US3853711A

US3853711A - Installation for automation of microbiological work techniques

Info

Publication number: US3853711A
Application number: US00153286A
Authority: US
Inventors: C Heden
Original assignee: Biotec AB
Current assignee: Biotec AB
Priority date: 1970-06-22
Filing date: 1971-06-15
Publication date: 1974-12-10
Anticipated expiration: 1991-12-10
Also published as: DE2130705B2; DE2130705A1; GB1360622A; JPS535397B1

Abstract

The present invention relates to a method for automating microbiological analysis of samples on gel substrates and to an installation for the automation of microbiological work technique. The method is mainly characterized by separately putting each substrate into a protective tube or cassette after the addition of active substance and/or sample, whereby the substrate is so shaped that diffusion, growth or other spreading in the substrate essentially occurs in the longitudinal direction of said substrate, and that the environmental conditions in the protective tubes are individually selected for each tube. The installation is mainly characterized in that it constitutes an interconnection of two or more of the following components as modules in the modifiable installation: a cutter, an incubating station, a thermostatic chamber, a refrigerated storage chamber and a reading station.

Description

United States Patent [191 Heden [75] Inventor: Carl Goran Heden, Solna, Sweden [73] Assignee: Ab Biotec, Stockholm, Sweden [22] Filed: June 15, 1971 [21] Appl. No.: 153,286

[] Foreign Application Priority Data June 22, 1970 Sweden 8592/ June 4, 1971 Sweden 7264/71 [52] U.S. Cl 195/127, l/103.5, 195/139 [51] Int. Cl C12b 1/24 [58] Field of Search 195/139, 108, 127, 103.5,

[56] References Cited UNITED STATES PATENTS 3,065,150 11/1971 Kravitz /139 3,205,151 9/1965 Landau 195/103.5 3,416,998 12/1968 Streitfeld 195/103.5 3,616,264 10/1971 Ray 195/127 3,661,718 5/1972 Rose 195/l03.5 3,728,277 4/1973 Elson et a1. 252/309 3,772,154 11/1973 Isenberg et a]. 195/127 OTHER PUBLICATIONS Oxoid Ltd., Food Engineering, August, 1967, p. 138.

INSTALLATION FOR AUTOMATION OF MICROBIOLOGICAL WORK TECHNIQUES [451 Dec. 10, 1974 LS. Gall, Partially Automated System for Microbiological Analysis," Dev. in Industrial Microbiology, Vol. 11, pp. 460469.

Primary ExaminerLionel M. Shapiro Assistant Examiner-T. G. Wiseman 5 7] ABSTRACT The present invention relates to a method for automating microbiological analysis of samples on gel substrates and to an installation for the automation of microbiological work technique. The method is mainly characterized by separately putting each substrate into a protective tube or cassette after the addition of active substance and/or sample, whereby the substrate is so shaped that diffusion, growth or other spreading in the substrate essentially occurs in the longitudinal direction of said substrate, and that the environmental conditions in the protective tubes are individually selected for each tube. The installation is mainly characterized in that it constitutes an interconnection of two or more of the following components as modules in the modifiable installation: a cutter, an incubating station, a thermostatic chamber, a refrigerated storage chamber and a reading station.

25 Claims, 5 Drawing Figures F|G.I

INCUBATING STATION SAMPLE/STAN OARO SOLUTIONS IN RACKS (OR CAROUSELI SPOOL OF STAIN- LESS WIRE MAGAZINE FOR CASSETTES {'AGAR STRIPS WITH APPLIEO SPIRALS I IN PROTECTIVE TUBES I gTHE'UNE CASSET E" YSTEMI J I MACHINE FOR .1 APPARATUS FOR I I I I I I MAKING SPIRALS HANDLING SPIRALS I AGAR BLOCK IN GLASS STRIPS WITH CUTTING MACHINE -I- LAYER AND APPLYING ED I I I I I AGAR LAYER ON I THERMOSTAT- STRIPS MECHANISM FOR I MAGAZINE STEPWISE FEEDING I CONTINUOUS ED'NG MAGAZINE FOR I OF STRIPS INTO (ADJUSTABLE NE I CAPACITY FOR 0 STRIPS P R O T E CT I\LE TLLBES I CASSETTE Imin= T I500 CASSETTES/24hrs WASHING STATION I REFRIGIERATED FOR STRIPS READING STATION STORAGE SPACE I ERII AGE EIIV E A SCRAPING AGAR LAYER OFF OF TUBES STRIPS FOR CASSEI IlzS CALCULATOR REGISTERING ANO IS UPPRAISING OF RESULTS MEASUREMENT RESULTS PAIENIEBBEM M 3.853.711

