IE44169B1 - Nutrient media for microbiological testing - Google Patents

Abstract

The test set for determination of the causative organism genera in inflammations of the urogenital tract consists of 8 selective nutrient media. These nutrient media are: modified lactose-sodium heptadecyl sulphate-P agar, citrate-G agar, modified ampholyte-cadmium sulphate-glycerol agar, phenylalanine-lithium chloride-G agar, modified DNase-ampholyte agar, mannitol-sodium chloride-thiocyanate agar, modified T.T.C-azide-P agar and peptone-liver hydrolysate-thiocyanate agar The second, fourth, sixth and eighth of the abovementioned nutrient media are novel.

Description

The present invention relates to a method of determining the genera of pathogens capable of causing infections of the urogenital tract and to a kit comprising certain nutrient media for use in the determination of the genera of such pathogens.

Methods and kits for determining the nature of microorganisms in various test materials have been described, for example, in German Offenlegungsschrift No. 2,408,167.

The methods and kits described hereto, however, have ]0 involved the use of known nutrient media, and the selectivity of these methods and kits have not been sufficient to enable the desired determination of the genera of pathogens.

The methods and kits of the present invention avoid, at least in part, the disadvantage of insufficient selectivity.

According to one feature of the present invention there is provided a method for determining the genera of pathogens capable of causing urogenital tract infections which comprises inoculating each of the following nutrient media: ’ Lactose-P-Nutrient (as herein defined), Citrate-G-Nutrient (as herein defined), Cadmium Nutrient (as herein defined), Phenylalanine-lithium-G-Nutrient (as herein defined), DNase Nutrient (as herein defined), Mannitol-Thiocyanate Nutrient (as herein defined), T.T. - Nutrient (as herein defined), Peptone-Thiocyanate Nutrient (as herein defined) with a sample of the said pathogen, incubating each of the nutrient media, and determining the genera of the said pathogen by evaluation of the growth if any on each of the nutrient media.

The nutrient media designated:- citrate-G-nutrient, phenylalanine-G-nutrient, mannitol-thiocyanate nutrient and peptone-thiocyanate nutrient are novel per Se and have not, hitherto,been described in an equivalent or similar composition. The remaining nutrient media are based on known media modified by altering the quantities of the components and/or adding additional substances.

The method of the present invention, enables those pathogens which cause a very high percentage of urogenital infections (Escherichia, Proteus, Klebsiella, Enterobacter, Pseudomonas, Serratia, Providencia, Citrobacter, Staphylococcus , Streptococcus, Candida) to be quickly and accurately identified. Moreover with regard to sensitivity determination according to German Offenlegungsschrift P 2,301,211, the method according to the invention provides a basis for successful therapy.

The microorganisms may, if required, be examined by means of simple trials.

The composition, preparation and characteristics of 8 nutrient media are described herein. Deviations from the indicated compositions may be made if these do not impede selectivity and growth of the microorganisms.

The term Lactose-P-nutrient as used herein denotes a nutrient medium comprising lactose, an indicator such as bromothymol blue, an anionic surfactant in a concentration effective to inhibit the growth of gram-positive microorganisms, and pimaricine in a concentration effective to inhibit the growth of Candida. Using bromothymol blue as the indicator the composition of the medium is such that pathogens of the genera Citrobacter incubated on the said medium produce a blue to blue-green colouration, pathogens of the genera Enterobacter incubated on the said medium produce a yellowish green colouration and pathogens of the genera Escherichia incubated on the said medium produce a yellow colouration. Other indicators of equivalent pH 44160 range can be used instead of bromothymol blue.

The surfactant employed in Lactose-P-Nutrient (as hereinbefore defined) is preferably an anionic long chain alkyl sulphate e.g. heptadecyl sulfate preferably used as its sodium salt (Tergitol 7; Tergitol is a trade mark). In one preferred embodiment LactoseP-Nutrient comprises lactose, polypeptone, yeast extract, sodium heptadecyl sulfate, pimaricine, bromothymol blue, agar-agar and distilled water. In one especially preferred embodiment Lactose-P-nutrient has the following composition:Lactose about 10.0 g polypeptone about 5.0 g yeast extract about 3.0 g sodium heptadecyl sulfate (Tergitol 7) about 0.15 g about 0.1 g about 0.025 g about 18.0 g The nutrient medium has pimaricine bromothymol blue agar-agar per litre of deioniser or distilled water, a pH of 6.9+0.1.

Sodium heptadecyl sulfate in the indicated concentration inhibits the growth of the gram-positive microorganisms, 416 9 e.g. Staphylococcus and Streptococcus.

Pimaricine in the mentioned concentrations inhibits the growth of Candida and other yeasts and mould fungi.

The term Citrate-G-nutrient’’ as used herein denotes a nutrient medium comprising a water-soluble citrate, bile salts in a concentration effective to inhibit the growth of gram-positive microorganisms, an indicator dye such as phenol red and pimaricine in a concentration effective to inhibit the growth of Candida; the composition of the medium being such that pathogens of the genera Citrobacter, Enterobacter and Klebsiella incubated on the said medium produce a visible colour change, for instance a raspberry red colouration.when phenol red is the indicator.

Citrate-G-nutrient (as hereinbefore defined) preferably also contains glucose and yeast extract in a concentration effective to shorten the growth-lag-phase and quicken the break down of citrate. In general the use of yeast extract and glucose enable evaluation to take place within 24 hours.

The water soluble citrate employed is conveniently sodium or potassium citrate e.g. trisodium citrate dihydrate.

In a preferred embodiment Citrate-G-nutrient comprises 416 9 trisodium citrate dihydrate, a mixture of salts of bile acids, pimaricine, glucose, yeast extract, sodium chloride, hydrated magnesium sulfate, ammonium dihydrogen phosphate, hydrated sodium ammonium hydrogen phosphate, phenol red, agar-agar and distilled water. In one especially preferred embodiment of the present invention Citrate-G-nutrient1 has the composition:- Trisodium citrate dihydrate about 5.0 g mixture of salts of bile acids about 1.0 g pimaricine about O.'l g glucose about 0.05 g yeast extract about 0.25 g sodium chloride about 5.0 g magnesium sulfate heptahydrate about 0.2 g ammonium dihydrogen phosphate about 0.2 g sodium ammonium hydrogen phosphate tetrahydrate about 0.8 g phenol red about 0.02 g agar-agar about 15.0 g per litre of deionised or distilled water. The nutrient medium has a pH of 7.1 + 0.1.

The mixture of salts of bile acids in the abovementioned concentration inhibits the growth of gram-positive microorganisms, namely staphylococcus and Streptococcus.

Pimaricine in the above-mentioned concentration inhibits the growth of Candida and other yeasts and mould fungi.

In combination with Lactose-P-Nutrient and the mobility test the pathogen genera Citrobacter, Enterobacter, Escherichia and Klebsiella may be distinguished from one another.

The term “Cadmium nutrient as Used herein denotes a nutrient medium comprising N-alkyl-aminocrotonic acid and a water soluble cadmium salt in a concentration effective to inhibit the growth of gram-positive bacteria and Candida 15 but to promote the formation of pyoverdin (fluorescent pigments) on the growth of Pseudomonas; the composition of the medium being such that pathogens of the genera Pseudomonas incubated on the said medium produce, a green to yellow colouration.

The water soluble cadmium salt used is preferably cadmium sulfate.

In a preferred embodiment Cadmium nutrient comprises -1416 9 proteose peptone, sodium chloride, dipotassium hydrogen phosphate, potassium nitrate, hydrated calcium nitrate, hydrated magnesium sulfate, hydrated cadmium sulfate, glycerine, an N-alkyl-aminocrotonic acid, agar-agar and deionised or distilled water. In one especially preferred embodiment Cadmium nutrient has the composition:- Proteose peptone about 20.0 g sodium chloride about 1.2 g dipotassium hydrogenphosphate about 0.8 g 10 potassium nitrate about 0.6 g calcium nitrate tetrahydrate about 0.4 g magnesium sulfate heptahydrate about 0.6 g cadmium sulfate octahydrate about 0.005 g glycerine about 8.0 ml 15 N-alkyl-aminocrotonic acid (Ampholyte KKPK 55) about 0.5 g agar-agar about 15.0 g- per litre of deionised or distilled water. The nutrient medium has a pH of 7.2 + 0.1.

N-Alkyl-aminocrotonic acids and water soluble cadmium salts, e.g. cadmium sulfate, in the above-indicated concentrations will inhibit gram-positive and most gram-negative 44iG9 bacteria, as well as Candida and other yeasts and mould fungi. Of the gram-negative bacteria, that may grow on this nutrient medium, only Pseudomonas is in a position to form pyoverdin (fluorescent pigments).

The above-mentioned composition of the nutrient medium enhances formation of pyoverdin and enables Pseudomonas aeruginosa to be identified within 24 hours.

The term Phenylalanine-lithium-G-nutrient as used herein denotes a nutrient medium comprising salts of bile acids in a concentration effective to inhibit the growth of gram-positive microorganisms, pimaricine in a concentration effective to inhibit the growth of Candida, a water soluble lithium salt in a concentration effective to reduce the growth of Enterobacteria other than Proteus and Providencia, and phenylalanine in a concentration effective to allow conversion into phenylpyruvic acid by Proteus and Providencia; the composition of the medium being such that pathogens of the genera Proteus and Providencia incubated on the said medium produce a green colouration on the addition of a solution of a ferric salt.

Phenylalanine employed in Phenylalanine-lithium-Gnutrient is DL or more preferably L-phenylalanine and the 416 9 water soluble lithium salt may, for example, be, lithium chloride. The solution of a ferric salt employed to test for Proteus and Providencia may, for example, be ferric chloride.

In a preferred embodiment Phenylalanine-lithium-Gnutrient comprises proteose peptone, yeast extract, sodium chloride, disodium hydrogen phosphate, potassium hydrogen phosphate, L-phenylalanine, a mixture of salts of bile acids, lithium chloride, pimaricine, agar-agar and deionised or distilled water and in an especially preferred embodiment has the composition:Proteose peptone yeast extract sodium chloride disodium hydrogen phosphate potassium hydrogen phosphate L-phenylalanine mixture of salts of bile acids lithium chloride pimaricine agar-agar about 10 about 3 about 5 about 5 about 0 about 2 about 1 about 2 about 0 about 16 θ g g 0· g 6 g 2 g 0 g 0 g θ g 2 g 0 g per litre of deionised or distilled water. The nutrient medium has a pH of 7.2 + 0.1. Λ mixture of salts of bile acids in the above-mentioned concentration inhibits the growth of gram-positive microorganisms.

Pimaricine in the above-indicated concentration inhibits the growth of Candida and other yeasts and mould fungi.

Water soluble lithium salts e.g. lithium chloride reduces the growth of most of the gram-negative enterobacteria, other than Proteus and Providencia. Proteus and Providencia alone are in a position to convert phenylalanine into phenyIpyruvic acid. After addition of a water soluble ferric salt such as ferric chloride e.g. several drops of a 10% iron(III)-chloride solution a green colour appears (phenylalanine-desaminase test).

The term DNase-nutrient as used herein denotes a nutrient medium comprising DNase and N-alkyl-aminocrotonic acid in a concentration effective to inhibit the growth of gram-positive microorganisms, yeasts and mould-fungi; the composition of the medium being such that pathogens of the fl genus Serratia incubated on the said medium produce^clear zone around the area of growth surrounded by a turbid precipi12 tation on the addition of dilute hydrochloric acid e.g.

IN HCl.

The DNase-nutrient (as hereinhefore defined) may, if desired, include an indicator such as methyl green, in which case the test for Serratia with dilute hydrochloric acid may be dispensed with.

In a preferred embodiment DNase-nutrient comprises tryptose, desoxyribonucleic acid, sodium chloride, an N-alkyl-aminocrotonic acid, agar-agar and deionised or distilled water and in an especially preferred embodiment has the composition:15 Tryptose desoxyribonucleic acid sodium chloride N-alkyl-aminocrotonic acid (Ampholyte KKPK 55) agar-agar about 16.0 g about 1.6 g about 4.0 g about 0.15 g about 12.0 g per litre of deionised or distilled water. The pH of the nutrient medium is about 7.3. If desired methyl green may be included in the nutrient medium in a concentration of about 0.04 g per litre of distilled water.

N-alkyl-aminocrotonic acids (Ampholyte) in the above-mentioned concentration inhibits the growth of the gram-positive microorganisms yeasts and mould-fungi.

Only Serratia of all the pathogens of urinary infections 5 may grow on this nutrient medium and is capable of attacking DNase and of producing a turbid precipitation after the addition of dilute hydrochloric acid e.g. IN hydrochloric acid.

The term Mannitol-thiocyanate nutrient as used Ιθ herein denotes a nutrient medium comprising a mannitol and a water-soluble thiocyanate and preferably also a water soluble lithium salt, a water soluble potassium salt and a water soluble chloride in a concentration effective to inhibit the growth of flora accompanying the pathqgen but not effective to reduce the growth of Staphylococci, pimaricine in a concentration effective to inhibit the growth of Candida; the composition of the medium being such that pathogens of the genus Staphylococci incubated on the said medium produce acid detectable by a pH indicator.

The water soluble lithium salt may, for example, be lithium chloride, the water soluble potassium salt may, for example, be potassium chloride, the water soluble chloride may, for example be potassium and/or sodium chloride and the water soluble thiocyanate may for example, be potassium thiocyanate.

In order to measure the pH change resulting from the production of acid in the nutrient medium it is convenient to add a pH indicator e.g. phenol red to the nutrient medium. It is also possible to flood the incubated culture with an indicator solution.

In a preferred embodiment Mannitol-thiocyanate nutrient comprises casein peptone, meat extract, gelatin, D-mannitol, yeast extract, sodium pyruvate, disodium hydrogen phosphate, lithium, chloride, sodium chloride, potassium thiocyanate, a pH indicator, pimaricine, agar-agar and deionised or distilled water and in an especially preferred embodiment has the composition:- Casein peptone about 10.0 g meat extract about 5.0 g gelatin about 5.0 g D-mannitol about 10.0 g yeast extract about 5.0 g sodium pyruvate about 10.0 g disodium hydrogen phosphate about 6.0 g lithium chloride about 5.0 g sodium chloride about 5.0 g potassium thiocyanate about 30.0 g pH indicator about 0.02 g pimaricine about 0.2 g agar-agar about 16.0 g per litre of distilled water. The pH of the nutrient medium is about 7.2. If desired the pH indicator may be phenol red.

With regard to the preferred embodiment abovedescribed lithium chloride, potassium chloride and potassium thiocyanate in the above-indicated concentrations inhibit the growth of the flora accompanying the pathogen without exercising a negative influence upon the growth of the Staphylococci.

The mannitol-positive .Staphylococci grow within 2k hours, and the break down of mannitol to give acid may, for example, be indicated by a change in the pH-indicator (e.g. phenol red). Where phenol red'is used as indicator the nutrient medium becomes reddish-yellow to yellow.

The mannitol-negative Staphylococci grow in smaller colonies and the nutrient medium does not show any change in colour.

Thus it is possible to note a difference between microorganisms of the Staphylococci genera. The pathogenic species Staphylococcus aureus is mannitol-positive, while biotypes of the mostly non-pathogenic species Staphylococcus epldermidis and Staphylococcus saprophyticus are mannitolnegative.

The above described nutrient media do not contain substances which deteriorate rapidly or which can only be stored for a short time. The nutrient medium has, !0 as opposed to known nutrient media, a relatively long storage life and is in a ready-to-use, pre-poured form.

The term T.T. - Nutrient as used herein denotes a nutrient medium comprising a reducing agent, preferably a water soluble thiosulfate in a concentration effective to inactivate growth inhibiting hydrogen peroxide, alipoic acid in a concentration effective to promote growth of Streptococcus faecium,pimaricine in a concentration effective to inhibit the growth of Candida, a 2,3,5-triphenyltetrazolium salt in a concentration effective to be reduced by Streptococcus faecalis to red or reddish brown formazan, and a water soluble e.g. alkali metal azide in a concentration effective to inhibit the growth of gram-ne^tive f lora but to allow 44ΐθ9 uninhibited growth of Enterococci. The composition of the medium may be such that Streptococcus faecalis of serologic group D incubated on the said medium produces red to reddish brown, completely flat and small colonies with a metallic gloss on the said medium.

The water soluble thiosulfate is conveniently sodium thiosulfate, the 2,3,5-triphenyltetrazolium salt is conveniently 2,3,5-triphenyltetrazolium chloride and the alkali metal azide is conveniently sodium azide.

In a preferred embodiment T.T. - nutrient comprises tryptose, yeast extract, glucose, disodium hydrogen phosphate, sodium thiosulfate, α-lipoic acid, agar-agar, pimaricine, 2,3,5-triphenyltetrazolium chloride, sodium azide and deionised or distilled water and in an especially preferred embodiment has the composition:- Tryptose about 20.0 g yeast extract about 5.0 g glucose about 2.0 g disodium hydrogen phosphate about 4.0 g sodium thiosulfate about 0.3 g α-lipoic acid about 0.001 g Agar-agar about 15.0 g pimaricine about O.i g 2,3,5-1 r ipheny 11. e t razo J iuin chloride (T.T.C.) about 0.1 g sodium azide about 0.8 g per litre of distilled water. The pH of the nutrient medium is about 7.2.

The water soluble thiosulfate e.g. sodium thiosulfate inactivates the growth-inhibiting hydrogen peroxide, since the Enterococci do not possess any catalase with which to break H^O^ down. α-Lipoic acid in the above-mentioned concentration serves as a growth promotor for Streptococcus faecium.

Pimaricine in the above-indicated concentration inhibits the growth of Candida and other yeasts and mould 15 fungi.

T.T. e.g. T.T.C. is reduced by Streptococcus faecalis to the red to reddish-brown colour of formazan.

The alkali metal azide e-3· sodiura azide in the above mentioned concentration inhibits only the growth of the gram-negative accompanying flora and allows uninhibited growth of the Enterococci.

The term Peptone-thiocyanate-nutrient as used herein denotes a nutrient medium comprising a vegetable protein hydrolysate (peptone) in a concentration effective to enhance the growth of Candida, a surfactant in a concentration effective to suppress the growth of gram-positive microorganisms and a water soluble thiocyanate in a concentration effective to suppress the growth of gram-negative accompanying microorganisms; the composition of the medium being such that pathogens of the genera Candida incubated in the said medium grow in colonies of a dirty white colour.

The water soluble thiocyanate may, for example, be sodium thiocyanate and the surfactant is generally an anionic surfactant, especially a long chain alkyl sulphate such as heptadecyl sulfate preferably used as its salt e.g. sodium heptadecyl sulfate.

The pH of the nutrient medium is preferably 5.5 to 6.0 especially about 5.8.

In a preferred embodiment Peptone-thiocyanate-nutrient comprises vegetable protein hydrolysate, liver hydrolysate, glucose, yeast extract, sodium chloride, magnesium sulfate, manganese (II) chloride, sodium heptadecyl sulfate, potassium thiocyanate, agar-agar and deionised or distilled water and in an especially preferred embodiment has the following composition:Vegetable protein hydrolysate liver hydrolysate glucose yeast extract sodium chloride magnesium sulfate heptahydrate manganese(ll)chloride tetrahydrate about 15.0 g about 1.0 g about 5.0 g about 3.0 g about 3.0 g about 0.25 g about 0.001 g sodium heptadecyl sulfate (Tergitol 7) about 0.05 g potassium thiocyanate agar-agar about 20.0 g about 22.0 g per litre of distilled water. The pH of the nutrient medium is about 5.8. per litre of deionised or distilled water. The pH of the nutrient medium is Characteristics about 5.8.

Vegetable protein hydrolysates, especiallyllycopeptone 8 mainly enhances the growth of Candida and other yeast strains.

A surfactant e.g. sodium heptadecyl sulfate (Tergitol 7.) 20 suppresses the growth of all gram-positive microorganisms in this concentration and at this pH-value.

A water soluble thiocyanate e.g. potassium thiocyanate suppresses the growth of all gram-negative accompanying *Mycopeptone B is a trade name for a vegetable protein hydrolysate. organisms in the above-mentioned concentration and at the above-mentioned pH-value.

As described above, the method of the present invention is particularly concerned with the determination of the types of pathogens causing infections of the urogenital tract. The method of the present invention may however he employed for the examination of other biological material, in which case it may be necessary to include the use of further nutrient media, for example, for examination of faeces in order to identify Salmonella or Shigella.

The method of the present invention may also be extended to the identification of the particular species concerned by including further nutrient media in the investigation process.

The method of the present inventioft is conveniently effected by inoculating each nutrient medium with a urine sample, the pH of the urine sample having been measured prior to inoculation. Thus the method of the present invention may, for example, be effected as follows:20 the temperature of the cooled nutrient media is allowed to rise to room temperature. The pH-value of the middle portion of the morning urine is measured. An inoculation 416 9 loop filled with the urine sample is applied and spread on each nutrient medium. The sample is only spotted on DNase nutrient Medium in form of lines in the middle of the plate. The nutrient media are then incubated for 24 hours at about 37UC., a lid being placed over each nutrient medium.

Evaluation is effected as follows:For identification of the pathogen genera the following criteria are decisive for each individual type; the colour changes described below relate in general to the preferred pH indicators. Other indicator compounds will of course produce different hues.

Citrobacter a) In Lactose-P-nutrient (as hereinbefore defined), lactose is in general, fermented slowly; the nutrient medium turns blue to blue-green within 24 hours. b) in citrate-G-nutrient (as hereinbefore defined) the citrate is attacked; the nutrient becomes raspberry red.

Enterobacter a) In Lactose-P-nutrient (as hereinbefore defined) , lactose 20 is fermented rapidly; the nutrient turns yellowgreenish and the colonies are big and slimy. b) In citrate-G-nutrient (as hereinbefore defined) the citrate is attacked, the nutrient becomes raspberry red.

Escherichia a) In Lactose-P-nutrient (as hereinbefore defined) the lactose is fermented rapidly, the nutrient medium becomes mandarine yellow and the colonies are not slimy. b) In citrate-G-nutrient (as hereinbefore defined) the citrate is attacked. In the presence of yeast extract + glucose (0.25 + 0.05 g/ltr.) there may be only a very weak growth. The nutrient turns to a light pink to orange.

Klebsiella a) In Lactose-P-nutrient (as hereinbefore defined) the lactose is quickly fermented, in a similar manner to Enterobacter; the nutrient medium becomes yellowgreenish as with Enterobacter, or sometimes mandarine yellow as with Escherichia; the colonies are big and slimy as with Enterobacter. As opposed to Enterobacter and Escherichia, Klebsiella is not mobile. Mobility must therefore be checked, for example in hanging drops. 441G9 b) In citrate-G-nutrient (as hereinbefore defined) the citrate is attacked; the nutrient medium turns raspberry red.

Cadmium nutrient (as hereinbefore defined) turns greenish to yellow; fluorescent pigments (pyoverdin) are formed.

Proteus On obtaining a good growth on phenylalanine-G10 nutrient (as hereinbefore defined) a solution of a ferric salt e.g. ferric chloride for example approximately 0.3 ml of an iron(HI)-chloride solution is added to the nutrient medium. After no more than 3 minutes Proteus will show a typical green colouring of the nutrient medium.

All species of the genus Proteus produce the enzyme urease, which decomposes urea into ammonia. The production of ammonia shifts the pH-value to the alkaline end of the pH range (8.2 to 9.0) and in order to detect the pH change the pH of the urine sample should be measured.

Providencia On obtaining a good growth on phenylalanine-Gnutrient (as hereinbefore defined) a solution of a ferric salt e.g. ferric chloride for example approximately 0.3 ml of an iron(III)-chloride solution is added to the nutrient medium. After no more than 3 minutes, Providencia shows the same typical colour reaction as Proteus.

As opposed to Proteus, however, the pH-value of the urine is shifted only slightly.

Serratia Serratia marcescens often forms on DNase-nutrient (as hereinbefore defined) pink to cherry red pigments.

On visible growth upon DNase-nutrient (as hereinbefore defined) a dilute hydrochloric acid e.g. approximately 0.3 ml of 1 N hydrochloric acid is added to the medium and after several minutes a distinctly clear zone is formed around the growth, surrounded by a turbid precipitation. Serratia marcescens is 96.7% DNase-positive. Serratia liquefaciens, playing only a subordinate role with respect to urinary tract infections, is 69.4% DNase-positive.

Rods occurring with infections of the urinary tract are able to grow on this nutrient medium, however, they are DNasenegative.

Staphylococcus In Mannitol-thiocyanate-nutrient the mannitol is degraded by Staphylococcus aureus, Staphylococcus epidermldis, biotype 4 and several Staphylococci saprophyticus strains. Acid is produced as a result of this process and may be detected by a colour change if a pH-indicator is used.

The nutrient medium becomes reddish-yellow if phenol red is used as indicator. When prolonging incubation to 36 hours the colonies become bright yellow.

In suspicious cases it is recommended to carry out the quick coagulase test. Staphylococcus aureus as opposed to other Staphylococci - is coagulase-positive.

Streptococcus_(Enterococcus) Streptococcus faecalis of the serologic group D reduces T.T. to formazan and grows in red to reddish-brown, completely flat and small colonies with a metallic gloss on the T.T. nutrient (as hereinbefore defined).

Streptococcus faecium of the serologic group D - apart from the sub-genus casseliflavus - is not capable of reducing T.T. to formazan. The colonies are colourless, completely flat and small.

There are types of the genera Proteus and Serratia, which occasionally grow on T.T.-nutrient in single colonies, which are larger, elevated and red without metallic gloss. 416 9 These are mobile, as opposed to the above-mentioned Enterococci. It is therefore recommended that mobility is examined in suspicious cases, for example in hanging drops.

