The Australian Wine Research Institute

Isolation and enumeration of viable yeast and/or bacteria in wine

A procedure is described for the isolation and enumeration of viable microorganisms from wine and other similar beverages. A good knowledge of aseptic technique is required for the procedure. The optimum volume of sample to be used will depend upon the expected count of viable microorganisms, and to a lesser extent on the amount of undissolved solids in the sample. If the expected count is high, suitable dilution or adjustment of the volume of sample used should be made. Where the expected count is uncertain, it is suggested that two determinations be made using two different volumes. In this way, the probability will be increased that at least one count will be within the range 10-200. The optimum number of colonies on the filter is about 50.

Reagents

1. Yeast counts: prepare WL nutrient agar plates.
2. Bacterial counts: prepare MRS/A/C plates (de Mann, Rogosa and Sharpe (MRS) nutrient agar supplemented with apple juice and cycloheximide solution)
3. 70 % v/v ethanol

Apparatus

1. Laminar flow cabinet
2. Gelman 47 mm magnetic filter funnels, 300 mL capacity
3. Buchner flask, 1 L, to fit filter funnels
4. Pre-sterilised 47 mm diameter, 0.45 mm pore-size, gridded membranes
5. Forceps, stainless steel
6. Corkscrew, ‘waiter’s friend’ type, with plastic casing removed
7. Vacuum pump
8. Incubator
9. Stereo- and phase-contrast microscope, slides, cover slips, inoculating loops etc.

Procedure

1. Preparation
   1. Place filter funnels in sterilisation bags (eg. No. 13 bags or similar) and autoclave at 121°C for at least 15 minutes.
   2. Prepare laminar flow cabinet for use by initially exposing to ultra-violet light for about 30 minutes, and then swabbing all surfaces with 70% ethanol. Ensure hands are clean – rinse with 70% ethanol to reduce the risk of contamination.
2. Filtration
   1. Remove the capsule from the wine sample if present, then spray the cork with 70% ethanol. If significant mould is present, wipe off with a tissue dampened with 70% ethanol. After about 1 minute, shake off excess ethanol and flame cork surface with Bunsen burner, in order to kill any microorganisms on the exterior of the bottle which may cause contamination.
   2. Rinse the corkscrew in 70% ethanol, shake off excess ethanol, and then flame the helical portion briefly, before insertion and removal of the cork. The ‘foot’ of the corkscrew should also be flamed, to avoid contamination as it comes into contact with the bottle neck.
   3. Remove the autoclaved filter funnel from the sterilisation bag and assemble in the Buchner flask. Avoid touching the interior of the filter funnel assembly if possible. Connect the flask to the source of vacuum.
   4. Dip forceps into 70% ethanol in a beaker, then flame before centering a sterile membrane in the membrane support area of the filter funnel, with the grid-marked side upwards.
   5. Flame the neck of the bottle, then pour a known volume of sample into the filter funnel. Gently apply vacuum in order to draw sample through the membrane.Options: Although it is often convenient to use the graduations on the filter funnel to determine the volume of sample analysed, more accurate measurements may be made using a sterilised measuring cylinder, glass pipette, or for smaller volumes, autopipettor with sterilised tip.For very accurate work, the sides of the funnel may be rinsed with 20-30 mL of sterile phosphate buffer solution added from the graduated cylinder used to measure the volume of the sample.
   6. Immediately filtration has ceased, disconnect the vacuum and remove the membrane using sterile forceps. Prolonged exposure to vacuum may result in excessive drying of the membrane. Roll the membrane grid-marked side upwards on to the surface of the MRS/A/C agar, taking care to avoid entrapping air bubbles between the membrane and the surface of the agar. Replace the lid on the Petri dish and store upside down.
   7. Record the following information on the underside of the petri dish: sample reference number; type of medium (colour changes can result in the medium following growth, which may make it difficult to identify the type of medium used); the volume of sample filtered through the membrane; and the date.
   8. Place a second sterile membrane in the support area of the filter funnel, and filter a second aliquot of sample. After filtration has ceased, remove membrane with sterile forceps and place on the surface of WL agar. Record relevant information on underside of petri dish.
   9. Repeat procedure for other samples, using a new filter funnel assembly for each sample.Option:
      In some cases, where a yeast count only is required, incubation on MRS/A/C agar can be omitted.
3. Incubation
   1. Place the labelled petri dishes in ‘snap-lock’ type plastic sandwich bags and then place these in the incubator, with the petri dishes stored upside down so as to minimise the risk of aerial contamination. Incubate at 27°C ± 1°C.
4. Examination and enumeration
   1. Membranes are generally examined for growth after 3 and 8-10 days’ incubation, although this will depend on the rate of growth of any microorganisms isolated. A visual examination only is generally performed after 3 days, with microscopic examination of colonies after 8-10 days.
   2. Visual examination should record the following information:
   • the number of each of the different colony types present (a tally counter may be used to facilitate counting);
   • the size of the colony (diameter in mm can be quoted)
   • the form/shape, ie. round, irregular, rhizoid, filamentous etc.;
   • the elevation, ie. convex, flat, umbonate, raised, effuse (thin, spreading), etc.;
   • the texture, ie. shiny, smooth, rugose (wrinkled), etc.;
   • the colour of the colony; and
   • any abnormal aroma emanating from the colonies, ie. acetic, hydrogen sulfide, cheesy, etc.
   • any discolouration of the green-blue colour of WL agar (due to the presence of bromocresol green indicator), which is indicative of acid production

