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Dehydrated Culture Medium
Sodium propionate selective agar for isolating Streptomyces and other actinomycetes from soil, water and sediment
Cat. No. AS-110221.7 g/LpH 8.1 ± 0.2Selective agarSoil & water microbiology
At a glance
- Target organismsActinomycetes
- Incubation25–30 °C, aerobic
- Time7–21 days
- Sterilisation121 °C, 15 min
Manufactured in Melbourne. TDS and SDS available. Batch COA on request.
Overview
Actinomycete Isolation Agar (AC Agar) is a selective solid medium for isolating and cultivating actinomycetes, including Streptomyces and related genera, from soil, water, sediment and other mixed environmental samples.
Isolating these organisms by direct plating is difficult because they grow slowly. On a rich non-selective agar, a soil sample's fast-growing bacteria and fungi cover the plate within a day or two, long before an actinomycete has formed a visible colony. This medium solves that by holding the competitors back. Sodium propionate inhibits many Gram-negative bacteria and fast-growing fungi, the nitrogen supply is deliberately restricted to sources actinomycetes use well, and the pH is set at 8.1, above the comfortable range of many soil organisms.
Given a long enough incubation, actinomycetes then develop their characteristic small, dry, powdery colonies with aerial mycelium, and the plate stays readable.
Key features
- Sodium propionate as the selective agent, with no antibiotic required
- Alkaline pH 8.1 adds a second layer of selectivity
- Restricted nitrogen base favours actinomycetes over competing soil organisms
- Supports aerial mycelium and sporulation, so colony morphology is diagnostic
- Glycerol, added separately, is a preferred carbon source for Streptomyces
- Suits slants for stock maintenance as well as plates for isolation
Principle of the Medium
| Component | Function |
|---|---|
| Sodium caseinate | Primary nitrogen source; poorly used by many competing organisms |
| L-Asparagine | Supplementary nitrogen and amino acids |
| Sodium propionate | Selective agent; inhibits many Gram-negative bacteria and fungi |
| Dipotassium phosphate | Buffers the medium at alkaline pH |
| Magnesium sulfate | Enzyme cofactor source |
| Ferrous sulfate | Trace iron for respiratory and biosynthetic enzymes |
| Agar | Solidifying agent |
| Glycerol (added separately) | Carbon and energy source; supports aerial mycelium formation |
Selectivity comes from the formulation itself, not from an antibiotic. For samples with heavy mould loads, add filter-sterilised cycloheximide or nystatin after autoclaving, once the agar has cooled to 45–50 °C.
Typical Composition
| Ingredient | g/L |
|---|---|
| Agar | 15.000 |
| Sodium propionate | 4.000 |
| Sodium caseinate | 2.000 |
| Dipotassium phosphate | 0.500 |
| L-Asparagine | 0.100 |
| Magnesium sulfate heptahydrate | 0.100 |
| Ferrous sulfate heptahydrate | 0.001 |
| Total | 21.701 |
Final pH 8.1 ± 0.2 at 25 °C. Glycerol (5 mL/L) is not included in the dehydrated powder and is added by the user. Formula may be adjusted and/or supplemented to meet performance specifications.
Applications
| Field | Typical use |
|---|---|
| Soil microbiology | Isolation and enumeration of actinomycetes from soil and compost |
| Water and sediment testing | Recovery of actinomycetes from water and sediment samples |
| Natural-product screening | Screening soil isolates for strains producing bioactive compounds |
| Culture maintenance | Subculturing and stock maintenance of actinomycete isolates on slants |
| Teaching laboratories | Soil and environmental microbiology practicals |
Comparable Products
| Manufacturer | Product | Relationship |
|---|---|---|
| BD Difco | 212168 Actinomycete Isolation Agar | Same published formulation |
| HiMedia Laboratories | M490 Actinomycete Isolation Agar | Same published formulation |
Comparison is based on published formulation and intended use. Users should confirm performance under their own conditions.
Frequently Asked Questions
How much powder do I need, and where does the glycerol go?
