Microorganism identification technique

A method for the identification of microorganisms comprises adding to a sample containing an unknown microorganism an emissive agent such as a radioactive amino acid to produce a mix of emissive products that depends on the metabolic mechanism of the microorganism. After incubation, the reaction is arrested and the emissive products are separated, as by electrophoresis on a gel plate. The plate may then be autoradiographed by exposure to a photographic film to produce on the latter a characteristic band pattern functioning as an identifier for the microorganism. Identification can be effected by comparing the identifier for the unknown with a collection of identifiers for known microorganisms to find a match with one of these known identifiers. The comparison may be carried out by scanning the unknown identifier to produce a signal which is compared with signals representing known identifiers stored in a computer. Alternatively, the emissive products, after separation, may be detected by direct scanning to provide an identifier signal for computer processing .

FIELD OF THE INVENTION 
The invention relates to a method for the identification of microorganisms 
and especially to such a method wherein an identifier is generated for the 
microorganisms which is then compared with a collection of identifiers 
representing various known microorganisms to find whether there is a match 
with one thereof, the comparison preferably being automated. 
BACKGROUND TO THE INVENTION 
In microbiology, microorganisms are classified in taxonomic categories, 
nomenclature being used to name the units delineated and characterised by 
classification. Identification by classical procedures involves the use of 
criteria established for classification and nomenclature in order to 
identify microorganisms by comparing the characteristics of an unknown 
unit with known units. Thus with a newly isolated microorganism, its 
identification requires an adequate characterisation thereof and then a 
comparison with published descriptions of other similar microorganisms. 
To identify an organism of interest that is present in a specimen of 
mineral, plant or animal origin, it is first necessary to obtain an 
isolated colony of the microorganism. There are well developed procedures 
for growing or cultivating microorganisms in the laboratory on nutrient 
material, some of these procedures requiring special conditions such as 
the absence of free oxygen. By incubating a nutrient agar-type medium, 
using the streak-plate or pour-plate method, cells are individually 
separated. In incubation, individual cells reproduce rapidly to generate a 
visible colony of cells, each colony being a pure sample of a single kind 
of microorganism. 
In order to identify an unknown cell, classical techniques call for the use 
of high-magnification optical or electron microscopes in order to 
determine colony and cell morphology. In addition numerous other 
characteristics may have to be determined, including staining 
characteristics, susceptibility to antimetabolites and serological and 
biochemical properties. The procedures for the identification of bacteria 
are set forth in detail in chapter 5 of the text "Clinical 
Bacteriology"--Fifth Ed.--J. Stokes et al., published by Arnold (London) 
1980. 
Thus, the identification of microorganisms by classical techniques is 
time-consuming, labour-intensive and expensive and, not withstanding the 
high order of technical skill required, is liable to error. 
The identification of microorganisms to clearly of great importance in the 
medical and veterinary fields. However, in recent years the need for 
efficient and relatively rapid identification techniques has become even 
more pressing owing to the remarkable expansion of environmental and 
industrial microbiology. Thus, the cultivation of microorganisms in food 
processing, in the fermentation of alcoholic beverages, and in the 
manufacture of pharmaceuticals and of such industrial reagents as the 
alcohols and acetic acid is already well established. The use of 
microorganisms has been proposed not only for syntheses but also for 
counter-pollution measures; an interesting use of selected microorganisms 
to degrade products of industrial organic syntheses is described in 
GB-A-2010327. Furthermore, genetic engineering is expected markedly to 
increase the range of applications of microbiology in industry and 
agriculture (see Scientific American, September 1981). 
There have, of course, been attempts to improve upon the classical 
techniques for microorganism identification. For example, Tsukamura and 
Mizuno (Kekkaku 1980, 55 (12) pages 525-530) disclose a method by which 
certain selected microorganisms can be distinguished. The precultured 
organism was incubated for 24 hours in a reaction medium containing 
L-.sup.35 S-methionine, after which the cells were centrifuged, washed and 
extracted with ethyl ether/ethanol. The extracted material was subjected 
to further extraction using petroleum ether and the resultant material was 
subjected to thin-layer chromatography. Any radioactive spots in the 
thin-layer were detected by an automatic scannner. These Japanese workers 
were able to distinguish Mycobacterium nonchromogenicum (which gave one 
strong radioactive spot at an Rf value 0.70-0.80) from M.terrae and 
M.triviale (which two organisms produced no spot or only a trace spot). 
