DK155159B - Apparatus and process for culturing microorganisms - Google Patents
Apparatus and process for culturing microorganisms Download PDFInfo
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- DK155159B DK155159B DK045881AA DK45881A DK155159B DK 155159 B DK155159 B DK 155159B DK 045881A A DK045881A A DK 045881AA DK 45881 A DK45881 A DK 45881A DK 155159 B DK155159 B DK 155159B
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Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
DK 155159 SDK 155159 S
iin
Opfindelsen angår et apparat til dyrkning af mikroorganismer af den i krav l's indledning angivne art. Opfindelsen angår også en fremgangsmåde til dyrkning af mikroorganismer under anvendelse af det omhandlede appa-5 rat, idet fremgangsmåden ikke omfatter dyrkning af virus.The invention relates to an apparatus for growing microorganisms of the kind set out in the preamble of claim 1. The invention also relates to a method for culturing microorganisms using the apparatus according to the invention, the method not comprising cultivating viruses.
Hidtil er dyrkningen af anaerobe mikroorganismer blevet udført i dyrkningsbeholdere, der er fuldstændigt forseg-10 lede med elastiske propper, som f.eks. gummipropper, skruelåg, kronlukker eller lignende.Heretofore, the cultivation of anaerobic microorganisms has been carried out in culture containers which are completely sealed with elastic plugs, e.g. rubber stoppers, screw caps, crown closures or the like.
Når man ved dyrkningen af anaerobe mikroorganismer fører en injektionspodenål gennem proppen for at overføre mi-15 kroorganismer efter dyrkningen, har dyrkningssubstratet en tendens til at sprøjte gennem nålen på grund af det frembragte indre tryk. Yderligere har skruelåg, kronlukker eller lignende en tendens til at ryge af, når beholderne flyttes, således at laboratoriepersonalet ofte er 20 udsat for fare. Når sådanne uheld sker, kommer den ydre luft i kontakt med dyrkningssubstratet, hvorved bakterier fra luften kan forurene substratet.When culturing anaerobic microorganisms, an injection graft needle is passed through the plug to transfer microorganisms after culture, the culture substrate tends to spray through the needle because of the internal pressure generated. Furthermore, screw caps, crown closures or the like tend to smoke when the containers are moved, so that the laboratory staff is often at risk. When such accidents occur, the outside air comes into contact with the culture substrate, whereby bacteria from the air can contaminate the substrate.
Dersom man anvender propper med simple fastgørelsesdele, 25 kan propperne ryge af under dyrkningen på grund af det forøgede indre tryk i dyrkningsbeholderen, der frembringes af de gasarter, der udvikles af de anaerobe mikroorganismer, hvorved dyrkningssubstratet bliver forurenet med bakterier fra luften. Yderligere bliver omgivelserne 30 forurenet med anaerobe organismer, der spredes rundt omkring, når propperne ryger af.If plugs with simple fasteners are used, the plugs may fume during cultivation due to the increased internal pressure in the culture vessel produced by the gases produced by the anaerobic microorganisms, thereby causing the culture substrate to be contaminated with bacteria from the air. Further, the environment 30 becomes contaminated with anaerobic organisms which spread around as the plugs smoke.
Ved dyrkningen af aerobe mikroorganismer har man anvendt dyrkningsbeholdere, der er lukket med propper, der er 35 2In the cultivation of aerobic microorganisms, culture containers have been used which are closed with plugs which are 35 2
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permeable for luften, som f.eks. Morton-lukker, vatpropper, aluminiumlåg, papirkuglepropper eller lignende.permeable to the air, e.g. Morton shutter, cotton stopper, aluminum cap, paper ball plug or the like.
Dyrkningen af aerobe mikroorganismer har imidlertid forskellige ulemper: Dyrkningssubstratet kan ikke lagres 5 gennem længere tid på grund af proppernes luftpermeabi-litet; substratet forurenes let med bakterier fra luften, dersom proppen tages af, når man udtager prøver; det er vanskeligt at transportere beholderne på grund af udsivning af substratet og laboratoriepersonalet er ud-10 sat for fare på grund af denne udsivning.However, the cultivation of aerobic microorganisms has various disadvantages: The culture substrate cannot be stored for a long time due to the air permeability of the plugs; the substrate is easily contaminated with bacteria from the air if the plug is removed when sampling; it is difficult to transport the containers due to leakage of the substrate and the laboratory personnel are at risk because of this leakage.
Ved almindelige dyrkningsmetoder er det nødvendigt at fjerne propperne, når man udtager prøver. Dette gør imidlertid, at bakterier fra luften kan trænge ind i 15 dyrkningsbeholderen, således at bakterier udefra inkuberes sammen med de dyrkede mikroorganismer. Dette bevirker, at det sommetider er vanskeligt at skelne de dyrkede organismer fra de forurenende bakterier.In common cultivation methods it is necessary to remove the plugs when sampling. However, this allows bacteria from the air to enter the culture vessel so that bacteria from the outside are incubated with the cultured microorganisms. This makes it sometimes difficult to distinguish the cultured organisms from the polluting bacteria.
20 Når aerobe og anaerobe mikroorganismer dyrkes under anvendelse af samme dyrkningsbeholdere eller -apparater, er der yderligere de problemer, at dersom man anvender de ovenfor nævnte lufttætte propper, forhindres propageringen af de aerobe mikroorganismer, og dersom der an-25 vendes luftgennemtrængelige propper, forhindres propageringen af anaerobe mikroorganismer. Det har derfor været nødvendigt at anvende specielle dyrkningsapparater til dyrkning af henholdsvis aerobe og anaerobe mikroorganismer. Dyrkningen bliver besværlig, og det er vanskeligt 30 eller umuligt at udføre dyrkningen på en rationel, sikker og korrekt måde.When aerobic and anaerobic microorganisms are grown using the same culture containers or apparatus, there are further problems that using the above airtight plugs prevents the propagation of the aerobic microorganisms and if air-permeable plugs are used the propagation of anaerobic microorganisms. It has therefore been necessary to use special culture apparatus for growing aerobic and anaerobic microorganisms, respectively. Cultivation becomes cumbersome and it is difficult or impossible to perform the cultivation in a rational, safe and correct manner.
USA patentskrift nr. 3 033 408 omhandler et dyrkningsapparat således som beskrevet i indledningen til krav 1.U.S. Patent No. 3,033,408 discloses a culture apparatus as described in the preamble of claim 1.
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Dette kendte apparat er udstyret med et lukke, hvor en gummiprop er kombineret med en vatprop udelukkende til anvendelse ved aerob dyrkning. Vattet eller lignende materiale anvendes som et filter til opretholdelse af ste-5 rilitet. Det kendte apparat er særdeles kompliceret opbygget, og det er derfor også besværligt at fremstille.This known apparatus is provided with a closure wherein a rubber stopper is combined with a cotton stopper solely for use in aerobic cultivation. The wadding or similar material is used as a filter to maintain stability. The known apparatus is very complicated in structure and therefore it is also difficult to manufacture.
Opfindelsen har til hensigt at tilvejebringe et apparat til dyrkning af mikroorganismer, der muliggør, at den 10 indre atmosfære bibeholdes hermetisk forseglet inden dyrkningen af mikroorganismerne, medens de frembragte gasarter under dyrkningen kan bortledes, samtidig med at man bibeholder dyrkningsmiljøet.The invention aims to provide an apparatus for growing microorganisms which allows the inner atmosphere to be hermetically sealed before the cultivation of the microorganisms, while the gases produced during cultivation can be dissipated while maintaining the culture environment.
