Use of anti-capsule agents in microbiological testing

The present invention relates to growing and testing microorganisms in which an anti-capsule compound is used in order to prevent false positive results. The present invention is suited for the characterization of commonly encountered microorganisms which commonly produce capsules (e.g., Kkebsiella, Enterobacter, Escherichia, Burkholderia, Pseudomonas, Sphingobacterium, Chryseobacterium, Bacillus, Micrococcus, Staphylococcus, Haemophilus, Neisseria, Gordona, Kytococcus, Jonesia, Rhodococcus, Corynebacterium, Streptococcus, Cellulomonas, Brevibacterium, Arcanobacterium, Tsukamurella, Acinetobacter, Cryptococcus, etc.), as well as organisms of medical, veterinary, commercial, and/or industrial importance from various and diverse environments.

FIELD OF THE INVENTION 
The present invention relates to growing and testing microorganisms in 
which an anti-capsule compound is used in order to prevent false positive 
results. The present invention is suited for the characterization of 
commonly encountered microorganisms which commonly produce capsules (e.g., 
Klebsiella, Enterobacter, Escherichia, Burkholderia, Pseudomonas, 
Sphingobacterium, Chryseobacterium, Bacillus, Micrococcus, Staphylococcus, 
Haemophilus, Neisseria, Gordona, Kytococcus, Rhodococcus, Jonesia, 
Corynebacterium, Cellulomonas, Brevibacterium, Arcanobacterium, 
Tsukamurella, Acinetobacter, Cryptococcus, etc.), as well as organisms of 
medical, veterinary, commercial, and/or industrial importance from various 
and diverse environments. 
BACKGROUND OF THE INVENTION 
Under appropriate conditions, many bacterial species produce an 
extracellular polysaccharide-containing layer which forms a "capsule" up 
to 10 .mu.m in thickness that surrounds the cells in a tight matrix. Some 
organisms produce a loose, amorphous "slime layer" that is more easily 
deformed than the relatively tight matrix of capsules. Slime layers also 
tend to be more easily deformed than capsules, and unlike capsules will 
not exclude particles. While the terms capsule and slime layer are often 
used in reference to these extracellular polysaccharide containing layers, 
some researchers refer to both structures as a "glycocalyx" (ie., 
glycocalyx is a more general term). 
In addition to various polysaccharides and polysaccharide derivatives, the 
capsule may also contain glycoproteins. This capsule can be stained 
(e.g.,with Alcian blue) or be observed as a clear zone around the cells in 
an India ink wet mount. If the polysaccharide is soluble, it may diffuse 
throughout the culture media in which organisms are growing, forming 
slime, and making liquid media very viscous. The capsule may also help 
prevent desiccation of cells. 
In vivo, capsular material is anti-phagocytic, and plays a role in 
pathogenicity of some organisms, including species such as Streptococcus 
pyogenes, S. pneumoniae, and Bacillus anthracis. In addition, glycocalyx 
material often plays important roles in pathogenesis, as it is involved in 
the attachment of microorganisms to host cells. In addition, capsular 
material allows microorganisms to attach to other surfaces, such as 
catheters and implants. Also, capsular material helps block the action of 
antimicrobials and causes biofouling in industrial processes. Thus, the 
production of extracellular capsular material provides microorganisms with 
virulence mechanisms, as well as allowing them to survive better in 
certain environments. 
In addition to the pathogenesis implications of capsule production, 
capsular material often interferes with the metabolic reactions used to 
identify bacterial strains. With some organisms, such as Bacillus species, 
the problem is especially severe and the capsule can make it difficult to 
even obtain uniform suspensions of organisms for testing. Despite advances 
in technology, there remains a general need for systems that provide rapid 
and reliable biochemical identifications of microorganisms. For example, 
difficulties in identifying organisms of importance such as mucoid strains 
of Pseudomonas cepacia (now Burkhrolderia cepacia) in cystic fibrosis 
patients have occurred (See e.g,. Roman et al, ASM Abstracts, Abstract 
C-222, American Society for Microbiology, Washington, D.C., [1991], p. 
379). In particular, it has been very difficult to develop an 
identification system which is capable of identifying various diverse 
types of organisms, while avoiding problems associated with the presence 
of capsular material produced by various organisms. 
SUMMARY OF THE INVENTION 
The present invention relates to growing and testing microorganisms in 
which an anti-capsule compound is used in order to prevent false positive 
results. The present invention is suited for the characterization of 
commonly encountered microorganisms which commonly produce capsules (e.g., 
Klebsiella, Enterobacter, Escherichia, Burkholderia, Pseudomonas, 
Sphingobacterium, Chryseobacterium, Bacillus, Micrococcus, Staphylococcus, 
Haemophilus, Neisseria, Gordona, Kytococcus, Rhodococcus, Jonesia, 
Corynebacterium, Streptococcus, Cellulomonas, Brevibacterium, 
Arcanobacterium, Tsukamurella, Acinetobacter, Cryptococcus, etc.), as well 
as organisms of medical, veterinary, commercial, and/or industrial 
importance from various and diverse environments. 
The present invention provides methods inhibiting capsule production by 
microorganisms comprising the steps of: a) providing a sample suspected of 
containing microorganisms, wherein the microorganisms produce capsules; 
and an anti-capsule agent selected from the group consisting of 
thioglycolate, thioglycolate salts, thioglycolate esters, 
dihydroxyterephthalates, dihydroxyterephthalate salts, ibuprofen, and 
ibuprofen salts; and b) exposing the sample to the anti-capsule agent 
under conditions that production of capsules by the microorganisms is 
inhibited. In some particularly preferred embodiments, the 
dihydroxyterephthalate is 2,5-dihydroxyterephthalate. In further preferred 
embodiments, the concentration of anti-capsule agent is 1 to 10 mM, while 
in other particularly preferred embodiments, the concentration of 
anti-capsule agent is 2.5 to 5 mM. 
In some preferred embodiments, the methods further comprise the step of 
testing the microorganisms. In alternative preferred embodiments of the 
methods, the testing comprises identifying the microorganisms. It is 
intended that the testing and/or identification steps incorporate any 
method suitable for the testing, detection, and/or identification of the 
microorganism. In other preferred embodiments of the method, the testing 
comprises determining the antimicrobial susceptibility of the 
microorganisms. It is intended that any number of antimicrobials will be 
tested using the methods of the present invention. Thus, it is not 
intended that the testing, identification, and or detection of the 
microorganisms in the methods of the present invention be limited to any 
particular format or testing panel. In particularly preferred embodiments 
of the methods, the microorganisms are selected from the group consisting 
of gram-negative bacteria, gram-positive bacteria, and fungi. 
