Introduction and growth of microorganisms in food

6/6/2020 Dr. Sujeet Kumar
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Dr. Sujeet Kumar Mrityunjay, PhD
Assistant Professor
Department of Life Science
School of Sciences
ITM University,Gwalior (Turari Campus)
Madhya Pradesh-474001 (India)
Introduction to microbiology: Microbiology in daily life,
Characteristics and morphology of bacteria, fungi, virus,
protozoa and algae. Control of micro-organisms- Growth
curve; Influence of environmental factors on growth-
PH, Water activity, O2 availability, Temperature, Pressure
and Radiation.

BSFT 203:-Food Microbiology
Unit I: - Introduction to microbiology: Microbiology in daily life, Characteristics and morphology of
bacteria, fungi, virus, protozoa and algae. Control of micro-organisms- Growth curve; Influence of
environmental factors on growth- PH, Water activity, O2 availability, Temperature, Pressure and
Radiation.
GENERAL CHARACTERISTIC OF FUNGI (MOULDS AND YEAST)
Classification and identification of moulds
Molds: - Mold growth on foods, with its fuzzy or cottony appearance, sometimes colored, is familiar to
everyone, and usually food with a moldy or "mildewed" food is considered unfit to eat. Special molds are
useful in the manufacture of certain foods or ingredients of foods. Thus, some kinds of cheese are mold-
ripened, e.g., blue, Roquefort, Camembert, Brie, Gammelost, etc., and molds are used in making Oriental
foods, e.g., soy sauce, miso, sonti, and other discussed later. Molds have been grown as food or feed and
are employed to produce products used in foods, such as amylase for bread making or citric acid used in
soft chinks. Some molds do produce various toxic metabolites (mycotoxins). The term "mold" is a
common one applied to certain multicellular filamentous fungi whose growth on foods usually is readily
recognized by its fuzzy or cottony appearance. Colored spores are typical of mature mold of some kinds
and give color to part or all of the growth. The thallus, or vegetative body, is characteristic of thallophytes,
which lack true roots, stems, and leaves.
Morphological Characteristics of molds:-
Hyphae and Mycelium - The mold thallus consists of a mass of branching, intertwined filaments called
hyphae (singular hypha), and the whole mass of these hyphae is known as the mycelium. The hyphae may
be submerged, or growing within the food, or aerial, or growing into the air above the food. Molds are
divided into two groups: septate, i.e., with cross walls dividing the hypha into cells; and noncoenocytic,
septate with the hyphae apparently consisting of cylinders without cross walls. The non-septate hyphae
have nuclei scattered throughout their length and are considered multicellular. Special, mycelial structures
or parts aid in the identification of molds. Examples are the rhizoids, or "holdfasts," of Rhizopus and
Absidia, the foot cell in Aspergillus, and the dichotomous, or Y-shaped, branching in Geotrichum.
Reproductive Parts or Structures: - Molds can grow from a transplanted piece of mycelium.
Reproduction of molds is chiefly by means of asexual spores. Some molds also form sexual spores. Such
molds are termed “perfect" and are classified as either Oomycetes or Zygomycetes if nonseptate, or Asco-
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mycetes or Basidiomycetes if septate, in contrast to "imperfect" molds, the Fungi Imperfecti (typically
septate), which have only asexual spores.
Asexual Spores:-The asexual spores of molds are produced in large numbers and are small, light, and
resistant to drying. They are readily spread through the air to alight and start new mold thallus where
conditions are favorable. The three principal types of asexual spores are (1) conidia (singular conidium),
(2) arthrospores or oidia (singular oidium), and (3) sporangiospores. Conidia are cut off, or bud, from
special fertile hyphae called conidiophores and usually are in the open, i.e., not enclosed in any container,
in contrast to the sporangiospores, which are in sporangium (plural sporangia), or sac, at the tip of a fertile
hypha, the sporangiophore. Arthrospores are formed by fragmentation of a hypha, so that the cells of the
hypha become arthrospores. Examples of these three kinds of spores will be given in the discussion of
important genera of molds. A fourth kind of asexual spore, the chlamydospore, is formed by many species
of molds when a cell here and there in the mycelium stores up reserve food, swell, and forms a thicker
wall than that of surrounding cells. This chlamydospore, or resting cell, can withstand unfavourable
conditions better than ordinary mold mycelium can and later, under favorable conditions, can grow into a
new mold.
Figure:- Rhizopus
Sexual Spores: The molds which can produce sexual spores are classified on the basis of the manner of
formation of these spores and the type produced. The non-septate molds (Phycomycetes) that produce.
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Conidial head of Aspergillus Geotrichum
1. Oospores are termed Oomycetes: - These molds are mostly aquatic; however, included in this group
are several important plant pathogens. The oospores are formed by the union of a small male gamete and a
large female gamete.
2.Zygospores: - Zygomycetes form zygospores by the union of the tips of two hyphae which often appear
similar and which may come from the same mycelium or from different mycelia. Both Oospores and
zygospores are covered by a tough wall and can survive drying for long periods.
3.Ascospores: - The Ascomycetes (septate) form sexual spores known as ascopores, which are formed
after the union of two cells from the same mycelium or from two separate mycelia. The ascospores,
resulting from cell division after conjugation, are in an ascus, or sac, with usual eight spores per ascus.
4.Basidiospores: - The Basidiomycetes, which include most mushrooms, plant rusts, smuts, etc., form a
fourth type of sexual spore, the basidiospore.
Cultural Characteristics: - Some molds are loose and fluffy; others are compact. Some look velvety on
the upper surface, some dry and powdery, and others wet or gelatinous. Definite zones of growth in the
thallus distinguish some molds, e.g., Aspergillus niger. Pigments in the mycelium-red, purple, yellow,
brown, gray, black, etc. - are characteristic, as are the pigments of masses of asexual spores; green, blue-
green, yellow, orange, pink, lavender, brown, gray, black, etc. The appearance of the reverse side of a
mold on an agar plate may be striking, like the opalescent blue-black or greenish-black color of the
underside of Cladosporium.
Physiological characteristics: - The physiological characteristics of molds are discussed as follows:-
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Moisture Requirements: - In general most molds require less available moisture than do most yeasts and
bacteria. An approximate limiting total moisture content of a given food for mold growth can be estimated,
and therefore it has been claimed that below 14 to 15 percent total moisture in flour or some dried fruits
will prevent or greatly delay mold growth.
Temperature Requirements Most molds would be considered mesophilic i.e., able to grow well at
ordinary temperatures. The optimal temperature for most molds is around 25 to 300 C, but some grow well
at 35 to 370C or above, e.g., Aspergillus spp., and some at still higher temperatures. A number of molds
are psychrotrophic; i.e., they grow fairly well at temperatures of refrigeration, and some can grow slowly
at temperatures below freezing. Growth has been reported at as low as - 5 to - 100C. A few are
thermophilic; i.e., they have a high optimal temperature.
Oxygen and pH Requirements Molds are aerobic; i.e., they require oxygen for growth; this is true at
least for the molds growing on foods. Most molds can grow over a wide range of hydrogen-ion
concentration (pH 2 to 8.5), but the majority are favored by an acid pH.
Food Requirements Molds in general can utilize many kinds of foods, ranging from simple to complex.
Most of the common molds possess a variety of hydrolytic enzymes, and some are grown for their
amylases, pectinases, proteinases, and lipases.
Inhibitors Compounds inhibitory to other organisms are produced by some molds, such as penicillin from
Penicillium chrysogenum and clavacin from Aspergillus clavatus. Certain chemical compounds are
mycostatic, inhibiting the growth of molds (sorbic acid, propionates, and acetates are examples), or are
specifically fungicidal, killing molds.
Classification and identification of molds: - Molds are plants of the kingdom Myceteae. They have no
roots, stems, or leaves and are devoid of chlorophyll. They belong to the Eumycetes, or true fungi, and are
subdivided further to subdivisions, classes, orders, families, and genera. The following criteria are used
chiefly for differentiation and identification of molds:-
1. Hyphae septate or non-septate
2. Mycelium clear or dark (smoky)
3. Mycelium colored of colorless
4. Whether sexual spores are produced and the type: oospores, zygospores, or ascospores
5. Characteristics of the spore head
a) Sporangia: size, color, shape, and location
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b) Spore heads bearing conidia: single conidia, chains, budding conidia, or masses; shape and
arrangement of sterigmata or phialides; gumming together of conidia
6. Appearance of sporangiophores or conidiophores: simple or branched, and if branched the type of
branching; size and shape of columella at tip of sporangiophore; whether conidiophores are single
or in bundles
7. Microscopic appearances of the asexual spores, especially of conidia: shape, size, color; smooth or
rough; one-, two-, or many-celled
8. Presence of special structures (or spores): stolons, rhizoids, foot cells, apo-physis,
chlamydospores, sclerotia, etc.
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GENERAL CHARACTERISTICS OF YEASTS, CLASSIFICATION AND IDENTIFICATION
OF YEASTS
Yeasts and yeast like fungi: - Like mold, the term "yeast" is commonly used but hard to define. It refers
to those fungi which are generally not filamentous but unicellular and ovoid or spheroid and which
reproduce by budding or fission. Yeasts may be useful or harmful in foods. Yeast fermentations are
involved in the manufacture of foods such as bread, beer, wines, vinegar, and surface ripened cheese, and
yeasts are grown for enzymes and for food. Yeasts are undesirable when they cause spoilage of sauerkraut,
fruit juices, syrups; molasses, honey, jellies, meats, wine, beer, and other foods.
General characteristics of yeasts: - Yeasts are classified chiefly on their morphological characteristics,
although their physiological ones are more important to the food microbiologist.
Morphological Characteristics: - The morphological characteristics of yeasts are determined by
microscopic examination.
Form and Structure: - The form of yeasts may be spherical to ovoid, lemon shaped, pearshaped,
cylindrical, triangular, or even elongated into a false or true mycelium. They also differ in size. Visible
parts of the structure are the cell wall, cytoplasm, water vacuoles, fat globules, and granules, which may be
metachromatic, albuminous, or Starchy. Special staining is necessary to demonstrate the nucleus.
Reproduction: - Most yeasts reproduce asexually by multilateral or polar budding, a process in which
some of the protoplasm bulges out the cell wall; the bulge grows in size and finally walls off as a new
yeast cell. In some yeasts, notably some of the film yeasts, the bud appears to grow from a tube like
projection from the mother cell. Replicated nuclear material is divided between the mother and daughter
cells. A few species of yeasts reproduce by fission, and one reproduces by a combination of fission and
budding. Sexual reproduction of "true" yeasts (Ascomycotina) results in the production of ascospores, the
yeast cell serving as the ascus. The formation of ascospores follows conjugation of two cells in most
species of true yeasts, but some may produce ascospores without conjugation, followed by conjugation of
ascospores or small daughter cells. The usual number of spores per ascus and the appearance of the
ascospores are characteristic of the kind of yeast. The ascospores may differ in color, in smoothness or
roughness of their walls, and in their shape (round, oval, reniform, bean- or sickle-shaped, Saturn- or hat~
shaped, hemispherical, angular, fusiform, or needle-shaped). "False" yeasts, which produce no ascospores
or other sexual spores, belong to the Fungi Imperfecti. Cells of some yeasts become chlamydospores by
formation of a thick wall about the cell, for example, Candida, Rhodotorula, and Cryptococcus.
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Cultural Characteristics: - Growth as a film on the surface of liquid media suggests an oxidative or film
yeast, and production of a carotenoid pigment indicates the genus Rhodotorula. The appearance of the
growth is important when it causes colored spots on foods. It is difficult to tell yeast colonies from
bacterial ones on agar plates; the only certain way is by means of microscopic examination of the
organisms. Most young yeast colonies are moist and somewhat slimy but may appear mealy; most colonies
are whitish, but some are cream-colored or pink. Some colonies change little with age, but others become
dry and wrinkled. Yeasts are oxidative, fermentative, or both. The oxidative yeasts may grow as a film,
pellicle, or scum on the surface of a liquid and then are termed film yeasts. Fermentative yeasts usually
grow throughout the liquid and produce carbon dioxide.
Physiological Characteristics: - Most common yeasts grow best with a plentiful supply of available
moisture. But since many yeasts grow in the presence of greater concentrations of solutes (such as sugar or
salt) than most bacteria. Most yeast requires more moisture than molds, however. on the basis of water
activity or aw, yeasts may be classified as ordinary if they do not grow in high concentrations of solutes,
i.e., in a low aw, and as osmophilic if they do. Lower limits of aw for ordinary yeasts range from 0.88 to
0.94. Osmophilic yeasts have been found growing slowly in media with aw as low as 0.62 to 0.65 in
syrups, although some osmophilic yeasts are stopped at about 0.78 in both salt brine and sugar syrup. The
aw values will vary with the nutritive properties of the substrate, pH, temperature, availability of oxygen,
and presence or absence of inhibitory substances. The range of temperature for growth of most yeasts is
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25 to 30°C and the maximum about 35 to 47° C. Some kinds can grow at 0°C or less. The growth of most
yeasts is favored by an acid reaction in the vicinity of pH 4 to 4.5, and they will not grow well in an
alkaline medium unless adapted to it. Yeasts grow best under aerobic conditions, but the fermentative
types can grow anaerobically, although slowly. In general, sugars are the best source of energy for yeasts,
although oxidative yeasts, e.g., the film yeasts, oxidize organic acids and alcohol. Carbon dioxide
produced by bread yeasts accomplishes the leavening of bread, and alcohol made by the fermentative
yeasts is the main product in the manufacture of wines, beer, industrial alcohol, and other products. The
yeasts also aid in the production of flavors or "bouquet" in wines. Nitrogenous foods utilized vary from
simple compounds such as ammonia and urea to amino acids and polypeptides. In addition, yeasts require
accessory growth factors. Yeasts may change in their physiological characteristics, especially the true, or
ascospore-forming, yeasts, which have a sexual method of reproduction. These yeasts can be bred for
certain characteristics or may mutate to new forms. Most yeasts can be adapted to conditions which
previously would not support good growth. Illustrative of different characteristics within a species is the
large number of strains of Saccharomyces cerevisiae suited to different uses, e.g., bread strains, beer
strains, wine strains, and high-alcohol-producing strains or varieties.
Classification and identification of yeasts: - The true yeasts are in the subdivision Ascomycotina, and
the false, or asporogennous, yeasts are in the subdivision Fungi Imperfecti or Deuteromycotina. Certain
yeasts are actually represented in two different genera based on whether they reproduce sexually.
The principal bases for the identification and classification of genera of yeasts are as follows:-
1. Whether ascospores are formed.
2. If they are spore-forming:-
a) The method of production of ascospores: - (1) Produced without conjugation of yeast,
cells (parthenogenetically). Spore formation may be followed by (i) Conjugation of
ascospores. (ii) Conjugation of small daughter cells. (2) Produced after isogamic conjugation
(conjugating cells appear similar). (3) Produced by heterogamic conjugation (conjugating cells
differ in appearance).
b)Appearance of ascospores: shape, size, and color. Most spores are spheroidal or ovoid, but
some have odd shapes, e.g., most species of Hansenula, which look like derby hats
c) The usual number of ascospores per ascus: one, two, four, or eight.
1. Appearance of vegetative cells: shape, size, color, inclusions.
2. Method of asexual reproduction: - a. budding. b. Fission. c. Combined budding and fission.
d. Arthrospores (oidia).
5. Production of a mycelium, pseudo mycelium, or nomycelium.
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6. Growth as a film over surface of a liquid (film yeasts) or growth throughout medium.
7. Color of macroscopic growth.
8. Physiological characteristics (used primarily to differentiate species or strains within a
species):- a. Nitrogen and carbon sources. b. Vitamin requirements. c. Oxidative or
fermentative: film yeasts are oxidative; other yeasts may be fermentative or fermentative and
oxidative. d. Lipolysis; urease activity, acid production, or formation of starch like
compounds.
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CHARACTERISTICS AND MORPHOLOGY OF BACTERIA
Morphological characteristics important in food bacteriology/microbiology: - One of the first steps in
the identification of bacteria in a food is microscopic examination to ascertain the shape, size, aggregation,
structure, and staining reactions of the bacteria present. The following characteristics may be of special
significance.
Encapsulation: - The presence of capsules or slime may account for sliminess or ropiness of a food. In
addition, capsules serve to increase the resistance of bacteria to adverse conditions, such as heat or
chemicals. To the organism they may serve as a source of reserved nutrients. Most capsules are
polysaccharides of dextrin, dextran, or levan.
Formation of Endospores: - Bacteria of the genera Bacillus, Clostridium, Desulfotomaculum,
Sporolactobacillus (rods), and Sporosarcina (cocci) share the ability to form endospores. Bacillus -
aerobic and some facultative anaerobic and Clostridium - anaerobic. Endospores are formed at an
intracellular site, are very refractile, and are resistant to heat, ultraviolet light, and desiccation.
Formation of Cell Aggregates: - It is characteristic of some bacteria to form long chains and of others to
clump under certain conditions. It is more difficult to kill all bacteria in intertwined chains or sizable
clumps than to destroy separate cells.
Cultural characteristics important in food bacteriology: - Bacterial growth in and on foods often is
extensive. Pigmented bacteria cause discolorations on the surfaces of foods; films may cover the surfaces
of liquids; growth may make surfaces slimy; or growth throughout the liquids may result in undesirable
cloudiness or sediment.
Physiological characteristics important in food bacteriology/microbiology: - These changes include
hydrolysis of complex carbohydrates to simple ones; hydrolysis of proteins to polypeptides, amino acids,
and ammonia or amines; and hydrolysis of fats to glycerol and fatty acids. O-R reactions, which are
utilized by the bacteria to obtain energy from foods (carbohydrates, other carbon compounds, simple
nitrogen-carbon compounds, etc.), yield such products as organic acids, alcohols, aldehydes, ketones, and
gases.
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CHARACTERISTICS AND MORPHOLOGY OF ALGAE
The term "morphology" describes the shape, form or growth habit of an organism and its parts. The term
algae (Latin — seaweeds) was first introduced by Linnaeus in 1753, meaning the Hepaticeae. The algae
comprise of a large heterogeneous assemblage of plants which are diverse in habitat, size, organization,
physiology, biochemistry, and reproduction. It is an important group of Thallophyta (Gr. Thallos — a
sprout; phyton — a plant), the primitive and simplest division of the plant kingdom. The orderly system-
atic study of algae is called Phycology (Gr.phycos — seaweeds; logos — study or discourse). Algae are a
group of eukaryotic oxygenic photosynthetic microorganisms that contain chlorophyll a (as seen in plants).
Algae range from single-celled organisms to complex multicellular organisms like seaweeds. Algae inhabit
a wide range of habitats from aquatic environments (freshwater, marine, and brackish) to soils and rocks;
only inadequate light or water seems to limit the presence of algae. Algae are most commonly found in
saturated environments either suspended (planktonic), attached to surfaces, or at the air–water interface
(neustonic). Endolithic algae can be found in porous rock or as surface crusts on desert soils. Algae are
often the predominant microorganisms in acidic (below pH 4) habitats, as seen with the red alga
Cyanidium that can grow below pH 2. Generally free-living, some algae have symbiotic relationships with
fungi (lichens), mollusks, corals, and plants, and some algae can be parasitic.
Morphological Characteristics of Algae: - Algae exhibit a very wide range of morphological diversity.
The simplest forms are unicellular, microscopic, motile or non-motile eukaryotic cells. They may be
spherical (Protococcus, Chlorella), or pyriform (Chlamydomonas). When motile (Volvox,
Chlamydomonas) the cells are generally provided with a pair of eukaryotic flagella. Diatoms show a
characteristic type of non-flagellar locomotion. Motile or non-motile algae may form a colony, known as a
coenobium. There are also many multicellular algae. These may form uniseriate or multiseriate filaments
which may be branched or un-branched. The branched filaments may have prostrate and erect branches
(heterotrichous habit). The multiseriate filaments may form a cylindrical thallus or sometimes a flat
thalloid structure. The siphonaceous algae have coenocytic body (multinucleate, without septa) which may
be simple or complex and elaborate. The brown algae which are exclusively marine and always
multicellular, often have large complex thalli. Diatoms are unicellular algae, but they have a cell which is
unique. It consists of two overlapping halves or valves, like those of a petridish. Some lower forms of
algae have a doubtful systematic position. Many of them, like the chrysomonads are amoeboid.
Euglenoids, have a flexible cell-covering. They are without a rigid cell wall and resemble protozoa in
many ways. The dinoflagellates are also peculiar in having a typically flattened cell with an equatorial
constriction, known as a girdle. However, all such atypical organisms are photosynthetic which justifies
their inclusion in algae.
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Characteristics of Algae:-
1. Algae are chlorophyll-bearing autotrophic thalloid plant body.
2. Almost all the algae are aquatic.
3. The plant body may be unicellular to large robust multicellular structure.
4. The multicellular complex thalli lack vascular tissue and also show little differentiation of tissues.
5. The sex organs are generally unicellular but, when multicellular, all cells are fertile and in most
cases the entire structure does not have any protection jacket.
6. The zygote undergoes further development either by mitosis or meiosis, but not through embryo
formation.
7. Plants having distinct alternation of generations. Both gametophyte and sporophyte generations
— when present in the life cycle are independent.
Occurrence of Algae:- The algae are ubiquitous (present everywhere) in distribution, i.e., they are found
in fresh water as well as marine water, on soil, on rock, as epiphytes or parasites on plants and animals, in
hot springs, in desert, on permanent snow-fields etc. But they mainly dwell in aquatic environments. Based
on habitat the algae may be categorized as:-
1. Aquatic algae.
2. Terrestrial algae, and
3. Algae of remarkable habitats.
1. Aquatic Algae: - Aquatic algae may be fresh water (when salinity is as low-as 10 ppm) or marine
(when salinity is 33-40%). Again, certain algae grow in brackish water which is unpalatable for
drinking, but less salty than sea water. The fresh water algae usually grow in ponds, lakes, tanks,
ditches etc. The very common fresh water algae are Chlamydomonas, Volvox, Ulothrix, Chara,
Oedogonium, Spirogyra, Nostoc, Oscillatoria etc. Some of the very common marine algae are
Sargassum, Laminaria, Ectocarpus, Polysiphonia, Caulerpa, Bangia, Padina etc. Fresh water
algae may be termed as planktonic when they grow and remain suspended on the upper part of
water (e.g., Volvox, diatom), while the benthic algae are bottom-dwellers. The algae that grow at
air-water interface are called neustonic. The benthic algae may be epilithic, that grow on stones;
epipelic attached to sand or mud; epiphytic — growing on plants; and epizoic — growing on
animal body surface. The marine algae may be supralittoral or sub- aerial, as they grow above the
water level and in the spray zone. The intertidal algae grow in such a depth so that they are
exposed periodically due to tides. Other marine algae are sublittoral, meaning that they are
constantly submerged at depths as great as 30-60 metres (100-200 ft). Again, the supralittoral
algae may be edaphic— that grow in and on the soil, epilithic— growing on stones, epiphytic —
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growing on plants, epizoic— growing on animal body surface, and corticolous — growing on tree
barks and parasitic on plants and animals. Some algae (e.g., Chlorella) live endozoically in various
protozoa, coelenterates, molasses etc.
2. Terrestrial Algae: - Some algae are found to grow in terrestrial habitats like soils,’ rocks, logs
etc. The algae that grow on the surface of the soil are known as saprophytes. Many blue-greens,
on the other hand, grow under the surface of the soil, and are called cryptophytes. The algae
growing in the desert soil may be typified as endedaphic (living in soil), epidaphic (living on the
soil surface), hypolithic (growing on the lower surface of the stones on soil), chasmolithic (living
in rock fissures) and endolithic algae (which are rock penetrating). The common terrestrial
members are Oscillatoria sancta, Vaucheria geminata, Chlorella lichina, Euglena sp., Fritschiella
sp. and Phormidium sp.
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CHARACTERISTICS AND MORPHOLOGY OF PROTOZOA
Protozoa are microscopic unicellular eukaryotes that have a relatively complex internal structure and carry
out complex metabolic activities. They are small organisms, ranging from a few microns in length up to
about 1 mm. Some protozoa have structures for propulsion or other types of movement.
General Characteristic Features:-
1. Protozoans are usually microscopic and unicellular individuals.
2. They exhibit all types of symmetry.
3. Most species occur as single but many are colonial.
4. Body is bounded by a cell membrane or plasmalemma.
5. Body may be naked or is covered by a pellicle or a test, made of silica or calcium carbonate.
6. A filamentous network of the cytoskeleton may form a dense supportive structure, called the
epiplasm.
7. Usually uninucleate, but may be more than single nucleus in some forms.
8. Locomotor organelles may be flagella (e.g., Euglena; the species of the euglena often serves as
the best member for the study of the algae as well as the protozoa), cilia (e.g., Paramoecium),
pseudopodium (e.g., Amoeba) or absent in parasitic forms (contractile myonemes are present in
the body).
9. Nutrition may be holozoic, e.g., Amoeba (animal-like), holophytic (e.g., Euglena), saprophytic,
mixotrophic or parasitic.
10. Intracellular type of digestion occurs within the food vacuoles.
11. Respiration performs generally through the outer surface of the body, but may be few obligatory
or facultative anaerobes.
12. Excretion performs generally through the body surface, and water regulation of the body is
accomplished by contractile vacuole.
13. Asexual reproduction occurs by fission (mitosis), plasmotomy or budding. In certain forms sexual
reproduction may occur either by conjugation or fusion by gametes (syngamy).
14. They never develop from blastula stage during development.
15. Mainly aquatic but many are parasitic, commensal or mutualistic.
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CHARACTERISTICS AND MORPHOLOGY OF PROTOZOA
The name ‘virus’ came from a Latin word virus which means venom or poisonous fluid. Although plant
diseases like leaf roll of potato and human diseases like yellow fever, small pox etc., were known for long
time, the nature of causative agent was known to us quite later. Adolph Meyer (1886), an agriculture
chemist of Holland, observed a diseased tobacco plant showing mottling of leaf and named it mosaic. He
was able to demonstrate the infectious nature of the sap of infected plant by grinding, filtering through
double filter paper and then applying the sap to the healthy plants.
Characters of Virus:-
1. They are non-cellular, self-replicating agents.
2. They can grow and multiply intracellularly as an obligate parasite (i.e., grow only in living host)
or remain inert outside the host.
3. Depending on the symmetry, they are of three types: cubical, helical and complex.
4. The viruses consist of two parts: the centrally placed nucleic acid, covered by protein coat.
5. The nucleic acid is either DNA or RNA, but both do not remain together.
6. The nucleic acid may be single or double stranded.
7. The outer covering i.e., shell or capsid is made up of protein units, called capsomeres; except
some animal viruses which are with additional polysaccharides.
8. They have no machinery of their own for protein synthesis and thereby they use host machinery
for the synthesis of protein.
9. During replication their nucleic acid directs the host cell to make different parts of virus and when
these parts assemble together they form a complete infectious particle, the virion.
10. They are transmitted very easily from one organism to another organism.
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CONTROL OF MICRO-ORGANISMS
Control of Microorganisms: - Control of microorganisms is essential in order to prevent the transmission
of diseases and infection, stop decomposition and spoilage, and prevent unwanted microbial
contamination. Microorganisms are controlled by means of physical agents and chemical agents. Physical
agents include such methods of control as high or low temperature, desiccation, osmotic pressure,
radiation, and filtration. Control by chemical agents refers to the use of disinfectants, antiseptics,
antibiotics, and chemotherapeutic antimicrobial chemicals. The basis of chemotherapeutic control of
bacteria is selective toxicity. Selective toxicity means that the chemical being used should inhibit or kill
the intended pathogen without seriously harming the host. A broad spectrum agent is one generally
effective against a variety of Gram-positive and Gram-negative bacteria; a narrow spectrum agent
generally works against just Gram-positives, Gram-negatives, or only a few bacteria. As mentioned above,
such agents may be cidal or static in their action. A cidal agent kills the organism while a static agent
inhibits the organism's growth long enough for body defenses to remove it. There are two categories of
antimicrobial chemotherapeutic agents: antibiotics and synthetic drugs. Antibiotics are metabolic products
of one microorganism that inhibit or kill other microorganisms. Chemotherapeutic synthetic drugs are
antimicrobial drugs synthesized by chemical procedures in the laboratory. Many of today's antibiotics are
now actually semi-synthetic and some are even made synthetically. Antibiotics are metabolic products of
one microorganism that inhibit or kill other microorganisms. Why then do bacteria produce antibiotics?
There is growing support for multiple actions for microbial antibiotic production:-
1. If produced in large enough amounts, antibiotics may be used as a weapon to inhibit or kill other
microbes in the vicinity to reduce competition for food.
2. Antibiotics produced in sublethal quantities may function as interspecies quorum sensing
molecules enabling a number of different bacteria to form within a common biofilm where
metabolic end products of one organism may serve as a substrate for another. All the organisms
are protected within the same biofilm.
3. Antibiotics produced in sublethal quantities may function as interspecies quorum sensing
molecules enabling some bacteria to manipulate others to become motile and swim away thus
reducing the competition for food.
4. Antibiotics action may result in the degradation of bacterial cell walls or DNA and these products
can act as cues that trigger other bacteria to produce a protective biofilm.
5. Antibiotics produced in sublethal quantities may trigger intraspecies quorum sensing. Exposure to
low concentrations of an antibiotic may trigger bacteria to produce quorum sensing molecules
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that trigger the population to produce a protective biofilm. The biofilm then protects the
population from greater concentrations of the antibiotic.
THE CONTROL OF MICROORGANISMS (Physical Agents to Control of Microorganisms)
Microorganisms are controlled by means of physical agents and chemical agents. Physical agents include
such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration.
Control by chemical agents refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic
antimicrobial chemicals.
Basic terms used in discussing the control of microorganisms include:-
1.Sterilization: - Sterilization is the process of destroying all living organisms and viruses. A sterile object
is one free of all life forms, including bacterial endospores, as well as viruses.
2.Disinfection: - Disinfection is the elimination of microorganisms, but not necessarily endospores, from
inanimate objects or surfaces.
3.Decontamination: - Decontamination is the treatment of an object or inanimate surface to make it safe
to handle.
4.Disinfectant: - A disinfectant is an agents used to disinfect inanimate objects but generally to toxic to
use on human tissues.
5.Antiseptic: - An antiseptic is an agent that kills or inhibits growth of microbes but is safe to use on
human tissue.
6.Sanitizer: - A sanitizer is an agent that reduces, but may not eliminate, microbial numbers to a safe
level.
7.Antibiotic: - An antibiotic is a metabolic product produced by one microorganism that inhibits or kills
other microorganisms.
8.Chemotherapeutic antimicrobial chemical: - Chemotherapeutic antimicrobial chemicals are synthetic
chemicals that can be used therapeutically.
9. Cidal: - An agent that is cidal in action will kill microorganisms and viruses.
10. Static: - An agent that is static in action will inhibit the growth of microorganisms
Physical Agents to Control of Microorganisms
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1. TEMPERATURE: - Microorganisms have a minimum, an optimum, and a maximum
temperature for growth. Temperatures below the minimum usually have a static action on
microorganisms. They inhibit microbial growth by slowing down metabolism but do not
necessarily kill the organism. Temperatures above the maximum usually have a cidal action, since
they denature microbial enzymes and other proteins. Temperature is a very common and effective
way of controlling microorganisms.
1. High Temperature: - Vegetative microorganisms can generally be killed at temperatures
from 50°C to 70°C with moist heat. Bacterial endospores, however, are very resistant to heat
and extended exposure to much higher temperature is necessary for their destruction. High
temperature may be applied as either moist heat or dry heat.
A.Moist heat: - Moist heat is generally more effective than dry heat for killing
microorganisms because of its ability to penetrate microbial cells. Moist heat kills
microorganisms by denaturing their proteins (causes proteins and enzymes to lose their three-
dimensional functional shape). It also may melt lipids in cytoplasmic membranes.
(I)Autoclaving: - Autoclaving employs steam under pressure. Water normally boils at 100°C;
however, when put under pressure, water boils at a higher temperature. During autoclaving,
the materials to be sterilized are placed under 15 pounds per square inch of pressure in a
pressure-cooker type of apparatus. When placed under 15 pounds of pressure, the boiling
point of water is raised to 121°C, a temperature sufficient to kill bacterial endospores. The
time the material is left in the autoclave varies with the nature and amount of material being
sterilized. Given sufficient time (generally 15-45 minutes), autoclaving is cidal for both
vegetative organisms and endospores, and is the most common method of sterilization for
materials not damaged by heat.
(II) Boiling water: - Boiling water (100°C) will generally kill vegetative cells after about 10
minutes of exposure. However, certain viruses, such as the hepatitis viruses, may survive
exposure to boiling water for up to 30 minutes, and endospores of certain Clostridium and
Bacillus species may survive even hours of boiling.
(II). Dry heat: - Dry heat kills microorganisms through a process of protein oxidation rather
than protein coagulation. Examples of dry heat include:-
1. Hot air sterilization: - Microbiological ovens employ very high dry temperatures: 171°C
for 1 hour; 160°C for 2 hours or longer; or 121°C for 16 hours or longer depending on the
volume. They are generally used only for sterilizing glassware, metal instruments, and other
inert materials like oils and powders that are not damaged by excessive temperature.
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2. Incineration: - Incinerators are used to destroy disposable or expendable materials by
burning. We also sterilize our inoculating loops by incineration.
(III). Pasteurization: - Pasteurization is the mild heating of milk and other materials to kill
particular spoilage organisms or pathogens. It does not, however, kill all organisms. Milk is
usually pasteurized by heating to 71°C for at least 15 seconds in the flash method or 63-66°C
for 30 minutes in the holding method.
1.2. Low Temperature: - Low temperature inhibits microbial growth by slowing down microbial
metabolism. Examples include refrigeration and freezing. Refrigeration at 5°C slows the
growth of microorganisms and keeps food fresh for a few days. Freezing at -10°C stops
microbial growth, but generally does not kill microorganisms, and keeps food fresh for several
months.
2. DESICCATION: - Desiccation, or drying, generally has a static effect on microorganisms. Lack
of water inhibits the action of microbial enzymes. Dehydrated and freeze-dried foods, for
example, do not require refrigeration because the absence of water inhibits microbial growth.
3. OSMOTIC PRESSURE: - Microorganisms, in their natural environments, are constantly faced
with alterations in osmotic pressure. Water tends to flow through semipermeable membranes, such
as the cytoplasmic membrane of microorganisms, towards the side with a higher concentration of
dissolved materials (solute). In other words, water moves from greater water (lower solute)
concentration to lesser water (greater solute) concentration. When the concentration of dissolved
materials or solute is higher inside the cell than it is outside, the cell is said to be in a hypotonic
environment and water will flow into the cell. The rigid cell walls of bacteria and fungi, however,
prevent bursting or plasmoptysis. If the concentration of solute is the same both inside and outside
the cell, the cell is said to be in an isotonic environment. Water flows equally in and out of the
cell. Hypotonic and isotonic environments are not usually harmful to microorganisms. However, if
the concentration of dissolved materials or solute is higher outside of the cell than inside, then the
cell is in a hypertonic environment. Under this condition, water flows out of the cell, resulting in
shrinkage of the cytoplasmic membrane or plasmolysis. Under such conditions, the cell becomes
dehydrated and its growth is inhibited. The canning of jams or preserves with a high sugar
concentration inhibits bacterial growth through hypertonicity. The same effect is obtained by salt-
curing meats or placing foods in a salt brine. This static action of osmotic pressure thus prevents
bacterial decomposition of the food. Molds, on the other hand, are more tolerant of hypertonicity.
Foods, such as those mentioned above, tend to become overgrown with molds unless they are first
sealed to exclude oxygen. (Molds are aerobic.).
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4. RADIATION: - Electromagnetic radiation is a form of energy that propagates as both electrical
and magnetic waves traveling in packets of energy called photons. In the context of sterilization,
ionizing radiation is a type of short wavelength, high intensity radiation that is used to destroy all
microorganisms during sterilization. The forms of ionizing radiation used for sterilization are
known as gamma irradiation, electron irradiation and x-ray irradiation.
1. Ultraviolet Radiation: - The ultraviolet portion of the light spectrum includes all radiations
with wavelengths from 100 nm to 400 nm. It has low wave-length and low energy. The
microbicidal activity of ultraviolet (UV) light depends on the length of exposure: the longer
the exposure the greater the cidal activity. It also depends on the wavelength of UV used. The
most cidal wavelengths of UV light lie in the 260 nm - 270 nm range where it is absorbed by
nucleic acid. In terms of its mode of action, UV light is absorbed by microbial DNA and
causes adjacent thymine bases on the same DNA strand to covalently bond together, forming
what are called thymine-thymine dimers. As the DNA replicates, nucleotides do not
complementary base pair with the thymine dimers and this terminates the replication of that
DNA strand. However, most of the damage from UV radiation actually comes from the cell
trying to repair the damage to the DNA by a process called SOS repair. In very heavily
damaged DNA containing large numbers of thymine dimers, a process called SOS repair is
activated as kind of a last ditch effort to repair the DNA. In this process, a gene product of the
SOS system binds to DNA polymerase allowing it to synthesize new DNA across the
damaged DNA. However, this altered DNA polymerase loses its proofreading ability resulting
in the synthesis of DNA that itself now contains many misincorporated bases. In other words,
UV radiation causes mutation and can lead to faulty protein synthesis. With sufficient
mutation, bacterial metabolism is blocked and the organism dies. Agents such as UV radiation
that cause high rates of mutation are called mutagens. The effect of this improper base pairing
may be reversed to some extent by exposing the bacteria to strong visible light immediately
after exposure to the UV light. The visible light activates an enzyme that breaks the bond that
joins the thymine bases, thus enabling correct complementary base pairing to again take place.
This process is called photo-reactivation. UV lights are frequently used to reduce the
microbial populations in hospital operating rooms and sinks, aseptic filling rooms of
pharmaceutical companies, in microbiological hoods, and in the processing equipment used
by the food and dairy industries. An important consideration when using UV light is that it has
very poor penetrating power. Only microorganisms on the surface of a material that are
exposed directly to the radiation are susceptible to destruction. UV light can also damage the
eyes, cause burns, and cause mutation in cells of the skin.
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4.2. Ionizing Radiation: - Ionizing radiation, such as X-rays and gamma rays, has much more
energy and penetrating power than ultraviolet radiation. It ionizes water and other molecules
to form radicals (molecular fragments with unpaired electrons) that can disrupt DNA
molecules and proteins. It is often used to sterilize pharmaceuticals and disposable medical
supplies such as syringes, surgical gloves, catheters, sutures, and petri plates. It can also be
used to retard spoilage in seafoods, meats, poultry, and fruits.
5. FILTRATION: - Microbiological membrane filters provide a useful way of sterilizing materials
such as vaccines, antibiotic solutions, animal sera, enzyme solutions, vitamin solutions, and other
solutions that may be damaged or denatured by high temperatures or chemical agents. The filters
contain pores small enough to prevent the passage of microbes but large enough to allow the
organism-free fluid to pass through. The liquid is then collected in a sterile flask. Filters with a
pore diameter from 25 nm to 0.45 µm are usually used in this procedure. Filters can also be used
to remove microorganisms from water and air for microbiological testing.
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USE OF CHEMICAL AGENTS TO CONTROL OF MICROORGANISMS
1. DISINFECTANTS, ANTISEPTICS, AND SANITIZERS
Disinfection is the elimination of microorganisms, but not necessarily endospores, from inanimate
objects or surfaces, whereas decontamination is the treatment of an object or inanimate surface to
make it safe to handle.
a) The term disinfectant is used for an agent used to disinfect inanimate objects or surfaces but is
generally to toxic to use on human tissues.
b) The term antiseptic refers to an agent that kills or inhibits growth of microbes but is safe to use on
human tissue.
c) The term sanitizer describes an agent that reduces, but may not eliminate, microbial numbers to a
safe level.
Because disinfectants and antiseptics often work slowly on some viruses - such as the hepatitis
viruses, bacteria with an acid-fast cell wall such as Mycobacterium tuberculosis, and especially
bacterial endospores, produced by the genus Bacillus and the genus Clostridium, they are usually
unreliable for sterilization - the destruction of all life forms. There are a number of factors which
influence the antimicrobial action of disinfectants and antiseptics, including:-
1. The concentration of the chemical agent.
2. The temperature at which the agent is being used. Generally, the lower the temperature, the longer
it takes to disinfect or decontaminate.
