Starting out with Microscopy

[Neils]

In lieu of doing a 'proper' course or attempting the BBKA/SBA module on it, does anyone have any good links for some reading material around how to best use the things and/or prepare slides?

If there are also answers seemingly to simple questions like what's the difference between my compound and stereo microscope, even better. Brunel helpfully tells me that the laws of physics prevent one microscope being used for both functions, but what laws and why?

I have a x100 objective [lens?] supplied with mine, which apparently requires some sort of oil that wasn't supplied, I'll chase up the suppliers next week to actually get hold of some, but it's pretty vague as to why I need to use this oil or what it is so I can get hold of some myself, I assume it's something to do with light refraction.

I was thinking a blob of honey might make a good first sample slide, and I dare say if it will stop raining long enough to get the hives a couple of dead bees will give ample things to look at to get the hang of things.

Other than the microscopes and slides I don't currently have any other gadgets/tools. A scalpel would seem to be an obvious other thing to obtain, but what else should I have around for when I start to try and play with them in anger?

[robin118]

Santa brought me the new beecraft book " Practical Microscopy for Beekeepers" by Bob Maurer. Having not even seen a microscope since I was at school never mind using one and thinking I should at least be able to test my own bees for Norsema. Not noing where to start and having read a couple of revues on this book I asked for it for Christmas. It describes the different types of microscopes needed their uses how to prepare slides for viewing and bees for dissection.

[Ruary]

In lieu of doing a 'proper' course or attempting the BBKA/SBA module on it, does anyone have any good links for some reading material around how to best use the things and/or prepare slides? Bob Maurer's book is quite good.
For a beginner Marson's practical microscopy is better.



If there are also answers seemingly to simple questions like what's the difference between my compound and stereo microscope, even better. Brunel helpfully tells me that the laws of physics prevent one microscope being used for both functions, but what laws and why? A stereo microscope is designed to give relatively low magnification and to have the view upright and not laterally inverted. It also has a large working distance. This allows you to manipulate objects under it as A0 the low magnification minimisess the effect of hand shake, B0 the upright etc. image means that if you move your hand to the right the image also moves to the right. This allows for dissection.
A compound microscope works on much larger magnifications the magnification being the ratio of the distance form the object to the lens and the distance of the lens to the image. This means that to keep the microscope a workable size the working distance must be small, and for the same reason they do not install the extra prisms or lenses to correct the image so the image under a compound microscope is inverted both vertically and laterally.


I have a x100 objective [lens?] supplied with mine, which apparently requires some sort of oil that wasn't supplied, I'll chase up the suppliers next week to actually get hold of some, but it's pretty vague as to why I need to use this oil or what it is so I can get hold of some myself, I assume it's something to do with light refraction. Unless you are looking at bacteria you don't really neeed the oil immersion lens, but the reason why oil is required is that the focal length of an X100 lens is tiny and to get that the lens needs to be of very small radius light without the oil will be reflected off the lens rather than refracted. If oil is used the oil is of the same refractive index as the lens and so the reflection does not occurr resulting in a much brighter and clearer image.


Other than the microscopes and slides I don't currently have any other gadgets/tools. A scalpel would seem to be an obvious other thing to obtain, but what else should I have around for when I start to try and play with them in anger? Slides cover glasses, water pestle mortar ( for nosema). glass rods etc. A scalpel is ot what you want at the moment.
Come to Gormanston and we will put you on the right track.

[Bumble]

A scalpel would seem to be an obvious other thing to obtain, but what else should I have around for when I start to try and play with them in anger?

You'll need more than one scalpel once you get started. You can get a dissecting kit for less than £20, or see if a friend who studied Biology has still got theirs tucked away in a drawer somewhere.

[Ruary]

One scalpel holder and a packet of blades for internal dissection but for that you need a stereo microscope, I gather the O.P. has a compound one as he has an oil imersion lens.
Dissection of external anatomy does not need a scalpel (see Marson's method), and can be done without stereo help.
Nosema testing does not need dissection kit. neither does pollen.
Pollen isample in honey on low budget equipment is best done by diluting a sample of about 10gm into 100cc of clean water and allowing the pollen to settle in a draft free location. Decant the supernatant liquid and smear the sediment onto a slide glass, stain with glycerine jelly and cover with cover glass.
I repeat the invitation come to Gormanston, we will show you everything and there is far more beekeeping information on offer there.