The present invention also provides methods for inhibiting capsule 
production by microorganisms comprising the steps of: a) providing a 
sample suspected of containing microorganisms, wherein the microorganisms 
produce capsules; a solid medium suitable for the growth of the 
microorganisms; and an anti-capsule agent; b) placing the anti-capsule 
agent on the solid medium to produce a treated medium; and c) inoculating 
the treated medium with the sample, under conditions that production of 
capsules by the microorganisms is inhibited, while the microorganisms 
grow. 
In some preferred embodiments, the methods further comprise the step of 
testing the microorganisms. In alternative preferred embodiments of the 
methods, the testing comprises identifying the microorganisms. It is 
intended that the testing and/or identification steps incorporate any 
method suitable for the testing, detection, and/or identification of the 
microorganism. Thus, it is not intended that the testing, identification, 
and or detection of the microorganisms in the methods of the present 
invention be limited to any particular format or testing panel. In 
particularly preferred embodiments of the methods, the microorganisms are 
selected from the group consisting of gram-negative bacteria, 
gram-positive bacteria, and fungi. In particularly preferred embodiments 
of these methods, the anti-capsule agent is selected from the group 
consisting of thioglycolate, thioglycolate salts, thioglycolate esters, 
dihydroxyterephthalates, dihydroxyterephthalate salts, ibuprofer, 
ibuprofen salts, salicylates, and salicylate salts. In some particularly 
preferred embodiments, the dihydroxyterephthalate is 
2,5-dihydroxyterephthalate. In further preferred embodiments, the 
concentration of anti-capsule agent is 1 to 10 mM, while in other 
particularly preferred embodiments, the concentration of anti-capsule 
agent is 2.5 to 5 mM. 
The present invention also provides compositions comprising microorganisms 
and an anti-capsule agent selected from the group consisting of 
thioglycolate, thiglycolate salts, thioglycolate esters, 
dihydroxyterephthalates, and dihydroxyterephthalate salts. In particularly 
preferred embodiments, the microorganisms are selected from the group 
consisting of gram-negative bacteria, gram-positive bacteria, and fungi. 
In some particularly preferred embodiments, the dihydroxyterephthalate is 
2,5-dihydroxyterephthalate. In further preferred embodiments, the 
concentration of anti-capsule agent is 1 to 10 mM, while in other 
particularly preferred embodiments, the concentration of anti-capsule 
agent is 2.5 to 5 mM. 
The present invention also provides microbial test kits comprising at least 
one anti-capsule agent and a suspension medium. In one embodiment, the 
anti-capsule agent of the test kit is selected from the group consisting 
of thioglycolate, thioglycolate salts, thioglycolate esters, 
dihydroxyterephthalates, dihydroxyterephthalate salts, salicylates, 
salicylate salts, ibuprofen, and ibuprofen salts. In other embodiments, 
the test kit also comprises control organisms (i.e., organisms with known 
characteristics used in order to ensure that the test kit is working 
properly). In some particularly preferred embodiments, the 
dihydroxyterephthalate is 2,5-dihydroxyterephthalate. In further preferred 
embodiments, the concentration of anti-capsule agent is 1 to 10 mM, while 
in other particularly preferred embodiments, the concentration of 
anti-capsule agent is 2.5 to 5 mM. In yet other embodiments, the 
suspension medium is suitable for use as a growth medium (i.e., 
microorganisms will grow in the medium), as well as a suspension medium. 
In still other embodiments, the suspension medium is suitable for use as a 
diluent, in which a microbial suspensions is prepared for inoculation of a 
test panel. In still other embodiments, the test panel comprises one or 
more carbon sources useful for the identification of the microorganisms 
present in the suspension. In yet other embodiments, the test panel 
comprises one or more nitrogen sources useful for the identification of 
the microorganisms present in the suspension. In further embodiments, the 
test panel comprises both carbon and nitrogen sources useful for the 
identification of the microorganisms present in the suspension. It is not 
intended that the present invention be limited to any particular testing 
format or panel. Rather, it is intended that the present invention 
encompass any format or panel suitable for identification of 
microorganisms. 
The present invention also provides methods for inhibiting biofilm 
formation, comprising the steps of: a) providing microorganisms, wherein 
the microorganisms are capable of forming a biofilm; a surface; and at 
least one anti-capsule agent selected from the group consisting of 
thioglycolate, thiglycolate salts, thioglycolate esters, 
dihydroxyterephthalate, dihydroxyterephthalate salts, ibuprofen, and 
ibuprofen salts; and b) exposing the surface to the anti-capsule agent 
under conditions such that biofilm formation by the microorganisms is 
inhibited. In some particularly preferred embodiments, the 
dihydroxyterephthalate is 2,5-dihydroxyterephthalate. In further preferred 
embodiments, the concentration of anti-capsule agent is 1 to 10 mM, while 
in other particularly preferred embodiments, the concentration of 
anti-capsule agent is 2.5 to 5 mM. In some embodiments of the method, the 
microorganisms are selected from the group consisting of gram-negative 
bacteria, gram-positive bacteria, and fungi. 
The present invention also provides methods for enhancing the effectiveness 
of an antimicrobial agent, comprising the steps of: a) providing 
microorganisms; at least one antimicrobial; at least one anti-capsule 
agent selected from the group consisting of thioglycolate, thiglycolate 
salts, thioglycolate esters, dihydroxyterephthalates, 
dihydroxyterephthalate salts, ibuprofen, and ibuprofen salts; and b) 
exposing the microorganisms to at least one antimicrobial and at least one 
anti-capsule agent under conditions such that the effectiveness of the 
antimicrobial against the microorganisms is enhanced. In some particularly 
preferred embodiments, the dihydroxyterephthalate is 
2,5-dihydroxyterephthalate. In further preferred embodiments, the 
concentration of anti-capsule agent is 1 to 10 mM, while in other 
particularly preferred embodiments, the concentration of anti-capsule 
agent is 2.5 to 5 mM. In some embodiments of the method, the 
microorganisms are killed, while in other embodiments, the microorganisms 
are inhibited. In preferred embodiments, the microorganisms are selected 
from the group consisting of gram-negative bacteria, gram-positive 
bacteria, and fungi. In yet other embodiments, the method also comprises 
identifying the microorganisms. In particular, it is contemplated that 
during in vitro testing of microorganisms for their susceptibility to the 
action of various antimicrobials, the microorganisms will concurrently be 
identified. In these embodiments, the testing panels used encompass 
identification substrates (e.g., carbon and/or nitrogen sources), as well 
as antimicrobial compounds suitable for in vivo and/or in vitro use. Thus, 
it is not intended that the methods of the present invention be limited to 
any particular setting. Indeed, it is intended that methods which increase 
antimicrobial effectiveness will find use in various settings, including 
but not limited to medical (e.g. in vivo treatment), veterinary (e.g., in 
vivo treatment), commercial, and/or industrial applications. It is 
intended that the methods of the present invention will find use in any 
number of settings in which it is desirable to increase antimicrobial 
effectiveness. 
