You are on page 1of 35

FOODS UNDER THE MICROSCOPE

Updated: February 2, 2009.


New information.

Introduction
Microscopy is a scientific discipline, the objective of which is to enlarge minute particles or
structures in order to study them. Depending on whether the objects are biological, geological, or
metallurgical, there is a wide variety of instruments and techniques suitable to achieve the
objective. A good starting point on the Internet is the Microscopy in Wikipedia.
There are many reasons to examine foods under a microscope. Low-magnification dissecting
microscopes function as magnifying glasses and may be used for better views of the food
structure. They show details of meat fibres or seed surfaces and spices which may be captured on
film or a video tape using various attachments. They are the first microscopes to be used when
examining a particulate food contaminant.
Microscopy at a higher resolution helps to understand what happens to the original biological
materials such as grains, muscles, or milk when they are processed into bread or pasta, ham or
steak, yogurt or cheese. With industrial food production, developers of new foods need to know
what causes a particular food to be elastic or spreadable (e.g.: string cheese and processed
cheese) or why some foods, which are smooth under normal conditions, become gritty (e.g.: ice
cream stored for too long in a freezer).
Optical microscopes provide a higher resolution than dissecting microscopes and open
completely new views at the specimens examined. They are relatively easy to use. Their price
makes them affordable to any laboratory - even individual enthusiasts and students.
Magnifications up to 2000x are achieved using this kind of microscopes. With a higher
magnification, greater details are revealed but smaller areas are shown. In addition to structural
details such as globules or fibres, specific staining procedures make it possible to distinguish
proteins from fats, starch, cellulose, mineral components etc. A book entitled Food Microscopy
by Olga Flint (Royal Microscopical Society Microscopy Handbook 30, Bios Scientific
Publishers, Oxford, UK 1994) presents instructions on food sample preparation for microscopy
and helps to interpret the results. It is illustrated with many micrographs.
Electron microscopes are the subject of this site. They provide a considerably higher
resolution than light (optical) microscopes. However, since electrons, and not photons, are used
to enlarge the views of the specimens, the resulting photographs (micrographs) are black and
white only. For education and illustration purposes, individual specimen components such as
starch granules, fat globules, muscle fibres, cell walls, microorganisms etc. in such micrographs
may be highlighted by false colours.
Electron microscopy provides a markedly higher magnification at a considerably better
resolution than light microscopy but is much more expensive to perform. Instead of light, a beam
of electrons generated from an incandescent tungsten or lanthanum hexaboride (LaB6) electrode
is used to magnify the image of the sample. The electrons are negatively charged particles which

behave like radiation having a very short wavelength. There are two major electron microscopy
modes - scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The
electron beam is focussed using magnetic lenses in both kinds of microscope. In the schematic
diagram on the left, 5 lenses are shown for TEM and only two for SEM. The specimen in placed
into the path of the electron beam (S) in the TEM but in the SEM it is placed at the end of the
focussed electron beam path. The image (Im) is produced in the form of a shadow on a
fluorescent screen in TEM. Greater detail on image formation in the TEM is also available. In
SEM, reflected and secondary electrons are processed by an electron detector to form a quasi
three-dimensional image on a monitor screen.
Since the electrons would be easily absorbed by air, the microscopic examination is carried
out in vacuo. To ensure that the electrons will penetrate a thin section of the specimen or its
replica, the electron beam is accelerated in the microscope. An anode with an orifice in its centre
is positively charged. The negative electrons rush toward it and those which are in the centre fly,
accelerated, through the orifice toward the specimen. Accelerating voltage of 3 to 20 kV has
been used to do SEM and 60 to 80 kV have been used in TEM of foods.
Environmental scanning electron microscopes (ESEM) can be used to examine hydrated
(water-containing) specimens at a reduced air pressure. In this way, there is no need to dry or
freeze the specimen. This is particularly useful with very fragile specimens such as moulds
(fungi and their spores) and plant specimens such as fruits and vegetables. ESEM micrographs
have been contributed by Mr. A.-F. Yang.
Traditional electron microscopy requires that the specimen must not release any gas or
vapour when inserted into the transmission or scanning electron microscope. Except for
powdered foods such as flour, sugar, or milk powder, most foods contain water. Drying or
freezing at a very low temperature of -100C ensure that the condition of not releasing gas or
vapour is met.
Transmission electron microscopy can by performed using various techniques. The most
common consists of embedding the specimen in a resin, cutting thin sections (15 to 90 nm thick)
using a microtome, staining the structures within the sections (using heavy metal salts such as
osmium, lead, and uranium), and placing the sections into the path of the electron beam.
Other TEM methods include negative staining, which is a relatively simple procedure
whereby minute particles such as casein micelles, dietary fibre, bacteriophage, or bacteria are
mixed with a heavy metal salt solution and applied on a thin electron-transparent film. The
specimen and the salt solution are dried and placed into the microscope. The heavy metal, which
surrounds the organic specimen, absorbs electrons but the electrons which pass through the
organic specimen form a 'negatively stained' image.
Metal shadowing is another simple technique whereby the specimen is dried on a translucent
film and then is 'shadowed' with platinum vapour in vacuo. The thickness of the coating (less
than 0.005 micrometer) depends on the angle of the surface shadowed. The image is formed by
the electron beam which passes through the different thickness of the coating which depends on
the topography of the specimen's surface.
Freeze-fracturing and freeze-etching techniques are the most laborious. They make it
possible to examine the specimen without altering it chemically (fixation) or physically
(dehydration, impregnation with a resin, drying). The specimen is rapidly frozen, then is freezefractured at a temperature below -110C and the fracture plane is replicated with platinum and

carbon either immediately or after a certain period of freeze-etching, during which a thin layer of
ice in the specimen sublimes off and reveals underlying structures. The specimen is thawed and
the replica is separated from the specimen, cleaned from specimen residues, and examined in the
microscope.
In scanning electron microscopy (SEM), the specimen is examined by a focussed electron
beam. Some of these electrons are reflected and others generates secondary electron from the
gold coating. (A great variety of other interactions also takes place). Secondary electrons (or, in
other applications, backscattered electrons) are used to form an enlarged image of the specimen
surface. The incident electrons carry a negative charge and in order to be 'neutralized' after they
have completed the examination, the specimen should be electrically conductive. This is
achieved either by chemical procedures which impregnate the specimen with osmium or, more
frequently, by physically coating its with gold, a gold-palladium, platinum, or iridium occasionally both procedures are combined.
Metal coating provides a path for the electrons. If this path is interrupted (by incomplete
metal coating or by cracks), the electrons sit in the area thus isolated and repel any electrons in
the incidental beam in accordance with the rule that electrically charged particles of the same
charge repel each other. Thus the area occupied by the stationary negative charge is by-passed
and cannot be examined. White spots or lines develop in such places and the image is
characterized as suffering from charging artifacts.
SEM shows specimen surfaces. To the uninitiated, such images may appear familiar, since this is
the way in which we perceive our environment, where we also see surfaces, for example, milk
powder particles. The images are observed on a monitor and photographed using a 35-mm
camera from a special monitor.
TEM is predominantly used to reveal internal structures. Mutual relationship between SEM and
TEM is explained on the left. Where SEM shows a chain of beads (top) in the same way as our
eyes would see it, TEM provides an enlarged shadow (bottom) of a section through the chain,
which had earlier been embedded in a resin. A scientist seeing only the section would assume
that there were several single beads (X) and a short chain (Y) present in the embedded sample.
The true structure could be revealed by using both electron microscopy modes.

Encapsulation of viscous foods for electron microscopy


Biological suspensions consisting of minute organisms are difficult to prepare for electron
microscopy as individuals. R. K. Salyaev (A method of fixation and embedding of liquid and
fragile materials in agar microcapsulae. Proc. 4th Europ. Reg. Conf. Electron Microsc. Rome 11,
37-38, 1968) suggested to concentrate them in agar gel capsules. When sealed, the capsules
would be handled as larger solid samples. This technique is very useful in other microscopic
disciplines including food science, particularly with viscous foods such as stirred yogurt, various
forms of cream, mayonnaise etc. It may be used for both scanning electron microscopy (SEM)
and transmission electron microscopy (TEM). For the latter technique, considerably smaller
capsules would be required than for SEM.

A modified encapsulation procedure is very simple to perform but it requires some


preliminary practice. The individual steps are shown in the diagram at left from top to bottom.
Agar (3%), dissolved by boiling in distilled water, stirred with a magnetic bar is allowed to cool
to approximately 40C and is then maintained at this temperature.
The sample to be examined by SEM is aspirated (approximately a 10 mm long column) into
a pasteur pipette, 1 mm in diameter. The sample is shown in blue colour inside the tip of the
pipette at the top of the diagram [Step 1]. The exterior of the pipette is wiped clean and its tip is
closed with a small droplet of the agar sol (yellow) [Step 2]. (Kalb M: Encapsulation of viscous
foods in agar gel tubes for electron microscopy. Food Microstructure 7, 213-214, 1988).
The pipette is then very briefly dipped into the agar sol and is immediately rotated and turned
up and down so that a thin sleeve (yellow) forms around the pipette [Step 3]. The sleeve should
be about 0.5 mm thick. It solidifies as the agar sol cools. With an increased experience, this
procedure becomes rapid and the sleeve is quite uniform. Dipping in agar sol may be repeated if
the first sleeve is too thin. Then the excess gel is trimmed at the upper level of the sample
(arrow) and is removed [Step 4].
The crucial Step 5 is to gently hold the agar gel capsule with fingers and pull the pipette out
from the sleeve using the other hand so that the sample fills the gel capsule. The capsule is then
positioned vertically (diagram: bottom line left) and a droplet of agar sol is used to close the
open end [Step 6]. When it cools, the sample is encapsulated and may be placed into a fixative. It
is then handled as a solid sample (dehydrated in a graded series of ethanol, defatted, and freezefractured).
For TEM, the sample is encapsulated using a thin (0.3-0.5 mm) capillary. The agar sleeve should
be as thin as possible. Capsules 2-3 mm long may be cut, after the sample had been fixed,
postfixed, and dehydrated, before embedding in a resin. At that stage, foods such as yogurt or
cream are sufficiently solidified.
In contrast to earlier assumptions, this procedure is not suitable for the encapsulation of milk.
During fixation, the milk serum is replaced with the fixative and casein micelles sediment at the
bottom of the capsule (Kalb M, Larocque G: Suitability of agar gel encapsulation of milk and
cream for electron microscopy. Lebensmittel-Wissenschaft und-Technologie 29(4) 368-371,
1996).
Encapsulation at low temperature There has been concern that dipping a heat-sensitive
sample such as mayonnaise or cream inside a thin-walled pasteur pipette into a warm agar sol as
mentioned above, would damage the microstructure of such food. A new procedure has,
therefore, been devised, which makes it possible to encapsulate viscous food samples including
egg yolk at low temperature (Veliky I A, Kalb M: Encapsulation of viscous high-fat foods in
calcium alginate gel tubes at ambient temperature. Food Structure 9, 151-154, 1990).
A double-needle assembly consists of a central needle, 1 mm in diameter, concentrically
located in a wider needle. The gap between the two needles is 0.3 mm. This assembly is
connected to two 5-mL syringes with pistons (gray) so that the food sample (blue) would flow
through the inner needle and a 3% sodium alginate solution (yellow) would flow from another
syringe to coat the food sample as shown in the diagram at left. The food sample and the sodium
alginate solution are extruded simultaneously into a 0.05 M calcium chloride solution, pH 6.5,
where sodium alginate immediately forms a gel and immobilizes the food sample. The rate of
extrusion depends on the viscosity of the food to be encapsulated. This procedure produces 100

to 200 mm long columns of encapsulated food, which may be cut into shorter segments and
transferred into a fixative for subsequent preparation for SEM.

