CA2237639C - Method for quantification of biological material in a sample - Google Patents

Abstract

Method for detection of a biological material in a sample. The method includes the steps of liquefying the sample (if necessary) and pouring the liquefied sample into the incubation plate. The incubation plate has a generally flat horizontal surface and the surface is divided into a plurality of at least 20 recessed wells. Each well is adapted to hold an aliquot of liquid and is sized and shaped, and formed of a suitable material, to hold the aliquot within the well by surface tension. Any excess liquid from the liquefied sample is poured from the surface of the plate. The method then involves incubating that incubation plate until the presence or absence of the biological material is determined.

Description

CA 02237639 1998-0~-12 W O 97/18455 PCTrUS96/18119 DESCRIPTION
r Method For Ouanti~ication of Bioloqical Material in a Sample Related A~lications This application is a continuation-in-part of Croteau et al., U.S. Serial No. 08/606,229 filed Feb~uary 23, 1996, which is a continuation-in-part of Croteau et al., U.S. Serial No. 08/557,529, ~iled November 13, 1995, both entitled "Method ~or Quanti~ication o~ Biological Material in a Sample" hereby incorporated herein by re~erence, including drawings.

Field o~ the Invention This invention relates to a method ~or quantification o~ biological material in a sample.

Backqround o~ the Invention Many industries need to detect and quanti~y the con-centration and level o~ biological material in a sample.
For example, the determination o~ bacterial concentration in food and water is an essential part o~ ~ood and water quality testing. EPA regulations require that no Coli-form such as Escherichia coli can be present in potable water. The "presence/absence" ~ormat o~ a testing medium, such as Colilert~ chemical mixture (IDEXX Laboratories, ME) which is used as a testing medium for Escherichia coli and all coli~orm bacteria, is very useful in making this determination. Colilert~ chemical mixture is based on the De~ined Substrate Technology described in Edberg, "Method and Medium ~or use in Detecting Target Microbes In Si tu in A Specimen Sample o~ A Possibly Contaminated Material,"
U.S. Patent Nos. 4,925,789 and 5,492,933. See also, Townsend et al., U.S. Serial No. 08/484,593 ~iled June 7, 1995 entitled, "Method and Composition ~or Detecting Bacterial Cont~m;nation in Food Productsn, hereby CA 02237639 1998-0~-12 incorporated by reference herein, describes a medium ~or the detection of bacteria in food and water samples.
However, there are areas where the ~uantification, not just the detection, of bacterial concentration is impor-tant. Examples of such areas include waste water, inco-ming water in water purification systems, surface water, and food testing. For example, numerous restaurant chains will only accept raw ground beef or poultry that contains less than a certain concentration of bacterial contamina-tion. Therefore, food processing plants must carry outthe necessary microbiological tests to determine the bacterial concentration of these food items before they can be released to customers.
The classical methods of quantification of biological material are the standard plate count method or the multi-ple tube fermentation (MTF) method. A quantity of sample being tested for microbial contamination is first dis-pensed in a Petri-dish. Then 15 ml of the appropriate media is poured over the sample. The Petri-dish is then swirled to mix the sample in the medium and the Petri-dish is left to solidify at room temperature for approximately 20 minutes. The medium is then incubated at a specific temperature for a specific time, and any resulting colonies are counted.
The multiple tube fermentation method is described in Recles et al., "Most Probable Number Techniques" published in "Compendium of Methods for the Microbiological ~m; ~ -tion of Foods", 3rd ed. 1992, at pages 105-199, and in Greenberg et al., "Standard Methods For the Ex~m;n~tion of Water and Wastewater" 8th ed. 1992). In this method, a volume of sample is dispensed into several tubes repre-senting this dilution range. The tubes are then incubated at the appropriate temperature so that the bacteria in each tube are allowed to grow. After incubation at a specific temperature for a specific time, the number of positive tubes is counted. The most probable number can CA 02237639 1998-0~-12 W O 97/18455 PCT~US96/18119 be determined from the formula described in Recles et al., - supra.
Water testing is mostly done by membrane filtration, where a certain volume of water is passed through the membrane and the membrane is incubated in a medium for a certain period of time. After appropriate incubation, the colonies are counted.