' saw 20F PATENTED DEE I 0 I974 SHEET 3 BF 3 INSTALLATION FOR AUTOMATION OF MICROBIOLOGICAL WORK TECHNIQUES Research has long been carried out to automate the work technique in the chemical field. For automatic chemical analysis, installations have been made having very large capacities thus facilitating a reorganization of hospital technique. By using such facilities, blood and urine samples can be quickly and inexpensively analyzed in such great quantities that these examinations have been able to be used to a far greater extent to support the clinical diagnostics.

Such an installation for use in the hospital most often consists of a chemical central processing unit which performs the actual analytical work, an electronic control unit which precisely determines all the work stages, an amplifier which transmits the results as electric impulses to a computer, and an electric teleprinter equipped with punched tape. Included in the central unit is a system for mechanically conveying samples and reaction solutions, pneumatic control of the table movement and the pipettes, reactant liquid supply, heating and cooling and colorimetric measuring of the mechanism of reaction, apparatus ventilation and washing of test tubes, pipettes and measuring heads. One model of the chemical central unit has 24 solid analysis channels and a maximum capacity of about 135 samples per hour, and a channel which makes possible at least 3,000 analyses per hour. The measuring occurs in a photometer in principle a 2-way photocell colorimeter having interference filters.

However, the problem with the usual chemical analysis is simpler than when microbiological work is involved. Instead of colour changes in the usual chemical analysis which occur instantaneously or shortly afterwards, the organisms in the microbiological analysis must be cultivated and have the possibility to grow and be developed under precisely defined conditions. Only thereafter can a reading be taken.

Within the field of medical, biochemical and pharmaceutical laboratory work, there arises a large number of analysis routines which build upon microbiological technique with solid substrates. These analyses include e.g., identification of strains, determination of microorganisms, resistance to antibiotics, microbiological determination of the quality of growth factors and growth inhibitors. Automation of these work routines facilitates an extensive rationalization of the work within a number of fields of work, such as medical and veterinary diagnostics, plant pathology, the pharmaceutical industry, nutritional research and ecology.

The present invention relates to a method for automating microbiological analysis of samples on gel substrates and is characterized in that each substrate is separately introduced into a protective tube after an active substance and/or sample has been added. The substrate is so shaped that diffusion, growth or other spreading therein basically occurs in its longitudinal direction. The environmental conditions in the protective tubes can be individually selected in each tube.

The invention also relates to an installation for automation of microbiological work technique, and consists of an interconnection ofa cutter for making agar strips, a machine for making spirals which transmit small amounts of liquid, an incubating station, a thermostatic chamber, a refrigerated storage chamber of agar strips and a reading station which possibly includes a computer and recorder.

The invention will be further described in conjunction with the accompanying drawings.

FIG. 1 is a block diagram of an embodiment of an installation according to the invention.

FIG. 2 shows protective tubes containing samples, and means for supplying a special atmosphere.

FIG. 3 is a cross section along the line 3-3 in FIG. 2.

FIG. 4 shows means for feeding different gel substrates, and

FIG. 5 shows a cutting machine for making gel layers from gel blocks.

The basic unit in the automation technique consists of long, narrow strips or shallow troughs of glass which are 2 X 50 cm and have a thin substrate layer l-2 mm thick. The glass strips are kept in sterile cassettes or tubes which can be provided with individual gas milieus, e.g., N air, 10% CO in air, etc. A module system has been worked out to extend from there. Said system is designed to perform the following functions:

A. Distribution, which aims at producing separate colonies from the micro-organisms in a mixture. From here, for example, the bacteria in a clinical sample can be isolated; micro-organisms in a fluid impinger used for testing the air can be isolated; and mutants from an irradiated, or an otherwise genetically manipulated population, can be isolated. The substrate strip consisting ofa gel hereafter called agar" is injected into an insulating tube, and the material from a testing wire is spread out along the middle line of said strip.

