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Dehydrated Culture Medium
Selective enrichment broth for recovering slow-growing actinomycetes from soil, compost and other heavily contaminated environmental samples
Cat. No. AS-11036.7 g/LpH 8.1 ± 0.2Selective brothEnvironmental microbiology
At a glance
- Target organismsActinomycetes
- Incubation25–30 °C, aerobic
- Time5–14 days
- Sterilisation121 °C, 15 min
Manufactured in Melbourne. TDS and SDS available. Batch COA on request.
Overview
AC Broth (Actinomycete Isolation Broth) is a selective liquid medium for the enrichment and cultivation of actinomycetes, including Streptomyces, Nocardia, Actinomadura and Micromonospora. It is the liquid form of Actinomycete Isolation Agar, using the same nutrient base without the agar.
The medium is built for samples where the target organisms are outnumbered. Soil, compost, sediment and plant material carry large populations of fast-growing bacteria and fungi that overgrow a plate long before a slow-growing actinomycete forms visible biomass. Sodium propionate, a restricted nitrogen supply and an alkaline pH together hold those competitors back, so the actinomycetes in the sample can multiply over an extended incubation.
AC Broth is used as a pre-enrichment step before plating on selective solid media, and in actinomycete ecology and natural-product screening programs.
Key features
- Alkaline pH 8.1 suits actinomycetes and suppresses many competing organisms
- Sodium propionate restricts Gram-negative bacteria and fast-growing fungi
- Low-nitrogen base favours slow-growing and rare actinobacterial genera
- Glycerol, added separately, is a preferred carbon source for Streptomyces
- Accepts cycloheximide or nystatin added after autoclaving for heavily mouldy samples
Principle of the Medium
| Component | Function |
|---|---|
| Sodium caseinate | Restricted peptide and nitrogen source; poorly used by many competitors |
| L-Asparagine | Additional nitrogen source favoured by actinomycetes |
| 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 |
| Glycerol (added separately) | Carbon and energy source |
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 broth has cooled to 45–50 °C.
Typical Composition
| Ingredient | g/L |
|---|---|
| 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 | 6.701 |
Final pH 8.1 ± 0.2 at 25 °C. Glycerol 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 and environmental microbiology | Enrichment of actinomycetes from soil, compost, sediment and plant material |
| Natural-product screening | Pre-cultivation of actinomycetes before screening culture extracts for bioactivity |
| Taxonomy and ecology | Recovery of rare genera such as Nocardia, Actinomadura and Micromonospora |
| Pre-enrichment | Liquid step ahead of plating on Actinomycete Isolation Agar or other selective agars |
Comparable Products
| Product | Relationship |
|---|---|
| Actinomycete Isolation Agar (AS-1102) | Solid form of the same published base, with 15 g/L agar added (21.7 g/L total) |
Frequently Asked Questions
How much powder do I need, and where does the glycerol go?
6.7 g of powder per litre of purified water, plus 5 mL of glycerol added separately before autoclaving. The glycerol is not part of the dehydrated blend, so it has to be measured in as a separate step.
Why is the pH alkaline rather than neutral?
Actinomycetes grow well around pH 8, while many competing soil bacteria and fungi prefer neutral to slightly acidic conditions. The alkaline pH is part of the selectivity, alongside the sodium propionate.
Do I need to add cycloheximide?
Not for every sample. The base formulation already 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.
How long should I incubate?
5 to 14 days with shaking at 100–150 rpm covers most work. Rare or slow-growing genera can need up to four weeks. Keep the flask loosely capped so it stays aerobic, and check for evaporation on long runs.
When should I use the broth rather than the agar?
Use the broth when the target organisms are too scarce to pick up by direct plating, and you want to increase their numbers first. Plate the enriched culture onto a selective agar afterwards to get single colonies.
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 a form that actinomycetes use readily and many competing organisms use poorly. Sodium propionate is the main selective agent, inhibiting a broad range of Gram-negative bacteria and fast-growing fungi. Dipotassium phosphate holds the medium at pH 8.1, which further restricts competitors, and magnesium and ferrous sulfate supply the cations needed for growth. Glycerol, added by the user, is the carbon and energy source and is particularly well used by Streptomyces.
Preparation
- Suspend 6.7 g of powder in 1 litre of purified or distilled water.
- Add 5 mL of glycerol and mix until the powder is fully dispersed.
- Check the pH and adjust to 8.1 with 1 N sodium hydroxide if required.
- 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.
- Dispense aseptically into sterile tubes or flasks, filling to no more than one third of the vessel volume so the culture stays aerobic.
Solid medium: Actinomycete Isolation Agar uses the same base with 15 g/L agar added, at 21.7 g/L plus glycerol.
Incubation and Interpretation
| Parameter | Condition |
|---|---|
| Atmosphere | Aerobic |
| Temperature | 25–30 °C |
| Agitation | 100–150 rpm |
| Time | 5–14 days; up to 4 weeks for slow-growing genera |
| Positive result | Turbidity, pellets or a surface pellicle; often an earthy odour |
Growth in the broth does not identify an organism. Plate the enriched culture on a selective agar to obtain single colonies, then confirm by colony morphology, microscopy and 16S rRNA gene sequencing as appropriate.
Limitations
- Enrichment changes the balance of the population, so the broth shows which organisms are present and does not give quantitative counts for the original sample.
- Fast-growing actinomycetes can outcompete rare genera during long incubation; plating at several time points gives better coverage of the diversity in a sample.
- Sodium propionate does not inhibit all competitors, and some propionate-tolerant bacteria and fungi will grow.
- Cycloheximide and nystatin are heat-labile; adding them above 50 °C reduces their activity.
- Long incubation in shaken flasks loses water by evaporation, which concentrates the medium and shifts the pH.
- Glycerol must be added by the user. Preparing the powder alone gives a medium with no usable carbon source.
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.
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 |
|---|---|---|
| Sodium propionate | 137-40-6 | 59.70 |
| Sodium caseinate | 9005-46-3 | 29.85 |
| Dipotassium hydrogen phosphate | 7758-11-4 | 7.46 |
| L-Asparagine | 70-47-3 | 1.49 |
| Magnesium sulfate heptahydrate | 10034-99-8 | 1.49 |
| Ferrous sulfate heptahydrate | 7782-63-0 | 0.01 |
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 with shaking at 28 ± 2 °C and read at 7 days.
| QC organism | Strain | Inoculum (CFU) | Expected result |
|---|---|---|---|
| Streptomyces griseus | ATCC 10137 | ≤ 100 | Good growth |
| 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 tan, homogeneous, free-flowing |
| Appearance, prepared medium | Clear to slightly opalescent, pale straw liquid |
| pH, prepared medium (25 °C) | 7.90–8.30 |
| Loss on drying | ≤ 6.0% w/w |
| Solubility | Complete in purified water |
| 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