15 Opfindelsen tilvejebringer således et apparat, der er velegnet til dyrkning af aerobe eller anaerobe mikroorganismer.Thus, the invention provides an apparatus suitable for growing aerobic or anaerobic microorganisms.
Desuden er det omhandlede apparat til dyrkning af mikro-20 organismer simpel i opbygning og let at håndtere.In addition, the apparatus for growing micro-organisms is simple in structure and easy to handle.
Apparatet er i følge opfindelsen ejendommeligt ved det i krav l's kendetegnende del angivne.According to the invention, the apparatus is peculiar to the characterizing part of claim 1.
25 Herudover tilvejebringer opfindelsen fremgangsmåder til dyrkning af aerobe og anaerobe mikroorganismer under anvendelse af apparatet. Fremgangsmåderne er ejendommelige ved det i den kendetegnende del af krav 10 og 11 angivne.In addition, the invention provides methods for growing aerobic and anaerobic microorganisms using the apparatus. The methods are peculiar to that of the characterizing part of claims 10 and 11.
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Dyrkningssubstratet kan være et, hvori man kan dyrke enten aerobe eller anaerobe mikroorganismer, men substrater, hvor man både kan dyrke aerobe og anaerobe mikroorganismer, foretrækkes.The culture substrate may be one in which either aerobic or anaerobic microorganisms can be grown, but substrates where both aerobic and anaerobic microorganisms can be grown are preferred.
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Opfindelsen forklares nærmere under henvisning til tegningen, hvor fig. 1 er et delvist snitbillede, set fra siden, af et 5 omhandlet apparat til dyrkning af mikroorganis mer, og fig. 2 er et delvis snit, set fra siden, der viser en tilpropningsdel, der indgår i det omhandlede ap-10 parat til dyrkning af mikroorganismer.The invention is explained in more detail with reference to the drawing, in which fig. 1 is a partial side elevational view of an apparatus for growing microorganisms; and FIG. 2 is a partial sectional side view showing a plug portion forming part of the apparatus for growing microorganisms.
Fig. 1 viser det omhandlede apparat til dyrkning af mikroorganismer med en beholder 1 med en åbning 3. Beholderen 1 kan være fremstillet af et hvilket som helst 15 passende materiale, f.eks. plast eller glas; imidlertid foretrækker man et transparent materiale, specielt glas, for at kunne se det indre af beholderen under inkubationen. Udformningen af beholderen er ikke begrænset til den i fig. 1 angivne rørform, men den kan være af en 20 hvilken som helst form, som f.eks. en flaske eller kolbe. Et ringformet fremspring 3a findes på den indre omkreds af den øverste del af beholderens åbning 3. Dette fremspring kan være dannet ved en hel række forskellige metoder, som f.eks. ved at opvarme den øverste del af 25 åbningen.FIG. 1 shows the apparatus for growing microorganisms with a container 1 having an opening 3. The container 1 may be made of any suitable material, e.g. plastic or glass; however, a transparent material, especially glass, is preferred in order to see the interior of the container during the incubation. The design of the container is not limited to that of FIG. 1, but it may be of any shape, e.g. a bottle or flask. An annular projection 3a is provided on the inner circumference of the upper portion of the opening 3 of the container. by heating the upper portion of the 25 aperture.
Beholderen 1 er lufttæt forseglet ved åbningen 3 ved hjælp af en tilpropningsdel 4. Tilpropningsdelen skal være uigennemtrængelig for luft og er fremstillet af et 30 for en nål gennemtrængeligt materiale, specielt af gum-miagtigt, elastisk materiale. Tilpropningsdelen 4 er udstyret, således som bedst vist i fig. 2, med en udsparing 4a i bunden, og udsparingen 4a danner en forholdsvis tynd ydre væg 6, der udgør tilpropningsdelens lege-35 5The container 1 is airtightly sealed at the opening 3 by means of a plug part 4. The plug part must be impermeable to air and is made of a needle-permeable material, especially of rubber-like elastic material. The plug part 4 is equipped, as best shown in FIG. 2, with a recess 4a at the bottom, and the recess 4a forms a relatively thin outer wall 6 constituting the play of the plug part 5
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me. Den ydre væg 6 passer lufttæt med åbningsdelen 3 af beholderen. Tilpropningsdelens legeme 6 er udformet ud i ét med en hoveddel 5, der har en lidt større diameter end selve legemet, og en ydre væg 5a af hoveddelen er 5 tilspidset bort fra legemet 6. Hoveddelen 5 er også udstyret med en udsparing 5c foroven, der reducerer hovedets tykkelse, således at den er lettere at gennemtrænge.Me. The outer wall 6 fits air tightly with the opening part 3 of the container. The body portion 6 of the plug is formed integrally with a body portion 5 having a slightly larger diameter than the body itself, and an outer wall 5a of the body portion 5 tapered away from the body 6. The body portion 5 is also provided with a recess 5c at the top which reduces the thickness of the head so that it is easier to penetrate.
10 Legemet 6 er udstyret med en gennemhulning 7, der står i forbindelse med den indre atmosfære i beholderen 1. Gen-nemhulningen 7 er, når den ses forfra, i form af en cirkel, der har en diameter på omtrent 0,3-3 mm eller en elipsoid form med en mindste akse på 0,3-2 mm og en 15 største akse på 2-3 mm. Der kan være adskillige gennem-hulninger 7 dannet i legemet 6. Fortrinsvis er symmetrisk anbragt to cirkulære huller, der hver har en diameter på 0,7-1,5 mm, med en afstand af 3-5 mm fra toppen af legemet 6. En ringformet rille 8 er dannet rundt om 20 legemet 6 under gennemhulningen 7.The body 6 is provided with a through-hole 7 which communicates with the inner atmosphere of the container 1. The through-hole 7, when viewed from the front, is in the form of a circle having a diameter of about 0.3-3. mm or an ellipsoid shape with a minimum axis of 0.3-2 mm and a 15 axis of 2-3 mm. There may be several holes 7 formed in the body 6. Preferably, two circular holes, each having a diameter of 0.7-1.5 mm, spaced 3-5 mm from the top of the body 6 are symmetrically arranged. An annular groove 8 is formed around the body 6 below the through-hole 7.
Tilpropningsdelen kan fremstilles ved at extrudere foropvarmet, gummiagtigt materiale under tryk i en form, hvorved der ved størkning dannes en formet tilpropning, 25 der endnu er uden nogen gennemhulning. Herefter laves hullet gennem sidevæggen med en varm nål, der er af forudbestemt størrelse og form, eller med en dorn eller lignende. Den således fremstillede tilpropning tørres herefter i luften eller ved opvarmning. Det er fordelag-30 tigt at overtrække tilpropningsdelene med en siliconolie for at lette indførslen i beholderen.The plug part can be made by extruding pre-heated rubbery material under pressure into a mold, thereby forming a molded plug, which is still without any through-hole. Next, the hole is made through the side wall with a hot needle of predetermined size and shape, or with a mandrel or the like. The plugging thus produced is then dried in the air or by heating. It is advantageous to coat the plug portions with a silicone oil to facilitate entry into the container.