B. Transferring to transmit material from a colony to a fluid substrate, to a glass strip for microscoping or to one or more special substrates. Examples of this are e.g., when large quantities of interesting microorganisms are produced in bouillon tubes for later serologic and other testing, when microscopic particles are spread out and coloured to facilitate identification, and when the object is to produce outgrowth on selective substrates, also for identification purposes. This is done by allowing the agar strip to pass a microscope where a sterile test wire sensitized by a foot pedal can transmit the material to a desired place. The clipped-off wire can, in certain instances, function as a magnetic agitator in the tubes, but is unnecessary in most cases.

C. Determination of resistance to antibiotics and inhibitors by means of measuring the size on the agar substrate of the inhibiting zones. By such a diffusion analysis, the sensitivity of micro-organisms to solutions of different substances can be determined. This can facilitate the indentification of unknown micro-organisms, but most important, it makes possible a determination of the sensitivity of the pathogenic agents to different types of antibiotics, and thereby also the choice of suitable therapy. The effect of a biologically active inhibitor on different known micro-organisms can also be determined. This is done by gradual feeding in an insulating tube whereby the actual micro-organism is spread over the agar surface with the help of a disposable needle, and in this connection small liquid samples which are sucked up in a suitable carrier (spiral) are applied.

D. Quantitative microbiological analysis with the help of bacteria embedded in the agar whose purpose is to determine, by a measurement of the size of the growth zone or the inhibition zone, unknown substances concentration in solutions with the effect on selected micro-organisms. This type of standardized diffusion analysis can be used for the quantitative determination of amino acids, vitamins and antibiotics in solutions. The procedure takes place as follows: Agar strips containing a suitable concentration of sample organisms are gradually fed into an insulating tube at the same time as small liquid samples sucked up in a suitable carrier (spiral) are applied. When determining amino acids and vitamins, so-called auxoautotrophic bacterial strains i.e., micro-organisms are used, the growth of which is dependent on the substance to be determined. This is of course excluded from the agar used.

E. Screening of micro-organisms which produce microbiologically active factors the purpose of which is to select the colonies from a mixture of micro organisms which are capable of producing a certain active factor. By means of such screening from e.g., soil samples or a mutant population, micro-organisms can be obtained which are capable of producing amino acids or antibiotics. In doing so, agar strips containing sample organisms in a suitable medium are injected into an insulating tube and the material with microorganisms from a testing wire is spread along the middle line of the strip.

In paragraph A above, a sample, consisting of microorganisms is added to the substrate, as is also the case in paragraph B. In paragraphs C and D, a certain microorganism is spread over the surface of the agar medium or embedded within the medium, and the sample consists of some microbiologically active substance which is added. In paragraph E, one microorganism is embedded in the substrate, and another microorganism is added as sample. The sample, which is added to initiate one of the various microbiological reactions to be studied by the present method and installation, is hereafter generally referred to as microbiologically active substance. Thus this expression is directed towards a culture of a microorganism or mixture of microorganisms, as well as a solution of some microbiologically active substance.

On the basis of these aims, a carefully thought out module system has been worked out for the required modules or components. This module coupling constitutes a closed, wholly. automatic analyzer which works continuously 24 hours per day. The block diagram on FIG. I shows an embodiment of an installation according to the invention consisting of a. a cutter for making agar strips from agar units which simultaneously lays said agar strips on the glass strips,

b. a machine for making spirals which transmit small amounts of liquid,

c. an incubating station,

d. a thermostatic chamber.

e. a refrigerated storage chamber for pre-incubated agar strips, and

f. a reading station possibly including manipulative operations, dependent on the type of analysis.

Special probiems are invited by embedding agar medium on glass strips in the form of mm wide and 1-2 mm thick strips. Agar melts when heated in boiling water baths, and solidifies again when a cultivating temperature (usually 37C) is neared. If a continuous embedding is to take place, the substrate must be kept at approximately 50C; this often causes a rapid worsening of the quality. Therefore, according to the invention, the embedding is done in units which can be kept at +4C. The cutter cuts up the unit into strips of desired thickness. These strips can then be directly laid on the glass strips. lOO20(l strips can be made from each agar medium unit. Previous traditional methods have provided for the embedding to be done manually in Petri dishes. These methods were time-consuming and not practically possible for the very large number of strips which were required for the machine according to the invention.

