Pond, lake, stream and river water (fresh water) microscopy

First trial of centrifuge on pond water 14/12/2017

“First Light” for my centrifuge – the second hand Eppendorf 5415c. I followed a procedure detailed on the Micscape website for using the centrifuge to concentrate the organisms in a pond water sample from Stowe Pool in Lichfield collected today.

The centrifuge worked well and did indeed concentrate the organisms (see photos below for procedure I followed) although the variety was limited in the sample.

Andy

1-The-Eppendorf-5145c-centrifuge

2-Pond-water-sample-including-piece-of-plant-root

3-Stirring-sample-to-release-organisms-into-water

4-Filtering-out-large-particles using tea strainer

5-Filling-Ependorf-1-5ml-tubes-with-pond-water – each one needs to have same amount fluid in order for centrifuge to balance:

6-Placing-Eppendorf-tubes-in-centrifuge- note that they are evenly spread around centrifuge to obtain balance at high G-forces – you should always use pairs and never one on its own:

7-Screwing-on-airtight-lid-onto-rotor:

8-Centrifuged-samples-on-homemade-stand:

9-Pipetting-off-excess-fluid-after-pellet-the idea is to carefully remove the fluid above the pellet of centrifuged solids at bottom of tube without disturbing the pellet. Ideally, the pellet and single drop fluid left after pipetting.

10-View-of-pellet-at-bottom-of-Eppendorf-tube. after pipetting – it is the brown spot at bottom of tube in picture. I used pipette to transfer the pellets and small amounts of fluid from 6 tubes to 2 tubes and centrifuged again to further concentrate the contents into two pellets:

11-Slide-after-pellet-and-drop-water-pipetted-onto-it-showing-concentrated-protozoa & debris. I pipetted the final 2 pellets onto a slide and the picture below shows many dark areas in the water drop – these are from the pellets:

12 Coverslip & nail varnish to seal it before viewing under microscope:

Photos of organism from sample today seen under Zeiss IM microscope x32 magnification. The first photo shows well one particular feature evident in many of the photos – which is that the centrifuge has caused chloroplasts to be concentrated on one side of the cell rather than spread throughout. Other photos show large vacuoles which I think are from damage to the cell. Most of the cells were inactive – I presume killed by the process, although the banana-shaped ciliated organism in the third photo below was alive and moving.

 

Damian processes Stowe Pool microscope slides for observing under Zeiss IM microscope with Andy

Damian came around this afternoon and took to the microscope like a pro!

He processed slides from a sample of pond water I collected yesterday and kept in my mini-aquarium (square glass vase of Ean Ean’s) overnight – producing live, heat fixed, heat fixed and H&E stained slides, which were then observed with x4, x10, x20, x32 and x40 objectives and photographs and video taken using the Mikrocam 9.0 camera on my Dell E4800 laptop.

Andy

Damian processing slides in my kitchen sink:

The slides Damian produced:

Micrometer eyepiece and calibration slide:

Microcamlab7 software screen photo grab with calibration slide with 20x objective:

The following photo was taken at the eyepiece using Samsung S7 through the LOMO micrometer eyepiece and with calibration slide in field of view:

Video from microscopy session, by Damian and Andy. Most of the video covers views of live slides, in direct and phase contrast transmitted light, using 30W www.retrodiode.com LED illuminator for Zeiss IM microscope. There is a clip at the end of the video which explores an H&E stained heat-fixed slide that Damian prepared (https://youtu.be/dySnojsTteM). In the first video look out for the vortices the organism (I think it is probably Vorticella but needs to be confirmed – see this video for comparison (not ours) https://youtu.be/193EpXPU6QM) is producing either side of its mouth-parts:

x20 objective, live view:

x20 objective, phase contrast Ph1 annulus, live views:

I suspect that the following is a photo of the dead carcass of one of the feeding organisms in the following photos:

x32 objective, live specimen, series of photos of organism feeding – notice how mouth-parts are closed in some photos and open in others. This organism is also seen at the beginning of the video above. In this video you can see the vortices at sides of mouth-parts – especially in phase contrast. I think it is probably Vorticella but needs to be confirmed – see this video for comparison (not ours) https://youtu.be/193EpXPU6QM).

x32 objective, live specimen, phase contrast Ph1 annulus:

x20 objective, H&E stained heat-fixed:

x40 objective, H&E stained, heat fixed:

Volvox slide from Chinese 91 slide selection under Zeiss IM microscope with different illuminators – Andy’s birthday present November 2017

Rachel and David, good friends of ours, bought me this selection of slides for my birthday this year. It is an eclectic collection plant, animal, insect and protozoal slides, mostly stained. At only £25, it was an absolute bargain – although the number of 91 is a bit strange.

My first forey into looking at these came today. It also provided my first opportunity to compare images between the LED illuminator and 100W halogen illuminator.

