Device for identifying microorganisms

A device for determining the presence and identity of a specific microorganism in a biological sample based on biochemical tests is disclosed comprising a set of containers, each of which contains a reagent for performing an individual biochemical test useful for the identification of microorganisms, the set of containers being assembled on a single supporting member which is imprinted with a series of marks adjacent the containers, which marks are indicative of the expected result of a specific microorganism's performance in each biochemical test using the reagent of each individual container, the series of biochemical test containers, reagents and corresponding marks being sufficient to identify a specific microorganism. Series of marks corresponding to the expected biochemical test results for several specific microorganisms are preferably imprinted on the supporting member, making possible the identification of a single microorganism species from among several possibly present. The marks may have different lengths, colors and graphical form or may be also replaced by symbols and/or numbers. In a preferred embodiment, a device is described for the identification of a single species from among the seven pathogenic species of the Candida genus.

This invention relates to a device for identifying microorganisms on the 
basis of biochemical tests. More particularly, the invention relates to a 
device for identifying microorganisms which is composed of a set of 
containers each of, which contain, reagents for the identification of said 
microorganisms and are assembled on a single supporting member on which 
there are also imprinted a series of marks correlating the biochemical 
test results observed in all the containers, in which all the biochemical 
tests take place, with known biochemical test results for one or more 
specific microorganisms so as to be sufficient to identify a specific 
microorganism. 
The marks have different lengths, colour and/or graphical form and can also 
be partially replaced by symbols and/or numbers. 
The device is preferably circular in shape and the containers are 
preferably in the form of test tubes. In order to make the use of the 
device more convenient, the name of the microorganism to be identified can 
be written adjacent to its identifying marks on the supporting member. 
The reagents for the biochemical tests which are placed in the containers 
may be in dried, freeze-dried, solid and also compressed in form, or in 
the form of a solution. 
The containers may be an integral part of the device or may be simply 
inserted into holes provided in the supporting member or adhered to the 
supporting member. The device may also be provided with a central bore, 
which is preferably circular. 
In order to illustrate the device of the present invention by way of 
example without limitation, the application of such a device to the 
identification of the pathogenic species of the Candida genus is described 
hereinafter. 
Many Candida species, and all the pathogenic species among them, are 
frequently occurring commensals in the natural cavities inside the human 
body. Such species, under the proper conditions, are capable of causing by 
endogenous mechanism a number of diseases. 
In addition to Candida albicans, which is the most frequently isolated 
species, at least six different Candida species, viz.: Candida 
stellatoidea, Candida krusei, Candida tropicalis, Candida 
pseudotropicalis, Candida quillermondii, Candida parapsilosis are also 
regarded as pathogenic. 
Therefore, from a diagnostic standpoint, once a Candida culture has been 
isolated, it is necessary to determine whether a species which is 
pathogenic in humans is present. 
Identification of the species to which the microorganism concerned belongs 
is made by resorting to biochemical tests, such as: 
(a) Assimilation of various sugars in a culturing medium which is devoid of 
any other carbon sources (C-auxanorgram); 
(b) Fermentation tests for various sugars, and 
(c) Growth in the presence of certain substances. 
The assimilation tests are performed by preparing a germ-agar, with the 
slurry of the strain to be identified and the specific C-auxanogram 
medium. On the germ-agar there are deposited either paper discs which have 
been soaked at the very moment of use with different sugar solutions, or 
paper discs carrying the sugars concerned in dry form. Alternatively, 
porcelain well which contain the sugar solutions may also be used. 
It is possible to use specialized plates, which have a sector partition on 
their lower surfaces. 
If such a specialized plate is used, separate containers for testing 
assimilation of different sugars are already present, and the practitioner 
need only label the individual containers or wells to indicate the sugar 
container in each. 
The results of the assimilation tests are read after an incubation at 
25.degree. C.-37.degree. C. for 24 to 48 hours. As growth halos are 
observed around the assimilation sugars, these halos are regarded as 
positive test results and indicated by symbols (+). The absence of growth 
is indicated with a contrasting symbol (-). 
Fermentation tests are usually carried out after having performed the 
auxanogram, because they complete the latter test: as a matter of fact a 
sugar which has not been assimilated cannot be fermented. Only the 
assimilated sugars are thus tested. 
The tests can be prepared in yeast water or in solutions which contain 
vitamin factors at a preselected concenration and with an appropriate 
concentration of the sugar to be tested. In order to evidence gas 
evolution Durham bubble caps can be used, or an admixture of paraffin wax 
and petroleum jelly. 
The performance of these tests requires the inoculation of the slurry to be 
tested, its incubation at 25.degree. C.-37.degree. C. and daily readouts 
for up to 10 days of incubation. 
Subsequently, it is possible to relate the test results to those reported 
for particular the species by referring to specially prepared tables as 
reported in many reference books, among which the following can be 
mentioned: J. Lodder, 1970 Edn., The Yeast, North Holland Publishing Co., 
Amsterdam. 