[gavin]

For folk a bit more local than Neil (you're welcome too if you want!), Alan Riach is bringing his microscope roadshow to Perth a week tomorrow. The SBA also organise microscopy workshops although this year's has now been cancelled due to insufficient interest. Look out for one next year instead if you don't fancy making the trip to see Ruary in action in Gormanston.

An excerpt from an email the other day from Linda, secretary of the Perth group:

The meeting will be held on 8th January 2013 at our usual venue of St John's Episcopal Church Hall, Princes Street, Perth, PH2 8LJ.

The meeting will start at 7.30pm.

The talk will be given by Alan Riach (the SBA's Education Convener) with the topic being "Microscopy" .
There will be chance to get "hands (or infact eyes!) on" various microscopes & subject material during this presentation.
Looking forward to seeing you at what promises to be a fascinating talk.

[Neils]

One scalpel holder and a packet of blades for internal dissection but for that you need a stereo microscope, I gather the O.P. has a compound one as he has an oil imersion lens.
I have both :)



I repeat the invitation come to Gromanston, we will show you everything and there is far more beekeeping information on offer there.

I'm still trying to sell this one to the Mrs :D

[Jon]

I'm still trying to sell this one to the Mrs :D

I'll be there this year so you can tell her you will be in good company!

[The Drone Ranger]

If you are interested in microscopy as a hobby there are a couple of links below
http://www.microscopy-uk.org.uk/index.html
http://www.quekett.org/
http://www.microscopy-uk.org.uk/mag/indexmag.html
some great ideas for those who like messing about with microscopes

I was hoping to try this
http://www.quekett.org/resources/making-rheinberg-illumination-discs

[mazza]

[QUOTE=gavin;15107 The SBA also organise microscopy workshops although this year's has now been cancelled due to insufficient interest. Look out for one next year instead if you don't fancy making the trip to see Ruary in action in Gormanston.

[/QUOTE]

I'm slightly confused Gavin... I just spent the weekend helping to host a 2 day SBA microscopy training workshop held at SASA! We had 21 (and a half!) people come along and i think everyone had a great time. The dissection section was the favourite, with a lot of fierce concentration going into preparing and mounting the bee specimins:)

[gavin]

Yeah - I'm confused too (obviously)! Don't know where I got that from but I thought it was right at the time. Did hear recently that there was one, but I never went back to correct the remark above. Apologies ....

Feel free to advertise them on here in future!

The half - was that a young person, someone with body parts missing, or was there an accident on the day?! I shuddered when I saw 'dissection' in the same paragraph .....

[mazza]

Ha ha... No, fortunately there were no major accidents or limb removals! (just some minor finger slicing;)) I'm surprised the organisers didn't advertise it on the forum... maybe it was published on the main SBA website? The half person was someone who could only attend on the Saturday...

[gavin]

Ah ... *that* kind of half person!

Were people bringing samples to look at on the day? These dwindling colonies (and the poor flying weather much of last year) could easily mean a resurgence of some of the traditional bee lurgies. I like that word, lurgie. The Bee Lurgie Unit, SASA ... has a certain ring to it?! :)

We have microscopes in the local association but don't get them out enough. Maybe we should do something with them this year.

[The Drone Ranger]

I've done the SBA training it was some years ago at Stirling Uni with Ian Craig and a cast of experts (including 2 from the bee disease unit)
It was excellent we used their gear and only had to take 30 bees and some flowers for pollen.
Most of my bees revived despite having been given IKF (Insect Killing Fluid) and 24 hours in the freezer
I think exposure to Neonic pesticides must have given them immunity or turned them into zombies

The links I gave earlier are great if you want to get some use from, and advice on, microscopes : you soon get fed up looking at the contents of bee abdomens :)

This site is good source of information about microscopes http://www.microscopemaster.com/stereo-microscope.html all very clear non jargon stuff

[The Drone Ranger]

We have microscopes in the local association but don't get them out enough. Maybe we should do something with them this year.

