|Talking about Electron Microscopy of Foods
with Miloš Kaláb
|Updated: April 25, 2013.|
January 19, 2005.
The world never looked the same after the Dutch biologist Antonie van Leeuvenhoek assembled glass lenses and created the first microscope.
When I was young (I was born in 1929), scientists like Robert Koch were our heroes - people, who were doing something beneficial for mankind. Dr. Koch was one of "the victors over death" - death caused by bacteria. He saw them, particularly the bacilli of tuberculosis (Mycobacterium tuberculosis), which were then called "Koch's bacilli". At the public school in Czechoslovakia which I attended, we even had theatre performances which showed us how the "bacilli" (represented by other children in white costumes) were dangerous to human health but at the same time were also helpless and unable to harm us if we behaved properly, ate a balanced diet, washed our hands after having used the toilet and before every meal, and never tried smoking.
There were many additional scientists such as Paul de Kruif and, in particular, Louis Pasteur, who used microscopes for discoveries beneficial for mankind. I find the reading exciting even in these days of high technology here and there and everywhere. Those early scientists had it very difficult, particularly if human prejudice opposed their ideas and experiments. One must admire their resourcefulness.
Are bacteria indeed so bad that we have to be on guard all the time? Of course, they are, at least a small but important group of them, called "pathogens". As all microorganisms, they are invisible to a naked eye, so we do not see as to whether our hands are contaminated, that means whether there are bad bacteria siting on them. They are the ones which can cause food poisoning or food-borne illness. Just look at some of them at left, enlarged several thousand times by a scanning electron microscope (right: a multisample stage holding 4 bacterial specimens).
| | The distance between these two vertical bars is 10 mm (i.e., 1 cm) on the monitor. One millimeter is one tenth of that distance. Divide that 1 mm into one thousand parts and you obtain 1 µm (one micrometer). The bar at the image of the Escherichia coli bacteria shows, how the 1 µm distance at that particular magnification compares to the dimensions of the bacteria. - The figure at right shows the monitor of a scanning electron microscope (Philips XL-30 ESEM) during an examination of a thin layer of bacteria on a bacterial filter.
Most bacteria are harmless and many are useful to humans (e.g. bifidobacteria, shown at left) by suppressing pathogens. Many other bacteria are used to make a variety of certain foods. Streptococcus thermophilus (shown at right) likes warm milk which it coagulates, with the help of Lactobacillus bulgaricus, into yogurt. Both bacteria belong to a large group of so-called "lactic acid bacteria" (lactobacilli and lactococci). This name has been given to them because they metabolize milk sugar (lactose) in milk and produce lactic acid. This acid coagulates milk proteins and thus produces yogurt, cultured buttermilk, and other fermented milk products. Most cheeses could not be made without bacteria. Lactic acid bacteria including leuconostocs also participate in the production of sauerkraut whereas acetic acid bacteria (Acetobacter aceti) are used to produce "organic" vinegar. Because of their favourable effects on human health, lactic acid bacteria and bifidobacteria are called "probiotic bacteria".
January 21, 2005.
During my young years, children did not have the luxury of having microscopes in their bedrooms and we had to wait until high school to learn about them and try them with a variety of subjects such as onion skin cells, home dust, pollen grains, or yeast cells (Saccharomyces cerevisiae).
At the Technical University in Brno, I studied technical microscopy as one of the subjects in the first year of chemical engineering. At that time (1949) it was, of course, optical or light microscopy and we examined various fibres such as cotton, wool, silk, etc. Modern polyester fibres only waited yet to be developed and electron microscopes were relatively new high-resolution instruments, too expensive for universities.
In 1952, I obtained my MEng. degree in chemical engineering, added more experience in food science to what I had learned as a summer student and went back to school (Slovak Academy of Sciences) to study pectic substances in crabapples. In 1957 I started earning for my living for good. The microscopes were forgotten for 15 more years - until the seventies.
At that time I was a research scientist at a government food research institute in Canada. I was assigned an objective to develop wieners from surplus skim milk powder. It was like in a Czech fairy tale - there the heroes had to find elusive golden-haired princesses, fireball birds, golden-mane horses, or princesses turned into gem stones burried deep at the bottom of the sea. However, each of these heroes had some kind of helper with supra-natural powers - but I even did not have a technician.