DESCRIPTION OF THE INVENTION 
The present invention relates to growing and testing microorganisms in 
which an anti-capsule compound is used in order to prevent false positive 
results. The present invention is suited for the characterization of 
commonly encountered microorganisms which commonly produce capsules (e.g., 
Klebsiella, Enterobacter, Escherichia, Burkholderia, Pseudomonas, 
Sphingobacterium, Chryseobacterium, Bacillus, Micrococcus, Staphylococcus, 
Haemophilus, Neisseria, Gordona, Rhodococcus, Jonesia, Kytococcus, 
Corynebacterium, Streptococcus, Cellulomonas, Brevibacterium, 
Arcanobacterium, Tsukamurella, Acinetobacter, Cryptococcus, etc.), as well 
as organisms of medical, veterinary, commercial, and/or industrial 
importance from various and diverse environments. 
In the development of the present invention, numerous compounds were tested 
for their ability to diminish capsule synthesis with their meg toxic to 
the organisms or their metabolic systems. In the most preferred 
embodiments, the present invention provides methods in which sodium 
thioglycolate is used to decrease capsular synthesis and facilitate 
identification of organisms. 
A wide range of species were surveyed during the development of the present 
invention. Indeed, the present invention provides methods suitable for use 
with both gram-negative and gram-positive species, including, but not 
limited to Klebsiella, Enterobacter, Burkholderia, Pseudomonas, 
Sphingobacterium, Chryseobacterium, Bacillus, Micrococcus, Gordona, 
Rhodococcus, Corynebacterium, Cellulomonas, Brevibacterium, 
Arcanobacterium, and Tsukamurella. Direct assays of capsule content of a 
mucoid strain of K pneumoniae throughout a range of thioglycolate 
concentrations indicated that thioglycolate at concentrations of 0.03% 
(approx. 2.5 mM) and greater, are effective at inhibiting capsule 
synthesis. Higher concentrations are effective at inhibiting capsule 
synthesis, but also exhibit some toxicity. 
In addition to growing cells in the presence of thioglycolate, in preferred 
embodiments of the methods of the present invention thioglycolate is 
spread onto the surface of agar culture media prior to streaking of cells 
(i.e., prior to inoculation), to enable the production of cell suspensions 
with evenly dispersed cells, without clumping or aggregation of cells. In 
other preferred embodiments of the methods, thioglycolate is added to cell 
suspensions prior to the inoculation of identification media and/or 
testing panels, in order to improve testing results. 
During the development of the present invention, it was observed that 
organisms that produced capsules tended to exhibit false positive 
reactions in various identification procedures. Although an understanding 
of the exact mechanism of action is not necessary in order to use the 
invention, it was thought that these capsule-producing bacteria utilize 
their capsules as a carbon source, causing the false positive results 
observed in carbon source utilization test panels. 
Although salicylate has been shown to be useful as an anti-capsule agent 
(See e.g., Domenico et al., J. Antimicrob. Chemother., 28: 801-810 
[1991]), and as an inhibitor of biofilm production, during the development 
of the present invention, it was determined that sodium salicylate was 
only partially effective in inhibiting false positive results with 
gram-negative bacteria such as Klebsiella and Enterobacter. The bismuth 
salt of salicylate was also tested during the development of the present 
invention. However, this compound was found to be too toxic and poorly 
soluble for use in the preferred embodiments of the present invention. 
In addition, some gram-negative bacteria, such as Burkholderia cepacia are 
capable of using salicylate as a carbon source. Thus, salicylate was 
determined to be less preferred agent for the elimination of false 
positive results with gram-negative bacteria. However, salicylate was 
found to be effective in reducing false positive reactions with 
gram-positive organisms. It was determined that none of the gram-positive 
organisms tested utilized sodium salicylate as a carbon source. Thus, 
sodium salicylate was found to be an excellent anti-capsule agent for 
various gram-positive organisms, including Micrococcus, Kytococcus, 
Rhodococcus, Jonesia, Cellulomonas, and Corynebacterium. 
With both salicylate and thioglycolate, the optimal concentration was found 
to be 1 to 10 mM in preferred embodiments (i.e., in suspension). In most 
preferred embodiments, the concentration range is 2.5 to 5 mM. These 
agents can be either added to the suspension medium (e.g. saline [e.g., 
0.85% NaCl], or PPS [Phytagel (0.01%), Pluronic F-68 (0.03%), and saline 
(0.45%)]), in which the cells are suspended prior to being inoculated into 
the testing panels, kits, materials, etc. In particularly preferred 
embodiments, 1 ml of a 50 to 100 mM stock solution of anti-capsule agent 
is added to 19 ml of a cell suspension. 
Several thioglycolate analogs were also tested, as well as other compounds, 
including octylthioglycolate, iso-octylthioglycolate, octadeyl 
thioglycolate, n-butyl thioglycolate, ethyl thioglycolate, 
2-mercaptopropionic acid, and 3-mercaptopropionic acid. However, most of 
these esters were not sufficiently water soluble, and all had a 
disagreeable odor. The only thioglycolate analog that was found to be 
partially effective was the ethyl ester; the structurally related acids, 
2- and 3-mercaptopropionic acid were not as effective. 
Ibuprofen, an anti-inflammatory agent with pharmacological effects similar 
to salicylate was also tested, and found to be partially effective as an 
anti-capsule agent for gram-negative and gram-positive bacteria. While 
ibuprofen (i.e., (S)-(+)-4-isobutyl-.alpha.-methylphenylacetic acid) was 
found to be a preferred compound, it is not intended that the present 
invention be so limited as other derivatives and analogs of the compound 
may be used in the present invention. Similarly, a wide variety of 
salicylate analogs were evaluated, the best of which was 
2,5-dihydroxyterephthalate. Thus, in particularly preferred embodiments, 
the dihydroxyterephthalate of the present invention is 
2,5-dihydroxyterephthalate. However, none of these agent worked as well as 
salicylate and thioglycolate for a wide range of bacteria. 
