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M17 AGAR

Code: CM0785

For improved growth of lactic streptococci and their bacteriophages and selective enumeration of Streptococcus thermophilus from yogurt.

Directions
Suspend 48.25g in 950ml of distilled water and bring gently to the boil. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and add 50ml of sterile lactose solution (10% w/v).

Lactose solution 10% (w/v)
Dissolve 10g of Lactose Code LP0070 in 100ml of distilled water. Sterilise by autoclaving at 121°C for 15 minutes or by membrane filtration through a 0.2µm membrane.

Description
M17 Agar is based on the formulation described by Terzaghi and Sandine¹ and is recommended as an improved medium for the growth and enumeration of lactic streptococci and their bacteriophages.

Because it supports better host growth it allows the demonstration of phenomena commonly associated with other bacterial virus systems but not previously reported for lactic streptococcal phages and makes possible detailed studies of plaque morphology and lysogeny.

Lactic streptococci are nutritionally fastidious and require complex media for optimal growth^2,3. Their homofermentative acid-producing nature requires that the medium is well buffered so that the culture pH is maintained above 5.7 during active growth. This maintenance of the pH is important as lower pH can result in injury and reduced recovery of lactic streptococci.

M17 Agar contains di-sodium-glycerophosphate which has sufficient buffering capacity to maintain the pH above 5.7 of actively growing cultures even after 24 hours at 30°C. This buffering agent also allows the addition of calcium without a precipitation complex being formed. The calcium-containing medium is used for the assay of bacteriophages of lactic streptococci¹.

Shankar and Davies⁴ reported that M17 Agar was suitable for the isolation and enumeration of Streptococcus thermophilus from yogurt as the high concentration of di-sodium-glycerophosphate resulted in suppression of Lactobacillus bulgaricus. M17 Agar has been recommended^5,6 by the International Dairy Federation for the selective enumeration of Streptococcus thermophilus from yogurt.

M17 Agar is also suitable for growing and maintaining starter cultures for cheese and yogurt manufacture as it has little deleterious effect on their subsequent acid-producing ability in milk at either 30°C or 22°C ¹.

One further useful property of this agar is its ability to detect streptococcal mutants which are unable to ferment lactose¹. These mutant Lac- strains form much smaller colonies than the parent lactose fermenting strain.

Technique
Bacteriophage assay
Microbiologists wishing to assay phage activity should consult the paper of Terzaghi and Sandine¹ for a comprehensive description of the method.

For the enumeration of Streptococcus thermophilus in yogurt
1. Mix or blend the yogurt sample to obtain a uniform homogenicity.
2. Weigh 10 ± 0.1g of the test sample into a 200ml round bottom centrifuge tube made of strengthened glass, or the container of the mechanical mixer.
3. Add sterile 0.1% (w/v) peptone solution ‡ to the test sample until the mass of the test sample and diluent is 50g.
4. Prepare a suitable series of decimal dilutions of the yogurt suspension in 9ml volumes of sterile 0.1% (w/v) peptone solution.
5. (i) Inoculate duplicate plates of M17 Agar with a loopful from each dilution and spread to obtain single colonies.
(ii) Add duplicate 1ml aliquots of the dilution into a Petri dish and prepare pour plates with 14ml of sterile M17 Agar cooled to 43°C ± 1°C.
6. Incubate at 35°C for 48 hours.
7. Examine the plates after 24 and 48 hours incubation. Streptococcus thermophilus colonies are visible after 18-24 hours and after 48 hours incubation form colonies of 1-2mm in diameter. Lactobacillus bulgaricus do not grow or produce very restricted colonies.
8. Carry out the counts on pour plates and express the results as the number of colony forming units per gram of sample.

0.1% peptone water⁶ can be prepared as follows: ‡

Sterilise by autoclaving at 121°C for 15 minutes.

Confirmation
Colonies isolated from milk products that are suspected to be Streptococcus thermophilus can be confirmed by the Gram stain (Gram positive cocci) and catalase test (negative).

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

* This organism is available as a Culti-Loop®

References
1. Terzaghi B. E. and Sandine W. E. (1975) Applied Microbiology 29. 807-813.
2. Anderson A. W. and Elliker P. R. (1953) J. Dairy Science 36. 161-167.
3. Reiter B. and Oram J. D. (1962) J. Dairy Res. 29. 63-77.
4. Shankar P. A. and Davies F. L. (1977) J. Soc. Dairy Technology 30. 28-30.
5. International Dairy Federation (1981) Joint IDF/ISO/AOAC Group E44.
6. International Organization for Standardization (1985) ISO/DIS 7889.