If memory serves me the nosema infected bees we sampled were brought by the staff from bee diseases
Ian Craig brought his pollen slides, lots of them to identify, and not many air bubbles unlike mine :)
We didn't have any previously stained and mounted nosema samples but everyone agreed that would have been helpful.

[mazza]

If memory serves me the nosema infected bees we sampled were brought by the staff from bee diseases
Ian Craig brought his pollen slides, lots of them to identify, and not many air bubbles unlike mine :)
We didn't have any previously stained and mounted nosema samples but everyone agreed that would have been helpful.

For nosema identification, the samples aren't usually stained in any way, so providing stained examples might not help people to recognise nosema sporeswhen testing their own bees at a later date. Gut contents of 30 bees are crushed in 15ml of distilled water, and a droplet is placed on a slide under a coverslip. This is then examined at x400 and the nosema spores are readily identified by their size, shape and colour. Next time however, we could make up some extra slides beforehand in a temporary mount and these can be passed around for examination:) It's always useful to get feedback on how we can improve the course, so thanks for the suggestion.

Gavin - Yes, there were a few people who had brought in bees which turned out to have some level of nosema infection (the first time i have seen this at the microscopy course; normally we supply infected material for analysis). We didn't see any acarine symptoms, but then again only a small number of bees were examined! I suspect the long winter confinement has contributed to an increase in the normal infection levels observed at this time of year...

[The Drone Ranger]

I'm an old school dry em and dye em guy :)
That's half the fun Lol!

Don't tell me I have to stop now and go cold turkey, what will I do with that Litre of stain ?

here's Randy Olivers method
http://www.microscope.com/education-center/articles/microscopy-nosema/

Ruary posted a method for making and staining more permanent slides on the BBKA forum
for the people interested in how it was done

[Neils]

Finally sent off the firms for one of the NDB short courses in July down in Devon. I'm taking a break from modules/assessments this year to, hopefully, concentrate on just beekeeping and preparing for the general husbandry assessment. But a weekend playing with microscopes seemed too good an opportunity to pass up, I've been putting off doing anything with them for far too long.

[The Drone Ranger]

Finally sent off the firms for one of the NDB short courses in July down in Devon. I'm taking a break from modules/assessments this year to, hopefully, concentrate on just beekeeping and preparing for the general husbandry assessment. But a weekend playing with microscopes seemed too good an opportunity to pass up, I've been putting off doing anything with them for far too long.

Hi Neils

Practical Microscopy by J. Eric Marsdon not only covers making slides of bee parts etc it has lots of other great info
When people talk about softening bees for slide mounts etc they are usually quoting from his book

It used to be sold by Nothern Bee books but I googled for them and had no luck
Amazon http://www.amazon.co.uk/Practical-Microscopy-J-Eric-Marson/dp/B001BP5NSW out of stock
Somebody on here will know where you can get a copy or the library might get one

[Ruary]

Hi Neils

Practical Microscopy by J. Eric Marsdon not only covers making slides of bee parts etc it has lots of other great info
When people talk about softening bees for slide mounts etc they are usually quoting from his book

It used to be sold by Nothern Bee books but I googled for them and had no luck
Amazon http://www.amazon.co.uk/Practical-Microscopy-J-Eric-Marson/dp/B001BP5NSW out of stock
Somebody on here will know where you can get a copy or the library might get one Try Brunell microscopes I have just looked and it seems to be in stock.

[The Drone Ranger]

Try Brunell microscopes I have just looked and it seems to be in stock.

Genius Well done that man :)
it's £12

Ruary
you had a post on BBKA a couple years ago on making stained permanent (ish) nosema slides
what is your latest advice on making them ? or is it not worth bothering with?