They were also able to differentiate between two particular Rhodococcus 
species and to distinguish Rhodococcus species from Nocardia species (the 
former giving a spot at Rf 0.10 or Rf 0.95, whereas Nocardia displayed no 
such spots). 
The method described in the Kekkaku article clearly does not qualify as a 
general method for the identification of microorganisms: thus, the 
Japanese workers were unable to differentiate M.terrae and M.triviale. 
Furthermore, the method required an initially high concentration of 
microorganism and a long incubation period in the .sup.35 
S-methionine-containing medium. An interesting observation is that, 
although methionine is an amino acid (such acids being the building blocks 
of proteins), the petroleum ether extraction would not have taken up 
proteins and thin-layer chromatography (TLC) is not a useful technique for 
resolving proteins. It would appear, therefore, that any radioactive spot 
that may be detected in the thin layer is not due to the incorporation of 
the .sup.35 S-methionine in a protein product of the metabolism of the 
organism, but is due to a product of a secondary reaction between the 
radioactive label and a compound derived from the organism. 
With a view to automating the identification of microorganisms, U.S. Pat. 
No. 4,288,543 to Sielaff et al. discloses a procedure in which the 
susceptibility of various strains of bacteria to antimicrobial agents is 
tested, this being done in conjunction with a determination of the 
light-scattering index of the organism being tested. The numerical growth 
data obtained by the light scatter comparisons are analysed by 
computer-assisted statistical techniques in order to identify the 
organism. The admitted drawback to this procedure is that one should use 
agents not in common therapeutic use in order to avoid errors resulting 
from strains that have become immune to various therapeutically utilised 
antibiotic agents. Furthermore, it is necessary to divide the initial 
sample of the microorganism (specifically a bacterium) into a number of 
sub-samples, each of which has to be inoculated with a respective 
growth-inhibiting agent, incubated and then tested. The Sielaff patent 
also makes of record other publications dealing with the automated 
identification of bacteria by computer analysis of growth inhibition 
patterns. 
The logical approach to the problem of identification is to find or create 
an indentifier, namely a characteristic by means of which the identity of 
an unknown can be determined. Thus, fingerprints are regarded as 
identifiers for human beings, since a person can be identified by his 
fingerprints alone, without reference to that person's other 
characteristics, such as sex, age, height, weight, shape, eye colour and 
the like. However, a problem in applying this approach to microbiology is 
the difficulty of selecting a microorganism characteristic that really is 
an identifier, that can be routinely utilised as such, and that is 
applicable throughout the group of microorganisms (especially bacteria) in 
question. The identifier, like a fingerprint, should be substantially 
universal. Obviously, like human fingerprints, there may be exceptions, 
but the generation of the identifier should be the rule and not the 
exception. 
One attempt to tackle this problem is described in GB-A-1489255. That 
specification describes a process for the identification of a 
microorganism which comprises inoculating a plurality of different .sup.14 
C-labelled substrates with an unknown organism and incubating the 
substrates for a time sufficient to cause metabolic breakdown of at least 
some of the substrates by the organism to produce the radioactive gas 
.sup.14 CO.sub.2. The gas that is evolved is analysed for radioactivity in 
order to obtain a "substrate radiorespirometric profile" for the unknown 
microorganism. Such a profile is said (page 3, lines 3-13) to serve as a 
fingerprint of the unknown microorganism, in that the profile can be 
compared to standard profiles obtained in the same manner from known 
microbes. That technique requires the unknown microorganism to be tested 
against a sufficient number of substrates taken individually in order to 
obtain a meaningful profile; for instance, in the specific Example of 
GB-A-1489255, thirty substrates are used. Thus, each unknown is subjected, 
in effect, to a series of complex tests and this must render it difficult 
to standardise the test procedure. 
