DE2326028A1 - MICROBIOLOGICAL GROWTH DETECTOR - Google Patents

Description

Patent attorney DIPL.-PHYS. DR. W. LANGHOFF Attorney B. LANGHOFF *

a MUNICH 81 WISSMANNSTRASSE 14. TELEPHONE 932774 ■ TELEGRAM ADDRESS: LANGHOFFPATENT MUNICH

Munich., May 22nd 1973 Our reference: 55-1235

William G. Glenn and Mildred H, Glenn, IOO65 Windstream, Apt, 3, Columbia _s Maryland, USA.

Microbiological growth detector

The invention relates to a microbiological growth detector for embedding with direct contact in a microbiological growth substance and for visual display microbiological growth in the same.

The microbiological growth detector of the invention is particularly suitable for embedding in food or other substances in which microbiological growth can occur.

It is known that the life processes of bacteria, fungi, yeasts and the like ₉ , in other words microorganisms, produce gaseous metabolic by-products, in particular carbon dioxide. These gaseous metabolic by-products of microbiological life processes originate from many complicated biochemical and enzyme reactions at the molecular level. This interaction causes degradation and conversion of available substances and substrates, for example proteins, carbohydrates and the like, as found in foods, pharmaceuticals and microbiologicals

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'Permanent general representative in accordance with § 46 of the PasstAnwO. Approved by the »Landgarlchtsn Munich I and II.

There are growth and nutrient substances - from which the living substances obtain energy, food as well as the thermodynamics and chemical equilibrium for their reproduction and the essential metabolism. The mechanism of decomposition and synthesis in various microbiological life forms is known per se ο For example, transaminase reactions result in proteins. Amino _{acids 3} alpha-keto acids and gaseous ammonia. The fermentative degradation of substrates by bacterial growth leads to a cleavage of pyruvic acid into oxaloacetic acid with the release of carbon dioxide. During transmethylation (transfer of methyl groups), acetylene is converted into ethylene, for example during nitrogen formation by soil bacteria. During decarboxylation, certain enzymes attack the carboxyl groups of amino acids and produce an amine and gaseous carbon dioxide = both aerobic and anaerobic microorganisms have many of these metabolic processes in common. In addition, strictly anaerobic organisms, for example Clostridia sp, can, under suitable conditions, deaminate combinations of amino acids (analin-glycine) and release both gaseous ammonia and carbon dioxide. The finding of these gaseous metabolic by-products in microbiological growth has already been the subject of much research, and it can be said that these microbiological forms are capable of reproduction and are therefore alive.

The detection of gaseous by-products in microbiological Growth is particularly useful in determining whether food is still available for human consumption are suitable or not. Such provisions are also particularly applicable to determine whether a microbiological Growth has occurred in a nutrient substance, such as for scientific research and clinical laboratories is used. In the case of food, it is essentially a matter of determining the presence of toxic bacteria,

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232602a

for example salmonella, staphylococci, streptococci, Escherichia coli etc .. It should be mentioned that the metabolic processes non-pathogenic bacteria, for example the various bacteria found in milk, result in similar metabolic by-products. Although the determination of a microbiological growth for scientific Working is of particular importance, since it forms the plague of the growth of. non-pathogenic Forms in food an indication of whether this is due to the passage of time or temperature effects for the human Enjoyment have already become unusable. It is generally believed that the presence of a large number of Bacteria, even non-pathogenic, raise serious doubts in the usability and health compatibility of food. , The plague of a noticeable microbiological growth in food can therefore be regarded as a reliable indicator that that the food in question is probably spoiled and no longer suitable for consumption.

Various methods are already known for visually indicating the bacterial status of food. According to One of these known methods (US-PS 2,485,566) is a small piece of paper with a neutral or faint alkaline solution of a pH indicator is added, whereby the paper is saturated or coated with a nutrient solution and with non-toxic, cold-resistant, acid-producing bacteria is vaccinated. The indicator package is then sealed and placed in the food container. If the whole thing goes on for a long enough time When exposed to high temperature, the bacteria in the indicator pack will multiply enough that they will do so much Acid form that changes the color of the indicator dye, indicating that similar bacterial growth is taking place could also have occurred in the food itself. This well-known growth indicator is obviously limited

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on chilled or frozen food and does not show any bacterial or other microbiological indicators Growth within the food itself. It is also necessary that the bacteria in large Close to the food.