21.7 g of powder per litre of purified water, plus 5 mL of glycerol added before autoclaving. The glycerol is not part of the dehydrated blend. Prepared without it, the medium has no usable carbon source and growth will be poor.
How long do the plates need to incubate?
7 to 21 days at 25–30 °C. Check periodically rather than reading once at the end, because colony morphology changes as aerial mycelium develops and because a plate left too long may be overgrown by a faster isolate.
What do actinomycete colonies look like on this medium?
Small, dry and powdery, often tough and firmly attached to the agar rather than easily picked off. Many develop a white to grey aerial mycelium as they sporulate, and some produce diffusible pigment that colours the surrounding agar.
Will the plates dry out over three weeks?
They can. Pour a full 20–25 mL per 90 mm plate, store the plates in sealed bags or a humidified container, and incubate them inverted. Plates poured thin will crack or shrink before the incubation is finished.
Should I use the agar or the broth?
Use the agar when you want single colonies and can plate the sample directly. Use AC Broth (AS-1103) first when the target organisms are too scarce to recover by direct plating, then plate the enriched culture onto this agar.
Do I need to add an antifungal?
Not for every sample. The base formulation suppresses many fungi. For soil or compost with a heavy mould load, add filter-sterilised cycloheximide at 50 mg/L after autoclaving, and nystatin at 50 mg/L as well if moulds still break through.
Intended use: For laboratory microbiological testing, quality control and research use only. Not intended for direct use in food, human or veterinary therapeutic applications, diagnosis, or consumption.
Mode of Action
Sodium caseinate and L-asparagine supply nitrogen in forms that actinomycetes use readily and many competing organisms use poorly. Sodium propionate is the selective agent, inhibiting a broad range of Gram-negative bacteria and fast-growing fungi. Dipotassium phosphate buffers the medium at pH 8.1, which further restricts competitors, and magnesium and ferrous sulfate supply essential cations. Glycerol, added by the user, is the carbon and energy source that drives aerial mycelium formation and sporulation at the agar surface.
Preparation
- Suspend 21.7 g of powder in 1 litre of purified or distilled water.
- Add 5 mL of glycerol.
- Heat to boiling with frequent mixing until the agar is completely dissolved.
- Sterilise by autoclaving at 121 °C for 15 minutes.
- Cool to 45–50 °C in a water bath.
- Add filter-sterilised cycloheximide (50 mg/L) and, if required, nystatin (50 mg/L), and mix gently.
- Pour 20–25 mL into each 90 mm Petri dish and allow to set on a level surface.
Liquid medium: AC Broth (AS-1103) uses the same base without agar, at 6.7 g/L plus glycerol.
Directions for Use
Inoculate the agar surface with 0.1 mL of a diluted soil or water suspension and spread with a sterile spreader. Allow the surface to dry before inverting. Incubate aerobically and examine periodically for small, dry, powdery colonies, which typically become visible after 5 to 7 days and continue to develop aerial mycelium over the following two weeks.
| Parameter | Condition |
|---|---|
| Atmosphere | Aerobic |
| Temperature | 25–30 °C |
| Time | 7–21 days |
| Positive result | Small, dry, powdery colonies, often with aerial mycelium |
Colony morphology on this medium is suggestive but not confirmatory. Identify isolates by microscopy, biochemical characterisation and 16S rRNA gene sequencing as appropriate.
Limitations
- Sodium propionate does not inhibit all competitors, and propionate-tolerant bacteria and fungi will grow.
- The long incubation leaves plates vulnerable to drying and to overgrowth by a single fast isolate; pour full plates and read them more than once.
- Cycloheximide and nystatin are heat-labile; adding them above 50 °C reduces their activity.
- Counts underestimate the actinomycete population in a sample, because the selective agents also restrict some target organisms.
- Glycerol must be added by the user. Preparing the powder alone gives a medium with no usable carbon source.
- Growth rate and morphology vary widely between genera, so a single reading time will not suit every isolate.
Literature and References
- Atlas RM. Handbook of Microbiological Media, 4th ed. CRC Press; 2010.