Med henvisning igen til fig. 1 forefindes et substrat 9 til dyrkning af mikroorganismerne i beholderen 1. Sub-35Referring again to FIG. 1, there is a substrate 9 for growing the microorganisms in container 1. Sub-35
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6 stratet 9 kan være et dyrkningssubstrat, der er velegnet til dyrkning af anaerobe mikroorganismer, som f.eks. hjerne-hjerte-infusionssubstrat, således som beskrevet side 895 i "Manual of Clinical Microbiology", 2. udgave, 5 udgivet af Edwin H. Lennette, Earle H. Spaulding og Joseph P. Truant (American Society for Microbiology, 1975), thioglycollatsubstrat beskrevet af Brewer i J.The substrate 9 may be a culture substrate suitable for growing anaerobic microorganisms such as e.g. brain-heart infusion substrate, as described on page 895 of "Manual of Clinical Microbiology", 2nd Edition, 5 published by Edwin H. Lennette, Earle H. Spaulding, and Joseph P. Truant (American Society for Microbiology, 1975), thioglycol substrate described by Brewer in J.
Amer. Ass. 115, 598 (1940), brucella bouillonsubstrat beskrevet side 896 i "Manual of Clinical Microbiology", 10 2. udgave, eller et substrat, der er velegnet til dyrk ning af aerobe mikroorganismer, som f.eks. Columbia bouillonsubstrat, der fås ved at udelade agaren fra substratet, beskrevet af Ellner et al. i Amer. J. Clin. Pathol. 45, 502 (1966), næringsbouillonsubstrat beskrevet 15 side 912 i "Manual of Clinical Microbiology", 2.. udgave, trypticase-sojabouillonsubstrat, beskrevet på side 241 af "Shin Saikin Baichi Gaku Koza", vol. 1, af Saka-zaki (K.K. Kindai Shuppan, 1978). Til den foreliggende opfindelse foretrækkes et substrat, hvori man kan dyrke 20 både anaerobe og aerobe mikroorganismer. Sådanne substrater er velkendte af fagmanden og omfatter f.eks. det anaerobe basale dyrkningssubstrat formuleret af Gifu University, beskrevet side 338 i "Anaerobic Bacteria and Anaerobic Bacteria Disease" af Kosaki og Suzuki (Igaku 25 shoin, 1968). Sammensætningen af substrater, der er velegnede til anvendelse ved fremgangsmåden ifølge opfindelsen, er vist nedenfor som substrat (A) og (B).Amer. Ass. 115, 598 (1940), brucella broth substrate described on page 896 of "Manual of Clinical Microbiology", 2nd Edition, or a substrate suitable for the cultivation of aerobic microorganisms such as e.g. Columbia broth substrate obtained by omitting the agar from the substrate described by Ellner et al. in Amer. J. Clin. Pathol. 45, 502 (1966), Nutrient Bouillon Substrate described on page 912 of "Manual of Clinical Microbiology", 2nd Edition, trypticase soybean bouillon substrate, described on page 241 of "Shin Saikin Baichi Gaku Koza", vol. 1, by Saka-zaki. (KK Kindai Shuppan, 1978). For the present invention, a substrate in which one can grow both anaerobic and aerobic microorganisms is preferred. Such substrates are well known to those skilled in the art and include e.g. the anaerobic basal culture substrate formulated by Gifu University, described page 338 in "Anaerobic Bacteria and Anaerobic Bacteria Disease" by Kosaki and Suzuki (Igaku 25 Shoin, 1968). The composition of substrates suitable for use in the process of the invention is shown below as substrates (A) and (B).
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77
Substrat (A)Substrate (A)
Sammensætning (g/literComposition (g / liter)
Ingredienser destilleret vand)_ 5Ingredients distilled water) _ 5
Trypton 5 -20Trypton 5 -20
Sojapepton 1 - 5 Kødekstrakt 2 - 5 Gærekstrakt 3 - 10 10 Leverfordøjelsesprodukt 0,5 - 2Soy peptone 1 - 5 Meat extract 2 - 5 Yeast extract 3 - 10 10 Liver digestion product 0.5 - 2
Glucose 2-3Glucose 2-3
Kaliumphosphat, dibasisk 0,1 - 3Potassium phosphate, dibasic 0.1 - 3
Natriumchlorid 2 - 5 L-cysteinhydrochlorid 0,35 - 0,5 15 p-aminobenzoesyre 0,04 - 0,06Sodium Chloride 2 - 5 L-Cysteine Hydrochloride 0.35 - 0.5 p-Aminobenzoic Acid 0.04 - 0.06
Natriumpolyanetholsulfonat 0,25 - 0,35 Hæmin 0,003- 0,007Sodium polyanetholsulfonate 0.25 - 0.35 Heme 0.003 - 0.007
Agar 0 - 0,01Agar 0 - 0.01
Gelatine 10 - 14 20 pH 7,3 ± 0,1Gelatin 10 - 14 pH 7.3 ± 0.1
Substrat (B)Substrate (B)
Til substrat (A) sættes 1,5-2,5 g natriumbicarbonat pr.To substrate (A) is added 1.5-2.5 g of sodium bicarbonate per ml.
25 liter destilleret vand.25 liters of distilled water.
I substrat (A) og (B) kan polypepton anvendes i stedet for trypton, kødpepton i stedet for kødekstrakt, dibasisk natriumphosphat i stedet for dibasisk kaliumphos-30 phat og dextran i stedet for gelatine.In substrates (A) and (B), polypeptone can be used instead of tryptone, meat peptone instead of meat extract, dibasic sodium phosphate instead of dibasic potassium phosphate, and dextran instead of gelatin.
Sammensætningen af særligt velegnede substrater (substrat (C) og (D)) er angivet nedenfor.The composition of particularly suitable substrates (substrates (C) and (D)) is given below.
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Substrat (C)Substrate (C)
Trypton 17 gTryptone 17 g
Sojapepton 3 g Kødekstrakt 3 g 5 Gærekstrakt 5 gSoy peptone 3 g Meat extract 3 g 5 Yeast extract 5 g
Leverfordøjelsesprodukt 1 gLiver Digestive Product 1 g
Glucose 2,5 gGlucose 2.5 g
Kaliumphosphat, dibasisk 2,5 gPotassium phosphate, dibasic 2.5 g
Natriumchlorid 5 g 10 L-cysteinhydrochlorid 0,45 g p-aminobenzoesyre 0,05 gSodium chloride 5 g L-cysteine hydrochloride 0.45 g p-aminobenzoic acid 0.05 g
Natriumpolyanetholsulfonat 0,35 g Hæmin 0,005 gSodium polyanethole sulfonate 0.35 g Heme 0.005 g
Agar 0,01 g 15 Gelatine 12 gAgar 0.01 g 15 Gelatin 12 g
Destilleret vand 1000 ml pH 7,3 ± 0,1Distilled water 1000 ml pH 7.3 ± 0.1
Substrat (D) 20 Trypton 10 gSubstrate (D) 20 Tryptone 10 g
Sojapepton 3 g Kødekstrakt 3 gSoy peptone 3 g Meat extract 3 g
Leverfordøjelsesprodukt 1 g Gærekstrakt 5 g 25 Glucose 2,5 gLiver digestion product 1 g Yeast extract 5 g 25 Glucose 2.5 g
Natriumchlorid 4,0 g L-cysteinhydrochlorid 0,45 g p-aminobenzoesyre 0,05 gSodium chloride 4.0 g L-cysteine hydrochloride 0.45 g p-aminobenzoic acid 0.05 g
Natriumpolyanetholsulfonat 0,25 g 30 Natriumbicarboant 2 g Hæmin 0,005 gSodium polyanetholsulfonate 0.25 g Sodium bicarbonate 2 g Heme 0.005 g
Gelatine 12 gGelatin 12 g
Destilleret vand 1000 ml pH 7,3 ± 0,2 35 9Distilled water 1000 ml pH 7.3 ± 0.2 35 9
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Substraterne (A) - (D) fremstilles på følgende måde. Ingredienserne bortset fra gelatine afvejes og opløses i 500 ml destilleret vand. Den anførte mængde L-cysteinhy-drochlorid og hæmin opløses i henholdsvis 0,01 N saltsy-5 re og i vand, inden de tilsættes opløsningen. Herefter afvejes gelatinen for sig, og gelatinen opløses fuldstændigt i 500 ml destilleret vand under opvarmning.The substrates (A) - (D) are prepared as follows. The ingredients except gelatin are weighed and dissolved in 500 ml of distilled water. The indicated amount of L-cysteine hydrochloride and hemin are dissolved in 0.01 N hydrochloric acid and in water, respectively, before being added to the solution. The gelatin is then weighed separately and the gelatin dissolved completely in 500 ml of distilled water under heating.
Begge opløsninger blandes grundigt og afkøles til stuetemperatur, hvorefter der indstilles med alkalisk opløs-10 ning, som f.eks. 2 N vandig natriumhydroxidopløsning, til den angivne pH-værdi, og der filtreres gennem absorberende vat. Filtratet filtreres herefter på et glasfilter under reduceret tryk, og filtratet anvendes som substrat.Both solutions are thoroughly mixed and cooled to room temperature, then adjusted with alkaline solution such as, e.g. 2 N aqueous sodium hydroxide solution, to the specified pH, and filtered through absorbent cotton. The filtrate is then filtered on a glass filter under reduced pressure and the filtrate is used as a substrate.
1515
Det indvendige af beholderen 1 holdes på et forudbestemt reduceret tryk, dvs. et tryk, således at det muliggør, at en forudbestemt mængde væske indeholdende mikroorganismerne suges ind. Et lukke 10 til at lukke det ydre af 20 tilpropningsdelen 4 og beholderåbningen 3 påsættes, idet der er et lille mellemrum mellem dette og det nedre fremspring 5b af hoveddelen 6 af tilpropningsdelen 4.The interior of the container 1 is kept at a predetermined reduced pressure, i.e. a pressure so as to allow a predetermined amount of liquid containing the microorganisms to be sucked in. A closure 10 for closing the exterior of the plug portion 4 and the container opening 3 is provided, there being a small gap between this and the lower projection 5b of the main portion 6 of the plug portion 4.
Lukket 10 forhindrer ikke bevægelse af tilpropningsdelen 4 og kan være udformet af et hvilket som helst materia-25 le, f.eks. plast eller aluminium.The closure 10 does not prevent movement of the plug part 4 and may be formed of any material, e.g. plastic or aluminum.
Det omhandlede dyrkningsapparat med den ovennævnte udformning kan fremstilles på følgende måde: 30 Et 20 ml reagensglas (diameter 15,5 mm, længde 165 mm) eller en passende beholder i form af en kolbe eller flaske fyldes med en forudbestemt mængde af et substrat (18 ml til et 20 ml reagensglas eller 45 ml til en 50 ml kolbelignende eller flaskelignende beholder). Herefter 35 10The above-mentioned culture apparatus of the above design can be prepared as follows: A 20 ml test tube (diameter 15.5 mm, length 165 mm) or an appropriate container in the form of a flask or bottle is filled with a predetermined amount of a substrate (18 ml to a 20 ml tube or 45 ml to a 50 ml flask or bottle-like container). Hereafter 35 10
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udluftes beholderen under reduceret tryk, og en blandet gasart (nitrogen:carbondioxid = 9,1) indføres langsomt for at give et næsten normalt tryk. Herefter reduceres trykket for at muliggøre, at en forudbestemt mængde 5 prøvemateriale kan suges ind. Tilpropningsdelen påsættes beholderen for at forsegle under reduceret tryk. Herefter steriliseres beholderen ved høj temperatur, idet man anvender en højtryksdampsterilisator ved en temperatur på 115-121 °C og et tryk på 1,5-2 kg/cm2 i 15-20 minut-10 ter.the vessel is vented under reduced pressure and a mixed gas species (nitrogen: carbon dioxide = 9.1) is introduced slowly to give an almost normal pressure. Thereafter, the pressure is reduced to allow a predetermined amount of sample 5 to be sucked in. The plug part is applied to the container to seal under reduced pressure. Thereafter, the container is sterilized at high temperature using a high pressure steam sterilizer at a temperature of 115-121 ° C and a pressure of 1.5-2 kg / cm 2 for 15-20 minutes.
Dyrkningen af mikroorganismerne under anvendelse af det omhandlede apparat kan udføres ved først at udtage en flydende prøve indeholdende mikroorganismer, f.eks. fra 15 en patients blod og derefter indføre prøven i beholderen. I dette tilfælde er det bekvemt at anvende en holder til vakuumblodopsamlingsrør (fremstillet af Terumo Corporation). Holderen består af et hult legeme, der er i stand til at optage apparatet til dyrkning af mikroor-20 ganismer, og en nål til udtagning af en prøve, hvilken nål er fastgjort ved den yderste del af det hule legeme og udformet således, at den danner en nålespids i begge ender. Efter at tilpropningsdelen af apparatet er blevet desinficeret med f.eks. alkohol eller iodtinktur, indfø-25 res dyrkningsapparatet i holderen, der har prøveudtagningsnålen. En af prøveudtagningsnålenes spidser indst ikkes i patientens åre, og den anden spids indstikkes i beholderen gennem tilpropningsdelen ved at indstikke udstyret dybt i holderen, således at en forudbestemt 30 mængde blod indsuges i dyrkningsapparatet af det reducerede tryk i apparatet. I en anden udførelsesform kan en injektionsnål anvendes til formålet. I dette tilfælde anvendes injektionsnålen til at udtage en prøve, og den indføres herefter i centret af den desinficerede til-35 11The cultivation of the microorganisms using the present apparatus can be carried out by first sampling a liquid sample containing microorganisms, e.g. from a patient's blood and then insert the sample into the container. In this case, it is convenient to use a vacuum blood collection tube holder (manufactured by Terumo Corporation). The holder consists of a hollow body capable of receiving the apparatus for growing microorganisms, and a needle for sampling, which needle is attached to the outermost portion of the hollow body and shaped so that it forming a needle tip at both ends. After the plugging part of the apparatus has been disinfected with e.g. alcohol or iodine tincture, the culture apparatus is inserted into the holder having the sampling needle. One tip of the sampling needle is inserted into the patient's vein, and the other tip is inserted into the container through the plug portion by inserting the equipment deep into the holder so that a predetermined amount of blood is sucked into the culture apparatus by the reduced pressure in the apparatus. In another embodiment, an injection needle may be used for the purpose. In this case, the injection needle is used to take a sample and is then inserted into the center of the disinfected tube.
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propningsdel af dyrkningsapparatet. Da dyrkningsappara-tet bibeholdes under reduceret tryk, suges en forudbestemt mængde af prøven automatisk fra injektionssprøjten og føres over i apparatet ved sugning.plug part of the cultivator. As the culture apparatus is maintained under reduced pressure, a predetermined amount of the sample is automatically sucked from the syringe and transferred to the apparatus by suction.
