PROCESS FOR ISOLATING BACTERIA IN BLOOD

Abstract: Bacteria can be isolated from blood by mixing blood to be examined with a liquid containing a hemolytic reagent and an anticoagulant. The resultant mixture is filtered through a filter having a pore size that does not allow passage therethrough of the bacteria to be separated. Nutrients are then supplied to the bacteria on the filter through the mixture so as to immedi­ ately culture the bacteria on the filter.

Description: Process for isolating bacteria in blood and an instrument for performing this process The present invention relates to a process for the isolation of bacteria in blood and an instrument for performing this process. Bacteria are present in blood of patients suffering from serious systemic infections such as septicemia or bacteremia. In the diagnosis of such systemic infections, blood tests for the identification of bacteria and antibiotics susceptibility are performed. In order to conduct such blood tests, isolation of bacteria from blood is required to be performed. In conventional processes for the isolation from blood of bacteria, a colony is grown in the following manner. Sampled blood is mixed with a liquid culture medium, and culture is performed until the culture medium becomes turbid with propagated bacteria. Subsequently, the propagated bacteria are recovered and transplanted onto another culture medium such as a blood or chocolate agar medium. In such conventional processes, it is difficult to directly isolate bacteria which is only present in blood in small numbers. These conventional processes require a pretreatment step of propagating bacteria before isolation of bacteria on an agar medium, resulting in complex and lengthy procedures. Bacteria propagation or multiplication in a medium as described above generally requires a propagation time of one to ten days, and colony growth requires a further one or two days. When the propagated bacteria are transplanted to a isolator medium, the bacteria under testing may contaminate the surrounding atmosphere or other bacteria may be mixed thereinto. It is an object of the present invention to provide a process for isolating blood bacteria, which allows direct sampling of the blood bacteria without requiring an additional process such as propagation of the blood bacteria, which allows isolation of the bacteria without transplantation into another culture medium, and in which a colony can be grown within a short period of time. It is another object of the present invention to provide an instrument to be used in the process for isolation of bacteria in blood as described above. According to an aspect of the present invention, there is provided a process for isolating bacteria from blood, comprising the steps of mixing blood of a patient with a liquid containing a hemolytic reagent and an anticoagulant; filtering the resultant liquid mixture with a filter having a pore size that does not allow passage of the bacteria therethrough; and supplying the bacteria remaining on the filter with nutrients contained in the liquid mixture so as to allow direct culturing of the bacteria on the filter without transplantation. According to another aspect of the present invention, there is provided an instrument for isolating bacteria from blood comprising: a vessel main body having an open top; a filter which is sustained in said vessel main body at an intermediate position along a height thereof so as to divide the interior of said vessel main body into upper and lower spaces and which has a pore size that does not allow passage of the bacteria therethrough; a water absorption member adhered on the entire bottom surface of said filter; a ventilation hole formed in a wall or the bottom of said vessel main body so as to allow ventilation between the lower space therein and a surrounding atmosphere; and a cover covering the open top of said vessel main body. According to another aspect of the present invention, there is provided a vessel for mixing a sampled blood with a mixture comprising a hemolytic reagent and an anticoagulant, which comprises a cylindrical body having an opening at one end, and a resilient cap detachably mounted on the opening, thereby hermetically sealing the opening, said vessel being preliminarily accomodated with said mixture. This invention can be more fully understood from the following detailed description when taken in conjunction with the accompanying drawings, in which Figs. 1 and 2 are sectional views showing an assembly for mixing sampled blood with a treatment liquid containing a hemolytic reagent, an anticoagulant and the like; Fig. 3 is a sectional view of an instrument for isolating bacteria from blood, according to an. embodiment of the present invention; and Figs. 4 and 5 are each a sectional view of an instrument for isolating bacteria from blood, according to modified embodiments of the present invention. The process of the present