Potted Bamboo Hand Sectioned

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mrsonchus
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Potted Bamboo Hand Sectioned

#1 Post by mrsonchus » Mon Aug 19, 2019 6:39 pm

Hi all,
I've been playing-around hand-sectioning some of the very small stems of a Bamboo (Phyllostachys aurea) plant standing in a large pot in my garden. These stems although very small, about 2mm diameter, are very tough and quite hard to cut through. I used a single-sided razor with the stem pieces held down by my thumb. The blade I aligned with the back of my thumb to move back the teeniest bit as successive sections were taken.

The potted Bamboo,
Image

The equipment is simple enough.
Image

The Safranin (red stain mainly for lignin - the hard reinforcing substance that supports plants) is a powder and for 5g (enough to make at least 500ml of stain!) is very inexpensive. This stain will stain just about any plant material and is simply mixed with water. I buy mine via e-bay from 'scuddlebut'....
Image

The mountant is a temporary one - months not days though. It's alcohol-based so specimens are best mounted from alcohol into this mountant. This little jar lasts years, and is very cheap from Brunel Microscopes - I think it's 'proper' name is 'Euparal'...
Image

After cutting some sections and placing them into some water, pick-up each section with a tweezer or froceps and dip into 1% Safranin for about 5 seconds, then put into water to remove excess stain. Then into 50% alcohol for about 30 minutes before placing into 95% alcohol for another 30 minutes before mounting.

Here's a nice section perfectly good enough to show the stem structure - in this case the stem of this Bamboo shows all the typical characters of a monocotyledonous plant, and a whole lot more....

This is a temporary-mount as described, with coverslip,
Image

Here's a 4x objective, 2-image stitch, in brightfield,
Image

Here at 10x,
Image

This is at 4x with the use of the 'DF' darkfield stop of the phase contrast condenser in my BX40,
Image

This is with the condenser set to the 'Ph3' setting, normally used for a high-power (above 40x) phase-contrast objective. Used as here with a normal BF 4x objective, the phase-stop of the condenser gives a quite godd darkfield,
Image

This is at 20x, fiber details begin to emerge....
Image
and,
Image

At 40x,
Image
and,
Image

60x, no wonder this is a tough stem to cut - fibers everywhere beneath the epidermis as well as surrounding the scattered vascular bundles seen in earlier images!
Image

Here's the 10x objective and the DF setting of the condenser,
Image

10x phase contrast objective with phase contrast condenser, not a very good phase result. Phase isn't known for being much use for sections that are stained, or more crucially, as thick as these. Phase performs as intended with thin and unstained or transparent tissue.
Image

I though some may like to see what may be easily, cheaply and quickly achieved with a razor, some stain, alcohol and quick & easy mountant.
Last edited by mrsonchus on Mon Aug 19, 2019 7:15 pm, edited 2 times in total.
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Re: Potted Bamboo Hand Sectioned

#2 Post by MichaelG. » Mon Aug 19, 2019 7:05 pm

mrsonchus wrote:... I used a single-sided razor with the stem pieces held down by my thumb. The blade I aligned with the back of my thumb to move back the teeniest bit as successive sections were taken. ...
You must be quite extraordinarily good at that, John :o
I bet you can slice Parma Ham to the requisite thickness too!

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Re: Potted Bamboo Hand Sectioned

#3 Post by mrsonchus » Mon Aug 19, 2019 7:13 pm

MichaelG. wrote:
mrsonchus wrote:... I used a single-sided razor with the stem pieces held down by my thumb. The blade I aligned with the back of my thumb to move back the teeniest bit as successive sections were taken. ...
You must be quite extraordinarily good at that, John :o
I bet you can slice Parma Ham to the requisite thickness too!

MichaelG.
Ha! It's really easy after a few goes, as long as the razor is new and sharp - you'd be surprised just how simple this is to do!
Have a go - you know you must! :D :D

Make several of different stems, e.g. a succulent, a docotyledon, a monocotyledon, a woody stem etc. Then get them together and observe their differences and similarities - then.... find out why they are this way.... then 4 weeks later you'll be hooked on Botany and have twigs, leaves and flowers everywhere, a couple of new microscopes and a load of chemicals on their way to you!! :D :D

Really, Botany is great for the microscope!
Thanks for looking Michael my friend.
John B

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Re: Potted Bamboo Hand Sectioned

#4 Post by Hobbyst46 » Mon Aug 19, 2019 7:15 pm

Fascinating protocol and images as ever, John B!

Tip: never get the Bamboo escape from the pot. Bamboo is wildly invasive. Digging out a small grown up Bamboo forest (done that) is hell of a job...
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Re: Potted Bamboo Hand Sectioned

#5 Post by mrsonchus » Mon Aug 19, 2019 7:21 pm

Hobbyst46 wrote:Fascinating protocol and images as ever, John B!

Tip: never get the Bamboo escape from the pot. Bamboo is wildly invasive. Digging out a small grown up Bamboo forest (done that) is hell of a job...
Ahhh Hobby, I know what you mean! I initially, in my sheer ignorance, bought this fine plant cheaply as it was clearly ailing in the garden-center....
I planted it by the fence, only to have my neighbour on my doorstep several days later almost hysterical and gesticulating, muttering words such as "invasive", phrases such as "taking-hold" and "overrunning my patio..."
Looking the beast up I soon discovered to my slight chagrin that the histrionic chap was quite correct.... My Wife quickly, very quickly in fact, 'enlightened me' as to what I must do - and this now beautiful specimen lives in a 3ft-ish pot on tarmac, well-away from any patios etc ripe for invasion!

Thanks for looking old chap. :D
John B

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Re: Potted Bamboo Hand Sectioned

#6 Post by einman » Mon Aug 19, 2019 7:46 pm

I always enjoy your work- so precise, and informative. Simply excellent!

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Re: Potted Bamboo Hand Sectioned

#7 Post by einman » Mon Aug 19, 2019 7:49 pm

mrsonchus wrote:
Hobbyst46 wrote:Fascinating protocol and images as ever, John B!

Tip: never get the Bamboo escape from the pot. Bamboo is wildly invasive. Digging out a small grown up Bamboo forest (done that) is hell of a job...
Ahhh Hobby, I know what you mean! I initially, in my sheer ignorance, bought this fine plant cheaply as it was clearly ailing in the garden-center....
I planted it by the fence, only to have my neighbour on my doorstep several days later almost hysterical and gesticulating, muttering words such as "invasive", phrases such as "taking-hold" and "overrunning my patio..."
Looking the beast up I soon discovered to my slight chagrin that the histrionic chap was quite correct.... My Wife quickly, very quickly in fact, 'enlightened me' as to what I must do - and this now beautiful specimen lives in a 3ft-ish pot on tarmac, well-away from any patios etc ripe for invasion!

Thanks for looking old chap. :D

My wife has been after me for sometime to plant Bamboo along our fence line. I have cause for not doing so. There is a local park here that has sections of Bamboo that seems to be doing quite well and is contained. I should look into what they are doing. The zoo also has many Bamboo plantings. Maybe they are simply spending a lot of time pruning etc...

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Re: Potted Bamboo Hand Sectioned

#8 Post by einman » Mon Aug 19, 2019 7:56 pm

YOu should consider writing a book using your postings here. The photos,explanations and technique are first rate. I would not hesitate to purchase a copy.

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Re: Potted Bamboo Hand Sectioned

#9 Post by MicroBob » Mon Aug 19, 2019 8:17 pm

Hi John,
what exactly was the reason why you bought your big microtome? Great hand sections and beautiful images!
It is always a good idea to keep in mind that it is possible to do microscopy with comparatively simple means.
A report like this shows every newbie that it is his personal fault when he doesn't get the same results! :mrgreen:

Bob

BTW: Our bamboo was easy to kill - once with the chain saw, pond foil on top and dead it was. But this isn't the rule.

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Re: Potted Bamboo Hand Sectioned

#10 Post by mrsonchus » Mon Aug 19, 2019 8:30 pm

Hi einman!
There must be many varieties with different growth habits - I'd take some advice as you say, the folk who seem to have this beastie 'under control' will very likely steer you right. I just happened-upon this lonesome and ailing specimen without realising what a tenacious and rapidly-spreading monster it could be!

I must say I really like it, especially as it's a rather interesting plant for comparison, especially with grasses and sedges I think.... I'm sure to return to this plant with a view to making some permanent slides I think. I'd like also to have a look at some longitudinal sections along those massive fibers..... I bet Toluidine-blue would stain this one well....

Thanks for your kind comment einman.

That reminds me actually, a few days ago I took a few quick hand sections of some ornamental grasses in a trough in my garden, but didn't bother to stain or mount them, but I did take a few images. I often do this to give me an idea of whether a plant wiull section in the orientation I would like.