Candida On peptone-thiocyanate-nutrient Candida grows in colonies of a dirty white colour. 441C9 According to a further feature of the present invention there is provided a kit for determining the genera of pathogens capable of causing urogenital tract infections which comprises each of the following nutrient media:5 Citrate-G-nutrient (as herein defined) Phenylalanine-G-nutrient (as herein defined) Mannitol-thiocyanate-nutrient (as herein defined) Peptone-thiocyanate-nutrient (as herein defined) each of said nutrient media being retained in a sterile container.

The kit preferably also contains Lactose-P-nutrient (as herein defined), Cadmium-nutrient (as herein defined), DNase-nutrient (as herein defined) and/or Τ.T.-nutrient (as herein defined).

In one embodiment of the present invention the kit comprises a plurality of sterile containers e.g. of plastics material preferably formed by deep drawing, each container being provided with a closure member for sealing the container and each container having therein one of the above-mentioned nutrient media.

In a further embodiment of the present invention the kit comprises a holder divided into a plurality of compartments adapted to receive a plurality of different nutrient media, the nutrient media being retained in sterile condition in the holder. In this connection the present invention also relates to a kit in which the nutrient media are contained in individual sterile sealed packages in which case each package may, for example, be retained in the abovementioned holder.

It is convenient to fill the sterilized nutrient media into the lower part of sterile deep-drawn packages equipped with a tightly sealing lid or other closure means. The lower part of these packages is divided into eight square cavities e.g. arranged in two columns of four, the base of each 2 cavity being, for example, 8 to 10cm and the depth of each cavity being 1 to 1.5cms. Several mis., preferably about 5 or 6mls of the above-mentioned nutrient media, is conveniently placed in each cavity.

The kit of the present invention may, of course, be employed in the method of the present invention by providing-all the eight above-mentioned nutrient media to the kit. - 30 44169 The preparation of the various nutrient media referred to in the specification is illustrated in the following Examples:Example 1 Lactose-P-Nutrient Composition Lactose 10.0 g polypeptone 5.0 g yeast extract 3.0 g sodium heptadecyl sulfate (Tergitol 7) 0.15 g pimaricine 0.1 g bromthymol blue 0.025 g agar-agar 18.0 g distilled water 1 litre final pH-value: 6.9 - 0.1 36.18 g of the above-mentioned mixture, except for pimaricine, are suspended in 900 ml of distilled water, steeped, thoroughly mixed with the aid of a magnetic stirrer and subsequently heated on a boiling water-bath for approximately to 15 minutes. The suspension is cooled to 50°C and the pH-value is measured (6.8 - 0.1), The nutrient medium is then sterilized in an autoclave at 121°C for 15 minutes, cooled to 55°C and mixed with 100 ml of a 0.1% sterile-filtered pimaricine solution, thoroughly mixed with the aid of a magnetic stirrer. The nutrient medium may then be dispensed into sterile deep-drawn containers, 5.5 to 6.0 mis of nutrient medium being dispensed into each container. The container may now be sealed with a tightly fitting lid.

Example 2 Citrate-G-Nutrient Composition Trisodiumcitrate dihydrate mixture of salts of bile acids pimaricine glucose yeast extract sodium chloride magnesium sulfate heptahydrate ammonium dihydrogenphosphate sodium ammonium hydrogenphosphate tetrahydrate phenol red agar-agar distilled water .0 g 1.0 g 0.1 ε 0.05 g 0.25 g 5.0 g 0.2 g 0.2 g 0.8 g 0.02 g .0 g litre final pH-value: 7.1 - 0.1 IlSBaration: 27.52 g of the above-mentioned mixture, except for pimaricine are suspended in 900 ml of distilled water, steeped and - 32 20 44163 thoroughly mixed with the aid of a magnetic stirrer and, subsequently, heated for approximately 10 to 15 minutes on a boiling water-bath. The suspension is cooled to 50uC and the pH-value is measured (7.3 - 0.1). The nutrient medium is then sterilized at 121 °C in an autoclave for 15 minutes, cooled to 55°C and thoroughly mixed with 100 ml of a 0.1% pimaricine solution, with the aid of a magnetic stirrer.

The nutrient medium may then be dispensed into sterile deepdrawn containers. 5.5 to 6.0 mis of nutrient medium being dispensed into each container. The container may now be sealed with a tightly fitting lid.

Example 3 Cadmium-Nutrient Composition Proteose peptone 20.0 g sodium chloride 1.2 g dipotassium hydrogen phosphate 0.8 g ' potassium nitrate 0.6 g calcium nitrate tetrahydrate 0.4 g 20 magnesium sulfate heptahydrate 0.6 g cadmium sulfate octahydrate 0.005 g glycerin 8.0 ml ampholyte KKPK 55 (N-alkyl-aminocrotonic acid) 0.5 g agar-agar 15.0 g 25 distilled water final pH-value: 7.2 T Q.l 1 litre - 33 4 416 9 Preparation: 0.50 g of ampholyte and 8 ml of glycerine are Well suspended in 1000 ml of distilled water with the aid of a magnetic stirrer. The suspension is mixed with 48.6 g of the remain5 ing components of the nutrient medium and then heated for 10 to 15 minutes on a boiling water-bath. The mixture is cooled to 50°C and the pH-value is measured (6.9 - 0.1), The nutrient medium is then sterilized for 15 minutes at 121 °C in an autoclave and cooled to 50°C.

The nutrient medium may then be dispensed into sterile deepdrawn containers, 5.5 to 6.0 mis of nutrient medium being dispensed into each container. The container may now be sealed with a tightly fitting lid.

Example 4 Phenylalanine-jd thi um-G-Nutrient Composition Proteose peptone 10.0 g yeast extract 3.0 g sodium chloride 5.0 g disodium hydrogen phosphate 5.6 g potassium hydrogen phosphate 0.2 g L-phenylalanine 2.0 g mixture of salts of bile acids 1.0 g lithium chloride 2.0 g pimaricine 0.2 g agar-agar 16.0 g distilled water 1 litre final pH-value: 7.2 - 0.1 Preparation 44.8 g of the above-mentioned mixture, except for pimaricine, are thoroughly suspended in 900 ml of distilled water with the aid of a magnetic stirrer and heated for 10 to 15 minutes on a boiling water-bath. The suspension is cooled to 50°C and the pH-value is measured (7.3 - 0.1). The nutrient medium is then sterilized at 121°C in an autoclave for 15 minutes, cooled to 55°C and thoroughly mixed with 100 ml of a 0.2% sterile filtered pimaricine solution, with the aid of a magnetic stirrer.

