Mycological media for food- and indoor fungi

For a more detailed description see Introduction to food borne fungi

RECOMMENDED MEDIA FOR CULTIVATION AND IDENTIFICATION OF COMMON FILAMENTOUS FUNGI

Acremonium OA, CMA, MEA (2% MEA CBS)
Alternaria MEA, PCA, HAY
Aspergillus Cz, CYA, 2% MEA (CBS);
for xerophilic species: Cz or MEA with additional sugar (20 or 40 %) or other low water activity media.
Aureobasidium MEA
Basidiomycetes 2% MEA CBS + 1 ppm benomyl
Botrytis PCA, MEA, HAY
Byssochlamys MEA, OA
Chaetomium OA (with lupine stem), CMA
Chrysonilia OA
Cladosporium MEA, OA, PCA
Emericella MEA, OA
Epicoccum MEA, OA, PCA, SEA
Eurotium MEA or Cz with sucrose 20 or 40% or other low water activity media
Fusarium PSA, PDA, SNA, CLA, YES
Geotrichum MEA
Monascus MEA, OA
Moniliella MEA, PCA
Mucorales MEA (4%)
for zygospore formation MYA (Absidia), CH (Mucor spp)
Neosartorya MEA, OA
Paecilomyces MEA, OA
Penicillium MEA, Cz, CYA, YES
Phialophora MEA, OA
Phoma OA (with lupine stem), MEA (4% MEA CBS) (+ UV)
Scopulariopsis MEA, OA
Stachybotrys MEA, OA, Hay
Talaromyces OA, MEA, CMA, YES
Trichoderma OA, MEA
Trichothecium MEA
Ulocladium MEA, PCA, HAY
Verticillium MEA
Wallemia MEA + additional sugar 20 or 40 % or other low water activity media
Xeromyces MY50G
CH = Cherry decoction agar; CLA = Carnation Leaf Agar; CMA = Cornmeal Agar; CYA = Czapek Yeast Extract Agar; CY20S = Czapek Yeast Extract Agar with 20% sucrose; Cz = Czapek Agar; C20S = Czapek Agar with 20% Sucrose; DG 18 = Dichloran Glycerol Agar Base; HAY = Hay infusion Agar; MEA = Malt Extract Agar; MEA + 20% sucrose = Malt Extract Agar with 20% sucrose; MYA = Malt Yeast Agar; MY50G = Malt Extract Yeast Extract 50 % Glucose Agar; OA = Oatmeal Agar; PCA = Potato Carrot Agar; SNA = Synthetischer Nährboden; YES = Yeast Extract Sucrose Agar.

MYCOLOGICAL MEDIA FOR DETECTION, ISOLATION AND IDENTIFICATION

Ingredients in grams are dissolved in one litre distilled water and sterilized by autoclaving at 121EC for 15 minutes, unless stated otherwise. Usually 15 or 20 g agar per litre are recommended in the original formulations. These quantities are mentioned in the formulations below. However, for some brands of agar smaller amounts (8 g) per litre might be sufficient. At CBS 8 g of powdered “Spanish agar 700” (EEC number E 406) is used for most media.
Addition of 1 ml trace metal solution (TMS) per litre medium is recommended for avoiding atypical colony growth and colour. Trace metal?solution: 1 g ZnSO4.7H2O and 0.5 g CuSO4.5H2O in 100 ml distilled water.
Several media are commercially available and indicated with the brand name between brackets. Media with the indication (CBS) are formulations used at the Centraalbureau voor Schimmelcultures.
Addition of mineral solution (MS), containing 5g KCL, 5 g MgSO4.7H2O, 0.1 g FeSO4.7H2O per 100 ml water is also recommended in some media (10 ml per litre medium).
For the suppression of bacterial growth, antibiotics should be added to the medium to give a final concentration of:
penicillin-G 50 ppm
streptomycin 30-50 ppm
aureomycin 20-50 ppm
neomycin 100 ppm
chloramphenicol 100 ppm
Only chloramphenicol withstands autoclaving without loss of activity.

ADYS: Acetic acid Dichloran Yeast extract agar
Yeast extract 20 g
Sucrose 150 g
MgSO4. 7 H2O 0.5 g
Dichloran 0.002 g
Agar 20 g
Distilled water 1000 ml
After autoclaving 0.5 % glacial acetic acid is added.