The cutting machine according to the invention is shown in greater detail on FIG. 5. The agar block 40 is embedded in a glass cassette 41 and is then laid on the base 48 of the cutting machine; the feeding belt from the piston 45 is coupled to the feeding mechanism 44. When a glass strip 47 is then placed in position, the desired strip thickness for the agar is set and the cutting machine is turned on. The agar block is then fed forward an amount equal to the desired thickness and a knife 42, operated by a drive mechanism 43, cuts off an agar sliver 47 which falls down onto the glass strip 46.

In previous works, a procedure has been described for taking up and transmitting small amounts of liquid with the help of textile thread. According to the present invention, this method has been improved whereby small spirals of stainless steel ribbon are used instead. In this way the possibilities are far greater of varying the volume, when necessary, on the transmitted amount of liquid. Furthermore, the steel spiral can be controlled by a magnetic field. After a large number of tests with rustproof spirals having different dimensions, ribbon thickness, spiral diameter, length and gradation, a spiral has been chosen which can transmit 13 ,ul solution with a relative standard deviation of 5 percent. The spiral can of course be widely varied depending on its different uses.

In the incubating station, the sample is transmitted with the help of the spiral or thread from the cutter to the agar strips. Said agar strips are then fed into insulating tubes which have a rubber membrane on the bottom. The insulating tubes are fed lying laterally onto the conveyor and are then moved in their longitudinal direction a few mm to the side of a guide rail. The lid which remains in the conveying direction thereby becomes free of the tube. The tube is then swung by another rail up 45, and the glass strip with the agar strip is allowed to slide into the insulating tube which then swings back. If the cultivation is to take place in a normal atmosphere, the tube is laterally moved back so that the lid is automatically put on. But if the cultivation is to take place in another atmosphere, the tube is instead moved further to the side a few cm, and away from the lid. In this way a cannula penetrates through the rubber bottom of the insulating tube. The desired gas, e.g,, nitrogen, carbon dioxide, etc., is blown in through the cannula for a desired time. Thereafter, the tube is laterally displaced back. With the return of the insulating tube, the cannula is pulled out, the bottom seals and the lid is reset automatically.

In an alternative procedure, the tube becomes axially fixed, and instead the lid and cannula are maneuvered e.g., hydraulically, pneumatically or electrically.

FIG. 2 illustrates the supply of controlled gas with a movable cannula 31 operated by a hydraulic cylinder 37. The gas is fed to the cannula through a pipe 30, and the gas supply after the penetration of the cannula 31 into the membrane 32 is controlled by a controlling device 38. Thereby the insulating tube 33 is fixed in tube brackets 34.

FIG. 3 is a section illustrating how the layer of agar 36 on the glass strip 35 is placed in the insulating tube 33.

It has also been shown that when there are from 1,000 to 2,000 tubes in the apparatus, the intermittent conveying with stops for each feeding of a glass strip causes vibrations and mechanical stresses. Therefore, a construction has been made where the tubes are conveyed suspended from clamps on a continuously moving chain. Said tubes are then lifted over on a chain having intermittent movement. Said chain takes care of the conveying operation through the incubating chamber. Immediately after this procedure, the tubes are again lifted over to another, or the same continuously moving chain. In this way, the intermittently moving mass will be so small that it cannot cause any vibrations in the machine.

The cassettes or tubes are incubated in the thermostatic chamber during the continuous feed through operation. The capacity is adjustable, and with a feeding rate of one cassette per minute, the installation has a capacity of 1,500 samples per 24 hours.

While awaiting the reading operation, the preincubated agar strips are kept in the refrigerated storage chamber.

The reading of the samples is finally taken in the reading station which possibly has optional work operations. The values are then transmitted to the computer to be registered; the net values are then assessed for final feeding as completed analysis results.

Supply and circulation lines are also included in the installation. Thus there is a chamber for rustproof wire for feeding said wire to the spiral machine and a washing station for cassettes from the reading station. Said cassettes are then fed to a chamber for renewed use. After the reading station, the agar strips are further scraped off from the glass strips. Said glass strips are washed, move on to a chamber and are then fed anew into the cutter.

Large quantities of inoculum are required for agar units which are used for standardized microbiological diffusion analyses. Tests have shown that several of the most common sample organisms for the microbiological determination of B-vitamins and amino acids can be kept without any drawbacks as a washed cell pasta at 20C for 4 weeks. In certain cases they can be kept for up to 8 weeks. In this way the supply of agar to the installation can take place without any trouble.

