# US 5,700,645 A · Methods and kits for separation, concentration and analysis of cells

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- Number: US 5,700,645 A
- Document ID: US5700645A
- Status: Granted
- Kind: A
- Application number: 08/485,428
- Filed: 1995-06-07
- Granted: 1997-12-23
- Earliest claimed: 1990-07-02
- Assignees: Promega Corporation
- Inventors: Edward E. Pahuski; Randall L. Dimond; John H. Priest; Lisa Zandt; Kathleen K. Stebnitz; Leopoldo G. Mendoza
- Web page: https://patentgrep.com/patent/US5700645A

## CPC

- C07K 1/36: by a combination of two or more processes of different types
- C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)}
- C12Q 1/10: Enterobacteria
- C12Q 1/24: Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- C12Q 2304/60: Chemiluminescent detection using ATP-luciferin-luciferase system
- Y10T 436/25375: Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Processes are provided for removal of cells as cell pellets from liquid milk samples, or from cultures or extracts of other food materials or other materials of biological origin. The concentrated cells in the pellet can be analyzed by various techniques to determine the relative cell count, such as by lysing of the cells followed by measurement of ATP. Nucleic amplification, as by the polymerase chain reaction method, can be carried out using the cellular pellet directly, without need for isolation of nucleic acid from the cells. After the amplification, an assay can be carried out for amplified nucleic acid segment indicative of the presence of cells of interest in the sample. The invention thus provides methods for obtaining cellular components from samples of milk, and cultures or extracts of other materials, including food materials, and for determining relative contamination of milk and such other materials by microorganisms. The invention also provides kits for carrying out its various methods.

## Claims

### Claim 1

A method of separating and concentrating cells from a liquid milk sample comprising the steps of:
(a) mixing a chelating agent and a microparticulate carrier wherein the microparticulate carrier sediments slightly slower than microbial cells, and wherein the microparticulate carrier serves as a visual indicator facilitating the removal of supernatant without disturbing the cell pellet in the milk sample,
(b) centrifuging the sample to form a cell pellet.

### Claim 2 (depends on 1)

The method of claim 1 wherein the microparticulate carrier is polystyrene beads with a diameter from about 0.5 .mu.m to about 1.5 .mu.m.

### Claim 3 (depends on 1, 2)

The method of any one of claims 1 or 2 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid and N-(2-acetamido)iminodiacetic acid.

### Claim 4 (depends on 1, 2)

The method of any one of claims 1 or 2 including the additional step of removing the supernatant from the cell pellet by aspirating the fluid above the pellet.

### Claim 5 (depends on 4)

The method of claim 4 including the additional step of resuspending the pellet in a liquid and testing the suspension to determine the concentration of microbial cells therein by determining ATP in the microbial cells using bioluminescence.

### Claim 6 (depends on 4)

The method of claim 4 including the additional step of resuspending the centrifuged pellet in a solution comprising a protease or a protease together with an ATPase.

### Claim 7 (depends on 4)

The method of claim 4 including the additional step of suspending the cell pellet in a liquid comprising a lysing agent which lyses somatic cells but not microbial cells.

### Claim 8 (depends on 7)

The method of claim 7 wherein said liquid comprising a lysing agent further comprises a protease or a protease together with an ATPase.

### Claim 9 (depends on 8)

The method of claim 8 including the additional step of centrifuging the sample after mixing the lysing agent therein, removing the supernatant, resuspending the remaining cell pellet in a liquid, adding an agent to the cell pellet which extracts ATP from microbial cells and quantitatively testing the sample for ATP.

### Claim 10 (depends on 8)

The method of claim 8 including the additional step of adding an agent to the cell pellet which extracts ATP from microbial cells and quantitatively testing the sample for ATP.

### Claim 11 (depends on 9)

The method of claim 9 wherein the step of testing for ATP includes the steps of adding a luciferase-luciferin reagent to the sample and measuring the relative amount of light emitted from the sample.

### Claim 12 (depends on 9)

The method of claim 9 wherein the agent which extracts ATP is a lysing agent which lyses microbial cells.

### Claim 13 (depends on 7)

The method of claim 7 including the additional step of centrifuging the suspension, and separating the supernatant from the cells in the pellet which will be primarily microbial cells.

### Claim 14 (depends on 13)

The method of claim 13 including the additional step of testing the supernatant liquid separated from the pellet for the concentration of ATP.

### Claim 15 (depends on 14)

The method of claim 14 wherein the step of testing for ATP includes the steps of adding a luciferase-luciferin reagent to the supernatant and measuring the relative light emitted from the sample.

### Claim 16 (depends on 13)

The method of claim 13 including the additional step of adding an agent to the cells in the pellet which extracts ATP from microbial cells and then testing for the quantitative level of ATP.

### Claim 17 (depends on 16)

The method of claim 16 wherein the agent which extracts ATP is a lysing agent.

### Claim 18 (depends on 16)

The method of claim 16 wherein the step of testing for the level of ATP comprises adding luciferase-luciferin reagent to the sample and measuring the relative light emitted from the sample.

### Claim 19 (depends on 1, 2)

The method of any one of claims 1 or 2 including, before the step of centrifuging, the additional step of mixing a lysing agent with the sample which will lyse somatic cells in the sample but not microbial cells.

### Claim 20 (depends on 19)

The method of claim 19 wherein the lysing agent is a non-ionic detergent.

### Claim 21 (depends on 19)

The method of claim 19 including the additional steps of removing the supernatant from the pellet, adding a lysing agent to the cell pellet to lyse the cells and testing the lysed sample to determine the relative amount of ATP which is present in the sample.

### Claim 22 (depends on 21)

The method of claim 21 wherein the step of testing the lysed samples includes the step of adding a luciferase-luciferin reagent to the sample and measuring the relative light output emitted from the sample.

### Claim 23 (depends on 19)

The method of claim 19 including the additional steps of removing the supernatant from the pellet, mixing the cells in the pellet with a liquid comprising a lysing agent which lyses somatic cells but not microbial cells, centrifuging the sample, removing the supernatant liquid, again adding a liquid comprising a lysing agent to the cells in the resulting pellet which lyses somatic cells but not microbial cells, centrifuging the sample, and removing the supernatant, and then testing the resulting pellet for the relative concentration of microbial cells.

### Claim 24 (depends on 23)

The method of claim 23 wherein at least the second of said liquids comprising a lysing agent further comprises a protease or a protease together with an ATPase.

### Claim 25 (depends on 24)

The method of claim 24 including the additional step of resuspending the centrifuged pellet in a solution that stabilizes the bacterial cells and prevents the loss of cellular metabolites.

### Claim 26 (depends on 25)

The method of claim 25 wherein the stabilizing solution contains magnesium ions.

### Claim 27 (depends on 26)

The method of claim 26 wherein the stabilizing solution also comprises a protease or a protease together with an ATPase.

### Claim 28

A microbial test kit for use in testing milk samples, comprising:
(a) a clearing solution comprising a chelating agent, a microparticulate carrier wherein the microparticulate carrier sediments slightly slower than microbial cells, and wherein the microparticulate carrier serves as a visual indicator facilitating the removal of supernatant without disturbing the cell pellet in the milk sample and a somatic cell lysing agent;
(b) a solution comprising an ATP extractant which releases microbial cell ATP; and
(c) an ATP detection reagent.

### Claim 29 (depends on 28)

The test kit of claim 28 wherein said microparticulate carrier is polystyrene beads with a diameter from about 0.5 .mu.m to about 1.5 .mu.m.

### Claim 30 (depends on 28, 29)

The test kit of any one of claims 28 or 29 including (1) a solution for washing a cell pellet resulting from centrifugation, (2) a buffer solution, and a solution for treating the cells in the pellet, said treatment solution comprising a protease or a protease together with an ATPase.

### Claim 31 (depends on 28, 29)

The test kit of any one of claims 28 or 29 wherein the ATP detection reagent is luciferin-luciferase, the ATP extractant is a lysing agent which lyses microbial cells, and the somatic cell lysing agent is a non-ionic detergent.

### Claim 32 (depends on 28, 29)

The test kit of any one of claims 28 or 29 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid and N-(2-acetamido)iminodiacetic acid.

### Claim 33 (depends on 28, 29)

The test kit of any one of claims 28 or 29 further including a filter for filtering cells from milk components.

### Claim 34 (depends on 28, 29)

The test kit of any one of claims 28 or 29 wherein the ATP extractant is selected from the group consisting of trichloroacetic acid, chlorhexidine, and CTAB.

### Claim 35 (depends on 34)

The test kit of claim 34 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid and N-(2-acetamido)iminodiacetic acid.

### Claim 36

A test kit for use in testing milk samples for somatic cells, comprising:
(a) a clearing solution comprising a chelating agent and microparticulate carrier wherein the microparticulate carrier sediments slightly slower than microbial cells, and wherein the microparticulate carrier serves as a visual indicator facilitating the removal of supernatant without disturbing the cell pellet in the milk sample;
(b) a solution comprising an agent which lyses somatic cells but not microbial cells; and
(c) an ATP detection reagent.

### Claim 37 (depends on 36)

A test kit according to claim 36 wherein the microparticulate carrier is polystyrene beads with a diameter from about 0.5 .mu.m to about 1.5 .mu.m.

### Claim 38 (depends on 36, 37)

The test kit of any one of claims 36 or 37 wherein the ATP detection reagent is luciferin-luciferase.

### Claim 39 (depends on 36, 37)

The test kit of any one of claims 36 or 37 wherein the somatic cell lysing agent is a non-ionic detergent.

### Claim 40 (depends on 36, 37)

The test kit of any one of claims 36 or 37 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid and N-(2-acetamido)iminodiacetic acid.

### Claim 41 (depends on 36, 37)

The test kit of any one of claims 36 or 37 further including a filter for filtering cells from milk components.

### Claim 42

A method of treating a liquid milk sample whereby cells in the sample are separated from other milk components and concentrated, comprising the steps of:
(a) mixing a chelating agent and a microparticulate carrier wherein the macroparticulate carrier sediments slightly slower than microbial cells, and wherein the microparticulate carrier serves as a visual indicator facilitating the removal of supernatant without disturbing the cell pellet with the milk sample;
(b) separating the cells in the sample from milk components.

### Claim 43 (depends on 42)

A method according to claim 42 wherein the microparticulate carrier is polystyrene beads with a diameter from about 0.5 .mu.m to about 1.5 .mu.m.

### Claim 44 (depends on 42, 43)

The method of any one of claims 42 or 43 wherein the step of separating the cells includes the step of filtering the sample with a filter which blocks cells and passes other milk components bound by the chelating agent.

### Claim 45 (depends on 42, 43)

The method of any one of claims 42 or 43 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid and N-(2-acetamido)iminodiacetic acid.

### Claim 46 (depends on 42, 43)

The method of any one of claims 42 or 43 wherein the step of separating is carried out by centrifuging the sample to form a cell pellet.

### Claim 47 (depends on 46)

The method of claim 46 including the additional step of removing the supernatant from the cell pellet.

### Claim 48

A clearing solution for use in testing milk samples, comprising a chelating agent, a microparticulate carrier wherein the microparticulate carrier sediments slightly slower than microbial cells, and wherein the microparticulate carrier serves as a visual indicator facilitating the removal of supernatant without disturbing the cell pellet in the milk sample and a somatic cell lysing agent.

### Claim 49 (depends on 48)

A clearing solution according to claim 48 wherein the microparticulate carrier is polystyrene beads with a diameter from about 0.5 .mu.m to about 1.5 .mu.m.

### Claim 50 (depends on 48, 49)

A clearing solution according to any one of claims 48 or 49 wherein the somatic cell lysing agent is a non-ionic detergent.

### Claim 51 (depends on 48, 49)

A clearing solution according to any one of claims 48 or 49 wherein the chelating agent is selected from the group consisting of nitrilotriacetic acid and N-(2-acetamido)iminodiacetic acid.

### Claim 52

A clearing solution comprising an aliquot of a culture or extract of a material of biological origin, said solution combined with a microparticulate carrier wherein the microparticulate carrier sediments slightly slower than microbial cells, and wherein the microparticulate carrier serves as a visual indicator facilitating the removal of supernatant without disturbing the cell pellet of the culture or the extract, wherein said microparticulate carrier is in suspension in the solution.

### Claim 53 (depends on 52)

A solution of claim 52 wherein the microparticulate carrier is polystyrene beads with a diameter from about 0.5 .mu.m to about 1.5 .mu.m.

### Claim 54 (depends on 52)

A solution of claim 52 wherein the material of biological origin is a food material other than milk or a milk product.

### Claim 55 (depends on 54)

A solution of claim 54 wherein the material of biological origin is a meat and the clearing solution comprises an aliquot of a culture prepared from the meat.

### Claim 56

A method of separating and concentrating cells from an aliquot of a culture or extract of a material of biological origin comprising the steps of:
(a) combining said aliquot with an aqueous suspension of a microparticulate carrier to form a clearing solution;
(b) centrifuging the clearing solution to form a cell pellet wherein the microparticulate carrier sediments slightly slower than microbial cells, and wherein the microparticulate carrier serves as a visual indicator facilitating the removal of supernatant without disturbing the cell pellet in the culture or extract.

### Claim 57 (depends on 56)

The method of claim 56 wherein the microparticulate carrier is polystyrene beads with a diameter from about 0.5 .mu.m to about 1.5 .mu.m.

### Claim 58 (depends on 56)

The method of claim 56 wherein the material of biological origin is a food material other than milk or a milk product.

### Claim 59 (depends on 58)

The method of claim 58 wherein the material of biological origin is a meat.