WO1987006955A1

WO1987006955A1 - Plate screens

Info

Publication number: WO1987006955A1
Application number: PCT/US1987/001128
Authority: WO
Inventors: James L. Hartley; James H. Campbell
Original assignee: Life Technologies, Inc.
Priority date: 1986-05-14
Filing date: 1987-05-14
Publication date: 1987-11-19
Also published as: EP0268651A1; EP0268651A4; AU7488787A

Abstract

A device (10) for use with culture media for uniformly spreading a liquid suspension thereon comprising a flat, sterile open fabric adapted to lie on the surface (12) of the medium (14), wherein the fabric produces a capillary effect on the liquid to distribute the liquid uniformly across the medium surface (12).

Description

^'

-1-

PLATE SCREENS

FIELD OF THE INVENTION

The present invention relates to organism culturing methods and articles, and more particularly to a device for spreading a liquid suspension of microorganisms on the surface of a culture medium. The invention is particularly applicable to enumeration of microorganisms in water, foods and the like, and will be described with particular reference thereto although it will be appreciated that the invention has other applications.

BACKGROUND OF THE INVENTION

Enumeration of microorganisms in water, foods, pharmaceuticals, etc., requires the application of samples to a culture medium, such as a petri plate containing mixtures of nutrients and agar, wherein individual organisms from a sample multiply on the surface of the agar to form visible colonies which are then counted. To ascertain the number of organisms in a test sample it is conventionally known to prepare a series of dilutions of the original sample and then apply 0.1 ml of each dilution of the surface of a

** culture medium, i.e. the agar. A spreading device. typically a sterilized bent glass rod, is used to spread the 0.1 ml of bacterial suspension over the surface of the agar. It is important that the sample be spread as evenly as possible over the agar, in that the more evenly the bacterial suspension is spread on the agar surface, the more information can be derived from each plate. A puddle of cells on one part of the plate results in clusters of colonies which are hard to count and likely to overlap with each other. In this respect, the number of colonies per standard (10 cm) petri plate should be above 30 (for statistical significance) and below 300 (above which overlapping colonies become more likely) . Consequently, the most accurate and reliable information will be obtained when the liquid samples are uniformly spread across the surface of the agar.

Spreading the liquid samples with a glass rod cannot ensure a uniform distribution of the sample on the agar surface. In addition, use of an instrument such as a glass rod requires sterilization before such use. Generally, sterilization of a glass rod consists of dipping the rod into alcohol, igniting the alcohol, and then allowing the rod to cool. As will be appreciated, such sterilization is a time consuming step in the overall procedure.

The present invention solves these and other problems, and provides a device and method for spreading a liquid suspension on the surface of a culture medium, which method and device are simple and easy to use and provide an even distribution of the suspension along the surface of the medium. SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a device for uniformly spreading a liquid suspension of bacteria along the surface of a sterile culture medium. Generally, the device is comprised of a sterile, flat, non-absorbent fabric having a shape commensurate with the surface profile of the culture medium to lie in registry therewith. The fabric is comprised of relatively fine strands disposed relative to the surface of the medium such that a cooperative capillary attraction between the surface of the culture medium and the strands on the liquid suspension is produced and effects an even distribution of the suspension along the surface of the culture medium.

More specifically the invention is generally for use in circular petri plates containing mixtures of nutrients and agars. In this respect, the invention is preferably comprised of a circular piece of nylon netting which is sterile and is placed on the surface of the agar plate on which the bacteria are to be spread. An appropriate volume of liquid containing the bacteria is applied to the netting, which serves to spread the liquid over the surface of the plate by capillary action. After the liquid soaks into the plate, the netting is removed, and the bacteria are allowed to grow into colonies in the usual manner.

Importantly, the netting uniformly spreads the liquid suspension thereby forming an even layer of liquid. This provides a more evenly spaced or spread colony formation which allows more reliable counting of

^* colonies at high densities. Likewise, the netting eliminates the need for manually spreading of the suspension on the surface of the culture medium thereby reducing labor costs.

In accordance with another aspect of the present invention, the fabric is coated with a substance soluble in the liquid suspension, which substance is to be applied to the surface of the culture medium simultaneously with the spreading of the liquid suspen¬ sion.

An object of the present invention is to enumerate bacteria from a liquid in an accurate and efficient manner»

Another object of the present invention is to provide a device which will spread a liquid suspension aver the surface of a culture medium forming an even layer thereon.

A still further object of the present invention is to provide a device as described above, wherein manual spreading of the liquid suspension is not required.

These and other objects and advantages of the invention will become apparent from the following description on an embodiment thereof taken together with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may take physical form in certain parts and arrangement of parts, a preferred embodiment of which will be described in detail in this specifica¬ tion and illustrated in the accompanying drawings wherein:

FIGURE 1 is a perspective view of a device in accordance with the present invention in exploded relationship to a culture medium; FIGURE 2 is an enlarged partial view of the device depicted in FIGURE 1 showing details thereof; and

FIGURE 3 is a greatly enlarged sectional view of the device shown in FIGURE 1 in operative relationship to the surface of the culture medium.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention is particularly applicable for enumerating microorganisms in a sample by spreading a liquid suspension of such sample on the surface of a culture medium and will be described with respect thereto, although it will be appreciated that the present invention has other broader uses and applica¬ tions. As set forth previously, enumeration of micro¬ organisms in water, food, etc. requires application of samples to a culture medium. Important to the results of such procedure is that the suspension be spread as evenly as possible on the agar surface to ensure even separation of colonies thereon. The present invention provides a device and method of spreading liquids over the surface of the medium in an even layer without the need for the traditional spreading method.

Referring now to the drawings wherein the showings are for the purpose of illustrating a preferred embodi¬ ment and not for the purpose of limiting same, FIGURE 1 shows a flat, screen-like element 10 which is provided to lie on a surface 12 of a culture medium 14. Element 10 is an open fabric or screen or netting comprised of relatively fine strands 16 with openings 18 there¬ between, as seen in FIGURE 2. When placed on culture medium 14, element 10 provides surface area opposed to surface 12 of medium 14. When liquid is placed on the medium surface 12, liquid contacts the surface area of strands 16 of element 10 to produce a capillary effect which spreads the liquid along strands 16 of element 10 and along surface 12 of medium 14.

To ensure complete spreading of the liquid across the entire surface of the medium, the screen-like element is preferably shaped commensurate with the profile of the culture medium such that the liquid is spread to the edge of the screen-like element. It will of course be appreciated that the shape of the screen¬ like element may assume any configuration which is positionable on the culture medium.

More specifically, the present invention is particulary applicable for use with culture mediums comprised of circular petri plates 20 of nutrients and agar. In the preferred embodiment, screen-like element 10 is comprised of tulle which is a commercially available nylon netting with about 11 squares per centimeter which is a mesh width of approximately. 1 millimeter. Circles of the tulle, cut or otherwise formed to match the surface profile of the agar in the petri plate, are sterilized by autoclaving. The tulle circles are then applied in a sterile fashion to the surface of agar plates. Preferably, the agar plates are of sufficiently low moisture content such that the volume of liquid to be applied (for example, 1 ml) will soak into the agar within a convenient period of time (for example, 30 minutes) . Prepared agar plates sold by GIBCO Laboratories, Life Technologies, Inc. have been found to be particularly suitable for use with the present invention. The liquid suspension of bacteria is applied to the agar surface through the tulle, and the suspension spreads over the agar of the tulle, forming an even layer of liquid. When the liquid has soaked into the agar, the dry tulle circle is removed.

Materials other than the nylon netting (tulle) may be used to perform the spreading functions. For example, fiberglass screening has been found to be suitable. Importantly, these materials must be very flat when applied to the agar surface, or they must become flat when the liquid is applied through the netting; otherwise uneven spreading of the liquid will result. As will be appreciated, the netting must allow spreading of the liquid. In this respect, complete contact of the netting with the agar surface does not spread the liquid satisfactorily, in that complete surface contact does not provide passages between the tulle, and the agar through which liquid may pass. Accordingly, the tulle netting is not pressed onto the agar surface, but rather rests thereon. The light weight of the tulle, and its construction, provide openings or gaps 22, best seen in FIGURE 3, which gaps permit passage of the liquid suspension therethrough.

In accordance with another aspect of the invention, the tulle or netting is coated with a substance (e.g., an antibiotic) which is soluble in the liquid suspen¬ sion. In this respect, the substance can be applied to the culture medium surface simultaneously with the spreading of the liquid.

The utility of the tulle screen method for enumerating bacteria was demonstrated as follows. A sample of Escherichia coli bacteria estimated to contain about 10⁸ organisms per ml was prepared. A series of dilutions was prepared to give an estimated 30 - 10,000 bacteria per ml. Aliquots of these dilutions were applied in triplicate to agar plates by the traditional spreading method (0.1 ml aliquots) or using screens (1.0 ml aliquots) . The bacteria were allowed to grow into colonies and the colonies were counted. The concentra¬ tion of bacteria in the original preparation was calculated for each aliquot and the results were as follows:

Number of

Colonies (Avgerage) Calculated .

Estimated Original Cone.

(bacteria/ml) Screens Spread

Concentration (1 ml) (0.1 ml) Screens Spread

10000/ml ND 221 _.— 2.3X10⁷

3000/ml ND 69 _-_._ 2.3X10⁷

1000/ml 237 23 2.4X10⁷ 2.3X10⁷

300/ml 72 5 2.4X10⁷ 2.3X10⁷

100/ml 24 ND 2.4X10⁷

30/ml 8 ND 2.7X10⁷

*ND = not determined

The data show that the results obtained using the screens are reproducible and are essentially the same as those obtained using the traditional spreading methods. In addition, the labor required for the tulle circle method was less than or equal to that of the traditional manual spreading method. Most important however is that the resulting colonies were spread very evenly over the agar surface, which enabled more reliable and accurate counting of colonies at higher densities. The uniform colony distribution also allowed calculation of the approximate colony total by counting only a sector of the total plate, such as one quarter, and multiplying by the appropriate factor (four, in this case) . This simplification is not possible with traditional spread¬ ing methods. Modifications and alterations of the present invention will occur to others upon their reading and understanding of this specification. For example, the netting may be comprised of a material which, after a period of time sufficient to evenly spread the liquid, will dissolve, thereby eliminating the necessity of removing the netting. Also, incorporation of a tab or other protrusion from the edge of the screen will facilitate handling of the screens. It is intended that all such modifications and alterations be included insofar as they come within the scope of the claims or the equivalence thereof.

Claims

Having thus described the invention, it is claimed:

1. A device for uniformly spreading liquid suspensions of bacteria along the surface of a sterile culture medium operative to detect the presence of said bacteria, comprising: a flat, sterile fabric shaped to lie on the surface of said culture medium, said fabric comprised of relatively fine strands disposed relative to said surface, said fabric cooperative with said surface to produce a capillary attraction on said liquid to effect an even distribution of said liquid along the surface of said medium.

2. A device as defined in claim 1 wherein said fabric is nylon having a mesh width of approximately 1 mm.

3. A device as defined in claim 1 wherein said fabric is coated with a substance soluble in said liquid to be applied to the surface of said culture simul¬ taneously with the spreading of said liquid.

4. A device as defined in claim 3 wherein said substance is an antibiotic.

5. A device as defined in claim 1 wherein said medium is a standard circular agar plate.

6. A device as defined in claim 1 wherein said fabric is soluble.

7. A construction for enumerating microorganisms found in a liquid suspension, said construction compris¬ ing: a sterile culture medium favorable for the growth of a predetermined microorganism, said medium having a generally planar surface and a predetermined profile, a removable, non-absorbent, wafer-like element shaped commensurate with said profile disposed on said surface in engagement therewith, said element coopera¬ tive with said surface to produce a capillary attraction on said liquid to effect an event distribution of said liquid along the surface of said medium.

8. A construction as defined in claim 7 wherein said element is comprised of a sterile fabric including relatively fine strands of nylon having a mesh width of approximately 1 mm.

9. A construction as defined in claim 7 wherein said element is coated with a substance soluble in said liquid to be applied to the surface of said culture simultaneously with the spreading of said liquid.

10. A construction as defined in claim 7 wherein said element is soluble in said liquid after a predeter¬ mined time.

11. In a method for enumerating microorganisms from a sample including the steps of: preparing dilutions of said sample; placing a predetermined amount of each dilution on the surface of a culture medium having a predetermined surface profile; incubating said culture medium, and, counting colonies formed on said surface of said culture medium, the improvement comprising the steps of:

(a) placing a flat, sterile netting of non-absorbent material on the surface of said culture medium prior to adding said dilutions, said netting shaped commensurate with said profile to lie in registry therewith;

(b) applying said dilution to said netting to spread the liquid evenly over the surface of said medium by cooperative capillary attraction of said dilution to said netting and said surface;

(c) allowing said dilution to soak into said medium; and,

(d) removing said netting and allowing said microorganisms to grow into colonies.