Microscope Slide vs Original Source

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DonSchaeffer
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Microscope Slide vs Original Source

#1 Post by DonSchaeffer » Sat Sep 18, 2021 2:58 pm

A few days ago, I drew a microscope slide out of my source (an aquarium). After some examination and time I found a large swarm of bacteria, fairly advanced, very dense, that seemed to form a boundary cutting off a group of several species of larger ciliates. The ciliates were sinning around and seemed to be in distress, not able to cross the swarm of bacteria. I made these interpretations of events on the slide.

My question is, does this represent anything that could be happening in the aquarium source? Is the slide somehow representative of events in the source? Another question: life forms, bacteria and protists find themselves on a slide--how long does the bacteria need to organize a swarm or did I just pick up a ready-made swarm when I took the sample?

apochronaut
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Re: Microscope Slide vs Original Source

#2 Post by apochronaut » Mon Sep 20, 2021 3:15 pm

No easy answers to those questions. Here are some thoughts, though.

Bringing a random sample to a slide brings a whole other set of conditions upon the organisms present. You now have a major change in current flow, a very thin vertical column, dramatically changed light penetration, changes in osmotic pressure and an extreme liklihood of rapid temperature change, In adfition to many other changes. Additionally, any large organisms brought with the sample , could now have a disproportionatly high effect upon the whole of the sample, primarily due to voracious feeding and physical disturbance of the environment.

Bringing that slide to the microscope table creates a more defined condition, with an almost assured temperature increase taking place, rapid deoxygenation of the environment and likely increased light levels. It is generally a recipe for first a dramatic change in behaviour then second a slow to rapid collapse of the captured ecosystem, one species after another. Sometimes however, one or two species will proliferate, changing the entire miniature ecosystem topsy turvy. Lysis of certain organisms can begin in minutes and those that are not immediately vulnerable to the changed conditions often undergo changed behaviour, usually frantic.

The only real way of determining whether the species mix and their relative behaviours on the slide are consistent with those factors in the native environment is to be able to set up some sort of microscopic observation of the native environment with the goal of comparing the two. Some older stereo microscopes had articulating arms and could be positioned to observe through the glass of a micro- aquarium. Unfortunately, 40 to 60X would usually not be adequate for such a comparison, obviously where truely micro-organisms are concerned, so another option would be to use a petri dish as your primary eco-system. Inverted microscopy with l.w.d. objectives through the bottom of the dish or water dipping objectives might offer some insight into the activities in that mini tank but such investigations would naturally be limited to no more than 600X : (40X l.w.d. objective x 15X w.f. eyepiece). for a standard petri dish or 1000X , using a cover slip bottom dish. Not everyone has an inverted scope though. An option would be to make a miniature tank using an oversized coverslip as a lid. This way one could examine the near coverslip community at least with high resolution microscopy and even deeper into the water column with 20 to 40X.

In order to be able to view both the bottom of the tank and the top, a design I thought of making in the past but never have had the time , is to make a sealed mini-aquarium with 4 partial slides for the sides and .17 covers top and bottom. This would allow the full aquarium to be inverted so a limited observation can take place after inversion. Much of the ecosystem clings to the bottom, despite gravity. I have really high quality 22 x 30mm .17 cover slips so that would be my dimmensions x a slide width, approx. 25mm high in the Z dimension. Whatever cover slip size one has would dictate the XY dimension. There would be a 3/8" hole drilled on the middle of the long dimension with a plastic plug to right angle elbow and a vertical extension pipe attached that rises to the upper surface. This allows the aquarium to be filled top to bottom with a small port for oxygen exchange. It is plugged during observation allowing for inversion . The hole is used to fill and empty the aquarium. That way the aquarium can be viewed in high N.A. immersion microscopy in upright mode or inverted mode. Top surface or bottom surface of the aquarium. I would like a better mechanism to seal the unit when needed but allow better oxygen exchange.
Probably a large piece of 3/4" plastic pipe cut at about a 15° angle and cemented in between two 15-16mm slide sections, thus centered in one of the long sides, would provide better oxygen exchange when the aquarium was not in microscopic use.