SUMMARY OF THE INVENTION 
The present invention now provides a method for identifying microorganisms 
comprising the steps of: adding to a specimen of microorganisms an 
emissive agent that is incorporated therein to produce a mix of emissive 
products in a manner that depends on the metabolic mechanism of the 
microorganisms; detecting the emissive products to derive a characteristic 
pattern functioning as an identifier for the microorganisms; and comparing 
the said identifier of the microorganisms with a collection of identifiers 
representing known microorganisms (the collection conveniently being in 
the form of stored information) to determine the identity of the 
microorganisms. 
Herein, the expression "metabolic mechanism" is to be broadly construed and 
includes catabolic and genetic mechanisms. 
Identification, for the purposes of this specification, encompasses the 
determination of whether the microorganisms in the specimen (which may be 
referred to as the "unknown" microorganisms) fall within a particular 
group. Identification can be carried out at various levels: thus, it may 
be sufficient to identify the microorganisms as being of a particular 
Family or Genus, although commonly one would wish to determine the Species 
or even the Subspecies, Strain or Serotype of the microorganisms in 
question. 
The present method can also be used to quantify the microorganisms since 
the overall intensity of the emission from the mix will be proportional to 
the quantity of the microorganism that produces the mix. 
The microorganisms in the specimen may, for example, be bacteria or fungi 
(which term includes the yeasts). 
However the method is also applicable to the identification of those 
microorganisms, in particular viruses or other obligate parasites such as 
Rickettsia and Chlamydia, that require a host cell for metabolic activity. 
Accordingly, the expression "mix of emissive products" is to be construed 
in such cases as referring to the mix produced by the microorganism and 
its host cell together. Here the identifier for the microorganism may be 
generated by altering the metabolism of the host cell as much as by the 
generation of its own products.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention is based on a discovery unexpectedly made in the 
course of research in which the function of messenger RNA (ribonucleic 
acid) in particular tissues was being investigated. In this investigation, 
messenger RNA was extracted from tissue and purified. Then the messenger 
RNA was translated into corresponding protein by adding thereto a cellfree 
system constituted by a ribosomal mixture with little or no genetic 
information of its own, but having the capacity to translate exogenous 
messenger RNA. As a consequence, the free amino acids in the translation 
medium was incorporated into the protein in a sequence dictated by the 
messenger RNA derived from the tissue. 
In order to identify the products of the translation system, a radioactive 
sulphur-containing amino acid (.sup.35 S methionine) was added to the 
system. 
After a translation period lasting about two hours, the translation medium 
was layered onto a gel plate and subjected to electrophoresis. 
Electrophoresis is the migration of colloidal particles in a liquid due to 
a potential difference established across immersed electrodes, the 
migration being toward the electrode having a charge opposed to that of 
the particles. Electrophoresis is applicable to a protein, for its 
molecules act as colloidal particles, and their charge is negative or 
positive, depending on whether the surrounding solution is acidic or 
basic. 
All free amino acids ran through the system, while all amino acids which 
had been incorporated into newly translated proteins were adsorbed on the 
plate at chromatographic positions characteristic of the protein into 
which they had been incorporated. Similarly, any .sup.35 S methionine 
which had been incorporated into a protein would be retained within it and 
thereby function as a marker for the molecules. 
The gel plate was then placed against a sensitive photographic plate. 
Appearing on the photographic plate as a result of radioactive emission 
(beta radiation) from the .sup.35 S methionine, were bands occupying 
positions thereon appropriate to the molecule in which the radioactive 
amino acid had been incorporated. 
In this plate, the bands had an intensity proportional to the amount of 
.sup.35 S methionine incorporated into the protein, i.e. to the number of 
methionine amino acids in the protein and to the quantity of the protein 
that had been synthesized. 
One would expect, when following this procedure, that in the absence of a 
message, no bands except for endogenous background translation would 
appear on the photographic plate. But contrary to this expectation, bands 
were developed which did not seem to correspond in any way to the 
messenger RNA that had been added to the translation system. Moreover, it 
was found that bands were created even when no RNA from any source 
whatever had been added to the system. 
The conclusion then reached was that the unexpected appearance of bands 
could only be imputed to bacterial contamination, and that it was 
bacterial RNA that was being translated. This conclusion was confirmed 
when the bands were caused to disappear by sterilizing the various 
solutions that were being used in this research programme. 
It was also observed that different solutions gave rise to different band 
patterns, each solution producing a unique pattern quite distinct from 
those emanating from the other solutions. It was discovered that these 
distinctive band patterns occurred because the solution from which they 
were derived were contaminated with different types of bacteria, each 
generating a different pattern of translated proteins. This led to a 
principle underlying the present invention, namely that microorganisms, 
for example bacteria, can be identified by their pattern of emissive 
products, for example radioactive proteins. 
In principle, any emissive agent can be used in the practice of this 
invention, provided that the agent is incorporated into products of 
metabolism that will serve as identifier for the microorganism in 
question. Thus, use could be made of emissive nucleic acids, long-chain 
fatty acids, carbohydrates and membrane units to produce a mix of emissive 
products. As long as this mix is the result of the specific metabolism of 
the microorganism and can thereby be used to identify the microorganism, 
it does not matter where or how the metabolic mechanism is being 
harnessed. However, the use of an emissive amino acid is preferred, since 
in general it will be incorporated in a range of protein products which 
will serve as a particularly useful identifier for the microorganism under 
investigation. 
The use of an emission, such as radioactivity, fluorescence or the like, is 
necessary to be able to detect the newly translated products, such as 
proteins, that are present in minute quantities, in general of the order 
of picograms of femtograms. Thus, it is preferred that the emissive agent 
should contain a radioactive element such as .sup.35 S, .sup.32 P, .sup.14 
C, .sup.3 H or .sup.125 I. The use of a radioactive amino acid as the 
emissive agent is especially preferred, examples being .sup.3 H leucine, 
.sup.14 C lysine and, especially, .sup.35 S methionine. 
The emissive agent may be added to the specimen by placing the 
microorganisms in a medium, to which the emissive agent is added (before, 
during or after the placement of the organisms), incubating the resultant 
medium for a period and arresting the incubation reaction at the end of 
that period. 
For example, a culture medium containing the microorganism to be 
identified, if appropriate after dilution, may have added thereto the 
emissive agent, if appropriate as a solution thereof. Alternatively, the 
culture step can be bypassed and the sample containing the microorganism 
can be obtained from source and the emissive agent added directly. After a 
predetermined incubation period (for example two hours), the reaction is 
arrested, for example by the addition of one or more appropriate chemical 
agents, such as sodium dodecyl sulfate (SDS) and mercaptoethanol. In the 
preferred embodiments, SDS also serves a useful function, in that it acts 
to break down aggregates of proteins that may have formed, as by hydrogen 
bonding. 
In general, it will be necessary to subject the resultant mix of emissive 
products to separation in order to "display" the unique characteristics 
thereof. An acceptable method for protein separation is isoelectric 
focusing, in which proteins are caused to separate across a substrate for 
example in the form of a plate, and to fix themselves at their isoelectric 
points. Affinity, molecular-sieve and ion-exchange chromatography may also 
be used for separation, as may isotachophoresis. 
In preferred embodiments, separation is effected by electrophoresis, since 
this is a highly discriminating method for segregating proteins. Thus, 
after the incubation reaction has been arrested, the inoculated culture 
medium may be layered on to a suitable substrate, for example a 
polyacrylamide gel, conveniently in the form of a plate, where it is then 
subjected to electrophoresis. 
While each emissive product in a mix obtained from a given organism may be 
found in mixes derived from other organisms, it appears that the mix 
obtained from a given organism is uniquely characterised by the nature of 
the products present and by their relative proportions. To that extent, 
the identifier for the microorganism under investigation is already 
implicit in the mix of emissive products. However, it is necessary to 
detect the emissive products in order to obtain a characteristic pattern 
that will permit the comparison step necessary to effect identification. 
As indicated above, the detection is generally preceded by a separation 
step in order, as it were, to "reveal" the components of the mix. It may 
in some cases be sufficient for identification purposes simply to 
determine the relative positions of the components in the resultant array 
or "spectrum" of separated or segregated emissive products. In other cases 
it may also be necessary to determine the relative proportions of the 
products, as by detecting the relative intensity of the emissions from the 
individual products. 
