SOME UNIVERSAL MEDIA FOR THE ISOLATION, GROWTH AND PURITY CHECK OF A BROAD SPECTRUM OF MICROORGANISMS

Khursheed A. Malik, Ph.D.

DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen
Mascheroder Weg 1B, D-3300 Braunschweig
Federal Republic of Germany

 

Media are generally required for the enrichment, optimum growth, isolation, and purity check of microorganisms. The main aim is to create a suitable growth environment near to the natural one which assures proper functioning of the enzymatic machinery of the microorganisms. The basic requirement of all culture media is that these should include the source of energy, the source of carbon, the source of trace elements and major elements (for details see Table 1). To permit the desired microbes to grow prominantly so that they can easily be distinguished from others, it is necessary to supplement the general media with additional growth factors and use optimal growth conditions. Proper pH, growth temperature and oxygen tensions are some further requirements that should be fullfilled to suit the needs and limits of the desired microorganisms (Table 1). For many bacteria the optimum pH range is between 6.5 to 7.5 and optimum growth temperature is about 25-30C.

In this report seven different universal media are described for aerobic growth of bacteria, fungi and yeasts. The types of a few microorganisms which have shown good growth on these media are listed in Table 2. However, as these are universal media, many other related microorganisms should also show growth on these.

Medium 1, is a universal nutrient medium which is routinely used at the DSM for the isolation, growth and purity check of many microorganisms. Only some genera which have shown good growth on this medium have been listed in Table 2 (for more details see DSM Catalogue of Strains).

Medium 2, (H₃P) , is a universal medium for heterotrophic microorganisms which has been developed by the author and has successfuly been used over more than 12 years for a broad spectrum of aerobic bacteria (Malik, 1988 a). It is highly buffered and the final pH of this medium results to 6.8 without adjustment which remains almost constant during normal growth.

Medium 3 (RBA), lacks the source of nitrogen and it was especially developed by the author for the isolation, growth and purity check of a broad spectrum of diazotrophic bacteria which are able to utilize atmospheric nitrogen. It has successfuly been used for more than 10 years for the genera Azomonas, Azorhizophilus, Azotobacter, nitrogen-fixing Bacillus, Xanthobacter, and few Rhizobium and Agrobacterium (Malik, 1988 b). This medium is transparent as compared to the opaque conventional media used for nitrogen-fixing bacteria. The advantage is that the observation of colony morphology and detection of impurities is much easier and all biochemical tests can clearly be evaluated. The new medium has proved to be superior to other media due to its increased nutritional quality. Almost all diazotrophic microorganisms will grow on this medium under nitrogen-fixing conditions. For microaerophilic nitrogen-fixing bacteria semi-solid medium with 0.3% (w/v) of agar can be used and the cultures should be incubated in a mixture of 10% (v/v) air and 90% (v/v) N₂.

Medium4, is a universal medium for the genera Rhizobium. Almost all Azorhizobium, Bradyrhizobium, Rhizobium, Sinorhizobium and few Agrobacterium have shown growth on this medium.

Medium 5 (H₃), is a mineral medium with high buffering capacity and it lacks the source of carbon. It has been developed by the author for a broad spectrum of chemolithoautotrophic hydrogen bacteria (Malik, 88b). During the last 13 years, it has successfuly been used for the growth of almost all chemolithoautotrophic hydrogen bacteria maintained in the DSM. For chemolithoautotrophic growth the cultures should be incubated in an atmosphere of 2% (v/v) O₂, 10% CO₂, 60% H₂ and 28% N₂. For growth under autotrophic nitrogen fixing conditions ammmonium chloride should be omitted and the cultures incubated in 5% (v/v) air, 10% CO₂, 30% H₂ and 55% N₂. For growth under hetrotrophic nitrogen fixing conditions the medium should be supplemented with 0.2% (w/v) glucose or sodium pyruate and incubated in 10% (v/v) air and 90% N₂. For more details see Malik and Schiegel, 1980; 1981.

Medium No. 6 and 7 were taken from the DSM Catalogue of Strains and are used successfully for the growth and purity check of a broad spectrum of fungi and yeasts held at the DSM (Dr P. Hoffmann, personal communication). When preparing media for fungi, it is helpful to note that most fungi grow best in media with a high C:N ratio at a pH of 5-6 and temperature between 20 and 25C. Tap water can be a source of useful trace elements provided it is not treated with chlorine as it may be slightly toxic for some species.

Yeasts will grow on simple synthetic media under both aerobic and anaerobic conditions. Optimum pH range is between 4.5 to 6.5 and optimum growth temperature for many yeast species is 20-25C.

