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THE DIATOM TEST SLIDES BY KLAUSE KEMP

AUTHOR: Barry Sobel

INTRODUCTION:
diatom
Diatoms are single-celled microalgae with a salicaceous skeleton. They are an important type of plankton called phytoplankton. They are bivalved and are symmetrical in one or more planes. Most amateurs are interested in the silicaceous structure so the organic matter is usually removed before they are permanently mounted on the slide. The valves are covered with periodic structures of varying size which on close examination are arranged in rows. These rows are called striae. The striae lay inside an areola.

On closer examination the striae are found to be composed of pores called puncta. The size of these structures vary between species and therefore resolving these may be possible with a relatively low power objective, or may require the highest power-high n.a. objective with oil immersion, or may not be resolvable with a light microscope even with DIC imaging or oblique illumination. In general, the finer the spacing of details, the more difficult to resolve. Resolution of details is limited in the light microscope to about 200 nm. This means that anything or any detail separated from another that is closer than 200 nm cannot be distinguished even with the best light microscope. Other factors influence the ability to see details so close to one another including contrast and the wavelength of light used. For this reason, special methods of increasing contrast are often needed to see details that are theoretically well within the limits of resolution. This is particularly important for diatoms, as can be seen in the images of the most difficult examples below. The puncta of Amphipleura pellucida are only 200-270 nm apart, therefore very close to the theoretical limit of resolution of the light microscope, and therefore a famous object for testing the upper limits of resolution of a light microscope. All of the images seen below except for the dark ground images were taken utilizing Kohler illumination.

kemp slides
Klaus Kemp was one of the most prolific diatom mounters of the second half of the twentieth century. He produced slides of individual diatoms, type slides, strews, beautiful arrangements, and test slides like those shown to the left. The reader will note that these are all labeled slightly differently. The one on the far left shows the diatoms mounted in ZRAX, also known as Nephrax, with the diatoms mounted on the underside of the coverslip rather than on the slide. The middle slide was also mounted in ZRAX, but the diatoms were mounted on the slide. The one on the right was mounted in Hyrax. Kemp made many test slides, but mounted most of the diatoms on the slide; mounting on the coverslip was done only by special request. This is most important for resolution of the puncta of the most difficult diatoms, where proximity to the objective and high contrast are a must for maximal resolution. Both the Zrax and Hyrax mountants have a refractive index of about 1.75, but for the most difficult diatoms a refractive index over 2 is better, though almost impossible to come by nowadays. Nephrax is apparently still available but contains toluene which if inhaled in any significant amount can cause pancreatitis and metabolic acidosis. This stuff costs about $450 for 200 ml in 2025 and it can be purchased without the toluene (which the user must add (under a fume hood!). High refractive index mountants in the late 19th and early 20th century used even more toxic ingredients and produced toxic fumes when being prepared, so things like Realgar and other similar mountants are no longer available.


Shown here is a micrograph of the diatoms on one of his test slides shown in one low power view, along with higher magnification views of parts of the diatoms to show details.

kemptestslide

Enlarged images of examples of the individual organisms are shown in the images below, most taken by the author from the diatom test slide made by the late Klaus Kemp. These were taken with an Olympus Vanox microscope using the objectives noted in the table at the end of this web page. Diatoms generally require oblique or NIC illumination for optimal visualization. The highest resolution of Amphipleura pellucida was an NIC image of a mount donated to James Solliday by the late Bill Sokol. The diatom was mounted on the coverslip and a very high refractive index mountant was used (refractive index of over 2), image by James Solliday. In the nineteenth century, notable methods involved using realgar (with arsenic sulfide), which could produce mountants with refractive indices above 2.0. Other approaches included using gum arabic formulas with sucrose, fructose, or glucose. Additionally, some researchers explored the use of natural oils and plastics as base components for mountants. Nowadays with the scanning electron microscope, diatom puncta from A. pellucida and similar difficult diatoms are resolved without the need for toxic mountants. Using light microscopy, almost all diatoms are better visualized using oblique or DIC(NIC) illumination, although darkground and phase contrast can also help in some cases. In order to get the results shown below, the user must understand how to properly adjust the condenser, aperture diaphragm, field diaphragm, and orientation of off-axis (oblique) illumination. The following images are enlarged to show details and they are not all the same scale. They were all taken with a Amscope 5.1 MP model MU503 camera using Amscope software.


GYROSIGMA BALTICUM: The diatom is large and the puncta are easily resolved, even with a 20X objective using oblique illumination or DIC. diatom diatom


NAVICULA LYRA: This medium size diatom has striae readily resolved at 20X. The puncta are well resolved at 40X. diatom diatom diatom

STAURONEIS PHOENICENERON: The striae for this diatom were weakly resolved with a 20X objective and oblique illumination, but the puncta are poorly resolved at 20X. This diatom requires oblique illumination or DIC with a 40X objective for better resolution ; when using oblique lighting, the best visualization requires rotating the source of the oblique light to a position optimal to get the best contrast. The Apices are rounded and the central raphe ends straight proximally. diatom diatom diatom

NITZCHIA SIGMA: I was able to resolve the striae weakly at 20X and better at 40X using DIC, but 40X oblique illumination with this objective did not resolve them quite as well. 100X Oil immersion DIC gave the best results for visualizing the puncta. diatom diatom diatom diatom

SURIRELLA GEMMA: The striae could be just barely resolved with oblique illumination at 40X. Puncta required 100X DIC, 100X oblique or 100X darkground illumination. diatom diatom diatom

PLEUROSIGMA ANGULULARUM: Easily resolved puncta are characteristic of this diatom. The puncta were resolved with oblique illumination and a 20X objective. Oblique illumination works well but the characteristic straight and angled striae rows show up best with higher power. Under 100X the walls around the puncta take on a three dimensional appearance even with oblique illumination. diatom diatom

FRUSTULIA RHOMBOIDES: This diatom was not resolved at 40X but with DIC it shows the characteristic blending of the two ribs into one on each end and a distinct raphe in between them. Oil immersion was required to resolve the puncta. diatom diatom

AMPHIPLEURA PELLUCIDA: The striae of this diatom, invisible at 40X, and were not easily resolved except with 100X oil immersion technique using oblique illumination but ONLY when the diatom was mounted on the coverglass. With Zrax media and coverglass mounting the puncta were barely visible at 100X. With a higher refractive index mounting medium, (not my slide) a much sharper view of the puncta was obtained. Such a high refractive index medium is not easily obtained these days, and I know of no commercial mountant available with a refractive index close to 2.0. diatom diatom diatom diatom


SUMMARY OF THE RESOLUTION RESULTS OF THE KEMP TEST SLIDE:
DIATOMOBJECTIVE
ILLUMINATION
STRIAE RESOLVEDPUNCTA RESOLVEDCOMMENTS
G.balticum20X
oblique
DISTINCTWELLGood Diatom for 20X
N.lyra20X
oblique
FAIRLY WELLPOORStriking diatom at 40X
40X
oblique or DIC
DISTINCTDISTINCT
S. phoenicenteron20XDETECTEDPOORDifficult striae at 20X but striae and puncta resolved at 40X.
40X Oblique Illum.
or DIC
DISTINCTDISTINCT
N.Sigma20X Oblique Illum.POOR-just barely seenABSENTThe striae of this diatom are not visible at 20X and are fairly resolved with proper DIC illumination at 40X as are the puncta; oblique illumination at 40X worked also, but not as well as DIC. This is a diatom where the visual resolution was clearly better than the photograph.
40X Oblique Illum.FAIRGOOD
40X NICFAIR TO GOODGOOD
100XGOODDISTINCT
S.gemma20XNONENONEThe striae of this diatom are not visible at 20X. Striae can be resolved at 40X. Puncta require 100X.
40XGOODNONE
100X DIC
OR OBLIQUE
DISTINCTDISTINCT
P. angularum40XDISTINCTDISTINCTPleurosigma angularum has long been a favorite diatom of users of the microscope. It is not resolved at low powers. At 40X both the striae and puncta are clearly resolved, but not at 20X. 100X objectives reveal the walls of the puncta.
100XDISTINCTDISTINCT
F. rhomboides40XDISTINCTNONEF. rhomboides reveals striae at 40X but puncta require 100X.
100XDISTINCTDISTINCT
A. Pellucida40XNONENONEThe most difficult to resolve diatom on the Kemp slide and cannot be resolved without 100X and even then requires both mounting on the coverslip and very high refractive index mountant (refractive index over 2) along with extremely oblique illumination and/or DIC.
100XGOOD ONLY IF MOUNTED ON COVERGLASSNONE
100X WITH VERY HIGH R.I. MOUNTANT(OVER 2.0), WITH COMBINED OBLIQUE AND DIC ILLUMINATIONGOOD*FAIR*


So, G. balticum is a good test diatom for lower powers e.g. 20X. N lyra striae can be seen with a good 20X. Good tests for 40X were the puncta of S. phoenicenteron, N. sigma, and especially P. angularum. The puncta of S. gemma, and F. rhomboides are good tests for a 100X. A. pellucida cannot be easily resolved unless mounted on the coverslip using a very high refractice index mountant(2.0 or higher).

I have summarized the results of the above work, including how I suggest to use the slide for testing using the objectives noted. I have not yet tested the results using other objectives but consider this a starting point for further work.
OBJECTIVESUGGESTEDMORE DIFFICULT
20X S-plan n.a.0.46G. balticum PunctaS. phoencenteron Striae, N. Sigma Striae.
40X S-plan n.a. 0.70N. lyra & S. phoenceteron PunctaS. gemma Striae, N. sigma puncta
100X S-plan Apo n.a. 1.35Puncta of S. gemma & F. rhomboidesStriae and Puncta A. Pellucida*

*For A. pellucida, visualizing the striae require mounting on coverslip; puncta require, in addition to coverslip mounting, mountant with Refractive Index of at least 2.0.