Summarv of the Invention The present invention provides a simple method for more accurate quantification of the number of micro-organism in a sample, or for quantification of any other type of discrete particulate biological material within a sample. Such biological materials include fungi or other living organisms, as well as aggregates of proteins, such as enzymes, or even co-factors, using reaction mixtures well known to those in the art. The invention generally makes use of a novel article which is designed to hold a liquid sample in which chemical and/or microbiological reactants are provided. For example, such chemical reactants may be a specific growth medium for bacteria.
The device used is generally in the form of an incubation plate having a multitude of wells able to hold separate aliquots of liquid. Generally, the device i8 designed to hold between 5 and 100 ml of liquid in total, and the wells are designed to form separate incubation chambers for each aliquot of sample. The wells can ~e of same size or of different size and shape to increase counting range and/or simulate dilution effects. See, Naqui et al., U.S.
Serial No. 08/201,110, filed February 23, 1994, entitled "Apparatus and Method for Quantification of Biological Material in a Liquid Sample", incorporated by reference herein.
Thus, in a first aspect the invention features a method for detection of a biological material in a sample.
The method includes the steps of liquefying the sample (if necessary) and pouring the liquified sample and reagent CA 02237639 1998-0~-12 W O 97/18455 PCTrJS96/18119 into the incubation plate. The incubation plate has a generally flat horizontal sur~ace and the surface is divided into a plurality of at least 20 recessed wells.
Each well is adapted to hold an aliquot of liquid and is sized and shaped, and formed of a suitable material, to hold the aliquot within the well by surface tension. Any excess liquid from the liqui~ied sample is poured ~rom the surface of the plate due to the hydrophobicity of the material used to form the plate. The method then involves incubating that incubation plate until the presence or absence of the biological material is determined. In a preferred embodiments the wells are cham~ered to allow liquid, that is above the horizontal plane, to be poured off easily (see Fig. 3).
As noted above, the biological material that can be detected is any material that forms a discrete particle, such as a microorganism, which may be quantified by determining the presence or absence o~ such a biolo~ical material within each well of the incubation plate. The sample may be any biological sample or en~ironmental sample such as waste water, food, a sur~ace swab, or swabs from other surfaces, such as a throat, or other samples well known to those in the art. This sample may be a liquid sample, or may be dissolved in a liquid to form the liquified sample. Thus, the term "liquefying" in the above paragraph re~ers to providing the sample in a liquid that once combined with a microbiological reagent can be rapidly aliquoted within the incubation plate. The liquidi~ied sample may remain as a liquid or may be solidified in the wells.
The incubation plate may be ~ormed of any desired material, but in particular it is desirable that a plastic be used which allows separate aliquots o~ the liqui~ied sample to be held by surface tension within each well without cross contamination of the wells. Preferably, the material is hydrophobic. The sur~ace can be untreated or CA 02237639 1998-0~-12 W O 97/1845~ PCT~US96/18119 treated chemically or physically to enhance retention of liquid within the wells.
The shape of the incubation plate is not relevant, and in preferred embodiments is a generally circular shape (such as that of a Petri-dish). Indeed, the incubation plate can be used to take the place of a Petri-dish.
Specifically, the method of this invention can be used to replace those existing tests that are generally run on Petri dishes to score the number of bacterial colonies.
lo Since discrete aliquots of the sample are provided in the plate, one of ordinary skill in the art need only score the number o~ positive wells in the plate to define the ~uantity of biological material within the original sample, as with the MPN test discussed above.
The generally flat horizontal surface is designed to allow the li~uid to be aliquoted readily between the wells and then excess liquid to be poured from the plate. In a preferred embodiment, a lip or pouring spout is provided for the plate. Those in the art will recognize that the depth and shape of the wells, as well as the material used to make the wells and the plate, are chosen such that surface tension can be used to hold the aliquots within each well dependent on the type of the liquid used in the liquified sample.
In other preferred embodiments, the surface defines at least 40, 60, 80 or even 200 or more recessed wells; the plate is formed of any formable plastic; a lid is also provided to prevent contamination of liquid within the wells; and the plate is provided in a sterile form so that no positive aliquots are noted unless at least one biological material particle is present in the sample.
In yet other preferred embodiments, the incubation plate is clear or colored, for example, white or yellow (to enhance the appearance of color (e.g., blue)) within the incubation plate) and the well has a diameter of about 0.15 inches, and the plate a diameter of about 3 or 5 inches.

CA 02237639 1998-0~-12 W O 97/18455 PCTnUS96/18119 In still other preferred embodiments, the incubation plate has a "pour-of~ pocket" adjacent to the sur~ace of the plate. The pocket has sufficient capacity to contain the excess liquid to be removed from the plate surface.
As an aid in preventing the excess fluid from spilling back onto the plate surface, it is preferable that the pocket contain an absorbent material, e.g., a gauze-like material. In a particular embodiment, the plate has both a pour-of~ pocket and a "landing pad". Th~e "landing pad"
i9 described below.
In a related aspect, the invention features a sterile incubation plate having a generally flat horizontal sur-face. The surface de~ines a plurality of at least 20 recessed wells (in preferred embodiments, at least 40, 60, 90 or even 200 recessed wells are provided) and each well is adapted as described above to hold aliquots of liquid by surface tension.
In preferred embodiments, the invention features the sterile incubation plate much as described above but having incorporated therein a "landing pad", which is a generally central area of the plate lacking wells, which can receive the sample prior to that sample being diluted in, for example, an incubation medium. Thus, a volume of 0.01 to 5 ml of sample liquid may be applied in the "landing pad" area (depending on its size and shape) and then that liquid dispensed into each well by applying the diluent and growth supporting medium (e.g., the ColilertTM
chemical mixtures noted above) and that liquid will simul-taneously dilute the sample and allow dispersion of the sample throughout the wells.
In addition, a pour spout can be provided within the incubation plate to allow pouring of~ excess liquid within the plate. Such a pour spout can be matched with a suit-able lid having a slit which allows liquid in the incuba-tion plate to be poured from the incubation plate onlywhen the slit is lined up with the pour spout, as des-cribed below.

CA 02237639 1998-0~-12 As indicated for the method above, the incubation - plate may also have a "pour-off pocket" adjacent to the surface of the plate. The pocket has sufficient capacity to contain the excess liquid to be removed from the plate surface, and preferably the pocket contains an absorbent material, e.g., a gauze-like material. In a particular embodiment, the plate has both a "pour-off pocket" and a "landing pad".
Applicant provides an extremely useful method which allows unskilled personnel to rapidly determine the quantity of biological material within a sample. Since the sample is readily liquified by people without siyni-ficant training in microbiology, and the materials for any specific tests can be provided by the manufacturer, such people can readily per~orm the tests with accuracy. The incubation plate is generally provided in the sterile form so that no }nappropriate detection of biological material can occur.
While it is known to provide plastic containers which can hold liquid within a plurality of recesses, applicant believes that this device provides a new automatic aliquo-ting method. This is an improvement over the existing products used to detect and quantify microorganisms because the liquid migrates to the individual wells without individual dispensing.
The present device can replace the use of a Petri-dish and can be used particularly in ~ood analysis and in testing of clinical samples. The separation of the wells of the present device prevents crosstalk or contamination between each aliquot. Because of this, many of the tests can be performed by observing fluorescence (which is not readily performed in an agar-containing Petri-dish). The device is particularly useful when there is a large quan-tity of microorganisms present in a sample, such as more than one organism per one ml or per ten ml.