invention requires a step of homogeneously mixing sampled blood with a liquid containing a hemolysin and an anticoagulant and, if necessary, a liquid culture medium and the like. This step is preferably performed in a sealed system so as to prevent both contamination of the surrounding atmosphere with the sampled bacteria and the introduction of foreign bacteria in the separated bacteria. Figs. 1 and 2 show an assembly for performing this step. The assembly shown in Fig. 1 consists of a vessel 1 and a rubber stop 3. The vessel 1 holds a liquid mixture 2 containing at least a hemolytic reagent and an anticoagulant, and is kept at a reduced pressure. The rubber stop 3 can be detachably mounted at the top opening of the vessel 1 and can liquid-tightly seal the vessel 1. In order to mix sampled blood with the mixture 2 using this assembly, the cannula of a syringe (not shown) is pierced through the rubber stop 3 so as to introduce blood into the vessel 1 utilizing the reduced pressure therein. After the blood has been introduced into the vessel 1, the cannula is pulled out from the rubber stop 3, and the vessel is turned upside down so as to mix the blood with the liquid mixture 2 held in the vessel 1. The hemolytic reagent may be saponin; the anticoagulant may be sodium amylo sulfate or sodium polyanethol sulfonate. A liquid culture medium can also be added to the liquid mixture 2 if needed. If so, the need for impregnating a water absorption member of an isolation instrument with a culture medium is obviated. An assembly shown in Fig. 2 comprises a cylinder 4 and a press member 5 which is capable of liquid-tightly sliding inside the cylinder 4. A rubber stop 3 is detachably and liquid-tightly fitted in the upper opening of the cylinder 4. A liquid mixture 2 containing a hemolytic reagent and an anticoagulant as described above is held in the space defined by the cylinder 4 and the press member 5. The liquid mixture 2 can also contain a liquid culture medium. When sampled blood is to be mixed with the mixture 2 using this assembly, the cannula of a syringe which has been used to sample blood is thereafter pierced into the rubber stop 3. As the blood is forcibly discharged into the cylinder 4, the press member 5 is slid downward inside the cylinder 4. After the blood has been introduced into the cylinder 4, the assembly is turned upside down so as to mix the blood with the hemolytic reagent and the anticoagulant. In order to prevent contamination of the blood by other bacteria, the assemblies as described above are preferably sterilized by gamma ray radiation or autoclaving. The blood treatment as described above damages red blood cells and also prevents blood coagulation, thereby allowing efficient subsequent blood filtration and blood bacterium culture. When a liquid culture medium is to be mixed with the liquid mixture as described above, the liquid culture medium can be simultaneously mixed into the blood containing the bacterium during this treatment. After the blood is treated with one of the above-described assemblies, the rubber stop is removed and the blood is poured onto a filter of an isolation instrument to be described below. Fig. 3 shows an embodiment of an instrument for isolating bacteria from blood according to the present invention. Referring to Fig. 3, reference numeral 11 denotes a cylindrical vessel with a bottom and an open top. An annular step 12 is formed in the wall at a position intermediate along the direction of depth of the vessel. The periphery of a water absorption member 13 is supported on the upper surface of the step 12. A filter 14 having a pore size that does not allow passage of the bacteria to be cultured therethrough is placed on the upper surface of the member 13 throughout the entire open top of the vessel 11. Thus, the filter 14 integral with the water absorption member 13 divides the interior of the vessel 11 into an upper space 15 and a lower space 16. In order to prevent blood leakage into the lower space 16 through any gap between the wall of the vessel 11 and the periphery of the filter 14, a sealing member 17 is arranged to press down upon the periphery of the filter 14. Alternatively, the periphery of the filter 14 can be adhered to the wall of the vessel 11 with an adhesive. A ventilation hole 18 for allowing the passage of air between the lower space 16 and the outer atmosphere is formed at an upper position of the wall of the vessel 11. Reference numeral 19 denotes a cover covering the open top of the vessel 11. Any material inert to blood can be used as the material.of the filter 14. Examples of such an inert material include nitrocellulose, polycarbonate, polyamide, cellulose ester and the like. Examples of such materials which are commercially available include "Millipore" (product of Millipolar Corp.), "Metricell" (product of German Instrument Company) and the like. The pore size of the filter 14 is determined in