. SAS user's guide. SAS Institute, Inc., Cary, NC. 494 pp. JOURNAL OF FOOD PROTECTION. VOL. 47, JULY 1984