55
Efter at prøven er opsamlet og suget ind i apparatet, inkuberes den anaerobe kultur ved 27-37 °C i 1-14 dage.After the sample is collected and sucked into the apparatus, the anaerobic culture is incubated at 27-37 ° C for 1-14 days.
Dersom det er nødvendigt, kan inkuberingen yderligere fortsættes. Frembragte gasarter kan let bortledes ved at 10 dreje tilpropningsdelen én omdrejning for at forøge gli-deevnen af propdelen.If necessary, further incubation can be continued. Generated gases can be easily discharged by rotating the plug part one turn to increase the slippage of the plug part.
Når man dyrker anaerobe kulturer, frembringer i det mindste en del anaerobe mikroorganismer, der er indført 15 uden at bryde den hermetiske forsegling, gasarter under inkuberingen, hvilket forøger det indre tryk af beholderen 1, fordi gennemhulningen 7 er lukket med den indre væg af beholderen. Når trykket forøges over et vist niveau, vil det tvinge tilpropningsdelen op til et punkt, 20 hvor gennemhulningen 7 når eller let overskrider toppen af åbningen 3 af beholderen 1. Herved vil de frembragte gasarter i beholderen bortledes gennem gennemhulningen 7 til det ydre af beholderen, og tilpropningsdelen standser der. Selv om tilpropningsdelen 4 eventuelt skulle 25 blive tvunget yderligere op af de frembragte gasarter, vil det ringformede fremspring 3a af beholderåbningen passe ind i den ringformede rille 8, der findes rundt om legemet 6, hvorved yderligere bevægelse af tilpropningsdelen 4 standses. Efter at gasarterne er bortledet, kan 30 tilpropningsdelen 4 tvinges ned manuelt for at lukke gennemhulningen 7 med den indre væg af åbningsdelen af dyrkningsbeholderen 1, således at beholderen 1 atter holdes lufttæt tillukket. Således forhindrer det omhandlede apparat, der muliggør bortledning af frembragte 35When cultivating anaerobic cultures, at least some anaerobic microorganisms introduced without breaking the hermetic seal generate gases during the incubation, which increases the internal pressure of the container 1 because the through-hole 7 is closed with the inner wall of the container. . As the pressure increases above a certain level, it will force the plug portion up to a point where the bore 7 reaches or slightly exceeds the top of the aperture 3 of the container 1. The gases produced in the container thereby are discharged through the bore 7 to the outside of the container. and the plug part stops there. Although the plug portion 4 should optionally be forced further by the gases produced, the annular projection 3a of the container opening will fit into the annular groove 8 found around the body 6, thereby stopping further movement of the plug portion 4. After the gases have been drained, the plug part 4 can be forced down manually to close the through-hole 7 with the inner wall of the opening part of the culture container 1, so that the container 1 is again kept airtight closed. Thus, the present apparatus prevents the discharge of generated 35
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12 gasarter, medens der samtidigt opretholdes en i det væsentlige lufttæt tilstand, at tilpropningsdelen 4 ikke ryger af under eller efter inkuberingen på grund af de frembragte gasarter, og substratet bibeholdes i beholde-5 ren uden udsivning, således at man med sikkerhed kan håndtere denne.12 gases while maintaining a substantially airtight condition that the plug portion 4 does not smoke during or after the incubation due to the gases produced, and the substrate is retained in the container 5 without leakage so that it can be safely handled. .
Ved aerob dyrkning kan dyrkningen på den anden side udføres under samme dyrkningsbetingelser som ved anaerob 10 dyrkning ved at udtage og indføre en prøve, således som nævnt ovenfor, trække tilpropningsdelen 4 op til et niveau ved hvilket gennemhulningen 7 delvis kommer ud af eller helt er over topdelen af åbningen 3 af dyrkningsbeholderen 1, og påsætte lukket 10. Det omhandlede dyrk-15 ningsapparat muliggør således forbindelse af det indre af beholderen med den ydre atmosfære, som f.eks. luften uden for beholderen, medens tilpropningsdelen 4 knyttet til åbningen 3 af dyrkningsbeholderen 1 stadigvæk forhindrer substratet i beholderen i at blive forurenet.In aerobic cultivation, on the other hand, the cultivation can be carried out under the same cultivation conditions as in anaerobic cultivation by taking and introducing a sample, as mentioned above, pulling the plug part 4 up to a level at which the through hole 7 partially comes out or is completely over the top portion of the aperture 3 of the culture container 1, and attach the closure 10. The present culture apparatus thus enables connection of the interior of the container with the external atmosphere, such as e.g. the air outside the container, while the plug portion 4 attached to the opening 3 of the culture container 1 still prevents the substrate in the container from being contaminated.
2020
Eksempler på en hel række mikroorganismer, der dyrkedes i det omhandlede apparat, vist i fig. 1, er angivet nedenfor.Examples of a whole range of microorganisms grown in the present apparatus, shown in FIG. 1 is listed below.
25 I efterfølgende tabeller er følgende forkortelser anvendt i forbindelse med de enkelte bakteriestammer.In the following tables, the following abbreviations are used in connection with the individual bacterial strains.
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13 NIHJ = The National Institute of Health, Japan ATCC = The America Type Culture Collection IID = The Institute of Infections Diseases IAM = The Institute of Applied Microbiology 5 RIMD = The Reserach Institute for Microbial Diseases IFO = The Institute for Fermentation, Osaka EKSEMPEL 1 10 Til anaerob dyrkning fremstilledes en suspension ved at opslæmme forskellige gasproducerende mikroorganismer angivet i tabel 1 og 2 i en sådan mængde steriliseret vand, at man fik fra 101 til 102 CFU/ml, og man udtog 2 cnr* af suspensionen og anbragte i beholderen 1, og der 15 dyrkedes ved 37 °C. Apparatet undersøgtes dagligt for vækst af mikroorganismer i dyrkningsbeholderen og for tilpropningsdelens bevægelse, forårsaget af gastrykket.NIHJ = The National Institute of Health, Japan ATCC = The America Type Culture Collection IID = The Institute of Infectious Diseases IAM = The Institute of Applied Microbiology 5 RIMD = The Reserach Institute for Microbial Diseases IFO = The Institute for Fermentation, Osaka EXAMPLE 1 For anaerobic culture, a suspension was prepared by suspending various gas-producing microorganisms listed in Tables 1 and 2 in an amount of sterilized water to obtain from 101 to 102 CFU / ml and extracting 2 ccn * of the suspension and placing in the container 1 and 15 were grown at 37 ° C. The apparatus was checked daily for growth of microorganisms in the culture vessel and for the movement of the plug part caused by the gas pressure.
Man fik de i tabel 1 og 2 angivne resultater. Bevægelsen af tilpropningsdelen er angivet som antal dage, der kræ-20 ves for, at en del eller hele tilpropningsdelen bevæger sig til dyrkningsudstyrets øverste åbning. Man så ikke yderligere bevægelse af tilpropningsdelen efter de dage, der krævedes for den ovenfor angivne bevægelse, ved fortsat inkubering.The results given in Tables 1 and 2 were obtained. The movement of the plug part is indicated as the number of days required for part or all of the plug part to move to the top opening of the cultivation equipment. No further movement of the plug part was seen after the days required for the above movement, with continued incubation.