   A stereomicroscope can be used to assist in the examination of colonies if required.

   3. The following information should be recorded during microscopic examination:
   1. Yeast:
   • the cell morphology, ie. spherical, ovoid, lenticular (lens-shaped), elongated, apiculate;
   • the size of the cells (the ocular micrometer can be used to estimate the dimensions of typical cells – see Appendix 1);
   • nature of budding, ie. polar, bipolar, lateral, multilateral, etc., or fission (e.g. in Shizosaccharomyces sp.); and
   • any other characteristics, eg. chain formation (pseudomycelium), presence of ascospores, etc.;
   2. Bacteria:
   • the cell morphology, i.e. rod-shaped, coccoid, diplococcoid, etc.;
   • the size of the cells;
   • the presence of pairs, chains (indicate approximate chain length, i.e. short, long, or indicate the approximate number of cells in each chain); and
   • any other characteristics, eg. the presence of spores, flagella/motility, encapsulation of chains, etc.
   3. If the count is greater than about 200, the test should be repeated if an accurate count is required, using either a smaller sample volume or a dilution designed to produce a count in the range of 10-200. Counts significantly in excess of this shall be recorded as ‘TNTC’ (too numerous to count).
   4. From the actual count calculate the number of organisms per mL of the sample, making due allowance for any dilution factors involved. The count should be expressed as ‘cfu/mL’ (colony-forming units per mL).
   5. Where two tests have been done on the one sample involving two different volumes or dilutions, and each membrane has a count within the range 10-200, calculate the two counts separately and report the mean of the two as the result.
   4. Microorganisms commonly isolated from wine
   1. It is important to stress that it is very difficult to identify microorganisms based on their colony and cell morphology only. Further physiological tests should be carried out to obtain identification to genus or species level. The following should only be used as a guide to the genera which are commonly present in wine.
   2. Yeasts found in wine include Saccharomyces sp., the yeast most commonly associated with alcoholic fermentation in wine, which are typically spherical to ovoid, and do not grow on a medium containing cycloheximide. Other genera, including Dekkera/Brettanomyces, Kloeckera, Saccharomycodes, Zygosaccharomyces, Candida and numerous others may also be found under certain conditions.
   3. Lactic acid bacteria of the following genera are commonly found in wine:
   • Lactobacillus sp. are typically rod-shaped cells;
   • Pediococcus sp. are coccoid, and may be present as tetrads; and
   • Oenococcus oeni, the organism most commonly associated with malolactic fermentation, are small, rod-shaped to diplococcoid cells.
   4. Acetic acid bacteria, including Acetobacter and Gluconobacter spp., are responsible for aerobic spoilage of wine, and are associated with the production of acetic acid (ie. volatile acidity) and ethyl acetate. These are typically rod-shaped cells, and may be tentatively differentiated from lactic acid bacteria by the fact that they exhibit catalase activity (see Appendix 2).
   5. Bacillus sp. are occasionally isolated from wines, but due to their relatively poor growth in wine their presence is generally interpreted as indicating that contamination has occurred, either from inadequately sterilised media and equipment, or from such locations as the cork or the neck of the bottle. The ability of this organism to sporulate means that it is able to survive in relatively hostile environments. It is characterised by large rod-shaped cells, often in chains, and is occasionally mistaken for yeast due to its relatively large size. Colonies are frequently observed to spread rapidly across the membrane surface, making enumeration difficult.
   6. Various moulds are occasionally observed to grow on agar, but they are inhibited by even low concentrations of ethanol, and their presence usually indicates contamination. Moulds are easily recognised by their fibrous mycelial growth.
   7. Any suspect microorganisms requiring further identification should be purified by streaking out onto a fresh agar plate, and then subjected to further diagnostic tests.

Reference:
Zoecklein, B.W.; Fugelsang, K.C.; Gump, B.H.; Nury, F.S. Production wine analysis. New York: Van Nostrand Reinhold; 1990: 270-274.

Appendix 1:
However, microscopic examination does not usually give sufficient information to identify the microorganisms responsible for an instability.

Appendix 2:
For example, most yeast found in wine are ovoid to spherical in shape, so examination of them under the microscope does not enable species identification.