Fixation of fat for TEM


Glutaraldehyde fixes only proteins. Unsaturated lipids in foods may be fixed using osmium
tetroxide following glutaralldehyde fixation. This procedure is called postfixation. Although
double bonds (-C=C-) in unsaturated fatty acids react with osmium tetroxide, osmium is easily
removed by hydrolysis. This reaction leads to the formation of a diol and osmium trioxide
(OsO3). Hydrolysis does not take place if a heterocyclic base such as pyridine or imidazole is
present in the fixative (the diagram below was drawn according to G. Greyer: Lipid fixation,
Acta Histochem. Suppl. XIX:202, 1977). Based on this information and a paper by S.
Angermller and H. D. Fahimi (Imidazole-buffered osmium tetroxide: An excellent stain for
visualization of lipids in transmission electron microscopy. Histochem. J. 14:823, 1982), P.
Allan-Wojtas and M. Kalb developed a procedure to fix milkfat globules (Milk gel structure.
XIV. Fixation of fat globules in whole milk yogurt for electron microscopy. Milchwisssenschaft
39:323, 1984).

Saturated fat in milkfat globules crystallizes at refrigerator temperatures. This fat does not
interact with osmium tetroxide (and also does not react with subsequent stains) and is easily
distinguished by light colour and sharp crystalline outlines from unsaturated lipids.
Imidazole-buffered osmium tetroxide may also be used with specimens destined for SEM. In
this case, the specimens are rapidly dehydrated in a graded ethanol series and are not treated with
chloroform or n-hexane but are subsequently freeze-fractured, returned into absolute ethanol at
20C, and immediately critical-point dried. Failure to either fix fat thoroughly (e.g., with
imidazole-buffered osmium tetroxide) or to remove it completely (no postfixation with osmium
tetroxide but extraction of the fat from ethanol-impregnated samples using chloroform or nhexane) would lead to artifacts: fat residues in the form of minute globules would be found in the
sample.
Note: This section on the fixation of fat has been added to this page in response to requests from
several food scientists.
Immunolocalization in electron microscopy
Immunolocalization of b-lactoglobulin in a milk product embedded in LR White resin
1. Thin section is treated with rabbit anti-b-lactoglobulin antibody (shown in pink colour) and
washed. The rabbit antibody adheres only to areas filled with b-lactoglobulin.
2. Then the section is treated with anti-rabbit IgG (immunoglobulin G - shown in green colour)
conjugated with gold granules (shown as yellow discs) and washed. Anti-rabbit IgG adheres only
to areas covered with the rabbit anti-b-lactoglobulin antibody. The areas which contain b-

lactoglobulin are thus labelled with gold granules. TEM shows the gold particles as minute black
discs.
Coloured areas in the action diagram at left are explained above: b-lactoglobulin (blue) is shown
as part of a milk product (brown) embedded in a resin (light yellow).
The final stage which is subjected to electron microscopy is presented below. Anti-blactoglobulin antibody is shown in pink and anti-rabbit IgG is shown in green with gold granules
in rich yellow:
Immunolocalization in electron microscopy is a technique which makes it possible to identify
a particular protein or polysaccharide among other proteins and polysaccharides by marking it
with minute gold granules. Examples may include the presence of b-lactoglobulin in curd which
consists mostly of casein or, in case of a fictitious adulteration, bovine milk proteins in sheep
cheese. Any substance which evokes immunological response as a 'foreign' body may be
detected. Dying the specimen, as is usually done in optical microscopy, is irrelevant because
electron micrographs do not reveal colours. Often the chemical composition of the minor
substance to be identified is very close to the major substance, so immunolocalization using
colloidal gold is used even in optical microscopy. These techniques are common in medicine and
biology and are increasingly useful in food science.
The food (milk product) under study is embedded in a resin such as LR White or Lowicryl
K4M and sectioned. An example of a thin section is in the diagram at right showing blactoglobulin inside a milk product. The crucial point is to react the section with a specific
antibodies against the substance to be marked (in this case, b-lactoglobulin). These antibodies
must be obtained from an animal different from cattle (for example, rabbit, goat), if cow milk
product is the main sample.
Thus, rabbit anti-b-lactoglobulin antibodies become attached only to the sites where there is
b-lactoglobulin in the sample. The section is washed to allow only these antibodies to remain
attached to the labelled sites. These antibodies are shonw in pink colour in the animation at left.
Next, antibodies against anything of rabbit origin are applied. However, these antibodies
(anti-rabbit IgG) are conjugated with gold granules (colloidal gold). The gold granules are very
small, 5 to 150 nm in diameter. Their diameter may be controlled, so there is a choice to use 5
nm, 20 nm, 50 nm or larger 'gold markers'. In the animated diagram at left, the anti-rabbit IgG is
shown in green colour and the gold markers are yellow. The anti-rabbit IgG reacts only where
there is a rabbit-made substance at that is the anti-b-lactoglobulin antibody. Excess of the marker
is washed off, the section is dried and examined in the electron microscope. There is a word of
caution: To show the principle and the procedure, nothing in these schematic diagrams is shown
to scale.
When the electron beam passes through the gold-labelled section, enlarged shadows of the
gold granules are shown where there is also b-lactoglobulin in the micrograph. Work is in
progress to show such micrographs on this page.
For more information, see, for example
Biotechnology and Bioapplications of Colloidal Gold. Ralph M. Albrecht and Gisele M. Hodges
(editors). Published by Scanning Microscopy International, Chicago 1988; ISBN 0-931288-39-8.
A paper by B. L. Armbruster and N. Desai in Food Structure Vol. 12, pp. 289-299 (1993) (search
through EDIT - FIND for the authors or immunolocalization).

Freezing samples for electron microscopy?


Development of ice crystals in a casein micelle suspension. Ice displaces the casein micelles.
The need to prevent hydrated samples (such as moist foods - meats, cheeses, fruits and
vegetables, etc.) from releasing water vapour when they are prepared for electron microscopy
has already been mentioned. Since a beam of electrons is used to obtain enlarged images and
electrons are easily absorbed by any molecules in the atmosphere, electron microscopy requires
that the specimen releases neither gas nor vapour when inserted in the evacuated stage chamber.
This applies to SEM as well as TEM (with the exception of so-called environmental scanning
electron microscopes - also see these micrographs.
Dehydration (drying) of the samples is one of the procedures to prevent the generation of
water vapour, freezing is another procedure, and replication of either dehydrated or frozen and
freeze-fractured samples is yet another possibility. Each of these procedures has some
advantages and some drawbacks.
Hydrated food samples that had been frozen may further be processed for SEM or TEM. It is
usually claimed that freezing fixes the specimen without need of using any chemicals so the
development of artifacts is reduced. This may be true only under very specific conditions - if the
aqueous phase present in the specimen is frozen rapidly to form "vitreous" ice.
Examples of ice crystal development in an ice crean mix. Bar in left image: 2 m, bar in right
image: 1 m)
Vitrification of water occurs when the freezing has occurred so fast that ice crystals have no
time to form. Otherwise, there is a great probability that the specimen will be distorted by ice
crystals which develop when water freezes "slowly". For pure water to form vitreous ice, the
freezing rate would have to be more than 1x106K/s and for biological samples the rate should be
greater than 1x104K/s. Heat can only be removed from the specimen by conduction through its
surface. This means that the high freezing rate may be achieved only in a very thin (up to 3 m
thick) superficial layer. For TEM the specimen thickness is limited to less than 10 m and
droplets must be smaller than 20 m in diameter. Beyond the zone of vitreous ice, ice crystals
grow and push other constituents in the specimen aside (schematically shown at left), be they
simple molecules such as sugars, suspended particles such as casein micelles, or solid structures
such as coagulated proteins or a polysacharide matrix. "Slow" freezing occurs even if a relatively
small sample, e.g., 2 mm in diameter, is dropped into liquid nitrogen. Although the temperature
of liquid nitrogen is very low (-196C), this is its temperature of boiling. Any object dropped
into it vapourizes the liquid around and generates a thin layer of nitrogen gas which insulates the
object and reduces the heat transfer from it. Freons (in the past) and n-hexane or liquid ethane
nowadays, cooled with liquid nitrogen are considerably better cryogens. Various rapid freezing
procedures, particularly high-pressure freezing, have also been developed - details may be found
in books listed at the end of this section - in fact, at present, the principles and applications seem
to be better explained in the older books than on the Web).

Negative staining of influenza virus with ammonium molybdate by M. Nermut [2]:


Top: Specimen freeze-dried
Bottom: Specimen air-dried
Bars indicate 0.1 m
Frozen specimens are usually freeze-fractured in order to examine the internal structure by
SEM. Freeze fracturing may be done in a special apparatus called the cryo-stage, attached to the
scanning electron microscope. A brief period of freeze etching removes a thin layer of the frozen
ice by sublimation thus allowing the solid structures to emerge. The frozen specimen is then
coated with gold.
Freeze fracturing may be combined with conventional SEM. The specimen is chemically
fixed, dehydrated in absolute ethanol, frozen and freeze-fractured in liquid nitrogen (ethanol does
not produce crystals when frozen). The fragments are thawed in absolute ethanol, critical-point
dried, mounted on SEM stubs, coated with gold, and examined by conventional SEM. The
benefits of this procedure have been outlined several paragraphs below and elsewhere.
Specimens destined for TEM may or may not be freeze-etched and then replicated with
platinum and carbon. The specimen is then removed and the replica is cleaned, captured on a
grid, and examined. Freezing is also advantageous to negative staining of some specimens. Airdrying used most frequently distorts even minute particles such as spherical viruses. Freezing
them with the thin layer of the negative stain still on the supporting film and freeze-drying
preserves the globular shapes of the viruses as had been shown by M. Nermut in [2]. Freezedrying is similarly useful with specimens destined for metal shadowing as had also been shown
by the same author. These procedures would be useful with casein micelles and other minute
hydrated corpuscular food components.
Could frozen ice cream be embedded in a resin? H. D. Goff provides the answer and decribes an
important preparatory procedure called freeze substitution.
Information in greater detail may be found in these and other books:
1. Electron microscopy of foods M. Kalab in: Physical Properties of Foods, M. Peleg and E. B.
Bagley (eds.), pp. 43-104, AVI Publ. Co., Inc., Westport, CT, USA, 1983.
2. Freeze-Etching Techniques and Applications E. L. Benedetti and P. Favard (eds.), 274 pp.,
Socit Franaise de Microscopie lectronique, Paris, France, 1973.
3. Freeze fracture: Methods, Artifacts, and Interpretations J. E. Rash and C. S. Hudson (eds.),
204 pp., Raven Press, New York, USA. 1979.
4. Freeze-Fracture Replication of Biological Tissues C. Stolinski and A. S. Breathnach, 173 pp.,
Academic Press, 1975.
5. Replica, Shadowing and Freze-Etch Techniques J. H. M. Willison and A. C. Rowe (A. M.
Glauert, ed.), 301 pp., North-Holland Publ. Co., 1980.
6. The Science of Biological Specimen preparation for Microscopy and Microanalysis J.-P.
Revel, T. Barnard, and G. F. Haggis (eds.), 245 pp., Scanning Electron Microscopy, Inc., AMF
O'Hare, IL, USA, 1984.
7. Cryotechniques in Biological Electron Microscopy R. A. Steinbrecht and K. Zierold (eds.),
297 pp., Springer-Verlag, Berlin-Heidelberg, 1987.

