U.S. patent number 3,856,628 [Application Number 05/254,193] was granted by the patent office on 1974-12-24 for method and apparatus for the identification of microorganisms.
This patent grant is currently assigned to Richard P. Crowley. Invention is credited to Anthony J. Sbarra.
United States Patent |
3,856,628 |
Sbarra |
December 24, 1974 |
METHOD AND APPARATUS FOR THE IDENTIFICATION OF MICROORGANISMS
Abstract
A method useful in identifying microorganisms and an apparatus
useful to identify microorganisms, which apparatus comprises a flat
sheet of absorbent material, the material characterized by a
plurality of separate test zones therein, each zone including a
substrate and an indicator, the substrate and indicator of each of
the zones so selected on the sheet material so as to provide, by
the reaction or a nonreaction of the microorganism in question with
the substrate in the zone, an indication of the identification of
the microorganism.
Inventors: |
Sbarra; Anthony J. (Milton,
MA) |
Assignee: |
Crowley; Richard P. (Wellesley
Hills, MA)
|
Family
ID: |
22963288 |
Appl.
No.: |
05/254,193 |
Filed: |
May 17, 1972 |
Current U.S.
Class: |
435/34; 435/38;
435/849; 435/805; 435/879 |
Current CPC
Class: |
C12M
23/10 (20130101); C12M 25/02 (20130101); Y10S
435/849 (20130101); Y10S 435/805 (20130101); Y10S
435/879 (20130101) |
Current International
Class: |
C12M
1/16 (20060101); C12M 1/20 (20060101); C12k
001/04 () |
Field of
Search: |
;195/13.5R,127,139 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Primary Examiner: Monacell; A. Louis
Assistant Examiner: Warden; Robert J.
Attorney, Agent or Firm: Crowley; Richard P.
Claims
What I claim is:
1. Means useful to identify a microorganism through a change in
abosrbent by the reaction or nonreaction of the microorganism with
a carbohydrate comprising:
a. an essentially flat sheet of absorbent material adapted to be
placed onto the top surface of a culture medium containing a
microorganism where identity is to be determined;
b. the absorbent material free of restriction on at least one
surface thereof and having a plurality of distinct and separate
test zones thereon in a sufficient number such that the
microorganism can be identified;
c. each test zone containing absorbed thereon in combination a
carbohydrate and a pH color indicator of phenol red, which
indicator changes color about the periphery of the test zone on the
fermentation of a carbohydrate by the microorganism; and
d. the indicator employed in each test zone being the said phenol
red and the carbohydrate in each test zone a different
carbohydrate, whereby the absorbent sheet may be placed on top of a
culture medium containing a microorganism in a culture plate, and
the change in color and lack of color change about the periphery of
the test zones used to identify the presence or absence of a
particular microorganism.
2. The means of claim 1 wherein the absorbent material comprises a
paper sheet having from four to eight test zones thereon.
3. The means of claim 1 wherein the carbohydrates are selected from
the group consisting of lactose, mannitol, adonitol, dulcitol,
glucose, sorbitol and inositol.
4. The means of claim 1 wherein the absorbent material is a
circular paper sheet adapted to fit within and cover substantially
the entire surface of a culture medium in a culture plate.
5. The means of claim 1 wherein the test zones are generally
circular, and a means of identification employed to identify the
test zones or the color change in fermentation by the
microorganism.
6. The means of claim 1 wherein the test zones comprise separate
absorbent discs secured to the absorbent sheet material.
7. A method of identifying a microorganism in a culture medium,
which method comprises:
a. placing into contacting engagement onto the top surface of a
culture medium containing or suspected of containing the
microorganism where identity is to be determined an essentially
flat absorbent sheet material, the sheet material having a
plurality of distinct and separate test zones thereon in a
sufficient number such that the microorganism can be identified,
each test zone containing a substrate and a pH indicator which, on
reaction between the microorganism in the culture medium and the
various substrates in the test zones, causes a change in color of
the indicator about the periphery of the test zones, or no change
in color by nonreaction, the test zones all having the same pH
indicator and different substrates;
b. incubating the culture medium and the sheet material with the
test zones for a period of time to permit the reaction or
nonreaction of the microorganism with the substrates in the test
zones; and
c. observing the change in color, if any, about the periphery of
each test zone as a means for identification of the
microorganism.
8. The method of claim 7 wherein the substrate is a carbohydrate
selected from the group consisting of lactose, mannitol, adonitol,
dulcitol, glucose, sorbitol and inositol.
9. The method of claim 7 wherein the sheet material is an absrbent
paper disc, and which includes wetting the surface of the sheet
material with water prior to incubation.
10. The method of claim 7 wherein the substrate is selected from
the group of lysine, ornithine and phenylalanine.