accordance with the application. However, the pore size of the filter 14 is generally 0.75 um or less and preferably 0.45 um or less. The filter 14 is preferably treated to give good hydrophilic properties by a known method to provide good blood wettability. The water absorption member 13 absorbs and holds therein the blood filtered through the filter 14 and thereby supplies the nutrients to the bacteria held on the filter 14. The water absorption member 13 preferably has a capacity to absorb the total amount of blood filtered through the filter 14. The material of the member 13 is preferably a filter paper of cellulose type, non-woven fabric or the like. The member 13 must be securely fixed either directly or through an adhesive to the rear surface of the filter 14. When the member 13 is not properly fixed to the filter 14, efficient filtering cannot be expected, and the supply of water and nutrients to the bacteria on the filter 14 becomes unsatisfactory. The adhesive to be applied between the filter 14 and the member 13 is conveniently a heat sealable adhesive of low-melting point polymeric fiber which does not impair filtration. The member 13 can be impregnated with a liquid culture medium which is dried upon impregnation, as needed. In this case, the dried medium is dissolved in the filtered blood absorbed and held by the water absorption member 13, which supplies water and nutrients to the bacteria on the filter 14. The upper space 15 above the filter 14 is for storing the sampled blood, and the lower space 16 is for storing the portion of blood which is not absorbed and held by the member 13. Fig. 4 shows a modification of an instrument for isolating bacteria from blood according to the present invention. The instrument of the modification is different from that described above with reference to Fig. 3 in the following respects. In the instrument shown in Fig. 4, a ventilation hole 18' is formed in the bottom of the vessel 11. A bacterial filter 21 (e.g., cotton stop) for preventing the entry of contaminating bacteria is fitted in the hole 18'. In order to prevent blood leakage through the hole 18', a cylindrical wall 22 is formed to surround the hole 18'. Buffer walls 23 are concentrically formed on the bottom surface of the vessel 11 so as to allow concentration of the blood only around the hole 18'. Thus, the same reference numerals as used in Fig. 3 denote the same parts in Fig. 4, and a detailed description thereof will be omitted. Fig. 5 further shows another modification of an. instrument for isolating bacteria from blood according to the present invention. This modification is much similar to that shown in Fig. 4, but differs in the following respects. The height of sealing member 17' is made higher than that shown in Fig. 4 and provided with a collar on its top. Accordingly, the cover 19 is mounted on the collar portion of the sealing member 17' thereby sealing the interior of the upper space 15. Further, a cylindrical wall 24 is projected downward at the center portion of the bottom of the vessel 11 surrounding the hole 18', and a bacterial filter 21 is provided to cross the cylindrical wall 24 so as to prevent bacteria from passing therethrough. Other constructions are substantially the same as those shown in Fig. 5. Thus the same reference numerals as used in Fig. 4 denote the same parts in Fig. 5. The instrument having the construction described above is sterilized by gamma ray radiation or ethylene oxide gas so as to improve the reliability of the test. Although the shape of the instrument according to the present invention is not particularly limited, it is preferably circular. Neither is the size of the instrument limited. When 2 m± of blood are to be tested, the vessel main body should have a diameter of about 60 mm. A process for isolating bacteria from blood according to the present invention using the instrument described above will now be described. A blood sample of the required amount is obtained and is then mixed with a liquid mixture containing a hemolytic reagent and an anticoagulant and, as needed, a liquid culture medium. Saponin is preferably used as the hemolytic reagent. Sodium amylo sulfate or sodium polyanethol sulfonate is used as the anticoagulant. Any liquid culture medium which is generally used for propagating bacteria may be used. The above process is a pretreatment to be followed by the filtration process and is performed so as to damage red blood cells and prevent blood coagulation. The blood treated in this manner is poured onto the filter 14 of the instrument and the cover 19 is closed. The blood is filtered by its own weight; the bacteria remain on the filter 14 while the blood is passed through the filter 14 and is absorbed by the water absorption member 13. Any excess blood which cannot be absorbed by the member 13 drips into the lower space 16 of the vessel. The air compressed by the filtered blood is exhausted through the