U.S. patent number 3,912,596 [Application Number 05/412,479] was granted by the patent office on 1975-10-14 for multiple inoculating system.
This patent grant is currently assigned to International Foundation of Microbiology. Invention is credited to Riaz-ul Haque, Richard A. Murphy.
United States Patent |
3,912,596 |
Haque , et al. |
October 14, 1975 |
Multiple inoculating system
Abstract
A multiple inoculation system is provided for the transfer,
cultivation, maintenance and identification of microorganisms. The
system includes a multiple needle inoculating plate, a separate
plate including multiple wells corresponding to the needles for
holding inoculum and transferring it to the needles, and a culture
dish having a plurality of compartments interconnected with each
other in the central portion of the dish. The culture dish is
filled with a suitable solidifiable liquid culture medium by
pouring the medium onto the central or any portion of the dish so
that the medium readily flows into each of the compartments. After
the culture medium has solidified, the compartments remain
separated from one another so that after the medium is cultivated
the spreading of microorganisms between compartments is minimized
and mutual interference in test reactions is avoided.
Inventors: |
Haque; Riaz-ul (Glen Ellyn,
IL), Murphy; Richard A. (Oak Park, IL) |
Assignee: |
International Foundation of
Microbiology (Chicago, IL)
|
Family
ID: |
26926561 |
Appl.
No.: |
05/412,479 |
Filed: |
November 2, 1973 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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233023 |
Mar 9, 1972 |
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Current U.S.
Class: |
435/309.1;
435/30; 435/34 |
Current CPC
Class: |
C12M
33/06 (20130101); C12M 23/12 (20130101); C12M
23/10 (20130101); C12M 33/04 (20130101); C12M
23/34 (20130101); C12M 29/00 (20130101); C12M
23/38 (20130101) |
Current International
Class: |
C12M
1/16 (20060101); C12M 1/32 (20060101); C12M
1/26 (20060101); C12M 1/20 (20060101); C12B
001/02 () |
Field of
Search: |
;195/139,127 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Parker, Esq.; John L.
Parent Case Text
DESCRIPTION OF THE INVENTION
This is a continuation of our application Ser. No. 233,023 filed
Mar. 9, 1972 and now abandoned.
Claims
We claim as our invention:
1. A multiple inoculation system comprising, in combination,
inoculation means carrying a plurality of inoculating needles,
means for contacting said inoculating needles with inoculum, and a
culture dish for holding a medium suitable for cultivation by
inoculum carried by said needles, said culture dish having a
bottom, an upstanding outer wall surrounding its periphery, a
plurality of upstanding, inner divider walls extending from the
outer wall inwardly toward the center of the dish to thereby define
a plurality of dish compartments, and intercommunication openings
in the inner divider walls forming means between said compartments
whereby a liquid culture medium poured onto the dish readily flows
by gravity into and is retained by each of the compartments.
2. The multiple inoculation system defined in claim 1 in which the
means for contacting the needles with inoculum comprises a plate
carrying a plurality of wells for holding inoculum corresponding to
the needles, and the inoculation means is adapted to be
superimposed over the holding plate so that the inoculating needles
contact and thereby pick up inoculum carried by the holding
wells.
3. The multiple inoculation system defined in claim 1 in which the
inner divider walls terminate short of the center of the dish to
define compartment openings toward the center of the dish.
4. The multiple inoculation system defined in claim 3 in which the
bottom of the culture dish is of flat, circular shape and the inner
divider walls are straight and generally radially disposed with
respect to the circular bottom so that the divider walls define a
plurality of sector-like dish compartments.
5. The multiple inoculation system defined in claim 4 in which the
compartment openings into the center of the dish are narrow in
width relative to the average width of the compartments
themselves.
6. In a multiple inoculation system including an inoculation plate
carrying a plurality of inoculating needles and a separate plate
carrying a corresponding plurality of wells for holding inoculum,
with the inoculation plate being adapted to be superimposed over
the holding plate so that the inoculating needles contact and
thereby pick up the inoculum carried by the holding wells, and in
which a culture dish is provided for holding a medium suitable for
cultivation by inoculum carried by the needles, the improvement
comprising a culture dish having a flat bottom, an upstanding outer
wall surrounding its periphery, and a plurality of upstanding,
inner divider walls extending from the outer wall inwardly toward
the center of the dish but terminating short of said center to
thereby define a plurality of dish compartments each opening toward
the center of the dish, the compartments thereby being in
intercommunication with one another so that a liquid culture medium
poured onto the dish readily flows by gravity into and is retained
by each of the compartments.
7. The multiple inoculation system defined in claim 6 in which the
bottom of the culture dish is circular in shape and the inner
divider walls are straight and generally radially disposed with
respect to the circular bottom so that the divider walls define a
plurality of sector-like dish compartments.
Description
The present invention relates to an inoculating system for
identifying microorganisms, and particularly to such a system for
achieving simultaneous inoculation of multiple samples.
For many years, an inoculating loop, inoculating needle, test
tubes, and Petri plates were the only tools available to the
microbiologist involved in the identification of microorganisms. He
began his work by spreading (streaking) the sample, using the
inoculating loop, onto a solid agar culture medium contained in a
Petri dish. The dish was then incubated, and the isolated colonies
of organisms which generally appeared after 12-24 hours of
incubation were picked-up with the inoculating needle and
transferred to an agar slant (agar medium which had been allowed to
solidify in a tube in a slanted condition). The resulting slant
culture was regarded as the stock culture and was subjected to
further tests to determine the identification of the disease
producing or other microorganisms.
In recent years, various multiple inoculation techniques have been
used for allowing the simultaneous inoculation of several cultures
onto a single substrate, thereby permitting more rapid
identification of the microorganisms. Multiple inoculation
techniques have been used with cultures of fungi, yeasts, bacteria,
and various other organisms. The tests generally involve the
determination of the biochemical activities of the culture, such as
the ability to use certain sugars, hydrolyse proteins,
polysaccharides, fats, and nucleic acids and the ability to produce
certain intermediary or end products such as indole, acetylmethyl
carbinol, acid, or acids and gas. Multiple inoculation is much less
tedious and time consuming than the sequential inoculation of
individual cultures previously carried out by hand.
We previously described a multiple inoculating device in The
American Journal of Clinical Pathology, Vol. 47, No. 4, p. 554
(1967) in which a plate carrying multiple inoculating needles is
used with an agar culturing dish. In one form, the plate carrying
the needles is pressed onto the surface of the agar to create
slight depressions in the agar corresponding to the imprint of the
needles, and bacteria from isolated colonies is then inoculated
into each of the multiple depressions using the standard
inoculating needle or loop. After incubation of the agar substrate,
the multiple inoculator is sterilized, re-pressed into the agar,
and then pressed into ten or more replicate test plates.
In another form which we described, an additional plate is utilized
containing multiple wells corresponding to each of the needles
carried by the inoculating plate. Colonies of organisms are placed
in the wells, and organism transfer to the inoculating needles is
achieved by lowering the needle carrying plate over the wells until
the needles are in contact with and pick up the respective
organisms carried in the wells. Then, multiple inoculation is
achieved by contacting the plate carrying the needles with the agar
substrate. A similar arrangement is described in U.S. Pat. No.
2,956,931 to Goldberg.
Other descriptions of devices for multipoint inoculation of agar
plates are also found in the literature. But these techniques and
devices suffer the serious disadvantage that diffusion of organisms
often takes place through the agar substrate or other culture
medium, and sometimes organism colonies spread along the surface of
the agar. Such migration of different organisms into one another
may impair or completely nullify the results of the identification
test reactions.
As might be expected, the prior art has tried to overcome these
problems in various ways but with only limited success. For
example, one technique that has been utilized with multipoint
inoculators is the so-called "divided Petri dish" (Journal of
Applied Bacteriology, Vol. 30, p. 495 (1967) by Sneath et al.) in
which the culture media is placed in a multi-compartmented Petri
dish which looks much like an ice tray. Each compartment in the
tray is completely separated from the next, and corresponds to a
particular prong or needle on the multipoint inoculating plate.
While such compartmented tray devices manage to prevent overgrowth
and spreading of organisms as well as mutual interference in test
reactions, the step of filling each of the compartments with agar
or other culture media is found to be unduly time consuming. Each
individual compartment must be separately filled, usually one at a
time. It can take as long as 5 or 10 minutes to fill a tray having
20-30 compartments, a situation which significantly limits the
productivity of the modern day microbiology laboratory.
It is an object of the present invention to provide a multiple
inoculation system for the transfer, cultivation, maintenance and
identification of microorganisms which eliminates cumbersome and
tedious multiple manipulations required by prior practices. A
related object is to provide such a multiple inoculation system in
which a laboratory technician may carry out many microorganism
identification tests simultaneously in less time than heretofore
required. Still another related object is to provide such a
multiple inoculation system in which the culture media may be
quickly and easily prepared for simultaneous cultivation of
multiple test samples without the need for specially trained
laboratory personnel.
Another object of the invention is to provide a technique and
devices for multiple inoculation of culture media in which the
media may be speedily and simply prepared for cultivation of
multiple test samples and yet in which positive separation of the
individual test samples from one another is assured. Yet another
object is to provide such a multiple inoculation system in which
multiple test samples of culture media may be rapidly and
simultaneously cultivated with assurance that neither the
microorganisms contained in adjacent samples nor their reaction
products with the culture media will migrate or otherwise
intersperse into each other. An ancillary object is to provide such
a multiple inoculation system in which positive, non-interfering
test results may be achieved in minimum time.
Othe objects and advantages of the present invention will become
apparent upon reading the following detailed description and upon
reference to the drawings, in which:
FIG. 1 is a bottom plan view of an illustrative inoculation plate
which may be used in carrying out the invention, showing a typical
arrangement of inoculating needles on the plate.
FIG. 2 is a top plan view of an illustrative holding plate for the
inoculum, showing a typical arrangement of wells on the plate for
holding the inoculum.
FIG. 3 is a top plan view of an illustrative culture dish suitable
for use in carrying out the invention.
While the invention is described in connection with certain
preferred embodiments, it will be understood that we do not intend
to limit the invention to those embodiments. On the contrary, we
intend to cover all alternatives, modifications and equivalents as
may be included within the spirit and scope of the invention as
defined by the appended claims.
Turning now to the drawings, a three component system for the
multiple inoculation and cultivation of microorganisms is shown.
The system includes a plate or other device 10 for inoculation
(FIG. 1), a separate plate or similar device 11 for preparing and
holding inoculum to be used (FIG. 2), and a culture dish or other
device 12 for culturing the inoculum (FIG. 3).
In the form shown in the drawings, the inoculation means 10 takes
the form of a flat circular plate or disc carrying a plurality of
depending inoculating needles or pins 14 arranged in spaced apart
circular relationship around the plate. As illustrated, ten needles
14 are utilized, although the number of needles may be varied as
desired. The inoculation plate 10 may also, if desired, include one
or more guides (not shown) for aligning and positioning the device
for preparation of inoculum and for inoculation. Preferably the
plate 10 also carries a handle (not shown) located on the plate
side opposite the needles 14 to facilitate handling of the plate by
a laboratory technician.
The means 11 for preparing and holding the inoculum, and for
contacting the inoculating needles with inoculum, is shown as a
flat, circular plate or disc having a plurality of depressions or
wells 13 for holding the inoculum. In this case, there are 10 wells
spaced apart on the plate 11 in circular relationship corresponding
to the number and location of the inoculation needles 14 carried by
the plate 10. The inoculum holding plate 11 also may include one or
more guides (not shown) corresponding to the guides on the
inoculation plate for assuring proper alignment with respect
thereto.
As will be apparent, the sizes and materials of construction of the
plates 10 and 11 may be varied within wide ranges. We have found
33/4 inch diameter thin metal plates to be satisfactory.
In carrying out the invention, the culture dish 12 which is a part
of our multiple inoculation system is specially shaped to permit
rapid filling of the dish with a solidifiable liquid culture
medium, and yet to provide insulation of multiple inoculated
samples from one another once the culture medium has solidified, so
that migration of microorganisms between compartments in minimized
and mutual interference in test reactions is avoided.
To this end, the illustrative culture dish 12 is shown having a
flat, circular bottom 15, with an upstanding outer wall 16 secured
to the dish bottom and surrounding its periphery. For the purpose
of defining a plurality of dish compartments 18, in this case ten
compartments corresponding to the ten inoculating needles 14, a
plurality of upstanding, inner divider walls 19 are secured to the
dish bottom 15 and/or to the outer wall 16, and extend from the
outer wall inwardly toward the center of the dish. The number of
dish compartments 18 used in carrying out the invention may vary
from only two to as many as 20 or more. The compartments in a
single dish usually will be, but need not be, uniform in size and
shape.
Attention is drawn to the fact that intercommunication means is
provided between the dish compartments 18 to permit ready flow of
liquid culture medium between compartments. In the drawings, the
inner divider walls 19 terminate short of the center of the dish,
so that the resulting dish compartments 18 each open toward the
center of the dish. As shown, the inner walls 19 are straight and
generally radially disposed with respect to the circular dish
bottom 15, with the result that the divider walls define a
plurality of sector-like (i.e. akin to the sector of an annulus)
dish compartments which are in intercommunication with one another
in the central portion of the dish. The inner ends 19a of the walls
thus define therebetween a plurality of compartment
intercommunication openings or interruptions 20 which are
relatively narrow in width compared with the average or maximum
width of the pie-shaped compartments themselves.
Such limited intercommunication between compartments, i.e. via the
relatively small openings 20, is preferred because the openings are
sufficient in size to permit solidifiable liquid culture medium
poured onto the dish to readily and quickly flow by gravity into,
and be retained by, each of the compartments 18; yet once the
poured culture medium has solidified, the inner divider walls 19
function to enclose virtually the entirety of the compartments and
to thereby isolate them so that adjacent tests will not interfere
with one another. The intercommunication means may, of course, take
various other forms. For example, the inner walls of the dish could
extend all the way to the dish center but contain one or more
perforations or other type interruptions to permit flow of liquid
culture medium therethrough.
The size of the culture dish 12 may be selected as desired, but it
will be advantageous to select a size corresponding to that of the
inoculation plate 10. The dish 12 may be formed of metal, plastic,
or other suitable construction materials. It is preferred to also
provide a lid (not shown) sized to fit over and cover the dish when
being incubated.
It will be understood, of course, that the shape and configuration
of the culture dish 12, and of the compartments 18 therein, may
vary as desired. While a circular dish shape has been depicted in
the drawings, other shapes may also be used in practicing the
invention. For example, the culture dish may have a square,
rectangular, or other polygonal shape. In such event, the inner
divider walls will extend from the outer, peripheral wall inwardly
toward a center line of the rectangle or square. This center line
may then be considered to be the center of the dish. The dish also
need not have a flat bottom, e.g. a cone shaped bottom may be
advantageous in some applications.
In preparing our multiple inoculation system for use, the
inoculation needles 14 are sterilized by momentarily holding them
in an open flame. Melted agar or other suitable culture medium is
poured onto the center or other portion of the culture dish 12 and,
as explained above, since the dish compartments 18 are connected at
the center of the dish, the medium flows and fills all of the
compartments simultaneously to whatever height is desired. The
inoculum is prepared by streaking a sample on an appropriate agar
plate with an inoculating loop, and incubating the medium. The
resulting isolated colonies of organisms are transferred to the
separate wells 13 of the holding plate 11, where they are suspended
in a sterile broth, saline, water, or other diluent.
When the inoculation needles 14 have cooled, the inoculation plate
10 is superimposed over the holding plate 11 and the needles are
inserted into the respective wells 13 containing the inoculum. The
needles 14 so charged with the inoculum are then aligned with the
respective compartments 18 of the culturing dish 12 and touched to
or pressed into the culture medium. Similarly, but without
necessarily recharging with the inoculum, the inoculation plate may
be used to inoculate as many as 10 to 15 different media contained
in separate culturing devices. Finally, the contents of the culture
dish 12 are incubated and the growth and biochemical reactions of
the organisms on or in the various media are then interpreted to
identify the organisms.
One of the advantageous features of the invention is that the
inoculation plate 10, inoculum holding plate 11 and culture dish 12
may be sequentially used together as a system, as described above
for the identification of an unknown bacterial culture, or they may
be used individually for the transferring, cultivation,
maintenance, and identification of microorganisms. Utilization of
the invention will thus significantly speed up and improve
microbiological laboratory procedures. The laboratory technician or
microbiologist may now transfer as many as ten or more cultures at
one time, utilize single isolated colonies as the inoculum, and
grow ten or more cultures simultaneously yet in isolation from each
other in a single culture dish.
By use of the specially shaped culture dish 12 of the invention,
the laboratory technician finds it possible to fill the dish with
agar jelly or other liquid culture medium, obtaining an even
distribution of jelly in all dish compartments, in a matter of only
a few seconds time (e.g. 10 seconds) as compared with 5 or 10
minutes required to fill the individual cups of the "divided Petri
dish" used previously. Use of the invention avoids the tedium of
prior practices, and minimizes the problems of contamination
inherent in time consuming filling of inidvidual cups thereby
obviating the need to use specially trained personnel.
Moreover, we find that after inoculation of the culture medium
contained in the dish compartments 18 has taken place, the
compartments are so well isolated from one another that there is no
migration of microorganisms or reaction products from one
compartment to another, or at least no such migration for a period
long enough (i.e. severals days) for the reactions and
identifications to have been completed.
Other advantages also inhere in the invention. The three main
components 10, 11 and 12 used in carrying out the invention may be
wholly or partly disposable or reusable and used aseptically or
otherwise. The components are suitable for either manual or
mechanically automatic operation.
* * * * *