8. Electron microscopy of milk products: A review of techniques M. Kalab, Scanning Electron


Microscopy/1981/III, pp. 453-472 (146 bibliographies).
***
Dry-fracturing and freeze-fracturing
Many food microscopists have not yet discovered the benefits of freeze-fracturing and open
their specimens by dry-fracturing to reveal their internal microstructure. However, there is an
important difference between the two preparatory procedures:
When a dried sample is fractured, the fracture runs through areas of the lowest resistance. If
the sample contains dense inclusions (calcium crystals in cheese, compacted protein clusters in
yogurt), such inclusions will not be fractured but will be either removed or will stay intact in the
sample. Even in a relatively uniform sample such as smooth yogurt or cheese, the topography of
the fracture will be coarse. Its images will be unsuitable even to roughly estimate the diameters
of the air cells, fat globules, or other constituents.
Dry-fracturing of a cheese sample from which fat has been removed (left diagram) shows 2
fractures (red lines). In both cases, the sample has fractured through the thinnest walls. The
resulting image is that of a very porous structure.
Freeze-fracturing of the same sample (right diagram) impregnated with absolute ethanol is
characterized by fracture planes (blue lines) running straight irrespective of cheese protein or
void spaces which, in this case, are filled with frozen ethanol. The resulting image is that of a
sizeable proportion of cheese protein.
Freeze-fracturing is carried out in liquid nitrogen on specimens that have been impregnated
with absolute (100%) ethanol. There are no air pockets in such specimens. Whereas freezing
produces ice crystals in most hydrated specimens, no crystals develop in specimens impregnated
in absolute ethanol. This means that there is no risk that the microstructure of the specimens will
be severely damaged by freezing if the aqueous phase is replaced with ethanol. Wherever
possible, it is advisable to trim each specimen into 2 or 3 small blocks (1x1x10 mm) before
fixation. When the blocks are fully dehydrated, they are immersed one after another in liquid
nitrogen using a pair of insulated needle-point tweezers. There should be a small metal block
immersed in the insulated box where liquid nitrogen is held. Freeze-fracturing is done on that
metal using a pair of tweezers and a scalpel (both insulated and precooled in liquid nitrogen). A
sudden increase in pressure on the scalpel tip at the edge of the sample chips off a small particle.
The entire specimen block may be fractured into more than 10 particles within a few seconds.
Each frozen fragment is then dropped into absolute ethanol held at ambient temperature and all
fragments are subsequently critical-point dried.
The fragments have smooth fracture planes as if an ultrasmooth cut has been made through
the specimen - a marked difference from the rugged surface of a dry-fractured specimen. Dense
as well as porous regions are all fractured in the single plane. Distribution and dimensions of
void spaces and ingredients and inclusions such as fat globules, microorganisms, calcium

phosphate crystals etc. may easily be evaluated. Micrographs of yogurt and various cheeses have
been obtained with freeze-fractured specimens.
***
Artefact in transmission electron microscopy of starch
Electron micrographs of thin sections of starch granules embedded in a resin typically resemble
the image shown at left. The dark 'spikes' have long puzzled researchers and some of them have
attempted to find correlations between the density, shapes, and dimensions of the 'spikes' and the
presence of other materials, such as proteins, inside the starch granules. However, the origin of
the 'spikes' was explained more than 26 years ago by D. Gallant and A. Guilbot (Artefacts au
cours de la prparation de coupes de graines d'amidon. Etude par microscopie photonique et
lectronique - Artefacts developed during the preparation of starch granule sections. An optical
and electron microscopy study (in French). Die Strke/Starch 23(7) 244-250, 1971). Both
diagrams have been drawn on the basis of the authors' paper.
During embedding, the dense starch granules resist impregnation with the resin and so remain
unimpregnated. While thin sections are floated on water during sectioning (diagrams at right),
the starch granule sections become hydrated and they swell (figures 2 and 3 from top). The
swollen section eventually develops folds (bottom figure). Thus, the nature of the 'spikes', i.e.,
the folds, is rather related to the dimensions of the starch granules, the thickness of the section
and the time, which the section spent in contact with water. Authors D. Gallant and A. Guilbot
documented the fold formation by SEM micrographs.
Many papers on the microscopy of starch have been published in Food Structure. Search for
them using the EDIT - FIND function of your browser. The journal may be found at universities
which have food science libraries.
Very interesting information about the structure of foods, their sources and ingredients and about
its importance in food preparation was published by Harold McGee: On Food and Cooking (The
Science and Lore of the Kitchen) at Charles Scribner's Sons, New York, 684 pages, 1984. The
role of starch in a variety of foods is discussed in great detail.
***
SEM of microorganisms
Microorganisms are present on purpose in many foods, such as cultured (fermented) dairy
products, leavened bakery products, sauerkraut, pickles, and even some special salami. Lactic
acid bacteria and bifidobacteria are part of a group of so-called probiotic bacteria which means
useful bacteria which may protect us from food-borne pathogens. Yeasts such as Saccharomyces
cerevisiae, known for their ability to ferment sugar and produce alcohol, are part of Fungi. For
their ability to produce carbon dioxide they are also used in leavened bakery products. Other
fungi found in foods are, e.g., Penicillium camemberti in Camembert cheese, P. rocqueforti in
Blue cheese. In contrast to these microscopic fungi, mushrooms can be seen by a naked eye and
are being collected for food in many parts of the world.

Nuclepore-type filters, 13 mm in diameter, pore size 0.4 m, are most suitable for SEM of
bacteria. The stability of the filters in absolute ethanol and liquid carbon dioxide is essential.
Wetting the filters with 0.1% polylysine hydrobromide solution may improve their holding
power for some bacteria during fixation, dehydration, and critical point drying. The lower part of
a KS-13 stainless steel syring holder (MicroFiltration Systems, Dublin, California) (catalog no.
301000) was used to hold the polycarbonate filters. A wet bacterial filter was placed on the dense
steel mesh and mild suction through an erlenmeyer flask (see the figure at left) was applied to
drain the polylysine solution. A dilute bacterial suspension was placed from a pasteur pipette on
the filter and a mild suction was applied after one or two droplets passed through the filter. The
wet filter was then transferred on the surface of 3-4 mL of a cacodylate buffered 2.5%
glutaraldehyde solution (pH ~7) for about half an hour and then immersed in the fixative. It is
useful to check (from the clarity of the fixative) whether the bacteria adhere to the filter. Yeast
cells do not adhere well.
The filters were then dehydrated in a graded ethanol series (20, 40, 70, 95, 100, 100, 100%
ethanol) and were critical-point dried. Following the finding that the sticky material of doublesided sticky tapes penetrates the filter pores and eventually covers the bacteria and makes them
unavailable to microscopy has prompted a change in the mounting procedure (figure at right).
Only a narrow strip (>1 mm wide, yellow) is now placed on an SEM aluminum stub (light blue).
A bacterial filter (gray) is cut in half and the cut is attached to partially over the souble-sided
sticky tape. Another half obtained from another filter is placed opposite to the first half. Small
beads (blue) of a thick conductive silver cement are used to attach the round edges of the filters
to the stub. The stubs are then sputter-coated with gold for SEM. Occasional zigzag outlines of
the bacteria were the result of vibrations inside the microscope. This defect was corrected by
replacing the original multispecimen stub holder.
Links to a gallery of SEM images of microorganisms may be found here.
***
Technical terms not explained on this page may be found elsewhere.
Information on other kinds of microscopic techniques such as confocal laser scanning
microscopy (CLSM) are also available on the Internet.
WWW Virtual Library: Microscopy provides links to various techniques, equipment suppliers
etc.
Another site explains various kinds of microscopy and refers the readers to various suppliers.
Links to various microscopy sites.
Links to various scientific libraries.
Suggestions for authors of scientific reports and manuscripts which may improve their writing
skills are provided at another Web site.

MILK

Milk and Its Constituents


FOODS UNDER THE MICROSCOPE

Everybody knows that milk is liquid. But why is it opaque and white? Or does it have a
yellowish tint? Why and when does it curdle? What is actually curdling? If yogurt and cheese are
both made from milk, why are they so different from each other? Is it true that there are bacteria
in both yogurt and cheese? Why are they there? Answers to these questions may already be
found on the Internet.

Milk is produced by all mammal females to feed their newborns and the liquid form is most
convenient. This means that the concentration of nutrients is relatively low compared to foods
consumed by adults. Cow milk contains 3 to 4% protein, 4 to 4.5% fat, and approximately 4.5%
milk sugar (lactose). It is opaque because it contains very small particles of casein (milk protein),
approximately 100 nm in diameter. (That is 1/10 of a micrometer; 1 micrometer is 1/1000 of a
millimeter). It also contains larger fat globules. These particles do not allow the light to pass
through (except for a very thin layer) and scatter it in all directions making the milk to look
opaque. The fact that all wavelengths of visible light are scattered at the same extent makes the
milk white.

Whereas fat globules may be observed under a light (optical) microscope, casein particles are too
small to be seen using this kind of microscope. They are also too small to be captured on most
laboratory filters but they may be concentrated using various ultrafiltration techniques. This is
now done on a commercial scale.

The yellowish tint of milk comes from fat. It is in the form of much larger globules, with an
average of 3 to 5 micrometers in diameter as shown by the diagram at the title. Some large fat
globules may be up to 15 micrometers in diameter. Cows which are fed a carotene-rich feed,
such as grass or hay, pass the lipophilic, orange-coloured pro-vitamin A into the milkfat and that
makes the milk slightly yellowish.

