Microscopy

Changing the Zeiss 47 17 61 flourescence epi-illuminator condensor on the Zeiss IM microscope

When I purchased my Zeiss IM inverted microscopy just over one year ago, there was no epi-fluorescence condenser present. I purchased one off ebay (Zeiss part 47 17 61 – in this post I will call it the EFC). Unfortunately, the filter slider was stuck. Luckily another in full working condition came up very cheaply recently and I have now replaced the first one. However, the technique for taking one out and putting in another was not obvious so this post is to record the process for future reference, should I or anyone else want to make this change on the Zeiss IM or IM35 microscope (both use same component). I will probably wish to do this myself. After I find a way to fix the stuff filter slider, I also have a Zeiss IM35 and will probably put the spare one on that, although epi-fluorescence requires use of the fluorescence cube for the microscope and I only have one of those and they are quite expensive second hand, so I doubt I will be acquiring a second one soon!

It is worth noting that the EFC provides epi-illumination but was only designed to be used for fluorescence microscopy. I am hoping to adapt the fluorescence filter cube to remove one of the filters and replace with plain glass so that I can use it for more general epi-illumination. Today’s project was the first step in this process as it allowed me to slide out of the way a blue filter in the filter slider which was stuck in the light path. In fact this needed to be done prior to the removal of the old EFC and – given that the reason for Today’s project is that it was stuck – this required the use of a hammer to be achieved! I was gentle and put something between hammer and microscope with softening covering (spare piece metal coveted in kitchen absorbent roll) and managed to succeed without causing further damage, although my heart was in my mouth while I did it.

Andy

Zeiss 47 17 61 epi-flourescence condenser  (EFC). This is the one that I removed today. You will notice that there is one slight difference between this one and the new one I installed in its place, which is shown in the later photos on this post – the adjustment screws for centering the illuminator light are longer in the new version. These can be seen in the later views from the back of the microscope.

Back of microscope – epi-illuminator is central round bit. The illuminator will be attached here (not attached in this photo):

The following pictures show an illuminator attached to the back of the Zeiss IM35 microscope. It is the square box hanging off the back, opposite the eyepieces (oculars). It would be exactly the same with the IM microscope as the only difference between the two varieties of microscope is that the IM35 has an inbuilt camera port whereas the IM does not have one.

View of epi-fluorescence condenser (EFC) from next to the eyepiece turret. This view shows the filter slider within the epi-fluorescence condenser component that was stuck on the first one I installed on the microscope. The epi-fluorescence condenser comprises both the white and black round parts on left side of the picture. The black part is seen to contain a circular filter holder with small black handle. This can be removed. For installation of the whole component or taking it out again, both the filter slider and the round filter holder need to be removed. This allows the whole component to then be slid out through the hole in the casing on the left – that hole is currently obscured by the black circular component which sits over it.

The the following photo shows the EFC from the other side. The long thin silver metal handle controls the aperture control within the EFC. To install or remove the EFC from the microscope, this handle must be first removed so that the EFC can slide out through the hole, otherwise it will obstruct it. The handle is removed by twisting it anti-clockwise, which will unscrew it from the EFC.

PROCESS OF REMOVAL OF EFC:

1. Start at back of microscope.

2. Locate the four retaining screws on the back which hold the etc In place and remove them.

3. Unscrew and remove long thin handle.

4. Locate and remove tiny screw from end of filter slider. This screw prevents filter slider being removed from EFC. Once screw is remembered bed, slide filter slider completely out of the EFC.

5. Remove content recluse filter holder (left below) from EFC. Note in this picture, filter slider is also shown as present but of course by now you will have already removed that in previous steps.

 

6. By now the EFC should be quite loose. Note it only pushed again eyepiece turret and not attached to it so there are no screws etc that need to be loosened there. The EFC should now be easy to thread through the hole in the back of the microscope and removed.

7. Insert new EFC by reversing the above.

Lichfield micro-meteorite project – making and setting up collector

On BBC Sky at Night TV show a few months ago, they collected micro-meteorites from roof collection at the Norman Lockyer Observatory in Sidmouth, UK. Rhys and I ordered some neodychromium magnetd and today we mounted them on a piece of spare wood using some small screws through their central holes. We put the wood and magnets, magnet side down, in the guttering on the front of our house in Lichfield, UK.

Hopefully, this will extra small metallic debris from the water running off the roof when it rains over the next few months. Again hopefully some of this will turn out to be micro-meteorites.

Andy and Rhys

Fixing broken timer on Eppendorf 5315c centrifuge



 



The bargain centrifuge has one problem.. …the timer didn’t work – it was like this when I bought it from ebay. I purchased a new timer from America (see previous posts). It arrived today and I spent an enjoyable hour dismantling the centrifuge and replacing the timer. A lot of dirt and much inside after 30-40 years and the timer was all rusted up. It is spring driven – I know, unbelievable in this digital age! The new timer is supposed to be the same but in fact the casing is slightly different shape so out came my hammer and pliers to bend the bracket it fixes to in the centrifuge to a accommodate the new one. After modification the bracket could only be reattached at one end so my trusty glue gun sorted out the other side. Sounds Heath Robinson but all worked well enough and you can’t see anything once the case is put back together.