The nutrient medium may then be dispensed into sterile deepdrawn containers, 5,5 to 6.0 mis of nutrient medium being dispensed into each container. The container may now be sealed with a tightly fitting lid.

Example 5 DNase-Nutrient Composition Tryptose desoxyribonucleic acid 20 sodium chloride ampholyte KKPK 55 methyl green agar-agar distilled water final pH-value 7.3 - 0.1 16.0 g 1.6 g 4.0 g 0.15 g 0.04 g 12.0 g litre “ 35 441-69 Preparation 0.15 g of ampholyte are thoroughly suspended in 1000 ml of distilled water with the aid of a magnetic stirrer, mixed with 33.6 g of the remaining substances, and then heated for 10 to 15 minutes on a boiling water-bath. The suspension is cooled to 50°C and the pH-value measured (7.0 - 0.1). The nutrient medium is then sterilized for 15 minutes in an auto clave and cooled to 50°C.

The nutrient medium may then be dispensed into sterile deepdrawn containers, 5.5 to 6.0 mis of nutrient medium being dispensed into each container. The container may now be sealed with a tightly fitting lid.

Example 6 Mannitol-Thiocyanate-Nutrient Composition Casein peptone meat extract gelatin D-mannitol yeast extract sodium pyruvate disodium hydrogen phosphate lithium chloride sodium chloride .0 g .0 g 5.0 g .0 g .0 S .0 g 6.0 g 5.0 g 5.0 g 416 9 potassium thiocyanate phenol red pimaricine agar-agar distilled water .0 g 0.02 g 0.2 g 16.0 g litre final pH-value 7.2 ί 0.1 Preparation 107.02 g of the hydrated nutrient medium are thoroughly suspended in 900 ml of distilled water with the aid of a mag10 netic stirrer and heated for approximately 10 to 15 minutes on a boiling water-bath. The suspension is cooled to 50°C and the pH-value is adjusted by means of a 10% sodium hydroxide solution to 7.4. The nutrient medium is then sterilized in an autoclave at 121°C for 15 minutes, cooled to 50°C and then mixed with 100 mis of a 0.2% solution of pimaricine in water.

The nutrient medium may then be dispensed into sterile deepdrawn containers, 5.5 to 6.0 mis of nutrient medium being dispensed into each container. The container may now be sealed with a tightly fitting lid.

Example 7 T.T.C,-Nutrient Composition Tryptose 20.0 g yeast extract 5.0 g glucose 2.0 g disodium hydrogen phosphate 4.0 g sodium thiosulfate 0.3 g α-lipoic acid 0.001 g Agar-Agar 15.0 g pimaricine 0.1 g 2,3,5-triphenyltetrazolium chloride (T.T.C.) 0.1 g sodium azide 0.8 g distilled water 1 litre final pH-value: 7.2-0.1 Preparation 46.3 g of the above-mentioned mixture, except ; for pimaricine, 2,3,5-triphenyltetrazolium chloride and sodium azide, are suspended in 890 ml of distilled water, steeped and thoroughly mixed with the aid of a magnetic stirrer. The mixture is then heated for 10 to 15 minutes on a boiling water-bath.

The suspension is cooled to 50°C and the pH-value adjusted to 7.2. Subsequently, the medium is sterilized for 15 minutes 441G9 at 121°C in an autoclave and cooled Lo 50°C. The following solutions, in sterile filtered condition, are then added: 100 ml of a 0.1% pimaricine solution, 2.5 ml of a 4% T.T.C.solution and 10 ml of a 4% sodium azide solution. All solutions should be prepared 30 minutes before addition to the remaining nutrient medium constituents at the outside, and should be shelved protected from the light. The mixture is stirred thoroughly with a magnetic stirrer.

The nutrient medium may then be dispensed into sterile deepdrawn containers, 5.5 to 6,0 mis of nutrient medium being dispensed into each container. The container may now be sealed with a tightly fitting lid. Example 8 Peptone-Thiocyanate-Nutrient Composition Vegetable protein hydrolysate (Mycopeptone B) liver hydrolysate glucose yeast extract sodium chloride magnesium sulfate-heptahydrate manganese(il)chloride-tetrahydrate sodium heptadecyl sulfate (Tergitol 7) potassium thiocyanate agar-agar distilled water final pH-value: 5.8 - 0.1 .0 g 1.0 g 5.0 g 3.0 g 3.0 g 0.25 g 0.001 g 0.05 g .0 g 22.0 g litre ά 16 9 Preparation 69.3 g of the above-mentioned mixture are suspended in I ltr. of distilled water, steeped, mixed together with the aid of a magnetic stirrer and subsequently heated on a boiling water-bath for 10 to 15 minutes. The solution is cooled to 4. 50ϋ0 and the pH-value adjusted to 5,8 - 0.1. The medium is then sterilized at 121°C in an autoclave for 15 minutes, and cooled to 50°C.

The nutrient medium may then be dispensed into sterile deep10 drawn containers, 5.5 to 6.0 mis of nutrient medium being, dispensed into each container. The container may now be sealed with a tightly fitting lid.

The pimaricine contained in some of the media described herein may, of course, be replaced wholly or in part by another material with similar characteristics of inhibiting Candida and other yeasts and. mould fungi while permitting the growth of the microorganisms to be tested for.

Claims (71)

1. A method for determining the genera of pathogens capable of causing urogenital tract infections which comprises inoculating each of the following nutrient media: 5 Lactose-P-Nutrient (as herein defined), Citrate-G-Nutrient (as herein defined), Cadmium Nutrient (as herein defined), Phenylalanine-lithium-G-Nutrient (as herein defined), DNase Nutrient (as herein defined), 10 Mannitol-Thiocyanate Nutrient (as herein defined), T.T.-Nutrient (as herein defined), Peptone-Thiocyanate Nutrient (as herein defined), with a sample of the said pathogen, incubating each of the nutrient media, and determining the genera of the said 15 pathogen by evaluation of the growth if any on each of the nutrient media.

2. A method as claimed in claim 1 wherein lactose-Pnutrient (as herein defined) contains bromothymol blue as indicator. 20

3. A method as claimed in claim 1 or claim 2 wherein Lactose-P-nutrient (as herein defined) contains an anionic long chain alkyl sulfate as the anionic surfactant.

4. A method as claimed in claim 3 wherein the Lactose-P- 41 4416° nutrient comprises a heptadecyl sulfate as anionic surfactant.

5. A method as claimed in claim 4 wherein the heptadecyl sulfate is sodium heptadecyl sulfate.

6. A method as claimed in any of the preceding claims wherein Lactose-P-nutrient comprises lactose, polypeptone, yeast extract, sodium heptadecyl sulfate, pimaricine, bromothymol blue, agar-agar and deionised or distilled water.

7. A method as claimed in any of the preceding claims wherein Lactose-P-nutrient has the composition:- Lactose about 10.0 g polypeptone about 5.0 g yeast extract about 3.0 g sodium heptadecyl sulfate about 0.15g pimaricine about 0.1 g bromothymol blue about 0.025 g agar-agar about 18.0 g per litre of deionised or distilled water.

8. A method as claimed in any of the preceding claims wherein Citrate-G-nutrient (as herein defined) additionally comprises yeast extract and glucose.

9. A method as claimed in any of the preceding claims wherein the water-soluble citrate is sodium or potassium citrate.

10. A method as claimed in claim 9 wherein the watersoluble citrate is trisodium citrate dihydrate.

11. A method as claimed in any of the preceding claims wherein Citrate-G-nutrient (as herein defined) contains phenol red as indicator.

12. A method as claimed in any of the preceding claims wherein Citrate-G-nutrient (as herein defined) comprises trisodium citrate dihydrate, a mixture of salts of bile acids, pimaricine, glucose, yeast extract, sodium chloride, 10 hydrated magnesium sulfate, ammonium dihydrogen phosphate, phenol red, agar-agar and deionised or distilled water.

13. A method as claimed in any of the preceding claims wherein Citrate-G-nutrient (as herein defined) has the composition:- Trisodium citrate dihydrate about 5.0 g mixture of salts of bile acids about 1.0 g pimaricine about 0.1 g glucose about 0.05 g yeast extract about 0.25 g sodium chloride about 5.0 g magnesium sulfate heptahydrate about 0.2 g ammonium dihydrogen phosphate about 0.2 g sodium ammonium hydrogen phosphate tetrahydrate about 0.8 g phenol red about 0.02 g agar-agar about 15.0 g per litre of deionised or distilled water. 5

14. A method as claimed in any of the preceding claims wherein the Cadmium nutrient (as herein defined) comprises cadmium sulfate.

15. A method as claimed in claim 14 wherein the Cadmium nutrient (as herein defined) comprises proteose peptone, 10 sodium chloride, dipotassium hydrogen phosphate, potassium nitrate, hydrated calcium nitrate, hydrated magnesium sulfate, hydrated cadmium sulfate, glycerine, an N-alkylaminocrotonic acid, agar-agar and deionised or distilled water. 15 16. A method as claimed in any of the preceding claims wherein Cadmium nutrient (as herein defined) has the composition:-

Proteose peptone about 20.0 sodium chloride about 1.2 dipotassium hydrogen phosphate about 0,8 potassium nitrate about 0.6 calcium nitrate-tetrahydrate about 0.4 magnesium sulfate-heptahydrate about 0.6 g cadmium sulfate-octahydrate about 0.005g glycerine about 8.0 ml N-alkyl-aminocrotonic acid (Ampholyte KKPK 55) about 0.5 g agar-agar about 15.0 g per litre of deionised or distilled water.

17. A method as claimed in any of the preceding claims wherein Phenylalanine-lithium-G-nutrient (as herein defined) 10 comprises L-phenylalanine.

18. A method as claimed in claim 17 wherein the watersoluble lithium salt is lithium chloride.

19. A method as claimed in claim 18 wherein Phenylalaninelithium-G-nutrient (as herein defined) comprises proteose 15 peptone, yeast extract, sodium chloride, disodium hydrogen phosphate, potassium hydrogen phosphate, L-phenylalanine, a mixture of salts of bile acids, lithium chloride, pimaricine, agar-agar and deionised or distilled water.

20. A method as claimed in claim 19 wherein Phenylalanine20 lithium-G-nutrient (as herein defined) has the composition:- Proteose peptone about 10.0 g yeast extract about 3.0 g sodium chloride about 5.0 g 4 410 9 disodium hydrogen phosphate about 5.6 g potassium hydrogen phosphate about 0.2 g L-phenylalanine about 2.0 g mixture of salts of bile acids about 1.0 g lithium chloride about 2.0 g pimaricine about 0.2 g agar-agar about 16.0 g per litre of deionised or distilled water.

21. A method as claimed in any of the preceding claims 1q wherein the DNase-nutrient (as herein defined) comprises an indicator.

22. A method as claimed in claim 21 wherein the indicator is methyl green.

23. A method as claimed in any of claims 1 to 20 wherein 15 DNase-nutrient (as herein defined) comprises tryptose, desoxyribonucleic acid, sodium chloride, an N-alkyl-aminocrotonic acid, agar-agar and deionised or distilled water.

24. A method as claimed in claim 23 wherein DNase-nutrient (as herein defined) has the composition:- 20 Tryptose about 16.0 g desoxyribonucleic acid about 1.6 g sodium chloride about 4.0 g N-alkyl-aminocrotonic acid about 0.15 g agar-agar about 15.0 g per litre of deionised or distilled water.

25. A method as claimed in claim 24 wherein the DNase5 nutrient (as herein defined) contains about 0.04 g of methyl green per litre of deionised or distilled water.

26. A method as claimed in any of the preceding claims wherein Mannitol-thiocyanate nutrient (as herein defined) contains a water-soluble lithium salt. 10

27. A method as claimed in any of the preceding claims wherein Mannitol-thiocyanate nutrient (as herein defined) contains a water-soluble potassium salt.

28. A method as claimed in any of the preceding claims wherein Mannitol-thiocyanate nutrient (as herein defined) 15 contains a water-soluble chloride.

29. A method as claimed in any of the preceding claims wherein Mannitol-thiocyanate nutrient (as herein defined) contains potassium thiocyanate.

30. A method as claimed in any of the preceding claims 20 wherein Mannitol-thiocyanate nutrient (as herein defined) contains a water-soluble sodium salt.

31. A method as claimed in any of the preceding claims wherein Mannitol-thiocyanate nutrient (as herein defined) - 47 441G9 contains casein peptone, meat extract, gelatin, D-mannitol, yeast extract, sodium pyruvate, disodium hydrogen phosphate, lithium chloride, sodium chloride, potassium thiocyanate, a pH indicator, pimaricine, agar-agar and deionised or 5 distilled water.

.32. A method as claimed in any of the preceding claims wherein the pH indicator in Mannitol-thiocyanate nutrient (as herein defined) is phenol red.

