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Claims  |
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I claim:
1. A device for use in the identification of microorganisms, comprising a
unitary tray member having a plurality of integrally formed test wells, at
least some of said wells serving as identification test wells for
microorganisms and containing biochemical test media which in hydrated
form permits growth of said microorganisms with the generation of a
volatile color-forming compound, and corresponding negative control wells
in close proximity to each of said test wells, said negative control wells
containing negative control media including an inhibitor which in aqueous
solution prevents color formation otherwise occuring therein from said
volatile color forming compound, and wherein both said biochemical test
media prior to inoculation with said microorganisms and said negative
control media have the same color in hydrated form.
2. The device according to claim 1 wherein said inhibitor is a buffer.
3. A device according to claim 2 wherein said microorganism identification
test wells contain said biochemical test media selected from the group
consisting of phosphodiesterase medium, Voges-Proskauer medium,
o-nitrophenyl-.beta.-D-galactopyranoside medium, tryptophan deaminase
medium and indol medium.
4. The device according to claim 3 wherein said buffer is selected from the
group consisting of KH.sub.2 PO.sub.4, and NaOH plus glycine.
5. The device according to claim 4 wherein at least one of said negative
control wells contains phosphodiesterase negative control medium which
comprises peptone buffered to a pH of about 4.8 to 5.3.
6. The device according to claim 4 wherein at least one of said negative
control wells contain o-nitrophenyl-.beta.-D-galactopyranoside negative
control medium which comprises o-nitrophenyl-.beta.-D-galactopyranoside
and peptone buffered at a pH of about 4.8 to 5.3.
7. The device according to claim 4 wherein at least one of said negative
control wells contains Voges-Proskauer negative control medium which
comprises methyl red Voges Proskauer medium and creatine monohydate
buffered to a pH of about 5.6 to 6.0.
8. The device according to claim 4 wherein at least one of said negative
control wells contains tryptophan deaminase negative control medium which
comprises tryptone, L-tryptophan, and NaCl to a pH of about 4.3 to 5.2.
9. The device according to claim 4 wherein at least one of said negative
control wells contains indol negative control medium which comprises
tryptone and NaCl buffered to a pH of about 9.8 to 10.2.
10. A device according to claim 1 wherein each of said microorganism
identification test wells contains a different biochemical test media, the
color of which after inoculation with said microorganisms can be compared
with the color of said media in said corresponding negative control wells
to provide indicia for the accurate identification of said microorganisms.
11. A device according to claim 1 wherein said biochemical test media and
said negative control media are present in said wells in dried form.
12. A device according to claim 1 wherein at least some of said wells serve
as antibiotic test wells containing a predetermined concentration of an
antibiotic, and antibiotic control wells, said control wells containing no
antibiotic.
13. A device according to claim 12 wherein said biochemical test media,
said negative control media and said antibiotic are present in dried form.
14. A device according to claim 12 wherein said wells are disposed in a
number of generally parallel rows.
15. The device of claim 14 wherein:
(a) said antiobiotic test wells are disposed in several vertical rows, each
of said vertical rows containing a different antibiotic with a different
concentration of said antibiotic present in each of said test wells of
said row; and
(b) said microorganism identification test wells and said negative control
wells are disposed in pairs as horizontal lines of wells running
perpendicular to said vertical rows of antibiotic test wells with each
horizontal line of negative control wells lying adjacent to a horizontal
line of said microorganism identification wells.
16. The device of claim 15 wherein:
(a) said vertical rows of antibiotic test wells number sixteen, each
vertical row having seven antibiotic test wells, constituting seven
horizontal lines of wells; and
(b) said pairs of horizontal lines of test wells and negative control wells
number two with one of said pair of horizontal lines of rows running
perpendicular and above said vertical antibiotic test rows and the other
of said pairs running perpendicular and below said vertical antibiotic
test rows; with said horizontal lines of rows of microorganism test wells
and negative control wells constituting sixteen wells in each of said
lines.
17. A method for the identification of microorganisms comprising:
(a) providing a unitary tray member having a plurality of integrally formed
test wells, at least some said wells serving as microorganism
identification test wells containing, in dried form, biochemical test
media which in hydrated form permits growth of said microorganisms with
the generation of a volatile color-forming compound and corresponding
negative control wells in close proximity to each of said test wells, said
negative control wells containing, in dried form, negative control media
including a buffer which in aqueous solution prevents color formation
therein from said volatile color forming compound, and wherein both said
biochemical test media prior to inoculation with said microorganisms and
said negative control media have the same color when in hydrated form;
(b) reconstituting the contents of said identification test wells and said
negative control wells with water;
(c) inoculating said identification test wells with said microorganism;
(d) incubating said tray member; and
(e) comparing said identification test wells with said negative control
wells to determine whether there has been a detectable change in said
biochemical test media in said identification test wells, indicating
activity of said microorganism.
18. A method for the identification of microorganisms according to claim 17
wherein an indicator capable of producing a color change in said
biochemical test media, in response to microorganism growth, is added to
said biochemical test media after inoculation with said microorganism.
19. The method of claim 17 including:
(a) providing some of said wells to serve as antibiotic test wells,
containing in dried form, a predetermined concentration of an antibiotic,
and antibiotic control wells containing no antibiotic;
(b) reconstituting the contents of said antibiotic test wells and said
antibiotic control wells with a nutrient broth;
(c) inoculating said antibiotic test wells and said antibiotic control
wells with said microorganism;
(d) incubating said antibiotic test wells and said antibiotic control
wells;
(e) visually comparing said antibiotic test wells with said antibiotic
control wells; and
(f) determining the minimum concentration of antibiotic necessary to
inhibit the growth of said microorganism. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a device for use in the identification of
microorganisms and more specifically to a means for eliminating the
problem of false-positive reactions in negative control wells of
microorganism identification test containers.
The large number of strains of highly pathogenic bacteria and their varying
susceptibility to antibiotics has resulted in the development of
techniques for microorganism identification and antibiotic susceptibility
testing including the determination of the minimum inhibitory
concentration of antibiotic, i.e., the lowest concentration of antibiotic
which completely inhibits the growth of the organism. These techniques
typically involve the use of test trays which comprise, for example, a one
piece plastic shell containing a plurality of receptacles or incubation
wells. The wells may be arranged in parallel rows such as described in
U.S. Pat. Nos. 3,826,717 and 3,356,462, or in circular patterns such as
described in U.S. Pat. No. 4,010,078.
During the antibiotic susceptibility test, the wells contain the
microorganisms to be inhibited, nutrient broth and antibiotic. Typically
before inoculation each of the antibiotic test wells contains a different
concentration of antibiotic and each row of such wells contains a
different antibiotic. Each of the wells is inoculated with a standard
amount of microorganism. The contents of the wells are incubated for a
period of time and are then examined to determine the minimum inhibitory
concentration of the appropriate effective antibiotic, U.S. Pat. No.
3,826,717. Those wells which contain a sufficient concentration of a
growth inhibiting antibiotic contain a clear solution, while those wells
which contain an insufficient concentration of antibiotic or an
ineffective growth inhibiting antibiotic have either a turbid solution or
a small turbid area or dot surrounded by a clear solution, indicating
growth of the microorganisms. Cross-contamination between test wells on
the same plate is minimized by the use of barrier elements between rows of
adjacent wells and by overlaying the openings of the wells with a cover or
other sealing means.
During the microorganism identification test the wells contain the
microorganism to be identified, nutrient broth and a biochemical indicator
which changes in color in response to the appropriate microorganism, U.S.
Pat. No. 4,010,078.
U.S. Pat. No. 3,107,204 discloses the use of an antibiotic test tray having
wells containing fibrous discs. The discs contain dehydrated
bacteriological media, a dye color indicator in accordance with bacterial
growth or activity and various chemotherapeutic agents. The discs are
arranged in pairs, each pair containing a high and a low concentration of
the same antibacterial agent. Color changes are compared against a control
to measure antibiotic activity, but not for identification of any
particular active microorganism. The problem of cross-contamination of the
materials in adjacent wells is recognized and overcome by the use of a
sealing lid.
U.S. Pat. No. 4,010,078 provides a device for use in the identification of
microorganisms comprising an open-topped multi-compartmented microorganism
culture media receiving portion and a cover member. Several of the types
of media employed contain an indicator which changes color in response to
the growth of a particular microorganism. The use of a standard or
negative control well is not discussed.
Heretofore the prior art has not provided an inexpensive microbial test
tray which gives rapid results and not only provides antibiotic
sensitivity information but further provides an accurate and reproducible
means of microorganism identification through comparisons between negative
control wells and biochemical test wells. An additional problem presented
by microorganism identification is that many of the known biochemical
tests involve only subtle color changes in the test media in response to
microorganism growth. Determinations that these subtle color changes have
occurred are difficult to make in a reliable manner. It is desired to
improve the reliability of microorganism identifications based on subtle
color changes in the biochemical test media.
SUMMARY OF THE INVENTION
The present invention provides a device and method which significantly
improves the ability to detect the results of microorganism identification
tests involving the detection of color changes in test media. The
invention substantially reduces the possibility of an improper result in
the identification of a microorganism due to failures to perceive subtle
color changes in the test media.
The present invention provides increased reliability in microorganism
identification by providing negative control wells which have an
appearance identical to the test wells when the media in both types of
wells are reconstituted with water and before the test wells are
inoculated. The negative controls maintain the original color of the test
media and provide a standard against which to compare the test media after
inoculation and incubation with the microorganisms.
To be useful in a compact test tray, particularly one having several
microorganism identification tests, the negative control wells must be
closely adjacent to the corresponding test wells. The addition of a series
of negative control wells lying adjacent to the test wells has been found
to produce a problem of cross-contamination heretofore not experienced.
Volatile products generated in the inoculated test wells can contaminate
the negative control wells through vapor transport means and produce a
false visible color change in the negative control wells. It is difficult
to determine whether or not a true color change has occured in the test
wells in response to microorganism activity without reference to a
negative control which retains the standard color of the test well media.
The present invention does not eliminate the problem of cross-contamination
of the negative controls, but does however, prevent color changes in the
negative controls, by the inclusion of an inhibitor capable of preventing
the volatile color forming compounds generated in the inoculated test
wells from changing the color of the negative control media. The inhibitor
is most conveniently provided as a buffer included in the negative control
media at the time of filling and packaging the test tray. The present
invention provides self-correcting negative controls, not requiring
further operator manipulation, which enable the negative controls to
retain the standard color of the test media.
The present invention provides negative control compositions which maintain
their original color and are not affected by cross-contamination resulting
from volatile products generated in the inoculated microorganism
identification test wells. Thus, test results obtained through practice of
the present invention are reproducible and accurate, i.e., free of false
positive reactions in negative control wells.
The preferred embodiment of the present invention provides means wherein a
multiplicity of tests employing many microorganism identification systems
and many concentrations of numerous antibiotics may be performed in a
single container in a rapid and efficient manner. The preferred embodiment
is a tray containing microorganism identification test wells and negative
control wells in addition to antibiotic test wells, with media in all the
wells being furnished in a dry state ready for rehydration.
The present invention provides a device and method of identifying
microorganisms which gives quick results and is readily employed in a
simple and inexpensive way in hospitals, medical laboratories and doctor
offices. In addition, since the present invention provides a more reliable
and reproducible microorganism identification system, it significantly
reduces the handling of potentially health-hazardous microorganisms by
laboratory personnel.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a partially schematic top plan view showing one embodiment of the
combination antibiotic and microorganism test container of the present
invention;
FIG. 2 is a side view of the antibiotic and microorganism test container of
the present invention with a partial cross-section of the microorganism
identification wells taken along 2--2 of FIG. 1.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring now more specifically to FIGS. 1 and 2, the embodiment of the
invention there illustrated will be considered in greater detail. As is
shown in FIG. 1, a tray member 2 is, in the form shown, of generally
rectangular peripheral configuration. The tray member is preferably formed
as a unitary article, as by molding. It is provided with a plurality of
integrally formed upwardly open wells. In the form illustrated, the plate
member 2 has sixteen vertical rows of wells and eleven horizontal lines of
wells which are designated by the letters "A" through "K". The wells are
downwardly directed cylinders having rounded bottoms.
The tray member 2 with wells may be made from a wide range of materials
such as glass, plastic, or even a lightweight metal such as aluminum. It
is generally preferred to mold these elements from a strong, liquid
impermeable transparent plastic material. Among the materials particularly
suited for such use are polyethylene, polypropylene, polystyrene, or the
like. These materials not only possess the desired properties, but can
also be economically manufactured into the desired shape by modern
conventional plastic fabricating techniques. In addition, the use of such
materials makes disposability after use economically feasible.
The preferred embodiment of tray member 2 is of generally rectangular
configuration and has a length of 17.5 centimeters and a width of 11.0
centimeters. Wells in a given row are spaced ten millimeters apart and
wells in adjacent rows are spaced nine millimeters apart. Each well is of
rounded-bottom cylindrical shape and has a depth of 10 millimeters, a
diameter of 7 millimeters at the opening of the well and a curvature
radius of 3 millimeters at the bottom, closed end of the well.
While the specific structure disclosed in detail herein has a generally
rectangular periphery in plan and such geometry is preferred, the
invention is not so limited and other external peripheral shapes may be
employed while retaining the benefits of this invention. Also, while for
purposes of illustration there have been shown the preferred
rounded-bottom cylindrical wells, other configurations such as flat-bottom
cylindrical wells or rectangular wells may be employed if desired.
Referring now to FIGS. 1 and 2, it is noted that each of the wells is
filled with the appropriate substance (preferably by automatic filling
means in a sterile environment) in such fashion that the upper surface 24
of the well contents will be disposed generally below the opening of the
well 26. In the preferred embodiment the contents of the wells are air
dried and must be rconstituted before use. It will be appreciated that
while for purposes of convenience of description herein a tray member 2
having 16 rows each containing 11 wells is being shown, different numbers
of rows containing different numbers of wells may be provided, depending
upon the number of antibiotics and identification media used. Also, for a
particular test, only those rows of wells which are to be employed in the
test may be filled.
Referring to FIG. 1 in the preferred embodiment of the present invention,
horizontal lines of wells "C" through "I" are antibiotic test wells (for
example, well 22), each vertical row 1-16 containing a different
antibiotic and each horizontal line C-I in each row containing a different
concentration of the particular antibiotic. It is preferred that the wells
of line "I" have the highest concentration of antibiotic and that the
concentration be progressively decreased through line "C" which will
contain the lowest concentration. Antibiotic control wells containing no
antibiotic are also provided. Growth of microorganisms in the antibiotic
control wells will confirm the fact that the microorganism will grow in
the test system and will provide a basis for comparison with antibiotic
test wells containing differing concentrations of antibiotic.
The effect of different types and concentrations of antibiotics on
microorganism growth is normally not measured by a color change but rather
is exhibited as a turbidity change when compared to adjacent clear or "no
growth" wells having inhibitory concentrations of antibiotic.
Referring now to FIGS. 1 and 2, in the preferred embodiment of the present
invention, horizontal lines of wells "A", "B", "J" and "K" are employed in
microorganism identification tests. The wells in lines "A" and "K" are
microorganism identification control wells (for example, well 18), also
referred to as negative control wells, containing negative control media.
The wells in lines "B" and "J" are microorganism identification test wells
(for example, well 20), containing microorganism identification media.
FIG. 2 illustrates that in the preferred embodiment negative control wells
(e.g. 18) are placed in close proximity and adjacent to identification
test wells (e.g. 20) which are adjacent to antibiotic test wells (e.g.
22).
Table I lists the type of media used in each microorganism identification
test well (i.e. test media) of the preferred embodiment although different
arrangements of the media and the use of additional or alternative media
are not beyond the scope of the invention. Optimum concentrations may be
determined for each ingredient in the various media listed in Table I, as
is well known to the skilled artisan. Each of the test wells in lines "J"
and "B" and therefore each of the corresponding negative controls in lines
"A" and "K", contain a different medium depending on the microorganism to
be identified, and each medium is capable of sustaining specific metabolic
activity of a particular microorganism. Indicators are either added to the
microorganism identification test media after inoculation with
microorganism, as is known, or are generated in the test media by
metabolism of the test media by the microorganism. The indicator produces
a change in the color of the media in the test wells in response to
microorganism metabolism. The activity of the organism under investigation
is determined by a color change in the test media, as compared with the
corresponding negative control.
TABLE I
______________________________________
Row Line Medium (Abbreviation)
______________________________________
1 J Mueller-Hinton broth
(MH)
2 J Lysine decarboxylase
(Lys)
3 J Arginine dihydrolase
(Arg)
4 J Ornithine decarboxylase
(Orn)
5 J Sulfide detection (H.sub.2 S)
6 J Urease (Ur)
7 J Tryptophan deaminase
(TDA)
8 J Indol (I)
9 J Voges-Proskauer (VP)
10 J Citrate utilization
(Cit)
11 J DNAse detection (DNA)
12 J o-nitrophenyl-.beta.-d-
galactopyranoside (ONPG)
13 J Glucose fermentation
(Glu)
14 J Arabinose (Ara)
15 J Inositol (Ino)
16 J Sorbitol (Sor)
1 B Mueller-Hinton broth
(MH)
2 B Sucrose (Suc)
3 B Mannitol (Man)
4 B Rhamnose (Rha)
5 B Raffinose (Raf)
6 B Melibiose (Mel)
7 B Dulcitol (Dul)
8 B Adonitol (Ado)
9 B Malonate utilization
(Mal)
10 B Esculin hydrolysis (Esc)
11 B Phosphodiesterase detection
(PDE)
12 B MacConkey (Mac)
13 B Oxidation fermentation glucose
(OFG)
14 B Oxidation-fermentation maltose
(OFM)
15 B Oxidation-fermentation xylose
(OFX)
16 B Pyocyanin (Pyc)
______________________________________
In the preferred embodiment it is essential that the original color of the
test media contained in the test wells of lines "B" and "J" be maintained
in the media of corresponding adjacent negative control wells in lines "A"
and "K". When the organism to be tested is inoculated into the test wells
of lines "B" and "J", a color change in the media contained in these wells
will confirm the fact that the organism has metabolized in that particular
test media. Negative control wells "A" and "K" provide a standard for
comparison with the test wells (rows "B" and "J") in order to make
possible the detection of even subtle color changes. If the original
colors in the negative control wells are altered by cross-contamination,
interpretations of microorganism identification tests may be erroneous.
Especially difficult problems have been observed in regards to the
following negative control wells, line "A", row 11 (Phosphodiesterase
medium), line "K", row 7 (Tryptophan deaminase medium), line "K", row 8
(Indol medium), line "K", row 9, (Voges-Proskauer medium), line "K", row
12 (o-nitrophenyl-.beta.-d-galactopyranoside medium). The negative control
media contained in these wells is clear and colorless and therefore is
most susceptible to any cross-contamination by volatile color-forming
compounds, resulting from the microorganism identification reactions
occuring in adjacent test wells. Table II lists the cross-contaminating
volatile color-forming compounds produced in the test wells.
TABLE II
______________________________________
Negative
Test Control Volatile Color-
Well Well Test forming
Row Line Row Line Medium Compound
______________________________________
11 B 11 A Phosphodiesterase
p-nitrophenol
7 J 7 K Tryptophan Indol
deaminase
8 J 8 K Indol Indol
9 J 9 K Voges-Proskauer
Acetoin
12 J 12 K o-Nitrophenyl-.beta.-
o-nitrophenol
d-galactopyranoside
(ONPG)
______________________________________
In accordance with this invention, it has been found not only that color
changes in negative control wells result from contamination by volatile
color-forming compounds generated in the adjacent test wells upon
inoculation but also that such undesirable color changes can be prevented
by including in the negative control well media an inhibitor which in
aqueous solution prevents color formation therein from the volatile color
forming compound. The preferred inhibitor is a buffering system, i.e. one
or more buffering compounds, capable of controlling the pH in the negative
control wells.
p-Nitrophenol and o-nitrophenol are pH indicators which in acid solutions
are colorless but which turn yellow when in the presence of alkaline
solutions. Thus, negative control media which change their color when
contaminated by o-nitrophenol or p-nitrophenol do so due to the color
change of these pH indicators when exposed to a basic pH. On the other
hand, negative control media which exhibit a color change when
contaminated with indol or acetoin do so due to a chemical color-forming
reaction between the negative control media and these cross-contaminants.
p-Nitrophenol and o-nitrophenol generated in phosphodiesterase and ONPG
test medium after inoculation with microorganism may impart a yellow color
to any medium having a basic pH. Therefore these volatile contaminants may
change the color of even unrelated or noncorresponding negative control
media having a basic pH. Negative control media lying adjacent to
inoculated phosphodiesterase or ONPG test wells must either have a pH of
6.0 or below, or be buffered to such a pH in order to avoid color changes
in the negative control media during microorganism identification testing.
Indol, generated in both the tryptophan deaminase test medium and the indol
test medium will react with the indol negative control media to which is
added the color forming coreactant p-dimethylaminobenzaldehyde.
In the preferred embodiment of the present invention the negative control
wells having media corresponding to the Phosphodiesterase detection media,
the Voges-Proskauer media and the
o-nitrophenyl-.beta.-d-galactopyranoside, must be maintained at a pH of 6
or below while microorganism identification reactions are occuring in
adjacent phosphodiesterase and ONPG test wells in order to prevent color
changes from occuring and thus false positive reactions. The preferred
inhibitor for use with these media is monobasic potassium phosphate
(KH.sub.2 PO.sub.4).
The pH of the indol negative control media must be 9.8 or greater in order
to prevent color changes from occuring in that negative control due to
contamination from the adjacent corresponding inoculated indol test
medium. The preferred inhibitor for the indol negative control is NaOH
plus glycine.
After determining the identity of the volatile color forming contaminant, a
suitable inhibitor useful in preventing color changes caused by that
volatile contaminant in a given negative control can be selected by known
laboratory procedures before the various media are introduced into the
tray wells. For example, a series of runs under varying pH conditions can
be used to select an appropriate buffering system so that no color change
will occur in the negative controls.
In accordance with a preferred embodiment of the invention, the antibiotic
test wells have antibiotic solutions introduced into them and the
microorganism identification wells have the microorganism identification
media, e.g. those of Table I, introduced into them. The contents of all
wells are then air dried, and the device is packaged in a sealed, moisture
proof wrapper or envelope for shipment to laboratory personnel. The
antibiotic well contents are reconstituted before use with a nutrient
broth. The microorganism test well contents are reconstituted with
distilled water.
In order to provide a specific illustration of the use of the microorganism
identification test system of this invention, four examples will be
considered. These will illustrate the specific compositions of five
particular microorganism identification test wells and the compositions of
their corresponding negative control wells. The compositions of the
negative control wells enable meaningful comparisons to be made between
the test wells and the negative control wells due to retention in the
negative control wells of the standard color of the corresponding
microorganism identification test media. Because the purpose of the
negative control is to provide a standard against which to compare color
changes in the test medium, the negative control medium need contain only
the buffering agent and those ingredients present in the test medium which
add to the color of the test medium. The compositions listed illustrate
the well contents prior to air drying.
EXAMPLE 1
The preparation of phosphodiesterase test medium and its negative control
medium will be considered first. Phosphodiesterase test medium (PDE) is
prepared by combining in a flask:
______________________________________
*Peptone 3.0 g
Thymidine-3'monophospho-p-nitrophenyl ester
0.2 g
0.1 M Na.sub.2 HPO.sub.4 100 ml
______________________________________
*Peptone is available under the tradename "BactoPeptone" from Difco
Laboratories, Inc., Detroit, Michigan.
The solution is dissolved with stirring and, if necessary, the application
of gentle heat (.ltoreq.50.degree. C.). After the components are
dissolved, the solution is filter sterilized through a 0.20 membrane
filter. The filtrate is aseptically decanted into a sterile container for
storage. The medium is stored, refrigerated and protected from light. The
pH of the test medium is about 8.0.+-.0.2.
The corresponding phosphodiesterase negative control medium is prepared by
combining in a flask:
______________________________________
Peptone 3.0 g
KH.sub.2 PO.sub.4 10.0 g
Distilled water 100 ml
______________________________________
The solution is dissolved, filter sterilized and stored according to the
procedure used for the phosphodiesterase reaction medium. The pH of the
negative control medium is in the range of about 4.8 to 5.3, and in this
example is about 5.0.
25 Microliters of the test medium and 25 microliters of control medium are
introduced into separate adjacent wells of a test tray. The media in the
wells are air dried. After rehydration with 100 microliters of sterile
distilled water both the uninoculated test medium and the negative control
medium are nearly colorless to pale straw in color. The pH of the
rehydrated test medium is about 8.0.+-.0.2 and the pH of the rehydrated
negative control medium is in the range of about 4.8 to 5.3, and in this
example is about 5.0.
After the test wells are inoculated with Serratia marcescens and incubated
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