My first attempt at using a hand microtome

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pippo1234
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Joined: Thu Dec 24, 2020 8:21 am

My first attempt at using a hand microtome

#1 Post by pippo1234 » Fri Apr 02, 2021 8:02 am

After various false starts and useful handholding from (Micro)Bob and John B I have finally got myself a bench microtome (the one from Brunel) and Olfa LBB blades.

This is my first attempt with a rosemary leaf (phase contrast and dark field). Apart from the air bubbles, the result far exceeded my expectations about what I could achieve with a hand microtome.

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Greg Howald
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Joined: Tue Oct 20, 2020 6:44 am

Re: My first attempt at using a hand microtome

#2 Post by Greg Howald » Fri Apr 02, 2021 11:13 am

That's a really good job especially when new to the process. I'm gonna send all my specimens to you so you can cut and mount them for me. It will give you lotsa practice.😃😃😃.
Well done.
Greg

Voyager-1
Posts: 27
Joined: Fri Mar 12, 2021 7:48 pm

Re: My first attempt at using a hand microtome

#3 Post by Voyager-1 » Fri Apr 02, 2021 11:26 am

Pippo1234,

Most excellent specimen!
I have to jump on the bandwagon! And in the process of ordering one of those Brunel bench microtomes.
Could not locate a supplier in the US, got in touch with Brunel Microscope Ltd.
Would like to know which blades from Olfa LBB blades you are using.... saw many types Olfa makes.
I do believe I watched a youtube video by John B,

https://www.youtube.com/watch?v=_7WhkEvj19U

Again, Great slices!

V

pippo1234
Posts: 81
Joined: Thu Dec 24, 2020 8:21 am

Re: My first attempt at using a hand microtome

#4 Post by pippo1234 » Fri Apr 02, 2021 2:49 pm

Would like to know which blades from Olfa LBB blades you are using.... saw many types Olfa makes.
These https://www.amazon.co.uk/gp/product/B00 ... UTF8&psc=1

as suggested by MicroBob. LBB is the model.

MicroBob
Posts: 3154
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Location: Northern Germany

Re: My first attempt at using a hand microtome

#5 Post by MicroBob » Fri Apr 02, 2021 7:14 pm

Hi,
nice sections! I like the dark field image better than the phasec ontrast one. For botanic sections phase doesn't offer additional detail and adds artifacts. Next you might try staining. Wacker W3A is quick and nice.

Bob

Voyager-1
Posts: 27
Joined: Fri Mar 12, 2021 7:48 pm

Re: My first attempt at using a hand microtome

#6 Post by Voyager-1 » Sat Apr 03, 2021 12:39 am

Pippo1234,

Thank you for the blade information! Will be ordering a few packages.
Hoping my attempts may some day come close, yours looks like top of the line!
Thanks again!

V

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mrsonchus
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Location: Cumbria, UK

Re: My first attempt at using a hand microtome

#7 Post by mrsonchus » Sat Apr 03, 2021 11:31 am

Very nice start!
A book on plant anatomy would add many layers of interest to your sectioning. The more you look over such revealed details the more the complexity adds to the pleasure and mystery!

Congratulation for these fine sections & images!
John B

pippo1234
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Joined: Thu Dec 24, 2020 8:21 am

Re: My first attempt at using a hand microtome

#8 Post by pippo1234 » Wed Apr 07, 2021 6:57 am

Some clumsy attempts at staining (no previous fixing). Parsley petiole using (a) fast green and (b) safranin and safranin-stained yew leaf. The Nikon 10x achro is a bit soft. After realising that 10x is possibly the most useful magnification for botanic work I have successfully bidded on its plan counterpart. I am looking forward to seeing what difference it makes. My plan 20x and 40x run rings around their standard achro counterparts.

Could somebody please point me in the direction of some reference on processing, including staining, for aqueous mounts? The most useful information I have found was a thread started by John B, but all the textbooks I have consulted describe procedures involving lots of alcohol processing for final mounting in Canada balm. It all feels a bit daunting for a beginner....

@ John: you are right about the usefulness of a plant reference. I have downloaded Bowes which you mentioned in a previous reference. It's fantastic! It seems written for microscopists. Thanks for the heads up!

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MicroBob
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Re: My first attempt at using a hand microtome

#9 Post by MicroBob » Wed Apr 07, 2021 7:34 am

Hi,
nice sections and first stains! That parsley petiole is also a very nice object with a good mix of different structures.
Safranine is a regresive stain when used in higher concentrations, it stains everything and you have to remove from where you don't want it. If used in very low concentrations (1 drop of 1% solution on 100ml of water, just faintly red) and applied for one hour cold, it works a bit more precisely.
In Germany we use this method to get permanent slides:

- Fixing in AFE 1 solution http://www.aeisner.de/rezepte/fixant2.html 1 day for 1cm pieces or 20 minutes for sections
- 70%, 50%, 30% ethanol into water
- staining
- 100% isopropanol two times, first very quick to get all water aout quickly as the mix tends to differentiate the stains quickly. I do this on the slide and remove Iso with paper tissue.
- Euparal (wich can cope with a little residual water)
- Drying with M6 bolt on top of cover slip, preferably at 45°C to increase drying speed, still wakes a week

This is fairly easy to do when you do steps 2 and 3 in a well slide or watch glass and step 4 on the slide. For the alcohols I use 10ml dropper bottles, for Euparal a real balsam glass. The ethanol can be the modern non stinky non-drinkable ethanol. The slides age fairly well, the slight acidity of the Euparal can decrease some stains over time, but no crystallisation, darkening, popping of cover slips etc. A very simple and good method and very clear slides after some drying time.

AFE1: Leaving the formaldehyde out is not ideal but acceptable, you end up mixing household non-drinkable ethanol with concentrated vinegar.

Bob

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