AFPA: Aspergillus flavus/A.parasiticus selective medium (Oxoid)
Peptone 10 g
Yeast extract 20 g
Ferric Ammonium Citrate 0.5 g
Dichloran 0.002 g
Chloramphenicol 0.1 g
Agar 15 g
Distilled water 1000 ml
Final pH 6.3 ± 0.2
NOTE: Dichloran and Chloramphenicol can be added before autoclaving.

CHERRY-DECOCTION AGAR (CBS)
1 kg cherries (without stones and stalks) in one litre water is heated to boiling and simmered gently for 2 h. Strain through cloth and sterilize at 110°C (= 0.5 atm) for 30 min. Dissolve 15 g agar in 800 ml water and sterilize at 121°C for 15 min. Add 200 ml cherry extract and mix well. Sterilize again for 5 min at 102°C (=0.1 atm). Final pH 3.8?4.6

CLA: Carnation leaf agar (Fischer et al., 1982)
Carnation leaves are cut into pieces, dried gently and sterilized by means of gamma irradiation or propylene oxide fumigation. A few sterile pieces can be placed on 1.5?2% water agar (nearly solid). Instead of carnation leaves also pieces of sterile filter paper can be used
NOTE: Suitable medium for cultivation of Fusarium.

CMA: Cornmeal agar (CBS)
Add 60 g freshly ground cornmeal to 1 litre water, heat to boiling and simmer gently for 1 h. Strain through cloth and sterilize for 15 min at 121°C overpressure. Fill up to 1 litre and add 15 g agar and sterilize at 121°C for 15 min. Also commercially available.

CREA: Creatine Sucrose agar
Creatine(1 H2O) 3 g
Sucrose 30 g
KCl 0.5 g
MgSO4.7H2O 0.5 g
FeSO4.7H2O 0.01 g
K2HPO4.3H2O 1.3 g
Bromocresol purple 0.05 g
Agar 15 g
Distilled water 1000 ml
Final pH 8.0 ± 0.2 (adjust after medium is autoclaved).
NOTE: a modification of CREA (=CRE) with 1.6 g
K3PO4.7H2O can also be used.

CREAD: Creatine Sucrose Dichloran agar
Creatine (1 H2O) 3 g
Sucrose 30 g
KCl 0.5 g
KH2PO4 1 g
FeSO4.7H2O 0.01g
MgSO47 H2O 0.5 g
Chloramphenicol 0.05 g
Dichloran 0.002 g
Bromocresole purple 0.05 g
ZnSO4.7H2O 0.01 g
CuSO4.5H2O 0.005 g
Agar 20 g
Distilled water 1000 ml
After autoclaving 0.05 g chlortetracycline is added and pH is adjusted to 4.8.

CZ: Czapek agar (CBS)
Sucrose 30 g
NaNO3 3 g
K2HPO4 1 g
KCl 0.5 g
MgSO4.7H2O 0.5 g
FeSO4.7H2O 0.01 g
Agar 15 g
Distilled water 1000 ml
Final pH 6.2 ± 0.2

CZ2OS: Czapek agar with 20% sucrose
The same as Czapek agar but containing 200 g sucrose.
NOTE: addition of 1 mL of the trace metal solution (see under formulation TMS) is recommended

CYA: Czapek yeast (autolysate) extract agar (Samson and Pitt, 1985)
NaNO3 3 g
K2HPO4 1 g
KCl 0.5 g
MgSO4.7H2O 0.5 g
FeSO4.7H2O 0.01 g
Yeast extract 5 g
Sucrose 30 g
Agar 20 g
Distilled water 1000 ml
Final pH 6.0?6.5
NOTE: addition of 1 mL of the trace metal solution (see under formulation TMS) is recommended

CZID: Czapek Dox Iprodione Dichloran agar
Czapek?Dox Broth (Difco) 35 g
CuSO4.5H2O 0.005 g
ZnSO4.7H2O 0.01g
Chloramphenicol 0.05 g
Dichloran (0.2% in ethanol) 1 ml
Agar 20 g
Distilled water to 1000 ml
After autoclaving and cooling to 50°C, add chlortetracycline solution, 10 ml and iprodione suspension, 1 ml. Chlortetracycline solution is 0.5% aqueous, and iprodione suspension (which should be shaken before addition to CZID) contains Rovral 50WP (Rhone?Poulenc, Agro Chemie, Lyon, France) 0.3 g in sterile water, 50 ml. CZID was developed for the selective detection of Fusarium species.