The "hot dog" I made from milk powder looked very fake. Only the pink colour and the spicy smell could probably fool a potential customer. Otherwise it was brittle - not elastic - and instead of being juicy like the comminuted meat product, the milk powder imitation felt like a piece of bad cheese in the mouth. "If I only knew how to look at the microstructure of the two products", I sighed hopelessly, "then, maybe, I would understand, what makes them so different".
Suddenly it appeared that there was a helper with an almost supra-natural power - the power to show the smallest protein particles in milk. They are called "casein micelles" and their dimensions are between 10 and 20 nanometers (nm). If 1 mm can be divided into 1000 µm, 1 µm can be divided into 1000 nm. The micelles are so small that not even the best optical microscope would show them. It would be as helpless as are our eyes when looking down at bacteria.
A colleague, an electron microscopist, examined my product and a real "meat-based" wiener. Whereas the wiener consisted of meat fibres, its fake duplicate made from milk powder consisted of tightly packed tiny globules. Therefore, there was no elasticity whatsoever. "Please teach me how to use your electron microscope, so I can investigate these foods in greater detail and learn how their microscopical structure relates to the manner we feel them in the mouth", I asked my colleague.
January 23, 2005.
My colleague's willingness to instruct his technicians to teach me started a new direction in my research. Since the early seventies I have been using electron microscopes to study the development of structure in milk products until the middle of the nineties. Later on, there was a greater interest in the microorganisms which contribute to the manufacture of milk and many other food products.
The high resolution of electron microscopy has helped to make advances
in medicine and in biology. It was introduced into food science relately
late. There was a rapid increase in the number of scientists using
electron microscopy in their studies. A journal "Food Microstructure"
was established in 1982 and later renamed "Food Structure". It was doing
very well, appearing quarterly, but in 1994 it vanished as the
publisher, "Scanning Microscopy International" in Chicago (USA) abruptly
stopped publishing it without giving reason. The findings of several of
my colleagues from those times may be accessed in the "Guest Food
Microscopists" sections 1, 2, and 3.
There are many conditions to be met if meaningful images (micrographs) of foods are to be obtained from an electron microscope. The specimens placed in a scanning or transmission electron microscope must not release any gases or vapours in vacuo because volatile substances would absorb electrons which are used to magnify the specimens (it is certainly obvious that specimens destined for light microscopy should not release smoke). There is one exception with so-called "environmental scanning electron microscopes" (ESEM) which are constructed and operated to examine hydrated biological samples. These microscopes are relatively new to the field of electron microscopy.
Preparing the specimens for electron microscopy means retaining their original structure as much as possible and making them resistant to the harsh conditions inside the microscope. Proteins are fixed (denatured under controlled conditions) using specific fixatives, e.g., glutaraldehyde and/or osmium tetroxide, or applying controlled heat-denaturation in a special microwave oven. However, it makes no sense to subject non-proteinaceous specimens such as starches or fats to a similar treatment.
January 24, 2005.
As the Editor-in-Chief of the scientific journal Food Structure, I reviewed a few manuscripts based on some kind of an idea as if there was a uniform procedure for the fixation of all foods irrespective of the composition, size, and microstructure of the specimens. Common sense suggests, however, that it will take considerably more time for glutaraldehyde or osmium tetroxide to penetrate a compact processed cheese sample than a thin fat-free yogurt sample or that, of two processed cheese samples, the one in the form of a prism with a cross section of 1x1 mm may require 24 hours to get fixed with osmium tetroxide whereas the other, in the form of a 5x5 mm cube, will not be fixed even after 3 or 4 days. "If in doubt, get the answer to your question by an experiment", I used to remind the authors. Osmium tetroxide is particularly useful for that purpose. Leaving 1x1x10 mm prisms of yogurt, low-fat cheese, and high-fat processed cheese in an OsO4 fixative and fracturing a small part of each prism each day will show the penetration of the fixative into the sample as a gradually diminishing light core of the sample's cross section.
This approach also concerns other procedures such as extraction, dehydration etc. It is true that foods are altered biological tissues - and this fact has to be respected. Ham is much denser than a live or dead muscle, cheese is much denser than yogurt.
January 25, 2005.
As a microscopist who has spent many years studying the microstructure of yogurt and cheese, I should be able to produce images which would show their true microstructures. They would probably be diagrams - but not micrographs. It is not possible to place a small piece of yogurt or cheese under an electron microscope and to expect an image of the true microstructure.