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Re: Microscope Slide vs Original Source

#3 Post by BramHuntingNematodes » Mon Sep 20, 2021 4:13 pm

Another limitation of the inverted scope is that in order to have a big dish, even with a slip-bottom dish, you will need a lwd condenser these are usually lower NA, commonly .5 but often even lower. A 40x achromat is a out as good as I have gotten to work with inverted
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apochronaut
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Re: Microscope Slide vs Original Source

#4 Post by apochronaut » Mon Sep 20, 2021 4:34 pm

You don't need to go l.w.d., if you use a cover slip bottom petri dish : very expensive they are but open to d.i.y'ing. A plastic petri dish could be drilled and cutout to receive a cemented on cover. An oil immersion higher N.A. condenser and objective combination can then be practical for inverted.

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Re: Microscope Slide vs Original Source

#5 Post by BramHuntingNematodes » Mon Sep 20, 2021 6:05 pm

I have made a few slip bottomed dishes, but the water is so deep in them the condenser can't quite close enough...I don't think I want to dip the condenser, do I?
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apochronaut
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Re: Microscope Slide vs Original Source

#6 Post by apochronaut » Mon Sep 20, 2021 6:43 pm

BramHuntingNematodes wrote:
Mon Sep 20, 2021 6:05 pm
I have made a few slip bottomed dishes, but the water is so deep in them the condenser can't quite close enough...I don't think I want to dip the condenser, do I?
Water dipping condenser. That's a thought now! What is the microscope?

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Re: Microscope Slide vs Original Source

#7 Post by BramHuntingNematodes » Mon Sep 20, 2021 8:12 pm

It is a invertoscop from the Opton era. The top of it was essentially sheared off so while I got it for pennies I have been doing many experiments to arrive at the current configuration. It has a I believe Motic lwd condenser in it currently.
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Re: Microscope Slide vs Original Source

#8 Post by apochronaut » Mon Sep 20, 2021 11:17 pm

What is the diameter of the dovetail ? How deep are your petri dish samples?

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Re: Microscope Slide vs Original Source

#9 Post by DonSchaeffer » Mon Sep 20, 2021 11:22 pm

The real answer is: You can't!
The whole idea of describing the behavior of microbes is fiction and poetry. I'll take poetry.

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Re: Microscope Slide vs Original Source

#10 Post by BramHuntingNematodes » Mon Sep 20, 2021 11:30 pm

The dovetail is 11mm wide, and the dish about that deep, maybe as shallow as 8mm. Originally I had the B&L insert condenser which was pretty effective in that it has a long reach. I swapped it for a 1.25 abbe for awhile but had trouble getting the annular diaphragms in focus (I just rest the diaphragms on top of the inverted condenser) and it had that look where the condenser wants to be closer to the slide. I acquired this Motic lwd condenser for I think 5 or 6 bucks and it has been just the ticket. Very nice views and the diaphragms are now a perfect fit.
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Re: Microscope Slide vs Original Source

#11 Post by BramHuntingNematodes » Mon Sep 20, 2021 11:31 pm

oh and the dovetail bracket is not super important as I have already fashioned a new one out of ipe and the pinion from the old Selsi microscope.
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apochronaut
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Re: Microscope Slide vs Original Source