The present invention also provides methods for preparation of suspensions 
of organisms that are suitable for use in inoculation of testing panels, 
kits, etc. These methods are particularly useful with organisms which tend 
to form aggregates or clumps when placed in suspension using methods known 
in the art. In these methods of the present invention, the 
capsule-inhibiting compound (e.g., thioglycolate) is swabbed onto the 
surface of the agar culture medium used to grow the culture to be tested 
prior to inoculation of the medium with the culture. The 
capsule-inhibiting compound prevents or inhibits the formation of 
pellicles (i.e., biofilms), allowing the production of uniform suspensions 
when the organisms are harvested from the solid medium and placed in 
solution. Embodiments of the present invention are particularly useful 
with spore-forming gram-positive organisms including but not limited to 
species such as those within the genus Bacillus. In preferred embodiments, 
0.6% (50 mM) sodium thioglycolate was used. In other preferred 
embodiments, the methods use special media suitable for growing Bacillus, 
such as "BUG+maltose" (available from Biolog). 
Thus, it is also contemplated that the present invention will find use in 
the inhibition or prevention of biofilms. For example, the present 
invention contemplates the use of thioglycolate, thioglycolate salts and 
analogs (e.g., octylthioglycolate, iso-octylthioglycolate, octadeyl 
thioglycolate, n-butyl thioglycolate, ethyl thioglycolate), as well as 
2-mercaptopropionic acid, and 3-mercaptopropionic acid to prevent biofilm 
formation on medical devices such as catheters (See e.g., U.S. Pat. No. 
5,716,406 to Farber, herein incorporated by reference). 
It is further contemplated that other compounds with characteristics 
similar to those of salicylate and thioglycolate will find use in the 
methods of the present invention. For example, it is contemplated that 
amphiphilic, mucolytic, keratolytic, and anti-inflammatory small molecules 
with the ability to bind metals will be useful in the methods of the 
present invention. Indeed, it was determined that ibuprofen is also 
somewhat effective as an anti-capsule agent. Furthermore, it is 
contemplated that these compounds will find use in alternative embodiments 
of the present invention, such as in industrial and medical applications 
in prevention of biofilm formation (See e.g., Muller et al., J. Infect. 
Dis., 177: 501-503 [1998]). 
It is also contemplated that the present invention will find use in 
providing compositions and means to increase the effectiveness of 
antimicrobials (e.g., treatment of cystic fibrosis patients colonized by 
mucoid organisms; See e.g., Domenico et al., J. Antimicrob. Chemother., 
28: 801-810 [1991]). Thus, it is contemplated that in these embodiments, 
patients will be treated with anti-capsule agents in conjunction with 
other antimicrobials (e.g., antimicrobials in common use). In this manner, 
the anti-capsule agent facilitates the action of the antimicrobial by 
reducing the interference of the capsule to the action of the 
antimicrobial(s). In preferred embodiments, the anti-capsule agent is 
provided to the patient in a concentration of 1-10 mM, with a particularly 
preferred concentration range being 2.5-5 mM. These anti-capsule agents 
are provided to the patient using a regimen that provides the best 
conditions for antimicrobial activity. It is not intended that the present 
invention be limited to any particular disease condition or setting. 
Indeed, it is intended that the present invention be used in any disease 
condition or setting in which the administration of anti-capsular agents 
in vivo is warranted. It is also not intended that the present invention 
be limited to use with any particular antimicrobial compound(s). Thus, it 
is intended that the present invention be used in conjunction with any 
appropriate antimicrobial or antimicrobial combination, such that the 
activity of the antimicrobial is enhanced and/or the organisms associated 
with the animal's (e.g., a human patient) disease are killed or inhibited. 
It is further contemplated that the present invention will be used in 
vitro, in order to assess the role of capsular material in pathogenesis, 
as well as other aspects of capsule production by various microorganisms. 
It is also intended that the present invention will be used alone or in 
conjunction with antimicrobial agents in industrial and commercial 
settings, in order to prevent or reverse biofilm formation. It is not 
intended that the present invention be limited to any particular setting 
or study. Rather, it is intended that the present invention will find use 
in any studies on capsular material, defects in capsule production, etc., 
including, but not limited to metabolic and other studies. 
Although embodiments have been described with some particularity, many 
modifications and variations of the preferred embodiment are possible 
without deviating from the invention. 
Definitions 
The terms "sample" and "specimen" in the present specification and claims 
are used in their broadest sense. On the one hand, they are meant to 
include a specimen or culture. On the other hand, they are meant to 
include both biological and by environmental samples. These terms 
encompasses all types of samples obtained from humans and other animals, 
including but not limited to, body fluids such as urine, blood, fecal 
matter, cerebrospinal fluid (CSF), semen, and saliva, as well as solid 
tissue. These terms also refers to swabs and other sampling devices which 
are commonly used to obtain samples for culture of microorganisms. 
Biological samples may be animal, including human, fluid or tissue, food 
products and ingredients such as dairy items, vegetables, meat and meat 
by-products, and waste. Environmental samples include environmental 
material such as surface matter, soil, water, and industrial samples, as 
well as samples obtained from food and dairy processing instruments, 
apparatus, equipment, disposable, and non-disposable items. These examples 
are not to be construed as limiting the sample types applicable to the 
present invention. 
As used herein, the term "primary isolation" refers to the process of 
culturing organisms directly from a sample. Thus, primary isolation 
involves such processes as inoculating an agar plate from a culture swab, 
urine sample, environmental sample, etc. Primary isolation may be 
accomplished using solid or semi-solid agar media, or in liquid. As used 
herein, the term "isolation" refers to any cultivation of organisms, 
whether it be primary isolation or any subsequent cultivation, including 
"passage" or "transfer" of stock cultures of organisms for maintenance 
and/or use. 
As used herein, the term "presumptive diagnosis" refers to a preliminary 
diagnosis which gives some guidance to the treating physician as to the 
etiologic organism involved in the patient's disease. Presumptive 
diagnoses are often based on "presumptive identifications," which as used 
herein refer to the preliminary identification of a microorganism based on 
observation such as colony characteristics, growth on primary isolation 
media, gram stain results, etc. 
As used herein, the term "definitive diagnosis" is used to refer to a final 
diagnosis in which the etiologic agent of the patient's disease has been 
identified. The term "definitive identification" is used in reference to 
the final identification of an organism to the genus and/or species level. 
Whether biological or environmental, a sample suspected of containing 
microorganisms may (or may not) first be subjected to an enrichment means 
to create a "pure culture" of microorganisms. By "enrichment means" or 
"enrichment treatment," the present invention contemplates (i) 
conventional techniques for isolating a particular microorganism of 
interest away from other microorganisms by means of liquid, solid, 
semi-solid or any other culture medium and/or technique, and (ii) novel 
techniques for isolating particular microorganisms away from other 
microorganisms. It is not intended that the present invention be limited 
only to one enrichment step or type of enrichment means. For example, it 
is within the scope of the present invention, following subjecting a 
sample to a conventional enrichment means, to subject the resultant 
preparation to further purification such that a pure culture of a strain 
of a species of interest is produced. This pure culture may then be 
analyzed by the medium and method of the present invention. 
As used herein, the term "culture" refers to any sample or specimen which 
is suspected of containing one or more microorganisms. "Pure cultures" are 
cultures in which the organisms present are only of one strain of a 
particular genus and species. This is in contrast to "mixed cultures," 
which are cultures in which more than one genus and/or species of 
microorganism are present. 
As used herein, the term "organism" is used to refer to any species or type 
of microorganism, including but not limited to bacteria, yeasts and other 
fungi. As used herein, the term fungi, is used in reference to eukaryotic 
organisms such as the molds and yeasts, including dimorphic fungi. 
As used herein, the term "spore" refers to any form of reproductive 
elements produced asexually (e.g., conidia) or sexually by such organisms 
as bacteria, fungi, algae, protozoa, etc. It is also used in reference to 
structures within microorganisms such as current and former members of the 
genus Bacillus (e.g., species which were previously included within the 
genus Bacillus, but have now been moved to another genus such as 
Paenibacillus), which provide advantages to the individual cells in terms 
of survival under harsh environmental conditions. It is not intended that 
the term be limited to any particular type or location of spores, such as 
"endospores" or "exospores."Rather, the term is used in the very broadest 
sense. 
As used herein, the term "capsule" refers to extracellular material in a 
relatively tight matrix that resists the passage of materials into the 
cell, while the term "slime layer" refers to an amorphous layer 
surrounding the cell that does not prevent the passage materials into the 
cell. As used herein, the term "glycocalyx" refers to both capsules and 
slime layers, as well as any other polysaccharide-containing extracellular 
material that surrounds some cells. It is not intended that the present 
invention be limited to any particular capsule-producing species. Indeed, 
the present invention encompasses capsule-producing bacteria as well as 
fungi. 
As used herein, the terms "microbiological media" and "culture media," and 
"media" refer to any substrate for the growth and reproduction of 
microorganisms. "Media" may be used in reference to solid plated media 
which support the growth of microorganisms. Also included within this 
definition are semi-solid and liquid microbial growth systems including 
those that incorporate living host organisms, as well as any type of 
media. 
As used herein, the term "carbon source" is used in reference to any 
compound which may be utilized as a source of carbon for bacterial growth 
and/or metabolism. Carbon sources may be in various forms, including, but 
not limited to polymers, carbohydrates, acids, alcohols, aldehydes, 
ketones, amino acids, and peptides. 
As used herein, the term "nitrogen source" is used in reference to any 
compound which may be utilized as a source of nitrogen for bacterial 
growth and/or metabolism. As with carbon sources, nitrogen sources may be 
in various forms, such as free nitrogen, as well as compounds which 
contain nitrogen, including but not limited to amino acids, peptones, 
vitamins, and nitrogenous salts. 
As used herein, the term "antimicrobial" is used in reference to any 
compound which inhibits the growth of, or kills microorganisms. It is 
intended that the term be used in its broadest sense, and includes, but is 
not limited to compounds such as antibiotics which are produced naturally 
or synthetically. It is also intended that the term includes compounds and 
elements that are useful for inhibiting the growth of, or killing 
microorganisms. "Antimicrobial susceptibility tests" are conducted in 
order to determine whether a microorganism is susceptible to the effects 
of an antimicrobial. 
As used herein, the term "testing substrate" is used in reference to any 
carbon and/or nitrogen source that may be utilized to differentiate 
bacteria based on biochemical characteristics. For example, one bacterial 
species may utilize one testing substrate that is not utilized by another 
species. This utilization may then be used to differentiate between these 
two species. It is contemplated that numerous testing substrates be 
utilized in combination. Testing substrates may be tested individually 
(e.g., one substrate per testing well or compartment, or testing area) or 
in combination (e.g., multiple testing substrates mixed together and 
provided as a "cocktail"). 
Following exposure to a testing substrate such as a carbon or nitrogen 
source, or an antimicrobial, the response of an organism may be detected. 
This detection may be visual (i.e., by eye) or accomplished with the 
assistance of machine(s) (e.g., the Biolog MicroStation Readers.TM.). For 
example, the response of organisms to carbon sources may be detected as 
turbidity in the suspension due to the utilization of the testing 
substrate by the organisms. Likewise, growth can be used as an indicator 
that an organism is not inhibited by certain antimicrobials. In one 
embodiment, color is used to indicate the presence or absence of organism 
growth/metabolism. 
As used herein, the terms "chromogenic compound" and "chromogenic 
substrate," refer to any compound useful in detection systems by their 
light absorption or emission characteristics. The term is intended to 
encompass any enzymatic cleavage products, soluble, as well as insoluble, 
which are detectable either visually or with optical machinery. Included 
within the designation "chromogenic" are all enzymatic substrates which 
produce an end product which is detectable as a color change. This 
includes, but is not limited to any color, as used in the traditional 
sense of "colors," such as indigo, blue, red, yellow, green, orange, 
brown, etc., as well as fluorochromic or fluorogenic compounds, which 
produce colors detectable with fluorescence (e.g., the yellow-green of 
fluorescein, the red of rhodamine, etc.). It is intended that such other 
indicators as dyes (e.g., pH) and luminogenic compounds be encompassed 
within this definition. 
As used herein, the commonly used meaning of the terms "pH indicator," 
"redox indicator," and "oxidation-reduction indicator," are intended. 
Thus, "pH indicator" encompasses all compounds commonly used for detection 
of pH changes, including, but not limited to phenol red, neutral red, 
bromthymol blue, bromcresol purple, bromcresol green, bromchlorophenol 
blue, m-cresol purple, thymol blue, bromcresol purple, xylenol blue, 
methyl red, methyl orange, and cresol red. The terms "redox indicator" and 
"oxidation-reduction indicator" encompass all compounds commonly used for 
detection of oxidation/reduction potentials (i.e., "eH") including, but 
not limited to various types or forms of tetrazolium, resazurin, methylene 
blue, and quinone-imide redox dyes including the compounds known as 
"methyl purple" and derivatives of methyl purple. The quinone-imide redox 
dye known as methyl purple is referred to herein as "redox purple." In a 
particularly preferred embodiment, "redox purple" comprises the compound 
with the chemical structure shown in FIG. 5, VI. It is contemplated that 
analogous derivatives of the reagent (e.g., alkali salts, alkyl O-esters), 
with modified properties (e.g., solubility, cell permeability, toxicity, 
and/or modified color(s)/absorption wavelengths) will be produced using 
slight modifications of the methods described in Example 13. It is also 
contemplated that various forms of redox purple (e.g., salts, etc.), may 
be effectively used in combination as a redox indicator in the present 
invention. 