ventilation hole 18 or 18', so that efficient blood filtering is performed. After filtration, the instrument of the present invention is kept at a constant temperature so as to grow a colony on the filter 14. Since the vessel has a top opening of a size substantially the same as that of the filter, recovery of the bacteria is easy. After recovery of the bacteria, various bacterial tests such as identification or antibiotics susceptibility test can be performed. If the water absorption member 13 is impregnated with a liquid culture medium which is dried after impregnation, addition of a liquid culture medium in the pretreatment process can be omitted. As can be seen from the above description, when both the process for isolating bacteria from blood and the instrument according to the present invention are used, many advantages to be described below are obtained. With conventional bacterial tests, propagation and culture of blood bacteria is first performed with a liquid culture medium. This process is lengthy. The instrument of the present invention does not require this process and can therefore shorten the overall test time. Furthermore, since the step of transplanting the bacteria from the propagation medium to the isolation medium is not required, the overall process is simple. Contamination of the surrounding atmosphere by the bacteria and introduction of foreign bacteria into the vessel which may be caused upon bacteria transplantation can thus be prevented. When the instrument of the present invention is used, all the blood bacteria can be trapped and directly isolated so that the bacteria detection ratio is high and the number of bacteria in blood can be counted, if so desired. Example A membrane filter (manufactured by Toyo Roshi K.K.) of nitrocellulose having a pore size of 0.45 iim was used as the filter. A cellulose paper filter "NO-63F" (Toyo Roshi K.K.) was used as a water absorption member. The filter and the water absorption member were adhered together by placing a low-melting point nylon adhesive between the filter and the member and heat sealing. The filter and the water absorption member had a diameter of 50 mm and were assembled as shown in Fig. 3. A comparison between this process and conventional processes was made. In the first conventional process, 2.0 mQ of blood was poured into a vessel holding a culture medium, an anticoagulant and a hemolytic reagent (total amount: 1.0 m±). The resultant mixture was poured into an isolating instrument for culturing. In the second conventional process, liquid culture was performed with "Eiken No. 5" (manufactured by Eiken K.K.). In the third conventional process, a "Vacutainer 50" (manufactured by BD company) was used. The obtained results are shown in Table 1 below. With N. Meningitidis, the conventional liquid culture medium process did not allow bacterium detection. However, the process of the present invention allowed detection and counting of the bacteria within one day. With the process of the present invention, since the bacteria are isolated in the form of a colony, identification and antibiotics susceptibility test could be performed immediately. The process of the present invention also provided better results than the conventional processes for other types of bacteria. In Table 1 below, "+" denotes positive and "-" denotes negative. Table 1 EMI11.1 Names <SEP> of <SEP> Inoculated <SEP> After <SEP> 1 <SEP> day <SEP> After <SEP> 2 <SEP> days <tb> bacteria <SEP> number <SEP> of <SEP> * <SEP> ** <SEP> * <SEP> ** <tb> <SEP> bacteria <SEP> Filter <SEP> Eiken <SEP> Vacutainer <SEP> Filter <SEP> Eiken <SEP> Vacutainer <tb> <SEP> method <SEP> No. <SEP> 5 <SEP> 50 <SEP> method <SEP> No. <SEP> 5 <SEP> 50 <tb> Neisseria <SEP> 30 <SEP> 22 <SEP> - <SEP> - <SEP> 22 <SEP> - <SEP> meningit- <SEP> 22 <SEP> 17 <SEP> - <SEP> - <SEP> 17 <SEP> - <SEP> idis <tb> Candida <SEP> 14 <SEP> 15 <SEP> - <SEP> - <SEP> 15 <SEP> - <SEP> + <tb> adbiceps <SEP> 2 <SEP> 3 <SEP> - <SEP> - <SEP> 3 <SEP> - <SEP> Bacteroides <SEP> 15 <SEP> 14 <SEP> - <SEP> + <SEP> 14 <SEP> + <SEP> + <tb> fragilis <SEP> 4 <SEP> 7 <SEP> - <SEP> + <SEP> 7 <SEP> + <SEP> + <tb> Peptostrep- <SEP> 17 <SEP> 0 <SEP> - <SEP> - <SEP> 18 <SEP> + <SEP> + <tb> tococcus <SEP> 6 <SEP> 0 <SEP> - <SEP> - <SEP> 1 <SEP> + <SEP> + <tb> parvulus <tb> Note : * ... A cultivation system for microorganisms in blood, which is developed by Eiken Kagaku Co. (Japan), and includes, in 45 ml of its aqueous solution, 0.3 g vegatable extract, 0.2 g yeast extract, 0.14 g meat extract, 0.45 g pepton, 0.14 g soy pepton, 0.45 g tryptone, 0.14 g grape sugar and etc. **... A cultivation system for microorganisms in blood, which is developed by Pecton-Dickinson & of Peptone Broth 11 medium. The Peptone accomodated with 45 ml of Peptone Broth 11 medium. The Peptone Broth 11 medium includes per one liter 20 g quadtone, 12 g gelatin, 5 g sodium chloride, 5 g dextrose, 2.2 g sodium bicarbonate and etc.