As already mentioned, the samples examined by EM must not release gas or vapour. That means that water and other volatile substances must be removed from the sample. This can be accomplished in several ways (including samples in the form of fine suspensions) such as
1. dehydrating and drying the sample and
None of these procedures can show the structures of yogurt or cheese as they exist in the "fresh" samples. Fresh samples contain water with various substances (including lactose, lactic acid, inorganic and organic salts, milkfat globules, and even bacteria) dissolved and dispersed in it. When we look at a small piece of yogurt, we only see the "wet" surface.
Preparation procedures always alter the microstructure of the samples destined for electron microscopy. Drying for SEM and embedding for TEM remove water, freezing changes the water into ice and may cause ice crystal formation. If the change is intentional, it may allow certain features (e.g., details of the protein structure) to stand out. Such changes are not called "artifacts". This term is reserved for unintentional changes which occur due to negligence or omission.
January 31, 2005.
One of the best procedures to obtain excellent SEM images of the internal micro- structure of foods such as yogurt, curd, cheese, and tofu consists of fixing the specimen, preferably in the form of 1x1x10 mm prisms. The prisms are then dehydrated in a graded ethanol series. When they are impregnated with absolute (100%) ethanol, they may be frozen directly in liquid nitrogen although, contrary to general belief, liquid nitrogen is a poor cryogen. If a subject is immersed into it, liquid nitrogen immediately forms a layer of gas around it thus insulating it and reducing the freezing rate. Freezing hydrated specimens of this size in liquid nitrogen always leads to the development of ice crystals. As they grow, they ruin the original microstructure. Unlike water in hydrated specimens, ethanol does not crystallize when immersed in liquid nitrogen and forms glass-like ice. The frozen prisms are then fractured using a pair of insulated tweezers (they are not available commercially but it is easy to insulate them using short strips of duct insulation) and an insulated scalpel. The 1x1 mm cross section of the specimens allows rapid fixation and impregnation and the 10 mm length makes it possible to obtain several good fragments. The fragments are returned into absolute ethanol to thaw. Finally, they are critical point dried (CPD) and mounted on SEM stubs.
Specimens such as curd or cheese which contain fat, need to be defatted (the micrograph of Mozzarella cheese above was obtained in that way). The specimens impregnated with absolute ethanol are extracted using three changes of chloroforn or n-hexane followed by three changes of absolute ethanol. The specimens are then rapidly frozen, one by one, fractured, and CPD. There is a crucial step when the fragments have to be mounted on the SEM stubs: The fractured plane should be facing up for an easy SEM examination. The fresh plane can be recognized as being shiny in reflected light.
Why such a complicated procedure? Wouldn't it be easier to examine the surface obtained by cutting the specimen using a blade or a scalpel? No - the milk or soy proteins would be smeared by the blade or scalpel on the cut surface and the images obtained would show artifacts.
February 6, 2005.
Last week, I was showing an environmental scanning electron microscope to a food scientist. He was excited to learn that such microscopes make it possible to examine hydrated specimens. "How can it be done?" he wondered, knowing well that specimens destined for electron microscopy must not release any vapours or gases while they are being examined.
It is a tricky arrangement. In biological applications, the stage is cooled to around the freezing point of water (0°C) to reduce the vapour tension above the hydrated specimen and the air pressure is somewhat but not fully reduced (<9 torr) around the specimen. This space of somewhat reduced air pressure is surrounded with space where the pressure is considerably lower. The electrons thus pass through high vacuum and only the last few millimetres above the specimen they encounter higher pressure.
The presence of water molecules in the atmosphere around the specimen makes it possible to examine specimens not coated with gold and yet, "charging artifacts" are no problem.
"This is great news for milk products and other foods", my colleague happily exclaimed, thus repeating the reactions of other food scientists. I am sorry to cool down this excitement. Environmental scanning electron microscopes (ESEM) are excellent in structural studies of hydrated specimens in which water is enclosed in cells, such as insects or plants. Most foods, however, are not of this kind. They contain "free" water at their surfaces which covers the solid matrix composed of proteins (cheeses, meats), polysaccharides (fruits, vegetables, starch gels), fats (butter, mayonnaise) or their mixtures. Enlarging such surfaces by ESEM would not show the immersed solids. In addition, many hydrated foods also contain volatile substances other than water and may also contain free fats or oils. Such substances would soon contaminate the column of the microscope and reduce its efficiency. As I have already experienced it, subsequent examination of dry, gold-coated specimens such as microorganisms at high vacuum at much higher magnifications around 20,000x does not produce high-quality images.
In contrast, it is actually almost impossible to show bacteria properly because they live in aqueous media. Their surfaces may be covered with capsules which consist of polysaccharide gels, or the bacteria have a variety of minute structures such as fimbriae or flagella on their surfaces, all of which would retain free water. ESEM would show the aqueous envelope. Because of the minute size of the bacteria, it would be extremely difficult to adjust the temperature of the specimen and the air pressure above it, as these two factors determine whether water will evaporate from the sample (due to increased temperature or reduced air pressure) or will condense on it (decreased temperature and increased air pressure).
October 6, 2005.
A few days ago, a colleague asked me why I do not show bacteria on the tip of a needle, on a computer keypad, on the edge of a drinking straw and similar objects which "must be full of microorganisms". Of course, it would be instructive to show such images - other people asked similar questions. So let's look at the letters n or o on the monitor in this text. They are a little over 1 mm wide. Enlarging them 1000 times would make them 1000 mm = 1 m (about 3 feet) wide. If these letters were coated with bacteria, they would look, at that magnification, very small, like tiny dots or very short lines. To show bacteria as three-dimensional objects with flagella, I use magnifications between 8,000 and 20,000 times. The letters "n" and "o" would each be 8 to 20 metres (24 to 60 feet) wide. The bacteria would then look like the fingers of a little child. The tips of needles would look like a very wide hills - not sharp at all. The background, to which the bacteria were attached, would be irrelevant. To make viewers believe that salmonella were photographed on chicken skin, it would be necessary to assemble a montage (collage), where a micrograph would be superimposed over a photograph.
Scientists express magnification either by stating the width of the image in micrometers (1 µm = 1/1000 mm) or by showing a bar of a certain length (1 µm, 2 µm, 5 µm, 10 µm, etc.). Lactobacillus acidophilus bacteria are shown at two magnifications. At the higher magnification (image at left), the 2 µm bar is 16 mm long on my monitor. This means, that a 1 µm long bacterium would appear 8 mm, i.e., 8000 µm long. The bacteria would, therefore appear to be magnified 8000x. If your monitor shows a different length, the true magnification can be calculated in the following way:
M = 1000A/B where
M = the true magnification in a print, a book, on a poster
If the width of the image is given in micrometers, a similar formula is used to calculate the true magnification:
M = 1000D/E where
M = the true magnification in a print, a book, on a poster
October 12, 2005.
In the summer, I received a request for SEM micrographs of cultured buttermilk and stirred yogurt from an author who wanted to illustrate his chapters on these products in his new book on cultured milk products.
Semisolid products would disintegrate in the
aqueous solution of glutaraldehyde during fixation. There is, however, a
procedure whereby viscous samples may be fixed and further prepared for
SEM without any problem after they are encapsulated in agar gel capsules (schematically shown at left). This procedure, initially developed in 1968 by R. K. Salyaev (Proc. 4th Europ. Reg. Conf. Electron Microsc., Rome, II, 37-38) is suitable for various suspensions and emulsions including very soft centrifugation pellets.
March 21, 2006.
The presence of bacteria in chicken intestines may seem to be only distantly related to foods. Every living creature has bacteria in its guts, so why to be concerned?. Experts who care for the safety of foods for human consumption would prefer if the bacteria in chicken intestines be "friendly to humans" rather than pathogenic. There are indeed many "friendly" bacteria and they are called "probiotic", such as lactic acid bacteria (lactobacilli, lactococci) which utilize milk sugar lactose and oxidize it into lactic acid. This acid then coagulates milk and halps to produce sour milk, yogurt, kefir, cultured buttermilk and other dairy product. In cheeses, lactic acid bacteria also participate in their ripening. Leuconostocs are also part of lactic acid bacteria. They may be found in sauerkraut and various pickled vegetables. Bifidobacteria are another group of probiotic bacteria. Food safety experts are interested to know whether probiotic bacteria could be used to colonize the intestines and thus replace harmful pathogenic bacteria such as salmonella, staphylococci etc. Cultivation and microbiological identification would be used in such studies. Yet, it appeared interesting for an electron microscopist to find the bacteria in their niche. My objective was thus to find bacteria in the mucus which adheres to the intestinal mucosa and even directly on the intestinal villi. This means that I was not interested in the intestinal contents.
Traditional preparation methods for SEM remove the intestinal mucus and show a clean surface of the mucosa. Thus fixation of the mucus is essential. The mucus consists of polysaccharides, so fixatives different from glutaraldehyde must be used. In their studies of intestinal mucus in young pigs, Paula Allan-Wojtas et al. (Microscopy Research and Technique 36:390-399, 1997) successfully used Ruthenium red (RR) or Alcian blue (AB) added to a glutaraldehyde fixative to retain the mucus. The authors illustrated their paper with 30 micrographs, all taken at low magnifications to show the mucus but not the microorganisms.
In our experiments, 1 cm2 intestinal samples were excised and fixed according to Allan-Wojtas et al. Alcian blue produced beige specimens with very little mucus retained. Ruthenium red produced purple specimens and both the mucus and the resident bacteria were easy to find. The fixed samples were cut into 1 mm x 1 mm x 10 mm strips, dehydrated in ethanol, and freeze-fractured. Examination of the fractures showed both the mucus and plentiful bacteria, particularly in the caecum (see the illustration) and the colon. Various shapes of the bacteria indicated than no single species prevailed, although we are aware that bacteria cannot be identified from their shapes alone. Microbiological procedures have been used for that purpose. SEM showed bacilli (rods), cocci, clubs, spirochetes etc. during an extensive search as the micrograph above at left shows. Freeze-fracturing made it also possible to photograph the microvilli of the intestines.
March 27, 2006.
While examining long-grain rice starch by scanning electron microscopy for another scientist, I obtained specimens of many other kinds of rice from my Chinese colleague, Mr. Ann-Fook Yang. Thus I also examined black glutinous rice, short-grain rice, glutinous rice, scented rice, red jasmin rice, and red yeast rice.
Rice starch differs by its shape from starch granules isolated from other sources such as wheat, corn, potatoes, beans etc. Whereas the latter starches produce oval shapes as cross sections of the granules, rice starch granules produce polygons (image at far left; bar: 5 µm). Rice starch is very tightly packed in the rice grain cells. The starch granules have sharper edges in long-grain rice than in glutinous rice. A fractured grain may reveal minute globular protein bodies among the starch granules or their imprints may be seen on the starch granules as minute dimples.
Red yeast rice has a grain structure similar to that of the other rice varieties
but the starch-holding cells are loosened from each other and the starch
granules appear to be degraded or porous (image at left; bar: 10 µm).
Red yeast rice is produced in Asia by culturing a red fungus (Monascus
purpureus) on the rice grains and then drying the red product.
July 20, 2006.
Recently I have received several requests for advice on negative staining of casein micelles for subsequent transmission electron microscopy (TEM). The writers complained that they have been obtaining fuzzy images. Regrettably I have been unable to find exemplary micrographs of negatively stained casein micelles on the Internet. However, I reprinted images of negatively stained casein micelles (obtained from E. Uusi-Rauva at the State Control Office for Dairy Products in Helsinki, Finland) in 1977 in an article entitled "Electron Microscopy in Dairy Research" (MSC-SMC Bulletin, November 1977, pp. 4-10 - i.e. the Microscopical Society of Canada Bulletin). Then, 4 years later, I published my own micrographs (see below).
The text from my own publication (Electron Microscopy of Milk Products: A Review of Techniques by M. Kalab, Scanning Electron Microscopy 1981/III, 453-472 - SEM Inc., AMF O'Hare (Chicago), IL 60666, USA) reads as follows:
2. Transmission Electron Microscopy
TEM comprises all techniques in which the specimen is placed in the electron beam and the enlarged shadow is examined. There are various methods of preparing the specimen for this kind of study.
2.1. Negative Staining
References related to negative staining:
29. Carrol RJ, Thompson MP, Nutting GC: Glutaraldehyde fixation of
casein micelles for electron microscopy. J. Dairy Sci. 51, 1903-1908,
Additional information on negative staining:
February 28, 2007.
The image at left is an anaglyph - a stereogram. When viewed through a pair of red-and-green glasses, this image will reveal the three dimensional structure of the yeast cells and bacteria on the surface of a kefir grain.
At Christmas, my 7-years old grandson Adam showed me a book full of photographs of spiders, where many of them were in the form of anaglyphs. Using a pair of red-and-green glasses it was possible to see the spiders as 3-dimensional creatures. I knew anaglyphs from various musea where they have frequently been used to show aerial photographs of mountains.
In the past, I published pairs of stereo micrographs of food structures in scientific papers. The easiest way to view them was through a simple optical apparatus but many people have learned to obtain the 3-dimensional effect simply by focussing their eyes behind the journal's page.
Although I have been retired for 12 years, I rushed to the electron microscope on January 2, 2007 to take micrographs of various samples at two angles differing by 12 degrees. I assumed that if I tinted one image red and the other green and superimposed them in Adobe Photoshop one over the other as layers at 50% opacity, I would have created an anaglyph. My assumption was incorrect. I obtained fuzzy brown images. Fortunately I have found free software on the Internet and that has made the task easy. Examples of SEM anaglyphs and my advice how to make them may be accessed here. Anaglyphs enhance the ability of the scanning electron microscope and make it easier to evaluate vertical distribution of structural elements, for example bacteria, spores, blood cells etc.
May 10, 2007.
Escherichia coli bacteria were found as regular harmless inhabitants of the human intestines in 1885 but one hundred years later, some of these bacteria were found to produce Shiga-like toxins called verotoxins. Such bacteria are called toxigenic and are marked as Escherichia coli O157:H7. Lately they caused food poisoning in North America when they appeared on leafy vegetables which are consumed raw, such as lettuce and spinach.
SEM was used to visualize the presence of E. coli on the leaves. For illustration purposes, live nonpathogenic E. coli at very low concentrations were reacted with lettuce and spinach leaves and then fixed and photographed. It is interesting to note that particularly on lettuce leaves the bacteria congregate at leaf stomata and even enter them, thus hiding inside the leaves. It is evident that it would be impossible to wash the bacteria out from the stomata. Compared to lettuce leaves, spinach leaves (image at right taken at a lower magnification) are considerably more convoluted and the bacteria may hide in the convolutions. It would be equally impossible to wash them out from their hiding places. Their nature, i.e., their adaptation to life in the intestines makes it very easy for them to propagate in the intestines if they are ingested with the leaves in the form of raw salads.
Bacteria may not be identified from their SEM images, like in the micrograph
of human feces shown at left but this micrograph shows how plentiful the
bacteria are in the excrements and what proportion of the entire mass they
represent. It is no wonder that a small piece of an excrement may contaminate
a large volume of water. The micrograph also indicates that it is very
important to thoroughly wash one's hands after the use of a toilet.
January 30, 2009.
Proper preparation of specimens is essential to obtaining excellent SEM micrographs. The need to work with very small specimens (~1.5 mm in diameter) was emphasized in various connections, particularly if such specimens need to be fixed and dehydrated. However, even small dry particles may be quite difficult to prepare properly for SEM. Charging artefacts at left (white arrows; bar=100 µm) are probably the best known problem. They originate when the conductive gold coating on the specimen has gaps. Electrons hitting the specimen cannot be discharged and their charge consequently deflects the incident electron beam thus creating the artefact. This has already been mentioned above in this webpage.
A double-sided sticky tape has been used to attach small particles to the SEM stub. Additional painting of the particle side walls with a conductive silver cement improves the contact between the particle and the support. However, powder and dust particles are often very small (<10 µm) and they sink into the dense sticky material of the sticky tape.
One possibility to alleviate this problem is to dissolve the sticky material in an organic solvent such as acetone or toluene and to spread a droplet over a glass cover slip, 13 mm in diameter. Evaporation of the solvent leaves a considerably thinner sticky layer which may provide a good contact without submerging the particles too deep (left image, bar: 200 µm).
Another possibility is to avoid the use of any binding substance. The glass cover slip mounted on an SEM stub is sputter-coated with gold while a small amount of the powder (such as very small pollen grains, plant protein bodies, starch granule fragments) spread in a single layer on another cover slip is only half-coated with gold (10 nm), the coating is stopped, the particles are stirred and spread again, and gold-coating is completed. Then the particles are transferred onto the gold-coated cover slip and a droplet of absolute ethanol is used to spread the particles over the entire cover slip. Ethanol is then allowed to evaporate and the particles are coated with a thin layer of gold (~10 nm). This procedure ensures that there will be an electrically conductive contact between the particles and the support so that charging artifacts will not develop (right image, bar: 200 µm).
November 25, 2009.
Digestion breaks down all food constituents. Although our body extracts most nutrients, there is still enough of them to support microorganism in the lower digestive tract, i.e., the large intestine. In fact, some bacteria are even beneficial for our health. A wide variety of bacteria represent about one third of the volume of the intestinal contents. This subject was already mentioned in May, 2007. To view the bacteria under the SEM, the excrements need to be prepared in the same way as other samples but dry fracturing would be preferred to freeze fracturing.
Animal feces and bird droppings contain microbial pathogens which appear in our environment due to the lack of hygienic measures. The bacterium Escherichia coli is present in the intestinal contents of most animals and is thus called the "intestinal rod". When it is found in other environments, it is an indicator that fecal matter is present.
The swine flu epidemic in 2009 has renewed the interest in protecting our health. SEM images of unhygienic conditions may, therefore, be educational.
The foods which we bring home from a foodstore also contain bacteria. Leaving a cut of meat to lie at room temperature instead of placing it into a refrigerator ensures that bacteria will grow on it and make it slimy and unfit for human consumption.
The role of bacteria in the digestive system has been the subject of scientific studies. Nowadays the tendency is to colonize the system with probiotic, including lactic acid bacteria and bifidobacteria.
October 2, 2011.
The Royal Microscopical Society in the United Kingdom welcomes submission of articles of general interest to microscopists. Full articles or reviews ideally between 3000 and 4000 words contain images/figures, where relevant. There is no limit of the number of colour images. All micrographs must include scale bars or field widths and are expected to be submitted in CMYK colour mode. To view the full guidelines for authors, please visit www.infocus.org.uk.
As the author, I thank Scientific Editor, Dr. Adrian Burden, and Editor, Ms. Laura Kingsbury, and her team for the beautiful form which they have given to my three contributions. They may be contacted at firstname.lastname@example.org.
August 30, 2012.
SEM images of six strains of toxigenic (hemorrhagic) E. coli bacteria (O26, O45, O103, O111, O121, and O145) studied by Ms. Kyriaki Chatzikyriakidou at the University of Wisconsin were obtained in sufficient quantities to make statistical evaluation of their dimensions possible. Characteristic images show differences expressed also in the form of a diagram below. The lengths of the bacteria are expressed in micrometers with the scale at left. Ms. Chatzikyriakidou will publish her study when it is completed. Any questions concerning that study should be directed to her.
She collects microorganisms in caves and photographs them using an optical microscope.
November 16, 2012.
Mr. Ann-Fook Yang, Chief Technician at the Electron Microscopy Unit (EMU), Agriculture and Agri-Food Canada in Ottawa is retiring at the end of November 2012 after 34 years of work in that position. In the past several months his group was quite small as shown in the snapshot at right taken at a new arival in the EMU - an FEI Quanta 600 ESEM. Ms. Denise Chabot, who will take over Ann-Fook's position and duties, is pointing to a puzzling finding on the microscope's monitor while technician Mr. Keith Hubbard stands behind. The author of this site managed to press his camera's remote control button and turn his attention to the monitor.
Ann-Fook is taking with him a large amount of experience in a great number of electron microscopy techniques. He took over the position of Chief Technician in 1996 from Mr. Sierk Itz. He will be remembered for many good deeds at EMU, for his assistance to a large number of scientists and technicians with whom he co-authored many scientific papers (some of which are listed below) and also for two important additions to the EMU - a Hitachi 7000 transmission electron microscope acquired from the Food Inspection Agency, AAFC-AAC, and the newer FEI Quanta 600 ESEM transferred from Communications Canada.
List of most important publications:
• Allan-Wojtas, P. and Yang, A.F. (1987). Solution to difficulties encountered in low temperature SEM of some frozen hydrated specimens. Examination of Penicillium nalgiovense cultures. Journal of Electron Microscopy Technique 6:325-333.
• Deng, C., Zhang, P., Vulesevic, B., Kuraitis, D., Li, F., Yang, A.F., Griffith, M., Ruel, M., and Suuronen, E.J. (2009). A collagen-chitosan hydrogel for endothelial differentiation and angiogenesis. Tissue Engineering Part A:16(10) 3099-3109.
• Greco, C., Martincic, I., Gusinjac, A., Kalab, M., Yang, A.F., and Ramirez-Arcos, S. (2007). Staphylococcus epidermidis forms biofilms under simulated platelet storage conditions. Transfusion 47(7):1143-1153.
• Hampson, M.C., Yang, A.F., and Bal, A.K. (1994). Ultrastructure of Synchytrium endobioticum resting spores and enhancement of germination using snails. Mycologia 86:733-740.
• Kalab, M. and Yang, A.F. (1985). Bacteria-related artefact in replicas of freeze-fractured yoghurt. Milchwissenschaft 40(7):385-387.
• Kodama, H., Nelson, S., Yang, A.F., and Kohyama, N. (1994). Mineralogy of rhizospheric and non-rhizospheric soil in corn fields. Clays and Clay Minerals 42:755-763.
• Patel, J.J. and Yang, A.F. (1981). Light and electron microscopic studies of nodules structure of alfalfa. Canadian Journal of Microbiology 27:36-43.
• Yang, A.F. and Hamilton, R.I. (1974). The mechanism of seed transmission of tobacco ringspot virus in soybean. Virology 62:26-37.
• Kaláb, M., Yang, A.F., and Chabot, D. (2008). Conventional scanning electron microscopy of bacteria. Infocus Magazine - The Proceedings of the Royal Microscopical Society, Issue 10 June 2008: 44-61.
• Yang, A.F. Guest Food Microscopist 3.
Contributions from the following dates may be viewed:
January 19, 2005
March 21, 2006
Feb. 28, 2007
Jan. 30, 2009
Nov. 25, 2009
Sep. 20, 2010
Oct. 2, 2011
Aug. 30, 2012
Nov. 16, 2012
Learn more about the subjects mentioned using the links below.
Any comments? Please send them to
Milos Kaláb, and mark the subject line "Microscopy comment". Thank you.
Antonie van Leeuwenhoek (1632 - 1723)
Robert Koch - one of the two fathers of modern bacteriology
The life and times of Louis Pasteur.
Paul de Kruif - a man of science
Escherichia coli are common in the intestines of humans and animals and if found in foods, they indicate fecal contamination. Very dangerous O157: H7 and similar mutants can cause severe infections, even death.
Saccharomyces cerevisiae ferment sugar and produce carbon dioxide and alcohol. They are used to make leavened bread, beer, wine and other spirits.
Light (a stream of photons) is used in optical microscopes to magnify the subject. A stream of electrons is used for this purpose in electron microscopes.
The first transmission electron microscope was developed between 1937 and 1939 by two postgraduate students working in the Physics Department of the University of Toronto with their physics professor.
Electron microscopy explained
TEM and SEM compared. Links to other kinds of microscopy.
There are two basic modes of electron microscopy (EM): scanning and transmission. Specimens destined for EM must not release gases or vapours during examination
Environmental scanning electron microscopy (ESEM) makes it possible to examine hydrated (water-containing) specimens if the water is confined to cells. Excellent images are obtained with fresh insects and plants or their parts. ESEM is, however, not suited for specimens which contain free water, such as cheeses, meat products, juicy fruits, etc.
Scanning tunneling microscopy, Atomic force microscopy and other techniques to study the microstructure of surfaces.
Microstructural Principles of Food Processing and Engineering - a book by Jose M. Aguilera and David W. Stanley.
Structure of Dairy Products - a book by Adnan Tamime.
Viewers interested in microscopic images of various subjects may wish to visit some of the following sites which specialize in science.
Images from the Microscope. Tabulated 29 links provide a cross section of different imaging modes and subjects.
Microscopy Websites compiled by the National Health Museum, Resource Center.
Dennis Kunkel Microscopy, Inc. The science image library holds over 1,500 light microscopy images and electron microscopy images (colorized and black & white).
Molecular Expressions TM offer one of the Web's largest collections of optical microscope images (micrographs) in colour.
MicroAngela Electron Microscope Image Gallery. Explore familiar and unexpected views of the microscopic world with colourized images from electron microscopes at the University of Hawaii.
Microscopes and Microscopy on the Web Links to microscopy resources.
Electron Microscopy Black and white transmission electron microscopy images of normal cells and organs and neoplasms.
Electron Microscopy Image Index by Indigo Instruments.
Microscopy UK Resources Links to various resources
David Scharf's Images The author is known in the scientific and photographic commu- nities for his work which has been shown in numerous magazines and museums.
There are also strictly commercial sites called photo banks which have large quantities of micrographs in their searchable archives, e.g.:
Visuals Unlimited is a leading source with a wide variety of images including micrographs.
Mediscan Mediscan is a photographic agency specialising in medical, health, scientific and natural history images, managed by Medical-On-Line Ltd.
Science Photo Library presents special photo techniques, images in action, press releases, feature stories etc.
Complete book of the microscope. Step-by-step microscope activities for children include preparing slides and observing everyday objects, insects and even your own cheek cells.
The images are protected by copyright - please contact the author for information.
© SCIMAT 2013