Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

Подождите немного. Документ загружается.

disease. The ingestion of B. bifidum together with

lactulose was shown to assist in reestablishing the

balance of the gut flora, which is usually disturbed

in liver cirrhosis, and this was accompanied by a

decrease in fecal pH and by a reduction of ammonia

and free phenols in the blood. (See Liver: Nutritional

Management of Liver and Biliary Disorders.)

0029 The beneficial effects of ingested bifidobacteria

and/or lactobacilli may be obtained if: (1) large

numbers of viable cells (10

–10

cells day

1

) are

introduced; (2) they survive gastric transit and, pref-

erably, can adhere to epithelial surfaces and grow; (3)

a fermentable carbohydrate is available in the gut;

and (4) they have a strong antagonistic effect against

harmful microorganisms.

Improved Lactose Utilization

0030 It was shown that lactase-deficient persons can digest

lactose in yogurt better than the same amount of

lactose in unfermented milk. Yogurt bacteria,

Streptococcus salivarius subsp. thermophilus and

L. delbrueckii sp. bulgaricus, produce the enzyme,

lactose (beta-galactosidase), which hydrolyzes lactose

to glucose and galactose. It is possible that they

supply preformed lactase to the gut, thus allowing

digestion of lactose. L. acidophilus and L. casei also

allow digestion of lactose by producing lactase. These

organisms survive in the gut better than yogurt bac-

teria, thus suggesting that lactase from the bacterial

cells remains in the small intestine for a longer time.

Possible Anticarcinogenic Activity

0031 The potential anticarcinogenic activity of some lacto-

bacilli and bifidobacteria has been shown in

many studies. The effect of L. acidophilus on Ehrlich

ascites tumor was studied using mice as an animal

model. Feeding acidophilus milk resulted in a smaller

number of tumor cells in mice than in those not receiv-

ing L. acidophilus. Similar results were obtained

with mice, transplanted with Meth-A ascites tumor

cells, that received a suspension of B. infantis,

repeated four to six times. (See Cancer: Diet in Cancer

Prevention.)

0032 Other studies with mice indicated a possible role of

macrophages in suppressing the growth of tumor

cells. Intraperitoneal injection of L. casei in mice

was shown to increase the phagocytic activity of peri-

toneal macrophages and their acid phosphatase activ-

ity. In addition, the consumption by mice of milk

containing L. casei resulted in activation of the

macrophages, measured by increased levels of lactic

dehydrogenase activity.

0033 Some of these effects may be due to substances

produced by the organism during growth, and/or to

the stimulation of the host immune response. It is also

possible that some lactobacilli and bifidobacteria in-

hibit the growth of organisms which may convert

procarcinogens into carcinogens in the gut. The oral

administration of L. acidophilus (10

cells day

1

)to

meat-fed rats was shown to reduce the activity of

azoreductase, beta-glucuronidase, and nitroreduc-

tase. These fecal enzymes can catalyze procarcinogen

conversion to a proximal carcinogen. In a study

with human subjects it was found that feeding

500 ml of milk containing 2 10

cells of

L. acidophilus ml

1

day

1

for 4 weeks significantly

reduced beta-glucuronidase, azoreductase, and

nitroreductase activities. When feeding with lactoba-

cilli had ceased, fecal enzyme levels returned to

normal after 4 weeks.

0034Another mechanism for the anticarcinogenic effect

refers to nitrosamines (potent carcinogens) which are

synthesized in vivo from amines and nitrite. The syn-

thesis of nitrosamines may be reduced by enzymatic

degradation carried out by some strains of lactobacilli

and bifidobacteria. (See Nitrosamines.)

0035Apparently, various mechanisms may be involved

in the potentially anticarcinogenic effects of lactoba-

cilli and bifidobacteria.

Control of Serum Cholesterol

0036The influence of gut bacteria on sterol metabolism

may be of significance in human nutrition in prevent-

ing the accumulation of cholesterol. The feeding of a

milk formula supplemented with L. acidophilus to

infants was shown to result in lower levels of blood

cholesterol than those recorded in infants receiving

the milk without L. acidophilus. Similar results were

reported on rats supplementary-fed skim milk fer-

mented with L. acidophilus, compared to those fed

unfermented skim milk.

0037Laboratory studies have shown that strains of

L. acidophilus originating from humans actively

assimilate cholesterol, but there are considerable

differences in the activity of strains. These results

are supported by experiments with pigs using pig

isolates of L. acidophilus. It was shown that supple-

mentary-feeding L. acidophilus to pigs resulted in

lower blood cholesterol than in those not receiving

L. acidophilus.

0038Strains of L. casei and B. bifidum were also shown

in laboratory studies to assimilate cholesterol to vary-

ing degrees. In a study with human subjects it was

found that the ingestion of large numbers of

B. bifidum daily during 6 weeks, significantly reduced

the levels of serum cholesterol and serum triglycerides

in subjects suffering from elevated serum lipids. This

treatment was without effect in subjects with normal

values of serum lipids. (See Bile.)

3914 MICROFLORA OF THE INTESTINE/Probiotics

Stimulation of the Immune System

0039 There are indications that the ingestion of lactic acid

bacteria and/or bifidobacteria stimulates the immune

response of the host. A significant increase in the

number of B lymphocytes and T lymphocytes, was

observed, as well as an augmentation of some im-

munoglobulins in mice given yogurt or other lactic

acid bacteria. Also, phagocytic activity of macro-

phages was shown to be enhanced in mice supplemen-

tary-fed with lactic acid-producing bacteria (e.g.,

L. acidophilus, L. casei, B. longum).

0040 Studies with human subjects also showed the

immunogenical effects of lactic acid bacteria. For

example, (1) a significant increase of phagocytic ac-

tivity of leukocytes in peripheral blood, and an in-

crease of immunoglobulin A (IgA) were found in

subjects given fermented milk containing L. acido-

philus strain Lal for 3 weeks. This effect was observed

at least 6 weeks after termination of the consumption

fermented milk; (2) a significant increase in numbers

of B lymphocytes and natural killer cells, the augmen-

tation of IgG, and an increase of the serum levels of

gamma-interferon were observed in subjects given

large numbers of yogurt bacteria for 28 days.

0041 The immunogenic effects of lactic acid bacteria

and/or bifidobacteria may be influenced by: (1) the

bacterial strain characteristics; (2) the numbers of

culture bacteria ingested; and (3) a duration of

ingesting culture bacteria. Further research is needed.

Selection of Microbial Strains

0042 Development of improved microbial supplements in-

volves selection of proper strains of the organism. If

the ingested bacteria are to survive, they must be

resistant to all antimicrobial factors which exist in

the gut. Gastric acidity is an important barrier to

gut colonization, but some protection is afforded by

the buffering effect of the food and food boli will tend

to raise the pH of the stomach. Various strains of

lactobacilli and bifidobacteria were shown to differ

significantly in acid tolerance. Other factors affecting

bacterial survival in the gut include bile acids, lyso-

zyme, and organic acids. The ability of bacteria to

adhere to the gut epithelium is also an important

factor. Adhesion is host-specific, e.g., only lactobacilli

and bifidobacteria originating from the human gut

have the potential to attach to human epithelial

cells. The adhesion ability varies between bacterial

species, and even different strains of the same species

show variations. It was also shown that growth in

milk enhances the ability of lactobacilli to adhere to

epithelial cells.

0043 Various strains of lactobacilli and bifidobacteria

were shown to differ significantly in the antagonistic

effect against harmful bacteria. Other desirable prop-

erties include immunogenic effects, assimilation of

cholesterol, and hydrolysis of lactose.

0044The selection of probiotic strains may include the

following criteria: (1) not pathogenic; (2) of human

intestinal origin; (3) survival of intestinal passage;

(4) adhesion to epithelial cells; (5) clinically tested

for the specific therapeutic effect; and (6) applicable

for manufacturing food products.

Evidence of Influence of Ingested

Bacteria

0045The maximum effect of ingested probiotic bacteria is

obtained when the protective indigenous flora have

been changed by diet, antibiotic therapy, disease, or

some other factors, as the following examples illustrate.

0046A disturbed balance of the gut flora in 28 patients

with leukemia was shown to be improved by

ingesting 2 10

bifidobacteria and 2 10

L. acidophilus in 200 ml of milk daily for 3

months. The counts of Klebsiella spp., Proteus

spp., Candida spp., and Pseudomonas spp. in the

feces decreased, and the levels of urine indican and

blood endotoxin reduced, compared to patients

not receiving supplements.

0047Fifteen patients (mean 2.5 years) received antibi-

otics, such as cephema, penicillins, and aminogly-

cosides, for curing septicemia and respiratory tract

infections. During treatment, diarrhea occurred

and lasted for 1–10 weeks. The counts of bifido-

bacteria decreased in the feces, and those of Can-

dida and Enterococcus increased. The ingestion of

bifidobacteria and L. acidophilus improved the

stool frequency within 3–7 days, along with the

restoration of the gut flora.

0048The beneficial effects of probiotics may be en-

hanced by including special ingredients (prebiotics),

and thus improving host health.

0049Probiotic bacteria can affect the composition of the

gut flora and/or its metabolic activity during the

period of ingestion and for some time after. For

example:

0050Milk fermented with L. acidophilus fed to human

volunteers reduced the E. coli count and increased

the lactobacillus count, but when the supplement

ceased, the bacterial counts returned to normal

after 9 days.

0051Five healthy volunteers ingested 3 10

B. longum

daily for 5 weeks; during feeding, counts of bifido-

bacteria increased and those of clostridia decreased;

in addition, ammonia levels and beta-glucuronidase

activity decreased in the feces and serum.

MICROFLORA OF THE INTESTINE/Probiotics 3915

3.0052 As already mentioned, feeding L. acidophilus to

humans reduced beta-glucoronidase, azoreduc-

tase, and nitroreductase activities, but when the

supplement ceased, enzyme levels returned to

normal after 4 weeks.

0053 It is difficult to achieve a permanent colonization of

ingested probiotic bacteria. However, their ingestion

continuously or at regular intervals may insure the

desired benefits.