With the methods described above, the free content of seven different amino acids in the urine has been determined. Furthermore, eight different B-vitamins have been determined by using the same basic medium as for the amino acids. This method of analysis has shown itself to be so sensitive that the percentage of free biotine in rat plasma could be determined to l nanogram/ml.

With the above mentioned installation, inoculated agar strips are placed in sealed tubes which are continuously fed through a thermostat unit. However, it may often be desirable to have so-called buillon cultures where micro-organisms are cultivated in liquid substrate. The specimen tubes according to the above also permit the setting in of continuous so-called test tube racks which are led through the installation in the same way as the agar strips and are similarly assessed at the reading station. Such test tube racks are preferably in the form of a plastics strip with indented cavities having a capacity of 0.1 or 1 ml, even if the size of the tubes makes it possible to use significantly larger cavities. In the extreme case of chromatography, a single depression can be provided which runs the entire length of a filter paper strip or, in the alternative, a thin layer bed. If, in the test tube rack used, each cavity has a capacity of 0.1 ml, there will be room for 99 cavities in each rack, a number which allows for a large series of tests in a quite limited space.

That there is need for a vast number of test series and great experimental capacity is evident from the fact that a teaspoon of earth can contain hundreds of different types of microorganisms. To determine more than half of these with present methods requires the use of a large and well equipped laboratory for a week or so. Therefore, it would be almost unthinkable to systematically follow the ecological effects of different controlling agents, etc. Apart from the costs, there are simply not enough resources available for the studies.

Separate colonies can e.g., first be isolated from the sample by cultivation on agar strips. Material fromthe colonies can then be easily transferred to liquid substrate portions in the test tube rack cavities which are then sealed with tape or, in the alternative, rice paper or fibre glass paper. A similar technique may be used in genetic selection work.

Cultures in liquid substrates quite often occur in dilutions and the tubes ought to be usable for serological work where constant amounts of an antigen or an antibody are allowed to react with opposite components in the dilutions l, 2, 4, 8, 16, 32, etc. In order to obtain optimum culture substrates, a number of different substances, amino acids, vitamins, salts, etc. are admixed. For example, when tissue cells are cultivated, up to 50 different components must be tested in the admixture where not only a large number of different components included are varied, but also their relative, mutual concentrations.

Both the dilution of samples and the additives are carried out with pipettes. One must be painstaking to avoid every impurity or non-desirable transferring, such as moving a sample to some of the master solutions with nutrients, transferring one nutrient to another, etc., things that may easily happen when dilution with pipettes occurs in a normal manner. The procedure shall also not demand too much time. This problem can possibly be solved by the system of handling small amounts of liquid by steel spirals, as mentioned above. There are also other well-known supply methods and arrangements for handling liquid samples.

As an alternative to the use of spirals according to the above for supplying liquid samples to gel layers, filter paper slips may be used which have been impregnated with the liquid to be supplied to the gel.

Application of the slips on agar plates has usually been effected manually but, in certain cases, has been rationalized by having each little round strip form the point on a jag of a punched-out star of filter paper. The preparing in patches of a complete filter paper cannot be effected since a sharp boundary for the spreading out of the active preparation is not then obtained.

With all such research work, it is most important to maintain sterile conditions. Because of this, handling of the small slips becomes time-consuming and difficult. To simplify the handling by pasting a number of slips in the form of disposable matrices is not suitable since the glue used could diffuse into the filter paper slips and damage the active substance.

In one improvement, the slips could be secured on an adhesive tape with only surface contact, where the sticky surface is of such nature that it yields neither vapors nor fluids. The sticky surface is preferably made ofa known per se, uniform, solid, sticky substance such as a high-molecular, sticky polymer.

With the practical application, the slips may be punched out of prepared filter paper and either remain stored in the hollow punchers or brought into contact with adhesive tape in special tubes. For example, such tubes with slips containing separate preparations are set up beside each other at a distance of about 15-30 mm with the opening facing up. The slips are forced up against the opening with a spring while they are prevented from being squeezed out by a tongue or guard in the mouth of the tube. A strip of tape is placed above the mouth of the tube, the slips stick when contacted with the sticky surface and the top slip in each tube is lifted up and is fixed onto the strip. The next portion of the tape can then be similarly handled, etc. The tape is manually rolled up into a roll containing a large number of batches of prepared slips protected from the air. The tape may then be stored for future use.

If the gel diffusion technique is to be used, the tape strip can e.g., be perforated with holes corresponding in number to the serum samples which are suspected to contain a certain antigen (e.g., Au in hepatitus). On the sides of each hole are a slip prepared with antibodies (Anti Au) and a slip containing antigen (-l-Au). if a certain serum sample now applied on the agar via the tape hole results in a line of precipitation towards the antibody slip, this indicates an early infection stage. If, on the other hand, it results in a line of precipitation towards the antigen slip, this means that the patient has developed antibodies of the type occurring after the termination of an infection.

When used, the tape with the sticky surface and the slips are laid down on an agar strip made e.g., as described above. This is done either manually or automatically. Incubation then occurs in a normal manner in a thermostat cabinet for a suitable period of time, e.g., 24 hours.

Of course, all work is effected in a known per se manner under sterile conditions but the handling and care taken are improved in comparison with earlier methods since the tape with analysis batches can be fully preprepared with the use of the most suitable tools and instruments.

The method is not confined in principle to these loose slips but, according to another embodiment, narrow strips of filter paper magazined in rolls are prepared instead. These strips are then pulled out parallel to each other from the rolls at a distance from each other of 15-30 mm. The tape is placed perpendicular to the direction of the strips and the latter are then lopped off in line with the tape edge. The method may also be modified in numerous other ways, e.g., with a wide tape which is lopped off together with the strips to a narrower width by supplying the small round filter paper slips with automatic applications, etc.

Registration and evaluation of the samples then take place in a normal manner by measuring the stain formed around each slip where the growth is hindered or stimulated, respectively, or by studying the position and dispersion of the precipitate which may occur with slips impregnated with different antigens and antibodies.

Despite the fact that the tape covers the agar, as a rule enough oxygen penetrates, due to the agar being strip-like, so that even aerobic bacteria will be able to grow. However, oxygen diffusion can be further facilitated by making the surface of the agar layer serrated. This is most easily done when the cutting in the cutter is effected with a thin wire or edge which, during the actual slicing operation, is made to oscillate in a known per se manner by mechanically, electrically or otherwise produced vibrations.

By serrating the agar surface. reading of the samples may also be facilitated under certain conditions (moist agar surface) since the growth preferably occurs in the formed grooves which makes the limits for the growth inhibition appear more clearly. This sharper boundary is especially important when reading is done automatically.

A technique somewhat different than the one described above for applying the active substances may also be used. Here bands or wires prepared with various substances are rolled up on spools mounted beside each other on a common shaft. The length of the shaft corresponds approximately to that of the agar strip. The narrow bands or wires are then simultaneously pulled out right across the agar strip and are then cut on both sides of said strip to lie in intimate contact with the surface.

Different types of agare are required for different purposes and micro-organism, and a number of various agar types can be arranged in a magazine, a certain arbitrary number of which can be coupled in. The agar block is propelled forwards a bit, corresponding to the desired thickness of the preparation, and is then sliced with transverse knife.

FIG. 4 shows an arrangement where there is a row 1 of spools with rolled-up bands or wires 2 on a shaft. The row of spools lies with its axis parallel to the agar strip and the bands are pulled out directly over said strip by means of a grip. The bands or wires are then cut off by a cutter which may consist of either long knives being of the same length as the agar strip or slitting knives or the like which run on a guide along the side edge of the agar strip.

On the figure, the row 1 of spools is placed above a stack with six cassettes 3-8 containing different types of agar. In the first step, a suitable length of agar is pushed out from the desired cassette by e.g., a hydraulic cylinder, and a slice is cut off with a knife 9. The slice is laid on a glass strip which rests on a hoist 10.

The glass strip is held in a magazine 12 and is fed from there on a track 11 to the hoist 10 which is controlled by e.g., a hydraulic cylinder 13 and is provided with a driver 14 that, at a determined height, may either feed or extract agar strips to or from protective tubes 15. The arrangement is designed to keep agar strips without protective tubes in sterile, well moistened air. But, in certain cases, it may be necessary to have a protective gas, a special atmosphere, a temperature other than room temperature, etc. Therefore, the agar strips, at this stage, may even be fed out for e.g., treatment with trichloracetic acid, reaction with fluorescent antibodies or to storage at 37C in special protective tubes.

