Microscopy

Circular Oblique Lighting (COL) on microscopic images of bacteria and algae

Using the slide from my previous post http://roslistonastronomy.uk/algae-superfood-after-two-days-in-water, I tried out Circular Oblique Lighting on the Zeiss IM today using appropriate COL annuli.

All of the following use Zeiss condenser NA 1.4 and low NA Leitz 25x objective (NA 0.22).

Andy

The following photo is a BRIGHT FIELD image of part of the slide – the bacteria are just visible as the blue/black mottling top right/and at bottom left:

The following picture is of the same field of field of view with same condenser and objective but adding in COL annulus – I don’t think there is a deep sky astronomy filter that works quite as well as this!

The following photo uses a COL filter with smaller hole for light to pass through – do you think there is a meaningful difference between the two?

I think there a difference – the bacteria stand out a bit more BUT so do the larger hill-like mounds in the background – these are due to dust on the slide or optics.

The following is a dark field photo (changed to greyscale in GIMP2) – again the contaminants stand out as well as the bacteria which is a real nuisance, although the bacteria are easily seen – same condenser and objective as above:

 

Algae superfood after two days in water

Ean Ean has bought for me a “superfood” based on Spirulina algae – apparently Kate Middleton eats it! It is sold as dry powder and I wondered if it was dead or alive. I put some in a cup of water for two days in the sun. In the photos below you can see algae cells – however lots of bacteria too which suggests that the algae are killed and the bacteria quickly take up residence with plenty of food available.

[You may also wish to view a following post where further work was done on this slide using COL, http://roslistonastronomy.uk/circular-oblique-lighting-col-on-microscopic-images-of-bacteria-and-algae]

Andy

Four algae cells showing cilia around edge from Hopwas Canal

Ean Ean and I collected the sample of water from Hopwas canal 19/4/2018 and today I viewed it in the Zeiss IM microscope using 40x objective. The picture below shows multiple cilia (hair-like structures) around edge of the cells which the cells use to move around. I have used Curves function in GIMP2 to enhance the image to demonstrate these cilia more effectively.

Andy

Attempts at dark field microscopy using x50 infinity-corrected objective with diaphragm

I still can’t seem to get dark field to work well!

Photos below using infinity-corrected 50x objective with iris diaphragm, curves adjusted in GIMP2.

Andy

Algae from my pot pond 12/4/2018:

Volvox on commercial slide viewed by myself today using same setup as above:

 

Home pot pond algae showing cilia

I have created a pond at home using a very large plastic pot from Homebase. It even obtained a new resident yesterday (a fish) who has joined the frogspawn and Elodea and some other water plants obtained from a friend’s pond – whence the fish also originated. The friend in question insists that the fish should not be lonely so another will be joining it later in the week!

The photos below are of an algal sample from the pot pond. 18 x Eppendorf 1.5ml centrifuge tubes were centrifuged at 10000 revolutions/minute for 10 minutes. I pipetted off excess water above the centrifuge pellet. I then combined the pellets into a single Eppendorf tube and centrifuged again using the same settings. This concentrated the algae to make microscopic observation easier.

The main findings today are of cilia on most of the algal cells. I have annotated the photos below to show whether these can be seen. In some cells the green chloroplasts are bunched up at one end and I suspect this is due to the centrifuging process.

Finally bacteria abound – not sure why I used to have such difficulty seeing bacteria initially but now I see them all the time – I suspect it is a bit like training yourself to observe faint fuzzies in the night sky – once it clicks it becomes a lot easier – perhaps it is also just about knowing what to look for!

Andy

Live bacteria amongst the algae:

https://youtu.be/WsejNez8AwQ

 

Commercial slide of Volvox showing daughter colonies inside parent colony

The following photos show daughter colonies inside Volvox colonies – blue circles within the blue circles.

I used x10, x20 and x40 objectives for these photos in bright field. Unable to get dark field even with x10 objective for this slide for some reason. Best I obtained was a sort of partial oblique illumination which you can see on photo with the partially dark background.

Andy