Fresh milk contains lactic acid bacteria and other microorganisms as contaminants. Their
dimensions are also shown in the diagram. Pasteurization (heating; usually at 63C for a
minimum of 30 minutes) kills the bacteria and makes the milk safe and more stable. This means
that the shelf life of the milk is increased. A considerably more severe heating (e.g., 110C for
30-40 min., 130C for 30 seconds, or 150C for <1 s) leads to the production of UHT or
sterilized milk with a markedly extended shelf life. Milk coagulates (curdles) particularly when it
is acidified - either by lactic acid produced by lactic acid bacteria or by stirring an acid (such as
citric acid) into it.

Milk also contains ingredients so small that they cannot be seen - not even under an electron
microscope - yet they are very important both in the diet and in the manufacture of milk
products. They are whey proteins (-lactalbumin and -lactoglobulin), the milk sugar called
lactose, and minerals and vitamins.

Casein micelles, whey proteins, and milkfat globules are the most important constituents of most
milk products. These products may be divided into

products based on proteins (yogurt, cheeses) and


products based on fat (various forms of cream including whipped cream and ice cream and
butter).
Casein micelles are stable in fresh milk. They do not sediment at the bottom and they do not
concentrate at the milk surface. They are in constant motion, travelling short distances, colliding
with each other and with the fat globules and then changing direction. In fresh milk, their free
path is only 3 casein micelle diameters long before they hit another particle and this distance is
reduced to only 1 micelle diameter in condensed milk. It is really a dense medium as the name
implies.

Casein micelles consist of smaller particles called submicelles. A model explains the
ultrastructure of the micelles and their aggregation. Casein micelles form the base of the majority
of milk products such as yogurt and cheeses.

Two major whey proteins are -lactalbumin and -lactoglobulin. Both have a high nutritional
value partly because they contain sulfur-containing amino acids, cysteine, cystine, and
methionine.

Alpha-Lactalbumin An interesting structural property of -lactalbumin has recently been


discovered in Sweden by Dr. Catharina Svanborg. Her research group studies bactericidal
properties of human milk. To test the way, in which breast milk prevents pathogenic bacteria
from infecting other cells, Dr. Svanborg has also used cancer cells. In her experimental setting,
the nuclei of the cancer cells started to shrink and die under the effect of breast milk. Their
"programmed" death in this case is called apoptosis - the cells fall apart to be recycled by the
body. The substance which causes the death of the cancer cells has been identified as lactalbumin. This protein may be converted into a protein which induces apoptosis.

Proteins are long chains consisting of individual 'links' called 'amino acids'. There are 20
different amino acids which may form different sequences. The human body contains about
50,000 different proteins. In their native state they are very flexible. When -lactalbumin is
completely folded, it serves as a nourishment. When it is partially unfolded, it destroys
pathogenic microorganisms and cancer cells. In the case of -lactalbumin there is an interesting
connection between its nutritious and body-protective roles. Apart from emphasizing the
importance of nursing, this study shows a new way of fighting infections and malignancy.

Another link to apoptosis and protein folding:


Apoptosis: Dance of Death.
Beta-Lactoglobulin is the most abundant protein in whey (about 6 g/l) and is responsible for
functional properties such as the ability of whey proteins to form a gel on heating.

Fat globules in fresh milk are encased ('packaged') in membranes which are lipophilic ('fat
loving') on the inside where they are in contact with fat and hydrophilic ('water loving') on the
outside where they are in contact with the aqueous medium. Membranes prevent the fat globules
from coalescing (aggregating). Since fat has a lower specific gravity (is 'lighter') than the milk
serum, fat globules slowly raise to the milk surface. Thus, even under primitive conditions, fat
can be collected (skimmed) from the milk. The fat thus collected is called 'cream' and the milk
deprived of fat is called 'skimmed milk' or 'skimmilk'. Fat can be prevented from raising in milk
by breaking the fat globules into much smaller droplets so that they would be subjected to the

Brownian motion. [In another model, the blue large particle may be seen as representing a fat
globule and the minute red balls may be considered as casein micelles.]

The process which makes this possible is called homogenization of milk. It consists of breaking
the original large fat globules into much smaller droplets, 0.1 to 2 micrometers in diameter.
Homogenization has two effects:

It reduces the dimensions of the fat globules thus stopping them from raising to the surface and
it breaks the fat globule membranes.
The disintegration of the large fat globules results in a 6-fold increase in the total surface of all
fat globules in a unit volume. The 'naked' fat droplets immediately attract various milk proteins
which then form artificial membranes and, thus, even the fragmented fat globules are protected
from aggregation.

Milk solids in meat binders


Comminuted meat is held together in meat products such as wieners and salami with the help of
so-called meat binders. They consist of cereal products (wheat flour, baked crumb, and starches),
milk products (nonfat dry milk), salt, and spices (which may include mustard flour). ["Kosher"
meat products must not contain any milk solids.] Meat binders must not introduce any
undesirable properties into the finished meat products. Milk powder is one of the most important
ingredients. However, this powder should not contain buttermilk solids because they could alter
the flavour of the meat products. Companies which produce meat binders are, therefore, careful
about the quality of the milk powders which they use and want to be sure that there is no
buttermilk present in them.

Chemical analysis cannot be used to detect small amounts of buttermilk solids in the bulk of
skimmilk powder because the major ingredients in both products are the same. During drying,
buttermilk solids may get into skimmilk solids unintentionally, if sweet uncultured buttermilk is
added to whole milk destined for cream separation in order to retrieve the residual fat from the
buttermilk.

Casein micelles are the only particles found by TEM in skimmilk solids

Fat globule membrane fragments (red arrows) are present in buttermilk solids in addition to the
globular casein micelles
An electron microscopy procedure has been developed for the detection of buttermilk solids. The
analytical principle is based on the peculiar composition of buttermilk. It is a byproduct of butter.
Butter is made from cream, i.e., from milkfat globules. Each globule is covered with a membrane
and the membranes on all fat globules break as the cream is churned to produce butter. A small
proportion of the membrane fragments goes into the butter but a considerably larger part remains
in the aqueous phase and forms "churn" buttermilk. Buttermilk has a high nutritional quality and
is used in a variety of food products, particularly baked goods, where it improves their
properties. It is not desirable in meat binders.

Transmission electron microscopy (TEM) can be used to detect milkfat membrane fragments in
milk solids. They may even be detected in the finished meat binders. Low-speed centrifugation
(415 g for 30 min) of an aqueous suspension of the binders separates coarse particles such as
spices, flour, and starch. Only milk solids remain in the supernatant. Ultracentrifugation at high
speed (8x104 g) causes even the casein micelles and the milkfat globule membrane fragments to
sediment. The pellet is fixed, embedded in a resin, and the resulting bloc is sectioned, stained,
and examined.

Pure skimmilk powder reveals only the presence of casein micelles. Casein micelles are also
present in buttermilk solids and there are fat globule membrane fragments clearly visible in
addition. Studies in greater detait revealed that as little as 1 part of buttermilk solids can be
detected in 20 parts of nonfat milk solids using TEM.

CHEESE
Cheeses are the most common dairy products. The basic procedure is simple and is based on
spontaneous processes which, thousands of years ago, probably led to the development of
cheese. Milk from various animals, particularly cattle, buffaloes, goats, and sheep is used to
make cheese. One of the best descriptions of cheesemaking procedures is easy to follow. Various
books may be found in libraries. Some scientific journal papers dealing with the development of
structure in cheese are richly illustrated with micrographs, e.g., in
The following links lead to individual topics on this page:
Coagulation of milk, Cottage cheese, Unripened cheeses, Cream cheese, Low-fat cheeses, Curd
granule junctions, Can I see curd granule junctions? Processed cheese
Coagulation of milk
To make cheese, milk is curdled using a bacterial starter culture and an agent called rennet
present in the tissue of the calf stomach. Rennet (chymosin) is a proteolytic enzyme and its role
in cheese making is to destabilize casein micelles and make them to coagulate. Similar enzymes
are also found in digestive tract tissues of other animals including chickens. Global shortage of
animal-based enzymes and various kinds of aversion of some people to such sources, have led to
the use of proteases isolated from plants and microorganisms to make cheeses, some of which
are called vegetarian cheeses.
The proteases break down -casein present on the surfaces of casein micelles in milk.
Deprived of its protective action, casein micelles coagulate and form a gel. When examined by
electron microscopy, the coagulum consists of casein micelle clusters and short chains. They
encapsulate fat globules - the natural large corpuscular particles present in milk. Void spaces in
the casein matrix are filled with the liquid milk serum called whey which is a solution of lactose,
minerals, and vitamins, and a suspension of whey proteins. The subsequent steps in the cheese
manufacture are aimed at separating the curd from the whey and ripening the curd into cheese.
There are many photographs showing, how cheese is being made.
Freshly coagulated milk is cut or broken into smaller particles using wire knives, stirrers or
other tools and the cut or broken milk gel is slowly heated and stirred. Casein micelle clusters
gradually shrink as they expel the whey and the protein matrix becomes compacted.
Another milk component - fat globules - stays with the proteins in the curd. A small number
of fat globules, which were exposed during curd cutting, are washed away with the whey. The
micrograph at left shows an early stage of milk coagulation. Fixation in a glutaraldehyde solution
preserved the solid constituents allowing water to be removed to show the microstructure of the
milk coagulum.

The removal of the whey makes the casein matrix (shown blue to black) more compact and
also brings the fat globules (yellow) closer together. The curd shown was made from fresh fullfat milk. The fat globules appear as separate entities in the curd - they do not interact with the
casein micelles in the fresh milk and they may be characterized as nonreacting inclusions in the
curd. This is true of fat globules unaffected by homogenization.
Homogenization of milk is a process during which large fat globules are disintegrated into
considerably smaller particles. Their total surface is up to 6-fold larger than was the total surface
of the original fat globules. Since the original fat globule membranes were fragmented by
homogenization, there is a large area of unprotected exposed fat surface. Consequently, the small
fat particles react immediately with any available proteins in the medium until all bare fat is
again well covered. Even entire casein micelles are used for this purpose. Homogenized milk
produces a different kind of curd. It is firmer than the curd made from nonhomogenized milk
provided that all other parameters have been left unchanged (enzyme, total solids, pH). This
phenomenon was known even before the use of electron microscopy, but now the reason for the
difference is clear: The minute fat globules with casein micelles anchored on their surfaces have
become part of the protein matrix. Fat is no more an inert inclusion but has become a structural
constituent. This example is one of many which show the benefits of electron microscopy in
elucidating interactions among food constituents and the development of food microstructure.
Each individual parameter used during the cheese manufacture has some effect on the cheese
produced. This is probably one of the reasons for the great variety of cheeses. On the other hand,
it is a great challenge for the cheese producer to keep sensory attributes of a particular product
constant in a world where, for example, the original rennet has to be replaced with a similar
product from a different source because there is a global shortage of calf stomachs or because the
consumers request that no constituents of animal tissues be used in cheese production. Enzymes
with a high proteolytic activity may be efficient in quickly curdling the milk but they may also
disintegrate a large proportion of the milk proteins which would consequently be lost for the
cheese production. High proteolytic activity may also lead to a weakened casein matrix and alter
the characteristic consistency of the cheese. Similar constrains are encountered quite frequently.
However, new technologies and new ingredients offer new directions in cheese manufacture.
Examples will be presented as this home page is further developed.
Rennet is added to milk along with a lactic bacteria culture. The role of the bacteria is to
assist curdling by decreasing pH of the milk. This is achieved as the bacteria oxidize lactose into
lactic acid. After the whey had been removed and the curd salted and pressed, the next stage ripening - takes place at a lower temperature for several weeks or months. This is the time when
bacteria slowly degrade the milk proteins and produce substances which give the cheese its
characteristic structure (carbon dioxide eyes in Swiss-type cheeses) and flavour (e.g., a low
concentration of propionic acid), The great variety of cheeses is made possible by the
combinations of many varieties of specific bacteria. However, some cheeses are made with
Penicillium moulds (fungi) such as Penicillium camemberti and P. roqueforti. rather than
bacteria. A small group of cheeses (Paneer, Queso Blanco, White cheese) is made by coagulating
milk while it is hot, with an acid, such as lactic acid. Such cheeses are not ripened.
Pressing and ripening

The increase in the density of the curd matrix as a result of whey removal and pressing has
been followed in various cheeses by TEM. The micrograph at left shows a thin section of
cheddared curd in a cross section. The casein micelle clusters are now more compacted (dark
areas) than in the preceding micrographs. The fat globules are in contact with each other, having
retained their fat globule membranes. An occasional bacterium (brown) may also be found.
Cheddaring is a process, during which slabs of the warm curd are piled up in the cheese vat,
subjecting the curd to a slow flow. It aligns the proteins and fat globules into a 'fibrous' structure
reminiscent of a baked chicken breast. A section cut parallel with the 'fibres' shows the internal
organization of the curd (micrograph at right). Similar kinds of structuring ('stretching') may be
found in Italian-style Mozzarella cheese and, in particular, in 'string cheeses'.
Cottage cheese
Cottage cheese represents an early stage product in the cheese manufacture. Curdled milk,
cut into cubes, is heated and gently stirred. The cubes shrink and expel whey, as has already been
explained. The whey is drained off and the curd is cooled so that its grains are prevented from
'matting', i.e., fusing with each other. The microstructure of Cottage cheese, as seen by SEM at
left, is still porous - consisting of distinct casein micelle clusters. Three bacteria (greenish) can
be seen as if attached by filaments to the protein network. These filaments developed during
sample preparation from a thin layer of polysaccharide mucus (bacterial capsules) which
surrounded the bacteria in the fresh curd. Highly hydrated polysaccharides are produced by some
lactic acid bacteria. They are beneficial in our diet as a source of dietary fibre. In addition, the
high viscosity of these polysaccharides modifies the mouthfeel of the food products such as
Cottage cheese or yogurt. A high water-holding capacity of the bacterial polysaccharides is
another beneficial property. However, the polysaccharides cannot be fixed during preparation of
the specimen for electron microscopy and the mucus shrinks during dehydration in ethanol into
filaments (at higher mucus concentrations it would shrink into scales). The filaments thus did not
exist in the original Cottage cheese and are artifacts.
Unripened cheeses
Some cheeses (Indian Paneer cheese, South American Queso Blanco cheese, American
White cheese, North American Ricotta cheese) are made by coagulating hot milk with an acid
and separating the curd from the whey.
All have some features in common: the milk is first heated to at least 85C and then is
coagulated using an acid such as citric, lactic, acetic, or hydrochloric acid (or an acid precursor
such as glucono--lactone) to a final pH value of 5.5 making the curd mildly acidic. The
coagulated milk is then cooled and the whey is separated. The microstructure of the casein
particles has a characteristic 'core-and-shell' structure (micrograph at left).
How the microstructure develops has not yet been fully explained but it is known that three
essential conditions must be met:

The milk must be coagulated at a temperature higher than 85C so that whey proteins may
interact with -casein;
Whey proteins and the milk salt system must be present in the milk;
The final pH value must be 5.5 0.1.
Cream cheese
As the name indicates, Cream cheese is made from pure cream or from mixtures of cream
and milk. It has a rich, mildly acidic flavour and a smooth buttery consistency.
In the traditional system of manufacturing, the cream mixture is pasteurized, homogenized, and
coagulated using a lactic bacterial culture. The curd is then heated to 52-63C, drained, and hotpacked or cold-packed. This kind of manufacturing procedure yields whey which has to be
disposed off.
In a newly formulated method of 'whey-less' manufacturing, the cream-and-milk mixture has
the total solids composition of the cheese. The mixture is also pasteurized, homogenized, and
incubated with a lactic bacterial culture at ~30C. Then the solidified mixture is homogenized
again and packed without cooling. Products which have not been made by the traditional
procedure may not be called 'cream cheese' and terms such as 'cream cheese food' or 'cream
cheese spread' are used.
Microstructure of traditional Cream cheese Fat globule clusters (dark yellow) are covered with
protein (dark blue) in the aqueous medium (light yellow).
Microstructure of newly formulated Cream cheese Large fat particles (brown) are not closely
associated with protein (dark blue) in the aqueous medium (yellow).
Microstructure of Cream cheese made from high-fat cream and acid-coagulated hot milk Fat
globules (brown) and the curd (which shows the core-and-shell ultrastructure of casein particles dark blue) are the major ingredients dispersed in the aqueous phase (yellow).
Another new procedure has been suggested by H. W. Modler. Curd is first produced by
coagulating hot milk with a 2.5% citric acid acid solution until pH of 5.3-5.5 is reached. The
whey is drained off. Whey proteins are retained in the curd depending on how high the milk was
heated prior to coagulation. The curd is mixed with high-fat (58%) cultured cream and the
mixture is homogenized at ~70C and the resulting cream cheese spread is hot-packed.
Structural differences between the cheeses are best observed using TEM of thin sections. This
technique makes it possible to examine the interior of the cheese particles whereas SEM shows
surfaces. This is useful, too, because surfaces may be formed by breaking (fracturing) cheese
particles; the structure of the protein matrix may thus also be observed. Preparation of the cheese
specimens for electron microscopy may preserve the fat globules or remove them.
In the traditional Cream cheese, TEM reveals a very high fat content in the form of minute fat
globules. Their surfaces are covered with protein particles. The protein frequently covers fat
globule clusters rather than each individual fat globule.

The newly formulated Cream cheese spread structure is different. This is noticeable at the first
glance: the fat is present in the form of relatively large fat particles which are not associated with
protein. Protein is relatively evenly distributed through the body of the spread in the form of
small clusters attached to the fat particles at random.
Cream cheese spread made by homogenizing high-fat cream with fresh curd differs from the
products mentioned above and reflects the manufacturing procedure. Fat is mostly in the form of
small globule clusters and the protein is in the form of relatively large particles. The structure of
the curd also reveals its origin - acid-induced coagulation of hot milk to pH 5.5. The 'core-andshell' ultrastructure of the casein particles is preserved (figure above at right), although the curd
undergoes homogenization during production.
Information on Cream cheese products presented in this section is based on earlier
experimental work described in several papers co-authored by D. A. Froehlich, M. Kalb, H. W.
Modler, and A. G. Sargant, in Food Structure and in Milchwissenschaft 40(4):193-196 (1985)
(Milk gel structure. XV. Electron microscopy of whey protein-based Cream cheese spread).
Low-fat cheeses
An example of low-fat cheese.
Electron microscopy has also been used in studies of low-fat or fat-free cheeses, where fat
has been replaced with one of the so-called fat replacers. They may be based on proteins,
polysaccharides, or even on indigestible fats and oils. Their action is based on the fact that our
tongue receives stronger signals about the dimensions of the particles than about their chemical
nature. Particles 1 to 3 m in diameter are perceived as fat. In the micrograph at left, a proteinbased fat replacer has been incorporated in cheese. It affects, because of its protein nature, only
sensory attributes and instrumental measurements of the cheese. Protein- and polysaccharidebased fat replacers do not melt and their use is limited to specific situations, for example frozen
desserts, salad dressings, and some other applications.
In the low-fat cheese featured at left, a fat substitute based on protein (light green-coloured
globular aggregates of microparticulated protein) was used to replace a small portion of milkfat.
Fat globules originally present in the cheese were extracted from the sample while it was
prepared for scanning electron microscopy. Initially they occupied the spaces which now appear
empty in the protein body of the cheese.
The low-fat cheese sample shown at left was freeze-fractured. This procedure makes it
possible to fracture (break) even minute particles and study their internal structure by scanning
electron microscopy.
Additional scientific papers are being processed for this page. Images of fat substitutes used
in cheese, the structure of cheeses made without the aid of microorganisms, Cottage cheese and
other cheeses such as Mozzarella will be gradually presented at this site. Processed cheese is
another important dairy product in which interesting discoveries have been made using electron
microscopy.

Brick cheese vs. Cheddar cheese


Two different structures compared
Differences between cheeses can not only be tasted but also seen, because manufacturing
processes impart special features on the cheese microstructure.
To make cheese, curdled milk is cut using steel wire knives or the milk gel is broken into
small particles using a propeller stirrer. Subsequent heating shrinks and compacts the particles,
as whey is drained off. The curd particles are pressed together and they fuse to make a uniform
body of cheese.
The sites of contact, where adjoining curd particles meet, are called curd granule junctions.
Their development is schematically shown in the diagram. The gelled (nonhomogenized) milk
which contains fat globules (yellow disks) is cut (red vertical line). The fat globules thus exposed
are washed out from the curd (arrows). The surface of the granules heals (middle figure).
Pressing of the 2 granules together (last figure) causes the superficial layers depleted of fat to
fuse. The junction is shown as an area devoid of fat globules.
If homogenized milk is used where the fat globules are considerably smaller, the width of the
junctions is markedly reduced.
The junctions can easily be seen by scanning electron microscopy (SEM) as compact zones.
To obtain the micrograph shown, a small Brick cheese sample (1x1x10 mm) was fixed in a
glutaraldehyde solution, dehydrated in ethanol, defatted in n-hexane, returned into absolute
ethanol, and rapidly frozen in liquid Freon 12. The frozen sample was transferred into liquid
nitrogen, where it was freeze-fractured, returned into absolute ethanol, where it thawed, and then
it was critical-point dried from liquid carbon dioxide. Defatting and freeze-fracturing were the
essential steps to show the junctions. Removal of fat makes the fracture plane of the sample
'rough' - full of cavities (initially occupied by fat) - in the area showing the interior of the curd
granules. The protein walls separating the cavities scatter light in all directions. Light scattering
causes this area to appear lighter than the compact structure of the curd granule junctions, which
is mostly free of fat globules. The contrast between both structures creates the curd granule
junction patterns.
The junctions are visible even to a naked eye. Brick cheese (left figure below) made from
curd obtained by knife cutting shows granules relatively similar in size. Such images may be
obtained even at a high school chemical laboratory and instructions on how to proceed are given
below. They are also characteristic of other 'stirred-curd' cheeses such as Farmer's, Eidam,
Gouda, etc.
Cheddar cheese manufacture involves considerably more work. The fused curd granules
(curd slabs) are piled one over the other in the cheese vat in traditional Cheddar making. Heat
and the presence of fat in the curd make it to slowly flow down. This cheddaring process
elongates the granules. The slabs are gradually replaced so that all are exposed to maximum flow
and then the slabs are milled into finger-like pieces. These are salted and pressed together.

Milling produces new cuts and thus new junctions (right figure). They are called milled curd
junctions and are noticeably thicker than the curd granule junctions.
Also mechanized and automated cheddaring produces both kinds of junction. Different
equipment leaves its marks in the junction patterns.
Do these findings have any practical importance? Yes. For example, they show that any
attempts to alter cheese processing would easily be detected. The findings ensure that the higher
price which the consumers pay for Cheddar cheese compared to Farmer's cheese is justified and
that they buy a product which really is Cheddar cheese.
Readers who would like to see the junctions in their piece of cheese and who have access to a
chemical laboratory, will find that the procedure is relatively simple.
Needs:
fume hood
3 to 4 petri dishes
cheese slicer or a sharp knife
a piece of filter paper
a pair of tweezers
2 glass or metal plates (at least 10x10 cm)
fine sand paper
2 to 5% glutaraldehyde solution
96% (denatured) alcohol
n-hexane or acetone

weights

Procedure:
Obtain thin (~2 mm) slices of the cheese under study, about 5x5 cm (2"x2") large, and
immerse them in an aqueous 2 to 5% glutaraldehyde solution in separate petri dishes overnight.
This treatment will fix the cheeses and make them easy to handle. It will also increase the
contrast between the junctions and the curd interior. Next day, replace the glutaraldehyde
solution with 96% denatured alcohol, about 3 times after 30 minute periods. Then replace
ethanol with n-hexane or acetone (twice) to extract fat from the cheese. Using a pair of tweezers,
place the cheese slices between two filter papers, place them, with the papers, between two glass
or metal plates, place a weight on them, and let the cheese slices dry overnight. All this work
must be done in a fume hood.
When the cheese slices are dry, they are light and brittle. Sand them carefully with a fine
sand paper and see the junctions emerge as sanding progresses. Why is it necessary to sand the
slices? Because cutting had smeared the cheese protein on the slices and it is necessary to
remove it.

Stretched Mozzarella, cheeses with very low fat contents, and processed cheeses do not
reveal curd junction patterns.
Processed cheese
Processed cheese has a relatively short history. First experiments started at the end of the last
century but success was achieved only in 1912, when citric acid was introduced as a melting salt.
This happened in Switzerland. A few years later, sodium phosphates were added to sodium
citrate and have been used since that time.
The initial idea of processing cheese was to increase the shelf life of cheese and, more
importantly, to utilize cheeses which may have had various defects. If it was possible with butter
by rendering it, some people believed, it should also be possible with cheese. However, when
cheese is melted without any additive, fat separates from protein and the result is terrible. The
secret of 'processing' cheese is in keeping the fat in the protein matrix. Heating, however,
decreases the ability of the cheese proteins to keep the fat globules in the dispersed state, which
means that the emulsifying capability of the proteins has been reduced. Melting salts restore it by
binding (sequestering) calcium which is present in the caseins. Melting salts with very strong
calcium-binding ability (affinity for calcium) lead to the production of hard processed cheeses
which contain fat in the form of very small globules. For those readers who like chemistry, the
affinity increases in the following order:
NaH2PO4 (monosodium phosphate)
Na2HPO4 (disodium phosphate)
Na2H2P2O7 (disodium pyrophosphate)
Na3HP2O7 (trisodium pyrophosphate)
Na4P2O7 (tetrasodium pyrophosphate)
Na5P3O10 (pentasodium tripolyphosphate).
It has to be emphasized that the melting salts are not emulsifiers but they restore the
emulsifying ability of the milk proteins very efficiently.
The principles of cheese processing are simple: Various natural cheeses are shredded and
then blended with the melting salts and other ingredients such as various kinds of milk solids
such as milk powder, whey powder, coprecipitates, cream, butter or butter oil, and sometimes
also previously processed cheese. Vegetables and spices may also be added and some processed
cheeses may contain 'muscle food ingredients' such as ham, salami, or fish. Other additives such
as preservatives, colouring and flavouring agents, binders, and salt and water complete the list of
the ingredients.
The blend is heated with constant stirring until a smooth mass is formed. In a continuous
processed cheese production, the temperature is increased to 140C for a few seconds to destroy
harmful bacteria (such as clostridia) if they happen to be present in some of the ingredients.
TEM of processed cheese. Undissolved melting salt crystals (white), fat being emulsified
(yellow), and a calcium phosphate crystal (red) are all clearly noticeable. Bar: 5 m

SEM of processed cheese. Cavities left in the protein matrix by undissolved melting salt crystals
(blue arrow), dark globular cavities initially occupied by fat, and a calcium phosphate crystal
(red) are also noticeable by SEM. Bar: 20 m
From the structural viewpoint, many features characteristic of natural cheeses are destroyed,
for example, the curd granule junction patterns and the original fat globule membranes. On the
other hand, new features are formed. In most processed cheeses, undissolved melting salt crystals
may still be evident. Adding the salts in crystalline form rather than in the form of an aqueous
solution to the cheese blend leaves some crystals undissolved. Interactions between the melting
salts and calcium in the natural cheeses lead to the formation of insoluble calcium phosphates.
Emulsification of fat - that means disintegration of large fat globules into smaller droplets - is
also often noticeable in processed cheese.
Processed cheese rework
Overheating affects the microstructure of processed cheese.
The amount of heat absorbed by processed cheese blends during processing may vary and
may even be excessive at times. A blend may receive too much heat during continuous
processing if, for example, packaging is delayed for some reason and the flow of the viscous
processed cheese blend in the pipes is reduced. The blend eventually thickens and stops moving.
Then it is called hot melt. It is removed from the pipes and is frozen for future use. Reworking or
reprocessing consists of thawing and shredding the hot melt and adding a small quantity of it to a
fresh blend. This is often done on purpose to modify the melting properties of processed cheese
in a desired manner. Hot melt is thus a type of process cheese food that is not packaged for sale
although it would meet product specifications. If it is re-used, it is called rework.
Electron microscopy revealed structural changes in the proteins in rework in the form of
small (<1 &micr;m in diameter) dark areas in the micrographs of thin sections. Darkening may
be the result of compaction of the cheese proteins or alterations in their chemical structure
whereby the heat-modified proteins would react more intensively with heavy metals used during
fixation and staining. Tests, in which heavy metals (Os, Pb, U) were omitted from the fixatives
and stains during sample preparation for electron microscopy indicated that the proteins in the
dark areas were rather compacted than chemically altered. The submicrostructure of the
compacted areas was found to be related to the melting salt used to make the processed cheese.
Hot melt contained considerably less undissolved melting salt crystals apparently because the
crystals had time to dissolve during the entended exposure to heat. Rework dispersed rapidly in
the freshly processed cheese blend. This visual observation was confirmed by optical
microscopy.
The upper image at left shows compact electron-dense structures (purple arrow) which
developed in processed cheese made with 2.7% trisodium phosphate, used as the melting salt,
due to excessive heating (82C for 5 hours).
The lower image at left shows two kinds of electron-dense structure developed in process
cheese made with 20% rework consisting of hot melt. The hot melt was obtained by processing

cheese with 2.7% sodium citrate and heating it at 82C for 5 hours (green arrow). The shredded
hot melt was added to a fresh cheese blend and the mixture was processed (using 2.7% trisodium
phosphate) and excessively heated. Purple arrows point to structures developed in the fresh
cheese blend.
What do these findings mean in processed cheese production? They show that the loss of
meltability is associated with structural changes in cheese proteins. They make it possible to
detect if rework was used in cheese processing; even at 10% rework, there was a high
concentration of the dark areas in the micrographs. Finally, these findings point to interesting
thermal effects on processed cheese, which may eventually be studied in greater detail and
explained.
Additional links:
Books (publishers, addresses, prices)
Cheese glossary
Dairy technology - explanation in greater detail
Bacteriophage-resistant bacteria in Cheddar cheese production (PDF file)
Milk ultrafiltration and whey processing.
Information from the food industry
The Basics of Making Cheese
The original 2005 version of "Cheese: Development of structure"
Scanning electron microscopy of cheese
How to prepare cheese samples for SEM
As with any sample to be examined by electron microscopy, the scientist/technician must have
knowledge of the composition and structure of the material to be examined. Curd and cheese
consist of a milk protein matrix which is interspersed with fat globules and lactic acid bacteria
from the starter culture. Other structures such as calcium phosphate crystals or ingredients used
in the cheese production may also be present. As curd changes into cheese, the matrix become
gradually more compact which means that whey and air pockets are reduced in size. It also
means that fixation, dehydration, and defatting is decelerated as the diffusion of the respective
agents is hampered.
Sample preparation starts by cutting a slice approx. 1.5 mm thick. It is subsequently cut into
prisms, approx. 1.5 mm wide, 10-15 mm long using two razor blades. The prisms are first fixed
in a 2.5% glutaraldehyde solution in 0.1 M sodium cacodylate buffer, preferably at about 6C
(refrigerator) for 2 h (milk coagulum, fresh curd) to 24 h (hard cheese, processed cheese).
At this stage the researcher needs to decide whether also the fat globules are of interest. They
may either be removed to show void spaces in the protein matrix which had initially been
occupied with fat (example: Micrograph of processed cheese), or fat may be retained (example:
Coagulation of milk). Retention of fat may be achieved by postfixing the specimens with
imidazole-buffered osmium tetroxide. It is described in Fixation of fat for TEM. That procedure
will be described later.

In the procedure described here, the fat will be removed. The prisms fixed with glutaraldehyde
need to be dehydrated in a graded ethanol series (20%, 40%, 60%, 80%, 96%, and 100%).
Depending on the compactness of the sample, the individual steps may take from 15 min
(coagulated milk, fresh curd) to 30 min. with denser specimens. Fat in the prisms in absolute
(100%) ethanol may then be extracted using n-hexane (initially, chloroform was used for this
purpose). Three changes of n-hexane are advisable to prevent residual fat from contaminating the
specimens (M. Kalb: Artefacts in conventional scanning electron microscopy of some milk
products. Food Microstruct. 3(2), 95-112, 1984). Replacing n-hexane with 2 changes of absolute
ethanol prepares the specimens for freeze-fracturing. The reason why freeze-fracturing is
important for SEM of milk products and why dry-fracturing is not suitable for such work is
explained a few paragraphs below the information about osmium tetroxide.
Freeze-fracturing starts by rapidly freezing the specimen in liquid nitrogen (LN) and breaking it
there thus producing smooth fracture planes. LN, however, would not prevent ice crystal
formation in hydrated specimens. In contrast to water, absolute ethanol does not form crystals on
freezing. It is used, therefore, to impregnate the minute prism specimens.
To freeze-fracture the specimens, the author uses a black cardboard box (110 mm x 90 mm x 50
mm) well insulated with polystyrene. Inside is a low rectangular copper dish. The dark colours
make it easy to see specimen fragments. The dish is half filled with LN and when vigorous
boiling has ceased, two or three prisms are immersed using a pair of insulated tweezers (made by
glueing insulation to the tweezers - a scalpel is insulated in a similar way). While holding the
prism with the tweezers, the scalpel tip is pressed a mm from the end of the prism. In this way,
several fragments are obtained. They are then picked with the tweezers and dropped into 10-20
mL of absolute ethanol in a small beaker. The thawed fragments are then critical-point dried
using liquid carbon dioxide as the transitional medium.
Suitable fragments of even thickness with well developed fracture planes are selected for
mounting on an SEM stub. Unlike the prism sides, the fracture plane is shiny when examined
under a dissecting microscope. The fragment is mounted with the fracture plane facing up. The
author uses a double-sided sticky tape attached to the SEM stub because it conveniently holds
the fragment in place while a small volume of a cement is painted around the base using two
needles to provide an unterrupted condutive path after the specimens are coated with gold. With
highly porous coagulated milk specimens it is advisable to paint the cement up to the upper edge
of the fragment. Care must be taken that the cement is neither too "thin" (the solvent would soak
into the porous fragment and ruin it) or it is too "thick" and would not adhere to the surface of
the fragment.
Coating with gold is the last preparatory step preceding SEM.
SEM of cheese with fat retained in the protein matrix
Fixation of cheese specimens with glutaraldehyde fixes only the proteins. Fat globules
surrounded with protein may be preserved but dehydration of the specimen with absolute ethanol
and subsequent critical-point drying from liquid carbon dioxide may result in partial extraction
of the fat. Fixation of milkfat in cheese and other milk products is based on the fact that such fat

contains unsaturated fatty acids which may react with osmium tetroxide. However, the reaction
in a regular (cacodylate, veronal-acetate etc.) buffer ultimately leads to conversion of the
unsaturated double bond to a diol while osmium is released in the form of osmium trioxide.
Stable fixation may be achieved using imidazole-buffered osmium tetroxide (Allan-Wojtas, P.,
Kalb, M.: Milk gel structure. XIV. Fixation of fat globules in whole milk yoghurt for electron
microscopy. Milchwissenchaft 39, 323-327, 1984). That procedure is based on a paper by S.
Angermller, H. D. Fahimi: Imidazole-buffered osmium tetroxide: an excellent stain for
visualization of lipids in transmission electron microscopy. Histochem. J. 14, 823-835, 1982

The original paper by Allan-Wojtas and Kalb on yogurt may be difficult to obtain. The
procedure may be adapted for cheese. It is easy to check as to whether a 24-h postfixation with
osmium tetroxide was sufficient: when fractured, the sample should be dark throughout its entire
diameter. However, it is not necessary to wash the samples for 48 h as mentioned in the text from
the Electron Microscopy section reproduced below:
(a) Thin sectioning Samples (<1 mm3) were taken from approx. 0.5 cm below the surface of
the yoghurt and were fixed for 24 h at 22C with a 1.4% glutaraldehyde solution in 0.1 M
cacodylate buffer (pH 7.4), containing 0.05% CaCl2 and 4% polyvinylpyrrolidone (w.w.
40,000). The samples were then washed for 48 h in several changes of the buffer and postfixed
for 24 h in an imidazole-buffered 0.5% OsO4 solution. This solution was prepared by dissolving
0.5 g of crystalline OsO4 in 50 mL of a 0.05 M veronal-acetate buffer (pH 7.4), and mixing this
stock solution with an equal volume of a 0.2 M imidazole solution adjusted to pH 7.4 with 1 N
HCl. The buffered solution was used immediately (~5 mL/1 particle). After 24 h, the samples
were removed from the fixative, washed with several changes of plain imidazole buffer, rinsed
briefly in distilled water, dehydrated in a graded ethnol series, and embedded in SPURR's low
viscosity embedding medium. Thin section (~90 nm) were stained with uranyl acetate and lead
citrate solutions and examined in a Philips EM 300 electron microscope oeprated at 60 kV.
Micrographs were taken on 35 mm film.
(a) SEM Samples (3x3x0.5 mm) were cut into pieces 3x1x0.5 mm) after fixation in the
buffered 1.4% glutaraldehyde solution as described above. The samples were then postfixed in
the imidazole-buffered 0.5% OsO4solution, washed, dehydrated in a graded ethanol series,
rapidly frozen in liquid Freon cooled to its freezing point with liquid nitrogen, and fractured
under liquid nitrogen. The fragments were thawed in absolute ethanol, critical-point dried from
carbon dioxide, mounted on aluminum stubs, coated with gold (~20 nm) and examined in a
Cambridge Stereoscan Mark II electron microscope operated at 20 kV; micrographs were taken
on 35-mm 125-ASA film.
Additional information:
MICROSCOPY: Scanning Electron Microscopy by J. Webb and J. H. Holgate
MICROSCOPY: Transmission Electron Microscopy by J. H. Holgate and by J. Webb
Characterization of nanomaterials in food by electron microscopy by A. Dudkiewicza, K. Tiede,
K. Loeschner, L. H. Soegaard Jensen, E. Jensen, R. Wierzbicki, A. B.A. Boxall, and K. Molhave

YOGURTH
Yogurt: Electron Microscopy
FOODS UNDER THE MICROSCOPE
New information: June 3, 2011.
Author: M. Kalab
Yogurt has been part of the diet in southeastern Europe and the Middle East for millennia
and is now part of the dairy counters even in the smallest grocery stores in many countries. It is a
cultured milk product easy to make, so there are many other websites with advice on how to
proceed. The most important part is to heat the milk at 85 to 95C for 5 to 10 minutes. Lactic
acid bacteria are essential to making yogurt from milk.
Compared to other milk products such as cheese, ice cream, or butter, yogurt contains most
milk constituents except lactose, which the bacteria convert into lactic acid. This acid gives the
yogurt a pleasant acidic flavour and, at the same time, the sweetness caused by lactose is
reduced. Live 'friendly' lactic acid bacteria protect the yogurt from harmful pathogenic
microorganisms and thus give it a longer shelf life. The fat content in some yogurts may be
reduced for dietetic reasons. Some yogurts may contain so-called thickening agents, such as
various plant polysaccharides (gums), gelatinized starch or gelatin. Their role is to firmly hold
water in the body of the yogurt.
Yogurt has traditionally been made from milk that had been partially condensed by
evaporation while it had been heated almost to boiling. Coagulation of the milk proteins is
induced by thermophilic bacteria, such as Lactobacillus delbrueckii subsp. bulgaricus and
Streptococcus salivarius subsp. thermophilus, i.e., bacteria which propagate well at an elevated
temperature of 40 to 45C. More recently, Lactococcus thermophilus is used together with L.
bulgaricus, with which it is synergistic, which means that each produces metabolites required for
the growth of the other. However, some lactococci produce bacteriocins called lactococcins
which kill other lactococci and are incompatible for use in mixtures.
The milk is coagulated by a slowly increasing concentration of lactic acid as the bacteria
metabolize lactose. The proteins do not precipitate (as would happen following an addition of a
large amount of lactic acid) but form a gel. Its ability to retain all the water present in the milk is
the result of a peculiar microstructure of the protein network. It consists of short branched chains
of casein micelles and resembles a sponge with very small pores.
Yogurt is unique from both the structural as well as compositional viewpoints, because it is solid
and has the highest water content of all solid milk products. Yogurt that has been stored for a
long period of time may show some syneresis as the separation of a liquid phase from a gel is
called. This is only a minor cosmetic defect and the liquid soaks back into the body of the yogurt
as soon as the yogurt is stirred.
Electron microscopy reveals interesting features in the development of yogurt structure. The
crucial condition in yogurt making is the heating of the milk. Its temperature must reach at least

85C (90C is more commonly used) and held at this temperature for at least 10 min. This
treatment alters the casein micelles and prepares them for the unique structure to form.
Casein micelles are described as protein globules about 100 nm in diameter, which consist of
yet smaller submicelles. Of several different casein molecules, kappa-casein (-casein) on the
surface of the micelles has a pivotal role in their stability. As long as this protein is intact, the
micelles stay in milk as individual entities. Any change in the integrity of -casein destabilizes
the micelles and they aggregate.
Casein micelles in unheated milk have relatively smooth surfaces with very small humps
caused by the submicelles. Heating above 85C leads to an interaction between -lactoglobulin
(one of the whey proteins) and -casein on the casein micelle surface. The result is a complex
which makes the casein micelle surface markedly coarser (figure at upper left). Casein micelles
with the -casein-lactoglobulin complex formed on their surfaces have a limited ability to
aggregate (figure at left). Consequently, short branched micelle chains are formed (upper
diagram at right).
In cheese manufacture, where -casein disintegrates under the effect of a proteolytic enzyme
called rennet, the micelles aggregate into large clusters (lower diagram at right).
Increasing the total solids content, particularly the amount of protein in yogurt, generally
increases the density of the protein network and decreases the pore sizes. Consequently, water is
more firmly bound in the product. This fortification of yogurt may be achieved by adding milk
powder, whey powder, milk protein concentrate, whey protein concentrate, or sodium caseinate.
Differences in the microstructure of yogurt containing 12.5%, 20%, and 30% total milk solids
from added skim milk powder are shown below, where shorter casein particle chains are
noticeable:

Milk solids: 10%


Milk solids: 15%
Milk solids: 20%
Whey protein concentrate which forms very fine dispersions and does not contain casein
micelles, forms bridges between the casein particles in yogurt. Transmission electron microscopy
(TEM) is better suited to show the fine bridges.
The image at left is a TEM micrograph of regular yogurt made from unfortified milk in
which the protein network consists of casein particles. The micrograph at right was obtained with
a yogurt made from skim milk fortified with 1.5% of an ultrafiltered whey protein concentrate.
These experiments have been published by H. W. Modler and M. Kalb.
Watery yogurt?
Separation of the liquid phase in gels is called syneresis. In yogurt, it is undesirable and it
occurs when the protein network is unable to firmly retain water. There are several reasons why
syneresis may develop. One of them is insufficient preheating of the milk destined for yogurt
production which means that casein micelle clusters are formed in addition to branched chains
and the clusters do not hold as much water as the porous structure based on chains. The total
solids content also has a great effect on the ability of yogurt to hold water. This is evident from
the micrographs above which show the structures of yogurt samples made with 10, 15, and 20%

total solids. The higher the total solids content, the denser the protein network, the smaller the
pores, and the stronger the water-holding force of the yogurt. Traditionally, milk used for yogurt
making was evaporated by heating but nowadays the total solids content is controlled by the
addition of milk solids such as milk powder or by concentrating the milk by ultrafiltration or
reverse osmosis. The acidity (expressed as pH which indicates the concentration of hydrogen
ions) of the yogurt also plays an important role. Most yogurts have their pH value in the range of
4.0 to 4.4. The presence of thickening agents such as gelatinized starch, gelatin, or various plantbased agents such as carrageenan or locust bean gums reduces syneresis but may impart sensory
properties which are not welcome by some consumers whereas others may like them (e.g.,
increased viscosity). Vibrations, to which goods are exposed during transportation, also increase
susceptibility of set-style yogurt to syneresis.
Susceptibility to syneresis can be measured in laboratory conditions using a drainage method
suggested as early as 1959 by D. B. Emmons et al. (Journal of Dairy Science Vol. 42, pp. 866869). The method is based on making yogurt in 250-mL beakers, cutting it into 4 parts, and
transferring the contents onto a stainless 120-mesh screen. The volume of the whey separated is
measured at 5 min. intervals for 60 min.
During their studies of milk gel structure, V. R. Harwalkar and M. Kalb designed a
centrifugation method (Scanning Electron Microscopy 1981:III, 503-513) which was later
applied to yogurt (Milchwissenschaft Vol. 38, No. 8, pp. 517-522, 1983). The relationship
between microstructure and susceptibility to syneresis was explained there.
Yogurt is made in 15-mL centrifugation test tubes. The test tubes are then subjected to
centrifugation for 10 min at forces ranging from 30 to 2000 xg. The volume of the separated
whey is measured and plotted against the centrifugal force applied. The g-force of the inflection
point on the resulting S-shaped curve has been used as an arbitrary measure of susceptibility to
syneresis. Two yogurts are shown in the diagram at left, where 'V' is the volume (in % at steps of
0, 10, 20, 30, and 40%) of the whey collected from each individual sample and g is the
centrifugation force at steps of 500, 1000, and 1500 xg). The blue curve was obtained with
yogurt containg 10% total solids. At the maximum centrifugation force, the whey released was a
little over 40% of the total volume of the yogurt. The red curve was obtained with a yogurt
fortified with skim milk powder to 15% total solids. The inflection point occurred at a higher gforce and the volume of the whey released was markedly lower than in the former yogurt.
An electron microscopy study of yogurt samples subjected to centrifugation and comparison
with the drainage test suggested that lowering pH increased the rigidity of the protein network
thus reducing susceptibility to deformation. Thus, at least two factors control syneresis - density
of the network and resistance of the protein chains to deformation.
Labneh
Labneh is strained yogurt popular in the Middle East. Unlike yogurt, it is made from whole milk.
Apart from cow's milk, sheep's and goat's milks are used in countries such as Lebanon, Syria,
and Turkey, where the climatic conditions are not favourable for cows to keep.
In the United Kingdom, labneh is marketed under the name of Greek cheese and in North
America it is often called yogurt cheese. In Arab countries, labneh is garnished with dried herbs
and olive oil and is served with bread.

Transmission electron microscopy of sheep's milk labneh: Fat particles (yellow - small arrows)
are encapsulated in aggregated protein particles (dark structures - large arrows). Bar: 1 m.
Scanning electron microscopy of nonhomogenized goat's milk labneh (left) made by the
traditional procedure and nonhomogenized labneh made by the ultrafiltration of cow's milk
yogurt (right). Protein is light, pores are dark. The labneh protein matrices were denser than
those of commercial yogurt shown higher up on this page. Bars: 25 m.
The traditional method of producing labneh consists of straining whole-milk yogurt in a
cheese cloth bag. Modern procedures are now increasingly used to make labneh. Excess liquid
from traditional yogurt can be removed in mechanical separators.
Changes to the traditional production of the yogurt have also been introduced, e.g., retentates
obtained by the ultrafiltration (UF) of milk are cultured to produce a yogurt with a higher solids
content, thus avoiding the need for concentrating it. In another procedure, warm yogurt may be
ultrafiltered, as suggested by Adnan Y. Tamime, the coauthor of several scientific papers on
labneh and a book on yogurt.
Protein matrices in labneh made by straining yogurt (traditional method) and in labneh
obtained by ultrafiltration of warm yogurt were examined by electron microscopy. The labneh
samples were made from cow's and also goat's and sheep's milks. The total solids contents of
labneh were between 20.5 and 22.5%, protein was 6.7-8.2% and fat was 7.8-8.9%.
Homogenization, which was used to make the product smoother, cannot be recommended
because it would decrease the firmness of labneh (cow's milk product was less affected than the
other two labnehs, where the pore sizes were found by microscopy to be larger). Homogenization
(single-stage ALM homogenizer at 7C and a pressure of 8 MPa) resulted in the disintegration of
the fat globules and association of their fragments with milk proteins, which could be observed
only by transmission electron microscopy (TEM) (figure at left).
There were differences in the microstructures of labnehs made from nonhomogenized
yogurts depending on the milk and the method of concentration used.
New information:
Examine yogurt by scanning electron microscopy: There are two yogurt styles concerning
consistency: "Set-style yogurt" is solid and may be cut, whereas "stirred yogurt" is viscous. Fat
may or may not be present in yogurt.
If only the protein matrix and the bacteria present in yogurt are of interest, it is advisable to
extract the fat from the specimens with 3 changes of n-hexane after they had been fixed with
glutaraldehyde and dehydrated in a graded ethanol series as shown below. Following the fat
extraction, the specimens are placed again in absolute ethanol for freezing and subsequent
freeze-fracturing.
If the fat globule distribution is also of interest, the fixation protocol must be modified to
ensure that protein as well as the fat are fixed. In that case, fixation in glutaraldehyde is followed
by post-fixation in buffered 0.5% osmium tetroxide solution which contains imidazole. The
"post-fixation" solution is prepared fresh by mixing a 1% OsO4 stock solution with an equal
volume of a 0.2M imidazole solution adjusted to pH 7.4. Both solutions are in the same buffer at
pH 7.4. The post-fixation may last 2-4 h (but must be extended to 24 h when cheese is

examined). The fixed and post-fixed specimen is washed with 2-3 changes of the plain buffered
imidazole solution before it may be dehydrated and further processed.
Failure to either remove fat completely or to fix it properly will result in partial retention of
fat and the micrographs will not show the proper structure and composition of the yogurt
specimen.
Set-style yogurt is trimmed into minute prisms, 1 mm 1 mm 10-15 mm. The prisms are
fixed for ~1 h in 2.5% glutaraldehyde solution in 0.1 M sodium cacodylate buffer (pH 6.8 or
lower - pH of yogurt is actually below 5.5). To retain fat, the specimens are then post-fixed with
imidazole-buffered osmium tetroxide as mentioned above. They are then dehydrated in a graded
ethanol series (20, 40, 60, 80, 95, and 100% (absolute ethanol) for 5-10 min at each
concentration. If fat had not been fixed, it must be extracted with 3 changes of n-hexane at this
stage and the specimens returned into absolute ethanol for 2 or 3 changes. The prisms
impregnated with absolute ethanol (with fat extracted or post-fixed) are frozen in liquid nitrogen.
Although this cryogen is not efficient to freeze hydrated specimens for electron microscopy,
ethanol does not form ice crystals on freezing, so there is no risk of artifact development.
Using an insulated scalpel and an insulated pair of tweezers (see Fig. 5), the prisms are
freeze-fractured into fragments. They are then dropped one by one into absolute ethanol (at room
temperature) to thaw. In the next step, the fragments are critical point-dried. Dried fragments are
mounted on SEM stubs with their fracture planes parallel to the base of the stub. Fracture planes
are shiny in reflected light. Double-sided sticky tape attached to the stub may facilitate
positioning of the fragments.
It is advisable to "paint" the side walls of the porous structure of dried yogurt with a
conductive silver cement up to the top edge to ensure electric conductivity when the specimens
are coated with gold.
A short working distance, somewhat higher accelerating voltage and a smaller beam spot size
improve the images compared to settings used with less demanding specimens.
Stirred yogurt may not be obtained in the form of solid particles unless it is aspirated into an
agar gel tube and prepared for SEM in this "encapsulated" form. The procedure is easy to
perform but requires some practice. The crucial point is the formation of a thin agar gel sleeve
around the pasteur pipette's narrow part. The height of the yogurt column in the tube may be 15
mm because the column in the gel tube will be shorter since it will be wider.
The proper concentration (~3%) and temperature (40C) of the agar sol are the major
contributing factors to success as is rapid turning of the pasteur pipette when forming the gel
sleeve.
After the tube is sealed with one or 2 droplets of agar sol, it is fixed and further processed as
mentioned above. The same steps are used as with set-style yogurt.
Unlike dry-fracturing, freeze-fracturing leads to micrographs which allow comparison with
other yogurt specimens concerning various parametres such as casein particle branched chains,
fat globule dimensions and distribution, and the distribution and dimensions of void spaces
around lactic acid bacteria. A dry-fractured particle breaks at the weakest places and the
topography of the fracture "plane" does not allow any meaningful conclusions.

Transmission electron microscopy may be done with either defatted specimens or specimens
with fat retained by post-fixation. The dimension of the specimens must be considerably smaller.
Post-fixed specimens may sometimes show a "ground pepper artifact" in the form of minute
black dots in the micrographs, the cause of which is not fully understood.
A book on yogurt:
A. Y. Tamime and R. K. Robinson: Yogurt - Science and Technology, 2nd edition - 1999
Published in the United Kingdom by Woodhead Publishing Limited, Abington Hall, Aginton,
Cambridge CB1 6AH England
ISBN 1 85573 399 4
Published in North and South America by CRC Press LLC, 2000 Corporate Blvd., NW Boca
Raton, Florida 33431
ISBN 0-8493-1785-1
Scientific papers on labneh microstructure:
Tamime AY, Kalb M, Davies G: Microstructure of set-style yoghurt manufactured from cow's
milk fortified by different methods. Food Microstructure 3:83-92 (1984).
Tamime AY, Kalb M, Davies G: Rheology and microstructure of strained yoghurt (Labneh)
made from cow's milk by three different methods. Food Microstructure 8:125-135 (1989).
Tamime AY, Davies G, Chehade AS, Mahdi HA: The production of Labneh by ultrafiltration - a
new technology. J. Soc. Dairy Technol. 42:35-39 (1989).
Tamime AY, Kalb M, Davies G, Mahdi HA: Microstructure and firmness of labneh (high solids
yoghurt) made from cow's, goat's and sheep's milks by a traditional method or by ultrafiltration.
Food Structure 10:37-44 (1991).