When the height of the row 1 of spools is set for feeding out the bands, this application takes place at the level 16. The strip is then hoisted one more step and carried onto a conveyor chain 17 which feeds it upwards stepwise.

Procedures are taken hereunder for the substances, with which the band is prepared, to diffuse out into the agar. After a predetermined, but optional period of time, the strip is transferred by a driver 18 to a downwards moving conveyor belt where inoculation is carried out by spreading the bacteria strains and the like, present here, as a film over the agar surface. At the same time, the bands set on can be removed. After a suitable period, which is similarly determined by a driver 19, the strip is fed up a certain height to an upwards moving conveyor chain where it finally reaches the level 20.

Here it is fed by means of a carrier 21 to a table 22 where it may either be carried to the left for optical reading at the arrow 23, or to the right for treatment with trichloroacetic acid in a protective tube before continuing on to optical reading.

With this installation, the possibility now exists of carrying out large scale taxonomic work with microorganisms. To this end a systematic identification process can be carried out with the help of optic or isotope-technical zoneand colony analysis based possibly on a monochromatic laser technique whereby a computer is coupled into the system. The module system has the great advantage of making possible a complete reproductive treatment scheme of inoculation, incubation, etc. The apparatus also performs a considerable time-saving function, e.g., in an epidemic situation. Because of its compact construction, it requires very little laboratory space and fewer personnel compared with the requirements for the same work contribution with conventional technique.

What is claimed is:

1. Apparatus for automatic performance of microbiological analyses of samples applied to a gel substrate layer, comprising a gel substrate preparation station, an inoculating station, an incubating station, a reading station capable of detecting microbiol growth, and conveyor means, said gel substrate preparation station comprising a solid block of said gel having a generally uniform elongated rectangular transverse cross-section with respect to at least one axis, cutter means for slicing successive transverse thin strips of gel from said block, a supply of similar glass strips; each having an area sufficient to provide a support for at least one of said thin sliced strips of gel, said conveyor means including means to relatively position glass strips with respect to sliced strips of gel for deposit of the slices of gel on successive glass strips at a first position and to move said glass strips in a sequential manner to other said stations after receiving a slice of gel.

2. The invention according to claim 1 wherein said block of gel comprises a rectangular slab the dimensions in cross-section in one plane having a ratio greater than l: 1 and said apparatus includes means to move said slab towards said cutter means.

3. The invention according to claim 1 wherein said conveyor means includes means to transfer said glass strip and gel layer to a plurality of stations.

4. The invention according to claim 1 wherein said plurality of stations includes a station for inoculating the gel strip prior to a station for inserting a glass strip and inoculated gel layer into a protective tube.

5. The invention according to claim 4 wherein one of said plurality of stations includes means to introduce a gas into the interior of a protective tube containing an inoculated gel layer.

6. The invention according to claim 5 wherein said protective tube includes an rubber membrane and said means to introduce said gas includes a cannula for penetrating said membrane.

7. The invention according to claim 4 wherein said plurality of stations includes a refrigerated storage space.

8. The invention according to claim 2 wherein said plurality of stations are located at at least two vertical levels with respect to each other and said conveyor means includes means to move a glass strip selectively upwardly and downwardly.

9. The invention according to claim 2 wherein said conveyor means includes two adjacent sections, each of said sections being movable in opposite directions, and transfer means to shift a glass strip between one section and the other section to move a glass strip between two of said stations.

10. The invention according to claim 9 wherein said conveyor means includes a plurality of endless chain means disposed generally in longitudinal alignment and said plurality of stations are disposed generally at locations in planes spaced from each other in the direction of said alignment.

11. The invention according to claim 10 wherein said apparatus also includes means to store a plurality of gel blocks generally in the form of rectangular thin slabs in stacked superposed relation to each other, means to selectively position a glass strip adjacent the end of any one of said slabs and cutter means to'slice a strip from said one end of a slab for placement on said strip.

12. The invention according to claim 11 wherein said apparatus includes means to supply a plurality of microbiologically active substances to slice of gel'on a glass strip at one end of said plurality of stations.

13. The invention according to claim 12 wherein said means to supply a plurality of microbiologically active substances includes a wire coil, the active substance being contained within said coil, and means to sever the wire coil in a length proportional to the amount of substance desired.

14. The invention according to claim 12 wherein said means to supply a plurality of microbiologically active substances includes a porous band having a substance contained therein.

