PROCESS AND APPARATUS FOR MEASURING THE PARAMETERS CHARACTERIZING THE MICROBIOLOGICAL INTERACTIONS BETWEEN PHAGES AND BACTERIA

Abstract: A process and an apparatus are described for determin­ ing the quantities characterizing tne phage development cycle starting at the interaction between virulent phages and host-bacteria within the duration of approx. one single burst. These parameters enable determination of expectable phage-yield, duration and standard deviation of phage development, multiplicity of infection. which are determined by the apparatus from signals in correlation with the number of bacteria (e.g. optical scattering). By collecting a proper programme the apparatus is well suitable for the quantitative determination of the biological response (parameter-change) occuring at the change in the environment resp. state of the phages behaving as unliving obiects, the bacteria and complexes and as a result of their interaction.

Description: PROCESS AND APPARATUS FOR MEASURING THE PARAMETERS CHARACTERIZING THE MICROBIOLOGICAL INTERACTIONS BETWEEN PHAGES AND BACTERIA The invention relates to 8 process and an apparatus for the realization of the process according to the invent ion with the purpose two determine during the approximate cycle- time of phage development the parameters of phage development cycle having been defined in the course of the interactions between virulent phages and carrier-bacteria (so e.g. the velocity constant of adsorbtion, T the time of phage development, and standard deviation theieof z , phage-yield C to be expected, multiplicity index of infection mi), i.e. to measure biological changes realized in the change of parameters caused by the physical and/or chemical effects affecting the phages or bacteria or the complexes thereof. The process and the equipment accordIng to the invent ion can be advantageously applied e.g. In the pharms- ceutical and chemical industry, in agriculture, in the field of ecology and everywhere, there there is a demand for te quantitative evaluation of the biological (resp. molecular biological) effects of physical and/or chamical agents. About phages, as biological objects and macromoleoules a versatile knowledge stays at disposal Dot- in respect to biological function and structure. In contraet to that, application of the phages for practical (biotechnological') purposes - as a possibility - has been slightly utilized up to now. Taking the increased demand for quantitative biological measurings and tests into consideration, it seems to be most probable that application of phage-bactris complexes - due to their well definable and measurable, quantitatively characterizable interactions - will come into the limelight. Quantities characterizing phage development cycle and determination thereof were defined and summarized in the classical work of Adams (Adams, J.',1. 3acteriophages, Intersci. Publ. New York). The methods described therein have been used ever since, however, with more or less modification. Grossi et al. (Groasi G.i. Cesarini, G. and Liello, F.1977. Development of phage populations in a bacterial culture: A mathematical model.Z.Naturforsch. 32 644-849) elaborated the method for the determination of the parameters characterizing the cycle of phage development, being more accurate, than the previous one. The diesdvantage of this work, however, lies in that their method is based on the experimental results of other authors, so the lack of the own experimental background inhibited further development of the model as well as propagation of application. Gáspár et al. (S. Gáspár, Gy. Rontó and G. Müller 1979. Determination of the biological parameters of bacterium-phage complexes. Zeitschrift für Allg.Mikrobiol. 19 163-169) performed experiments directed to measuring the parameters characterizing the phage development cycle and mathematical models co-ordinated with experimental conditions were prepared. In such a manner parameter-values having been defined by said process were reliable and accurate but at the same time preparatory works and performance of measurements weeded were utmost complicated and evaluation of result was time connumptive. Concentration measuring based on the interaction between bacterium and phage, the determination of the so-called plague forming activity has been frequently used for detecting certain physical reap. chemical damaging effects. So e.g. Hontó et. al. examined the damaging effect of UV radiation and radiomimetics on phages, wherein the experimental data given in the reporting publication, i.e. measurements,represent the result of minimally several months of research activity (Rontó, Gy., Sarkadi,K. und Tarjin, I.: 1967. Zur Analyse der UV-Dosiswirkingskurven der T7-Phagen, Strahlenter. 134 151-157 ; Karczag, A., Rontó, Gy. and Tarján, I. : 1972. Study of the UV-effect on MS2 phages. Acta Biochem. Biophys. Hung 7 173-177; ont6, Gy., Smotryseva, M, Kruglya kova, K., Emmanuel, N., and Tarján, I.: 1975. Intactivation of bacteriophages T7 and \F9 X 174 by radiomimetics. Acts Bio chem.Biophys. Rung. 10 115-122). The Hungarian Patent Application SE-1938 relating to a measuring (qualifying) process based on the lethal mutstion of the phage T7 of the mutagenic effect of chemical substances, led back the measuring process to the determination of plague forming activity in a traditicna' Lanoer. Parameters characterizing the phage development cycle can be used as reliable measuring informations for characterizing different processes, if goos reproducibility of measuring, reliability and quick automatic performance can be ensured. For the sake of reproducibility with certain part-processes time constancy of the bacterium-suspension, the biological state being suitable for measuring, as well as concentration needed for measuring must be ensured. A further task lies in the elimination of the statistic errors of manual processes according to original descriptions (plague counting titration ). Simultaneously, in order to be able to eliminate subjective errors resulting from plague counting it seems to be expedient to search macroscopic characteristics, which are in an unsmbiguous correlation with the number of bacteria and can be measured in an automatized manner. Furtheron, it seems to be expedient to measure the parameters - compared to known methods - more accurately and within the possible shortest time (if possible, during the burst time). Timely constancy of the bacterium suspension and concentration needed for measuring can be ensured by a continuouslg operated fermentor. A further task of the fermentor is to maintain the proper biological state of the bacteria to be able to separate phage development cycles following each other in interest of simple evaluation. In order to be able to eliminate statistical errors infection with a high multiplicitJ was used whIch led within a shorter tize (during one burst) to change which could be detected also by physical /chemical characteristic. In this state namely the phages dissolve the bacteria (lysis). The process can be measured by detecting the number of bacteria, or any light-scattering, light absorption, ionenviroment etc. being in an unambigous connection with the number of bacteria. In case of an infection of high multiplicity (about 1) ,the ratio signsl/noise will be optimal which enables accurate determination of multiplicity and by the derivation of the measured signals measuring of the parameters T and . or the quick determination of the expectable phage-yield bacteria used to be infected with a multiplicity of mi > 3, in this case at the end of the phage development cycle a complete lysis takes place. On the lysate thus obtained - containing also the newly developed phages - measuring of the multiplicity mi can be repeated by the known dilution. The product of the multiplicity having been determined in such a manner and of the dilution applied give the expectsble phage-yield C. Accordingly, independently of the bacterium concentration the meaeurement determines the expectable numbers of phages arising from one bacterium during the period of two bursts. The advantage of the specified process lies in that for its realization the equipment can be organized unit per unit in a separated form, principal layout thereof can be seen in the drawing. The equipment for performing the process according to the @ invention consists of the ir dependent fermentor 1, wherefrom bacteria in the reeves quantity arrive through the charger 2 into the biological reactor 5; from the recipient 3 storing the phages'the suitable phage quantity was already led into said reactor via the diluting unit 4. Gas-charger 6 ensures mixing, eventual oxygen supply or closing from oxygen. At the beginning of the measuring cycle a part of the sample arrives by the aid of the liquid forwarding unit 8 into the measuring unit 7; if it is possible, in order to ensure continuous measuring, unit 7 should be designed with a flow-type character or so, that it could be used as a reaction space (introduction of gas, thermostating). At the end of the completed phage development cycle the sample returns to the measuring unit 7, and after the measurement it arrives to the collector 9. The process and the equipment according to the invention - m8 specified above - is measuring the characterizing parameters of the phage-bacterium complex, i.e. the measuring process can be repeated, if the critical pert of the system - the biological reaction space - is flushed between two measuring cycles. Distilled water is well suitable for this purpose, as the majority of the active phages disturb- ing the following measuring cycle is irreversibly inactivated in the ion-free waster. Both