Claims: Claims: 1. A process for isolating bacteria from blood, comprising the steps of: mixing blood to be examined with a liquid containing a hemolytic reagent and an anticoagulant, filtering the resultant mixture through a filter having a pore size that does not allow passage therethrough of the bacteria to be separated; and supplying nutrients to the bacteria on said filter through the mixture so as to immediately culture the bacteria on said filter. 2 The process according to claim 1, characterized in that the mixture also contains a liquid culture medium. 3. The process according to claim 1, characterized in that a water absorption member is arranged in tight contact with a lower surface of said filter so as to allow said water absorption member to absorb the mixture, in order to subsequently supply the nutrients to the bacteria. 4. The process according to claim 1, characterized in that said water absorption member contains a dried culture medium. 5. The process according to claim 1, characterized in that said step of mixing the blood to be examined. with the liquid is performed in a closed system. 6. An instrument for isolating bacteria from blood, comprising: a vessel main body (11) having an open top; a filter (14) which is sustained in said vessel main body ( at an intermediate position along a height thereof so as to divide the interior of said vessel main body into upper and lower spaces (15, 16), and which has a pore size that does not allow passage of the bacteria therethrough; a water absorption member (13) arranged in tight contact with a lower surface of said filter (14); a ventilation hole (18, 18') formed in a wall or the bottom of said vessel main body (11) so as to allow ventilation between the lower space and an outer atmosphere; and a cover (19) for closing the open top of said vessel main body (11). 7. The instrument according to claim 6, characterized in that said filter (14) and said water absorption member are formed integrally with an adhesive. 8. The instrument according to claim 6, characterized in that said ventilation hole (18, 18') is formed in the bottom of said vessel main body (11) which partially defines the lower space. 9. The instrument according to claim 6, characterized in that said ventilation hole (18, 18') is formed in the bottom of said vessel main body (11) and is defined by a cylindrical wall. 10. The instrument according to claim 9, characterized in that buffer walls are concentrically formed on the bottom of said vessel main body around said cylindrical wall as the center thereof. 11. The instrument according to claim 6, characterized in that a bacterial filter (14) is fitted in said ventilation hole. 12. The instrument according to claim 6, characterized in that said filter (14) and said water absorption member are fixed to the side wall of said vessel main body (11) by means of a caulking member. 13. A vessel for mixing a sampled blood with a mixture comprising a hemolytic reagent and an anticoagulant, which comprises a cylindrical body (1) having an opening at one end, and a resilient cap (3) detachably mounted on the opening, thereby hermetically sealing the opening, said vessel being preliminarily accomodated with said mixture. 14. The vessel according to claim 13, characterized in that the mixture further comprises a culture medium. 15. The vessel according to claim 13, characterized in that the intereior of the vessel is kept at a reduced pressure for introducing blood. 16. The vessel according to claim 13, which further comprises a plunger (5) slidably inserted inside the cylindrical body.

Inventors: KAMINAGAYOSHI, SATOSHI
Applicants: TERUMO KABUSHIKI KAISHA TRADING AS TERUMO CORPORATION
Application year: 1984
Main classification: C12M1
IPC codes: C12M1, C12Q1