33. A method as claimed in any of the preceding claims 10 wherein Mannitol-thiocyanate nutrient (as herein defined) the composition:- Casein peptone about 10.0 g meat extract about 5.0 g gelatin . about 5.0 g D-mannitol about 10.0 g yeast extract about 5.0 g sodium pyruvate about 10.0 g disodium hydrogen phosphate about 6.0 g lithium chloride about 5.0 g sodium chloride about 5.0 g potassium thiocyanate about 30.0 g phenol red about 0.02 g pimaricine about 0.2 g agar-agar about 16.0 g per litre of deionised or distilled water.

34. A method as claimed in any of the preceding claims 5 wherein T.T.-nutrient contains a reducing agent.

35. A method as claimed in claim 34 wherein T.T.-nutrient (as herein defined) contains sodium thiosulfate.

36. A method as claimed in any of the preceding claims wherein T.T,-nutrient (as herein defined) contains 2,3,510 triphenyltetrazolium chloride.

37. A method as claimed in any of the preceding claims wherein T.T.-nutrient (as herein defined) contains an alkali metal azide.

38. A method as claimed in claim 37 wherein the alkali metal 15 azide is sodium azide.

39. A method as claimed in any of the preceding claims wherein T.T.-nutrient contains tryptose, yeast extract, glucose, disodium hydrogen phosphate, sodium thiosulfate, α-lipoic acid, agar-agar, pimaricine,’ 2,3,5-triphenyltetra20 zolium chloride, sodium azide and deionised or distilled water.

40. A method as claimed in claim 39 wherein T.T.-nutrient has the composition:49 Tryptose about 20.0 g yeast extract about 5.0 g glucose about 2.0 g disodium hydrogen phosphate about 4.0 g sodium thiosulfate about 0.3 g α-lipoic acid about O.OOlg agar-agar about 15.0 g pimaricine about 0.1 g 2,3,5-triphenyltetrazolium chloride (T.T.C.) about 0.1 g sodium azide about 0.8 g per litre of deionised or distilled water.

41. A method as claimed in any of the preceding claims wherein Peptone-thiocyanate-nutrient (as herein defined) contains sodium thiocyanate. 15

42. A method as claimed in any of the preceding claims wherein Peptone-thiocyanate-nutrient (as herein defined) contains an anionic long chain alkyl sulfate as surfactant.

43. A method as claimed in any of the preceding claims wherein Peptone-thiocyanate-nutrient (as herein defined) 20 contains a heptadecyl sulfate.

44. A method as claimed in claim 43 wherein the heptadecyl sulfate is sodium heptadecyl sulfate.

45. A method as claimed in any of the preceding claims wherein the pH of Peptone-thiocyanate-nutrient (as herein defined) is about 5.8.

46. A method as claimed in any of the preceding claims 5 wherein Peptone-thiocyanate-nutrient (as herein defined) contains vegetable protein hydrolysate, liver hydrosylate, glucose, yeast extract, sodium chloride, magnesium sulfate, manganese (II) chloride, sodium heptadecyl sulfate, potassium thiocyanate, agar-agar and deionised or distilled water. 10 47. A method as claimed in claim 46 wherein Peptone-thiocyanate-nutrient (as herein defined) has the composition:-

Vegetable protein hydrolysate about 15.0 g liver hydrolysate about 1.0 g glucose about 5.0 g 15 yeast extract about 3.0 g sodium chloride about 3.0 g magnesium sulfate heptahydrate about 0.25g manganese (II) chloride tetrahydrate about O.OOlg sodium heptadecyl sulfate about 0.05 g 20 potassium thiocyanate about 20.0 g agar-agar about 22.0 g per litre of deionised or distilled water. 441G9

48. A method as claimed in any of the preceding claims wherein the pathogens proteus and providencia are identified by the phenylalanine desaminase test on the incubated sample of pathogen. 5

49. A method as claimed in claim 48 wherein the pathogens proteus and providencia are distinguished by pH measurement in order to detect presence of ammonia.

50. A method as claimed in any of claims 1-47 wherein the pathogen serratia is identified by adding dilute acid to the 10 incubated nutrient medium.

51. A method as claimed in any one of the preceding claims ' substantially as herein described.

52. A kit for determining the genera of pathogens causing urogenital tract infections which comprises in combination 15 each of the following nutrient media:Citrate-G-nutrient (as herein defined), Phenylalanine-G-nutrient (as herein defined), Mannitol-thiocyanate-nutrient (as herein defined), and Peptone-thiocyanate-nutrient (as herein defined) 20 each of said nutrient media being retained in a sterile container.

53. A kit as claimed in claim 52 which also contains lactose P-nutrient (as herein defined), Cadmium-nutrient (as herein defined), DNase-nutrient (as herein defined) and/or T.T.nutrient (as herein defined).

54. A kit as claimed in claim 52 or claim 53 which comprises 5 a plurality of sterile containers, each container being provided with a closure member for sealing the container and each container having therein one of the said nutrient media.

55. A kit as claimed in claim 54 wherein the container is of a plastics material. 10

56. A kit as claimed in claim 55 wherein the containers are formed by deep drawing.

57. A kit as claimed in claim 52 or claim 53 which comprises a holder divided into a plurality of compartments adapted to receive a plurality of different nutrient media, 15 each compartment retaining a different nutrient medium in sterile condition.

58. A kit as claimed in claim 52 or claim 53 wherein each nutrient medium is contained in an individual sterile sealed package. 20

59. A kit as claimed in any one of claims 53 to 58 wherein the Lactose-P-nutrient is as defined in any one of claims 2-7. 441 69

60. A kit as claimed in any one of claims 52 to 59 wherein the Citrate-G-nutrient is as defined in any one of claims 8-13.

61. A kit as claimed in any one of claims 52 to 60 wherein 5 the Cadmium-nutrient is as defined in any one of claims 14. -16.

62. A kit as claimed in any one of claims 52 to 61 wherein the Phenylalanine-lithium-G-nutrient is as defined in any one of claims 17-20. 10

63. A kit as claimed in any one of claims 52 to 62 wherein the DNase-nutrient is as defined in any one of claims 21-25.

64. A kit as claimed in any one of claims 52 to 63 wherein the Mannitol-thiocyanate-nutrient is as defined in any one of claims 26-33. 15.

65. A kit as claimed in any one of claims 52-64 wherein the Τ.T.-nutrient is as defined in any one of claims 34-40.

66. A kit as claimed in any one of claims 52 to 65 wherein the Peptone-thiocyanate-nutrient is as defined in any one of claims 41-47. 2o

67. A kit as claimed in claim 52 substantially as herein described.

68. Citrate-G-nutrient (as herein defined). - 54 44169

69. Phenylalanine-lithium-G-nutrient (as herein defined).

70. Mannitol-thiocyanate-nutrient (as herein defined).

71. Peptone-thiocyanate-nutrient (as herein defined).