DG18: Dichloran 18% Glycerol agar
Peptone 5 g
Glucose 10 g
KH2PO4 1 g
MgSO4.7H2O 0.5 g
Dichloran (0.2% in ethanol) 1.0 ml
Glycerol 220 g
Chloramphenicol 0.1 g
Agar 15 g
Distilled water 1000 ml
Add minor ingredients and agar to ca. 800 ml distilled water. Steam to dissolve agar, then make to one litre with distilled water. Add 220 g glycerol and sterilize by autoclaving at 121°C for 15 min. The final aw is 0.955: final pH 5.6 ± 0.2. Commercially available (Oxoid).

DRBC: Dichloran rose bengal chloramphenicol agar
Peptone 5 g
Glucose 10 g
KH2PO4 1.0 g
MgSO4.7H2O 0.5 g
Dichloran 0.002 g
Rose Bengal 0.025 g
Chloramphenicol 0.1
Agar 15 g
Distilled water 1000 ml
Final pH 5.6 ± 0.2
NOTE: Original formula according to King et al. (1979) contains chlortetracycline. Hocking (1981) replaced this antibioticum with chloramphenicol.

DRYES: Dichloran Rose Bengal Yeast Extract Sucrose agar (Frisvad, 1983)
Yeast extract 20 g
Sucrose 150 g
Dichloran (0.2% in ethanol) 1.0 ml
Rose bengal (5% soln., w/v) 0.5 ml
Chloramphenicol 0.1 g
Agar 20 g
Water to 1000 ml
Final pH 5.6 (adjusted after medium is autoclaved). This medium detects Penicillium verrucosum and P. viridicatum by production of a purple reverse colour. Can be modified by adding 0.5 g MgSO4.7H2O.

DYSG: Dichloran Yeast Extract 18% Glycerol agar
Yeast extract 20 g
Sucrose 150 g
K2HPO4 1 g
MgSO4.7 H2O 0.5 g
Chloramphenicol 0.05 g
Dichloran (0.2% in ethanol) 1.0 ml
ZnSO4.7H2O 0.01 g
CuSO4.5H2O 0.005 g
Agar 20 g
Glycerol 220 g
Water 1000 ml
After autoclaving 0.05 g chlortetracycline is added.

HAY: Hay infusion agar (CBS)
Sterilize 50 g hay in one litre water at 121°C for 30 min. Strain through cloth and fill up to one litre. Adjust pH to 6.2 with K2HPO4 and take 1000 ml extract and add 15 g agar. Autoclave for 15 min at 121°C.

MEA: Malt extract agar (according to Blakeslee)
Malt extract (powdered) 20 g
Peptone, bacteriological 1 g
Glucose 20 g
Agar 20 g
Distilled water 1000 ml
Final pH 5.0?5.5

MEA: Malt extract agar (Oxoid)
Malt extract 30 g
Mycological peptone 5 g
Agar 15 g
Distilled water 1000 ml
Final pH 5.4 ± 0.2
Sterilize by autoclaving at 115°C for 10 min.

MEA: Malt extract agar (Difco, Bacto)
Malt extract, Difco 30 g
Bacto agar 15 g
Distilled water 1000 ml
Final pH 5.5 ± 0.2

MEA: Malt extract agar (Merck)
Malt extract 30 g
Peptone (from soya flour) 3 g
Agar 15 g
Final pH 5.6 ± 0.1

MEA: Mout extract agar 4% (2%) (CBS)
Malt extract 400 ml
(200ml for 2% MEA)
Distilled water 600 ml
(800ml for 2% MEA)
Agar 15 g
Add water to malt extract from the brewery until it contains 10% sugar (measurement with areometer). Mix 400ml (200ml) of this solution with 15 g agar and 600 (800) ml water. Malt agar may also conveniently be prepared with malt syrup (10?40 g/l) or malt powder (10?20 g/l).
M20 or M40
The same as MEA but containing 20 or 40% sucrose.