Another well-known indicator for showing spoiled Food in containers (US-PS 3 0β7 015) an indicator disc made of porous paper is used, which is impregnated with a weak solution of an alkaline material and a suitable indicator dye .. The The indicator disk is separated from the food by a diffusion disk made of porous parchment paper. There is also a perforated rear protection between the parchment paper and the food lies. Such a growth detector is placed in a wall of the food container so that one can see when the indicator disc changes color due to the generation of carbon dioxide. One such growth indicator not only requires a paper disk and an alkaline wetting solution, but is also relatively costly, has a short shelf life and must not come into contact with the food itself, especially then not when it comes to acidic products for which the palatability is to be monitored. The well-known The indicator disc is essentially suitable for milk. It is not intended to be used for acidic foods nor is it set out how this could be done. The intention to prevent the water in the Milk or the food passes through the diffusion disk and that the large dye molecules or Potassium hydroxide or the like pass through the diffusion disk cannot be realized in this way, so that the uselessness of this growth indicator is obvious.

There is also known an elongated indicator pen which

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impregnated or coated with a chemical indicator agent (US Pat. No. 2-626,855). This indicator pen is inserted into the food to be tested, with the indicator agent in the presence of substituted Amines or other amino condensation products such as them occur in the case of putrefaction or due to an acidic pH value or in the case of reduction conditions due to the spoiled Food itself, showing a color change. The use of this indicator pen requires direct Contact between the indicator agent and the food to be tested. This pen cannot therefore be used as an accessory to a food itself, since it is also necessary during only temporary contact some indicator substance is leached and ingested by the food. Another difficulty lies in treating the indicator pens so that they show a sufficiently sharp color change without too much Indicator substance gets into the food. Furthermore, the indicator pen must not lose its effectiveness, if it is exposed to the surrounding atmosphere for a long period of time.

For these reasons, the previously known growth indicators can only be used to a greater or lesser extent.

The invention is based on the object of creating a microbiological growth detector that extends over can be kept in constant contact with a food for a long time without affecting this one harmful. or even exert toxic effects. '

The solution to this problem is given by an indicator solution that consists of metabolic by-products of the microbiological Growth is discolored, and by an impermeable to bases and acids, to the metabolic by-product but permeable cover for the indicator solution,

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Such a growth detector can be used in direct Bringing into contact with the food to be monitored, for example on the surface of the same or in a limited area Air space above a packaged food or the like, and can be in the oven or in an autoclave treat or cook according to the desired method of preparation or preservation of the food.

According to a particular embodiment, the indicator solution used is Alizarin ^ red S, which changes color from red to yellow in the presence of carbon dioxide, and this indicator solution is housed in a composite envelope made of a plastic film, which has special permeability properties, as described below is. Such a growth detector is useful in microbiological wafers in foods such as baby foods, canned soups, canned vegetables, home-made foods, and the like.

According to another embodiment, the indicator solution a frost-resistant mixture or compound is used, which is particularly suitable for frozen food and the like, to detect microbiological growth at low temperatures as well as at ambient temperature.

The invention is described below with reference to schematic drawings described in addition to several exemplary embodiments.

Figure 1 is a top plan view of a growth detector according to the invention.

FIG. 2 is a section along line 2-2 of FIG.

The growth indicator according to the invention comprises an indicator

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material pointing to one or more metabolic by-products responds to microorganisms in order to produce a visible color change in the indicator material. This itself is in an envelope that is permeable to the gaseous by-products, but impermeable to the Indicator material and for any constituents of the substance in which microbiological growth is detected so that an erroneous display cannot occur and a correct display cannot be prevented.

The microbiological growth detector shown in Figure 1 comprises an envelope 10 which has a container for Indicator solution 11 forms. The envelope consists of two envelope halves 20 made of a plastic, which along their edge 14 are tightly connected to one another. Form and the outline of the envelope for the indicator solution can of course be chosen as desired. The envelope can e.g. consist of pieces of circular or flat tubes that only need to be sealed at the ends. Of the For example, the outline may be circular, square, or any other desired shape.

The indicator material is enclosed in the envelope for example by heat sealing, using a solvent as a binding agent or in some other way, as it is known for joining plastics. Polyethylene can be sealed with heat, for example, polyester can be combined with a solvent such as benzyl alcohol, and other materials can be combined with the Connect a wide variety of adhesives to one another. In the present case, a heat seal is preferred applied to bas ingress of potentially toxic substances in the growth base, although non-toxic adhesives and the like can also be used.

The envelope 10 is preferably transparent or translucent, so that the color change of the indicator material

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can be easily determined without opening the envelope. The latter must be permeable to gaseous metabolic by-products so that the color change can occur, such as carbon dioxide. Since the carbon dioxide concentration in the surrounding taffeta is usually relatively small, the addition of a small amount of carbon dioxide results in a relatively larger proportionate increase in concentration than, for example, the addition of nitrogen, since the nitrogen concentration in the atmosphere is almost 80 % . The material of the casing should also be resistant to the temperatures occurring during the treatment of food, for example when canning or freezing food, temperatures of at least 95 to 120 ° C. and pressures of about 1.1 bar can occur over an hour or longer .

As shown in Figure 2, the wrapper is composed of several layers, namely an inner layer 24 and an outer layer 22. The inner layer is about 40 microns thick and is made of polyethylene and the outer layer is ten thick Micron and consists of a polyester (polyethylene terephthalate). Such a cover has proven to be particularly suitable for the purposes of the invention. The inner polyethylene layer enables the envelope to be heat-sealed and has a particularly low permeability for water combined with a high permeability for carbon dioxide and other gases. The outer layer made of polyester improves the durability of the casing at higher temperatures, such as those that can occur when handling food, and results in high tensile and tear strength. Such a cover has a low water diffusion rate and enables the production of a growth detector with a satisfactory shelf life. When such a growth detector is treated in a pressure pot, the rate of water loss is 50 % slower compared to the samples not treated in a pressure pot, so that

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an increased shelf life and service life results »

This growth detector speaks practically only on gaseous ones Metabolic by-products of microbiological growth and not changes in pH in the growth base. The necessary isolation of the indicator solution from the acidic and basic components of the raw material is achieved by choosing the material of the envelope, which has a certain impermeability to acids and Bases must have in order to influence the indicator solution to be avoided by these substances «Of course, this impermeability cannot be completely achieved. It is enough However, what is assumed in the following, that the material of the envelope so far impermeable to acids and Bases is that the pH of the food or the like no influence on the indicator solution during the intended shelf life or lifetime of the growth indicator Has. *

It should be mentioned> ■ that the permeability for carbon dioxide of polyethylene is a hundred times larger than that of polyester. Accordingly, the total permeability of the multilayered Wrapping is determined to a large extent by changes in the thickness of the polyester layer. A relatively small one Changing the thickness from 2.5 to 5 microns will decrease the overall permeability the multi-layer coating against carbon dioxide so much that it allows for early detection spoiled food is no longer usable. On the other hand, there is a reduction in the thickness of the polyester layer to increase the carbon dioxide permeability increasing the sensitivity of the growth detector, the sensitivity is already too big for some applications, v / Ie is explained further below, and also the permeability The coating for acids becomes too strong, so that a premature Color shift of the indicator solution can occur when acidic foods are used, and thus the food

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the duration of the growth detector is reduced. Changes in the thickness of the polyethylene film also have, of course Influence, on the properties of the multilayer covering, but to a far lesser extent than changes in the Thickness of the polyester layer "

Of course, other substances which are permeable to carbon dioxide can also be used to implement the invention and impermeable to acids and bases, e.g. polypropylene can be used instead of polyethylene, especially where higher temperatures occur, as the sealing strength of polyethylene then depends on the Limit could be. Polypropylene is a bit more expensive, however.

The selection of the indicator material 11 depends not only on the environment in which the envelope is to be embedded, but also on the type of gaseous metabolic by-products by which the indicator material is to be influenced and on the concentration of this gas in the indicator material, at which a color change should occur. In turn, the gas concentration depends on a number of factors _s namely the nature of the microorganisms which produce the gas, the growth rate of the microorganisms, permeability, absorption and adsorption of the growth base for the generated gas, the volume of the between the growth base (food) and the Containers confined air, the permeability of the envelope to the gas, and numerous other factors. As mentioned above ₉ , the most common gas is carbon dioxide. If this is present in an aqueous indicator solution, carbonic acid is produced, which

in turn reduces the pH of the indicator solution. Useful Indicator solutions are those where a clearly visible color change occurs with a slight shift in the acidity of the indicator solution »

The indicator material can be a liquid solution which _s

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the required parbanding properties and others has physical properties. The less indicator solution is used per surface unit of the envelope, the more sensitive the growth detector can be made, bearing in mind, however, that a reduction of the indicator solution, the useful life of the growth detector shortened.

It has been found that a double-layer cover with a 10 micron thick polyester layer and a 20 micron thick polyethylene layer and a surface of about 1000 mm ² should contain 1000 milligrams of indicator solution if possible. The rate of water loss with such a polyester-polyethylene cover at a temperature of 22 to 27 ° C, 50 to 90 % relative humidity and 730 to 790 mm Hg air pressure was about 60 when the cover was filled with 1000 milligrams of indicator solution and the surface area indicated above Milligrams per m per hour. If such a multi-layer cover is placed in an autoclave at a temperature of 120 ° C and a pressure of 1.1 bar for 20 minutes, the water loss'

speed to a value of 30 ^ milligrams per 'm and Hour reduced. Converted to 1000 milligrams of the indicator

solution and an area of 1000 mm is the rate of water loss then 0.7 milligrams per day. Under these conditions there is a water loss of 500 milligrams (0.5 ml) acceptable, although the indicator solution is then more concentrated, but still works effectively, so that it is useful duration of 700 days is achieved.

The useful life of the growth detector according to the invention is influenced to a significant extent by the Rate of water loss from the indicator solution into the surrounding area of the envelope when it is placed in a container in which there is a microbiological growth base. Changes in the osmotic pressure of the water

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with respect to the various media that are in contact with the envelope cause corresponding changes of water loss, so that different foods or the like also have different rates of water loss have as a consequence. The higher the water concentration in the growth base, the lower the rate of water loss and the longer the useful life of the growth detector.

It has been found that solutions of about 0.01 to 0.08 % Alizarin'.Rot S (sodium alizarin sulfonate), hereinafter referred to as ARS, in distilled water are suitable for the purposes of the invention. An optimum of properties results at a value of 0.0 ¹ % ARS, as will be explained further below. This indicator solution has a pH of about 6 to 6.2 and a very sharp color change from red to yellow over the pH range of about 6.0 to 4.6 in the presence of carbon dioxide. Toxicity tests were carried out with 0.04 percent ARS solution on Swiss albino mice, and it was found that this solution is not toxic when administered intravenously, when introduced into the peritoneum or orally or combinations of these possibilities.

Another indicator solution that has many similar properties in common with the 0.04 percent ARS solution is bromocresol green solution, hereinafter referred to as BCG solution, which is obtained by dissolving 52.17 milligrams of BCG in one liter distilled water is produced. The pH of this solution is 6.0. BCG shows a color change from blue to light green in the presence of carbon dioxide.

Other indicators can also be used, but the ARS indicator gives the most visible color change, is economical, stable in the temperature range between frozen food and treatment temperatures

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of canned food and appears to be completely non-toxic at the concentrations in question. The main disadvantage of BCG is that the color does not change in the presence of carbon dioxide is pronounced. Bromcresol purple (BCP) can also be used to indicate a pH shift between 6.8 and 5> 2, however the reading is not as clear as when using ARS.

A modified indicator solution, which is particularly suitable for frozen food or for use at low temperatures, ends with a mixture of 30 to 90 % glycerine in distilled water. Such mixtures do not freeze at the usual freezing temperatures in the household. Depending on the water content In the basic indicator solution, concentrations of ARS or BCP between 0.01 and 0.08 % are used. When using BCP, the original pH value of the solution is approximately 6.4 or higher, whereby "the sensitivity of the indicator to carbon dioxide with increasing pH -Value decreases.

There can also be indicator solutions with higher or lower Concentrations of the indicator substance are used at Reducing the concentration, however, reduces the intensity of the color change accordingly, so that this possibly is no longer perceptible clearly enough. When the indicator concentration is increased, the color intensity also increases increased, but the amount required for a color change is due to the buffering effect of the indicator substances increased in carbon dioxide “Of course, he also takes Price of such a growth detector too.

Examples of cold-resistant indicator solutions are given below:

1. Mix 35 milliliters of distilled water and 3 _S 33

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mg BCP. 15 milliliters of glycerine are added to this, so that a 30 percent glycerine solution with a concentration of 0.0066 % BCP and a pH value of 7.0 is obtained.

2. Mix 15 milliliters of distilled water and 1.675 milligrams of BCP. To this end, 10 milliliters of glycerine are added. 25 milliliters of a ¹ JO percent glycerine solution containing 0.068 percent BCP and a pH of 7.0 are obtained.

3. Mix 15 milliliters of distilled water and 2.010 milligrams of BCP. This is done using 15 milliliters of glycerine given. You get 30 milliliters of a 50 percent glycerine solution that contains 0.0067 percent BCP and one Has a pH of 7.0.

Microbiological growth detectors according to the invention can be placed in or on the surface of a growth base, such as food, during or immediately after filling a container with the food. The growth detector then connects to the food container edited and included in this. When the processing is not done properly or when microorganisms later get into the container, for example because it is not completely tight, it is later damaged in the Factory or during transport, or the like, and if such a container is temporally and temperature-wise Fate that leads to the growth of microorganisms, carbon dioxide is produced as a metabolic by-product of such growth. Since the partial pressure of the As carbon dioxide rises in the container, carbon dioxide migrates through the envelope of the growth detector and reacts with the indicator solution, whereby carbonic acid is produced and the pH value of the indicator solution is reduced, so that the color of the indicator changes, if used as an indicator

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ARS is used, there will be a distinctive color change from red to yellow, indicating that the contents of the The container is probably no longer suitable for consumption. . . :

Alternatively, such growth detectors can also from Consumers themselves are added to the food containers after opening the same and after using one Part of the food. You can also use all manufactured foods, e.g. potato salad, coleslaw, Egg salad, crab salad or the like can be added. If the contents of the container have absorbed microorganisms and their growth has progressed so far that carbon dioxide was produced as a metabolic by-product., If the indicator solution changes color, this indicates that the remaining food is likely to be unusable will.

It is known that many fruit acids and the like are volatilized when foods are processed. Of the Growth indicator according to the invention is not influenced by such volatile fruit acids, not even by organic and inorganic acids, as the coating is impermeable to them.

Microbiological growth detectors according to the invention provide a visual indication of microbiological growth due to the generation of carbon dioxide regardless of the pH of the growth base, even when the growth indicator is in direct contact with a growth base whose pH value is significantly lower than the pH value of the indicator material at the color change point.

Growth indicators according to the invention can be used in connection with potential growth bases that are determined by Interrupted or infrequent 'use can spoil, for example

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Baby food and pet food. Another area of application are various growth bases, for example biological serums, pharmaceutical products and the like, in which conditions similar to those of food can arise.

The sensitivity of the growth detector depends on a number of variables. For example, it is not only used by the The thickness of the polyester layer of the envelope is influenced, but also by the type and thickness of the material of the other layer the coating, the concentration and the pH value as well as the type of indicator solution and the relationship between the volume of the indicator solution and the surface area of the envelope.

The sensitivity of the growth detector is preferably adapted to the intended use. Growth detectors for monitoring biological cultures for Research purposes can be relatively sensitive. However, if only the degree of bacterial growth is to be determined as a measure of the palatability of a food, a weaker sensitivity can be used to be appropriate.

The level of bacteria at which foods are commonly considered harmful is around 10 bacteria per gram of the food or higher. With a bacteria content of 10 or less per gram of the food, it is generally considered to be suitable for consumption. Often some health-promoting foods contain large numbers of non-pathogenic bacteria, ζ '.Β. Buttermilk, sour cream or similar dairy products. If a growth indicator for this is set to a display level that is too low, it could be simulated that the product in question is not suitable for consumption. On the other hand, the consumer would be poorly served

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be through an indicator that only responds when the bacterial content is so high that it looks, the smell or taste can be determined. One recognizes microbiological from the following examples Growth detectors for reliably indicating food spoilage before it is presented to the consumer otherwise becomes obvious without aids.

The visual indication of a spoiled state of food

6 Q mean at a content of 10 to ICK bacteria per Grams allows the use of colorimetric techniques to increase the rate of growth at a much lower Determine bacterial content. The finding that there is bacterial contamination leaves the food or the like appear unsuitable for consumption at a foreseeable future time, and much earlier, without the need for a change in color, which is clear even to an inexperienced observer. Numerous color comparators are known in analytical microbiology that are fully useful for the purposes of the invention can be used, for example in food factories for internal quality control. You don't need to do that End product to wait for full bacterial growth in order to be able to determine a color shift. In addition, the Inclusion of a microbiological growth detector in a can or the like provides additional security for the consumer ensure that the contents of the can have not been spoiled since it left the factory. . ·

Below are some examples where a Wrapping made of polyester-polyethylene layers is used, with a thickness of the polyester layer of about 10 to 11 microns and the polyethylene layer from about 33 to 41 Micron. The bacteria used in these examples come from standard laboratory cultures of the ATCC (American type culture collection).

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For example

A growth detector according to the invention is made by adding 0.75 to 1 milliliter of 0.04 percent ARS solution in a cover made of polyester-polyethylene

measured the figures 1 and 2 with a surface of 1000 mm. Approximately 100 grams of the following foods were each filled into two screw-top jars:

Cream of Chicken Soup

Potato cream soup

Celery cream soup

vegetable soup

Minestrone

Veal with vegetables as baby food Ham with vegetables as baby food Ham with vegetables as a toddler meal

A jar of each of these foods was oven treated and sealed. The other glass was made with Escherichia · coil inoculated and sealed and allowed to stand at 37 ° C. As a result, the indicator solution showed in all of the inoculated samples a color change to yellow or orange-yellow after 2 1/2 to 3 days, while the indicator solution in the non-vaccinated Glasses remained unchanged.

Beisgiel_2

An indicator package was produced with 0.75 to 1 milliliter of a 0.04 percent ARS solution in an envelope made of polyester-polyethylene with a surface of

ρ
about 1000 mm. The indicator package was then sealed in a glass which contained 0.1 - normal hydrochloric acid with a pH of 1.2. After 160 days there was still no visible change in the indicator solution. This clearly shows,

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that the display does not depend on the pH of the growth base.

Example_iel_3_

24 indicator packages were made from a polyester-polyethylene laminate and filled with about 1 milliliter of bromocresol green (BCG) solution, which was produced by mixing 1.5 milliliters of 0.04 percent BCG solution in water and 10 milliliters of distilled water had been. 100 grams of a mushroom soup were poured into each of the 12 glasses. The remaining 12 jars were each filled with 90 grams of chicken baby food. About 65 cm ^ in the upper part of the jars remained free of food. An indicator package was put into each of the grasses. For each food, two glasses were separated as control glasses, treated in a pressure pot at 120 ° C. for 20 minutes and then sealed with paraffin. The remaining jars were inoculated with about 100 to 1750 bacteria of the species Salmonella typhimurium, sealed and then stored at room temperature. In all cases, the BCG indicator solution showed a color change from blue to light green in the vaccinated jars after 8 days, while no color change occurred in the control jars. The bacterial count of the vaccinated jars at the end of the 8th day showed a bacterial density of 3 x 10 ^ to 4.8 χ 10 ^ bacteria per gram. The initial pH of the samples was between about 5.4 and 6, 2, while the last pH value for the inoculated jars was between about 5.1 and 6.0 and was unchanged for the control jars.

For example £ iel_4

Three samples were taken from each of 46 different soups with an initial pH between about 4.2 and 6.5. These soups were filled in screw-top jars. The indicator packages contained 1 milliliter 0.04

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percent ARS solution and were incorporated into the glasses. These were then treated at 120 ° C. and a pressure of 1.1 bar for about 20 minutes and then sealed. To There was no change in the color of the indicator solution for 16O days.

Example 5

Four samples from 44 soups were placed in screw caps vials, each sample containing approximately 100 grams. Two samples of each soup were made with Salmonella typhimurium vaccinated and the remaining samples with Staphylococcus aureus. The growth detector was encased in a polyester-polyethylene laminate one milliliter of a 0.04 percent ARS solution. An indicator pack was placed in each glass. The samples were then sealed and stored at room temperature for 8 to 10 days. At the end of that time it was Color of all growth detectors turned yellow.

The vaccinations were given with 10 to 56 salmonella per 100 grams and with 47 to 87 staphylococci per 100 grams. The initial pH of the soups was between 6.4 and 4.9, and the pH at the end of the treatment period was between 5, 9 and 4.5. The largest change in pH was a drop of 1.55 units, namely from 6.25 to 4.7. In the case of two soups, there was no significant drop in pH value during the experiment. The last bacterial plate count performed was 2.8 10 to 9.6 ο "bacteria per gram of soup.

Example_6

Small growth detectors were made with 0.2 milliliters of 0.04 percent ARS solution in a polyester

2 Polyethylene envelope with a membrane surface of approx. 200 mm. Candida albicans (yeast) and Aspergillus spp. separated twice on sloping surfaces of Sabaraud dextrose agar

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painted according to the usual microbiological techniques. Such a mini-growth detector was placed in each headspace (about 5 milliliters) above the agar slant. and then tightly closed the tube. In each case a color change of the indicator from red to yellow occurred after 72 to 6 hours at a temperature of 37 ° C, while similar mini detectors placed on separate sterile control surfaces made from the same substances remained red. Since the microbiological forms of yeast, mold and fungi, like bacteria, have an intercellular metabolism which leads to the production of carbon dioxide, the invention can also be used for monitoring the growth of such substances.

Example_7

Growth detectors were used with 1.0 milliliters of 0.04 percent ARS solution in a polyester-polyethylene envelope. The surface area was 1000 mm. In this experiment, which was repeated several times with modifications, "the growth detectors were either placed in a confined space containing inorganically produced carbon dioxide or in a space containing carbon dioxide produced by the metabolism of the growth of bacteria on food was created. When the color of growth detectors had changed, which in the organic generated carbon dioxide occurred after about ¹ J hours at the inorganic generated carbon dioxide, and after 1 to several days, with the latter depending on the original number of bacteria, to the food, temperature and the like, the growth detectors were removed and exposed to the surrounding atmosphere. In all cases it was found that the color change "was reversed and the original red color was created. These experiments showed that the growth detectors detect the carbon dioxide as a gas, because of its higher partial pressure within the

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closed space compared to the surrounding atmosphere, and that the carbon dioxide gas in the same how'der is released so that the color goes back from yellow to red. This also shows that in the application inorganic and organic acids (e.g. acetic acid, Citric acid, fumaric acid and the like) in the oven treatment of the food itself probably a color change would cause the indicator solution from red to yellow, but would not be allowed to pass through the membrane, rather, through which only gas, namely carbon dioxide, passes.

Example_8

Growth detectors were made with 0.3 and 1.0 milliliters, respectively, of 0.04 percent ARS solution with a pH of 6.2. A polyester-polyethylene laminate with a surface area of ^{300 or 1000 mm 2 was used as the cover.}

There was a growth detector in each of a number. Jars with macaroni, beef and ham baby food brought. The control jars were sealed and treated in a pressure pot. The remaining glasses were rated at about 7.2 Bacteria vaccinated per gram of Salmonella typhimurium type, then closed and kept at room temperature.

The percent light transmittance T was then spectrophotometrically for each indicator solution at a wavelength of 620 millimicrons measured and compared as a percentage with the control samples treated in the pressure pot. It resulted the following test results:

Exposure time indicator - A 25th Bacteria / gr volume 10, 3 Plate count Milliliters 7, 9 23 0.3 121 2.57 x 10 ⁵ * 23 1.0 2.2 χ 10 ⁵ 309849 /

33 O , 3 15th , 3 * 33 1 , 0 16 , 3 45 1 , 0 19th .5 *

² ^ average of three samples.

It should be noted that a percentage permeability of 10 % or more can be determined by an inexperienced eye and can easily be determined by using a Parb reference card . This also shows that such determinations can be carried out in less than 2k hours after the occurrence of the contamination, so that the invention can be used for quality control in many food factories where only a limited test time is available.

Example_9. .

K membrane envelopes were made of a plastic i Im® laminate with a polyester layer 12 microns thick and a polyethylene layer 50 microns thick. The envelope was filled with 0.04 percent ARS solution according to the data below.

Code volume and surface pH value (mm)

3-60 0.3 ml., 6.0 '350

3-62 0.3 ml., 6.2 350

1-60 1.0 ml., 6.0 950

1-62 1.0 ml., 6.2 950

These wrappings were then hung separately in a 250 milliliter side arm bottle each, which weighed 3.3 grams

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contained powdered calcium carbonate. The bottles were closed with rubber stoppers and evacuated through the side arm for 10 minutes (20 liters / min, 0.7 cfm). After the evacuation, 18 milliliters of diluted sulfuric acid (prepared from 30 milliliters of concentrated sulfuric acid diluted with 500 milliliters of distilled water) was slowly added to each bottle with a hypodermic needle from a hypodermic syringe. If appropriate, positive pressure was released from the generated carbon dioxide by loosening the top stopper of the bottle. When the production of carbon dioxide ceased, the plugs were pushed back into place and a rubber band was wrapped around as a backup and served as an improved safety valve. Since carbon dioxide is heavier than air, the bottles were saturated with carbon dioxide gas. All bottles were then kept at room temperature (22-25 ° C.) and the time until the color changed was measured. The values are:
Code 'Time to color change

3-60 186 min.,

3-62 'ended after 96 hours, no envelope.

1-60 'about 3.5 days,

1-62 finished after 96 hours, no change

Identical tests were carried out, but with an envelope made of a plastic film laminate, the thickness of the Polyester layer was about 11.5 microns and the thickness of the polyethylene layer was about 3 * f microns. The values are: Code time to color change

3-60 65 min.

3-62 69 min.

1-60 315 min.

i-62 351 min.

A comparison of these times between the growth detectors

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with different wrappings clearly shows that carbon dioxide is much slower through the thicker material diffuses, so that this is unfavorable or unusable as a microbiological growth detector.

Example_10

Chicken with vegetables, a high protein baby food, was used as the basis for the growth of Salmonella typhimurium bacteria. Sixteen jars were prepared containing approximately 90 grams of the food, with 15 cubic centimeters in the upper part of the room divided into four sections of four units. Each of the jars was inoculated with 0.52 bacterial cells per gram (a total of 46 _S 8 cells per jar). Before sealing, a growth detector was wrapped in a 10 micron thick polyester film and 50 micron thick polyethylene film and filled with 0.04 percent ARS solution with a pH of 6.0 or 6.2 and a volume of 0.3 or 1.0 milliliter placed in each glass. All glasses were then left at the ambient temperature of 20-25 ° C ".

After 120 hours, only the growth detector showed a volume of 0.3 ml and a pH of 6.0 a color change to yellow, with the subsequent Bakfeeriencounting 9.2 χ 10 'organisms per gram. Even after 15 days, there was still no final color change to yellow in the other samples.

In contrast, when using thinner film, namely a polyester film 10 microns thick and one 40 micron thick polyethylene film when using the same ARS solution and similarly treated foods, but only with a quarter of the bacteria of the Salmonella type typhimurium vaccinated, a hundred percent color change from red to yellow after only 5 to 8 days. Since even in

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15 days only 6 percent of the growth detectors (1/16) with the thicker foils, even at higher initial concentrations the bacterial inoculation did not show any color change, it can be seen that the use of a 12.5 thick polyester layer in conjunction with a 50 micron thick polyethylene layer as a coating and an indicator solution of 0.04 percent ARS solution with a pH of 6.0 or higher in a volume of 1.0 milliliter and with a surface area

of 1000 mm does not give satisfactory results for making spoiled food visible.

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Claims (13)

Claims Microbiological growth detector for embedding in a microbiological growth substance and for visual purposes Display of microbiological growth in the same, marked 'by an indicator solution, which is discolored by metabolic by-products of microbiological growth, and by a for Bases and acids impermeable to the metabolic by-product however, permeable envelope for the indicator solution. 2. Growth detector according to claim 1, characterized in that the envelope is made of transparent Made of plastic. 3. Growth detector according to claim 1 or 2, characterized through the use of an indicator solution that responds to carbon dioxide. 4. Growth detector according to claim 1 to 3, characterized in that the envelope has a .Water passage speed of a maximum of 60 milligrams ρ " per m and h. 5. growth detector according to claim 2 to A, characterized characterized in that the envelope is a laminate with a polyethylene terephthalate film of about 10 Micron thickness. 309849/1219 6. Growth detector according to claim 5, characterized in that the other .Schicht of the laminate is a film made of polyethylene or polypropylene. 7. growth detector according to claim 6, characterized in that the other layer is made of medium density polyethylene and is approximately 38 microns thick. 8. Growth detector according to claim JL to 7, characterized characterized in that the active surface 2 - - the envelope is about 1000 mm. 9. Growth detector according to claim 1 to 8, characterized by Alizarin Red S, bromocresol Green or bromocresol purple as an indicator. 10. Growth detector according to claim 9 .. characterized that the indicator solution is 0.0 ^ J Percent Alizarin Red S contains. 11. Growth detector according to claim 1 to 10, characterized geke.η nze. rejects that the indicator solution Contains 30 to 90 percent by volume glycerine. 12. Growth detector according to claim 1 to 11, characterized geken η indicates that the pH of the indicator solution is about 6.0. 13. Growth detector according to claim 9, characterized in that the indicator solution is 0.01 contains up to 0.08 percent Alizarin Red S. lh. Growth detector according to Claims 1 to 14, characterized by a solid indicator substance. 309849/1219