Dehydrated Culture Medium
Low-pH enrichment broth for isolating and maintaining acid-tolerant lactic acid bacteria from fermented foods and beverages
Cat. No. AS-110435.25 g/LpH 4.9 ± 0.1Enrichment brothFood & fermentation
At a glance
- Target organismsLactic acid bacteria
- Incubation30–37 °C, microaerophilic
- Time24–72 h
- Sterilisation121 °C, 15 min
Manufactured in Melbourne. TDS and SDS available. Batch COA on request.
Overview
Acid Broth is a rich liquid medium held at pH 4.9 for the isolation, enrichment and maintenance of acid-tolerant lactic acid bacteria, including Lactobacillus, Pediococcus, Leuconostoc and Oenococcus.
The nutrient base carries no antibiotic or chemical inhibitor. Selectivity comes from the pH alone. At 4.9 the medium is below the growth range of most Gram-negative bacteria and spore-forming Bacillus species, while the lactic acid bacteria that dominate fermented foods grow well. The base is a rich peptone, yeast extract and glucose formulation with the growth factors these organisms need: Tween 80 as a fatty-acid source, acetate and citrate, and manganese as an enzyme cofactor.
This makes it a practical enrichment broth for fermented dairy, vegetable and beverage samples, and a convenient maintenance medium for starter and probiotic cultures.
Key features
- Selective by pH rather than by antibiotic, so no supplement is needed
- Rich base supports dense growth of lactic acid bacteria within 24–72 hours
- Tween 80, acetate, citrate and manganese supply the growth factors this group requires
- Suits fermented dairy, vegetable, wine and beer samples
- Also used to test the acid tolerance of bacterial isolates
Principle of the Medium
| Component | Function |
|---|---|
| Tryptone | Peptides, amino acids and nitrogen |
| Yeast extract | B vitamins, nucleotides and growth factors |
| Glucose | Fermentable carbohydrate and energy source |
| Polysorbate 80 (Tween 80) | Fatty-acid source; stimulates growth of lactobacilli |
| Dipotassium phosphate | Buffer and phosphate source |
| Sodium acetate | Buffer and growth factor for lactic acid bacteria |
| Ammonium citrate | Nitrogen source; restricts some competing organisms |
| Magnesium and manganese sulfate | Cations required by lactic acid bacteria enzymes |
The acidity is the selective element. Because the pH is set after sterilisation, the final figure depends on your technique, so check it on each batch of prepared broth before use.
Typical Composition
| Ingredient | g/L |
|---|---|
| Tryptone (pancreatic digest of casein) | 10.00 |
| Glucose | 10.00 |
| Yeast extract | 5.00 |
| Sodium acetate trihydrate | 5.00 |
| Dipotassium phosphate | 2.00 |
| Ammonium citrate | 2.00 |
| Polysorbate 80 | 1.00 |
| Magnesium sulfate heptahydrate | 0.20 |
| Manganese sulfate monohydrate | 0.05 |
| Total | 35.25 |
Final pH 4.9 ± 0.1 at 25 °C, adjusted aseptically after sterilisation. Formula may be adjusted and/or supplemented to meet performance specifications.
Applications
| Field | Typical use |
|---|---|
| Food and fermentation microbiology | Isolation of Lactobacillus and related genera from yoghurt, cheese, sauerkraut and sourdough |
| Oenology and brewing | Cultivation of Oenococcus oeni in malolactic fermentation studies; recovery of lactic acid bacteria from wine and beer |
| Starter and probiotic cultures | Maintenance of acid-tolerant starter strains and evaluation of culture stability under acidic conditions |
| Research and development | Enrichment of lactic acid bacteria from mixed populations; acid-tolerance testing of isolates |
Comparable Products
| Product | Relationship |
|---|---|
| MRS Broth | Comparable nutrient base and growth factors, prepared at pH 6.2 rather than pH 4.9; the standard neutral-pH medium for the same organisms |
Frequently Asked Questions
How much powder do I need?
35.25 g per litre. Suspend it in 950–980 mL of water first, then make up to 1 litre, so the volume is accurate once everything has dissolved.
Why is the pH adjusted after autoclaving rather than before?
Autoclaving glucose at low pH partly hydrolyses and caramelises it, which darkens the medium and reduces the sugar available for growth. Sterilising near neutral and acidifying afterwards avoids that. Both methods are described under Preparation.
Should I use lactic acid or hydrochloric acid?
Either sets the pH. Sterile lactic acid is the usual choice for lactic acid bacteria work, because it acidifies with the same acid the organisms themselves produce rather than introducing chloride.
How is this different from MRS Broth?
The nutrient base and growth factors are comparable. MRS is prepared near pH 6.2 and gains its selectivity from acetate and Tween 80; Acid Broth is prepared at pH 4.9 so the acidity itself does the selecting. Use Acid Broth when the sample is heavily contaminated with organisms that would grow at neutral pH.
Do I need an anaerobic jar?
Not usually. These organisms are microaerophilic, so a filled, tightly capped tube or bottle with little headspace is normally enough. A jar or anaerobic gas pack helps for strict strains and for longer incubation.