CA 02237639 1998-0~-12 W O 97/18~5 PCTAUS96/18119 Other ~eatures and advantages o~ the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Descri~tion of the Prefer~ed Embodiment The drawing will first briefly be described.

Drawinqs Figs. 1 and 2 are diagrammatic representations of a formed plastic incubation plate.
Fig. 3 shows a cross section of a well, with or without a chamfer.
Fig. 4 is a diagrammatic representation of a formed plastic incubation plate having a pour spout and corresponding slit as well as a "landing padn.
Fig. 5 is a diagrammatic representation of a formed plastic incubation plate having a "landing pad" and a "pour-off pocket".

Structure Referring to Figs. 1 and 2 there is shown an incuba-tion plate 10 having a plurality of wells 12 each having a diameter of about 0.16 inches. The incubation plate 10 has a diameter of about 5 lnches. The incubation plate is made of formed plastic. Wells 12 are spaced apart suffi-ciently to prevent crosstalk between the wells. These wells may have a chamfer (Fig. 3) if desired to prevent liquid remaining at the upper edge of the well. Those in the art will recognize that incubation plate 10 can be readily formed by standard procedure and manufactured in the general shape of a Petri-dish, with or without a lip or pouring spout, and with or without a lid 14. This lid is provided with a dimple 16 to prevent contact of the lid with plate 10.
Referring to Fig. 4, there is shown in incubation plate 10 having a plurality of wells much as described above. The incubation plate also includes a "landing pad~

CA 02237639 1998-0~-12 W O 97/18455 PCTrUS96/18119 22 of size about one and one-half inch diameter which is - simply an area abLe to hold a defined volume of liquid.
Within the incubation plate is also provided a pour spout 24 which allows excess liquid to be removed from the incubation plate. Also provided is a corresponding lid 14 having a slit which can be matched with the pour spout to allow liquid to be removed from the incubation plate.
When the slit is not aligned with the pour spout, evapora-tion of liquid within the plate is decreased by reducing lo air flow over the liquid in the wells. A dimple 16 may also be provided in the lid to prevent the lid surface contacting the wells and thus preventing cross contamina-tion between the wells.
Referring to Fig. 5, there is shown an incubation plate lO having a plurality of wells much as described above, which also includes a "landing pad" 22 o~ size about one inch diameter. Within the incubation plate is also provided a "pour-off pocket" 26 adjacent to the sur-face of the plate which allows excess liquid to be removed from the incubation plate. As shown in the cross-sectional view, the "pour-off pocket" is formed by an elevated barrier 28 between the pocket and the plate surface. The barrier has a lower barrier section 30, which serves as a channel through which the excess fluid from the plate surface may be poured into the pocket.
Typically, the pocket will contain an absorbent material which will retain the fluid within the pocket, preventing back spill onto the plate surface.

Use In use, a reagent powder suitable for detection of a biological material can be rehydrated with an appropriate amount of sterile liquid and then inoculated with a known volume of a test sample. For example, 20 ml of sterile water can be inoculated with between lo and 1,000 micro-liter of sample. The inoculated reagent can then be added to incubation plate 10 and that liquid swirled within CA 02237639 1998-0~-12 W O 97/18455 PCTrUS96/18119 incubation plate 10 to distribute the inoculated liquid reagent to each of wells 12. Incubation plate 10 is then held at an angle of approximately 90 degrees to allow excess inoculated liquid reagent to be removed from the plate. A lid may then be placed on the incubation plate and that plate held in an incubator for the appropriate length of time, for example 18-48 hours. After that length of time, the presence or absence of a positive result can be scored in each well 12 of the plate. In addition, for plates having a "pour-off pocket", due to the larger volume of fluid contained in~ the pocket, a positive result in the pocket can serve as an early indication o~ high bacterial counts.

