Molecular Techniques and Methods

Identification of Bacterial Cells by In situ Hybridization

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

YT Broth (1 liter)
Tryptone ----------------------------------------- 10 g
Yeast extract ------------------------------------- 5 g
Glucose ------------------------------------------ 5 g
NaCl --------------------------------------------- 8 g
Add distilled H2O to make a final volume of ---- 1 L
  • Adjust pH to 7.2.


    Paraformaldehyde Solution (100 ml)
    4% Paraformaldehyde --------------------------- 4 g
    PBS -------------------------------------------- 100 ml


    Hybridization Solution (10 ml)
    0.9 M NaCl ------------------------------------- 1.8 ml of 5 M NaCl
    20 mM Tris-HCl (pH 7.2) ----------------------- 0.2 ml of 1 M Tris-HCl (pH 7.2)
    0.01% SDS ------------------------------------- 0.01 ml of 10% SDS
    Distilled H2O ------------------------------------ 8 ml


    Blocking Solution (10 ml)
    150 mM NaCl ----------------------------------- 0.3 ml of 5 M NaCl
    100 mM Tris-HCl (pH 7.5) ---------------------- 1 ml of 1 M Tris-HCl (pH 7.5)
    0.5% BSA (Bovine Serum Albumin) ------------- 50 mg
    0.5% Blocking Reagent -------------------------- 0.5 ml of 10% Blocking Reagent
    Distilled H2O ------------------------------------ 8.2 ml


    Washing Solution (10 ml)
    150 mM NaCl ------------------------------------ 0.3 ml of 5 M NaCl
    100 mM Tris-HCl (pH 7.4) ----------------------- 1 ml of 1 M Tris-HCl (pH 7.4)
    0.01 % SDS -------------------------------------- 10 ul of 10% SDS
    Distilled H2O ------------------------------------- 8.7 ml


    Lysozyme Solution (100 ml)
    Lysozyme ----------------------------------------- 100 mg
    TE ------------------------------------------------ 100 ml


    Peroxidase Substrate-Nickle Chloride Solution (10 ml)
    1.3 mM 3,3'-Diaminobenzidine -------------------- 2.8 mg
    0.02% H2O2 --------------------------------------- 67 ul of 3% Hydrogen Peroxide
    0.03% Nickel chloride ----------------------------- 100 ul of 3% NiCl2
    5 mM Tris-HCl (pH 7.4) --------------------------- 50 ul of 1 M Tris-HCl
    Add distilled H2O to make a final volume of ------- 10 ml




    PROCEDURES

    Preparation and Fixation of Bacterial Cells

    1. Grow E. coli in YT broth at 37oC.

    2. Pellet bacterial cells by centrifugation for 5 min at 3,000g.

    3. Resuspend bacterial cells in PBS.

    4. Add 3 volumes of Paraformaldehyde Solution to 1 volume of suspended cells.

    5. Fix bacterial cells for 3 hours at room temperature.

    6. Pellet cells by centrifugation for 5 min at 3,000g.

    7. Remove the supernatant.

    8. Wash the fixed cell pellet with PBS.

    9. Resuspend the cells in an aliquot of PBS.

    10. Spot these fixed cell suspensions onto cleaned glass slides and allow to air dry for at least 2 hour.

    11. Dehydrate the cell samples by immersing the slides successively in ethanol series as follow.

    Step
    Ethanol
    Time
    1
    50%
    5 min
    2
    80%
    5 min
    3
    98%
    5 min


    12. Prepare DIG-labeled oligonucleotides.



    Hybridization and Signal Detection using Fluorescently Labeled anti-DIG Fab Fragments

    13. Add 8-10 ul Hybridization Solution containing 50 ng of DIG-labeled oligonucleotide probe to the slide.

    14. Incubate the slide for 2 hours at 45oC in an isotonically equilibrated humid chamber.

    15. Dilute fluorescein- or rhodamine-labeled anti-DIG Fab fragments 1:4 in Blocking Solution.
  • Add 10 ul of the diluted antibody to the slide.

    16. Incubate the slide for 1 hour at 27oC in the humid chamber.

    17. Remove the slide from the humid chamber and immerse in 40 ml of a Washing Solution at 30oC for 10 min.

    18. Rinse the slides briefly with sterile water (which has been filtered through a 0.2 um filter).
  • Air dry and mount the slides.

    19. View the cell smears with a Plan-Neofluar 100 x objective (oil immersion) on the following setup:
  • A Zeiss Axioplan microscope fitted for epifluorescence microscopy with a 50 W mercury high pressure bulb and Zeiss filter sets 09 and 15 (Zeiss, Oberkochen, FRG).
  • For photomicrographs, use Kodak Ektachrome P1600 color reversal film and an exposure time of 15 s for epifluorescence or 0.01 s for phase contrast micrographs.



    Hybridization and Signal Detection using Peroxidase-conjugated anti-DIG Fab Fragments

    13. Prior to hybridization, incubate fixed cells for 10 min at 0oC with Lysozyme Solution.

    14. Remove lysozyme by thoroughly rinsing the slide with sterile H2O.

    15. Air dry the slide.

    16. Add 10 ul Hybridization Solution containing 50 ng of DIG-labeled oligonucleotide probe to the slide.

    17. Incubate the slide for 2 hours at 45oC in an isotonically equilibrated humid chamber.

    18. Dilute fluorescein- or rhodamine-labeled anti-DIG Fab fragments 1:4 in Blocking Solution.
  • Add 10 ul of the diluted antibody to the slide.

    19. Incubate the slide for 1 hour at 27oC in the humid chamber.

    20. Remove the slide from the humid chamber and immerse in 40 ml of a Washing Solution at 30oC for 10 min.

    21. Rinse the slides briefly with sterile water (which has been filtered through a 0.2 um filter).
  • Air dry and Mount the slides.

    22. Incubate slide with Peroxidase Substrate-Nickle Chloride Solution until a purplish-blue precipitate forms.

    23. View slides under a light microscope and photograph with Kodak Ektachrome P1600 color reversal film.




    NOTES

  • After adding 1 volume ethanol to the resuspended cells, you may store the cell suspension at -20oC for up to 3 months without apparent influence on the hybridization results.




  • KIT INFORMATION




    REFERENCES



  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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