TOPICAL TIPS

an annotated list of on-line links and bibliography on...

MINI-AQUARIUMS, MICRO-AQUARIUMS, HANGING DROPS, WET CHAMBERS, AND

ELEMENTARY COMPRESSORS

Second  part

Walter  Dioni                                                                                                               Cancun, Mexico

NOTE: The pictures without a credit, were made for this publication by Axel Quarchioni, using C4D. I am grateful for his generous contribution of personal time away from his family.

WET  CHAMBERS

Remember what the Wet Chambers are: Wet chambers  are closed preparations, in which a drop of liquid is supported in a cavity full of air, which ensures a much more prolonged observation time, allowing a free exchange of gases between the drop and the surrounding air.

Even if the principle is the same there are a number of different implementations, and as was the case with micro-aquariums, the physical construction can go from the simplest to more elaborate designs. Fantasy, ingenuity, available tools and materials, and handicrafts skills, determine the chamber a microscopist can use.

HANGING DROP WET CHAMBERS

This is a technique that has inspired a lot of variants from the time of its invention, which Simon Stricker in his “Manual of Histology” of 1872 assign to Recklinghausen (no date, but obviously before 1872).

Wet Chambers in all its forms are small closed spaces in which a drop of media is placed, surrounded by air, to assure a free interchange of respiratory gases.

EXCAVATED SLIDES - (well slides, excavated slides, concave slides)
There are professional slides with a concave excavation at its center, or even with multiple excavations, which can directly receive a "Hanging drop".

To use them a small and thin drop of fluid containing the observable subjects, is placed at the center of a coverslip bigger than the diameter of the excavation, of course. A thin ring of Vaseline is drawn around the concavity, one mm out of their boundary. The coverslip is swiftly but carefully overturned so the drop hangs from its underside and is lowered and applied to the Vaseline seal. The drop now hangs over an air supply that assures a more or less long term observation. If you plan to use an immersion objective, the four corners of the coverslip should be fixed with a drop of nail polish. This geometry allows for large powers to be used on mobile organisms that adhere to the underside of the coverslip (to count the toes of bdelloids, for example). The preferred point of observation is the peripheral boundary of the drop, where it is thinner and can be explored using the highest powers. Some prefer to reverse the procedure they put the Vaseline around the coverslip on the table, deposit a drop in the center of this, invert the hollow slide and apply it to the cover. Then the slide is inverted. Of course if you fail with the first technique you only need to start again by putting the drop on a cover. But if you fail with the second technique you must start with a new excavated chamber, and a new cover, and repeat all the steps.

In the 20th century someone suggested to personally create the concavities using a dentist’s drill for that task. Those that have one at their disposal can do a trial.

The commercial "excavated slides"  are expensive! To apply the useful technique of the "Hanging drop" several microscopists have developed substitutes equally or even more useful, and many of them very cheap.

THE SIMPLEST MOIST CHAMBER (cardboard)

In 1913, in his Précis de Microscopie, (available free on-line) Langeron proposed to cut a square sheet of cardboard, of the desired thickness, perforated with a punch, or cut with a scalpel, soaked in water and simply attached to the slide by capillary action.

So it was not only permeable to oxygen, but the chamber kept saturated with water vapor. An occasional drop of water added in the periphery can keep the chamber running for hours. Saving them in humid chambers with the atmosphere saturated with water, ( see Part 1) when not at the microscope, can last for days.

The most important problem in long lasting observations over some days is the possible contamination of the  cardboard frame  with bacteria or moulds.

MORE PERMANENT WET CHAMBERS

A modern and comprehensive article on the subject, full of creative ideas is that of Jean-Marie Cavanihac (published in English and French) at:

http://www.microscopies.com/DOSSIERS/Magazine/Articles/JMC-LAMES% 20A% 20PUITS/lamepuits.htm

http://www.microscopy-uk.org.uk/mag/artmar02/jmcwells.html

DEEP  CELLS - There are however, some preferred common structures. The commonest is a ring (in various thicknesses and depth) cut off any waterproof material (PVC, glass, aluminium, plexiglass, etc). 

The one at left is the 1913 proposal of Langeron.

Below there is a picture of two deep cells donated to the author by J.M. Cavanihac

O-RING CHAMBERS - O-rings  have even be proposed (the circular rings which are used as compressible washers in mechanics, which are available in various diameters and thicknesses - see a good description of this in G. Couger’s reference at the end of this paragraph) cemented to slides with Vaseline, or balsam, turpentine, cyanoacrylate, epoxy cements, or any other acceptable glue, deposited on the out side of the ring. Any of these cements are dissolved in solvents, toxic to our organisms, but they are very volatile and after a couple of hours in the air the chambers can be used with little danger. One exception is the silicone cement. Even that used for aquariums has acetic acid as a component. To use it flush the future deep cell with a large volume of water for at least a day, before using it.

Don’t yield to the temptation to use PVA, (polyvinyl acetate, white multipurpose cement used in houses and schools, because it is soluble in water and will cause a disaster).

A verbal discussion of most of the anterior deep wells, specially the O-Rings was made in 2006, at the Yahoo Group Microscope, with an appropriate summary by Gordon Couger

http://tech.groups.yahoo.com/group/Microscope/message/31110

OPTICAL  DEFECTS  OF  THE  "HANGUING  DROP" - Any hanging drop technique, with either excavated slides or humid chambers of any material, have a major flaw. The highest powers can be implemented only at the periphery of the drop, and even with the lowest powers the refraction and reflection of light on the convex surface of the drop leads to marked differences of illumination as the observer moves the slide under the objective.

To remedy this defect Ranvier designed in the 19th century the glass chamber that bears his name.

c, glass chamber, perforated, s, central disc, b circular excavation. (taken from Langeron, 1918)

 Even if there were some different designs, his chamber was made of glass and consisted of a thick slide (at least two times thickest than a normal slide) carved in the center with a relatively wide and deep ring (it was the supply of oxygen) leaving a central cylinder. The top of this is worn and polished to reduce its height by about 100 microns. The drops to be observed were placed on the central pillar. A very thin line of Vaseline was traced around the chamber and a slide placed on it pushing it down until it is supported on the slide. Of course, the central drop was reduced to a thin sheet of 100 microns, without optical defects. 

** FLAT HANGING DROP - lucky microscopists that own some of the expensive circular COVERSLIPS of approximately 1 cm in diameter, may resort to them for the next trick. Place on the table a coverslip greater than the diameter of the wet chamber. If you are working with caution, never mind if this is circular or square, except for aesthetic reasons. Deposit a sufficiently small drop in the centre.

With a tweezer take a circular coverslip and deposit it very carefully on the drop. If this was well calculated this is sufficient, if not, liquid must be absorbed with a long and narrow paper filter triangle until you get the desired thickness of the drop. Set the small cover with 3 or 4 small drops of Vaseline or Valap. Deposit a water drop in the bottom, at the internal chamber edge. Capillarity acts to stick it on the periphery. Spread the top edge of the chamber with a thin layer of Vaseline. Quickly reverse the ready pair of coverslips, and carefully adhere it to the chamber.

In the best case (experience facilitates it) the circular coverslip and a flat culture drop shall be suspended in the center of the chamber. The chamber can have the height you want. The drop does not evaporate because it is in a saturated atmosphere, and observation may be maintained for a long time, with the material distributed in a thin, uniform layer of water, which avoids the illumination problems of the hanging drop. If you have suitable sizes (10x10 or 12 x 12 mm) you can also use square coverslips, of course.

** Thick MICRO-AQUARIUM FOR LONG TIME OBSERVATIONS - Another micro-aquarium, more elaborate, but also relatively simple, suitable for somewhat larger organisms, observable at medium and low powers, can be prepared with circular plastic boxes, about 5 cm in diameter which are often sold in stores providing articles for children. In the center of the lid of the box a hole whose diameter must be smaller than the width of a coverslip (15 or 20 mm in diameter is good) is carefully made.

The outside of the hole is plugged with a coverslip carefully cemented. The inside is plugged with another coverslip, but cemented covering only half or just more, the diameter of the hole. Once the adhesive dry, it remains formed between the two coverslips a semicircular microacuaria, with a depth equivalent to the thickness of the plastic cap used. The interior coverslip can be adhered with Vaseline, reinforcing its corners with nail varnish, or paraffin to have an easier to clean aquaria.

To use this aquarium a ring of filter paper is placed at the bottom of the box (the center is left open for the passage of microscope light) which is saturated with water. Fill the cell with the sample containing the organisms to observe and cover the box. In this cell atmosphere, saturated with water vapor, the cell will host organisms for days, allowing a continued study of development, cycles, and various physiological phenomena.

As the external aquarium wall is a coverslip, this chamber could be used even with an inverted microscope. Cut a disc of thin plastic, or even paper, make a hole in the center similar to the aquarium hole, and stick this separator to the external wall of the box. This would protect the external coverslip from accidents over the inverted microscope stage. In this case, the interior coverslip wall can be dispensed, and the entire surface of the hole would be the research field. This wet-box is the only one that offer this opportunity.

Of course, uncovering the box, food can be added to micro-aquarium, or the medium can be changed in whole or in part. The box should be open up periodically to allow for a renewal of the indoor air, or to change the ring of moist paper if there are bacteria, algae or fungi growing on it.

COIN'S  BOX  MICRO-AQUARIUM - Martin Mach suggested the use of plastic boxes used to display coins, which are about 3.5 mm deep, allowing a cylindrical drop, that thick, to be centered inside the box. This big wet chamber was suggested to facilitate the observation of Tardigrada in the stereoscope. But it is clear that has many other possibilities. See

http://www.tardigrades.com/

Review items # 29 and # 30 of his "Tardigrades Magazine"

CONCLUSIONS

Therefore, if the microscopist is in need for a micro-aquarium (To answer for exmple: How long does the development of a Rotaria embryo last? Can I see the hatching of a rotifer egg? How often does Adineta lay an egg? How many times a day does a Coleps, or Euplotes divide? At which rate does an Aeolosoma produce its paratomic offspring?) it would be able to find easily in their home environment, or nearby, materials to convert their normal slides and coverslips into one or more versions of the instruments described in this two part article.

And ... is there still room for invention and discovery? Probably in terms of materials, but, as we have seen, the concepts are stable for much more than a century, as this advanced “growing slide” published in 1905, demonstrates:

http://www.archive.org/details/elementsapplied01winsgoog

Charles-Edward Amory Winslow  Elements of Applied Microscopy: A Text-boook for Beginners (1905)

It can be easily implemented with some standard slides, a glasscutter and some drops of cement. The in and out holes are provided with wicks, the right one with its end submerged in a flask with medium supported at some height over the stage, and obviously, the left one goes to a collection flask situated downstream.

There is even an electrically warmed slide which could be possibly imitated and adapted to modern electronics, by some of the many amateurs (who don’t live in Cancún of course, where such accessories are absolutely useless) described in the above cited book of S. Stricker.

I have left out many specialized wet chambers, designed from the end of the 19th century to now, to warm the samples (parasitological ones for ex.), to study thermotaxis, or galvanotaxis, or phototaxis, etc. The interested microscopist can find them in the above linked Striker’s and Amory’s and Davis's books, and others, explore the list of titles in Internet Archives.  Even if many materials are now outdated, or if there are new techniques to apply with advantages, their descriptions are inspiring.

I think that the opportunity open by INTERNET ARCHIVES publishing very old, but much interesting and fertile books on microscopy and related disciplines like histology, or parasitology, must be profited by amateur microscopists to know the published versions of useful techniques and devices. So they can devote his efforts to adapt them to our modern times.

You could discover that by 1882 ALL the most useful instruments used as auxiliaries for the bright field microscope were invented and were commercially produced. Read on line Microscopy by George Edward Davis, published that year.

WELL!  A lot is published here. Read it. Refer to the cited sources. Remember it. Use it. We have reviewed over a century of descriptions of microaquaria, wet chambers for hanging drops and flat hanging drops, humid chambers to hold the wet chambers, and microaquaria, etc. And I am sure that the story doesn’t end here...

APPENDIX

I would also draw attention to this revolutionary and highly specialized wet chamber, which those amateurs who like to explore the bacteria they found in their studied media would appreciate. As described in

If you study one of the very common mobile bacteria, the usual picture you can take without recourse to the techniques of stained smears, is like the upper half of the illustration below.

But if you use an agar bed laid over the slide, the liquid around the bacteria will be absorbed, the bacteria stop over the surface of the gel and the pictures you can take will be similar to the second half, which I like more than the common Gram stained smear technique. (Picture modified from the original paper.)

**It is possible to lay an agar film over the slide in more simple ways than the standardized methods the authors use. Make a two storey adhesive tape cell (100 microns deep). Fill it with 0.5 - 1% melted agar. Take a very cold and humid slide and apply to the surface. After a few seconds slip the upper slide away. You must have a good field of experimentation between the tapes.

Here the authors use Phase Contrast, but you can see by the following two pictures, one of which I took from my review of the Book of  Betsey Dexter Dyer “A field Guide to Bacteria” (http://www.microscopy-uk.org.uk/mag/artjan04/wdbacteria.html) that you can see the bacteria without it. Try the agar bed.