~xample 1: ~se of Incubation Plate For Bulk Testina For total plate count a plate as described above is used for the detection and quantification of the total bacterial concentration o~ food. It is based on a Multi-ple Enzyme Technology (Townsend and Chen, Method and Composition for Detecting Bacterial Contamination in Food Products, U.S. Serial No. 08/484,593 hereby incorporated by reference herein) which correlates enzyme activity to the presence of viable bacteria in ~ood. It utilizes multiple enzyme substrates that produce a blue ~luorescent color when metabolized by bacteria. When the liquid reagent is inoculated with a prepared food sample and dispensed into a plate as described herein the total viable bacterial concentration of that food product can be determined after 24 hours of incubation. The actual medium used herein is not critical to the invention, but is provided only for illustrative purposes.

Storaqe and Dis~osal Store bulk powder and unused Simplates at room temper-ature (4 to 25~C) away from the light. After use, the Simplate device will contain viable bacteria which must be handled and discarded appropriately. Once the powder is CA 02237639 1998-0~-12 W O 97/18455 PCT~US96/18119 rehydrated it is stable for up to 24 hours when stored at - 4 to 25~C.

Test Procedure 1. Pour an appropriate amount of bulk powder to a container of sterile deionized water. One vial contains enough powder for 10 tests. Each test has a ~inal volume o~ 10ml. For example: add 1 vial of powder to 100ml of sterile water to make enough media for 10 tests.

2. Place test sample on the center "landing pad" 22 of the plate 10 shown in Fig. 4. At the completion of this procedure half of the test sample will be poured off and discarded, there~ore, the size o~ the inoculum must take this into account. For example, if you wish to measure the bacterial concentration o~ 0.lml of test sample then you must place 0.2ml of test sample on the "landing pad". Place no more than 2ml on the center "landing pad".

3. Remove the lid from the plate and dispense 10ml of TPC media in the plate making sure to direct the liquid over the test sample on the center "landing pad". If the test sample is greater than 0.lml add enough TPC to achieve a final volume of 10ml in the plate. Note, if the liquid is not dispensed on the "landing pad" it may splatter.

4. Place the lid back on the plate. Note, to ensure that the liquid remains in the Simplate make sure that the slit on the lid is not lined up with the pour spout.

5. Distribute the liquid into the wells by swirling the plate as you would a standard pour plate.

6. Line up the slit on the lid with the pour spout and carefully pour off the excess liquid that did not end up in the wells. Holding the plate at an angle of approx-imately 90- from the work bench ensures proper pour off of excess liquid. Make sure that all liquid "cross bridges"
between wells are removed by gently tapping the plate.
Dispose of excess liquid appropriately.

CA 02237639 1998-0~-12 W O 97/18455 PCT~US96/18119 7. Slide the lid away from the pour spout to avoid drying the liquid in the wells during incubation and to avoid contamination from outside through the opening.

8. Place the plate in an incubator for 24 hours.
Plates can be inverted if desired. Incubation temperatures greater than 37 C are not recommended.

9. Count the number of fluorescent wells after 24 hours by placing a 6 watt 36nm W light within five inches of the plate. Do not read plate before 24 hours..
Results are stable to 48 hours.

10. Compare the number of fluorescent wells to an MPN
chart to determine the most probable number of bacterial present in the plate.

Test Procedure Usina Plate Havinq "Pour-Off Pocket"
1. Pour an appropriate amount of bulk powder to a container of sterile deionized water. One vial contains enough powder for 10 tests. Each test has a final volume of 10ml. For example: add 1 vial of powder to 100ml of sterile water to make enough media for 10 tests.
2. Place test sample on the center "landing pad" 22 of the plate 10 shown in Fig. 5. At the completion of this procedure half of the test sample will be poured off into the pocket, therefore, the size of the inoculum must take this into account. For example, if you wish to measure the bacterial concentration of 0.lml of test sample then you must place 0.2ml of test sample on the "landiny pad". Place no more than 2ml on the center "landing pad".
3. Remove the lid from the plate and dispense 10ml of media in the plate making sure to direct the liquid over the test sample on the center "landing pad". If the test sample is greater than 0.lml add enough media to achieve a final volume of 10ml in the plate. Note, if the li~uid is not dispensed on the "landing pad" it may splatter.

CA 02237639 1998-0~-12 W O 97/18455 PCTrUS96/18119 4. Distribute the liquid into the wells by swirling - the plate as you would a 5tandard pour plate, taking care that the fluid does not enter the "pour-off pocket".
5. Care~ully pour off the excess liquid that did not end up in the wells through the "pour-off pocket" barrier channel. Holding the plate at an angle of approximately go ~rom the work bench ensures proper pour off o~ excess liquid. Make sure that all liquid "cross bridges" between wells are removed by gently tapping the plate.
106. Place the plate in an incubator ~or 24 hours.
Plates can be inverted if desired if the "pour-off" pocket contains an absorbent material.
7. Count the number of ~luorescent wells after 24 hours by placing a 6 watt 36nm W light within five inches o~ the plate. Do not read plate before 24 hours. Results are stable to 48 hours.
8. Compare the number of fluorescent wells to an MPN
chart to determine the most probable number of bacterial present in the plate.

ExamPle 2: Use of Incubation Plate for Unit Dose Testinq The plate and media described in Example 1 are used for this test.
Test Procedure 1. Add 10ml of sterile water to the tube of predis-pensed powder. If greater than 0~lml of ~ood sample is tobe inoculated into the test, reduce the volume of sterile water appropriately to achieve a final volume of 10ml in the tube.
2. Inoculate the liquid reagent with the food sample being tested.
3. Shake tube several times to completely mix powder and inoculated food sample. Avoid excessive mixing which tends to foam up liquid reagent. Too much foam can complicate the distribution of the liquid into the plate.
The rest o~ the procedure is as in Example 1.
Other embodiments are within the following claims.

Claims (51)

What is claimed is:

1. A method for detection of the amount of a biological material in a sample, comprising the steps of:
liquefying said sample if necessary, and pouring the liquified sample into an incubation plate having a generally flat horizontal surface, said surface defining a plurality of at least twenty recessed wells, each well being adapted to hold an aliquot of liquid and being sized and shaped, and formed of a material suitable to hold said aliquot within each said well by surface tension or by gelling of said liquid;
pouring off any excess liquid from said incubation plate, and incubating said incubation plate until the presence or absence of said biological material in one or more of said wells is determined so that the amount of said biological material can be determined.

2. The method of claim 1, wherein said surface defines at least 40 recessed wells.

3. The method of claim 1, wherein said surface defines at least 60 recessed wells.

4. The method of claim 1, wherein said surface defines at least 90 recessed wells.

5. The method of claim 1, wherein said surface defines at least 200 recessed wells.

6. The method of claim 1, wherein said plate is formed of plastic.

7. The method of claim 6, wherein said plastic is PVC.

8. The method of claim 6, wherein said plastic is formed of a hydrophobic material.

9. The method of claim 1, wherein said plate is generally circular in shape.

10. The method of claim 1, wherein said plate is provided with a lid to prevent contamination of liquid in the wells.

11. The method of claim 1, wherein said plate is clear.

12. The method of claim 1, wherein said plate is colored.

13. The method of claim 12, wherein the color is yellow.

14. The method of claim 1, wherein each said well is about 0.15 inch in diameter.

15. The method of claim 1, wherein each said well is chamfered to aid in the removal of excess liquid.

16. The method of claim 1, wherein said plate is about three inches in diameter.

17. The method of claim 1, wherein said plate is about five inches diameter.

18. The method of claim 1, wherein said plate has a lip to aid removal of excess liquid not present in a well from said plate.

