GB2157708A - Collecting and preserving microorganisms - Google Patents

Abstract

A collecting medium for micro-organisms comprises a collecting layer for retaining micro-organisms and an incubation layer for conserving the micro-organisms of the collecting layer. Such provides for collection and preserving of micro-organisms, after which identification thereof can be achieved, which is of importance in medical applications.

Description

SPECIFICATION A collecting medium for micro-organisms This invention relates to an improved collecting medium and collection apparatus for micro-organisms.

Present methods of analysis of micro-organisms are unsatisfactory due to the slow response time of the analysis techniques and the relative difficulty in collecting samples in hostile or difficult environment conditions.

In accordance with one aspect of the invention a collecting medium for micro-organisms comprises a collecting layer for retaining micro-organisms that come into contact therewith and an incubation layer for conserving or enhancing the population of the micro-organisms on the collecting layer.

Such a medium allows simple and easy collection and preserving of micro-organisms, after which rapid methods of identification of species of the organisms is achieved by, for example, Pyrolysis Mass-spectrometry (Py-MS) acting singly or in combination with other techniques such as Gas Liquid chromatography (GLC).

Preferably one or both of the collecting and incubation layers are provided with a protective outer layer, the protective layers being removed when the medium is about to be used to collect microorganisms. Furthermore the collecting and incubation layers may be provided and borne on opposite sides of a common base-substrate.

The collecting layer may include two or more different constituent portions, with which two or more different groups or species of micro-organisms are collected. The collection or incubation layer may be composed of one or more organic substances, for example, Nutrient Agar, Nutrient Gelatin, Blood Agar, Tryptose Soya Agar and Gelatin in order to retain and preserve certain specific micro-organisms.

In accordance with another aspect of the invention, an apparatus for use with a collecting medium for micro-organisms in accordance with the invention comprises means for exposing the collecting layer for micro-organism collection and means for positioning the collecting layer in contact with the incubation layer such that the microorganisms are conserved or enhanced.

Preferably the apparatus is totally enclosed so that it may be used in difficult or hostile environments, for instance in the presence of corrosive metallic and non-metallic substances.

The exposing means may comprise an exposure window and associated shutter arrangement in a substantially enclosed apparatus. The positioning means is preferably in the form of a spool which in use matches the collecting layer of the medium after one complete revolution with the incubation layer thereof. Means may be provided to remove any protective layers from the collecting or incubation layers before exposure to and conserving/enhancing or micro-organisms respectively.

The invention may be applied to medical activities such as contagious disease control, monitoring of patients suffering from communicable disease, hospital cleanliness, immunisation clinics etc, in particular by checking the micro-organism species contained in the breath, salvia and tissue of patients and personnel. Animal and human micro-organism species may also be determined, which has particular application in the food processing industry, travel and immigration services, agriculture and the water supply and sewer industry.

Furthermore, when a micro-organism counter measure has been formulated and applied to a problem, the changes in the population of the organisms may be monitored by the repeated application of the invention. Accordingly the invention may be used for both the formulation of counter measures and assessing the efficiency with which they work.

The invention may also be used to monitor/regu- late the movements of certain species of micro-organisms to or from selected geographical locations. Furthermore, the invention may be used to monitor microbial induced corrosion in, for instance, marine environments and also in the fuel metal tanks of aircraft.

The invention will now be described, by way of example with reference to the accompanying drawings in which: Figure 1 shows a cross-section of a collecting medium in accorda.lce with the invention, Figure 2 shows an apparatus in accordance with the invention for use with a medium as shown in Figure 1, Figure 3 shows a detailed portion of the medium of Figure 1, and Figure 4 shows an alternative detailed portion to that shown in Figure 3.

The collecting medium 10 shown in Figure 1 comprises five individual layers. A baseisubstrate layer 12 formed from a film of PVC polyvinylchloride or other similar substance such as polyster film, supports or bears on the upper surface thereof a collecting layer 14 for retaining micro-organisms that come into contact therewith, the base layer 12 supporting a bearing on the lower surface thereof an incubation layer 16 for conserving or enhancing the population of the micro-organisms collected on the layer 14. Both the collecting layer 14 and the incubation layer 16 are provided with a protection layer 18 and 20 respectively.

The apparatus 22 as shown in Figure 2 for use with the collecting medium 10, is totally enclosed so that it may be used in difficult or hostile environments. On one side of the apparatus 22, exposing means in the form of an exposure window 24 and associated shutter arrangement 26 is provided.

A feed spool 28 and a recovery spool 30 are also shown, the arrangement being similar to that of a conventional optical camera. In similar fashion, the micro-organism medium 10 is first wound onto the feed spool 28 which is then fitted into the apparatus 22. A part length of the medium 10 is then wound off onto the recovery spool 30 after firstly having wound off a suitable length of the protective layers 18 and 20 from the collecting layer 14 and incubating layer 16 by means of a third spool 32 and fourth spool 34 respectively. The medium 10 is then set ready for exposure.

The operation of the apparatus 22 then follows the next sequence of events: Firstly, the shutter arrangement 26 is opened by means of a sliding member 36 which moves from left to right as shown in Figure 1 leaving an appropriate area of the collecting layer 14 exposed to the microbial environment. The member 36 is moved by means of the spool 32 as it winds off the requisite length of protective layer 18. This positions the member 36 on the right hand side (see 36') of the exposure of the window 24.

Secondly, after the desired exposure interval has elapsed, spool 30 is rotated so that the exposed section of the collecting layer 14 is wound therearound. At the same time, spool 34 removes the protective layer 20 from the lower side of the medium 10 thus exposing the incubation layer 16. The arrangement is such that layers 14 and 16 eventually come into intimate contact with each other, whereby the microbial population on the collecting layer 14 is conserved or enhanced by the organic materials in the incubation layer 16. Needless to say, a complementary rotation of the spool 28 takes place as this operation proceeds.

Thirdly as the medium 10 moves along the camera 'window' due to the rotation of the spools 28 and 30, the slidingimember/roller 36 moves back towards the left hand side of the exposure window 24, thus leaving a section of unexposed medium 10, the said film being protected from the microbial environment by another section of layer 18.

This layer 18 remains covering the layer 14 until the next exposure is required, and whereafter the above steps are repeated.

At the beginning of each recording medium 10 there are three special 'tags' 38 (wind-on strips) to facilitate medium loading and winding-on of the base layer 12 and the protective layers 18 and 20.

A slipping clutch roller spool may be fitted to the spools 32 and 34, so that any slack strip of layers 18 and 20 may be taken up. The normal windingon procedure may then be followed as the medium 10 is driven by the main drive spool 30 mechanism.

It will be noted that the chemical nature of the organic materials that comprise collecting and incubating layers 14 and 16 may be adjusted to suit the particular requirements of a user. In otherwords, the conservation or enhancement of populations of microbial species in which there may be a special interest could be selectively advanced by suitably altering the chemical composition of the two aforementioned layers. Many compositions are known which relate to different species and the enhancement of their growth rates, and needless to say, any one of these compositions could be employed in the practical application of this invention, including the collection of anaerobic species.

Preferably the collecting and incubation layers 14 and 16 are provided with a very thin layer of inorganic material (e.g., stearic acid) to serve to prevent the material of layer 14 and 16 from adhering to the protective layer 18 and 20 respectively as the medium 10 is operated through the exposure and winding- on procedures.

A wide range of different microbial species may be collected on one exposure by using a pattern or matrix on the receiving surface of collecting layer 14, the pattern or matrix comprising an array of different chemicals each of which suits a different range-type of species. In two preferred arrangements, the shape of the matrix is of either a circular or rectangular shape as shown in Figure 3 and 4, and the chemical organic substances are as listed below: Nutrient Agar, Nutrient Gelatin, Blood Agar, Tryptose Soya Agar, and Gelatin (the last is used in conjunction with the other materials except the Nutrient Gelatin).

It does not appear that any preferential effect upon microbial collection performance will accrue from variations in the geometry of the matrix, however, there should be provision for recording on one film the simultaneous collection of microbial species from different environments. This may be accomplished by partitioning (see I to IV in Figures 3 and 4) the recording medium and by providing separate conductance ducts from each environment. It should be noted that in the apparatus shown in Figure 2, there is an area (see y) of the incubation layer 16 that is exposed to the environment but not to the direct impingement of micro-organisms. This section of the layer 16 serves to record the gas atomic/molecular species which accompanied the microbial population during both the exposure time interval and also the time interval between consecutive recordings.This section of recording medium supplies the so- called 'background' molecular counts that have to be subtracted from the counts derived from the collecting layer 14 in order to account for the atomic/molecular species that are not necessarily part of the microbial population of interest.

The sizes of recording filmimedium 10 is preferably similar to those of conventional optical cameras, namely 35 mm and 70 mm. This enables many mechanical engineering tools and techniques to be employed in the production processes of the medium 10 as well as the exposure apparatus 22 itself.

For a land version of the apparatus 22, the interior thereof may be at slight excess pressure to that of the exterior, so as to prevent microbial penetration except at the exposure window 24. In these circumstances, the receiving surface matrix should include a microbially neutral surface composed of gelatin alone, to collect environmental gas atomic/molecular species for a background count.

For a marine version of the apparatus 20, a pressure compensating device should be fitted to enable the nechanical parts of the apparatus to function unhindered by differential stress that develops with increases in immersion depth. The apparatus 22 may be filled with an asceptic liquid which excludes the 'background count' of atomic/ molecular species and or the main atomic/molecular species of the sea are collected by a microbially inert surface on the matrix.

In an embodiment, the collecting surface matrix may be changed to include thin metal strips which may be accommodated on Z fold recording media.

This facility may be necessary because a number of micro-organisms exhibit highly preferential settlement rates for different metals and other substances. These settlement rates are not easily predictable so that an empirical approach to this aspect of microbial behaviour is the only practical approach that may be safely adopted.

When the medium 10 has been exposed and is ready to be analysed, Pyrolysis Mass-spectrometry (Py-MS) is used for quick analysis. Thereafter conventional biological assay techniques or indeed other slow analysis techniques maybe used for independant confirmation purposes.

For comparison it will be noted that Py-MS analysis takes a few minutes to complete while assay techniques may take upto three weeks to complete.

Rapid identification in certain fields maybe of critical safety or commercial importance and furthermore, maybe useful in a situation that requires semi-continuous monitoring.

Py-MS analysis requires only 5 micrograms of micro-organism material for determination of the species in each sample, while a small number of such samples around (30) are needed to statistically characterize the nature of the micro-organism population.

Py-MS instrumentation produces a pyrogram that represents a mixed microbial population sample and the data of the pyrogram have to be analysed with the aid of computer assisted calculation techniques. These techniques select species from a data bank containing the pyrograms of many different species. The selection of species together with an estimation of the relative proportions in which they comprise the pyrogram are determined by the computer program. An acceptable determination forms the basis upon which disease control is founded and the regulation of the transfer of species is made between different geographical locations.

The invention may also be applied to the typification and taxonomy of micro-organisms since the pyrolysis Py-MS technique may be used in full combination with a secondary molecular fragmentation technique that can yield further information about the molecular structure of any micro-organisms. This is to say that the pyrolysis building bloc fragmentation products may be differentiated as to internal structure and thus provide additional data regarding the initial macromolecules.

Claims (13)

1. A collecting medium for micro-organisms comprising a collecting layer for retaining microorganisms that come into contact therewith and an incubation layer for conserving or enhancing the population of the micro-organisms on the collecting layer.

2. A collecting medium as claimed in Claim 1 wherein one or both of the collecting and incubation layers are provided with a protective outer layer, the protective layer(s) being removed when the medium is about to be used to collect microorganisms.

3. A collecting medium is claimed in either Claim 1 or 2 wherein the collecting and incubation layers are provided and borne on opposite sides of a common baselsubstrate.

4. A collecting medium as claimed in any preceding claim wherein the collecting layer includes two or more different constituent portions, with which two or more different groups or species of micro-orgnisms are collected.

5. A collecting medium as claimed in any preceding claim wherein the collection and/or incubation layers are composed of one or more organic substances to retain and preserve certain specific micro-organisms.

6. A collecting medium as claimed in Claim 5 wherein the organic substance is Nutrient Agar, Nutrient Gelatin, Blood Agar, Tryptose Soya Agar or Gelatin.

7. An apparatus for use with a collecting medium for micro- organisms having both a collecting and incubation layer comprising means for exposing the collecting layer for micro- organism collection and means for positioning the collecting layer in contact with the incubation layer such that the micro- organisms are conserved or enhanced.

8. An apparatus as claimed in Claim 7 wherein the apparatus is totally enclosed.

9. An apparatus as claimed in either Claim 7 or 8 wherein the exposing means comprises an exposure window and associated shutter arrangement.

10. An apparatus as claimed in any one of Claims 7 to 9 wherein the positioning means is in the form of a spool which in use matches the collecting layer of the medium after one complete revolution with the incubation layer thereof.

11. An apparatus as claimed in any one of Claims 7 to 10 wherein means are provided to remove any protective layers from the collecting or incubation layers.

12. A collecting medium substantially as herein described with reference to Figures 1 to 3 or 4 of the accompanying drawings.

13. An apparatus for use with a collecting medium for micro- organism substantially as herein described with reference to Figure 2 of the accompanying drawings.