I chose the Volvox slide – these multicellular organisms are found in lakes and ponds and inland waterways world-over and can be amazing to look at under a microscope.

The following images show Volvox using the two different illuminators and at varying magnification.

Volvox is a polyphyletic genus of chlorophyte green globe algae in the family Volvocaceae. It forms spherical colonies of up to 50,000 cells. They live in a variety of freshwater habitats, and were first reported by Antonie van Leeuwenhoek in 1700 (Wikipedia).

The following diagram comes from Wikipedia and shows the structure of a Volvox colony – which can be seen in my photos below.

Andy

The dual illuminator setup on Zeiss IM microscope:

The 91 Chinese slide selection:

Chinese-Volvox-slide-x20-obj-Halogen-100W-illum-011217.bmp – whole Volvox colony showing many daughter colonies – with x20 objective. The picture also shows that it is not possible to get all of the spherical Volvox colony in focus at the same time:

Chinese-Volvox-slide-x63-obj-LED-30W-illum-011217II.bmp (below) – same field as above with LED illumination rather than halogen illuminatation (which was used above). Photo looks similar – in practice colour temperature was very different – much warmer with the halogen – and the similar colours reflect the effect of the auto-white balance adjustment present in the Bresser Mikrocamlab7 software:

Chinese-Volvox-slide-x32-obj-Halogen-100W-illum-011217.bmp (below) – Halogen, now magnification increased using x32 objective – which brings out detail in the daughter colonies inside the Volvox:

Same field and magnification as above with LED illumination (below):

The following three photos are a series of photos of the same field all using LED illumination but this time using the 63x objective to concentrate on one of the daughter colonies. Volvox is spherical so it is not possible to get the whole daughter colony in focus at the same time so the three photos are at different levels within the colony to bring different parts into focus. It looks to me as though the daughter colonies are already forming grand-daughter colonies within them, even before separating from their parent!

Microscopy sediment from lake next to new waterfront apartments at Barton Marina 28/10/2017 & attempts at H&E on dry-fixed specimen & 63x Zeiss Plan objective

My mother came up to celebrate my daughter’s birthday and we went for a walk around Barton Marina (A38 near Lichfield) and enjoyed a Thai lunch. Whilst there, I collected a sediment specimen from the lake next to the new waterfront apartments.

Photos below are using the Zeiss IM microscope and Bresser MikrOkular camera, unless otherwise stated – the Mikrocam needs a good clean!

This was also a chance to try heat-fixing drop of filtered sediment water on a slide and using H&E staining. I also recently obtained a Zeiss Plan objective x63 and had try with this.

H&E staining (from http://www.histology.leeds.ac.uk/what-is-histology/H_and_E.php):

The most commonly used staining system is called H&E (Haemotoxylin and Eosin). H&E contains the two dyes haemotoxylin and eosin.

Eosin is an acidic dye: it is negatively charged (general formula for acidic dyes is: Na+dye). It stains basic (or acidophilic) structures red or pink. This is also sometimes termed ‘eosinophilic’.
Thus the cytoplasm is stained pink in the picture below, by H&E staining.

Haematoxylin can be considered as a basic dye (general formula for basic dyes is:dye+ Cl). Haemotoxylin is actually a dye called hematein (obtained from the log-wood tree) used in combination with aluminium ions (Al3+). It is used to stain acidic (or basophilic) structures a purplish blue. (Haematoxylin is not strictly a basic dye, but it is used with a ‘mordant’ that makes this stain act as a basic dye. The mordant (aluminium salts) binds to the tissue, and then haematoxylin binds to the mordant, forming a tissue-mordant-haematoxylin linkage.)
Thus the nucleus is stained purple in the picture below, by H&E staining.

This means that the nucleus, and parts of the cytoplasm that contain RNA stain up in one colour (purple), and the rest of the cytoplasm stains up a different colour (pink).

What structures are stained purple (basophilic)?

DNA (heterochromatin and the nucleolus) in the nucleus, and RNA in ribosomes and in the rough endoplasmic reticulum are both acidic, and so haemotoxylin binds to them and stains them purple.
Some extracellular materials (i.e. carbohydrates in cartilage) are also basophilic.

What structures are stained pink (eosinophilic or acidophilic)?

Most proteins in the cytoplasm are basic, and so eosin binds to these proteins and stains them pink. This includes cytoplasmic filaments in muscle cells, intracellular membranes, and extracellular fibres.

Brown structures in the H&E photos below are sand (small particles of rock). I think that the blue ovals are single celled organisms killed by the heating process (holding the slide over a flame to dry the drop of sediment water pipetted onto it) and then stained by the H&E staining process.

For future reference, it turns out the 63x Zeiss Plan objective has the longest working distance in the Zeiss IM microscope when the adjustment collar is twisted to as far as it can go on the 22 side of the scale (in other direction from the 12 on the collar scale and towards and beyond the 22 on this scale as far as it can go). Set at this distance the objective can focus through the bottom of a slide as well as through the top.

Andy

Video of microscopy live sample x32 objective Phase 1 annulus showing singe celled organisms (unstained live sample):

Barton-Marina-sediment-sand-grains-x20-Mikrocam-281017.jpg (below):

Barton Marina sediment unstained live specimen x32 obj Zeiss IM Ph1 Single celled organisms MikrOkular camera 281017 (below):

Following photo is of Barton Marina sediment filtered through tea strainer and seen through Zeiss IM microscope with x32 objective. Slide is heat fixed and HE-stained (below):

Following photos are of Barton Marina sediment filtered through tea strainer and seen through Zeiss IM microscope with x63 objective. Slide is heat fixed and HE-stained (below):

The following is photo of skeleton of stack of algal cells with 63x objective. This gives a scale to the small blue-stained cells – supporting the idea that these H&E stained small blue circles are bacteria – in this photo and also in the above photos.

Microscopy Stowe Pool pond water – live photographs using Watec-120N video camera

All images using Watec 120-N B&W camera and Yawcam software, with detail of diatom.

Andy

x4 objective:

x20 objective:

x32 objective:

x100 objective:

One of 32x objective images upscaled using online image upscale website (http://www.imageenlarger.com/):

x40 phase contrast images – these show the segmentation of the diatom protozoal exoskeleton:

 

Microscopy of pond water from Stowe Pool, Lichfield, 2/9/2017

I have re-visited this pool several times since Christmas to see if the organisms visible have changed in any way. Certainly, some new ones today – but perhaps I am just getting better at this!

Some good video of various organisms, including Daphne, the water flea, below.

Andy

Video:

Photos:

x4 objective:

x4 objective – Daphnia:

x20 objective:

x32 objective – Daphnia exoskeleton and Daphnia eye:

H&E staining of pond water sample

This is my first attempt to do H&E staining – I used the pond water sample from John Brockley’s garden yesterday which showed so much Volvulus. Heat fixed it first then applied the Haematoxylin for around 20 mins, washed it off, and then eosin for 5 mins. The former should bring out nuclear material in purple and the latter cytoplasmic material – I will leave you to decide whether I was successful from the photos below!

Haematoxylin and eosin stain is one of the principal stains in histology. It is the most widely used stain in medical diagnosis and is often the gold standard; for example when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed “H&E section”, “H+E section”, or “HE section”. A combination of hematoxylin and eosin, it produces blues, violets, and reds (Wikipedia).

I used this staining technique a lot in my anatomy degree and most pathology slides use H&E.

Andy

For comparison non-stained photos from yesterday from same water sample (below – these first two are NOT stained with H&E):

Volvulus-from-John-Brockleys-pond-270817-HE-stain-10x-Planapo-obj-Zeiss-Photomic-III (below – reds and purples are evident but not as pronounced as I expected. However this is only using the 10x objective so higher magnification would be more insightful into whether stain is effective):

 

16x objective H&E stain:

40x objective H&E – panorama of 7 photos (below – try downloading this image and zooming in – it is a much bigger image than previous ones as it is created from 7 photos – there is a great amount of detail within the cytoplasms of the cells other than chloroplasts (which were visible in unstained images)):

Fixing pond water algae using heat & results of experiment in using fluorescein dye

I tried two new techniques today – one was to fix a pond water sample with heat. I purchased a cheap £3.99 spirit burner off ebay for the purpose but that simply blackened the slide. Turned out using the hob on the cooker worked perfectly but needed to use pair pliers as I burnt my fingers initially!

I then tried fluorescein dye on the fixed slide to see if it would be taken up by any of the cells – nothing appeared to fluoresce (I purchased a ring UV light for this purpose) under the Zeiss Photomicroscope at 16x.

Nevertheless, the fixed slide showed a wide range of species and some good photos from today below (Bresser MikrOkular camera). Lots of Volvulus. I had concentrated the sample first by filtering it through a coffee filter paper from Tescos.

Andy

Zeiss Photomicroscope III with 16x objective 28/08/2017 – sample of neighbour John Brockley’s pond water – fixed with flame – no staining (below):

As above, with 40x objective and NOT fixed showing detail of Volvulus and another algal species. In both, chloroplasts and cilia are easily seen (below):

Identifying two organisms in pond water sample 17/5/2017

The following are two photographs reproduced from my post 17/5/2017, which I would like to identify myself.

Looking at Patterson 1992, these might be different views of the same colony of single called organisms, the top being complete and the bottom a part of a colony. I would call this a Volvoid type organism, with potential identifications shown on scanned pages from that book below: