Staining E.coli with Hoechst 33342 and FM 4-64FX was too dim. Could anybody please suggest an optimal protocol?

06 September 2019 3 7K Report

Hello experts from Research Gate,

I am struggling with staining live E.coli with Hoechst 33342 and FM 4-64FX. The fluorescent images were too dim even at high concentration of the fluorescent dyes (2-10 times higher than the concentration recommended by Invitrogen). And I had to acquire images at high exposure time (1000-4000ms) which led to photobleaching, high phototoxicity, high noise and extremely slow image acquisition. It seemed that Hoechst could not penetrate all the live E.coli cells uniformly (a few were bright and many were too dim).

Could you please have a look at my protocols and point out the problem of the protocols.

Protocol 1. Staining with Hoechst 33342 (or SYTOX Green):

Grow E.coli in LB broth to reach OD600 ~ 0.3 (see note 1)

Aliquot 1mL of bacterial suspension to a microcentrifuge tube

Centrifuge at 12,000 rpm for 2 minutes

Remove supernatant

Resuspend in 20uL of Hoechst 33342 10ug/mL (or/and SYTOX Green 5uM/mL) working solution (see note 2)

Incubate at room temperature, in dark, for 10 minutes (see note 3)

Wash 03 times with PBS

Centrifuge at 12,000 rpm for 2 minutes and remove supernatant

Resuspend in 20uL of Prolong live antifade reagent

Incubate at room temperature, in dark, for 15 minutes. Go to step 12 for imaging or step 11 for fixing

Centrifuge at 12,000 rpm for 2 minutes and remove supernatant. Resuspend in 20uL of paraformaldehyde 4%. Incubate at room temperature, in dark, for 10 minutes. Go to step 12 for imaging on agarose pad

Imaging: gently spread 2uL of bacterial suspension onto a small piece of nutrient agar placed on a microscopy slides. Airdry for 1-5 minutes. Place a coverslip on top. Ready for imaging.

Protocol 2. Staining with FM 4-64 FX after staining with Hoechst (or SYTOX Green):

Start from step 10 of protocol 1

Centrifuge at 12,000 rpm for 2 minutes and remove supernatant. Resuspend in 20uL of of FM 4-64FX 5ug/mL (see note 4)

Incubate at room temperature, in dark, for 5, 10, 15, or 20 minutes (see note 5).

Go to step 6 for imaging or step 5 for fixing

Centrifuge at 12,000 rpm for 2 minutes and remove supernatant. Resuspend in 20uL of paraformaldehyde 4%. Incubate at room temperature, in dark, for 10 minutes. Go to step 6 for imaging on agarose pad

Imaging: gently spread 2uL of bacterial suspension onto a small piece of nutrient agar placed on a microscopy slides. Airdry for 1-5 minutes. Place a coverslip on top. Ready for imaging.

Protocol 3. Staining with FM 4-64 FX alone:

Start from step 4 of protocol 1

Centrifuge at 12,000 rpm for 2 minutes and remove supernatant. Resuspend in 20uL of of FM 4-64FX 5ug/mL (see note 4)

Incubate at room temperature, in dark, for 5, 10, 15, or 20 minutes (see note 5).

Go to step 6 for imaging or step 5 for fixing

Centrifuge at 12,000 rpm for 2 minutes and remove supernatant. Resuspend in 20uL of paraformaldehyde 4%. Incubate at room temperature, in dark, for 10 minutes. Go to step 6 for imaging on agarose pad

Imaging: gently spread 2uL of bacterial suspension onto a small piece of nutrient agar placed on a microscopy slides. Airdry for 1-5 minutes. Place a coverslip on top. Ready for imaging.

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Note:

I have also diluted the bacterial suspension at 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256

I have tried Hoechst at different concentrations including 10, 20, 50, 100ug/mL

Incubation time also was 15, 20, 30 minutes

Also FM 4-64FX at 20 and 100ug/mL

Also on ice

Fluorescent dyes were prepared in water (Sigma) or PBS and stored in dark microcentrifuge tubes at -20oC. All the dyes were purchased from Invitrogen

The microscope was Nikon Ni-e with mercury lamp, DAPI filter (Ex: 375/28, Em: 460/60) for Hoechst, FITC (Ex: 480/30, Em: 535/45) for SYTOX Green, and TRITC (Ex: 540/25, Em: 605/55) for FM 4-64FX

Tuan-Anh Tran

Vad Pérez thanks for your suggestion.

I've tried not drying the bacterial suspension when mounting on agarose pad. But the bacteria was floating around and I couldn't image them.

Could you please let me know more about how drying sample can impact on the quality of fluorescent images?

David Schad

Hello,

While I am not experienced with staining bacteria with Hoechst, have stained mammalian cell nuclei. Your images may be good with some modification. If you open the image in FIJI (ImageJ), you can change the levels. In FIJI, under the image tab, select adjust, then brightness/contrast. There, you will see a bell curve. To remove background pull the minimum from the left side to the center of the curve. After that, to increase the signal, pull the maximum from the right. You should continue using your protocol, maybe leave the stain on longer. But then take images at the maximum exposure time, as well as some at lower exposures where signal is still visible. From these images you can remove background and increase the signal in FIJI. You can also do this in Photoshop. The Hoechst 33342 may be less efficient at entering bacteria due to the capsule or even LPS. Also, perhaps some of the bacteria have more nucleic acids present.

Iago Grobas

Have you found a solution? I am actually interested in whether it is enough to stain the bacteria or you have to put the dyes in the agarose pads too.

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