Authors: Sriramulu Diraviam Dinesh
The artificial sputum medium (ASM) was formulated to mimic the sputum of cystic fibrosis (CF) patients. The intra- and inter-patient variation in the composition of highly viscous CF sputum complicates the achievement of a reproducible culture condition to study microbial biofilms, for example, Pseudomonas aeruginosa colonization in the CF lung. However, the ASM is a homogenous non-viscous medium, which can be prepared in bulk quantities using a fixed set of ingredients. The conventional biofilm models always had cells attached to a solid biotic or abiotic surface submerged in a steady-state or continuous flow of culture medium. In contrast, P. aeruginosa grows in the CF lung under micro-aerophilic to anaerobic conditions in the form of microcolonies eventually forming a macro-aggregate/colony in which bacteria adhere to each other and to sputum components. Similarly, in ASM, P. aeruginosa forms micro- and macro-colonies and showed various phenotypes specific to CF isolates.
Intact: intact biofilm in ASM, Disrupted: ASM-grown biofilm disrupted by repeated pipetting. No colouration in the crystal violet staining of the culture well shows that ASM-grown biofilm do not attach to the surface.
Ingredients for 1 litre of ASM:
- 5 g mucin from pig stomach mucosa (NBS Biologicals),
- 4 g low molecular-weight salmon sperm DNA (Fluka), 5.9 mg diethylene triamine pentaacetic acid (DTPA) (Sigma),
- 5 g NaCl (Sigma),
- 2.2 g KCl (Sigma),
- 1.81 g Tris base (Sigma),
- 5 ml egg yolk emulsion (Oxoid),
- 250 mg each of 20 amino acids (Sigma).
Note: 5 g/L casamino acids (Difco) can be used instead of 20 amino acids.
- magnetic stirrer
- pH meter
- Take 800 ml of distilled water in a beaker and place it on a magnetic stirring plate.
- Use a magnetic stirrer to dissolve the ingredients completely.
- Add mucin, DNA, DTPA, salts NaCl and KCl, and Tris base one after another with constant stirring.
- Add 250 mg of each amino acid except tryptophan.
- Stir the mixture continuously to dissolve amino acids.
- Insert a calibrated pH probe and check the pH of the medium continuously with constant stirring.
- Adjust the pH using Tris base until it stabilizes at 7.0.
- Make up the volume of the medium to 1000 ml.
- Sterilize the medium in an autoclave at 110oC for 15 minutes.
- Cool the sterilized medium and add filter-sterilized tryptophan (250 mg/L of medium) from the stock.
- Add 5 ml of egg yolk emulsion.
- Maintain sterile conditions for steps 10 and 11.
- Mix the medium well and store in a refrigerator for several months.
Note: For preliminary/routine experiments, casamino acids could also be used at 5 g/L of medium, instead of individual amino acids.
Stabilization of pH is an important factor in the preparation of ASM due to the presence of amino acids.
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Sriramulu Diraviam Dinesh, Division of Cell and Immune Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
Correspondence to: Sriramulu Diraviam Dinesh ([email protected])
Source: Protocol Exchange (2010) doi:10.1038/protex.2010.212. Originally published online 24 December 2010.