Here's a grass-leaf (ornamental, possible 'zebra grass') hand sectioned a few days ago, just in a bowl of water and 'stood up' for the camera....
Image

At first glance it looks very 'sedge-like',
and,
Image

It seems to have many features in common with my Carex.pendula (AKA 'Pendulous Sedge') sections structurally speaking....
Here's a Carex.pendula section, look at the uni-laterally-thickened fibrous cells surrounding the bundle - the grass-leaf has these also.
The Carex.pendula leaf TS looks very similar at this level,
Image

Here's a look at the grass-leaf hand-section bundle close-in,
Image

Here in Carex.pendula the similarly thickened (i.e. unevenly parallel to bundle sides). At first I thought this may be an artifact of an oblique microtome section, but now I think, having seen the same in the hande-sectioned grass above, that this is a genuine anatomical feature of the vascular bundle's border.... Fascinating!
Image

These bundles are very similar indeed, as is the gross morphology of the leaf sections I think...
John B

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Re: Potted Bamboo Hand Sectioned

#11 Post by mrsonchus » Mon Aug 19, 2019 9:45 pm

MicroBob wrote:Hi John,
what exactly was the reason why you bought your big microtome? Great hand sections and beautiful images!
It is always a good idea to keep in mind that it is possible to do microscopy with comparatively simple means.
A report like this shows every newbie that it is his personal fault when he doesn't get the same results! :mrgreen:

Bob

BTW: Our bamboo was easy to kill - once with the chain saw, pond foil on top and dead it was. But this isn't the rule.
Thanks Bob, I love making permanent sections, the ability to have a collection available is a sheer pleasure. The detail, ah... the finest of details!
John B

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Re: Potted Bamboo Hand Sectioned

#12 Post by 75RR » Tue Aug 20, 2019 3:37 pm

Nice job cutting that by hand (had to rephrase my original sentence) + some great images

Here is a link to a practical and effective method to slice samples:

http://www.microscopy-uk.org.uk/mag/ind ... icera.html
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Re: Potted Bamboo Hand Sectioned

#13 Post by mrsonchus » Tue Aug 20, 2019 3:50 pm

75RR wrote:Nice job cutting that by hand (had to rephrase my original sentence) + some great images

Here is a link to a practical and effective method to slice samples:

http://www.microscopy-uk.org.uk/mag/ind ... icera.html
Ah yes, I remember it well! When I started I read and tried all of the late Walter-D's protocols. He's a bit of a Hero as far as I'm concerned! I always point beginners to his superb articles, and the little book is a nice thing to have also.

The two blades held together has of course it's limits - OK for small soft leaves though. The W-D articles are simply superb, and any beginner following this thread would find a real treat if searching this forum for his material, just a good as when he wrote them.

Had a quick look at the mount today, all bubbles are gone and it's solidifying nicely - it'll be interesting to see how long it stays good for...
An image from the mount today, still looks good,
Image

Thanks for looking 75' :D
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Re: Potted Bamboo Hand Sectioned

#14 Post by MicroBob » Tue Aug 20, 2019 9:19 pm

Hi John,
your last image is really great! It is nice to see that you acheive such good images just after changeing microscopes.

I know Walter Dionis articles from the Micscape magazine which I have read up and down. You wrote "would find a real treat if searching this forum for his material" - did he post in this form?

Bob

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Re: Potted Bamboo Hand Sectioned

#15 Post by mrsonchus » Tue Aug 20, 2019 10:23 pm

MicroBob wrote:Hi John,
your last image is really great! It is nice to see that you acheive such good images just after changeing microscopes.

I know Walter Dionis articles from the Micscape magazine which I have read up and down. You wrote "would find a real treat if searching this forum for his material" - did he post in this form?

Bob
Hi Bob, I don't think he posted here, it's the Micscape articles I'm thinking of - it's been so long since I read them that I'd actually forgotten where I read them! :oops:

For others, here's the link to Walter D's articles...

I learned so much from them. I also gained a lot of confidence from Walter's basic and considered approach, which as my results improved, encourage me to the stage that I'm at now, making my own permanent Botanical slides.... In all honesty, the years of subsequent enjoyment with my 'scopes and Botany, started with this fine fellow's work.
John B

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Re: Potted Bamboo Hand Sectioned

#16 Post by einman » Tue Aug 20, 2019 11:48 pm

MicroBob wrote:Hi John,
your last image is really great! It is nice to see that you acheive such good images just after changeing microscopes.

I know Walter Dionis articles from the Micscape magazine which I have read up and down. You wrote "would find a real treat if searching this forum for his material" - did he post in this form?

Bob
My thoughts exactly!

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Re: Potted Bamboo Hand Sectioned

#17 Post by Dave S » Fri Aug 30, 2019 6:43 pm

Excellent work John, your skill is to be admired.