- Difco & BBL Manual: Manual of Microbiological Culture Media, 2nd ed. BD Diagnostics; 2009.
- Williams ST, Davies FL. Use of antibiotics for selective isolation and enumeration of actinomycetes in soil. J Gen Microbiol. 1965;38(2):251–261.
- Goodfellow M, Williams ST. Ecology of actinomycetes. Annu Rev Microbiol. 1983;37:189–216.
- Hayakawa M, Nonomura H. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J Ferment Technol. 1987;65(5):501–509.
- Shirling EB, Gottlieb D. Methods for characterization of Streptomyces species. Int J Syst Bacteriol. 1966;16(3):313–340.
Hazard Identification
Not classified as hazardous according to the GHS (Rev. 7) as adopted in Australia under the model WHS Regulations. Not Dangerous Goods under the ADG Code.
Fine powder can form combustible dust concentrations in air. Dust may cause mild mechanical irritation of the eyes and respiratory tract. Contains casein-derived material: people with milk protein allergy should avoid exposure.
Supplements added by the user are hazardous and are not covered by this section. Cycloheximide is acutely toxic and a reproductive toxicant. Nystatin is a sensitiser. Consult the supplier's SDS for each supplement and handle it according to that document before adding it to this medium.
Composition
| Ingredient | CAS No. | % w/w |
|---|---|---|
| Agar | 9002-18-0 | 69.12 |
| Sodium propionate | 137-40-6 | 18.43 |
| Sodium caseinate | 9005-46-3 | 9.22 |
| Dipotassium hydrogen phosphate | 7758-11-4 | 2.30 |
| L-Asparagine | 70-47-3 | 0.46 |
| Magnesium sulfate heptahydrate | 10034-99-8 | 0.46 |
| Ferrous sulfate heptahydrate | 7782-63-0 | 0.01 |
Percentages are nominal and calculated from the formulation.
First Aid
| Route | Action |
|---|---|
| Inhalation | Move to fresh air. Seek medical advice if symptoms persist. |
| Skin | Wash with soap and water. |
| Eyes | Rinse with water for several minutes. Seek medical advice if irritation persists. |
| Ingestion | Rinse mouth. Do not induce vomiting. Seek medical advice if unwell. |
Poisons Information Centre (Australia, 24 h): 13 11 26
Handling, Spills and Disposal
- Avoid generating and breathing dust. Weigh in a ventilated area.
- Spills: sweep up or HEPA-vacuum without raising dust; wash the area with water.
- Unused product: dispose of as non-hazardous waste under local regulations.
- Inoculated medium: treat as potentially biohazardous and decontaminate (for example by autoclaving) before disposal.
- Medium containing cycloheximide or nystatin: dispose of as chemical waste under local regulations, not as general waste.
Quality Control Specification
Each batch is tested for growth performance. Prepared with glycerol, incubated aerobically at 28 ± 2 °C and read at 7 days.
| QC organism | Strain | Inoculum (CFU) | Expected result |
|---|---|---|---|
| Streptomyces griseus | ATCC 10137 | ≤ 100 | Good growth; typical colonies with aerial mycelium |
| Escherichia coli | ATCC 25922 | ≤ 100 | Partial to complete inhibition |
Sterility of the prepared medium: no growth after 14 days at 30–35 °C.
Physical and Chemical Tests
| Test | Specification |
|---|---|
| Appearance, powder | Cream to light beige, homogeneous, free-flowing |
| Appearance, prepared medium | Pale straw, clear to slightly opalescent firm gel |
| Gel strength | Firm, comparable with a 1.5% agar gel |
| pH, prepared medium (25 °C) | 7.90–8.30 |
| Loss on drying | ≤ 6.0% w/w |
| Sterility, prepared medium | No growth at 14 days, 30–35 °C |
The information on this page is provided in good faith as a guide to the properties and use of this product and does not constitute a warranty of fitness for a particular purpose. Users are responsible for confirming suitability for their application. Ausamics Pty Ltd · ABN 56 676 640 467 · 36/25 Trafalgar Road, Epping VIC 3076, Australia · support@ausamics.com