15. The invention according to claim 12 wherein said means to supply a plurality of microbiologically active substances includes adhesive tape, and filter paper adhered to said tape, the active substance being contained by said filter paper.

16. The invention according to claim 12 wherein said slabs are vertically stacked in horizontal superposed planes, and the sections of said conveyor means each comprise a pair of vertically extending horizontally spaced endless chains.

17. The invention according to claim 3 wherein said station for the receipt of a microbiologically active substance includes means to supply a plurality of said substances, said means to supply a plurality of active substances including a horizontally aligned group of individual supplies of said substances to be dispensed to various locations along the length of a slice of gel.

18. The invention according to claim 17 wherein said means to supply said microbiologically active substances includes a plurality of revoluble spools mounted in axial alignment on an axis parallel to the length of said slice of gel.

19. The invention according to claim 1 wherein said apparatus includes means to store a plurality of gel blocks generally in the form of rectangular thin slabs, conveyor means to support a glass strip adjacent one end of any one of said slabs, and cutter means to slice off a thin strip of said one end of said slab for placement on said glass strip.

20. The invention according to claim 19 wherein said apparatus also includes means to store a plurality of said glass strips and means to successively position said strips to receive a thin strip of gel thereon.

21. The invention according to claim 20 wherein said conveyor means includes vertically reciprocal hoist means to support a glass strip to receive a slice of gel,

two series of vertically spaced elements movable concurrently in parallel paths adjacent said hoist means and transfer means to move glass strips horizontally from said hoist means to opposite pairs of said elements.

22. The invention according to claim 21 wherein said conveyor means includes a second two series of vertically spaced elements movable concurrently in parallel paths adjacent the paths of said first mentioned two series of elements, and transfer means to move glass strips horizontally between said first and second two series of elements.

23. The invention according to claim 3 wherein said station for examination of an inoculated gel slice comprises optical zone and colony analysis means including monochromatic laser means.

24. The invention according to claim 1 wherein said plurality of stations includes a station for removing said strips of gel from successive glass strips.

25. The invention according to claim 24 wherein said plurality of stations includes means for washing successive glass strips, and for returning said glass strips to said means to advance successive glass strips to said first position.

* i= l= l=

Claims

1. APPARATUS FOR AUTOMATIC PERFORMANCE OF MICROBIOLOGICAL ANALYSES OF SAMPLES APPLIED TO A GEL SUBSTRATE LAYER, COMPRISING A GEL SUBSTRATE PREPARATION SATION, AN INOCULATING STATION, AN INCUBATING STATION, A READING STATION CAPABLE OF DETECTING MICROBIOL GROWTH, AND CONVEYOR MEANS, SAID GEL SUBSTRATE PREPARATION STATION COMPRISING A SOLID BLOCK OF SAID GEL HAVING A GENERALLY UNIFORM ELONGATED RECTANGULAR TRANSVERSE CROSSSECTION WITH RESPECT TO AT LEAST ONE AXIS, CUTTER MEANS FOR SLICING SUCCESSIVE TRANSVERSE THIN STRIPS OF GEL FROM SAID BLOCK, A SIPPLY OF SIMILAR GLASS STRIPS, EACH HAVING AN AREA SUFFICIENT TO PROVIDE A SUPPORT FOR AT LEAST ONE OF SAID THIN SLICED STRIPS OF GEL, SAID CONVEYOR MEANS INCLUDING MEANS TO RELATIVELY

2. The invention according to claim 1 wherein said block of gel comprises a rectangular slab the dimensions in cross-section in one plane having a ratio greater than 15:1, and said apparatus includes means to move said slab towards said cutter means.

11. The invention according to claim 10 wherein said apparatus also includes means to store a plurality of gel blocks generally in the form of rectangular thin slabs in stacked superposed relation to each other, means to selectively position a glass strip adjacent the end of any one of said slabs and cutter means to slice a strip from said one end of a slab for placement on said strip.

12. The invention according to claim 11 wherein said apparatus includes means to supply a plurality of microbiologically active substances to slice of gel on a glass strip at one end of said plurality of stations.

21. The invention according to claim 20 wherein said conveyor means includes vertically reciprocal hoist means to support a glass strip to receive a slice of gel, two series of vertically spaced elements movable concurrently in parallel paths adjacent said hoist means and transfer means to move glass strips horizontally from said hoist means to opposite pairs of said elements.