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Bacterial Analysis

What is it and why is it important?

Bacterial analysis is commonly used to determine total cell abundance in a collected water sample. It may also be useful in comparing the relative phytoplankton cell counts at different depths of a water column. Samples are analysed under an epifluorescence microscope, after filtering and staining.

How was it done?

Filtration:

25ml of a sample was filtered using a vacuum pump through a top filter (mesh 0.2µm), and backing filter (mesh 0.45µm), at 300-400mbar.

Staining:

After filtration, 1ml of fridge-stored DAPI stain was added to the filter (4, 6-Diamidino-2-phenylindole). This binds in between the minor DNA base pairs on the DNA double-helix of any living cells present on the top filter. This filter was then incubated out of direct light for 5-10 minutes and then washed with milliQ water.

Slide Prep:

The filter was dried and added to a microscope slide between layers of immersion oil. Process then repeated on each of three samples.

Microscopy:

Slides were excited under an epifluorescent microscope using green light, wavelength 510-560nm, and UV light, wavelength 330-380nm. The green light caused photosynthetic pigments to emit red light, and UV caused white fluorescence from all cells with DAPI stained DNA. This was repeated at 5 random points on each filter, capturing images from the microscope detector at 630x magnification to be counted for epifluorescence signatures implying the presence of a cell.

Results/Analysis

The three bottle samples came from approximate depths of 8, 20, and 52 metres in the water column from station 29 at E1. Near the surface there was a moderately high estimated total phytoplankton biomass, which increased at the lower depth sample to a peak in biomass corresponding with the observed deep chlorophyll maximum at approximately 22m on our CTD data. Below this depth, the phytoplankton biomass declines significantly as expected from the the low CTD chlorophyll values also observed.

Due to problems with the formaldehyde preservative adding too much background fluorescence, bottles 7 and 11 did not provide useful DAPI stained images. However, bottle 22 from the bottom depth did undergo successful DAPI staining and hence allowed us to estimate a total microbial biomass at that depth of approximately 92475 fg C ml-1 (using Whitman, 1998, method). This meant that the phytoplankton biomass made up just 2.5% of the total biomass at 52m. This might be expected given the lack of light penetration and strong thermocline lying above.

Figure 1. A) Lens image of epifluorescence from phytoplankton. B) Processed image of fluorescence signatures from A, developed in image-j software. Both images exposed to green light of wavelength 510-560nm. C) Lens image of epifluorescence from all microbes stained with DAPI. D) Processed image of fluorescence signatures from C, developed in image-j software. Both images exposed to UV  light of wavelength 330-380nm. Images from 630x magnification.

Water samples were

filtered and stained, and  

epifluorescence analysis was carried out

to calculate microbial cell counts throughout

the water column.

References

W. Whitman, D. Coleman, W. Wiebe, 1998. Prokaryotes: The unseen majority, PNAS, Volume 95, Issue 12, pp 6578- 6583

Estimated total phytoplankton biomass with corresponding bottle sample and depth taken from

Bacterial Analysis Physics Chemsitry Zooplankton Phytoplankton

Nutrients Zooplankton Chlorophyll