M40Y: Malt yeast 40% sucrose agar
Malt extract powdered 20 g
Yeast extraxt 5 g
Sucrose 400 g
Agar 15 g
Distilled water 1000 ml
NOTE: Recommended for xerophilic fungi, e.g. Eurotium, Wallemia.

MS: mineral solution per 100 ml water
KCl 5g
MgSO4.7H2O 5 g
FeSO4.7H2O 0.1 g
Add 10 ml of the solution to 1 L medium

MSA: Malt Salt Agar
Malt extract 20 g
NaCl 75 g
Agar 20 g
Distilled water 1000 ml

MYA: Malt yeast agar (CBS)
Malt extract 10 g
Yeast extract 4 g
Glucose 4 g
Agar 15 g
Distilled water 1000 ml
Adjust pH ± 7.3

MYCK: Malt extract Yeast extract Chloramphenicol Ketoconazol agar (Baertschi et al., 1989).
Malt extract 20 g
Yeast extract 2 g
Chloramphenicol 0.5 g
Agar 15 g
Distilled water 1000 ml
Ketoconazol (1% wt./vol. 95% ethanol) 50 mg/l, filter?sterilized is added after autoclaving. Final pH 5.6. For the isolation of Mucor species.

OA: Oatmeal agar (CBS)
Heat 30 g oat flakes in 1 litre water to boiling and simmer gently for 2h. Filter through cloth and fill up to 1 litre. Add 15 g agar to one litre and sterilize by autoclaving at 121°C for 15 min. When using powdered oatmeal, filtering is superfluous. Lupine stems may be placed in slants with oatmeal agar. Also commercially available.

OGYE: Oxytetracycline?Glucose?Yeast?Extract?Agar
(Oxoid, modified)
Yeast extract 5 g
Glucose 20 g
Biotin 0.0001 g
Oxytetracycline 0.1 g
Agar 12 g
Distilled water 1000 ml
Final pH 7.0 (approx.). Addition of the oxytetracycline after autoclaving at a temperature of 50°C.

PCA: Potato?carrot agar (CBS)
40 g carrots and 40 g potatoes are separately washed, pealed, chopped, boiled in one litre water each for 5 min and filtered off. Sterilize for 15 min at 121°C. Take 250 ml potato extract and 250 ml carrot extract, 500 ml distilled water, 15 g agar and sterilize at 121EC for 15 min.

PDA: Potato?dextrose agar
Add 200 g scrubbed and diced potatoes to 1 litre water and boil for 1 h. Let it pass through a fine sieve, add 15 g agar and 20 g glucose (= dextrose) and boil until dissolved. pH 5.6 ± 0.1. Also commercially available.

PSA: Potato?sucrose agar
Same recipe as PDA, but replace glucose by sucrose. Adjust pH 6.7 ± 0.2

RA: Rice meal agar
Rice meal 75 g
Agar 20 g
Distilled water to 1000 ml. Boil for 2 hours before autoclaving.

SNA: Synthetischer nährstoffarmer agar (Nirenberg, 1976)
KH2PO4 1 g
KNO3 1 g
MgSO4.7H2O 0.5 g
KCl 0.5 g
Glucose 0.2 g
Sucrose 0.2 g
Agar 20 g
Distilled water 1000 ml
NOTE: Pieces of sterile filter paper may be placed on the agar. Recommended for the cultivation of Fusarium, but also for poorly sporulating Deuteromycetes.

TAN: Tannic acid?nitrate agar
NaNO3 0.30 g
Sucrose 3 g
K2HPO4.3H2O 1.3 g
Agar 20 g
Mineral solution (MS) 10 ml
Trace metal solution (TMS) 1 ml
Distilled water 850 ml
After autoclaving and cooling to 60°C add 150 ml tannic acid solution (TA), which is also 60°C ml; tannic acid solution contains 10 g tannic acid and 150 ml water. Pasteurize in boiling water for 10 min.
NOTE: used for identification of Fusarium.

TMS: Trace Metal Solution per 100 ml water
ZnSO4.7H2O 1 g
CuSO4.5H2O 0.5 g
Add 1 ml of the solution to 1 L medium just before autoclaving

YES: Yeast Extract Sucrose agar
Yeast extract 20 g
Sucrose 150 g
MgSO4.7H2O 0.5 g.
Agar 20 g
Distilled water 885 ml
Recommended for secondary metabolite analysis.

V?8 Agar
200 ml V?8 juice (vegetable juice, commercially available, Campbell), 3 g CaCO3, 20 g agar in 1000 ml distilled water. Autoclaving for 30 min at 110°C.

WATER AGAR 2%
Agar 20 g
Distilled water 1000 ml
SPECIFIC MEDIA USED FOR THE CULTIVATION AND IDENTIFICATION OF YEASTS

ACETIC ACID AGAR
Glucose 100 g
Tryptone 10 g
Yeast extract 10 g
Agar 20 g
Distilled water 1000 ml
The molten medium is cooled to approximately 45°C, glacial acetic acid added to a concentration of 1%, rapidly mixed, and poured into Petri dishes.
The medium is used for the test for resistance to acetic acid

GP: Glucose Peptone Yeast extract
Glucose 40 g
Peptone 10 g
Yeast extract 5 g
Agar 20 g
Distilled water 1000 ml

TGY: Tryptone Glucose Yeast Extract Agar
Glucose 100 g
Tryptone 5 g
Yeast extract 5 g
Agar 15 g
Distilled water to 1000 ml
Addition of antibiotics (Chloramphenicol or oxytetracycline 100 mg/l) are recommended. For enumeration of yeasts in products where moulds are not present.

YEAST MORPHOLOGY agar
Commercially available (Difco).

UREA BROTH
Commercially available as Urea R Broth. Urea broth is reconstituted according to the instructions on the package and 0.5 ml amounts are dispensed aseptically into sterile tubes. It may then be stored in a deep freezer for several weeks.

YEAST NITROGEN BASE and YEAST CARBON BASE
Commercially available. An appropriate amount as indicated on the package is dissolved in distilled water, for auxanogram tests it is solidified with 1.5% of agar.
Carbon sources used on this course are: D?xylose, sucrose, alpha, alpha?trehalose, lactose, raffinose, erythritol, D?mannitol, D?glucuronate, and DL?lactate. Nitrogen sources are: nitrate, L?lysine, and cadaverine.

YEAST MALT AGAR
Malt extract 3 g
Yeast extract 3 g
Peptone 5 g
Glucose 10 g
Agar 20 g
Distilled water 1000 ml
Commercially available

REFERENCES

ABILDGREN, M.P., LUND, F., THRANE, U. & ELMHOLT, S. 1987. Czapek?Dox agar containing iprodione and dicloran as a selective medium for the isolation of Fusarium species. Lett. Appl. Microbiol. 5: 83?86.
ANDREWS, S. 1992. Differentiation of Alternaria species isolated from cereals on dichloran malt extract agar. In Modern Methods in Food Mycology, eds R.A. Samson, A.D. Hocking, J.I. Pitt and A.D. King. pp. 351?355. Amsterdam: Elsevier.
ANDREWS, S. & PITT, J.I. 1986. Selective medium for the isolation of Fusarium species and dematiaceous Hyphomycetes from cereals. Appl. Environ. Microbiol. 51: 1235?1238.
BAERTSCHI, C., BERTHIER, J., GUIGUETTAZ, C. & VALLA, G. 1989. A selective medium for the isolation and enumeration of Mucor species. Mycological Research 95: 373?374
BOOTH, C. 1971. Fungal culture media. In Methods in Microbiology, ed. C. Booth. pp. 49?94. London: Academic Press.
DIFCO MANUAL. 1984. Dehydrated Culture Media and Reagents for Microbiology, 10th ed. 1155 pp. Detroit, Michigan: Difco Laboratories.
FRISVAD, J.C. 1983. A selective and indicative medium for groups of Penicillium viridicatum producing different mycotoxins in cereals. J. Appl. Bacteriol. 54: 409?416.
FRISVAD, J.C. 1985. Creatine?sucrose agar, a differential medium for mycotoxin producing terverticillate Penicillium species. Lett. Appl. Microbiol. 1: 109?113.
HOCKING, A.D. & PITT, J.I. 1980. Dichloran?glycerol medium for enumeration of xerophilic fungi from low?moisture foods. Appl. Environ. Microbiol. 39: 488?492.
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.
KING, A.D., HOCKING, A.D. & PITT, J.I. 1979. Dichloran?rose bengal medium for enumeration and isolation of moulds from foods. Appl. Environ. Microbiol. 37: 959?964.