[Ruary]

Ruary
you had a post on BBKA a couple years ago on making stained permanent (ish) nosema slides
what is your latest advice on making them ? Well I suppose I could say come to Gormanston and ask me there, but I am feeling kind so my process is to place a drop of distilled water containing the spores on the sldie and add nigrosin stain. Spread the drop so that it covers the extent which would be covered by the cover-glass. Dry it on a hot plate until almost dry and then mount in plain glycerine jelly. Check that it is OK. allow to set etc and seal with whatever varnish you use.
I am wondering whether it would work with drying the slide completely.

[The Drone Ranger]

I'm No expert Ruary


This method dries the specimen completely but they are fixed in ethanol so that would seem to be an important step and I don't think the slides would store

When air-dried, ethanol-fixed smears of infected tissue are stained with Giemsa’s stain (10% in 0.02 M phosphate buffer) for 45 minutes.
Nosema apis spores will have a distinctive appearance, with thick unstained walls and an indistinct blue interior, without visible nuclei. Insect cells, fungal spores and other
protozoa stained in this way will generally have thinner walls, blue/purple cytoplasm and magenta-coloured nuclei

Clipped from http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.02.04_NOSEMOSIS.pdf

[gavin]

No wish to spoil your fun, but wet (water) mounts of bee abdomen juice are fast and easy. When Nosema is there it is usually abundant and really obvious - those jiggling rice grains. OK, you can't keep the slides, but that's all I've ever done. Pollen is different. You can usually see the structure better if it is stained in one way or another.

It might be interesting to make dry mounts of Nosema to see if you can spot the difference between the fat ones (N. ceranae) and the thinner ones (N. apis). I think.

[Ruary]

No wish to spoil your fun, but wet (water) mounts of bee abdomen juice are fast and easy. When Nosema is there it is usually abundant and really obvious - those jiggling rice grains. OK, you can't keep the slides, but that's all I've ever done. Pollen is different. You can usually see the structure better if it is stained in one way or another.

It might be interesting to make dry mounts of Nosema to see if you can spot the difference between the fat ones (N. ceranae) and the thinner ones (N. apis). I think. Yes, fresh is best but what if you need to show nosema and don't have a sample of infected bees. That is what my method is designed to do.
DR I have a home lab no Giemsa but plenty of nigrosin, I tried the glycerine jelly as an experiment and it has worked. It won't cater for examination at X1000 but the thickness of the mount is OK for X400.


Gavin I think you might have your nosemas reversed apis is more square ended, nosema ceranae is slighly almond shaped. See next post

[Ruary]

1486 This shows the two nosemas at the same magnification the sample of nosema ceranae obtained via Dave Cushman from FERA, the Nosema apis from my collection and PCR'ed by Randy Oliver in USA who was looking for a sample of genuine Nosema apis.

[gavin]

Gavin I think you might have your nosemas reversed apis is more square ended, nosema ceranae is slighly almond shaped. See next post

I did indeed - thanks for putting me right.

[gavin]

Here's an interesting aside. N vespula is of course a wasp parasite.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779023/bin/nihms156154f5.jpg

[Neils]

My first attempt at looking for Acarine.

1521

[Ruary]

Nice dissection, you removed the collar so that the whole of the tracheae can be examined. Now the real question positive or not??

[Neils]

I'm going with "Not".

I thought it would be easier to locate a decent photo of an similar view showing Acarine but maybe not.

I'm not a biologist, doctor or whatever, but in my limited experience once you get past the initial yuk factor, nothing looks obviously wrong, discoloured, swollen or otherwise out of place.

Given that Acarine is a mite, an obvious thing to look for is, well, a mite. The bee looked normal, in rude health (apart from being dead) even. THe colour of the photo is not really representative of what we saw directly looking under the microscope. There is a lot of Blue going on and the Trachea looks brown in the photo compared to under the microscope where it was white and not, therefore, miscoloured which can be indicative of Acarine, there is no "patching" in evidence nor any other malformations of the Trachea.

I believe that technically you're supposed to sample "Crawling bees outside the hive". As we didn't have any, these were ultimately destined for our nosema sampling but we thought we'd have a go as well.