#12 Post by apochronaut » Tue Sep 21, 2021 1:19 pm

I don't know about phase . There are a specific set of parameters required and anything could throw things out. That definitely would take a little messing with. However for BF, if you keep the sample thickness about like a slide or slightly more ; 1 1/3 mm you are replacing the slide with water. Obviously you can't do oil immersion( perhaps with precisely meaured coverslip supports in each corner) but any condenser you can get close enough that has an N.A. of .90 should work to give you good to excellent high magnification at an N.A. of maybe 1.15 with some objectives.
If you can't get close enough, there were some condensers made with R.M.S. threads, so they could receive an objective to use as a condenser. It was particularly good for apochromat objective/condenser matching. Any achromat or better yet fluorite no cover .90 or .95 objective would work in one of those condenser bodies or an adapter could possibly be fashioned to thread in or onto some condenser body you already have. The extension required to reach and finely adjust the condenser lens was in the R.M.S. mount, which carried a locking nut. The condenser rack is the coarse focus and the condenser nose, the fine focus.
If some high N.A. water dipping objective came along? or maybe even one of those 70X 1.23 N.A. Lomo water immersion apochromats that always seem to have a seized correction collar would work as an immersion condenser. I have seen those sell pretty cheap when seized. It just needs to be wide open in this case.

The three companies that made mounts for objective condensers that I know of were Spencer, Baker and Leitz. The Spencer was a small circular dovetail and was the condenser supplied as a default 1.3 achromat with their research stands up until 1955 , the Baker I have never seen but I have a cardioid DF condenser with an R.M.S. thread that was used in one . The Leitz type has shown up on ebay from time to time and I think it is on an Akehurst slide. They are all nifty condensers because of their adjustability. Poetry doesn't do them justice.

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Re: Microscope Slide vs Original Source

#13 Post by zzffnn » Tue Sep 21, 2021 7:10 pm

Many protists swim up and down though (or just enough to make chasing and focusing on them difficult), when there is no thickness restriction from a cover slip, except for nematodes, heliozoa and moebas.

Otherwise great idea on using water dipping objectives on a RMS threaded condenser. It would be nice to have such a condenser to toy with dipping into mini-aquarium.

One minor correction on LOMO water dipping objectives:

I believe official LOMO dippers are 40x NA 0.75 (and the highly similar Zeiss version) and 60x NA 1.0 D=0 (no cover). Maybe with 30x NA 0.6-0.9. Not sure about 85x NA 1.0.

Probably not with 65x NA 1.1 or 70x NA1.23 (I have dipped those and got hazzy images - lots of spherical aberration when used without cover slips).

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Re: Microscope Slide vs Original Source

#14 Post by BramHuntingNematodes » Tue Sep 21, 2021 7:56 pm

good for removing distractions to focus on nematodes
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Re: Microscope Slide vs Original Source

#15 Post by apochronaut » Tue Sep 21, 2021 8:00 pm

Yes, Fan. Those are not dipping objectives but in this case they are being used as a condenser. I'm not sure the s.a. will be so lethal. An abbe 1.25 has lots of s.a. The idea of a supported cover slip, basically just contacting the sample would obviously be better. Even if the sample crept over the cover, the contact is w.i. anyway, so no particular harm done.

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Re: Microscope Slide vs Original Source

#16 Post by DonSchaeffer » Tue Sep 21, 2021 10:35 pm

All I really meant by the question was:
If you your examination of the microscope slide sample shows bacteria in a swarm and protists trapped by it, does that mean it is happening in the source?

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Re: Microscope Slide vs Original Source

#17 Post by apochronaut » Tue Sep 21, 2021 11:10 pm

Have a look.

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Re: Microscope Slide vs Original Source

#18 Post by apochronaut » Tue Sep 21, 2021 11:49 pm

1) Here is the Spencer cat.#315 1.3 N.A. achromat aplanat. The condenser body is a sleeve type as many older Spencer condensers were but were fitted inside a c.o.b. dovetail with one set screw. Thus the actual dovetail can be swapped out for another one to custom fit any microscope : as long as you can fabricate the dovetail.
2) The condenser lens pack removed. Note the locking ring for height fine adjustment.
3) Fitted with a 37mm parfocal F. Koristka 1/15" IMM ACQUA AP.N 1.20 Achromat
4) Fitted with an infinity corrected 100X 1.15 W. D.I.N. Plan Achro objective Olympus pattern.
5) Fitted with a Spencer 44X .95 dry apochromat with correction collar. 34mm parfocal.

R.M.S. male to female extensions can also be used .
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