Process and apparatus for ascertaining the concentration of microorganism in a water specimen

• US 4,018,652 A
• Filed: 01/09/1976
• Issued: 04/19/1977
• Est. Priority Date: 01/09/1976
• Status: Expired due to Term

• Current Assignee: Mcdonnell Douglas Corporation (The Boeing Co.)
• Original Assignee: Mcdonnell Douglas Corporation (The Boeing Co.)
• Inventors: Holen, James T., Fadler, Norman L., Lanham, James W.
• Primary Examiner(s): Jones, Raymond N.
• Assistant Examiner(s): Warden, Robert J.
• Application Number:
  US05/647,802
• Time in Patent Office: 466 Days
• Field of Search: 195/103.5, 195/127, 195/139, 23/253 R, 23/259
• US Class Current: 435/36
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: G01N 2035/00148 Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales
• CPC Class Codes: Y10S 435/848 Escherichia
• CPC Class Codes: Y10S 435/852 Klebsiella
• CPC Class Codes: Y10S 435/874 Pseudomonas
• CPC Class Codes: Y10S 435/885 Streptococcus

Abstract

The concentration of microorganisms in a known volume of a water specimen is ascertained by introducing the water specimen into a plurality of wells which have known volume and contain a nutrient medium. The mixture of water specimen and nutrient medium is incubated and the wells are observed for a change in the appearance thereof which indicates metabolic activity, that is, the existence of microorganisms in the wells. If all the wells change appearance, then it is known that the concentration exceeds a certain limit, that is, at least one cell per specific well volume. On the other hand, if none of the wells change, then it is most likely the concentration is less than one cell per total volume of specimen in the wells. A change in appearance of some but not all of the wells indicates a concentration between the foregoing limits, and this concentration is estimated by statistical evaluation based on proven statistical computations.

Claims

1. An apparatus for determining the concentration of microorganisms in a water specimen, said apparatus comprising a rigid body having a plurality of first wells and a plurality of second wells therein, the first wells being of equal volume and the second wells being of equal volume with the volume of the second wells being different from that of the first wells, the rigid body also having an inlet which opens outwardly from the body and forms an entry into which the water specimen may be introduced into the body, the rigid body further having filling channels leading up to and opening into the first and second wells, there being a separate filling channel for each first well and for each second well with each filling channel communicating with the inlet such that a water specimen introduced into the inlet passes into the filling channels and then into the wells, the upstream end of each filling channel being in communication with the inlet independently of the other filling channels so that the water specimen will flow into each well without passing through another well;

means closing the ends of the wells and the sides of the filling channels for isolating the interiors of the wells and filling channels from the surrounding atmosphere except through the inlet, said means being transparent; and

a culture medium in the wells.

2. An apparatus according to claim 1 and further comprising means at the inlet for isolating the channels from the surrounding atmosphere.

3. An apparatus according to claim 2 wherein the means at the inlet for isolating the channels comprises a septum in the inlet of the rigid body.

4. An apparatus according to claim 1 wherein the means for closing the ends of the wells is a light-transmitting tape adhered to those surface areas of the rigid body out of which the wells open.

5. A process for ascertaining the concentration of microorganisms in a water specimen, said process comprising:

placing the water specimen in communication with filling channels leading to sealed first cavities which are of known and equal volume;

placing the water specimen in communication with additional filling channels leading to sealed second cavities which are of known and equal volume, the volume of the second cavities being less than the volume of the first cavities;

placing the water specimen under a vacuum so that air is evacuated from the first and second cavities and filling channels through the water specimen;

releasing the vacuum and subjecting the water specimen to atmospheric pressure so that the water specimen is forced through the filling channels and into the first and second cavities to take the place of the evacuated air;

mixing the water specimen with a nutrient medium such that a mixture of water specimen and nutrient medium exists in the first and second cavities;

observing the water specimen in the first and second cavities for a change in the appearance thereof to determine the number of cavities which change appearance, and comparing the number of cavities which do change appearance with tabulated results derived from statistical evaluations to determine the concentration of microorganisms.

6. A process according to claim 5 wherein the water specimen and nutrient medium are mixed together in the first and second cavities.

7. A process according to claim 5 and further comprising placing the water specimen in communication with third cavities which are of known and equal volume, the volume of the third cavities being less than the volume of the second cavities, whereby when the water specimen is placed under a vacuum, the air is evacuated from the third cavities as well as from the first and second cavities, and when the vacuum is thereafter released, the water specimen is forced into the third cavities, and comparing the number of third cavities which change appearance with tabulated results derived from statistical evaluations to determine the concentration of microorganisms;

and wherein the step of mixing the water specimen with the nutrient medium is such that a mixture of water specimen and nutrient medium also exists in the third cavities.