the process and the equipment are well suitable for measuring biological consequences of certain (physical and/or chemical ) effects in the following manner: I. Inactivation test By dialysis the purified phages can be dissolved to have an optional solution and can be stored thereic practically for an unlimited time. In the solution the phages behave as unliving objects as long as they do not contact with their host cells (bacteria ). The phages having the properties of living matter but not showing symptoms of lie in the solution, can be subjected to well defined physical and/or chemical effects and in accordance with the process according to the invention we can test all the biological consequences of the structural change occurring under the influence o. treatment. The recipient 3 storing the phages is well suitable for treating the phages (e.g. at a given temperature, incubation with chemicals of given concentration and for a given period ), while the effect (resulting frequently in a reduced activits,i.e. in the change of multiplicity ) can be measured by the apparatus. 2. Cell-virus test In case that anactive phage arrives at a bacteria cell instead of the normal, informations relating to the function of bacteria informations needed for producing phages will be obtained. As already mentioned, parameters cf the phage development cycle depend considerably on the biological state of the bacteria. So e.g. bacteria having been subjected to different treatments in the fermentor can be tested on the basis of the parameters of phage-production. In case of the proper selection of the bacterium-phage system, the partprocess of other cell virus interactions can be also modelled. Intervention into the system is ta'x @g place through tl:e fertentor 1 of the apparatus, in the msasurerent where the phages are untreated. 3. rhage-programme test In case that in the course of the phage-bacterium interaction the adsorption rate constant is high, phage development process in the bacteria contained in the sample-spece is taking place approximately synchronized. The influences affecting the system are contacting the given phases of the phage-producing programme in the phage development cycle in the optional moment, accordingly, change in parameters is giving an information,which is characterizing for the influence effecting the complex. in such a manner photosensitizers, chemical and/or physical effects inhibiting resp. promoting metabolism - which can be activated in the optional moment of the phage development cycle and can be maintained in the activated state for the optional period - can be tested in a quite new manner. Wt; the apparatus according to the invention the effect of the photovsensitizer can be advantageously measured, if the measuring system work based on an optical principle and the activating light is perpendicular to the measuring light path. In the course of a composite measuring process - i.e. the system contains at least two ol fermentors 1 and/or recipients 3 storing the phages, between the fermentors 1 in itselves there is an OP- connection: from a,g of the fermentors 1 ur the recipients 3 storing the phages the materials can get through an AND-connection into one of the biological reaction spaces 5, which are then placed nezessarily in the aut@mstic sample changes 10. It goes without saying that the number of the chargers 2 and diluting units 4 must be in compliance with the number of the fermentors 1 end recipients 3. In the proper moment the sample arrives serially from the sample charger 10 into the measuring unit 7 and therefrom into the collector 9. As it becomes obvious, the composite system can be adventageously automatized and applied, while automatics may perform evaluAtion and recording of the measurement. Example The process and the apparatus according to the invention - which serve for measuring the interaction between phages and bacteria (parameters characterizing biological interaction) - will be detailed by illustrating the measuring of the mutagenic effect, as described in the Hungarian Patent Application 5E-1938, from 'which all the advantages of our method and apparatus, respectively, so speedness, simplicity, reproducibility and possibility of automatization become obvious. The tesk to be solved: folloning the remcant activity of T7 phages incubated with a chemical in dependence of time. Expediently, the apparatus can be realized In the following composition: A fermentor, functioning as a chemostat, that means, that the ratio of the volume of a culture vessel provided with an overflow and the velocity of the fe culture medium defines the growth rate of the bacteria, which is cnstant in time. A bacterium charger, which is delivering the bacterium suspension of a given volute into the biological reaction spaces. Two recipients for storing the phages, into which the phages having been treated with a reageot reap. those serving for control are arranged. Two parallel functioning diluting units, which are forwarding the diluted treated phages and control-phages, respectively into the biological reaction spaces. Obviously, the reaction spaces are arranged in an automatic sample charger and are thermostated to a temperature optimal for the host bacteria. Optionally, the gas feeder can lead 2 gas into any reaction space. The measuring unit is operating on optical basis, it is giving an optical signal proportional to the number of not-lyzed bacteria. For the performance of measuring process the apparatus is provided with a cuvette with the flow character and thermostated to the proper temperat re. A peristaltic pump delivers the samples from the reaetor via 8 cuvette into the collector. From the magnitude of the optical signals measured immediately after infecting the bacteria and at the end of the cycle multiplicity of phages ready to infect can be essi- ly deducted. In the course of the detumdnation it should be considered that partly the phages are adsorbed on the bacteria according to the Poisson distribution, partly two distortioning factors are to e considered, in so far as, te bacteria fall apart to not-completely transparent parts in course of lysis, furtheron during the lysis-period uninfected bacteria are further growing. In the fermantor the 3.coli B/r host bacteria are continuously incubated, e.g. division time amounts to 1,6 h with an td9 minial nutrient enriched with 1,5 g/l glucose and 1,5 g/l casaminoacid. The proper solution of the chemical to be measured and the control solution (containing merelg a solvent) are mixed with the needed quantity of purified bacteriophage (e.g.in a concentration of 1011/ml). Thereafter the solut- ions are led into the suitable recipients storing the phages. In proper moments of measuring the diluting units allow to flow identically diluted samples from both of the treated and control samples, respectively, into the reaction spaces (e.g. 1 ml each per sample space in a terfold dilution). As 8 luting solution the nutrient is used. Thereafter the bacterium charger is feeding in equal intervals (e.g, in every minute) host-bacteria into the reaction spaces (so e.g. 3 ml each into the reaction spaces of a six-member sample changer), meanwhile oxygen is bubbled through the substance to obtain proper mixing. The samples in the reaction spaces are freshened by a throughflow of C2 from time to time (e.g. at least once in every minute for a period of minimum 5 seconds), quite up to the beginning of the lysis (e.g. into each single sanple space from feeding the bacteria up to the 14th minute ) . pro: the reaction space containing a selected dilution of the treated phages, a part of the sample is transferred into the optical measuring device by the aid of a peristaltic pump. By the continuous measurement and derivation of the optical signal we are controlling, whether latency time and its standard deviation (e.g. 18 minutes and 3 minutes, resp.) comply with preacriptions (biological control). In case if the biological control does not meet the requirements, the diluted chemical may influence metabolism of the host bacteria, which excludes evaluation of measuring. In case of suitable values of biological control, after having finished lysis (e.g. in the 26th minute after bacteria feeding) the samples are suctioned one after the other - io the rhythm of bacteria feeding - through the measuring cuvette, and the magnitude of the optical signels is registered. After finishing the measurement and rinsing the reaction spaces with distilled water the process can be repeated from the next moment of measuring (e.g. in every forty minutes). At the end of a measurement series on basis of the magnitude of the optical signals having been defined in points of time belonging to the single samplings the change in the concentration of the treated sample related so the control sample can be calculated, i.e. ne mag obtain the inactivation kinetics of the chemical used. Considering the necessity of continuous control and strict time correlation of the afolementioned processes, it seems to be expedient to carry out measuring with an automatic control and data processing (microprocessored system). The process according to the invention: helps to solve actual biological prblems, as e.g. quick recognition of the mutagenic effect of chemicals i.e. quantitative evaluation theraof, as well as promotion of research of substances affect ing viri and bacteria i.e. physical effects exerted thereon. Quickly obtainable and reliable data of the (auto- matized ) apparatus having been assembled according to the given task, enables application for biotechnological purposes and useful application of dynamically developing biological science.

Claims: Claims : 1. Process for measuring the parameters of the phage development cycle occuring under the effect of the interaction between bacteria and phages by using methods of detection known in itselves c h a r a c t e r i z e d in that after having mixed the phages and host-bacteria by continuously measuring physical and/or chemical charac- teristics in a functional correlation with the number of bacteria, during one single process of interaction, by using the values of the measured signals detected at the beginning and end of a generating cycle the index of multiplicity (mi) can be determined; by means of the zero-point of the second derived function of the measured signal according to the period of the generating cycle latence time (T) can be determined, on basis of the third derived function standard deviation of latence time ($) can be obtained, i.e. by proper dilution of the lyeate obtained at the end of the first cycle of generation and starting a naw cycle and measuring repeatedly multiplicity, expectable phage-yield (C) can be determined. 2. Apparatus for measuring the microbiological interactions between bacteria and phages containing bacteria source(s) , spaces for storing the phages, reaction spaces for establishing the interaction and measuring device(s), c h a r a c t e r i z e d in that the bacterium roducirg fermentor Cl) is connected to the biological reaction space (5) through a charger (2), space (3) for storing the phages and diluting t:nit (4), according to necessity gas can be fed through a feeder (6) into said reaction space which is connected to the collector (9) vie the measuring unit (7) and the unit (8) forwarding the liquid. 3. Process as caimed in claim 1., c h a r a C t e r i z e d in that the change in the parameters of the phage development cycle occuring as a result of a physical and/or chemical effect exerted on the phages (inactivation test) is determined. 4. Process according to claim 1 C h a r a c t e r i z e d in that the change of the parameters of the phage development cycle occuring as a result of the physical and/or chemical effect exerted on the bacterium-phage complex (phage-progremme test) is determined. 5. Process as claimed in claim 1, c h a r a c t e r i z e d in that the change of the parameters of the phage development cycle occuring as a result of physical and/or chemical effect exerted on the bacteria (cell- virus test) is determined. 6. Apparatus as claimed in claim 2, c h a r a c t e r i z e d in that the fermentor is operated as a chemostat. 7. Apparatus as claimed in claim 2 end 6 c h 8 r a c t e r i z e d in that at least one measuring unit (7) is operated on optical principles. 8. apparatus as claimed in claim 2 and 6, c h a r a t e r i z e d in that at least one measuring unit (7) is operated on elsctrochemical principles. s. Apparatus as claimed in claim 2 and 6, c in a r a c t e r i z e d in that at least one measuring unit (7) is a cell-counter. 10. Apparatus as clained in claims 2 and 6 to 9, c h a r a c t e r i z e d in that for simultaneous related, resp. parallel measurings two or core fermentors (1) phage-charger@ (2), recipients (3) for atoring, dilut ing units (4) and reaction spaces (5), automatic sample changers (10) performing the connection between said components are used. 11. Apparatus as claimed in any of claim 2 and 6 to 1C, c h a r a c t e r i z e d in tat the surfaces contacting biological substances are made of a hydrophobic material, s.g. teflon. 12. Apparatus as claimed in claims 2 and 6 to 11, c h a r a c t e r i z e d in that the valves regulating the flow consist of pipes in a closed system and ere operated by pipe-break.

Inventors: DERKA, ISTVAN, ERDELYI, JANOS, GASPAR, SANDOR, MODOS, KAROLY, MULLER, HENRIK, RONTO, GYORGYI, DR. M.D., TARJAN,  IMRE, DR., Derka, István, Erdélyi, János, Gáspár, Sándor, Módos, Károly, Müller, Henrik, Ronto, Györgyi, Dr. M.D., Tarjan, Imre, Dr.
Applicants: MAGYAR TUDOMANYOS AKADEMIA TERMESZETTUDOMANYI KUTATOLABORATORIUMAI BIOFIZIKAI KUTATOLABORATORIUMA, RADELKIS ELEKTROKEMIAI MUSZERGYARTO IPARI SZOVETKEZET
Application year: 1984
Main classification: C12Q1
IPC codes: C12Q1, C12M1