For more details on cultivation media it is recommended to consult the catalogues of strains of major culture collections as these include information on incubation conditions and lists of growth media. Reference should also be made to standard textbooks for more information about special physiological groups of microorganisms requiring a special gaseous atmosphere, oxidation-reduction potential, growth factors, incubation time and incubation temperatures.

 

REFERENCES

Hawksworth, D.L. & Kirsop, B.E. (1988). Filamentous Fungi. Living Resources for Biotechnology. Cambridge University Press, Cambridge.
Hill, L.R. & Kirsop, B.E. (1991). Bacteria. Living Resources for Biotechnology. Cambridge University Press, Cambridge.
Kirsop, B.E. & Kurtzman, C.P. (1988). Yeasts. Living Resources for Biotechnology. Cambridge University Press, Cambridge.
Malik, K.A. (1988 a). Long-term preservation of some Rhodospirillaceae by freeze-drying. Journal of Microbiological Methods 8, 273-280.
Malik, K.A. (1988 b). A new freeze-drying method for the preservation of nitrogen-fixing and other fragile bacteria. Journal of Microbiological Methods 8, 259-271
Malik, K.A. & Schiegel, H.G. (1980). Enrichment and isolation of new nitrogen-fixing hydrogen bacteria. FEMS Microbiology Letters 8, 101-104.
Malik, K.A. & Schiegel, H.G. (1981). Chemolithoautotrophic growth of bacteria able to grow under N₂-fixing conditions. FEMS Microbiology Letters 11, 63-67.

Table 1. Basic requirements of all Media^a

Type of microbes	Energy	Source of carbon	Trace and major elements
Photosynthetic	Sunlight or artificial light	CO2, exogenous organic nutrients and growth factorsb	Source of sulfur, nitrogen phosphorous, trace elements
Chemoautotrophic	Reduced inorganic molecules or irons : H2, CO, H2S, S, NH4+, NO2-, N, Fe++, Mn++, etc.	CO2, Carbonate, growth factors	Same as above
Heterotrophic	Organic molecules	Organic supplements, compounds or exogenous organic nutrientsc	Same as above

a. In addition, proper pH, temperature and oxygen tensions must be provided to suit the needs and limits of desired microbes.

b. Water-soluble vitamins, purines, pyrimidines, aminoacids, lipids, minerals, etc.

c. Carbohydrates, sugars, sugar alcohols, compounds leading to TCA cycle, TCA cycle acids, etc.

Table 2. Microorganisms which show good growth in the following media

Microorganisms	Growth media		Microorganisms	Growth media
Acinetobacter	1		Micrococcus	1
Aeromonas	1		Microcyclusa	2 or 5
Agrobacterium	1 or 3		Paracoccusa	1 or 5
Alcaligenesa	1 or 5		Proteus	1
Ancylobacter	2 or 5		Pseudomonasa	1 or 5
Aquaspirillum	2		Rhizobium	4
Azomonas	1 or 3		Rhodobacterb	1 or 2
Azorhizobium	4		Rhodopseudomonasb	1 or 2
Azorhizophilus	3		Rhodospirillumb	2
Azospirilluma	2 or 5		Serratia	1
Azotobacter	3		Shigella	1
Bacillus	1		Sinorhizobium	4,2
Bradyrhizobium	4 or 5		Xanthobactera	1,2,3 or 5
Calderobacterium	5		Xanthomonas	1 (not all)
Derxia	1, 2, 3 or 5		Zoogloea	1
Entrobacter	1		Yeastsc	6
Erwinia	1		Fungic	7
Escherichia	1		Algaed	2
Flavobacterium	1

a. Only chemolithoautotrophic bacteria will grow on Medium 5.

b. Only photosynthetic bacteria (non-sulfur) which show growth under heterotrophic conditions.

c. Some species may require special conditions such as iron complexes, high temperature, etc.

d. Not all but many fresh water algae have shown good growth.

1. NUTRIENT AGAR MEDIUM

Peptone	5.0g
Meat extract	3.0g
Agar	15.0g
Distilled water	1000.0ml

Adjust pH to 7.0. For Bacillus strains the addition of 10 mg MnSO4 x H2O is recommended for sporulation. Autoclave at 121C for 15 minutes.

2. MEDIUM FOR HETEROTROPHS (H₃P)

Solution A:
KH2PO4	2.3g
Na2HPO4 x 2H2O	2.9g
Distilled water	50.0ml
Solution B:
NH4Cl	1.0g
MgSO4 x 7H2O	0.5g
CaCl2 x 2H2O	0.01g
MnCl2 x 4H2O	0.005g
NaVO3 x H2O	0.005g
Trace element solution SL-6 (see medium 5)	5.0ml
Distilled water	850.0ml
Agar (if necessary)	15.0g
Solution C:
Ferric ammonium citrate	0.05g
Distilled water	20.0ml
Solution D:
Yeast extract	1.0g
Sodium acetate	1.0g
Di-sodium succinate	1.0g
DL-Malate	1.0g
Distilled water	30.0ml
pH 7.0
Solution E:
Sodium lactate	1.0g
Sodium pyruvate	1.0g
D-Mannitol	1.0g
D-Glucose	2.0g
Distilled water	50.0ml
pH 7.0
Standard vitamin solution:
Riboflavin	10.0mg
Thiamine	50.0mg
Nicotinic acid	50.0mg
Pyridoxine HCl	50.0mg
Calcium pantothenate	50.0mg
Biotin	0.1mg
Folic acid	0.2mg
B12	1.0mg
Distilled water	100.0ml

Solutions A, B, C and D are autoclaved separately for 15min at 121C, cooled down to 50C and then mixed aseptically with filter-sterilized solution E (at 50 C) and 5.0ml of standard vitamin solution. The final pH of this medium should be 6.8 without adjustment. In the case of known microorganisms only one organic nutrient (0.2-0.5%) on which the organism grows can be used and it is not necessary to use all the organic compounds listed under Solution D and E.

3. MEDIUM FOR DIAZOTROPHS (RBA)

Solution A:
KH2PO4	0.1g
K2HPO4	0.9g
NaCl	0.1g
CaCl2 x 2H2O	0.1g
MgSO4 x 7H2O	0.1g
Na2MoO4 x 2H2O	0.005g
NaVO3 x H2O	0.005g
MnSO4 x H2O	0.005g
FeSo4 x 7H2O	0.01g
Trace element solution SL-6 (see Medium 5)	3.0ml
Distilled water	900.0ml
Agar (if necessary)	15.0g
Adjust pH to 7.3.
Solution B:
Yeast extract	0.05g
di-Sodium succinate	1.0g
DL-Malate	2.0g
Distilled water	50.0ml
Adjust pH to 7.3.
Solution C:
Sodium pyruvate	1.0g
D-Mannitol	2.0g
D-Glucose	2.0g
Distilled water	50.0ml
Adust pH to 7.3.

Sterilize solutions A and B separately at 121C for 15min., cool down to 50C and then mix aseptically with filter-sterilized solution C and 5.0ml of filter-sterilized standard vitamin solution (see Medium 2). For known microorganisms only appropriate carbon source (0.2-0.5%) can be used instead of solution B and C.

4. MEDIUM FOR RHIZOBIUM

Yeast extract	1.0g
Mannitol	10.0g
Agar	15.0g
Soil extract	200.0ml
Distilled water	800.0ml
Adjust pH to 7.0.
Soil extract:
Air-dried garden soil	80.0g
Na2CO3	0.2g
Distilled water	200.0ml

Autoclave soil suspension for one hour at 121C. To obtain a clear supernatant allow to settle and centrifuge. Adjust pH to 7.2. Sterilize the medium at 121C for 15min.

5. MINERAL MEDIUM (H₃)

KH2PO4	2.3g
Na2HPO4 x 2H2O	2.9g
NH4Cl	1.0g
MgSO4 x 7H2O	0.5g
NaHCO3	0.5g
CaCl2 x 2H2O	0.01g
Fe(NH4)citrate	0.05g
Trace element solution SL-6	5.0ml
Distilled water	980.0ml
Agar (if necessary)	15.0g
Adjust pH to 6.8.
Pfennig's Trace element solution (SL-6)
ZnSO4 x 7H2O	0.1g
MnCl2 x 4H2O	0.03g
H3BO3	0.3g
CoCl2 x 6H2O	0.2g
CuCl2 x 2H2O	0.01g
NiCl2 x 6H2O	0.02g
Na2MoO4 x 2H2O	0.03g
Distilled water	1000.0ml

Autoclave for 15 minutes at 121C. Sterilize Fe (NH4) citrate (0.05g in 20ml H2O) separately and then add to the medium. For more details see Malik, 1988 b.

6. UNIVERSAL MEDIUM FOR YEASTS

Yeast extract	3.0g
Malt extract	3.0g
Peptone	5.0g
Glucose	10.0g
Agar	15.0g
Distilled water	1000.0ml

Autoclave at 121C for 15 minutes. For Brettanomyces species add 0.5-1% CaCo3 to the above medium. For osmophilic species add 20-60% sucrose.

7. UNIVERSAL MEDIUM FOR FUNGI

Malt extract	30.0g
Soya peptone	3.0g
Agar	15.0g
Distilled water	1000.0ml

Adjust pH to 5.6. Sterilize at 121C for 10 min. For osmophilic species add 20-60% sucrose.

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Published by : UNESCO / WFCC-Education Committee 1991