What impresses me most is that I have managed to fix this without the help of Ed or Led or Pete – great guys, really helpful, but I am pleased to manage to do it myself this time.

Andy

Campo del Cielo meteorite thin section – microscopy using Zeiss IM microscope (not using polarisation)

I have waited some considerable time for this meteorite thin section to arrive – from the famous Campo del Cielo fall. Largely iron (black areas), there are also some minerals.

The following photos are NOT polarised.

Andy

x4 objective:

x10 objective:

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.

 

Eppendorf 5415 C Micro-Centrifuge Variable Speed with Timer – photos & manual

An old but sturdy piece of kit – useful for microscopy – allows samples to be concentrated (e.g. pond water samples) making it easier to find targets of interest. Only problem is that the timer does not work so I need to source one of those.

I have found a replacement timer – I intend to ask Ed whether he can take a look with me to see if we can work out how to replace it – not asked him yet – help please, Ed!
I also picked up a spare rotor – the screw on top was missing from mine.
All these problems with second hand stuff – its cheap but often needs work to get it working – but then that is the fun of the thing.
Below are photos of the centifuge, spare rotor and new timer.

Click below to read the manual for this machine (PDF file):

Centrifuge_Eppendorf_5415C manual

Andy

Diehl Series 600 T85 springwound timer – finding a replacement timer for the Eppendorf 5315c micro-centrifuge

Diehl Series 600 T85 springwound timer – finding a replacement for the 5315c centrifuge

Is this the correct one?

https://www.ebay.co.uk/itm/Poltic-or-Diehl-Springwound-Timer-30-MIN-Tanning-Bed-Type-600-T85-with-BELL/371656358069?epid=1376092377&hash=item568871f8b5:m:mjcQ-kmGpI3cfy5GJ-pL_oQ

or is this one a better choice?

https://www.ebay.co.uk/itm/Poltic-or-Diehl-Springwound-Timer-30-MN-Tanning-Bed-Type-600-HOLD/391491109081?_trkparms=aid%3D222007%26algo%3DSIM.MBE%26ao%3D2%26asc%3D49479%26meid%3Dac3b581f3858496789ad0cb084fb3974%26pid%3D100623%26rk%3D2%26rkt%3D6%26mehot%3Dag%26sd%3D371656358069&_trksid=p2047675.c100623.m-1

or this one?

https://spasandmore.com/catalog/timers/poltik-t85-typ-600-30-min-twist-timer-with-bell-8302.html

or possible UK source:

http://www.plccenter.co.uk/Buy/DIEHL/T85600?redirect=true

 

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:

Microscopy of 2 week Petri dish culture of bacteria from hair in my armpit

This may seem a weird experiment to have done – but 2 weeks ago Ean Ean snipped off some hair from my armpit directly into a Petri dish to see what we grew. The reason for this behaviour was that my T-shirts and jumpers tend to develop a lot of holes in the armpit areas and we wondered what bacteria was doing the damage. I do have reasonably hairy armpits!

Pictures below of the bacteria we grew – they are small round bacteria – this is known as coccus (cocci in pleural).

The Gram staining (by Rhys today) shows that the bacteria are Gram Positive.

A brief internet search shows that common axillary bacteria include Corynebacterium and Propionibacterium. Another bacteria that is often present but in smaller number is Staphylococcus.

Corynebacterium is a genus of bacteria that are gram-positive and aerobic. They are bacilli, and in some phases of life they are, more particularly, club-shaped, which inspired the genus name. The principal features of the Corynebacterium genus were described by Collins and Cummins in 1986. They are gram-positive, catalase-positive, non-spore-forming, non-motile, rod-shaped bacteria that are straight or slightly curved. Propionibacterium is a gram-positive, anaerobic, rod-shaped genus of bacteria named for their unique metabolism: They are able to synthesize propionic acid by using unusual transcarboxylase enzymes. Staphylococcus is a genus of Gram-positive bacteria. Under the microscope, they appear round, and form in grape-like clusters. The Staphylococcus genus includes at least 40 species (Wikipedia).

My bacteria appear to be Staphylococci – round and blue.

 

Preparing sample for microscopy today:

1. The Petri dish culture – axillary (armpit) hairs are visible!

2. The spatula was used to scrape off the bacterial culture into this container and then it was mixed with small amount water:

3. Small drop put onto a slide:

4. This is then dried:

 

Unstained heat-fixed slide, with 63x objective:

Gram stained slide, x32 objective:

 

Gram stained slide, x63 objective: