Chapter 27. Bacterial and biofilm iridescence.

Why were some of the bacteria I observed in my last post coloured? This video is digitally zoomed and as you can see the bacteria that form tetrads are not only coloured but are changing colour. My first thought was that it was some kind of digital imaging artefact. That's human nature I think. If you see something you can't explain you rub your eyes and throw the vodka bottle away. And there's reason to be concerned. The digital camera attached to my microscope has a CMOS detector which are apparently less sensitive and more susceptible to noise than alternatives. Also, entry level digital cameras take a probabilistic approach to estimating the colour of any given pixel and, when you watch the linked videos, there is a strange static oily rainbow pattern covering the field of view. Having said all that, if it is an imaging artefact, then it's an artefact that only effects a sub-set of the bacteria in the film, which is interesting anyway.

Firstly I needed to know if the observation could be repeated so I continued to allow the biofilm to form by detaching the venturi valve from my powerhead and angling its exhaust away from the tank surface. As before I sampled by dipping a microscope slide into the aquarium and scooping up a fragment of the film in situ. I then viewed at x100 magnification without a coverslip. This image was taken around two weeks after first contact.

Aquarium surface biofilm in situ x100 magnification. The almost continuous sheet of bacilli is punctuated by the occasional island of coloured bacteria.

The oily rainbow effect is much reduced in this image although there is still variation in background colour. I think the effect is a microscopy artefact. I can recreate and accentuate it by setting the condenser to about half way between the light and the stage of the microscope.

The coloured bacteria were much less common than in the previous sample but were still easy to find. And as before they change colour over time. To produce the video I used VirtualDub to crop from 1280x1024 to 484x448 (19%) but any media player I use expands it to full screen when playing and must do some interpolation or the video would be far more pixelated. Below is a still from the cropped video expanded to 1106x1024 using GIMP to do the interpolation.

Six day old aquarium biofilm x100 zoomed x5,  Iridescent bacteria?

I can't find any images like these on the internet. The bacteria seem to be all the colours of the spectrum, twinkling away. There is a genus of cyanobacteria that form similar grid like rafts. Merismopedia cells divide on two planes and so produce tetrads. They are reported to contain reddish, blue/green, violet or pink content but they seem to form much more orderly structures than these and there's no mention of cells changing colour. Importantly, the bacteria described here are not coloured when viewed under a cover-slip (Chapter 26 and subsequently confirmed) which suggests that pigmentation is not important. I might be the first person to ever observe this species so I thought I would have first go at describing them.

Stubby rods 1 to 1.5 micrometres wide and 1 to 3 micrometres long. Division by binary fission on two planes to form tetrads. Forms colonies in a freshwater/air interface aquarium biofilm. Colonies range from two to several hundred cells. Cells exhibit varied dynamic colouration under direct trans-illumination. 

Aquarium biofilm with a colony of coloured bacteria. Four frame .GIF. The frames were taken 1-2 seconds apart. 

This GIF (made with GIMP) shows the changing colours nicely. They look washed out to me when I upload my videos to YouTube, I guess YouTube has a different interpolator (which does make the point that interpolation may be accentuating the effect). The obvious question I had was; what is known about coloured bacteria? I couldn't find much information but I did find an article about bacterial iridescence from the blog "small things considered". In it the author explains that iridescence has been observed for bacterial colonies but is poorly understood and has only been studied in detail since 2012. Very helpfully she links the original research papers and they concern the iridescence of quite large colonies of bacteria cultured on growth media. There is no mention of individual cells being iridescent although there is some microscopy of the colony fringes. However the research is still interesting because it describes a category of iridescent bacteria colonies called 'rainbow diffuse' that

"exhibited all spectral colors ranging from red to blue only under the condition of trans-illumination"

I think they're talking about oblique trans-illumination from reading about their experimental set up, but the above description broadly fits my observations albeit at a much larger scale. So, what is iridescence, what causes it and could it explain what I have described here?

It seems iridescence is an optical phenomenon of surfaces in which hue changes with the angle of observation and the angle of illumination. Given that the angle of illumination and observation are fixed when looking down a microscope, if iridescence is responsible for the changing colour of these bacteria then it must be because the biofilm fragment I'm observing is floating across the surface of the drop of water on the microscope slide. You can see this movement in the videos I have linked (movement is not obvious in the above GIF because, for artistic reasons, I nudged the layers to align better). What struck me about the wikipedia page on iridescence was that one of the the examples used was an aquarium biofilm but the iridescent pattern didn't look anything like the oily effect I remembered from my aquarium or oily effects in general. The photo on wikipedia shows a perfect rainbow with all the colours in the expected order and that's not what comes to mind when looking at oil on wet tarmac. It's usually a bad idea to rely on memory so I thought I had better start photographing the biofilm as well as examining it under the microscope.

Aquarium biofilm iridescence under epi-illumination (left panel) and trans-illumination (right panel).

The photo on the left was taken the day before the microscopy I've described above and the biofilm has produced a series of rainbows, one for each of the five LED bulbs in my strip-light. The effect was angle dependent. In the left panel the rainbow effect was still visible under trans-illumination. So it seemed that either my memory was at fault or I was observing a different effect. Either way, I couldn't help wondering if the rainbow coloured bacteria I saw under the microscope had anything to do with the rainbow effect on the aquarium surface.

If they did then I would have expected them to be iridescent under epi-illumination just like the aquarium surface.

Aquarium biofilm x100 under epi-illumination. Two frame GIF digitally zoomed x4.

I captured these images by turning off the microscope lamp and illuminating the sample from the side using an LED torch. The cells in the colony of coloured bacteria in the centre seem to be different colours from the surrounding bacilli but there is no great range of colour or change in colour. I tried a variety of angles of illumination and couldn't see any evidence of iridescence in any of the tetrad colonies I examined. I wondered if converting a microscope to epi-illumination in the simplistic way I had could produce meaningful images. From what I read here there seem to be two concerns. First, my achromatic objectives don't have the anti-reflective coatings found on epi-illumination objectives. The linked video certainly has some strange arcs of light but seems reasonably well resolved. Second, all my achromats have a lens corrected to allow for a 0.17mm coverslip.

This made me think because all the images I've uploaded were captured without coverslips. What effect would not using a coverslip have under trans-illumination when using such an objective and could it be that the phenomenon I have described is a microscopy artefact after all? Achromatic lenses are designed to prevent chromatic aberration  (and spherical aberration) and they do this by focusing the different wavelengths of light on a single focal point under the expectation that the light entering the objective has been split into different wavelengths by passing through a coverslip. If you don't use a coverslip, I guess the light enters the objective in phase and is then split. As I mentioned earlier, when I put a coverslip on biofilm samples I loose the coloured effect from the tetrads so CA does make sense. However, and I feel a future retraction coming as I write this but, I don't think chromatic aberration could explain my observations because high numerical aperture objectives like the x100 I used are much less susceptible to CA than high power objectives and CA only seems to effect red and blue light.

So, if the coloured tetrads were not iridescent under epi-illumination then that suggested that they didn't contribute to the biofilm iridescence. I wanted to understand what did cause the biofilm iridescence in the hope that it would help me understand why the tetrads were coloured.

Coming soon: Imaging aquarium biofilms in situ at x400 and x1000 magnification and the mystery of the oily effect is solved.