11. The method of claim 7 wherein the indicator is phenol red.
12. The method of claim 7 which includes:
a. securing separate absorbent discs as test zones to the absorbent
sheet material; and
b. placing the absorbent discs into contacting engagement onto the
surface of a culture medium.
Description
BACKGROUND OF THE INVENTION
In general, the identification of various gram-negative and
gram-positive microorganisms by their biochemical reactions has
become a common procedure. Such techniques have proven most useful
in assigning a genus and species to an unknown microorganism. Its
identification is arrived essentially by the ability or inability
of the particular microorganism to attack a specific substrate, the
substrate being incorporated into a nutrient medium. Typically, the
culture medium would contain appropriate nutrients to encourage the
growth of particular microorganisms, a specific substrate (i.e.,
carbohydrate) and a suitable indicator, so that after a desired
incubation period, the specific biochemical activity of the sample
containing the microorganism may be determined. This would
generally take a period of 24 to 48 hours.
The identification of microorganisms is often detected based on
their ability or inability to cause fermentation of a carbohydrate
in the presence of an indicator. For example, the biochemical
identification of the genus Salmonellae may be accomplished by
employing a culture media containing a variety of different
substrates, such as lactose, glucose, ornithine, lysine and
phenylalanine. Microorganisms belonging to the Salmonella group
would not be expected to attack phenylalanine or lactose. They
would, however, be expected to attack lysine, ornithine and
glucose. Thus, any organisms suspected as belonging to the genus
Salmonellae would not attack either lactose or phenylalanine. It
should be pointed out that identification of microorganisms as
outlined above is attempted only on pure cultures. Thus, after
primary isolation (usually after a 24-hour incubation period) on an
appropriate nutrient medium (i.e., blood agar medium), isolated
colonies will appear, and these are transferred to individual,
specific identifying mediums as alluded to above.
During the past few years, many efforts have been directed toward
the development of rapid bacteriological identification techniques,
so that information concerning the identification of particular
microorganisms; for example, in urine samples, throat swabs and the
like, may be rapidly determined and proper preventive or corrective
action taken. All of the presently available identification
techniques are based, first, on isolating the microorganisms from
infected patient tissue on a primary culture. Isolation of the
microorganisms is typically accomplished by innoculating a primary
culture containing a nutrient with the bacteriological sample
material and incubating the innoculated primary culture for a
period of time, generally 24 hours, after which the resulting
isolated microorganism is removed and innoculated into a number of
different identification systems. Such identification systems
include a tubed media containing nutrient material and specific
identifying substrates, such as carbohydrates and appropriate
indicators. These different tubed media are innoculated with the
primary isolate and then incubated at 37.degree.C for an additional
24-hour period. After this incubation period, the reactions in each
of the tubed media are read and recorded. Identification of the
organism can be made by comparing the reactions noted with those
previously established for each organism.
An additional method employed to identify microorganisms, although
not frequently used, involves the impregnation of the particular
substrate and culture medium on a paper disc. An additional disc is
impregnated with an indicator. The prepared disc is now placed in
culture tubes, water is added, and the solution then innoculated
with the microorganism in question. As previously, the tubes are
incubated and read after a suitable incubation period. By employing
a series of culture tubes, the identification of the microorganism
in question can be determined. Such techniques as described, while
useful in identifying microorganisms, require a number of tubes, a
variety of individual observations of each tube and assessments of
the reaction and nonreaction of the products involved, and a
comparison of the reactions in order to arrive at a suitable
identification of the microorganism, all of which involve time and
the possibility of error. Accordingly, a method which provides for
a rapid and simple identification of microorganisms in a single
procedure would be most desirable.
SUMMARY OF THE INVENTION
My invention relates to a simple, rapid error-free method and
apparatus for identifying microorganisms. My method provides a
single-step procedure or technique by which the reaction or
non-reaction of a microorganism in question can be ascertained. My
apparatus useful in my method comprises an absorbent sheet material
characterized by a plurality of different zones, the zones selected
to determine by the reaction or nonreaction of a substrate and
indicator in each zone with the identification of the
microorganism. My method and apparatus allows a person to determine
a plurality of different biochemical reactions, all on a single
sheet of material, of the microorganism in question at the same
time from a single test procedure and on a single culture
plate.
My method comprises impregnating an absorbent sheet material, such
as paper in the form of a paper disc, with a substrate subject to
chemical reaction or nonchemical reaction with a microorganism and
an identification agent to detect the chemical reaction or
nonchemical reaction between the substrate and the microorganism in
question. For example, the substrate might be a carbohydrate
subject to fermentation, and an indicator subject to color change
upon fermentation of the carbohydrate. The sheet material is
impregnated in a plurality of zones, each zone containing a
different substrate and an indicator. The zones may be placed in
any position or form, and generally there will be from four to
eight zones in each paper disc, each zone separated from the other
zone so that the chemical reaction in one zone will not be
obscured, overlapping with the chemical reaction of another zone.
Impregnation of the paper disc may be accomplished by placing a
drop of the substrate and identifying agent on the paper disc and
drying the disc rapidly. If desired, each particular zone may be
identified and numbered, such as by placing a printed circle line
around each zone, and numbering the zone, providing a code
substrate identifying agent in each zone, or the color change in
each zone required to identify each particular microorganism.
My method comprises placing the impregnated sheet material
containing the various zones selected for a particular
microorganism onto the top surface of a culture plate on which the
microorganism in question has been streaked out, the culture plate
containing the streaked microorganism in question with the
absorbent sheet material contained in the zones, and the top
surface is then incubated, and after an appropriate incubation
period of time, the culture plate is removed and observation made
of the reaction occurring in the various zones of the absorbent
sheet material. Thus, in a rapid, simple and effective manner, on a
single sheet of paper and culture plate, the microorganism in
question could be rapidly identified. Since the substrate and
indicator would, by simple diffusion, diffuse into the medium, and
by observing the color change on the absorbent sheet material about
each zone, identification of the microorganism can be made. If
desired, the absorbent sheet material may be dampened with a little
water while being placed on the top surface of the culture plate in
order to promote diffusion and rapid reaction. Of course, it is
obvious that different multiple paper discs be prepared, each
containing a different set of zones and different substrates and
indicators, each paper disc selected to identify a particular
microorganism. The different substrates and indicators to be used
would be determined by the nature of the microorganism in
question.
FIG. 1 is a plan view of an impregnated paper disc of my
invention.
FIG. 2 is a cross-sectional view of my disc along lines 2--2 of
FIG. 1 in a culture plate.
FIG. 3 is a schematic illustration of my method of identifying a
microorganism in an inoculated culture plate.
DESCRIPTION OF THE INVENTION
The biochemical identification of, for example, the genus
Salmonellae is accomplished by preparing a generally circular paper
disc, which disc has been impregnated to form six generally
circular zones, each zone impregnated as follows:
A phenol red and lactose
B phenol red and mannitol
C phenol red and adonitol
D phenol red and dulcitol
E phenol red and glucose
F phenol red and sorbitol
A bacteriological sample isolated containing the microorganism in
question is then streaked out onto the surface of a culture plate
containing trypticase soy agar. The surface of the culture plate is
streaked with the sample, using a bacteriological loop, so that the
entire surface is contacted with the sample. The impregnated paper
disc which is generally designed to fit within the culture plate is
then placed on top of the streaked sample. The culture plate is
then incubated for about 24 hours at a temperature of 37.degree.C.
The culture plate is then removed, and the paper disc then observed
for a color change about the peripheral edges of each of the zones.
If the culture sample contains the microorganism Salmonella, then
the substrates in zones B, D, E and F would be fermented after
incubation, and fermentation of these zones is detected by a color
change in the indicator about the pheripheral edges of the zone. In
the present case, a yellow zone indicating fermentation would be
seen around the edges of the zone containing the fermented
compounds, while no color change would be observed for zones A and
C.
FIG. 1 shows an impregnated paper disc 10 of my invention which
comprises an absorbent circular paper sheet 12 having a diameter
less than the culture plate or Petri disc in which it is to be
used. The paper sheet 12 contains six zones as set forth above,
with a circular line 18 about the zones, and each zone identified
by the alphabetical letter shown.
FIG. 2 shows the use of the paper disc 10 (along the lines 2--2 of
FIG. 1) in a circular culture plate 14 containing an inoculated
culture medium 16. The culture plate and the disc subsequently are
incubated and the color changes of each zone observed to identify
the microorganism.
FIG. 3 is a schematic illustration of the method of using my paper
disc 10 to identify a microorganism in a manner set forth
above.
In a similar manner, the biochemical identification of Escherichia
coli may be accomplished by employing a paper disc with zones as
follows:
A phenol red and lactose
B phenol red and glucose
C phenol red and mannitol
D phenol red and dulcitol
E phenol red and adonitol
F phenol red and inositol
If the sample is E coli, then after incubation, a color change
would be observed around the peripheral edges of the zones
containing the fermented glucose, lactose, dulcitol and mannitol,
while inositol and adonitol would not be fermented by E coli.
My method and apparatus has been described by impregnating a single
sheet of paper with the substrate identifying agent; however, it is
also recognized that each zone may also be represented by a
separate paper disc or absorbent disc placed or adhered to a
carrier sheet, rather than having a single absorbent sheet
containing impregnated zones. The absorbent sheet material should
be selected to have a capillary attraction so that there may be
contact between the sample on the culture plate and at least the
peripheral edges of each zone so that some diffusion between the
substrate and identification agent and the sample will occur during
the incubation period to provide a reaction by which the
microorganism can be identified. To provide good contact, the
impregnated paper disc should be placed down firmly onto the
surface of the culture medium to promote diffusion and
reaction.
* * * * *