The emissive products may be detected by exposing an appropriately 
sensitive film to the emission from the said products. Thus, in 
embodiments wherein radioactively emissive products have been subjected to 
electrophoresis on a gel substrate, the latter may be autoradiographed by 
placing it adjacent to an X-ray film for a period sufficient to produce in 
that film a characteristic band pattern; the latter will in general 
resemble a bar code pattern and may be referred to as such. 
As indicated above, an unknown microorganism can be identified by finding a 
match of the identifier for that unknown with one of a collection of 
identifiers representing various known microorganisms. Thus, the band 
pattern in the film, once fixed, may be compared to the band patterns 
obtained in an analogous mannner from known microorganisms. 
The autoradiographs obtained as described above, although permitting visual 
comparisons to be effected, are also machine-readable, in that the band 
pattern may be sensed electrooptically by means adapted to provide a 
corresponding electrical signal pattern. 
It is also possible to dispense with the autoradiographing step and to 
detect directly the emissive products by sensing the emission therefrom by 
means adapted to provide a corresponding electrical signal pattern which 
can be processed to give a print-out in a desired graphical form for 
visual comparison with print-outs obtained analogously from known 
microorganisms. 
However, the electrical signal pattern obtained by direct sensing of the 
emissive products or by sensing of an autoradiograph may be processed in a 
computer to perform identification procedures; thus, the obtained 
electrical signal pattern may be compared in a computer with patterns 
stored in the computer memory, said stored patterns representing a 
collection of known microorganisms. The stored patterns constitute a 
library of identifiers which can be created by subjecting each of a number 
of known microorganisms to the emissive-agent addition, incubation, 
separation and detecting steps described above. The comparison may be 
effected by automated pattern recognition techniques known in principle. 
Pattern recognition entails the steps of feature extraction and then of 
classification using statistical analysis. Such techniques are described 
in "Pattern Recognition, a Statistical Approach" by P. A. Devijver and J. 
Kittler published by Prentice Hall, London (1982). 
Referring now to FIG. 3, there is shown a computerised system for scanning 
a band pattern formed on a radiographic plate 10, the pattern being an 
identifier for an unknown microorganism. 
This pattern is electro-optically scanned by a scanner 11. In principle, it 
is immaterial whether the scanner moves along a stationary plate, or the 
plate is moved beneath a stationary scanner. The scanner comprises a 
light-sensitive element, for example a photo-electric cell, and may be 
responsive not only to the absence or presence of bands but also to 
variations in the intensity of those bands. 
The output of scanner 11 is converted into digitised signals for processing 
in a computer terminal 12. Computer terminal 12 operates in conjunction 
with a memory 13 in which is stored a library of band patterns of known 
forms of microorganism. 
The function of the computer system is to identify the unknown 
microorganism whose band pattern has been scanned. To this end an 
electronic comparator or the equivalent software 14 is provided to find a 
match between the input signals and one of the stored patterns 
representing various known forms of microorganisms. When a match is found, 
information is conveyed to computer terminal 12 whose output then provides 
on a visual display 15 a read-out of the identified micro-organisms, and 
on an associated print-out 16, a hard copy of the reading. 
Direct scanning of the separated emissive products can be effected by means 
of apparatus analogous to that shown in FIG. 3, except that the plate 10 
would be replaced by a substrate bearing the separated emissive products 
and that, where the emission is radioactivity, the electro-optical scaner 
11 is replaced by a suitable radiation counter such as Geiger counter. 
Alternatively, a video camera may be used, whereby the emissions are caused 
to impinge on a plate sensitive thereto, the plate being scanned by an 
electron beam to generate a video signal. The video signal may then be 
digitised and processed in a computer to effect identification. 
In practice, the computer may store not only the names of known 
microorganisms, but also data in regard to the nature and characteristics 
thereof. These stored data can be printed out, so that the user of the 
system is informed not only as to the identity of the unknown 
microorganism, but also useful information relevant thereto. 
Another approach obviating the need for visual inspection of the band 
pattern in determining the identity of an unknown microorganism is to 
electrooptically scan the pattern to produce a signal which is converted 
into a multi-digit code number unique to the pattern. A scanning system 
capable of converting a pattern into a unique code number is disclosed in 
the U.S. Pat. No. 3,581,282 to Altman wherein the pattern is that produced 
by the palm of the hand, the Altman system serving to identify 
individuals. 
By converting band patterns into code numbers in the manner taught by 
Altman, one can then create a directory of code numbers and in which each 
code number is related to a known type or strain of microorganisms. Thus 
one who wishes to identify an unknown microorganism first produces a 
radiograph in the manner disclosed hereinabove, and then by means of a 
scanning system of the Altman type, converts the band pattern into a code 
number. Once he has the code number, it becomes a simple matter to consult 
the code directory to fix the identity of the unknown microorganism. 
As pointed out previously, for purposes of identification it may not always 
be necessary to find the exact identity of the microbe, and to determine 
for example that the microbe is E.coli, serotype 06. In some situations, 
it may be sufficient for purposes of identification to determine whether 
the unknown is of a particular genus. To this end the computer may be 
governed by an algorithm or programme operating in conjunction with an 
appropriate data bank so as to process the output of the scanner or 
detector to identify the unknown with respect only to genus or whatever 
other taxonomical description is required. 
Thus, the process of identification in the context of the present 
invention, lies in determining whether an unknown microbe lies within or 
matches a genus, a species, a strain or any other established or 
predetermined frame of reference. In that sense, a system in accordance 
with the invention may have a degree of resolution that depends on the 
task assigned thereto. 
It is desirable in order to provide a constant identifier unique to each 
microorganism, that the conditions under which the pattern is produced be 
standardised. To facilitate standardisation a reference standard may be 
established in a form which can be fed as reference data into a computer. 
Subsequent samples may thereafter be checked by the computer with the 
reference to see whether a search lies within an acceptable tolerance 
band. Under standardised conditions, the pattern from a given 
microorganism is always substantially the same. 
To overcome difficulties in maintaining absolutely standard conditions for 
determinations, the detection system may be calibrated, for example by 
detecting the emission products of a known control, which emission 
products have been obtained and separated under the same conditions as the 
emission products of the unknown. The signal of the unknown is then 
modified (to a degree determined by the difference between the control 
signal and a corresponding signal stored in the data bank) in order to 
obtain a proper comparison of the unknown with the stored data. 
Commonly, the microorganisms in the specimen to which the emissive agent is 
added will be of a single kind, i.e. the microorganisms will be 
substantially identical, as in a pure culture. However, the present method 
can in appropriate circumstances be applied to specimens, comprising a 
mixture of microorganisms. Thus, it would be possible to create a library 
of "composite identifiers" each representing a mixture of microorganisms 
that commonly occur together. Furthermore, it may be possible to identify 
the members of a mixture of microorganisms from a composite identifier 
obtained therefrom by using computer "subtraction" techniques already 
known in principle. 
The present invention shares the advantages of known automated 
identification techniques, for example it can dispense with the need for 
interpretation of results by experienced technicians, since the 
identification can be effected automatically. In addition, the present 
method exhibits a number of other advantages, not least in the simplicity 
and comparative speed with which the identifier can be obtained. Thus, in 
contrast to the techniques in US-A-4,288,543 and GB-A-1,489,255 mentioned 
above, the specimen of microorganisms need be reacted with only one 
substrate. 
The present invention permits the production of the identifier in the form 
of stored information (e.g. as an autoradiograph, as a printed graphical 
representation or as data in a computer memory) that admits of ready 
comparison with similarly obtained identifiers of known organisms. 
Also, in contrast to Isukamura et al. above, not only is the present method 
quick (compare the twenty-four-hour incubation period and extraction 
procedures employed by Tsukamura et al., despite the initial high 
concentration of the microbe), but also the present method has been used 
to identify all bacteria so far studied. Thus, Tsukamura et al. were 
confined to the further characterisation of a few specific organisms that 
had already been classified, since they investigated only the absence or 
presence of specific products, as detected by TLC. That approach depends 
on the finding of specific products to identify a microorganism. By 
contrast, in the present method it is the pattern of non-specific 
products, none of which have been selected and none of which may be 
unique, which provides the identifier. 
Another advantage of the present method is that is is possible to bypass 
the culture stage and directly to identify a microorganism taken from 
source (e.g. water, soil, urine, cerebrospinal fluid). 
A further advantage of the present method is that it can be used not only 
to identify known organisms, but also to discover new ones. Thus, if the 
identifier obtained from an unknown microorganism fails to match any 
identifier in the library of known microorganisms, it would be possible to 
assign a name to that unknown, whereupon its identifier may be added to 
the library in order to create a new "known" organism. For example, the 
organism C. difficle has proved difficult to classify and specimens have 
hitherto been referred to by isolate numbers. By means of the present 
method, it is possible to determine which of the isolates are, in fact, 
identical and then to classify the different strains. Thus, the present 
method can be used to classify microorganisms to create a taxonomy with 
reference to those subsequent identifications. 
The present invention is illustrated by the following specific Examples. 
EXAMPLE 1 
To identify different types of bacteria, a 1/100 dilution was made of 
culture mediums containing Proteus, E. coli, Pseudomonas and Klebsiella, 
each cultured in duplicate. 
Five microliters of solution was taken from each sample, to which was added 
five microliters of L-.sup.35 S methionine. No translation system was 
added. After a two-hour incubation period, the reaction was arrested by 
adding 10% SDS (sodium dodecyl sulfate) and 3% mercaptoethanol in 
trisbuffer. After heating, each solution was then layered onto a 
polyacrylamide gel plate. After carrying out electrophoresis for sixteen 
hours at 70 volts or 2.5 hours at 200 volts, the gel was fixed for three 
hours, dehydrated with acetic acid washes and exposed to a 
2,5-diphenyloxazole/acetic acid mixture for three hours. The plate was 
then water-washed and dried to provide the desired specimen. Finally the 
plate was autoradiographed by exposing the gel to an X-ray film overnight. 
A trace of the resultant autoradiograph is shown in FIG. 1 wherein channels 
1 and 2 are the duplicates of Proteus; channels 3 and 4, the duplicates of 
E.coli; channels 5 and 6, the duplicates of Pseudomonas; and channel 7, 
Klebsiella. 
It will be evident from an examination of FIG. 1 that the pattern of bands 
in channels 1 and 2 are identical so that duplicate samples of Proteus 
give rise to the same bar code identifier. This is also true of E.coli in 
channels 3 and 4 or Pseudomonas in channels 5 and 6; and Klebsiella in 
channel 7 is different from the other bar codes. The products are located 
by their position and the distribution around that position (as reflected 
by the resolution of the separation technique). 
EXAMPLE 2 
The procedure described in Example 1 was repeated for a second five 
microliters from each of the cultures. The resultant autoradiograph was 
indistinguishable from that shown in FIG. 1, thereby indicating that under 
standardised conditions, each bacterium gave rise to a distinctive band 
pattern readily distinguishable from those produced by the other bacteria. 
It is therefore possible with the naked eye to identify each bacterium by 
means of its autoradiographed identifier. 
EXAMPLE 3 
(A) To determine whether it was possible to identify different serotypes 
within a single bacterial type, three different serotypes of E. coli were 
cultured in duplicate (0106-075) and treated in the same manner as 
described in Example 1 to produce an autoradiograph. 
As shown in FIG. 2, the band patterns for the duplicate 01 serotype are the 
same, this being true of the band patterns for the duplicate 06 and 075 
serotypes. But there are significant differences in the band patterns for 
the different serotypes which serve to distinguish the identifiers from 
each other. Although the differences in this case are not as pronounced as 
when different genera of bacteria are involved, it is clear that the 
present identification method is capable of a fine degree of resolution. 
(B) In another experiment six different serotypes of E. Coli were cultured 
in duplicate. One set of duplicates was labelled with the name of the 
serotype (01, 06, 075, 040, 0108) and the other set was given a code 
number. 
Fifty microliters of sample were taken from each culture incubated with 
approx. 5 .mu.Ci (5 .mu.l) of .sup.35 S methionine (1385 Ci/mmol) for two 
hours at 37.degree. C. The incubation was stopped by the addition of 55 
.mu.l SDS/PAGE sample buffer (4% SDS, 6% mercaptoethanol, 4% glycerol in 
Tris/HCl, pH 6.7) and the sample was then heated. Twenty-five microliters 
from the mixture was loaded onto a 1% SDS/12.5% polyacrylamide gel (16 
cm.times.18 cm). Twelve samples were loaded onto one gel plate. The plate 
was electrophoresed at 70 volts overnight in a cold room after which it 
was fixed (20% acetic acid, 20% isopropanol in water), fluorographed 
(EN.sup.3 HANCE, New England Nuclear), dried and then exposed to X-ray 
film (Fuji RX) for three days at -70.degree. C. The six serotypes of 
E.coli in the named samples gave six distinct band patterns upon 
autoradiography, these patterns being different from one another. It 
proved possible for three investigators, who were ignorant of the code, 
independently to discern the correct match between each of these band 
patterns and the band pattern of the corresponding code-numbered sample. 
This experiment shows that it is quite possible to distinguish different 
serotypes of the same microorganism by means of the present invention. 
Ordinarily, the identification of serotypes is a time-consuming affair 
which requires the raising of antisera and the performing of agglutination 
tests against the O and the H antigens of the microorganism. 
In the procedure described above in Example 3 (B), the array of radioactive 
proteins obtained after electrophoresis is fluorographed. Fluorography is 
employed in order to enhance the exposure of the film and entails the 
addition of a substance which will emit light upon stimulation with 
radioactivity. Thus, fluorography may be utilised as part of the detection 
step in the present process. Fluorography may be particularly advantageous 
when the emissive agent comprises tritium (.sup.3 H) as the radioactive 
element. 
EXAMPLE 4 
In a manner analogous to that described in Example 3(B), six different 
serotypes of E. coli were incubated in a medium containing .sup.35 
S-methionine and then subjected to electrophoresis in order to obtain, for 
each serotype, an array of segregated emissive protein products. 
The segregated products were detected by direct scanning by mounting the 
polyacrylamide gel strips containing the products on a conveyor and 
passing the strips beneath a collimated geiger counter functioning as the 
scanner in an apparatus substantially as described above with reference to 
FIG. 3. The scanner was interfaced with an ITT 2020 microcomputer which in 
turn was interfaced with a visual display unit (VDU), floppy disc drives 
and a printer. 
The pattern for each serotype was displayed on the VDU and a hard copy was 
obtained from the printer, the pattern being presented in the manner of a 
histogram. The patterns from the serotypes were clearly distinguishable 
from one another. 
EXAMPLE 5 
The microorganisms mentioned in Examples 1 to 4 above are all gram 
negative aerobic bacteria. An analogous procedure to that described in 
Example 1 has been successfully employed for the identification of various 
gram positive aerobic bacteria, such as Staphylococcus aureus and 
Streptococcus faecalis, as well as gram positive anaerobic bacteria such 
as Clostridium perfringens, C bifermentans, C. butyricum and C. difficile 
(including subgroups of the isolation numbers) and gram negative anaerobic 
bacteria such as Bacteroides bivius, B. thetaiotamicron, B. vulgatus, B. 
corrodens, B. ovatus, B. distasonis and B. fragilis (including various 
serotypes thereof); for the identification of a yeast (Candida albicans); 
for the identification of Gonococcus species; and for the identification 
of a mixture of organisms (E.coli and Proteus sp.). 
EXAMPLE 6 
Host cells (human embryonic lung fibroblast cells) were grown for a period 
of 24-48 hours in specimen tubes using a minimum essential medium (1 ml 
for each tube). 5.varies.10 .mu.l (5 .mu.Ci) of .sup.35 S methionine were 
added to each tube, which was subsequently inoculated with a virus, either 
Herpes simplex 1 or Herpes simplex 2. The tubes were incubated for various 
periods of time ranging from 1 to 4 days. One tube was harvested daily as 
follows. The supernatent was first poured off and then trypsine was added. 
50 .mu.l of the resultant slurry was added to 50 .mu.l SDS/PAGE buffer 
solution. The sample was then loaded onto a polyacrylamide gel plate and 
subjected to electrophoresis as described above. 
The segregated, emissive protein products were detected by means of 
autoradiography. The band pattern obtained from the H. simplex 1/host cell 
entity was clearly distinguishable rom that obtained from the H. simplex 
2/host cell entity.