See also: Bile; Cancer: Diet in Cancer Prevention;

Carbohydrates: Classification and Properties;

Metabolism of Sugars; Cholesterol: Factors Determining

Blood Cholesterol Levels; Dietary Fiber: Properties and

Sources; Physiological Effects; Immunology of Food;

Infants: Breast- and Bottle-feeding; Liver: Nutritional

Management of Liver and Biliary Disorders;

Nitrosamines; Protein: Synthesis and Turnover

Further Reading

Alm L (1991) The therapeutic effects of various cultures –

an overview. In: Robinson RK (ed.) Therapeutic Proper-

ties of Fermented Milks, pp. 45–64. Essex: Elsevier Ap-

plied Science.

Fuller R (1992) Probiotics. The Scientific Basis. London:

Chapman & Hall.

Gilliland SE (1989) Acidophilus milk products: a review of

potential benefits to consumers. Journal of Dairy Science

72: 2483–2494.

Havenaar R and Huis in’t Veld JHJ (1992) Probiotcs: a

general view. In: Wood BJB (ed.) The Lactic Acid Bac-

teria: Vol. 1. The Lactic Acid Bacteria in Health and

Disease, pp. 151–170. Essex: Elsevier Applied Science.

Mitsuoka T (1989) Bifidobacterium microecology. In: Les

Laits Fermente

s, pp. 41–48. Paris: John Libbey.

Mollet B, Donnet A, Neeser JR et al. (1997) Effects of

probiotics on the immune system. In: Probiotics – Facts

and Opinions, pp. 35–40. Bern: Verlag Schweizerische

Milchkommission.

Playne MJ and Crittenden R (1996) Commercially available

oligosaccharides. In: Bulletin IDF 313, pp. 10–22. Brus-

sels: International Dairy Federation.

Ras

JLJ and Kurmann JA (1983) Bifidobacteria and their

Role, p. 87 Basel: Birkha

user Verlag.

Savage DC (1977) Interaction between the host and its

microbes. In: Clarke BTJ, Bauchop T (eds) Microbial

Ecology of the Gut, pp. 277–310. London: Academic

Press.

Sellars RL (1991) Acidophilus products. In: Robinson RK

(ed) Therapeutic Properties of Fermented Milks, pp. 81–

116. Essex: Elsevier Applied Science.

Simone CD, Bianchi Salvatori B, Jirilo E, Baldinelli L, Fabio

SD and Vesely R (1989) Yogurt and the immune re-

sponse. In: Les Laits Fermente

s, pp. 63–67. Paris: John

Libbey.

Microorganisms See Aeromonas; Antibiotic-resistant Bacteria; Bacillus: Occurrence; Detection; Food

Poisoning; Bifidobacteria in Foods; Campylobacter: Properties and Occurrence; Detection; Campylobacteriosis;

Clostridium: Occurrence of Clostridium perfringens; Detection of Clostridium perfringens; Food Poisoning by

Clostridium perfringens; Occurrence of Clostridium botulinum; Botulism; Escherichia coli: Occurrence; Detection;

Food Poisoning; Occurrence and Epidemiology of Species other than Escherichia col i; Food Poisoning by Species

other than Escherichia coli; Lactic Acid Bacteria; Microbiology: Classification of Microorganisms; Detection of

Foodborne Pathogens and their Toxins; Mycobacteria; Salmonella: Properties and Occurrence; Detection;

Salmonellosis; Shigella; Staphylococcus: Properties and Occurrence; Detection; Food Poisoning; Vibrios: Vibrio

cholerae; V i brio parahaemolyticus; V i brio vu l nificus; Viruses; Yeasts; Yersinia enterocolitica: Properties and

Occurrence; Detection and Treatment; Zoonoses

3916 MICROFLORA OF THE INTESTINE/Probiotics

MICROSCOPY

Contents

Light Microscopy and Histochemical Methods

Scanning Electron Microscopy

Transmission Electron Microscopy

Image Analysis

Light Microscopy and

Histochemical Methods

J H Holgate and J Webb, Reading Scientific Services

Ltd, Whiteknights, Reading, UK

Background

0001 Light microscopy has been in use in the food industry

since the first instruments were introduced. In the

early years, its primary application was for detecting

adulteration of foodstuffs. Identification was depend-

ent primarily upon the recognition of structural

features combined with the reaction to selected

microchemical tests. (See Adulteration of Foods: De-

tection.) Today, a wide range of routine and research

microscopes and microscopy techniques are avail-

able, providing the food scientist with microstruc-

tural information on the nature and form of

individual food components and on their distribution

within a product. This allows the role and inter-

actions of food ingredients to be examined and pro-

vides information valuable to the understanding of

the properties of finished products, cooking and

manufacturing processes, and the influence of both

the type and form of ingredients. Electron microscopy

techniques complement this approach, allowing the

microstructure of the same types of food materials to

be examined at greater magnifications and with dif-

ferent types of preparation procedures. (See Analysis

of Food.) The light microscope is probably one of the

most versatile laboratory instruments available to the

food scientist. A basic instrument, available today for

the same price as a good-quality balance, provides a

wide range of magnifications and other facilities to

provide structural information on almost any form of

food sample.

Principles

0002 The light microscope is an instrument for visualizing

fine detail of an object. It does this by creating a magni-

fied image through the use of a series of glass lenses,

which first focus a beam of light onto or through

an object, and convex objective lenses to enlarge the

image formed. In the majority of light microscopes,

the image is viewed directly through binocular eye-

pieces that act as a secondary lens in the form of a

magnifying glass to observe the projected image. Such

instruments are termed ‘compound microscopes,’ and

the total magnification is the sum of the objective

magnification and the eyepiece magnification. The

magnification range extends from 10 to 1000,

with a resolving power of the order of 0.2 mm,

depending on the type and numerical aperture (area

available for passage of light) of the objective lenses.

A number of books are available, providing compre-

hensive details on the theory of the light microscope

and guidance to the practical use of the instrument,

including methods of image enhancement and instru-

ment care. The reader is referred to these, in par-

ticular an extensive series of handbooks published

by the Royal Microscopical Society, for further in-

formation.

Microscopy Techniques

0003Microscopy techniques for use in the food industry are

frequently a combination of those used in biological

and materials sciences. Samples are viewed using either

transmitted or incident (reflected) illumination. Most

frequently, transmitted light is used where a beam of

light is allowed to pass through a relatively thin object.

The microstructural detail becomes visible due to the

absorption of a portion of the light. Incident light is

used mainly for the examination of solid, opaque

objects, although it now forms the basis for all modern

fluorescent microscopes, referred to as ‘epifluores-

cence.’ (See Spectroscopy: Fluorescence.)

0004The light source is often a single or multicoil tung-

sten lamp or quartz halogen lamp. However, xenon

arc or mercury vapor lamps provide more intense

beams, as required for fluorescence microscopy.

Contrasting Techniques

0005Though many structures are visible by standard

(bright field) transmitted light, there are components

MICROSCOPY/Light Microscopy and Histochemical Methods 3917

that are nonabsorbent and appear transparent or lack

any contrast. This can be overcome, or additional

information may be gained by using one of several

contrasting techniques available that will introduce

or enhance image contrast, frequently with minimal

disruption of the specimen. The most frequently used

are phase contrast, differential interference contrast,

and polarized light.

0006 When using phase contrast or differential interfer-

ence contrast (Nomarski) optics, for example, the

phase of part of the light is altered, and this is then

recombined with light that has passed through the

specimen and thus yields improved differentiation

within a specimen. Phase contrast images are

characterized by enhanced contrast and visibility of

unstained materials and are used routinely for exam-

ination of microorganisms isolated from food. Inter-

ference contrast provides a distinct relief appearance,

with a shallow depth of focus often giving the appear-

ance of a three-dimensional image. It has proved

particularly valuable for examining small particles

and emulsion droplets (Figure 1).

0007 A different type of contrast is generated in certain

samples when they are viewed under crossed polars.

Plane polarized light (light vibrating in only a single

plane) is generated using a polarized filter and allowed

to impinge upon the specimen. The light then travels

through a second polarization filter (the analyzer),

which is orientated at 90



to the first, effectively

blocking the uninterrupted plane polarized light. Any

material that is anisotropic or birefringent, e.g., crys-

tals, is capable of rotating the light plane so that the

polarization of emerging light will be altered and par-

tially extinguished and will have an orientation that

will pass through the analyzer filter. The resultant

image is of bright features against a black background.

0008 The use of polarized light has many applications in

the study of food. Food starches and many crystalline

food ingredients, e.g., sugar, have characteristic sizes,

shapes, etc., that can be observed using polarized

light. Intact starch granules are strongly birefringent

but lose this property during gelatinization (e.g.,

cooking), and polarized light microscopy is fre-

quently used to follow this process during, for

example, the baking of bread doughs. (See Starch:

Structure, Properties, and Determination.)

Fluorescence

0009Irradiation of samples with light of specific short

wavelengths can result in reemission of this energy

as light of longer wavelengths. This is referred to as

‘fluorescence.’ The emitted light, different in color

from the excitation light, may be separated by the

use of specific filters. Some components of food, for

example collagen and lignin, fluoresce naturally after

excitation with light (autofluorescence). However,

such fluorescence is often very weak, and it is more

common for selective, strongly fluorescent dyes

(fluorochromes) to be used to produce a high contrast

within a sample (Figure 2). (See Lignin.)

0010Almost all modern fluorescence microscopes use

epifluorescence, in which the specimen is illuminated

by a high-density mercury vapor source (via a beam

splitter through the objective) to excite only the

surface layers of the sample.

0011The technique has particular value in food micro-

scopy as it can be used to detect substances at low

concentrations and for rapid sample screening at

low magnification where the particles of interest are

in localized regions within the sample. Dyes in

common use in food microscopy are Acridine orange

for staining bacteria and milk proteins and Nile blue

for fats. However, the availability of fluorescent dyes

10 µm

fig0002Figure 2 Bacteria isolated from milk. Preparation is stained

with Acridine orange and viewed using epifluorescence.

10µm

fig0001 Figure 1 Emulsified droplets of fat in a salad cream. Prepar-

ation viewed using Nomarski interference contrast.

3918 MICROSCOPY/Light Microscopy and Histochemical Methods

is steadily increasing, especially in combination with

highly specific labeling agents such as antibodies

(immunolabeling) and lectins, and is likely to be one

of the main growth areas within food light micro-

scopy over the coming years.

Sample Preparation

0012 The majority of food materials and their ingredients

require some kind of preparation before the micro-

structure can be examined by light microscopy. The

choice of sample preparation will depend on the ma-

terial being examined and the kind of information

required. It may range from simple whole mounts

and smears, to more complex, sectioned prepar-

ations.

0013 Often, food samples or their ingredients, e.g., milk

powders, can be examined simply by direct viewing

on a glass microscope slide; alternatively, further

valuable information can be gained after supporting

the sample in a transparent mounting medium. Such

media are frequently inert as well as transparent and

include water, liquid paraffin, glycerol, chloral hy-

drate (used to ‘clear’ plant cells), and many commer-

cial mountants. The choice will depend on the nature

of the material to ensure that little or no change, for

example dissolution or swelling, occurs prior to

examination.

0014 Liquid or semisolid foods, for example viscous

pastes, fats and emulsions, are often viewed as

‘smears’ across a slide. However, it is important to

be aware of the potential disruption of the micro-

structure during this type of preparation and the dif-

ferences in detail provided by samples of different

thickness. Contrast is often enhanced by phase or

interference contrast. The more traditional prepar-

ation for light microscopy, and one frequently used

for food samples, is the preparation of thin slices or

sections through the material. Such sections are

mounted on to slides, their contrast often enhanced

by multiple staining before examination by bright-

field illumination.

Sectioning

0015 Sectioning techniques for light microscopy range

from the relatively simple freehand sectioning with

a knife or razor blade to the precision cutting of

embedded material using sophisticated bench micro-

tomes or ultramicrotomes. A few foods can be

sectioned with little or no prepreparation, and it is

still commonplace in a food laboratory to see initial

freehand sectioning. A bench microtome, however,

enables ribbons of sections of more uniform size and

thickness (5–50 mm) to be cut. Stainless-steel knives

are used most frequently, although more resilient ma-

terials, such as tungsten carbide, are often used to cut

hard materials and provide longer-lasting blades. Al-

ternatively, freshly cleaved glass knives, used rou-

tinely for ultrathin sectioning for transmission

electron microscopy, are used to cut thinner sections

(< 5 mm) for light microscopy. Food plant material,

particularly dried tissue and some solid composite

foods, can be sectioned successfully with no prior

preparation or treatment. However, the majority of

foods and their ingredients require some preparation

prior to sectioning to ensure that they are rigid

enough to enable sections to be cut with minimum

distortion. Wax or resin embedding and low-

temperature ‘cryofixation’ are the most frequently

used preparative techniques and have been used in

food microscopy for many years.

Fixation/embedding

0016Whatever the embedding media used, the food mater-

ial needs to be chemically preserved or ‘fixed’ prior to

infiltration and embedding with the wax or polymer.

Successful fixation depends on the type of food ma-

terial being prepared, in particular the ease of pene-

tration of the fixative into the material. The use of

traditional aldehyde fixatives (e.g., formaldehyde or

glutaraldehyde) is common, as is the use of osmium

tetroxide, in liquid or vapor form, particularly with

fats present. However, many different fixation pro-

cedures have now been adapted and developed for

specific foods in order to achieve optimum preserva-

tion of individual components. These include a

number developed more specifically for electron

microscopy. After fixation, the specimen is usually

dehydrated with solvent prior to being infiltrated

and then embedded in a wax or polymer. Solidifica-

tion or polymerization of the embedding media pro-

duces a hard, rigid preparation from which sections

can be cut. Wax embedding remains one of the

cheapest preparation procedures for light micro-

scopy; the procedure can be readily automated for

routine use, and considerable improvements have

been made to the waxes available. However, it is

considered an awkward, somewhat time-consuming

and messy procedure and is increasingly being re-

placed with resins and other polymeric materials for

preparing food samples. A wide range of different

types of resins and polymers are now available, pro-

viding high flexibility in viscosity and conditions for

polymerization, e.g., temperature and time and in-

cluding some acrylic polymers that provide low-

temperature hardening (often using ultraviolet light

polymerization). The use of the newer resins for

embedding samples generally offers a significantly

MICROSCOPY/Light Microscopy and Histochemical Methods 3919

improved safety in the form of the reduced hazards of

the solvents used compared with wax embedding and

in the handling of the resins compared with the previ-

ously used epoxy resins, etc.

Cryostat Sectioning

0017 Cryofixation of samples, followed by ‘cryostat’ low-

temperature sectioning, has the significant advantage

of speed. Samples are supported (using a gel or

commercial preparation such as Tissue-Tek) on

metal blocks (often after an initial chemical fixation),

and then fast-frozen, generally in liquid nitrogen

(196



C) to preserve or ‘cryofix’ their structure.