What was the set up for capturing those impressive, and inspiring images?
Brunel SP100 (with 4x, 10x, 40x,60x, and 100x (oil) plan objectives), and Canon EOS 4000d Camera (microscopy use only)

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Re: Potted Bamboo Hand Sectioned

#18 Post by mrsonchus » Fri Aug 30, 2019 7:26 pm

Hi Dave, the setup is a Canon 200D mounted atop the photo-tube of my Olympus BX40.
The photo-tube has within it an Olympus 'PE2.5x' photo-eyepiece that projects the image onto the camera's sensor.
The system is an infinity system with of course infinity PlanC UIS2 objectives, a 1.25 n.a. Abbe condenser and an LED 10W 6000K lamp conversion.
I control the camera with Canon's superb 'Utility v3' software, via U.S.B. tethering to a Windows 10 Linx tablet PC.

Here it is,
ws_BX40 with camera 1.jpg
ws_BX40 with camera 1.jpg (71.29 KiB) Viewed 1585 times
and,
Image

The photo-eyepice sits inside the tubes at the junction of the black and cream coloured parts,
Image

It works beautifully both for photography and eyepiece observation, especially with the LED converter I bought from 'RetroDiode' via e.bay.
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Re: Potted Bamboo Hand Sectioned

#19 Post by Hobbyst46 » Fri Aug 30, 2019 7:40 pm

Hi mrsonchus,
I notice that you cover the camera and tube top with a plastic bag. I would fear condensation, and for my microscope use covers made of heavy cloth.
The protocol and images are as attractive as ever and the darkfield ones shine among the brightness.
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Re: Potted Bamboo Hand Sectioned

#20 Post by Dave S » Fri Aug 30, 2019 7:47 pm

A very nice setup indeed John, which clearly produces some impressive results.

Having whetted my appetite for micro photography, with the Bresser Researcher Trino, I have started the process of looking at upgrading to a scope with Plan objectives, and Kohler illumination.

As such, I have provisionally sold the Bresser, and am looking at other options, without going over the £1k mark, and am making enquires with people like Bresser, Brunel, and GT Vision.

I will of course retain the EOS 4000d, which I bought specifically for microscopy use. I didn't want to put my 'pride and joy' EOS 5D MklV on a microscope, plus the fact it is a 'heavy beast'.
Brunel SP100 (with 4x, 10x, 40x,60x, and 100x (oil) plan objectives), and Canon EOS 4000d Camera (microscopy use only)

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Re: Potted Bamboo Hand Sectioned

#21 Post by mrsonchus » Fri Aug 30, 2019 8:43 pm

Hobbyst46 wrote:Hi mrsonchus,
I notice that you cover the camera and tube top with a plastic bag. I would fear condensation, and for my microscope use covers made of heavy cloth.
The protocol and images are as attractive as ever and the darkfield ones shine among the brightness.
Hi Hobby' - never had any problems with condensation. The bag isn't air-tight or even close. Just makes a good dust cover. I also use a great-big clear plastic bag to put over the BX40 as it's dust cover - again, loose and open at desk-level. Works well.
John B

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Re: Potted Bamboo Hand Sectioned

#22 Post by mrsonchus » Fri Aug 30, 2019 8:44 pm

Dave S wrote:A very nice setup indeed John, which clearly produces some impressive results.

Having whetted my appetite for micro photography, with the Bresser Researcher Trino, I have started the process of looking at upgrading to a scope with Plan objectives, and Kohler illumination.

As such, I have provisionally sold the Bresser, and am looking at other options, without going over the £1k mark, and am making enquires with people like Bresser, Brunel, and GT Vision.

I will of course retain the EOS 4000d, which I bought specifically for microscopy use. I didn't want to put my 'pride and joy' EOS 5D MklV on a microscope, plus the fact it is a 'heavy beast'.
Hi Dave, I'm interested to see how you choose, and what 'scope you find! Keep us updated!
John B

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Re: Potted Bamboo Hand Sectioned

#23 Post by Dave S » Sat Aug 31, 2019 8:56 am

I know that the majority of enthusiasts only buy used high-end gear, but I only ever buy new, whether it be a car, photography gear, or whatever. Its just something that I do, and enjoy doing.

So, it will be of Chinese origin, and new. That said, most of the ' big names' outsource their manufacturing to China. albeit I suspect with tight quality controls.

My Bresser Trino, did not disappoint, and its just that I have the desire to move up the Plan ojectives, and Kohler illumination.

The Bresser TRM301 was No.1 on my 'wants list', except that is uses Halogen illumination, albeit they are looking to bring out an LED version at some future date.

I'll 'weigh up' up all the options, and see where I go from there.

In the mean time, I'll continue to enjoy looking at the work of your good self, and others.
Brunel SP100 (with 4x, 10x, 40x,60x, and 100x (oil) plan objectives), and Canon EOS 4000d Camera (microscopy use only)

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Re: Potted Bamboo Hand Sectioned

#24 Post by 75RR » Sun Sep 01, 2019 5:28 am

Dave S wrote: "That said, most of the ' big names' outsource their manufacturing to China."

Source?
Last edited by 75RR on Mon Sep 02, 2019 9:00 am, edited 1 time in total.
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Re: Potted Bamboo Hand Sectioned

#25 Post by Hobbyst46 » Sun Sep 01, 2019 8:17 am

Dave S wrote:I know that the majority of enthusiasts only buy used high-end gear, but I only ever buy new, whether it be a car, photography gear, or whatever. Its just something that I do, and enjoy doing.

So, it will be of Chinese origin, and new. That said, most of the ' big names' outsource their manufacturing to China. albeit I suspect with tight quality controls.

My Bresser Trino, did not disappoint, and its just that I have the desire to move up the Plan ojectives, and Kohler illumination.

The Bresser TRM301 was No.1 on my 'wants list', except that is uses Halogen illumination, albeit they are looking to bring out an LED version at some future date.

I'll 'weigh up' up all the options, and see where I go from there.

In the mean time, I'll continue to enjoy looking at the work of your good self, and others.
I have recently tested a newunbranded 4X Plan objective and it wa not as plan as one expects. Not for high level photography. I think that this is not only a quality control issue but related to design as well. So, the fact that production lines have been moved to othdr countries does not mean that high quality is being spread and internalized around the new mfg neighborhoods.
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Re: Potted Bamboo Hand Sectioned

#26 Post by viktor j nilsson » Sun Sep 01, 2019 8:21 am

Actually, since in most cases you are going to stack the pictures anyway, it might not be a big deal if a 4x isn't plan. The Lomo 3.7x for example is exceptionally sharp and free of CA when used for macro work, but it is far from plan.

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Re: Potted Bamboo Hand Sectioned

#27 Post by mrsonchus » Sun Sep 01, 2019 11:17 am

Must agree with you viktor - the FOV image through a camera and 'scope is usually nowhere near that of the objective, for various reasons. The central portion of the image is very often indeed most relevant for photomicrography, planarity of objective notwithstanding....
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Re: Potted Bamboo Hand Sectioned

#28 Post by viktor j nilsson » Sun Sep 01, 2019 3:17 pm

That is true, but I also mean that stacking algorithm will find and combine the sharp parts, even if they occur in different pictures. So the central parts will be sharp in one picture, and the peripheral parts will be sharp in another picture.

Put another way, a lens that isn't plan may be very sharp across the entire field, it's just that the central and peripheral regions focus at different points. In that case, stacking can produce excellent results. Other non-plan lenses, however, may simply not produce a sharp image at the periphery, in that case stacking won't help. One has to try to find out.

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Re: Potted Bamboo Hand Sectioned

#29 Post by 75RR » Mon Sep 02, 2019 4:50 am

"Actually, since in most cases you are going to stack the pictures anyway, it might not be a big deal if a 4x isn't plan."

It is true that digital cameras and the ability to stack and stitch have changed microscope photography substantially, and as you say an objective that is sharp need not be plan as stacking provides a workaround.

It does not bode well however for the quality of said objective if it claims to be Plan and is sold as such and isn't.

There are also occasions where one may not want to stack, and having to, because an achromat or at best a semi-plan has been labeled plan is not really on.

One wonders if these manufactures like many resellers depend on buyer passivity/apathy (can't be bothered with the hassle to send it back) to sell a substandard product.
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Re: Potted Bamboo Hand Sectioned

#30 Post by MichaelG. » Mon Sep 02, 2019 7:37 am

viktor j nilsson wrote:
... Put another way, a lens that isn't plan may be very sharp across the entire field, it's just that the central and peripheral regions focus at different points. In that case, stacking can produce excellent results. Other non-plan lenses, however, may simply not produce a sharp image at the periphery, in that case stacking won't help. One has to try to find out.
.

You make an important point there, viktor

Curvature of field is nicely hidden by the stacking process, whereas other optical artefacts are more troublesome.

Note, however, my use of the word hidden
In the 'illustrative' images that we mostly produce, this is of no consequence ... but for anyone taking important dimensional measurements it could be a minefield.

MichaelG.
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