19. The method of claim 1, wherein said wells hold a total of between five to 100 ml.

20. The method of claim 1, wherein said plate is sterile.

21. The method of claim 1, wherein said plate further comprises a pour-off pocket adjacent to said surface, wherein said pocket is adapted to contain said excess liquid, and wherein said excess liquid is poured from said surface of said incubation plate into said pour-off pocket prior to incubation.

22. The method of claim 21, wherein said pour-off pocket contains an absorbent material.

23. The method of claim 1, wherein said plate comprises a "landing pad" area of size sufficient to hold the liquified sample prior to dispersal of the sample to said wells.

24. The method of claim 23 wherein said method comprises applying said sample to said "landing pad" and subsequently providing a further liquid to simultaneously dilute said sample and disperse said sample into said wells.

25. The method of claim 23, wherein said plate further comprises a pour-off pocket adjacent to said surface, wherein said pocket is adapted to contain said excess liquid, and wherein said excess liquid is poured from said surface of said incubation plate into said pour-off pocket prior to incubation.

26. The method of claim 1, wherein said incubation plate comprises a pour spout and wherein said plate further comprises a lid having a slit adapted to cooperate with said pour spout to allow excess liquid to be poured from said incubation plate and to allow separation of said slit in said lid and said pour spout in said incubation plate to thereby permit incubation of the incubation plate without significant loss of liquid from said wells by evaporation.

27. A sterile incubation plate for determination of the amount of a biological material having a generally flat horizontal surface, said surface defining a plurality of at least twenty recessed wells, each well being adapted to hold an aliquot of liquid and being sized and shaped, and formed of a material suitable to hold said aliquot within each said well by surface tension, wherein said incubation plate will not provide any positive response for said biological material in the absence of a said biological material present in a sample applied to said plate.

28. The plate of claim 27, wherein said surface defines at least 40 recessed wells.

29. The plate of claim 27, wherein said surface defines at least 60 recessed wells.

30. The plate of claim 27, wherein said surface defines at least 90 recessed wells.

31. The plate of claim 27, wherein said surface defines at least 200 recessed wells.

32. The plate of claim 27, wherein said plate is formed of plastic.

33. The plate of claim 32, wherein said plastic is PVC.

34. The plate of claim 32, wherein said plastic is formed of a hydrophobic material.

35. The plate of claim 27, wherein said plate is generally circular in shape.

36. The plate of claim 27, wherein said incubation plate is provided with a lid to prevent contamination of liquid in said wells.

37. The plate of claim 27, wherein said plate is clear.

38. The plate of claim 27, wherein said plate is colored.

39. The plate of claim 38, wherein the color is white or yellow.

40. The plate of claim 27, wherein each said well is about 0.15 inch diameter.

41. The plate of claim 27, wherein said plate is about three inches diameter.

42. The plate of claim 27, wherein said plate is about five inches diameter.

43. The method of claim 27, wherein said plate has a lid to allow removal of excess liquid not held in a well from the plate.

44. The plate of claim 27, wherein said wells hold a total of between five to 100 ml.

45. The sterile incubation plate of claim 27, further comprising a pour-off pocket adjacent to said surface of said plate, wherein said pocket is adapted to contain excess liquid removed from said surface of said plate.

46. The sterile incubation plate of claim 27, wherein said plate comprises a "landing pad" adapted to hold a sample prior to aliquoting of said sample to said wells.

47. The sterile incubation plate of claim 46, wherein said "landing pad" has a diameter of at least one inch.

48. The sterile incubation plate of claim 46, wherein said plate comprises a pour spout.

49. The sterile incubation plate of claim 48, wherein said plate further comprises a lid having a slit adapted to cooperate with said pour spout to allow removal of liquid said incubation plate.

50. The sterile incubation plate of claim 46, further comprising a pour-off pocket adjacent to said surface of said plate, wherein said pocket is adapted to contain excess liquid removed from said surface of said plate

51. The sterile incubation plate of claim 50, further comprising an absorbent material within said pour-off pocket.