Carbohydrate assimilation and fermentation tests can be carried out with 
commercially available kits ready for use. Among these, the "API 20 C 
Yeast System (Analytab Products, Inc., New York) and the "Uni-Yeast Tek 
System" (Flow Diagnostics) can be mentioned. 
API 20 C consists of a 20-cell tunnel, the cells containing dehydrated 
carbohydrates for the assimilation and fermentation tests. 
The cells are filled with a slurry of the fungus to be tested, as prepared 
in an agar furnished with the kit which is dissolved and cooled to 
50.degree. C. After an incubation at 30.degree. C. for 48 to 72 hours, the 
fermentation of the sugars is indicated by the colour change of an 
indicator and the production of gas, which is indicated by the formation 
of bubbles. 
The assimilation of the carbohydrates is indicated by the growth of the 
strain in the cells. 
The Uni-Yeast Tek System consists of two test tubes and a multipartitioned 
Petri dish. The tests tubes contain culturing media for the detection of 
germinative pseudotubules and assimilation of sucrose, respectively, and 
it possible to identify C. Stellatoridea. The dish has 11 peripheral 
compartments which contain solid media for the fermentation of 
carbohydrates, for the detection of urease and for the assimilation of 
carbohydrates and nitrates. At the centre of the dish there is a small 
cell which contains corn meal agar for detecting mycelium and 
chlamidopspores. 
The dish is inoculated with a drop of a slurry in distilled water of the 
fungus being tested and is incubated at 25.degree. C. from 2 to 7 days. 
The device of the present invention makes it possible to identify a 
particular species of microorganism contained in a biological sample. In a 
preferred embodiment, a specific pathogenic species of microorganism 
belonging to the Candida genus is identified, in a simple and efficient 
way using assimilation and fermentation tests of a few sugars, testing for 
growth in the presence of KNO.sub.3 and in the presence of cycloheximide, 
and for the ability to produce urease. 
The device in question, preferably having a circular shape, has on its top 
surface a set of containers, which are preferably in the form of test 
tubes or a set of hollow spaces. The number of containers or spaces is at 
least 16 for the particular embodiment which relates to the Candida genus. 
The containers hold the reagents for the biochemical tests, in any of the 
forms mentioned hereinabove. 
On the top surface, there are marks of different lengths, colour and/or 
graphical form (also partially replaced by symbols and/or figures) 
adjacent to the containers, a series of marks for each of the Candida 
species to be considered and which indicate the performance in each 
biological test that is characteristic of the individual Candida species, 
which series of marks together are sufficient to identify a specific 
microorganism from among the possible species considered. Along the mark 
it is possible to inscribe the name of the Candida species to be 
identified. 
Such a device provides a unified laboratory apparatus for testing a 
microorganism sample for the sugars assimilated and fermented, while 
simultaneously ascertaining growth with KNO.sub.3 being present, 
cycloheximide sensitivity, and the capacity of producing urease. 
It is thus rapidly and efficiently possible to relate the performance of a 
sample in a series of biological tests to the species to be identified.

In the device, there are seven arc-of-circle marks 17-23 having different 
lengths. The seven marks (17-23) are preferably of different colours to 
further differentiate the marks, however the colour type is not critical 
and in the list reported hereunder, the colours are indicated, by way of 
illustration, to correspond to the several identifiable species: 
Yellow: Candida guillermondii (17) 
Red: Candida albicans (20) 
Green: Candida parapsilosis (21) 
White: Candida pseudotropicalis (19) 
Sky-blue: Candida tropicalis (18) 
Violet: Candida krusei (23) 
Orange: Candida stellatoidea (22) 
In addition, if the mark adjacent a particular test container is continuous 
(--) the characteristic test result for the species corresponding to the 
marks is positive, whereas, if the mark is discontinuous (- - - -) the 
characteristic test result for that species is variable and, lastly, if 
the mark is lacking the test result characteristic of that species is 
negative. 
The device also reports symbols which have the following meanings: 
f: for fermentation 
a: for acidifying 
f/a: for fermentation or acidifying 
-/a: for negative or acidifying 
f/-: for fermentation or negative 
v: for variable 
A sugar is fermented if there is production of acid and carbon dioxide. 
A sugar is acidified if there is only a production of acid. 
At the centre of the device there is a central bore 24, which, at the 
instant at which the Petri dish is positioned, serves to deposit a certain 
volume of water so as to moisten the environment. The reagents for the 
biochemical tests are placed in the 16 containers from 1 to 16 inclusive 
in any of the forms which have already been described and can be 
identified after the symbols of the reagents. 
The 16 containers hold the following regents: 
Fermentation tests: 
Glucose (GLU) (13) 
Maltose (MAL) (14) 
Sucrose (SAC) (15) 
Lactose (LAT) (16) 
Growth tests: 
KNO.sub.3 (1) 
Cycloheximide+glucose (CEX) (12) 
Urease production: 
Urea (URE) (3) 
Assimilation tests 
Inositol (INO) (4) 
Maltose (MAL) (5) 
Trehalose (TRE) (6) 
Glucose (GLU) (7) 
Sucrose (SAC) (8) 
Cellobiose (CEL) (9) 
Raffinose (RAF) (10) 
Lactose (LAT) (11) 
Control tests: 
no reagent (2) 
(for the nitrogen assimilation test) 
pH indicators can be added to the reagents in order to make the reactions 
more conspicuous. 
For the identification of the pathogenic species of the Candida genus, the 
procedure scheme is as follows: the device of FIG. 1 is placed in a Petri 
dish and the eight containers which contain the sugars to be tested in the 
assimilation tests, as listed hereinabove, and the container which holds 
the cycloheximide are filled with a solidifiable culturing medium having 
no further carbon sources, with or without a pH indicator. The container 
for the nitrogen source assimilation test and the control container 
(comparison) are filled with another solidifiable culturing medium, with 
or without a pH indicator and devoid of any nitrogen sources. 
The slurry of the microorganism sample to be tested is then prepared, by 
taking it from an isolation plate (eg Sabourand) and with it there are 
inoculated all the containers for the fermentation tests as enumerated 
above, the urea-containing container is inoculated, and, in addition, a 
drop of the slurry is deposited in the containers with the already 
solidified culturing media. 
The containers intended for the fermentation tests are then closed with 
previously melted petroleum jelly. The entire assembly is then incubated 
at 25.degree. C.-30.degree. C. 
On completion of the incubation stage, the sugar assimilation tests are 
evaluated: if the tested sample the expected microorganism is present, 
there will be growth or colour change in the pH indicator, otherwise no 
change shall be observed. 
The colour is observed of the mark which unites the assimilation test to 
the cyclohexemide growth test which have given positive results (growth or 
colour change of the pH indicator) and the species of the Candida genus is 
determined. 
For example, if the assimilation tests in maltose (MAL), trehalose (TRE), 
glucose (GLU) and that of growth in cyclohexemide prove positive, this 
means that Candida stellatoidea (continuous orange line) is present. By 
following the coloured mark along its circumference, one reads the results 
of the biochemical tests which confirm the identification: in the case of 
Candida stellatoidea, the fermentation tests for glucose (GLU), and 
maltose (MAL) will be positive (f) whereas that of sucrose (SAC) will be 
negative (-/a). 
The control test serves as a reference for the nitrogen source assimilation 
test (KNO.sub.3). 
The device, the subject of the present application, for example the one 
shown in FIG. 1, can serve also for the identification of other pathogenic 
yeasts: their identification is based on similar tests made with the same 
procedure and takes place with the help of a table in which the results of 
such tests have been inscribed. 
Such tables are reported in many specific reference books, such as J. 
Lodder, 1970 Edn., The Yeast, North Holland Publishing Co., Amsterdam. 
EXAMPLE 
In the device shown in FIG. 1 the reagents for the sugar assimilation tests 
are represented by solutions of the several sugars (20% to 40%) in 
distilled water, with polyvinyl alcohol (1% to 0.5%). The reagent for the 
potassium nitrate assimilation test is a solution of KNO.sub.3 at the 
concentration of 10% in 1% to 0.5% polyvinyl alcohol. 
No reagents are present in the control solution for nitrogen assimilation. 
The reagent for the cycloheximide sensitivity test is a solution of glucose 
of 20% concentration in distilled water and polyvinyl alcohol (1% to 0.5%) 
and cycloheximide (0.5%). 
For the urease production test the reagent is a specific Christensen type 
broth with a pH indicator. 
The reagents for the fermentation tests are a conventional specific broth 
based on oxmeat extract, sodium chloride, and distilled water, which 
separately contains the individual sugars at the concentration of 2% and 
bromothymol blue is the pH indicator. Portions of 100 microliters of each 
solution are deposited in the respective containers. 
At this stage, the device is placed in an apparatus wherein the reagents 
undergo a drying cycle, for example under a vacuum. 
On completion of such a cycle, the device such be placed into an envelope 
of an appropriate material, such as a plastics material (alone or within a 
Petri dish), which is sealed. 
Sterilization can be carried out either with ethylene oxide or with gamma 
rays. After such a procedure, the device (alone or within a Petri dish) is 
slipped into an envelope of a material which gives sufficient safety 
against moisture effects. 
The devices so prepared can be stored also for a long time at +2.degree. 
C.-+8.degree. C. prior to being used for identifying the microorganisms. 
The culturing medium for the sugar assimilation test is based on Y.B.N. 
(Difco), 2% agar-agar, and bromocresol purple as the pH indicator. 
The culturing medium for the nitrogen source assimilation test is based on 
Y.C.B. (Difco), 2% agar-agar and bromothymol blue as the pH indicator.