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Dyrkning under anvendelse af Substrat CCultivation using Substrate C
TABEL 1 14TABLE 1 14
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Vækst af Bevægelse af 5 Navn på mikroorganisme mikroorganisme tilpropningsdel Escherichia coli NIHJ glimrende 2 dageGrowth of Movement of 5 Name of microorganism microorganism plug part Escherichia coli NIHJ excellent 2 days
Citrobacter freundii ATCC 8090 glimrende 1 dage n Salmonella typhimurium IID 971 glimrende 2 dageCitrobacter freundii ATCC 8090 excellent 1 days n Salmonella typhimurium IID 971 excellent 2 days
Salmonella paratyphi IID 605 glimrende 1 dagSalmonella paratyphi IID 605 excellent 1 day
Salmonella enteritidis IID 604 glimrende 2 dage 15Salmonella enteritidis IID 604 excellent 2 days 15
Arizona arizonae ATCC 13314 glimrende 2 dageArizona arizonae ATCC 13314 excellent 2 days
Salmonella pullorum 3H-2 glimrende 2 dage „n Klebsiella pneumoniae IID 875 glimrende 2 dageSalmonella pullorum 3H-2 excellent 2 days An Klebsiella pneumoniae IID 875 excellent 2 days
Enterobacter cloacae IAM 1624 glimrende 2 dageEnterobacter cloacae IAM 1624 excellent 2 days
Enterobacter aerogenes RIMD 0502001 glimrende 1 dag 25Enterobacter aerogenes RIMD 0502001 excellent 1 day 25
Hafnia alvei ATCC 13337 glimrende 3 dageHafnia alvei ATCC 13337 excellent 3 days
Proteus morganii IID 602 glimrende 2 dage _n Proteus mirabilis RIMD 1641002 glimrende 2 dageProteus morganii IID 602 excellent 2 days _n Proteus mirabilis RIMD 1641002 excellent 2 days
Bacillus macerans IFO 3490 glimrende 4 dageBacillus macerans IFO 3490 excellent 4 days
Clostridium tetani IID 524 glimrende 8 dage 35Clostridium tetani IID 524 excellent 8 days 35
Clostridium sporogenes IAM 19235 glimrende 2 dageClostridium sporogenes IAM 19235 excellent 2 days
Clostridium spheroides glimrende 1 dag _i_i_Clostridium spheroides brilliant 1 day _i_i_
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TABEL 2TABLE 2
Dyrkning under anvendelse af Substrat DCultivation using Substrate D
Vækst af Bevægelse afGrowth of Movement of
Navn på mikroorganisme mikroorganisme tilpropningsdelName of microorganism microorganism plug part
Escherichia coli NIHJ glimrende 2 dage 5Escherichia coli NIHJ excellent 2 days 5
Citrobacter freundii ATCC 8090 glimrende 1 dagCitrobacter freundii ATCC 8090 excellent 1 day
Salmonella typhimurium IID 971 glimrende 2 dage , n Salmonella paratyphi IID 605 glimrende 1 dagSalmonella typhimurium IID 971 excellent 2 days, n Salmonella paratyphi IID 605 excellent 1 day
Salmonella enteritidis IID 604 glimrende 2 dageSalmonella enteritidis IID 604 excellent 2 days
Arizona arizonae ATCC 13314 glimrende 2 dage 15Arizona arizonae ATCC 13314 excellent 2 days 15
Salmonella pullorum 3H-2 glimrende 2 dageSalmonella pullorum 3H-2 excellent 2 days
Klebsiella pneumoniae IID 875 glimrende 2 dage 9n Enterobacter cloacae IAM 1624 glimrende 2 dageKlebsiella pneumoniae IID 875 excellent 2 days 9n Enterobacter cloacae IAM 1624 excellent 2 days
Enterobacter aerogenes RIMD 0502001 glimrende 1 dagEnterobacter aerogenes RIMD 0502001 excellent 1 day
Hafnia alvei ATCC 13337 glimrende 3 dage 25Hafnia alvei ATCC 13337 excellent 3 days 25
Proteus morganii IID 602 glimrende 2 dageProteus morganii IID 602 excellent 2 days
Proteus mirabilis RIMD 1641002 glimrende 2 dage 3Q Bacillus macerans IFO 3490 glimrende 4 dageProteus mirabilis RIMD 1641002 excellent 2 days 3Q Bacillus macerans IFO 3490 excellent 4 days
Clostridium tetani IID 524 glimrende 8 dageClostridium tetani IID 524 excellent 8 days
Clostridium sporogenes IAM 19235 glimrende 2 dageClostridium sporogenes IAM 19235 excellent 2 days
Clostridium spheroides glimrende 1 dag 35 16 TABEL 2 (fortsat)Clostridium spheroides excellent 1 day 35 16 TABLE 2 (continued)
Dyrkning under anvendelse af Substrat DCultivation using Substrate D
DK 155159 BDK 155159 B
Vækst af bevægelse afGrowth of movement of
Navn på mikroorganisme mikroorganisme tilpropningsdel 5 Veillonella alcalle- cens ATCC 17745 udmærket 4 dageName of microorganism microorganism plug part 5 Veillonella alcalleens ATCC 17745 excellent 4 days
Fusobacterium necro- phorum RIMD 0623001 glimrende 1 dagFusobacterium necrophorum RIMD 0623001 excellent 1 day
Fusobacterium varium 10 ATCC 8501 glimrende 1 dagFusobacterium varium 10 ATCC 8501 excellent 1 day
Bacteroides melanino- genicus RIMD 0230004 glimrende 1 dag _I_I_ 15 20 25 30 35 17Bacteroides melaninogenicus RIMD 0230004 excellent 1 day _I_I_ 15 20 25 30 35 17
DK 155159 BDK 155159 B
EKSEMPEL 2EXAMPLE 2
Ved en aerob dyrkning opslæmmedes hver stamme i en passende mængde destilleret vand, således at man havde ca.In aerobic cultivation, each strain was slurried in an appropriate amount of distilled water so that there was about
1 x 10^-5 x 10"*· CFU/ml. Suspension af de respektive stammer indføres i de respektive substrater i det om-5 handlede apparat. Man inkuberede ved 37 °C efter at have trukket tilpropningsdelen op, således at tilpropningsdelens hul var over beholderens åbning. Antallet af levende organismer i dyrkningsvæsken måltes efter 2 dage på sædvanlig måde, og resultaterne er vist i tabel 3 og 4, 10 hvilket angav god vækst på 10^-10^ CFU/ml.1 x 10 6 -5 x 10 "* CFU / ml. Suspension of the respective strains is introduced into the respective substrates of the apparatus concerned. Incubate at 37 ° C after pulling up the plug part so that the hole of the plug part The number of living organisms in the culture fluid was measured after 2 days in the usual manner and the results are shown in Tables 3 and 4, 10 indicating good growth of 10 ^ -10 ^ CFU / ml.
Som en kontrol holdtes dyrkningssubstraterne ved 37 °C i 21 dage uden tilsat bakteriestamme. Dyrkningsapparatets indre var i forbindelse med luften som angivet ovenfor 15 med eller uden lukke. Man så ingen forurening med bakterier fra den fri luft.As a control, the culture substrates were maintained at 37 ° C for 21 days without added bacterial strain. The interior of the culture apparatus was in connection with the air as indicated above with or without closure. No pollution was seen with bacteria from the open air.
20 25 30 35 1820 25 30 35 18
DK 155159 BDK 155159 B
TABEL 3TABLE 3
Dyrkning under anvendelse af Substrat CCultivation using Substrate C
Antal udsåede Antal levende Navn på mikroorganisme mikroorga- mikroorganis- nismer/ml mer/ml 5 ---Number of seeded Number of living Name of microorganism microorganic microorganisms / ml more / ml 5 ---
Pseudomonas aeruginosa IID 1001 1,5 x 10 1 x 108Pseudomonas aeruginosa IID 1001 1.5 x 10 1 x 108
Escherichia coli NXHJ 1,0 x 10 7 x 108 , n Salmonella enteritidis IID 604 1,3 x 10 2 x 109Escherichia coli NXHJ 1.0 x 10 7 x 108, n Salmonella enteritidis IID 604 1.3 x 10 2 x 109
Shigella sonnei IID 969 1,0 x 10 4 x 108Shigella sonnei IID 969 1.0 x 10 4 x 108
Enterobacter cloacae IAM 1624 1,7 x 10 2 x 109 15Enterobacter cloacae IAM 1624 1.7 x 10 2 x 109 15
Proteus mirabilis RIMD 1641002 9,0 X 10 1 x 109Proteus mirabilis RIMD 1641002 9.0 X 10 1 x 109
Staphylococcus aureus ATCC 6538P 0,5 x 10 2 x 107 9n Streptococcus pyogenes υ IID 693 0,2 x 10 lx 109Staphylococcus aureus ATCC 6538P 0.5 x 10 2 x 107 9n Streptococcus pyogenes υ IID 693 0.2 x 10 lx 109
Kontrol (med lukke) — ingen vækstControl (with closing) - no growth
Kontrol (uden lukke) — ingen vækstControl (without closing) - no growth
Pseudomonas aeruginosa 25 IID 1001 4,5 x 10 4 x 108Pseudomonas aeruginosa 25 IID 1001 4.5 x 10 4 x 108
Pseudomonas maltophila IID 1275 4,7 x 10 4 x 108Pseudomonas maltophila IID 1275 4.7 x 10 4 x 108
Achromobacter xyloso- xidans ATCC 27061 1,6 x 10 1 x 109 30Achromobacter xylosoxidans ATCC 27061 1.6 x 10 1 x 109 30
Escherichia coli NIHJ 0,5 x 10 3 x 109Escherichia coli NIHJ 0.5 x 10 3 x 109
Shigella sonnei IID 969 1,3 x 10 1 x 109Shigella sonnei IID 969 1.3 x 10 1 x 109
Enterobacter cloacae .Enterobacter cloacae.
35 IAM 1624 2,3 x 10 3 x 10y35 IAM 1624 2.3 x 10 3 x 10y
Proteus mirabilis QProteus mirabilis Q
RIMD 1641002 1,9 x 10 3 x 109RIMD 1641002 1.9 x 10 3 x 109
Staphylococcus aureus QStaphylococcus aureus Q
ATCC 6538P 5,3 x 10 5 x 10° TABEL 4 (fortsat)ATCC 6538P 5.3 x 10 5 x 10 ° TABLE 4 (continued)
Dyrkning under anvendelse af Substrat DCultivation using Substrate D
1919
DK 155159 BDK 155159 B
Antal udsåede Antal levende Navn på mikroorganisme mikroorga- mikroorganis- nismer/ml mer/ml 5 Streptococcus pyogenes IID 693 1,2 x 10 5 x 10yNumber of seeded Number of living Name of microorganism microorganic microorganisms / ml more / ml 5 Streptococcus pyogenes IID 693 1.2 x 10 5 x 10y
Bacillus subtilis ATCC 6633 7,1 x 10 3 x 108Bacillus subtilis ATCC 6633 7.1 x 10 3 x 108
Listeria monocytogenes 10 IID 579 3,4 x 10 5 x 108Listeria monocytogenes 10 IID 579 3.4 x 10 5 x 108
Salmonella enteritidis IID 604 1,9 x 10 1 x 10ySalmonella enteritidis IID 604 1.9 x 10 1 x 10 y
Clostridium tetani IID 524 1,2 x 10 7 x 108 15 Pusobacterium necro- phorum RIMD 0623001 3,4 x 10 8 x 108Clostridium tetani IID 524 1.2 x 10 7 x 108 15 Pusobacterium necrophororum RIMD 0623001 3.4 x 10 8 x 108
Bacteroides fragilis ss. fragilis RIMD 2,1 x 10 6 x 10' 0230001 -n Neisseria meningitidis ATCC 13090* 6,0 x 10 5 x 107Bacteroides fragilis ss. fragilis RIMD 2.1 x 10 6 x 10 '0230001 -n Neisseria meningitidis ATCC 13090 * 6.0 x 10 5 x 107
Kontrol (med lukke) — ingen vækstControl (with closing) - no growth
Kontrol (uden lukke) — ingen vækst _I______I___ * En suspension af mikroorganismer fremstilledes ved O c at anvende menneskeblod i stedet for destilleret vand.Control (without closure) - no growth _ A suspension of microorganisms was prepared using O c using human blood instead of distilled water.
30 3530 35
DK 155159 BDK 155159 B
20 EKSEMPEL 3EXAMPLE 3
Hver stamme angivet i tabel 5 og 6 opslæmmedes i en passende mængde steriliseret, destilleret vand, og man ind-5 stillede opslæmningen således, at den indeholdt ca. 1 x 10-^-5 x 102 CFU/ml, hvorefter man anvendte den til forsøg; Inkuberingen påbegyndtes ved 37 °C, efter at tilpropningens hul var trukket op over dyrkningsapparatets åbning, dersom det drejede sig om aerobe kulturer, og 10 uden at trække proppen op, dersom det drejede sig om anaerobe kulturer. Antal levende mikroorganismer måltes daglig på sædvanlig måde, og antal dage nødvendige for at nå omtrent 5 x 10^ CFU/ml eller mere noteredes. Resultaterne er vist i tabel 5 (anaerobe kulturer) og ta-15 bel 6 (aerobe kulturer), hvilket angav, at visse af de dyrkede stammer voksede bedre i substrat D end i substrat C.Each strain listed in Tables 5 and 6 was slurried in an appropriate amount of sterilized distilled water, and the slurry was adjusted to contain approx. 1 x 10 - 5 x 102 CFU / ml and then used for testing; The incubation was started at 37 ° C after the plug of the plug was pulled up over the aperture of the culture apparatus in the case of aerobic cultures and 10 without pulling the plug in the case of anaerobic cultures. The number of living microorganisms was measured daily in the usual manner and the number of days required to reach approximately 5 x 10 5 CFU / ml or more was noted. The results are shown in Table 5 (anaerobic cultures) and Table 6 (aerobic cultures), which indicated that some of the cultivated strains grew better in substrate D than in substrate C.
20 25 30 \ 35 TABEL 5 2120 25 30 \ 35 TABLE 5 21
DK 155159 BDK 155159 B
Anaerobe kulturerAnaerobic cultures
Dager der kræves for 5 Navn på mikro- Antal udsåede vækst på 5 x 10' eller organisme mikroorganismer mere CFU/ml levende pr. ml mikroorganismerDays required for 5 Name of micro- Number of seeded growth of 5 x 10 'or organism microorganisms more CFU / ml live per day. ml microorganisms
Substrat C Substrat DSubstrate C Substrate D
Pseudomonas in aeruginosa 4,5 x 102 2 dage 1 dag u IID 1001Pseudomonas in aeruginosa 4.5 x 102 2 days 1 day u IID 1001
Pseudomonas fluorescens 3,4 x 101 2 dage 1 dag IFO 12055Pseudomonas fluorescence 3.4 x 101 2 days 1 day IFO 12055
Neisseria 15 meningitidis 6,0 x 10'1' 4 dage 2 dage ATCC 13090Neisseria 15 meningitidis 6.0 x 10'1 '4 days 2 days ATCC 13090
Neisseria gonorrhoeae 1,0 x lO'1^ 5 dage 3 dage IID 828 ______I_I_I___ 20 25 30 35Neisseria gonorrhoeae 1.0 x 100 1 5 days 3 days IID 828 ______I_I_I___ 20 25 30 35
DK 155159 BDK 155159 B
22 TABEL 622 TABLE 6
Aerobe kulturerAerobic cultures
Dage, der kræves forDays required for
Navn på mikro- Antal udsåede vækst på 5 x 10' eller organisme mikroorganismer mere CFU/ml levende pr. ml mikroorganismer 5 --Name of micro- Number of seeded growth of 5 x 10 'or organism microorganisms more CFU / ml live per ml microorganisms 5 -
Substrat C Substrat DSubstrate C Substrate D
Staphylococcus Ingen epidermidis 3,8 x 101 vækst 2 dage ATCC 12228 in Fusobacterium Ingen nucleatum 2,0 x 101 vækst 2 dage IID 891Staphylococcus No epidermidis 3.8 x 101 growth 2 days ATCC 12228 in Fusobacterium No nucleatum 2.0 x 101 growth 2 days IID 891
Fusobacterium Ingen varium 1,2 x 101 vækst l2' 1 dag ATCC 8501 15 Bacteroides Ingen fragilis ass. 3,4 x 101 vækst *2' 1 dag distasonis RIMD 00230002Fusobacterium No varium 1.2 x 101 growth l2 '1 day ATCC 8501 15 Bacteroides No fragilis ass. 3.4 x 101 growth * 2 '1 day distasonis RIMD 00230002
Peptostrepto- Ingen coccus micros 2,5 x 102 vækst '2' 3 dage 20 RIMD 1636001Peptostrepto- No coccus micros 2.5 x 102 growth '2' 3 days 20 RIMD 1636001
Peptostrepto- Ingen coccus 2,6 x 101 vækst '2' 3 dage parvulus RIMD 1637001Peptostrepto- No coccus 2.6 x 101 growth '2' 3 days parvulus RIMD 1637001
Bifidobac- 25 terium adole- 1,5 x 102 3 dage 1 dag scentis ATCC 15705 _I___ (1) Suspensionen af mikroorganismer fremstilledes ved at 30 anvende menneskeblod i stedet for destilleret vand.Bifidobacterium Adults 1.5 x 102 3 days 1 day scentis ATCC 15705 (1) The suspension of microorganisms was prepared using human blood instead of distilled water.
Observeret i 7 dage.Observed for 7 days.
3535
DK 155159 BDK 155159 B
2323
Som beskrevet udvikles der i det omhandlede apparat gasarter under inkuberingen af den anaerobe kultur, hvilke let bortledes gennem hullet i tilpropningsdelen. Med det samme apparat kan en aerob kultur også dyrkes simpelt 5 hen ved at trække tilpropningsdelen op, således at hele gennemhulningen eller en del heraf er over dyrkningsbeholderens åbningsdel. Apparatet, der har samme tilpropning og samme dyrkningssubstrat, kan således anvendes til både anaerobe og aerobe kulturer, inkuberingen kan 10 udføres sikkert og uden fare, fordi tilpropningsdelen ikke har tendens til at ryge af, og substratet herved blive spredt rundt omkring. Yderligere er andet udstyr ikke nødvendigt for at bortlede gasarterne eller luften fra apparatet, således at apparatet har den yderligere 15 fordel, at det er let at fremstille og billigt.As described, in the present apparatus, gases are developed during the incubation of the anaerobic culture which are readily discharged through the hole in the plug portion. With the same apparatus, an aerobic culture can also be simply cultivated by pulling up the plug portion so that all or part of the through hole is over the opening portion of the culture container. Thus, the apparatus having the same plugging and the same culture substrate can be used for both anaerobic and aerobic cultures, the incubation can be carried out safely and without danger because the plugging part does not tend to smoke, and the substrate is spread around it. Further, other equipment is not required to vent the gases or air from the apparatus, so that the apparatus has the additional advantage of being easy to manufacture and inexpensive.
20 25 30 3520 25 30 35
Claims (14)
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6971379A JPS55162977A (en) | 1979-06-04 | 1979-06-04 | Microorganism cultivating appliance |
| JP6971379 | 1979-06-04 | ||
| JP4261380A JPS56140781A (en) | 1980-04-01 | 1980-04-01 | Picture analyzing device |
| JP4261380 | 1980-04-01 | ||
| PCT/JP1980/000120 WO1980002694A1 (en) | 1979-06-04 | 1980-06-03 | Microorganism-culturing device |
| JP8000120 | 1980-06-03 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DK45881A DK45881A (en) | 1981-02-03 |
| DK155159B true DK155159B (en) | 1989-02-20 |
| DK155159C DK155159C (en) | 1989-07-17 |
Family
ID=26382333
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK045881A DK155159C (en) | 1979-06-04 | 1981-02-03 | APPARATUS FOR CULTIVATING MICRO-ORGANISMS AND PROCEDURES FOR USING THIS |
Country Status (1)
| Country | Link |
|---|---|
| DK (1) | DK155159C (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3033408A (en) * | 1958-12-10 | 1962-05-08 | Joud Andre | Arrangement for closing test tubes |
| FR2029242A5 (en) * | 1969-01-21 | 1970-10-16 | Alriq Christian | Self-perforable plug for bacteriological - culture tubes |
| US3898046A (en) * | 1973-04-23 | 1975-08-05 | Jintan Terumo Co | Stopper for sealing containers |
| US3904482A (en) * | 1972-01-06 | 1975-09-09 | Becton Dickinson Co | Method for the cultivation of microorganisms from body fluid |
| WO1980001047A1 (en) * | 1978-11-28 | 1980-05-29 | Dematex Dev & Invest | Tube and stopper for blood-sampling systems |
-
1981
- 1981-02-03 DK DK045881A patent/DK155159C/en not_active IP Right Cessation
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3033408A (en) * | 1958-12-10 | 1962-05-08 | Joud Andre | Arrangement for closing test tubes |
| FR2029242A5 (en) * | 1969-01-21 | 1970-10-16 | Alriq Christian | Self-perforable plug for bacteriological - culture tubes |
| US3904482A (en) * | 1972-01-06 | 1975-09-09 | Becton Dickinson Co | Method for the cultivation of microorganisms from body fluid |
| US3898046A (en) * | 1973-04-23 | 1975-08-05 | Jintan Terumo Co | Stopper for sealing containers |
| WO1980001047A1 (en) * | 1978-11-28 | 1980-05-29 | Dematex Dev & Invest | Tube and stopper for blood-sampling systems |
Also Published As
| Publication number | Publication date |
|---|---|
| DK155159C (en) | 1989-07-17 |
| DK45881A (en) | 1981-02-03 |
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