DAPI staining protocol

Philip Kerrison and Michael Steinke (Last update: 7 December 2010)

4',6‐diamidino‐2‐phenylindole (DAPI) is a fluorescent DNA stain useful for multiple purposes including,
cell cycle studies, determining the mitotic index of an organism or enumerating unicellular protists and
bacteria. This guide refers specifically to the qualitative or quantitative determination of bacterial cells
within a liquid culture following the methods presented in Sherr & Sherr (1993) and Sherr et al. (1993).

Fig 1. DAPI tray containing; A. filtration tower and

clamp; B. 25 mm filters (0.8 µm cellulose and 0.2 µm
black polycarbonate [Nuclepore]) and two sets of
tweezers; C. Glass slides, 22 x 22 mm coverslips,
alkaline Lugol’s; and D. immersion oil with a plastic
pipette.

Material:
• DAPI tray (see Fig 1).
• DAPI solution; 1 mg dissolved in 1ml of sterile‐filtered MilliQ (kept in small aliquots in Michael's
freezer drawer in lab 3.07). Keep in fridge while in use.
• 5% thiosulphate solution. Mix regularly – watch for bacterial contamination. Kept in syringe in
fridge. Replace disposable 0.2 µm Minisart filter unit before use.
• Sterile 1.5 mL centrifuge tubes.
• 10 or 25 mL syringe filled with Artificial Seawater (ASW) with a disposable filter unit.
Note: Wear gloves throughout procedure (see Risk Assessment).

Prepare filtration tower

• Attach the pump set‐up to the inhalant line of an air pump.
• Place a cellulose filter on the filtration tower. Wet it and let it soak with sterile‐filtered ASW.
• Place a black filter on top of it and briefly suck the filter flat.
• Clip on the glass filtration tower.

Fix cells
• Take a culture sample, usually 1 mL will be sufficient but the volume may need adjusting.
• For analysis immediately; place 1 mL of culture into a sterile MCT with 10 µl of Alkaline Lugol’s
and wait for a few minutes.
• For later analysis, store the sample with 10 µl of Lugol’s per mL in a sterile nearly full
glass container that is closed gas‐tightly. Samples should be checked fortnightly, if the
liquid is no longer stained to a weak tea colour, the culture will degrade. Add more
Lugol's if necessary.
DAPI staining protocol 2

Staining
• Add one drop of 5% thiosulphate to each 1 mL sample to remove staining.
• Add 10 µl of DAPI solution to each 1 mL sample using sterile pipette tips. Replace tip for each
aliquot to prevent cross‐contamination.
• Leave for a few minutes for the DAPI to stain.
• Pipette 1 mL from the MCT into the filtration tower. Turn the pump on. Before the filter is dry,
start running a few mL of sterile‐filtered ASW into the filtration tower to wash away excess DAPI
(about 5× sample volume), rinse any sample from the sides of the glass tube and ensure an even
distribution of cells on the filter.
• After the filter is dry, turn off the air pump.

Making slides
• Add a small drop of immersion oil onto a glass slide. Use Cargille Type DF or FF immersion oils.
• Unclip the filtration tower and carefully remove the black polycarbonate filter using the flat
edged tweezers, leaving the cellulose filter in place. Carefully position it, sample up, onto the
small drop of immersion oil.
• Add another drop of oil on top of the coverslip and the carefully place on top of filter.
• Press onto the coverslip with the tweezers to remove air bubbles.
• Two filters can fit on one slide.
• Use as little oil as you can get away with. Otherwise, leave the slides tilted on blue roll while
preparing other samples. This will allow excess oil to be wicked away.
• Store samples in the freezer in lab 3.07 in a red slide box.

Next sample
• Wash the filter tower with running tap water and then rinse with sterile‐filtered ASW.

Fluorescent Microscopy
• Take the slides to the Bioimaging lab 4.19 for analysis using the BX41 microscope. This can be
booked on Google Calendar (login: bioimaging1)
• Training for this microscope, run by Dr Philippe Laissue happens at least once a month.
• Details on the facility are available at
http://www.essex.ac.uk/bs/research/facilities/Bioimaging/default.aspx

Quantification of bacterial numbers

2
The area of the filter exposed to sample is 250 mm . Count the number of bacterial cells within a known
area of the slide then factor up to determine how many cells are on the filter. This equals the number of
bacterial cells per volume filtered (e.g. 1 mL).

References
Sherr EB, Sherr BF (1993) Preservation and Storage of Samples for Enumeration of Heterotrophic Protists. In: Kemp PF, Sherr BF, Sherr EB,
Cole JJ (eds) Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton
Sherr EB, Caron DA, Sherr BF (1993) Staining of Heterotrophic Protists for Visualization via Epifluorescence Microscopy. In: Kemp PF, Sherr
BF, Sherr EB, Cole JJ (eds) Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton