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.

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