The frozen block is sectioned at approximately 20

to 30



C using a conventional bench microtome

housed in a refrigerated unit (cryostat). The technique

obviously has particular application for frozen foods

such as icecream, but is also used frequently for foods

that have high water contents or that are difficult to

handle in any other way. However, some food prod-

ucts are unsuitable for sectioning when frozen, for

example, if they remain soft or become brittle. Also,

the initial capital expenditure can be high and, al-

though the sections can be preserved and permanently

mounted, the frozen block can only be kept if stored

under liquid nitrogen. Sections are collected on to

glass slides, sometimes aided by a thin coating/layer

of adhesive such as egg albumen, and allowed to

warm to room temperature. They are often further

chemically fixed at this stage prior to staining.

Staining Methods

0018 Other than using the optical contrasting techniques

described above, the use of dyes to stain components

specifically is the most common way of increasing

contrast for light microscopy. Staining techniques

rely on the interaction or absorption of the dyes with

the sample components. Many different stains are

now available, and procedures have been developed

for food and food ingredients in order to color spe-

cific individual components and thus produce en-

hanced contrast and enable identification of protein,

fat, starch, and sugar, for example, (See Protein:,

Determination and Characterization; Starch: Struc-

ture, Properties, and Determination.)

0019 Frequently, two or more stains can be used in a

single preparation. Within food microscopy, many

different stains are now readily available for the

differentiation of different types of protein, carbohy-

drate, and fats. Some of these are available in vapor

form, e.g., iodine for starch and osmium tetroxide for

fats, which has a particular advantage in causing little

disruption to the specimen. The selection of stains and

the procedures used will vary, depending not only on

the type of food material under examination but also

on the sample preparation technique employed and

the exact information required. However, stains are

still often classed according to the nature of their

binding to the specimen. Acidic or basic dyes are

used for attaching to positively or negatively charged

sites, respectively, and thus can be used, for example,

to distinguish between different types of proteins.

Solubility dyes rely on the dye being more soluble in

one component than another. The Sudan family of

stains use this property to stain fats in food.

0020Stains are described as ‘histochemical’ when a spe-

cific chemical reaction is used to attach the chromo-

phore to a particular chemical grouping in the

specimen. The most frequently used is the periodic

acid Schiff (PAS) reaction used to demonstrate the

presence of different polysaccharides by the forma-

tion of aldehyde groups following oxidation.

Applications of Light Microscopy

0021The versatility of the preparation procedures and tech-

niques for light microscopy makes them applicable to

a wide variety of different foods and ingredients.

Selected samples are presented below, although many

more are presented in other parts of the Encyclopedia

as well as numerous food microscopy publications.

Powders

0022A wide range of food materials, in particular raw

ingredients, exist in powdered form. Common

examples include ground spices, spray-dried flavors

and milk powders, and freeze-dried beverages and

proteins. Light microscopy provides a way of examin-

ing the size, shape, and often the internal structure of

individual powder particles. Liquid paraffin is fre-

quently used as a simple mountant, although glycerol

is more successful when fat is present. Examination

under polarized light shows the presence of crystalline

or other birefringent structures, enabling their size and

location to be determined. The presence of lactose

crystals, for example, is often demonstrated by their

characteristic tomahawk shape when viewed under

polarized light (Figure 3). In more complex powder

mixtures, individual components can also be discrim-

inated by selective staining. The use of an iodine/

potassium iodide solution or simply iodine vapor

will stain starch a distinctive blue/black color, whereas

fat appears pale brown, and proteins appear yellow.

Animal-based Products

0023Many meat and comminuted meat products are

readily prepared using cryostat sectioning. A variety

of staining procedures are available for identifying

3920 MICROSCOPY/Light Microscopy and Histochemical Methods

individual components including fats, muscle, con-

nective tissue, and bone. These range from single-

step stain mountants, for example, Toluidine blue,

to more complex sequential staining with three or

four different solutions (e.g., the Picro–Mallory tech-

nique). Polarized light can further complement the

use of stains in order to locate crystalline fatty regions

and connective tissues and to differentiate fresh and

processed muscle tissue.

Fruit and Vegetables

0024 Many cellular structures are visible using phase con-

trast, but more detailed information on changes to

cell wall and cell contents can be obtained following

staining for cellulose, pectins, etc. The understanding

of the effects of processing of fruit and vegetables by

brining, freezing, cooking, etc. has been greatly facili-

tated by microscopical studies on samples taken at

different stages of such processes (Figure 4).

Bakery Products

0025A combination of preparation and observation tech-

niques has been important in examining the complex

microstructure of many bakery products, e.g., bread,

cakes, and biscuits. Such techniques have enabled the

changes in ingredients, especially starch and protein,

to be followed and correlations to be made between

the microstructure and the physical and sensory (e.g.,

texture) properties of the finished products. One of

the key changes in baking is the gelatinization of

starch, which can be followed successfully through

examination of preparations using polarized light.

Photomicrography

0026The recording of images obtained by light microscopy

is an important area in its own right and thus,

unfortunately, beyond the scope of this article. The

advent of high-resolution digital cameras has facili-

tated image acquisition, enabling rapid recording of

images, and, where the investment has been made,

even short ‘videos’ can be made, e.g., for studying the

dissolution of particles. As always, the food micro-

scopist still needs to experiment to ensure that high-

quality images observed through binocular eyepieces

are permanently recorded for future reference.

Developing Areas within Light Microscopy

0027The two areas of most recent development in the field

of light microscopy have been the use of specific

labeling techniques, in particular immunolabeling,

and the availability of confocal instruments. In the

former, labeled antibodies are used as specific re-

agents for the detection/staining of specific specimen

components. Labeling is most frequently achieved by

fluorescent dyes. Although antibodies have been

raised for many biological and biomedical applica-

tions, they are currently limited for foods mainly

because the antigenic sites are modified as a result of

food processing and manufacture.

0028In a confocal microscope, a confocal image is built

up point by point by scanning a sample with a focused

beam of laser light. It is a noninvasive technique

enabling optical sectioning of a sample and producing

images with well-defined focal planes but without the

problems of out-of-focus blur. Combined with cur-

rent developments in image-processing techniques,

confocal microscopy is being applied widely in non-

food areas and is beginning to be applied to food, and

has considerable potential for the future.

0029Light microscopy offers versatility in the number of

optical techniques available for examining food

materials, combined with ease of preparation and

100 µm

fig0003 Figure 3 Characteristic tomahawk crystals of lactose viewed

under crossed polars.

100 µm

fig0004 Figure 4 Section through a fresh onion shoot; Toluidine blue

staining.

MICROSCOPY/Light Microscopy and Histochemical Methods 3921

operation at a relatively low cost. Although a decline

in its routine use was evident after the introduction of

electron microscopes, it has reemerged as an invalu-

able instrument for investigations into the science of

food and food processing.

See also: Adulteration of Foods: Detection; Analysis of

Food; Lignin; Protein: Determination and

Characterization; Spectroscopy: Fluorescence; Starch:

Structure, Properties, and Determination

Further Reading

Bradbury S (1982) An introduction to the optical micro-

scope. Royal Microscopical Society Microscopy Hand-

book No. 01. Oxford: Oxford University Press.

Cook HC (1974) Manual of Histochemical Demonstration

Techniques. Oxford: Butterworths.

Flint O (1994) Food microscopy. Royal Microscopical

Society Microscopy Handbook No. 30. Oxford: BIOS

Scientific Publishers.

Hartley WG (1979) Hartley’s Microscopy. Oxford: Senecio.

Heathcock JF (1988) The use of light microscopy and elec-

tron microscopy in the food industry. Microscopy and

Analysis 2: 81–90.

Heathcock JF and Holgate JH (1990) The Continued Im-

portance of Light Microscopy as a Complementary

Technique to EM in the Elucidation of Food Micro-

structure. Proceedings of RMS Conference Micro 90,

20, pp. 713–718.

Ploem JS and Tanke HJ (1986) Introduction to fluorescence

microscopy. Royal Microscopical Society Microscopy

Handbook No. 10. Oxford: Oxford University Press.

Vaughan JG (ed.) (1979) Food Microscopy. London:

Academic Press.

Winton AL and Winton KB (1935) The Structure and Com-

position of Foods, vols. 1 and 2. New York: John Wiley.

Scanning Electron Microscopy

J Webb and J H Holgate, Reading Scientific Services

Ltd, Whiteknights, Reading, UK

Introduction

0001 A scanning electron microscope uses a finely focused

beam of electrons to reveal the detailed surface char-

acteristics of a specimen and provide information

relating to its three-dimensional structure. It also

has a particular advantage of providing great depth

of field. With the introduction of the first commercial

instruments in the mid-1960s the scanning electron

microscope provided the link between the magnifica-

tion ranges offered by light microscopy and the higher

resolving capability of the transmission electron

microscope (TEM). Nowadays, following develop-

ment of electron guns, electromagnetic lens systems

and vacuum systems, scanning electron microscopes

can have a resolution of 1 nm. Therefore, there is now

considerable overlap between the resolving power

of the two forms of electron microscope available,

although the techniques are complementary and

different types of microstructural information are

obtained in each case.

0002Food and its ingredients have been examined by

scanning electron microscopy (SEM) ever since the

first instrument was available. In the early days the

main applications were in studies on the micro-

structural characterization of dry foods, in particular

powders. However, with the development of a range

of different preparation techniques able to handle

many different types of foods, almost every food

material has now been examined in some way by

SEM. (See Analysis of Food.)

Principles

0003When a fine beam of electrons is focused on to the

surface of a specimen, different interactions occur,

including the emission of secondary and back-

scattered primary electrons. If these are collected

and amplified they can be used to create an image

corresponding to the surface topography of the

specimen. The electron beam is scanned across the

specimen repeatedly in a raster pattern, which is syn-

chronized with the scan of a cathode ray tube such

that the image is presented in a digitized form built up

on a TV monitor. Magnification is achieved through

the electron beam scanning an increasing smaller area

of the same specimen and most modern scanning

electron microscopes have a magnification range

of 7 20, up to more than 300 000, and resolution

in the order of 2–4 nm or better. The great depth of

focus of the microscope (> 500 that of light micro-

scopy) is achieved by the convergence angle of the

primary electron beam and the relatively long

working distance between the final lens and the

specimen. This depth of focus enables production of

images that appear to be three-dimensional.

0004As with transmission electron microscopes, con-

ventional instruments must be operated under high

vacuum in order that the electron beam can travel to

the specimen surface without collision with gas

molecules. However, the introduction of specialized

vacuum systems with pressure-limiting apertures

brings a new perspective to SEM and enables samples

3922 MICROSCOPY/Scanning Electron Microscopy

to be examined at higher pressures. In conventional

SEMs, the source of electrons is generally a heated

tungsten filament, which provides ease of operation,

low cost, and simplicity. More intense sources include

lanthanum hexaboride (LaB6) tips and field emission

guns and these are used where greater brightness is

required. Accelerating voltages used typically range

from less than 1 kV up to 40 kV. The higher voltages

can provide greater resolving power, but at the risk

of excessive penetration into many specimens and

damage by the electron beam. Lower voltages are

used to image fine structure on sample surfaces.

0005 The generation of an image from secondary elec-

trons remains the most frequently used form of

imaging of biological samples within a conventional

SEM (CSEM). However, the interaction of a primary

electron beam with a specimen surface can also pro-

vide other information, for example, from back-

scattered or Auger electrons and X-rays.

Specimen Preparation

0006 In its simplest form, preparation for SEM involves

securing a specimen on to a metal support ‘stub’

and, if the sample material is nonconducting, coating

the surface with a conducting thin layer of metal. The

requirement for conductivity is to prevent the build-

up of electrical charge on the specimen surface once it

is bombarded with electrons within the instrument

and also to enhance the secondary image. Many dif-

ferent procedures have been developed for securing

dry food specimens on SEM stubs. These vary

depending on the size, shape, and composition of

the specimen and the type of information required.

One of the most frequently used support media

employs a colloidal silver cement (silver DAG),

which not only holds the specimen firmly but im-

proves conductivity. However, others include carbon

cement (carbon mixed with an acrylic carrier), glues,

gels, and adhesive tape.

Metal Coating

0007 In the early days of SEM, surface metal coats were

prepared by resistive evaporation in a high-vacuum

coating unit. However, this procedure often caused

localized heating of the specimen surface and, as the

metal was applied unidirectionally, it did not always

cover all the crevices in the specimen. It was soon

superseded by the introduction of diode sputter

coating, which is carried out under a lower vacuum.

The coating material forms the cathode and the spe-

cimen the anode. Sputtering is carried out in an argon

plasma generating a multidirectional spray of metal

particles that carry into crevices and around corners.

Relatively inexpensive sputter coaters are now avail-

able, which provide continuous, uniformly thin metal

coats of controllable thickness, often in automated

units. Such instruments deposit a metallic layer with

a thickness typically of 10 nm and a grain size of

approximately 2–5 nm to achieve a continuous film.

Thicker films, up to 20 nm, are used for specimens

with particularly irregular surfaces.

0008For conventional SEM, the most frequently used

metal has been gold, although others used include

silver, platinum, palladium, tungsten, chromium, or

mixtures of two of these. Chromium is now being

used increasingly with the requirement for thinner

and finer layers which do not mask the fine detail of

the specimen and provide ultrahigh-resolution images.

It is deposited under a higher vacuum to achieve a

finer grain size (0.5 nm) and a continuous, thin

(< 5 nm) layer.

Drying Techniques

0009Many food products and ingredients contain rela-

tively high levels of water. In the virgin state, such

products are unsuitable for examination in a conven-

tional SEM, as the high vacuum system causes rapid

desiccation and consequential damage to the fine

structure of the sample. For these types of specimens,

examination by conventional SEM has been carried

out successfully following some form of drying step.

In many cases, preparation has involved a brief chem-

ical fixation to stabilize the components of the sample

in situ (for example, with glutaraldehyde, which

cross-links proteins) before solvent dehydration

and drying using critical-point drying. Alternatively

freeze-drying has been used.

0010Water needs to be removed with great care in order

to minimize shrinkage or other artifacts and a

great deal of work has been undertaken to optimize

the procedures. Critical-point drying involves the

replacement of the organic dehydration fluid in the

specimen with liquid carbon dioxide and subsequent

conversion of the CO

into gas in a chamber pressur-

ized to the critical point and temperature where liquid

and gaseous CO

are in equilibrium. After gaseous

is released, the specimen is dry without having

passed through any phase boundary. It has been

shown to be a particularly useful technique for

single-cell preparations (Figure 1) and food with

high tissue contents such as meat, fish, and vege-

tables. However, the technique has now been shown

to cause shrinkage of greater than 30% in some

samples and to generate many surface distortions

and is now losing favor as a routine SEM procedure.

Furthermore, any solvent-extractable components

are lost during the dehydration process.

MICROSCOPY/Scanning Electron Microscopy 3923