Ampouled Media

m-Green Yeast and Fungi Broth, 2 mL

(6505)

Intended Use
Ampouled m-Green Yeast and Fungi Broth, 2 mL is used for the detection of yeast and fungi in beverages
by the membrane filtration method in a laboratory setting. Ampouled m-Green Yeast and Fungi Broth, 2 mL is
not intended for use in the diagnosis of disease or other conditions in humans.

Product Summary and Explanation

Ampouled m-Green Yeast and Fungi Broth, 2 mL is a prepared, ready to use medium for membrane filtration
testing. m-Green Yeast and Mold Broth is an improved modification of the formula, m-Yeast and Mold Broth.
The addition of Bromocresol Green aids in identification as it is absorbed into fungal colonies. m-Green Yeast
and Fungi Broth is a relatively complex formula compared to other media used for the isolation of fungi and
yeast. It is also rich in nutrients, providing an environment for excellent fungal growth.
1
Fungi have been found in potable water and on the inner surface of distribution system pipes. They can
1
survive water treatment or they enter the system after treatment and remain viable.

Principles of the Procedure

Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue provide nitrogen, carbon, and amino
acids in m-Green Yeast and Fungi Broth. Yeast Extract is the vitamin source. Dextrose is an energy source
for metabolism of fungi. Potassium Phosphate is a buffering agent. Magnesium Sulfate, Thiamine, and
Diastase (a mixture containing amylolytic (starch) enzymes) provide essential ions, minerals, and nutrients.
Metabolic by-products from fungal growth diffuse into the surrounding medium, lowering the pH which inhibits
bacterial growth, and producing an acid reaction that causes residual Bromocresol Green to change to
yellow, which is an acid reaction. The colonies are green due to diffusion of Bromcresol Green into the
colonies.

Medium Composition: Per Liter

Enzymatic Digest of Casein ...................................................... 5 g
Enzymatic Digest of Animal Tissue........................................... 5 g
Yeast Extract............................................................................. 9 g
Dextrose.................................................................................. 50 g
Magnesium Sulfate ................................................................ 2.1 g
Potassium Phosphate ............................................................... 2 g
Diastase ............................................................................... 0.05 g
Thiamine .............................................................................. 0.05 g
Bromcresol Green .............................................................. 0.026 g
Final pH: 4.6 ± 0.2 at 25°C
Formula may be adjusted and/or supplemented as required to meet performance specifications.

Physical Characteristics
Appearance of medium: Clear, dark green.
pH at 25ºC: 4.6 ± 0.2

Test Procedure
Preparation
1. Assemble the manifold or filtration flask that will supply the vacuum source, complete with rubber stopper.
2. Using a gentle twisting motion, secure the funnel adapter into the rubber stopper.
3. Using the same gentle twisting motion, secure the Neogen Filter onto the funnel adapter.

Revision: 4 Effective Date: 5/30/2017

Ampouled Media
Filtration Procedure
1. Remove filtration cover and carefully pour the sample onto the filter.
2. Apply vacuum just long enough to pull the sample through the filter (if using a manifold, open only one
valve at a time.)
3. Rinse the inside walls of the filter funnel with approximately 20 mL of sterile buffered solution. Apply
vacuum just long enough to pull the solution through the filter, and turn off vacuum. Note: This step is
optional if only water is being tested.
4. Briefly remove the filter and its funnel adapter from the rubber stopper to release any remaining
vacuum pressure, and then re-secure into the stopper.
5. Add m-Green Yeast and Mold Broth onto the top of the filter. When doing so, be careful not to touch
the filter with the tip of the ampoule.
6. Very briefly apply vacuum so that the media does not pool on top of the filter, and is visible underneath
the filter. (Note: The media has been soaked correctly into the filter if there is a small pocket of air
around the bottom port. The filter should be moist, but not oversaturated the dry.)
7. Remove and appropriately discard the plastic funnel. Place the filtration system cover over the
filter/base assembly converting the unit to a Petri dish for sample incubation.
8. Remove the filter from the funnel adapter, and place a plug on the open bottom port.
9. Place the Neogen filter into the incubator inverted so that the cover is on the bottom, and incubate at
25 - 30ºC. Read and record results after 2 and up to 7 days (see note 3 under Limitations of the
Procedure).
10. Dispose of test materials in accordance with all applicable local, state, and federal regulations.

Expected Cultural Response:

Sterile water was added to sterile filtration units and inoculated with the cultures listed below. The inoculum
was filtered followed by the ampoulized m-Green Yeast and Fungi Broth and the filtration housing removed.
Plates were incubated aerobically at 25 - 30ºC and examined for growth at 2 and up to 7 days (see note 3
under Limitations of the Procedure).

Approx. Inoculum Expected Results

Microorganism
(CFU)
Uninoculated Media NA No Growth
®
Aspergillus niger ATCC 16404 50 - 300 ≥ 85% recovery
®
Candida albicans ATCC 10231 50 - 300 ≥ 85% recovery
®
Penicillium roquefortii ATCC 10110 50 - 300 ≥ 85% recovery
®
Saccharomyces cerevisiae ATCC 9763 50 - 300 ≥ 85% recovery
®
Trichophyton mentagrophytes ATCC 9533 50 - 300 ≥ 85% recovery

Results
All colonies growing on the surface of the membrane should be counted. Mold colonies generally appear
white with a green tint and are filamentous. Yeast colonies are cream colored and opaque.

Storage
Store Ampouled m-Green and Yeast Mold Broth, 2 mL at 2 - 8 ºC.

Expiration
Refer to expiration date printed on the front of the box container.

Limitations of the Procedure

1. Analyze sample as soon as possible after collection.
2. Samples containing colloidal or suspended particulate material can clog the membrane filter, thereby
prevent filtration, or cause spreading of microbial colonies which could interfere with colony
identification.

Revision: 4 Effective Date: 5/30/2017

Ampouled Media

3. To establish that no growth is recovered, filters can be held up to 7 days or as established by internal
validation of the procedure. To establish that a test result is complete and the results can be recorded
for a positive recovery, internally validate the optimum time frame for holding the filters by testing the
recommended quality control organisms listed under Expected Cultural Response. Species growth
rates vary, so the optimum time frame may vary as well.
4. Some aciduric or acid-adapted bacterial species may occasionally be recovered on this medium.

Packaging
m-Green Yeast and Fungi Broth, 2 mL Code No. 6505 Box of 50
Neogen Filter “White” Code No. 6550 Box of 50
Neogen Filter “Black” Code No. 6555 Box of 50

References
1. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (eds.). 1998. Standard methods for the examination of water and wastewater,
20th ed. American Public Health Association, Washington, D.C.

Technical Information
Contact Neogen Corporation for Technical Service or questions involving Ampouled Media at (517)372-9200 or (800)-234-5333 or fax
us at (517)372-2006.

Revision: 4 Effective Date: 5/30/2017