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
Tryptone and yeast extract supply peptides, amino acids, B vitamins and nucleotides, and glucose is the fermentable energy source. Polysorbate 80 provides oleic acid, which lactobacilli need for membrane synthesis, while acetate, citrate and manganese act as buffers and cofactors for this group. Setting the final pH at 4.9 puts the medium below the growth range of most Gram-negative bacteria, spore-forming Bacillus species, yeasts and moulds, so the aciduric lactic acid bacteria in the sample dominate the culture.
Preparation
- Suspend 35.25 g of powder in 950–980 mL of purified or distilled water.
- Mix until dissolved, then make the volume up to 1 litre.
- Dispense into suitable tubes or bottles, filling them nearly full to limit headspace.
- Sterilise by autoclaving at 121 °C for 15 minutes.
- Cool to approximately 50 °C.
- Aseptically adjust the pH to 4.9 using sterile lactic acid or hydrochloric acid.
- Mix gently and confirm the pH of a sacrificial tube before use.
Alternative method: Some laboratories prepare and autoclave the medium at its natural pH of about 6.8–7.0 and acidify to pH 4.9 aseptically after cooling. This avoids acid hydrolysis of the glucose during sterilisation and is the preferred route where the carbohydrate content matters.
Incubation and Interpretation
| Parameter | Condition |
|---|---|
| Atmosphere | Microaerophilic or anaerobic |
| Temperature | 30–37 °C |
| Time | 24–72 hours |
| Positive result | Turbidity, often with a sediment and a further fall in pH |
Growth does not identify an organism. Subculture onto a suitable agar and confirm isolates by Gram stain, catalase test, carbohydrate fermentation profile or 16S rRNA gene sequencing as appropriate.
Limitations
- Selectivity rests entirely on the pH, so an inaccurate final pH changes how the medium behaves. Check the pH of each prepared batch.
- Acid-tolerant yeasts and moulds grow at pH 4.9 and are not inhibited. Samples with a heavy yeast load may need a separate antifungal step or a different medium.
- Some lactic acid bacteria, including strains of Oenococcus oeni, start slowly at this pH and need incubation beyond 72 hours.
- Enrichment changes the balance of the population, so results are qualitative and not counts for the original sample.
- Autoclaving the medium already acidified will darken it and reduce the available glucose; use the alternative method where this matters.
- Prepared broth continues to acidify as the culture ferments, so pH readings taken during incubation reflect growth rather than the starting condition.
Literature and References
- Atlas RM. Handbook of Microbiological Media, 4th ed. CRC Press; 2010.
- De Man JC, Rogosa M, Sharpe ME. A medium for the cultivation of lactobacilli. J Appl Bacteriol. 1960;23(1):130–135.
- Salfinger Y, Tortorello ML, eds. Compendium of Methods for the Microbiological Examination of Foods, 5th ed. American Public Health Association; 2015.
- Hammes WP, Hertel C. The genera Lactobacillus and Carnobacterium. In: The Prokaryotes, 3rd ed. Springer; 2006:320–403.
- Lonvaud-Funel A. Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie van Leeuwenhoek. 1999;76(1–4):317–331.
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.
The acid used to adjust the pH is hazardous and is not covered by this section. Lactic acid and hydrochloric acid are corrosive. Consult the supplier's SDS for the acid you use, and handle it with appropriate eye protection and gloves.
Composition
| Ingredient | CAS No. | % w/w |
|---|---|---|
| Tryptone (enzymatic digest of casein) | 91079-40-2 | 28.37 |
| Glucose | 50-99-7 | 28.37 |
| Yeast extract | 8013-01-2 | 14.18 |
| Sodium acetate trihydrate | 6131-90-4 | 14.18 |
| Dipotassium hydrogen phosphate | 7758-11-4 | 5.67 |
| Diammonium hydrogen citrate | 3012-65-5 | 5.67 |
| Polysorbate 80 | 9005-65-6 | 2.84 |
| Magnesium sulfate heptahydrate | 10034-99-8 | 0.57 |
| Manganese sulfate monohydrate | 10034-96-5 | 0.14 |
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.
Quality Control Specification
Each batch is tested for growth performance at pH 4.9. Microaerophilic incubation at 35 ± 2 °C for 48 hours.
| QC organism | Strain | Inoculum (CFU) | Expected result |
|---|---|---|---|
| Lactiplantibacillus plantarum | ATCC 8014 | ≤ 100 | Good to luxuriant |
| Lacticaseibacillus casei | ATCC 393 | ≤ 100 | Good to luxuriant |
| Escherichia coli | ATCC 25922 | ≥ 1000 | None to poor (inhibited) |
Lactiplantibacillus plantarum and Lacticaseibacillus casei are the current names for the organisms previously called Lactobacillus plantarum and Lactobacillus casei. The ATCC numbers are unchanged.
Physical and Chemical Tests
| Test | Specification |
|---|---|
| Appearance, powder | Cream to light tan, homogeneous, free-flowing |
| Appearance, prepared medium | Light amber, clear to slightly opalescent liquid |
| pH, before adjustment (25 °C) | 6.60–7.00 |
| pH, final after adjustment (25 °C) | 4.80–5.00 |
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