RESOLUTION OENO 8/95

MICROBIOLOGICAL ANALYSIS OF WINES AND MUSTS

THE GENERAL ASSEMBLY

IN VIEW of article 5, paragraph 4 of the International Convention for the Unification of Methods of Analysis and Appreciation of Wines of 13 October 1954,

ON THE PROPOSAL of the Sub-Commission on Unification of Methods of Analysis and Appreciation of Wines,

DECIDES:

TO SUPPLEMENT the International Oenological Codex with the monograph "Microbiological Analysis of Wines and Musts: Detection, Differentiation and Counting of Micro- organisms".

Objective:

Microbiological analysis is aimed at following alcoholic fermentation and/or malolactic fermentation and detecting microbiological infections, allowing the detection of any abnormality, not only in the finished product but also during the different phases of manufacture.

Comments :

All experiments must be carried out under normal microbiological aseptic conditions, using sterilized material, close to a bunsen burner flame or in a laminar flow room and in flaming the openings of pipettes, tubes, flasks, etc.

Before carrying out microbiological analysis, it is necessary to ensure that the samples to be analysed are taken correctly.

Field of application :

Microbiological analysis can be applied to wine, must, mistelles and all products even when they have been changed by bacterial activity.

Microbiological analysis techniques :

Microbiological analysis may be undertaken using the following techniques:

1. Quality tests

1.1. Air quality tests

1.2. Sterilizer tests

2. Detection, differentiation of micro-organisms and direct counting of yeasts

2.1. Microscopic examination of liquids or deposits

2.2. Staining with methylene blue

2.3. Gram staining

2.4. Catalase Test

2.5. Direct counting of yeasts by microscope

3. Counting of micro-organisms by culture

3.1. Culture in and on environment or solid medium

3.1.1. Plate culture

3.1.2. Membrane culture after filtration

3.2. Culture in liquid environment - "Most Probable Number" (MPN).

Equipment :

Laminar flow room Microscope

Incubator - for incubation between 20 and 35°C, with thermometer;

Incubator - for dry sterilisation, thermostatically controlled at 180°C, with thermometer; Incubator - in or in airtight jars with a generator, or other devices which produce an atmosphere rich in

pH-meter - calibrated in pH units with a precision of ± 0.1;

Autoclave - allowing steam sterilisation of equipment and culture media Centrifuge;

Water bath; Gas burner;

Balance - with a precision of 0.1 mg; Cell counter - Neubauer or equivalent;

Sterilising filtration devices - with sterilized filtering membranes of 13 mm or 25 mm in diameter, of 0.22 µm porosity, in cellulose ester or polyvinylidene fluoride or equivalent, with hydrophobic edges;

Bunsen burner;

Measured flask - 100 and 1000 ml;

Conical flask - 100, 150, 300, and 1000 ml stopped with cotton wool, sterilized; Test tubes - 50, 100, 300, 500 and 1000 ml;

Pipettes - 1, 10, 15, 20 and 25 ml sterilized; Centrifugal tubes - sterilized;

Autoclavable tubes - glass, rimless, dimensions 160 x 16 mm and 180 x 18 mm, stopped with cotton wool, or equivalent , before sterilisation;

Slides; Coverglasses;

Pasteur Pipettes - sterilized; Wire and loop - sterilized; Filter paper;

Immersion oil;

Alcohol - 70 and 95% volume.

Comment: Other pieces of equipment serving the same functions can be used. Techniques for the taking of samples :

It is necessary that samples be representative. Pipettes can be used for casks and long glass tubes for tanks. The samples must be taken in a sterilized area to avoid external contamination which could give false positives. Thus, to take samples from the "tasting taps" of casks, it is necessary first to allow several litres of sample flow. Surfaces in contact must be disinfected, for example with 70% alcohol or by gas burning.

Samples must be analysed as quickly as possible after they have been taken. They must be kept cool (4 to 5°C) during transportation and storage, if the microbial flora has to be stabilised.

1.      Quality tests

Objective

These tests are aimed at detecting the risk of microbial infection in advance. Principle:

This technique is based on organoleptic modifications (clouds, films, deposits, selected colours) shown by wine when subjected to certain aeration and temperature conditions which can bring about bacterial activity. The nature of the modification checked should be confirmed by microscopic examination.

Operating method:

1.1.      Air quality tests

A 50 ml wine sample after filtration on coarse sterile filter paper is placed in a 150 ml sterile conical flask stopped with cotton and left at an ambient temperature for at least 3 days. The clarity, colour and possible presence of clouds, deposits and films are examined in the course of time. A microscopic examination is carried out in the case of cloud, deposit or film or a colour attained.

1.2.      Incubator quality tests

A 100 ml wine sample after filtration on coarse sterilized filter paper is placed in a sterile conical flask stopped with cotton, put in a sterilizer at 30°C and examined after at least 72 hours. Organoleptic changes can be indicative of microbial development. A microscopic examination must therefore be made.

2.      Detection, differentiation of micro-organisms and direct counting of yeasts

2.1.      Microscopic examination of liquids or deposits

Objective:

Microscopic examination under cool conditions is aimed at detecting and differentiating the yeasts from the bacteria which might be present. However, microscopic observation cannot distinguish between viable and non-viable micro-organisms.

Comment:

With staining, an estimation of the viable yeasts can be made. Principle:

This technique is based on the magnification made by the microscope which allows the observation of micro-organisms, whose size is that of the micron.

Operation method:

Microscopic examination can be carried out directly on the liquid or on the deposit.

Direct observation of the liquid will only be useful when the population is sufficiently high (more than 5 x 105 cells/ml).

When wine shows a lower micro-organism population, it is necessary to concentrate the sample. Thus, about 10 ml of homogenized wine is centrifuged at 3000 - 5000 rpm for 5 to 15 minutes. After decanting the supernatant, the deposit is re- suspended in the liquid remaining at the bottom of the centrifuge tube.

To carry out the microscopic observation, a drop of the liquid sample or the homogenized deposit is placed on a clean glass slide with a Pasteur pipette or a sterilized wire. It is covered with a coverglass and placed on a slide on the stage of the microscope. Observation is made in a clear field, or preferably in phase contrast, which allows a better observation of detail. A magnification of x400 - 1000 is generally used.

2.2.      Vital Staining with methylene blue

Objective:

Vital staining with methylene blue is used to differentiate between viable and non-viable yeast cells.

Principle:

This coloration is based on the presence of viable cells which reduce the colouring. For example, methylene blue is reduced in leuco-derivatives by viable cells which remain colourless. Dead cells absorb the colouring, appearing blue.

Reagent:

Methylene blue solution of 0.1%

Preparation: Dissolve 0.1g of methylene blue with 100 ml of distilled water in a conical flask. Filter through paper. The solution must be freshly prepared.

Operating method:

Place a drop of sample and a drop of colouring on a slide.  Mix together with a wired after 5 minutes observe through a microscope in a clear field.

Results:

The viable cells remain colourless and the dead cells appear blue. Comments:

These colouring methods are not completely reliable and are only used for approximate enumerations. They are not recommended for bacteria.

To differentiate between viable and non-viable micro-organisms (both yeasts and bacteria) more elaborate techniques can also be employed, such as those using an epifluorescence microscope.

2.3.      Gram staining

Objective:

Gram staining is used to differentiate between lactic bacteria (Grams positive) and acetic bacteria (Grams negative) and also to observe their morphology.

Comments:

It must be remembered that Gram staining is not conclusive as other bacteria apart from lactic and acetic bacteria may be present.

Principle:

This colouring is based on the difference between Gram positive and Gram negative bacteria due to the differences in structure and chemical composition of their cellwalls. In Gram negative bacteria, the cell walls rich in lipids have a much reduced quantity of peptidoglucan which allows the penetration of alcohol which dissolves the gentian-violet-iodine complex, forming when the colourless cell is left, which will then be recoloured in red by saffron. Conversely, the cell walls of Gram positive bacteria contain a large quantity of peptidoglucan and a weak concentration of lipids. Thus, the thick peptiglucan wall and the dehydration caused by the alcohol do not allow the alcohol to penetrate the cell which maintains the violet or dark blue colouring of the gentian- violet-iodine complex.

There are several modifications to the Gram colouring technique. It loses its usefulness if it is performed on a culture which is too old. Thus, the strain must be in an exponential growth phase within 24 to 48 hours.

Solutions:

The water used must be distilled.

1. Gentian violet solution

 Preparation: Weigh 2g of gentian violet (or crystal violet), and put into a 100 ml conical flask and dissolve in 20 ml of 95% vol. alcohol. Dissolve 0.8g of ammonium oxalate in 80 ml of distilled water. Mix the two solutions together and only use after a period of 24 hours. Filter through paper at time of use. Keep out of light in a dark flask.

2. Lugol solution

Preparation: Dissolve 2g of potassium iodide in a minimal quantity of water (4 to 5 ml) and dissolve 1g of iodine in this saturated solution. Make the volume up to 300 ml with distilled water. Keep out of light in a dark flask.

3. Saffron solution:

Preparation: Weigh 0.5g of saffron in a 100 ml conical flask, dissolve with 10 ml of 95% vol. alcohol and add 90 ml of water. Stir. Keep out of light in a dark flask.

Operating method:

Smear preparation

Take a subculture of the bacteria in liquid or solid environment. Collect the young culture bacteria from the deposit (after centrifugation of the liquid culture) or directly from the solid environment with a loop or wire and mix in a drop of sterilized water.

Make a smear on a slide, spreading a drop of the microbial suspension. Let the smear dry, and then carry out fixation, rapidly passing the slide 3 times through the flame of a bunsen burner, or equivalent.

After cooling, perform staining.

Staining

Pour a few drops of gentian violet solution onto the fixed smear. Leave to react for 2 minutes and clean with water.

Pour in 1 to 2 drops of lugol solution. Leave to react for 30 seconds. Wash with water and dry on filter paper.

Pour in 95% vol. alcohol, leave for 15 seconds. Rinse with water and dry on filter paper.

Pour in a few drops of saffron solution, leave to react for 10 seconds. Wash with water and dry on filter paper.

Place a drop of immersion oil on the smear.

With the immersion objective, observe through a microscope in clear field. Results:

Lactic bacteria remain violet or dark blue coloured (Gram positive). Acetic bacteria are red coloured (Gram negative).

2.4.      Catalase Test.

Objective:

This test is aimed at making a distinction between acetic and lactic bacteria. The yeasts and acetic bacteria have a positive reaction. Lactic bacteria give a negative catalase.

Comments:

It must be taken into account that the catalase test is not conclusive as other bacteria apart from lactic and acetic bacteria may be present.

Principle:

The catalase test is based on the property that aerobic micro- organisms have of decomposing hydrogen peroxide with release of oxygen:

Reagent:

3 Volume hydrogen peroxide solution

Preparation: Measure 10 ml of 30 volume hydrogen peroxide in a 100 ml calibrated phial and fill with freshly boiled distilled water. Stir and keep in the refrigerator in a dark flask. The solution must be freshly prepared.

Operating method:

Place a drop of 3 volume hydrogen peroxide on a slide and on this place a small sample of young colony. If gas is released, it can be concluded that the culture contains catalase. It is sometimes difficult to observe gas clearing immediately, particularly with bacterial colonies. It is therefore advisable to examine the culture through a microscope (objective x10).

2.5.      Direct counting of the yeast cells with a microscope

Objective:

Direct counting with a microscope is aimed at evaluating the total yeast cell population (viable and non-viable)

Comment:

This technique is only recommended for the counting of yeasts. Enumeration of bacterial cells is both difficult and approximate due to their small size and, more importantly, because of the presence of colloidal particles. This technique can be used to estimate numbers of viable cells which are stained by methylene blue.

Principle:

The technique is based on the counting of micro-organisms in a sample of known volume through a microscope. Special slides of the haematimetre or cell counting type are used to measure this volume.

Comment:

The slides consist of a restricted square surface, which, covered by a thick, optically flat coverglass, contain a sample of known volume. There are various cell counters. For example, the Neubauer cell counter, considered here, consists of a central cavity 0.1 mm deep and 1 in surface area, of which

the base is divided into 400 small squares. Thus, the volume of a sample corresponding to a small square is 1/400 .

Operating method:

Place a drop of the homogenised sample on the central plane of the chamber, avoiding spillages. Cover with a thick coverglass, avoiding gas bubbles. Leave to stabilise for a few minutes.

The concentration must be neither too concentrated nor too dilute. Where very dense populations are concerned, the sample must be diluted. Conversely, where the number of cells to be counted is small, a concentration must be made by centrifugation.

Count the micro-organisms through a microscope at random so that the total is between 200 and

500. Avoid counting the same cell twice. To obtain a representative figure, count the cells in different areas. Thus, for example, five large squares can be counted (each consisting of 16 small squares), 1 in the centre and 4 on the edges of the square plane or 5 large squares on the diagonal.

It is advisable to perform the counting twice. Find the mathematical average of the two counts, record the results of the 400 small squares, and multiply by 104 to express the result in cells/ml. If the sample has been concentrated or diluted, the concentration or dilution factor must be taken into account.

3.      Counting of micro-organisms by culture

Objective:

The purpose of counting of micro-organisms by culture is to evaluate the level of contamination of the sample, that is to say, to estimate its microbial stability since only viable micro-organisms are capable of changing the product.

According to the culture media used and the culture conditions, three types of micro-organisms can be counted, namely, yeasts, lactic bacteria and acetic bacteria.

3.1.      Culture in solid medium

Principle:

This method is based on the pre-supposition that a viable micro-organism, cultivated in or on specific solid nutritive media and in suitable conditions, multiplies giving rise to a colony visible to the naked eye.

3.1.1.     Petri dish culture Objective:

This  technique  is  aimed  at  counting  micro-organisms  by  dilution  when  the  microbial concentration is high.

Equipment:

Apart from the equipment already mentioned, the following is required. Petri dishes (plates) - 57 (plastic) or 65 (borosilicate glass).

Diluants and solid culture media (see annexes 4 and 5) Operating method:

Enumeration in Petri dishes can be performed by inoculation in or on the surface of the appropriate medium after suitable incubation.

Preparation of dilutions:

Successive, decimal and increasing dilutions must be made in such a way that the appropriate number is obtained (between 30 and 300 colonies) for the counting and differentiation of colonies, as the microbial concentration is not known in advance.

An estimation of the dilutions can be made using a microscope. Thus, starting with an homogenized sample, prepare a series of decimal dilutions (1:10) in the diluant.

Take a 1 ml sample and add to 9 ml of diluant in the first tube. Homogenize. Take 1 ml of this dilution and add to 9 ml of diluant in the second tube. Continue this dilution protocol until the last suitable dilution, according to the presumed microbial population, using sterilized pipettes for each dilution (see diagram 1 of Annex 1).

Preparation of inoculations by incorporation in gel medium

Prepare Petri dishes in such a way that a dilution giving counts of between 30 and 300 colonies can be obtained.

Inoculate 1 ml of sample and 1 ml of each of the dilutions prepared and chosen according to the presumed microbial population, homogenized at the time, in two separate Petri dishes using different sterilized pipettes or automatic pipettes with autoclavable hydrophobic tips. Then pour 15 ml of appropriate gel culture medium, at 42 to 45°C, liquefied beforehand in a boiling water bath (or in microwave oven or on a heated magnetic gyratory shaker),avoiding prolonged heating. Homogenize immediately ,gently making circular movements in both directions, avoiding spillages and the formation of air bubbles. Leave to cool on a level surface until solidified.

Preparation of inoculations on the surface of the gel medium

Place 0.1 to 0.2 ml of chosen sample dilutions, so that there is no excessive liquid on the surface of the gel which would cause uneven spreading. Dilutions 10 times more concentrated than in the incorporation method must be used. A homogenic distribution is made on the surface of the medium, having been

placed and solidified in plates with a glass spreader sterilised with alcohol and flamed. Incubate the inverted plates in incubator:

• yeasts - 20 to 25°C, for 3 to 10 days, under aerobic conditions.

(Comment: for the counting of yeasts, 3 days incubation is usually sufficient. If the presence of Dekkera Brettanomyces is suspected, the incubation must be for 7 to 10 days)

• lactic bacteria - 25°C, 4 to 10 days, under anaerobic or microaerophilic conditions.
• acetic bacteria - 25 to 30°C for 2 to 4 days, under aerobic conditions.

Count the colonies with the naked eye or with a magnifying glass or a colony counter. If there is any doubt, confirm the identity of the colonies (yeast or bacteria) with a microscope.

Test the sterility of the medium, the diluant and the equipment by making a blank test with a sample of sterilized water for each series of tests.

Results:

Express the results in Colony Forming Units/ml - CFU/ml (rather than micro-organisms/ml, since each colony can be the result of a micro-organism or a cluster), in scientific notation with a decimal (for example 1100 would be reported as "1.1 x CFU/ml").

When the number of colonies is calculated by estimation, the results are expressed as ECFU/ml (E - estimated).

Count the plates containing between 30 and 300 colonies.

Calculate the CFU/ml by multiplying the mathematical average of the colonies affected by the dilution factor.

Specify incubation time.

Rules for calculating the CFU by counting and by estimation

1. Examine the dish of each of the dilutions containing between 30 and 300 colonies and calculate the mathematical average.
2. If only one of the two prepared dishes contains between 30 and 300 colonies, calculate the mathematical average of the two dishes corresponding to this dilution.
3. If two consecutive dilutions show dishes containing between 30 and 300 colonies, calculate the mathematical average, provided that the larger number is not more than double the smaller one. When the larger number is more than twice the smaller one, only use the smallest count (for example: 150 (dilution ) and 350 (dilution ); the ratio 350/150 is greater than 2. Thus, use the lower number, i.e. 150, and calculate the CFU/ml by multiplying by the dilution factor).

4.  If all the dishes have less than 30 colonies, consider only the dishes in which the dilution is the lowest (least diluted). Calculate the mathematical average and report the results as estimates.
5. If none of the dilution dishes contain colonies and there are no inhibiting substances present, consider the counts as "< 1.0" times the lowest dilution factor and report the results as estimates (for example, if there are no colonies in the dilution (lowest), the result will be reported as "< 1.0 x ECFU/ml").
6. If all the dishes contain more than 300 colonies, consider the count closest to 300 as an estimate. Thus, only consider dishes with the highest dilution, i.e. the most diluted, and report the results as estimated.
7. If the number of colonies significantly exceeds 300, report the result as "TNTC" (too numerous to count). If there are less than 10 colonies per , count in 13 squares (from the colony counter) having a representative distribution of colonies. Determine the mathematical average by square and multiply by the appropriate factor to calculate the estimated colonies per dish. Similarly, when there are more than 10 colonies/, count 4 representative squares, calculate the mathematical average per square and calculate as indicated previously. The factor is 57 for dishes with a surface area of 57 and 65 for dishes with a surface area of 65 .
8. When there is an overpopulation in the dishes (more than 100 colonies/) report the results as estimates. For the calculation, multiply 5700 or 6500 by the highest dilution factor. For example, for a dilution of , the ECFU will be reported as "> 5.7 x ECFU/ml" or "> 6.5 x ECFU/ml".

3.1.2.     Membrane filtration method Objective:

The purpose of this technique is to count the micro-organisms when the microbial population is low. A concentration of micro-organisms remaining on a membrane filter of suitable porosity is made after filtration of a sample of given volume.

Equipment:

In addition to equipment previously referred to, the following are required: Vacuum pump - or equivalent device;

Filtration unit (support-filter and funnel) - for membranes 47 mm diameter.  Can be made of stainless steel, borosilicate glass or autoclavable/sterile non-autoclavable plastic;

Filtration ramp or vacuum flask;

Trap - to be inserted between the flask and the vacuum pump; Tongs - for fixation;

Tongs with rounded sides - for membranes;

Pincers - to take hold of the filtration unit during sterilization (by flaming or by immersion in boiling water);

Stainless steel pot - for sterilization of the filtration unit in boiling water.

Filtering membranes - cellulose esters of 47 mm diameter with hydrophobic sides, preferably white and square with a porosity of 0.2 µm, 0.45 µm or 0.8 µm. Preferably, use membranes in proprietary individual sterile packages. Otherwise, sterilize them by autoclave for 10 mins at 121°C.

Petri dishes - made of borosilicate glass or 60 mm sterilized plastic; Sterilized absorbent stoppers.

Culture medium (see annex 5)

Operating method:

1. Preparation of plates

Prepare the necessary number of Petri dishes with the appropriate culture medium. The membrane filter can be placed on an suitable gel medium or on a solid stand - absorbent stopper soaked with suitable liquid in a nutritive medium.

1. Petri dishes with suitable gel media (see annex 5)

Deposit into 60 ml Petri dishes about 6 ml of solid medium, pre-liquefied in a water bath or similar, and allowed to cool to 45°C. Leave to solidify on a level surface. Invert the dishes and leave for at least 2 to 3 hours before use.

The prepared dishes can be used when they are free of contamination but not dry.

2. Petri dishes with suitable liquid medium(see annex 5)

If a liquid nutritive medium is preferred, deposit the membrane on an absorbent stopper soaked with the liquid medium in Petri dishes.

For the preparation of dishes with liquid medium, open the Petri dishes and place sterilized absorbent stoppers inside (there are sterilized dishes with sterilized absorbent stoppers commercially available).

Generally, phials of 2 ml of sterilized liquid nutritive media or equivalent are used. These are also available commercially.

Open a 2 ml phial of appropriate liquid medium and pour about 1.8 ml onto an absorbent stopper, spreading over the whole surface.

2. Filtration of the sample

Use autoclavable filtration equipment at the start of a series of filtrations. Between filtrations, for steel or borosilicate glass units, sterilise with alcohol and flame in a Bunsen burner flame or immerse in boiling water bath for 5 mins. For plastic equipment, alternative sterilization methods can be used, such as a UV. radiation with a two minute exposure or other appropriate chemical or physical agents.

Insert a trap between the vacuum flask and the vacuum pump. Attach the filtration unit onto the vacuum flask. Fix the funnel on the filter stand with appropriate tongs.

Introduce a few millilitres of sterile water to ensure a sound contact between the membrane and the filter support, and leave to drain completely.

Remove the membrane filter of suitable porosity from its sterile individual pack with flamed tongs which have been first allowed to cool.

Place on the base of the filter support the membrane filter, which must be centred with the square facing upwards.

Introduce an appropriate volume of homogenized sample, according to the presumed population. The figure considered for the colony count is between 20 and 200. However, for a good differentiation, the number of colonies should not exceed 60 - 80.

Make a vacuum. After filtration, break the vacuum carefully to avoid a backward surge. (The filtration volume varies, generally, from 25 to 500 ml. A sample of known volume must always be poured. If the volume of the sample is less than 20 ml, pour enough sterilized water into the funnel to cover the membrane and then, with a sterilized pipette, introduce the sample to be filtered.)