As used herein, the terms "testing means" and "testing device" are used in 
reference to testing systems in which at least one organism is tested for 
at least one characteristic, such as utilization of a particular carbon 
source, nitrogen source, or chromogenic substrate, and/or susceptibility 
to an antimicrobial agent. This definition is intended to encompass any 
suitable means to contain a reaction mixture, suspension, or test. It is 
intended that the term encompass microtiter plates, petri plates, 
microcard devices, or any other supporting structure that is suitable for 
use. For example, a microtiter plate having at least one gel-initiating 
agent included in each of a plurality of wells or compartments, as 
disclosed in pending U.S. Pat. No. 5,627,045, and U.S. patent appln. Ser. 
No. 08/762,656, both of which are herein incorporated by reference, 
comprise testing means suitable for use with the present invention. Other 
examples of testing means include microtiter plates without gel-initiating 
means included in the well (See e.g., U.S. Pat. No. 5,589,350, and pending 
U.S. patent appln. Ser. No. 08/685,695, both of which are hereby 
incorporated by reference). 
It is also intended that other compounds such as carbon sources or 
antimicrobials will be included within the compartments. The definition is 
also intended to encompass a "microcard" or miniaturized plates or cards 
which are similar in function, but much smaller than standard microtiter 
plates (for example, many testing devices can be conveniently held in a 
user's hand). It is not intended that the present invention be limited to 
a particular size or configuration of testing device or testing means. For 
example, it is contemplated that various formats will be used with the 
present invention, including, but not limited to microtiter plates, 
microcards, petri plates, petri plates with internal dividers used to 
separate different media placed within the plate, test tubes, as well as 
many other formats. 
As used herein, the term "gelling agent" is used in a broad generic sense, 
and includes compounds that are obtained from natural sources, as well as 
those that are prepared synthetically. As used herein, the term refers to 
any substance which becomes at least partially solidified when certain 
conditions are met. For example, one gelling agent encompassed within this 
definition is Gelrite.TM., a gellan which forms a gel upon exposure to 
divalent cations (e.g., Mg.sup.2+ or Ca.sup.2+). Gelrite.TM. is produced 
by deacetylating a natural polysaccharide produced by Pseudomonas elodea, 
and is described in U.S. Pat. No. 5,627,045, and pending U.S. patent 
appln. Ser. No. 08/762,656; and described by Kang et al. in U.S. Pat. Nos. 
4,326,052 and 4,326,053; all of which are herein incorporated by 
reference). 
Included within the definition are various gelling agents obtained from 
natural sources, including protein-based as well as carbohydrate-based 
gelling agents. One example is bacteriological agar, a polysaccharide 
complex extracted from kelp. Also included within the definition are such 
compounds as gelatins (e.g., water-soluble mixtures of high molecular 
weight proteins obtained from collagen), pectin (e.g., polysaccharides 
obtained from plants), carrageenans and alginic acids (e.g., 
polysaccharides obtained from seaweed), and gums (e.g., mucilaginous 
excretions from some plants and bacteria). It is contemplated that various 
carrageenan preparations will be used in the present invention, with iota 
carrageenan comprising a preferred embodiment. It is also contemplated 
that gelling agents used in the present invention may be obtained 
commercially from a supply company, such as Difco, BBL, Oxoid, Marcor, 
Sigmna, or any other source. 
It is not intended that the term "gelling agent" be limited to compounds 
which result in the formation of a hard gel substance. A spectrum is 
contemplated, ranging from merely a more thickened or viscous colloidal 
suspension to one that is a firm gel. It is also not intended that the 
present invention be limited to the time it takes for the suspension to 
gel. 
Importantly, it is intended that the present invention provides a gelling 
agent suitable for production of a matrix in which organisms may grow (i 
e., a "gel matrix"). The gel matrix of the present invention is a 
colloidal-type suspension of organisms produced when organisms are mixed 
with an aqueous solution containing a gelling agent, and this suspension 
is exposed to a gel-initiating agent. It is intended that this 
colloidal-type gel suspension be a continuous matrix medium throughout 
which organisms may be evenly dispersed without settling out of the matrix 
due to the influence of gravity. The gel matrix must support the growth of 
organisms within, under, and on top of the gel suspension. 
As used herein the term "gel-initiating agent" refers to any compound or 
element which results in the formation of a gel matrix, following exposure 
of a gelling agent to certain conditions or reagents. It is intended that 
"gel-initiating agent" encompass such reagents as cations (e.g., 
Ca.sup.2+, Mg.sup.2+, and K.sup.+). Until the gelling agent contacts at 
least one gel-initiating agent, any suspension containing the gelling 
agent remains "ungelled" (i.e., there is no thickening, increased 
viscosity, nor hardening of the suspension). After contact, the suspension 
will become more viscous and may or may not form a rigid gel (i.e., 
contact will produce "gelling"). 
As used herein, the term "inoculating suspension" or "inoculant" is used in 
reference to a suspension which may be inoculated with organisms to be 
tested. It is not intended that the term "inoculating suspension" be 
limited to a particular fluid or liquid substance. For example, 
inoculating suspensions may be comprised of water, saline, or an aqueous 
solution which includes at least one gelling agent. It is also 
contemplated that an inoculating suspension may include a component to 
which water, saline or any aqueous material is added. In particularly 
preferred embodiments, once the inoculating suspension contains 
microorganisms, this suspension is used to inoculate test panels for the 
identification, testing, and/or detection of the microorganisms present in 
the sample. In addition, it is intended that the inoculating suspension be 
used to determine the antimicrobial susceptibility of microorganisms 
and/or used in a manner so as to increase the effectiveness of 
antimicrobial agents (i.e., when the inoculating suspension also contains 
at least one anti-capsule agent). 
As used herein, the term "test kit" is used in reference to a combination 
of reagents and other materials. It is contemplated that the kit may 
include reagents such as carbon sources, nitrogen sources, chromogenic 
substrates, antimicrobials, diluents and other aqueous solutions, as well 
as microplates (e.g., GN, GP, YT, SF-N, SF-P, and other MicroPlates.TM., 
obtained from Biolog), inoculants, microcards, and plated agar media. The 
present invention contemplates other reagents useful for the growth, 
identification and/or determination of the antimicrobial susceptibility of 
microorganisms. For example, the kit may include reagents for detecting 
the growth of microorganisms following inoculation of kit components 
(e.g,tetrazolium or resazurin included in some embodiments of the present 
invention). It is not intended that the term "test kit" be limited to a 
particular combination of reagents and/or other materials. Further, in 
contrast to methods and kits which involve inoculating organisms on or 
into a preformed matrix such as an agar surface or broth, the present 
invention involves inoculation of a testing plate in which the organisms 
are suspended within a gel-forming matrix. 
As used herein, the term "test panel" refers to any combination of testing 
substrates useful for the identification microorganisms. For example, it 
is intended that the term encompass testing panels such as the Biolog 
MicroPlates.TM., in which the detection of carbon source utilization is 
used to identify organisms- In preferred embodiments, the test panel 
includes a multi-well test plate. The wells of the multi-well test plate 
contain various substrates useful for the detection and/or identification 
of the microorganism of interest. An "antimicrobial test panel" is used to 
determine whether a microorganism is susceptible to the effects of at 
least one antimicrobial. 
With any of the testing formats, the visual result that is detected by eye 
or by instrument can be any optically perceptible change such as a change 
in turbidity, a change in color, or the emission of light, such as by 
chemiluminescence, bioluminescence, or by Stokes shift. Color indicators 
may be, but are not limited to, redox indicators (e.g., tetrazolium, 
resazurin, and/or redox purple), pH indicators, or various dyes and the 
like. Various dyes are described in U.S. Pat. Nos. 4,129,483, 4,235,964 
and 5,134,063 to Bochner, hereby incorporated by reference. See also, 
Bochner, Nature 339: 157 (1989); and ASM News 55: 536 (1990). A 
generalized indicator useful for practice of the present invention is also 
described by Bochner and Savageau (See, Bochner and Savageau, Appl. 
Environ. Microbiol., 33: 434 [1977]). 
Testing based on the redox technology is extremely easy and convenient to 
perform. A cell suspension is prepared and introduced into the testing 
compartments of the device. Each compartment is prefilled with a different 
substrate. 
In a preferred embodiment, all wells are prefilled with test formula 
comprising a basal medium that provides nutrients for the microorganisms, 
and a color-change indicator, and each compartment is prefilled with a 
different carbon compound or "testing substrate," against which the 
microorganism is tested. "Basal medium," as used herein, refers to a 
medium which provides nutrients for the microorganisms, but does not 
contain sufficient concentrations of carbon compounds to trigger a color 
response from the indicator. "Carbon compound," "carbon source" and 
"testing substrate" are equivalent terms, and are used interchangeably 
herein to refer to a carbon chemical in sufficient concentration as to 
trigger a color response from the indicator when it is utilized 
(metabolized) by a microorganism (e.g., GN, GP, YT, and other 
MicroPlates.TM. commercially available from Biolog). In a particularly 
preferred embodiment, redox purple is used as a redox indicator in the 
present invention. 
One of the principal uses of the present invention is as a method and 
device for simple testing and speciation of microorganisms. The present 
invention contemplates microbiological testing based on the redox 
technology discussed above wherein a sample of a pure culture of 
microorganism is removed from a culture medium on which it has been grown 
and suspended in saline or water at a desired density. This suspension is 
then introduced into the compartments of the testing device which have 
been prefilled with basal medium, indicator, and substrate chemicals. The 
method is extremely easy and convenient to perform, and, unlike other 
approaches, the method and device do not require skilled personnel and 
cumbersome equipment. 
EXPERIMENTAL 
The following examples are provided in order to demonstrate and further 
illustrate certain preferred embodiments and aspects of the present 
invention and are not to be construed as limiting the scope thereof. 
In the experimental disclosure which follows, the following abbreviations 
apply: eq (equivalents); M (Molar); .mu.M (micromolar); N (Normal); mol 
(moles); mmol (millimoles); .mu.mol (micromoles); nmol (nanomoles); g 
(grams); mg (milligrams); .mu.g (micrograms); ng (nanograms); 1 or L 
(liters); ml (milliliters); .mu.l (microliters); cm (centimeters); mm 
(millimeters); .mu.m (micrometers); nm (nanometers); .degree. C (degrees 
Centigrade); (S)-(+)-4-isobutyl-.alpha.-methylphenylacetic acid 
(ibuprofen); BBL (Becton Dickinson Microbiology Systems, Cockeysville, 
Md.); DIFCO (Difco Laboratories, a division of Becton Dickson); Sigma 
(Sigma Chemical Co., St. Louis, Mo.); Aldrich (Aldrich Chemical Co., 
Milwaukee, Wis.); Biolog (Biolog, Inc., Hayward, Calif.); ATCC (American 
Type Culture Collection, Rockville, Md.); API (API Analytab Products, a 
division of Biomerieux); and Biomerieux (Biomerieux, Hazelwood, Mo.). 
The following Table lists the principal bacterial strains used in the 
following Examples. In addition to these organisms, other species were 
also tested during the development of the present invention; this list is 
simply intended to provide examples of organisms tested. The numbers used 
in this Table refer to the numbers assigned to these cultures in the 
Biolog culture collection. For some species, more than one strain was 
used. For example, one strain K pneumoniae (Biolog #5008) is a relatively 
non-mucoid strain, while another strain (Biolog #1043) is a highly mucoid 
strain. In addition, two P. aeruginosa strains were used; (Biolog #1191) 
is a non-mucoid strain, while 13363 is a mucoid strain. These organisms 
are available from Biolog. 
TABLE 1 
______________________________________ 
Organisms Tested 
Organism Biolog Number 
______________________________________ 
Enterobacter aerogenes 
4303 
Klebsiella pneumoniae 5008 
Klebsiella pneumoniae 1043 
Pseudomonas aeruginosa 1191 
Pseudomonas aeruginosa 13363 
Chryseobacterium indologenes 1791 
Micrococcus lylae 6823 
Micrococcus luteus 9061 
Kytococcus sedentarius 14080 
Bacillus mycoides 9257 
______________________________________

EXAMPLE 1 
Testing of Gram-Negative Organisms 
In this Example, 2.5 mM and 5 mM each of thioglycolate, sodium salicylate, 
and ibuprofen were tested with various Gram-negative organisms to 
determine the anti-capsule activity of the compounds. In these 
experiments, the test compounds were added to PPS suspensions of 
organisms, and the suspensions were then used to inoculate Biolog GN 
plates (Biolog) used to identify grain-negative organisms, incubated 
overnight at 35.degree. C. (or 30.degree. C. for C. indologenes). In the 
following Table, "++" indicates that a strong false positive reaction was 
observed in the negative control well of the GN plate, while "+" indicates 
a moderate false positive, "-" indicates that no false positive reaction 
was observed, "+/-" indicates an equivocal reaction, and "pi" indicates 
that the true positive reactions were partially inhibited. In this Table, 
the column labelled "No Additions" was the control in which no 
anti-capsule compound was added. 
TABLE 2 
______________________________________ 
Results With Gram-Negative Organisms 
Thioglycolate 
Salicylate 
Ibuprofin 
No 2.5 5 2.5 5 2.5 5 
Strain Additions mM mM mM mM mM mM 
______________________________________ 
E. aerogenes 
++ +/- - +/- +/- +/- +/- 
(4303) (pi) (pi) 
K. pneumoniae ++ - - - - + + 
(5008) (pi) (pi) (pi) 
K. pneumoniae ++ - - - - + - 
(1043) (pi) (pi) 
P. aeruginosa - - - - - - - 
(1191) (pi) (pi) (pi) (pi) 
P. aeruginosa - - - - - - - 
(13363) (pi) 
C. indologenes ++ + +/- ++ ++ +m/- - 
(1791) (pi) (pi) 
______________________________________ 
EXAMPLE 2 
Testing of Gram-Positive Organisms 
In this Example, various gram-positive organisms were tested using 2.5 mM 
and 5 mM thioglycolate, thioglycolate ethyl ester, salicylate, and 
ibuprofen. The same methods as described in Example 1 were used, with the 
exception being that Biolog GP plates were used, and all of the organisms 
were incubated at 30.degree. C. In addition, B. mycoides was grown on BUG 
medium containing 0.25% maltose, and BUGM medium containing 1% glucose 
(both of these media are available from Biolog). The results are shown in 
Table 3. The same indicators as used in Table 2, above (Example 1) are 
used in Table 3. In this Table, "NT" indicates that the compound was not 
tested, and "NR" indicates that no reaction was observed, due to the 
toxicity of the compound for the organism tested. 
TABLE 3 
__________________________________________________________________________ 
Results With Some Gram-Positive Organisms 
Thioglycolate 
No Thioglycolate Ethyl Ester Salicylate Ibuprofin 
Strain Additions 
2.5 mM 
5 mM 
2.5 mM 
5 mM 
2.5 mM 
5 mM 
2.5 mM 
5 mM 
__________________________________________________________________________ 
M. lylae 
+/- +/- + + + +/- +/- - - 
M. luteus +/- + + + + - to - to +/- +/- 
(pi) (pi) (pi) +/- +/- (pi) (pi) 
K. sedentarius + NT NT NT NT - +/- + - 
(pi) 
B. mycoides - - - - - - - NR NR 
grown on BUG (pi) (pi) (pi) (pi) 
with maltose 
B. mycoides ++ NT NT NT NT NT NT NT NT 
grown on BUGM 
with glucose 
__________________________________________________________________________ 
EXAMPLE 3 
Growth of Gram-Positive Organisms on Solid Media 
In addition to testing the anti-capsule compounds in suspensions used to 
inoculate the GP plates as described in Example 2, in this Example, 
anti-capsule agents were also tested for their ability to inhibit biofilm 
production by organisms that tend to form mucoid slime, pellicles, or 
crusty-appearing aggregates on solid media. 
In these experiments, sodium thioglycolate (0.6%; 50 mM) was swabbed onto 
the surface of the agar medium (BUG with maltose and BUGM with glucose, 
both of which are available from Biolog) used to grow Bacillus species 
prior to inoculating the plate with the culture to be tested. After 
swabbing, the anticapsular agent was allowed to be absorbed into the 
medium prior to inoculation with the microbial culture to be tested. 
Examples of Bacillus species that are among the most problematic due to 
capsule production are B. licheniformis (e.g. Biolog strains #9251, 9754, 
and 11101), which forms very mucoid growth often overlaid with a skin-like 
pellicle, and B. subtilis (e.g., Biolog strain #9265), which produces very 
dry, crusty aggregates. These strains (as well as numerous others) adhere 
tightly to the agar surface and/or clump strongly, making them difficult 
to remove from the agar and suspend in a solution. Thus, the growth 
characteristics of these and other organisms make it very difficult to 
test and/or identify these organisms. 
In these experiments, it was determined that thioglycolate greatly 
diminished the formation of mucoid slimes, pellicles, or crusty-looking 
aggregates (i.e., biofilms) on the agar surfaces. While an understanding 
of the mechanism is not necessary in order to use the present invention, 
it is thought that these types of capsular manifestations promote 
attachment and aggregation of microbial cells present on surfaces to form 
biofilms. Organisms grown using this method also provided uniform cell 
suspensions when placed in liquid media or diluents. In contrast, when 
these biofilm-producing organisms were grown using methods known in the 
art (i.e., without thioglycolate), the suspensions are not uniform as the 
cells tend to aggregate and/or form non-dispersable clumps. It is 
contemplated that these methods will find use with other biofilm-producing 
gram-positive and gram-negative organisms, as the present invention 
clearly provides methods to prevent or inhibit the formation of biofilms 
such as pellicles. 
EXAMPLE 4 
Testing of Fungi 
In this Example, anti-capsule agents are tested with various fungal 
species. In particular, fungi such as Cryptococcus and Aureobasidium, are 
used in these experiments. These fungi are grown on media suitable for 
their growth (e.g., malt extract agar, potato dextrose agar, or other 
media) under conditions suitable for fungal growth. A suspension of fungal 
cells is prepared by transferring some of the growth into a water, saline, 
or gelling (e.g., 0.25% phytagel) solution. The anti-capsule agent (e.g., 
thioglycolate, salicylate, ibuprofen) is added to the solution as 
described in the above Examples. This suspension is then used to inoculate 
a testing panel suitable for the testing and/or identification of fungi 
(e.g., Biolog YT MicroPlate.TM.), and incubated for approximately one to 
four days at approximately 26.degree. C. (i.e., incubation occurs for an 
amount of time suitable for the particular organism to be identified). The 
negative control wells (e.g., wells A1 and D1 in the Biolog YT 
MicroPlate.TM.) are used as a basis to assess the growth (e.g., turbidity) 
in the other wells of the testing panel (e.g., microplate); these wells 
contain a variety of carbon sources. 
From the above Examples, it is clear that the present invention provides 
unexpected and much improved methods for the rapid biochemical testing of 
microorganisms, in many uses and formats (or configurations) and in 
particular, provides a major advance in the testing of capsule-producing 
microorganisms. The present invention provides advantages to both 
automated and manual systems for identification of microorganisms. For 
example, the results may be observed visually (i.e., by eye) by the person 
conducting the test, without assistance from a machine. Alternatively, the 
results may be obtained with the use of equipment (e.g., a microplate 
reader) that measures transmittance, absorbance, or reflectance through, 
in, or from each well of a multitest device such as microplate or 
microcard. These advantages enhance the speed and accuracy of scoring test 
results in studies to characterize and/or identify microorganisms.