“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.
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:
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.
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):
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.
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:
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:
Rachel and David, good friends of ours, bought me this selection of slides for my birthday this year. It is an eclectic collection plant, animal, insect and protozoal slides, mostly stained. At only £25, it was an absolute bargain – although the number of 91 is a bit strange.
My first forey into looking at these came today. It also provided my first opportunity to compare images between the LED illuminator and 100W halogen illuminator.
I chose the Volvox slide – these multicellular organisms are found in lakes and ponds and inland waterways world-over and can be amazing to look at under a microscope.
The following images show Volvox using the two different illuminators and at varying magnification.
Volvox is a polyphyletic genus of chlorophyte green globe algae in the family Volvocaceae. It forms spherical colonies of up to 50,000 cells. They live in a variety of freshwater habitats, and were first reported by Antonie van Leeuwenhoek in 1700 (Wikipedia).
The following diagram comes from Wikipedia and shows the structure of a Volvox colony – which can be seen in my photos below.
The dual illuminator setup on Zeiss IM microscope:
The 91 Chinese slide selection:
Chinese-Volvox-slide-x20-obj-Halogen-100W-illum-011217.bmp – whole Volvox colony showing many daughter colonies – with x20 objective. The picture also shows that it is not possible to get all of the spherical Volvox colony in focus at the same time:
Chinese-Volvox-slide-x63-obj-LED-30W-illum-011217II.bmp (below) – same field as above with LED illumination rather than halogen illuminatation (which was used above). Photo looks similar – in practice colour temperature was very different – much warmer with the halogen – and the similar colours reflect the effect of the auto-white balance adjustment present in the Bresser Mikrocamlab7 software:
Chinese-Volvox-slide-x32-obj-Halogen-100W-illum-011217.bmp (below) – Halogen, now magnification increased using x32 objective – which brings out detail in the daughter colonies inside the Volvox:
Same field and magnification as above with LED illumination (below):
The following three photos are a series of photos of the same field all using LED illumination but this time using the 63x objective to concentrate on one of the daughter colonies. Volvox is spherical so it is not possible to get the whole daughter colony in focus at the same time so the three photos are at different levels within the colony to bring different parts into focus. It looks to me as though the daughter colonies are already forming grand-daughter colonies within them, even before separating from their parent!
Now that I have two working illuminators, this allowed me to rig them up today using this device, the Zeiss dual illuminator selector cube 46 70 78, to give the ability to quickly and easily change illumination method – in this case from the halogen 100 W bulb to the LED retrodiode.com 30W illuminator.
The following photos show the (now working as it has a new bulb) Zeiss 100 W illuminator attached to the epi-illumination port, and light coming through into the base of the objective turret through the epi-illumination port. Unfortunately, this is not sufficient to use epi-illumination yet. I have a flourescence cube holder which fits into the space under the objective turret and directs light up into the objective lens. This currently only has flourescence filters on it, which remove most of the light, seriously dimming the image. Hence I need to modify that cube holder to allow all light to be re-directed without filtering. I think this will involve purchasing a purpose-cut mirror…..but I still need to look into it.