Remove the funnel and take hold of the membrane with the cooled flamed tongs and place on the culture medium in the Petri dish with the square facing upwards.

Avoid the formation of air bubbles between the membrane and the medium, since they would prevent homogenic contact and, consequently, a correct microbial growth.

Invert the dish and incubate in an incubator for a suitable time and in suitable conditions according to the type of micro-organisms to be detected.

For yeasts:

Use membrane filters of 0.45 µm or 0.8 µm porosity, YEPD gel medium (see annex 5) and incubate under aerobic conditions, between 20 and 25°C, for 3 to 10 days. (Comment: for the counting of yeasts, incubation for 3 days is usually sufficient. If the presence of Dekerra/Brettanomyces is suspected, incubation must be for 7 to 10 days)

For lactic bacteria:

Use membrane filters of 0.2 µm or 0.45 µm porosity, suitable gel culture medium (see annex 5) and incubate under anaerobic or microaerophilic conditions, at 25°C. The length of incubation can be 10 days.

For acetic bacteria:

Use membrane filters of 0.2 µm or 0.45 µm porosity, suitable gel culture medium(see annex 5) and incubate under aerobic conditions between 25 and 30°C for 2 to 4 days. Count the developed colonies with the naked eye, with a magnifying glass or in a colony counter. Confirm the identity of the colonies (yeasts or bacteria) with a microscope, if there are any doubts.

Test the sterility of the media, the membrane filters and the equipment by performing a blank with a sample of sterilized water for each series of tests.

Results:

Express the results in Colony Forming Units/ml - CFU/ml (instead of micro-organisms/ml, since each colony can be the result of a micro-organism or a cluster).

When a large volume of wine is filtered, and even so there are insufficient numbers of developed colonies, the results can be presented relative to the volume used.

If there is no colony development and there are no inhibiting substances present, report the results as "< 1.0 CFU" by the highest volume of wine filtered.

Count all the colonies on the membrane when there are 1 to 2 colonies per square. If too many colonies per square have been obtained, the analysis of the sample should be repeated if possible with a smaller quantity of sample.

If the number of colonies is too high (above 200), estimates can be made when the colonies are not agglomerated and the distribution is representative.

If this is not possible, report the results as "TNTC" (too numerous to count). Refer to the porosity of the membrane used and the incubation period.

3.2.      Culture in liquid medium- "Most Probable Number" (MPN)

Objective:

The purpose of this technique is to evaluate the number of viable micro-organisms in wines having high contents of solid particles in suspension and/or high incidence of plugging.

Principle

This technique is based on the estimation of the number of viable micro-organisms in liquid medium, starting from the principle of its normal distribution in the sample.

Diluants and liquid culture media (see annexes 4 and 5)

Operating method:

Several quantitative and successive solutions are prepared and following this, after incubation, a certain proportion of tests will not lead to any growth (negative tests), while others will begin to grow (positive tests).

If the sample and the dilutions are homogeneous, and if the number of dilutions is sufficiently high, it is possible to treat the results statistically, using suitable tables (tables based on McCrady's probability calculations), and to extrapolate this result to the initial sample.

Preparation of dilutions:

Starting from a sample of homogenized wine, prepare a series of decimal dilutions (1:10) in the diluant.

Take 1 ml of wine and add to 9 ml of diluant in the first tube. Homogenize. Take 1 ml of this dilution to add to 9 ml of diluant in the second tube. Continue this dilution protocol until the last suitable dilution, according to the presumed microbial population, using sterilized pipettes for each dilution. The dilutions must be made until extinction, i.e. the absence of development in the last dilutions (see the diagram to annex 2).

Preparation of inoculations:

Inoculate 1 ml of wine and 1 ml of each of the prepared dilutions, homogenized at the time, in, respectively, 3 tubes with the appropriate culture medium(see annex 5). Homogenize.

Incubate the inoculated tubes in the incubator at 25°C for yeasts (3 days, up to 10 days), under aerobic conditions, and for lactic bacteria, under anaerobic or microaerophilic conditions (8 days, up to 10 days), making periodic observations up to the last day of incubation.

Results:

All those tubes which show a microbial development leading to the formation of a whitish deposit, more or less evident and/or with a more or less marked disturbance, are considered as positive. The results must be confirmed by observation through a microscope. Specify the incubation period.

The reading of the tubes is made by noting the number of positive or negative tubes in each combination of three tubes (in each dilution). For example, "3-1-0" signifies: 3 positive tubes in the 100 dilution (wine), 1 in the dilution and zero in the dilution.

