Method for isolating cells
The present invention relates to a method for isolating cells from a complex sample comprising the steps of: a) providing a complex sample, b) incubating said sample with: at least one chaotrope, a buffer and at least one detergent, c) isolating said cells from the resulting mixture by centrifugation or filtration.
1. A method for isolating viable cells with cell walls from a complex sample comprising:
a) providing a complex sample,
b) incubating said complex sample at 25° C. to 70° C. for at least 20 minutes with a lysis solution comprising:
at least one chaotrope in a concentration between 50 mM to 10M, that is urea, guanidine HCl, guanidine thiocyanate, guanidium thiosulfate, thiourea, sodium thiocyanate potassium thiocyanate, sodium iodide, sodium perchlorate or a combinations thereof
a buffer having a pH of 6 to 10 and
0.01% to 5% of at least one anionic, non-ionic and/or zwitterionic detergent until dissolution of said complex sample to form a mixture,
c) subjecting the resultant mixture consisting of said complex sample and said lysis solution from incubation and dissolution step b) to a centrifugation or a filtration and isolating viable cells with cell walls from the resultant mixture whereby said viable cells with cell walls are retained on a filter after the filtration or retained in a pellet after the centrifugation.
2. A method according to claim 1 , wherein the cells with cell walls are bacterial cells, fungal cells, archaeal cells, algae cells or plant cells.
3. A method according to claim 2 , wherein the cells with cell walls are Gram-positive bacterial cells or Gram-negative bacterial cells.
4. A method according to claim 1 , wherein said sample is a food sample, blood, or a tissue sample.
5. A method according to claim 4 , wherein the food sample is a milk product, a fish product, a raw fish or a meat product.
6. A method according to claim 5 , wherein the milk product is raw milk, milk powder, yoghurt, cheese or ice cream.
7. A method according to claim 4 , wherein said food sample is raw meat, meat rinse, sausage, chocolate, egg, an egg product or mayonnaise.
8. A method according to claim 1 , wherein the chaotrope is urea, guanidine HCl, guanidine thiocyanate, guanidium thiosulfate, or a combinations thereof.
9. A method according to claim 1 , wherein the sample is incubated with a solution comprising 1 M to 8 M of said chaotrope.
10. A method according to claim 9 , wherein the sample is incubated with a solution comprising 4M to 8 M, of said chaotrope.
11. A method according to claim 1 , wherein the buffer is a phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, TRIS buffered saline buffer (TBS) or TRIS/EDTA (TE).
12. A method according to claim 1 , wherein the detergent is an anionic detergent.
13. A method according to claim 12 , wherein that the anionic detergent is sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS) or deoxycholate (DOC).
14. A method according to claim 1 , wherein the zwitterionic detergent is 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO).
15. A method according to claim 1 , wherein that the non-ionic detergent is an ethoxylated aliphatic alcohol.
16. A method according to claim 15 , wherein that the non-ionic detergent comprises a C 13 to C 15 aliphatic alcohol.
17. A method according to claim 15 , wherein said ethoxylated aliphatic alcohol is a C 13 to C 15 aliphatic alcohol.
18. A method according to claim 1 , wherein the cells with cell walls are washed after the isolation step with buffer and/or detergent comprising solutions.
19. A method according to claim 1 , wherein the amount of the cells with cell walls in the sample is determined.
20. A method according to claim 1 , wherein the DNA or RNA of the cells is isolated.
21. A method according to claim 1 , wherein the sample is processed by a stomacher or mixer prior to incubation.
22. A method according to claim 1 , wherein the sample is spiked with a defined amount of cells.
23. A method according to claim 1 , wherein the sample is pre-incubated with a compound exhibiting osmotic stress-protective properties to the cells.
24. A method according to claim 23 , wherein said compound is glycine betaine or beta-lysine.
25. A method according to claim 1 , wherein the sample is further incubated with at least one biopolymer degrading enzyme.
26. A method according to claim 25 , wherein the biopolymer degrading enzyme is a protease, a cellulase or an amylase.
27. A method according to claim 25 , wherein the biopolymer degrading enzyme is incubated with the sample prior to step b) and/or after step c).
28. A method according to claim 1 , wherein said incubation is performed at 30° C. to 60° C.
29. A method according to claim 28 , wherein said incubation is performed at 35° C. to 50° C.
30. A method according to claim 1 , wherein the buffer has a pH value greater than 7 and lower than 9.
31. A method according to claim 1 , wherein said lysis solution comprises 0.2-2% of at least one detergent.