For a number of dilutions higher than 3, only 3 of these results are significant. To select the results adopting for the determination of the "MPN", it is necessary to determine the "typical number" according to the examples in the following table:

Calculation of the Most Probable Number (MPN)

Taking account of the typical number obtained, the MPN is determined through Table A (Annex 3) based on McCrady's probability calculations, considering the dilution made. If the dilution series is ; ; the reading is direct. If the dilution series is ; ; the reading is

0.1 times this value. If the dilution series is   ; ; ; the reading is 10 times this value.

Comment:

If there is a need to increase the sensitivity, a concentration of wine can be used. To obtain this concentration of micro-organisms in 1 ml, centrifuge 10 ml of wine and take 1 ml of deposit (after having taken 9 ml of excess liquid) and inoculate according to the previously described method.

Expression of Results:

The micro-organism content of wine must be expressed in cells per ml, in scientific notation to one decimal place. If the content is lower than 1.0 cells per ml, the result must be presented as "<1.0 cells per/ml".

Bibliography

Annex 1 - OENO 8/95

Annex 2 - OENO 8/95

Annex 3 - OENO 8/95

Annex 4 - OENO 8/95

Diluants:

Diluants are indicated by way of example. The water to be used must be distilled, double distilled or deionised, with no traces of metals, inhibitors or other antimicrobial substances.

1.      Physiological water

Preparation: Weigh 8.5g of sodium chloride in a 1000 ml calibrated flask. After it has dissolved in the water, adjust the reference volume. Homogenize. Filter. Distribute 9 ml in the test tubes. Stop with cotton wool and autoclave for 20 mins at 121°C.

2.      Ringer's solution 1/4

Preparation: Weigh 2.250g of sodium chloride, 0.105g of potassium chloride, 0.120g of calcium chloride (Ca) and 0,050g of sodium hydrogen carbonate in a 1000 ml calibrated flask. After it has dissolved in water, make up to the mark. Homogenize. Distribute 9 ml in the test tubes. Stop with cotton wool and autoclave for 15 mins at 121°C. (This solution is available commercially)

Annex 5 - OENO 8/95

Culture media

Culture media and antimicrobials are indicated by way of example.

The water to be used must be distilled, double distilled or deionised with no traces of metals, inhibitors or other antimicrobial substances.

1.      Solid culture media

1.1.      For yeasts

YEPD medium (Yeast Extract, Peptone, Dextrose), gel medium + chloramphenicol

Preparation: Weigh 10g of yeast extract (Difco or equivalent), 20g of peptone, 20g of glucose and 100 mg of chloramphenicol(1) in a 1000 ml Erlenmeyer. Dissolve in 450 ml of water.

Then separately dissolve 20g of gel medium with 500 ml of water, in a 1000 ml Erlenmeyer, in boiling water bath, stirring frequently, avoiding prolonged heating. After it has completely dissolved, add to the other solution and complete the 1000 ml volume with water and mix together. Distribute portions of 15 ml (for enumeration in plates) and 6 ml (for enumeration by membrane filtration) in the test tubes. Stop with cotton wool and autoclave for 15 mins at 121°C. Instead of chloramphenicol, penicillin, 20U/ml, and streptomycin, 40U/ml, can be added on the plate when the portions of media are poured onto the plates.

1.2.      For lactic bacteria

Medium of Lafon-Lafourcade et al , gel medium + actidione

Preparation: Weigh 5g of yeast extract, 10g of meat extract, 15g of trypsic peptone, 5g of sodium acetate, 2g of ammonium citrate, 0.05g of manganese sulphate, 0.2g of magnesium sulphate,

(1) To inhibit the growth of most of the bacteria sulphate, 20g of glucose and 50 mg of actidione(2) in a 1000 ml Erlenmeyer. Dissolve in 400 ml of water and correct the pH to 5.4 with 1N sodium hydroxide or 1N hydrochloric acid. Add 1 ml of Tween 80.

Then separately dissolve 20g of gel medium with 500 ml of water, in a 1000 ml Erlenmeyer, in a boiling water bath, stirring frequently, avoiding prolonged heating. After it has completely dissolved, add the other solution and complete the 1000 ml volume with water. Homogenize and distribute 15 ml (for enumeration in plates) or 6 ml (for enumeration by membrane filtration) in the test tubes. Stop with cotton wool and autoclave for 20 mins at 121°C.

Dubois Medium(Medium 104), gel medium + actidione

Preparation: Weigh 5g of yeast extract (Difco), 5g of peptone, 3g of L-malic acid, 0.05g of magnesium sulphate, 0.05g of manganese sulphate and 50 mg of actidione in a 1000 ml Erlenmeyer. Dissolve in 200 ml of water. Add 250 ml of tomato juice and correct the pH to 4.8 with 1N sodium hydroxide or 1N hydrochloric acid solution. Add a drop of Tween 80.

Then separately dissolve 20g of gel medium with 500 ml of water, in a 1000 ml Erlenmeyer, in boiling water bath, stirring frequently, avoiding prolonged heating. After it has completely dissolved, add to the other solution and make up the 1000 ml volume with water. Homogenize and distribute 15 ml (for enumeration in plates) or 6 ml (for enumeration by membrane filtration) in the test tubes. Stop with cotton wool and autoclave for 20 minutes at 121°C.

TJB medium (Tomato Juice Broth), gel medium + actidione

Preparation: Weigh 5g of glucose, 2g of tryptone (Difco), 5g of peptone (Difco), 5g of yeast extract (Difco), 1g of liver extract, and 50 mg of actidione in a 1000 ml Erlenmeyer. Dissolve in 400 ml of tomato juice. Correct the pH to 5.5 with 1N sodium hydroxide or a 1N hydrochloric acid. Add a drop of Tween 80.

(2) To inhibit the growth of most of the yeasts

Then separately dissolve 20g of gel medium with 500 ml of tomato juice, in a 1000 ml Erlenmeyer, in a boiling water bath, stirring frequently, avoiding prolonged heating. After it has completely dissolved, add to other solution and make up to 1000 ml volume with tomato juice. Homogenize and distribute 15 ml (for enumeration in plates) or 6 ml (for enumeration by membrane filtration) in the test tubes. Stop with cotton wool and autoclave for 20 mins at 121°C.

Comment:

The tomato juice used is diluted 4.2 times and filtered on Whatman No.1 (1000 ml).

1.3.      For acetic bacteria

medium, gel medium + actidione

Preparation: Weigh 1.2g of yeast extract, 2g of ammonium phosphate and 50 mg of actidione in a 1000 ml Erlenmeyer. Add 500 ml of cider and correct the pH to 5 with 1N sodium hydroxide or 1N hydrochloric acid.

Then separately dissolve 20g of gel medium in 450 ml of water in a 1000 ml Erlenmeyer, in boiling water bath, stirring frequently, avoiding prolonged heating. After it has completely dissolved, add to the other solution and complete the 1000 ml volume with water. Homogenize and distribute 15 ml (for enumeration in plates) or 6 ml (for enumeration by membrane filtration) in the test tubes. Stop with cotton wool and autoclave for 20 mins at 121°C.

Carr medium, gel medium + actidione

Preparation: Weigh 30g of yeast extract and 50 mg of actidione in a 1000 ml Erlenmeyer. Dissolve in 500 ml of water. Add 1 ml of 2.2% bromocresol green.

Then separately dissolve 20g of gel medium in 450 ml of water, in a 1000 ml Erlenmeyer, in boiling water bath, stirring frequently, avoiding prolonged heating. After it has completely dissolved, add to the other solution and make up the 1000 ml volume with water. Homogenize and distribute 15 ml (for enumeration in plates) or 6 ml (for enumeration by membrane filtration) in the test tubes. Stop with cotton wool and autoclave for 15 mins at 121°C. At the time of liquefaction and cooling to 45°C, add to the gel medium 20 ml per litre of alcohol sterilized by filtration (polyvinylidene fluoride membrane) and mix.

2.      Liquid culture media

2.1.      For yeasts

YEPD medium(Yeast Extract, Peptone, Dextrose) + chloramphenicol

Preparation: Weigh 10.0g of yeast extract (Difco or equivalent), 20g of peptone, 20g of glucose and 100 mg of chloramphenicol. Dissolve, make up to 1000 ml volume with water and mix.

Distribute 5 ml portions of this medium in the test tubes and autoclave for 15 mins at 121°C.

2.2.      For lactic bacteria

MTJ medium (50% MRS medium "Lactobacilli Man Rogosa and Sharpe Broth" + 50% TJB medium "Tomato Juice Broth") + actidione

Preparation: Weigh 27.5g of MRS "Lactobacilli Man Rogosa and Sharpe Broth" (Difco or equivalent). Add 500 ml of water, heat to boiling to permit complete dissolution and add 20.5g of TJB "Tomato Juice Broth" (Difco or equivalent). Add 50g of actidione. Dissolve with water in order to obtain 1000 ml of solution having first corrected the pH to 5 with 1N hydrochloric acid and mix.

Distribute 10 ml portions of this medium(3) in the tubes and autoclave for 15 mins at 121°C.

(3): The 10 ml volume is used instead of the 5 ml volume as with yeasts, due to the greater sensitivity of lactic bacteria to oxygen.