3. The kinds of microorganisms present. Endospore producers such as Bacillus species, Clostridium
species, and acid-fast bacteria like Mycobacterium tuberculosis are harder to eliminate.
4. The number of microorganisms present. The more microorganisms present, the harder it is to
disinfect or decontaminate.
5. The nature of the material bearing the microorganisms. Organic material such as dirt and excreta
interferes with some agents.
The best results are generally obtained when the initial microbial numbers are low and when the surface
to be disinfected is clean and free of possible interfering substances.
There are 2 common antimicrobial modes of action for disinfectants, antiseptics, and sanitizers:-
1.They may damage the lipids and/or proteins of the semipermeable cytoplasmic membrane of
microorganisms resulting in leakage of cellular materials needed to sustain life.
2.They may denature microbial enzymes and other proteins, usually by disrupting the hydrogen and
disulfide bonds that give the protein its three-dimensional functional shape. This blocks metabolism.
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A large number of such chemical agents are in common use. Some of the more common groups are listed
below:-
1.Phenol and phenol derivatives: - Phenol (5-10%) was the first disinfectant commonly used. However,
because of its toxicity and odor, phenol derivatives (phenolics) are now generally used. The most common
phenolic is orthophenylphenol, the agent found in O-syl®, Staphene®, and Amphyl®. Bisphenols contain
two phenolic groups and typically have chlorine as a part of their structure. They include hexachlorophene
and triclosan. Hexachlorophene in a 3% solution is combined with detergent and is found in PhisoHex®.
Triclosan is an antiseptic very common in antimicrobial soaps and other products. Biguanides include
chlorhexadine and alexidine. A 4% solution of chlorhexidine in isopropyl alcohol and combined with
detergent (Hibiclens® and Hibitane®) is a common hand washing agent and surgical handscrub. These
agents kill most bacteria, most fungi, and some viruses, but are usually ineffective against endospores.
Chloroxylenol (4-chloro-3,5-dimethylphenol) is a broad spectrum antimicrobial chemical compound used
to control bacteria, algae, fungi and virus and is often used in antimicrobial soaps and antiseptics. Phenol
and phenolics alter membrane permeability and denature proteins. Bisphenols, biguanides, and
chloroxylenol alter membrane permeability.
2.Soaps and detergents: - Soaps are only mildly microbicidal. Their use aids in the mechanical removal
of microorganisms by breaking up the oily film on the skin (emulsification) and reducing the surface
tension of water so it spreads and penetrates more readily. Some cosmetic soaps contain added antiseptics
to increase antimicrobial activity. Detergents may be anionic or cationic. Anionic (negatively charged)
detergents, such as laundry powders, mechanically remove microorganisms and other materials but are not
very microbicidal. Cationic (positively charged) detergents alter membrane permeability and denature
proteins. They are effective against many vegetative bacteria, some fungi, and some viruses. However,
bacterial endospores and certain bacteria such as Mycobacterium tuberculosis and Pseudomonas species
are usually resistant. Soaps and organic materials like excreta also inactivate them. Cationic detergents
include the quaternary ammonium compounds such as benzalkonium chloride, zephiran®, diaprene,
roccal, ceepryn, and phemerol. Household Lysol® contains alkyl dimethyl benzyl ammonium chloride and
alcohols.
3.Alcohols: - 70% solutions of ethyl or isopropyl alcohol are effective in killing vegetative bacteria,
enveloped viruses, and fungi. However, they are usually ineffective against endospores and non-
enveloped viruses. Once they evaporate, their cidal activity will cease. Alcohols denature membranes and
proteins and are often combined with other disinfectants, such as iodine, mercurials, and cationic
detergents for increased effectiveness.
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4.Acids and alkalies: - Acids and alkalies alter membrane permeability and denature proteins and other
molecules. Salts of organic acids, such as calcium propionate, potassium sorbate, and methylparaben, are
commonly used as food preservatives. Undecylenic acid (Desenex®) is used for dermatophyte infections
of the skin. An example of an alkali is lye (sodium hydroxide).
5.Heavy metals: - Heavy metals, such as mercury, silver, and copper, denature proteins. Mercury
compounds (mercurochrome, metaphen, merthiolate) are only bacteriostatic and are not effective against
endospores. Silver nitrate (1%) is sometimes put in the eyes of newborns to prevent gonococcal
ophthalmia. Copper sulfate is used to combat fungal diseases of plants and is also a common algicide.
Selinium sulfide kills fungi and their spores.
6.Chlorine: - Chlorine gas reacts with water to form hypochlorite ions, which in turn denature microbial
enzymes. Chlorine is used in the chlorination of drinking water, swimming pools, and sewage. Sodium
hypochlorite is the active agent in household bleach. Calcium hypochlorite, sodium hypochlorite, and
chloramines (chlorine plus ammonia) are used to sanitize glassware, eating utensils, dairy and food
processing equipment, hemodialysis systems, and treating water supplies.
7.Iodine and iodophores: - Iodine also denatures microbial proteins. Iodine tincture contains a 2%
solution of iodine and sodium iodide in 70% alcohol. Aqueous iodine solutions containing 2% iodine and
2.4% sodium iodide are commonly used as a topical antiseptic. Iodophores are a combination of iodine
and an inert polymer such as polyvinylpyrrolidone that reduces surface tension and slowly releases the
iodine. Iodophores are less irritating than iodine and do not stain.They are generally effective against
vegetative bacteria, Mycobacterium tuberculosis, fungi, some viruses, and some endospores. Examples
include Wescodyne®, Ioprep®, Ioclide®, Betadine®, and Isodine®.
8.Aldehydes: - Aldehydes, such as formaldehyde and glutaraldehyde, denature microbial proteins.
Formalin (37% aqueous solution of formaldehyde gas) is extremely active and kills most forms of
microbial life. It is used in embalming, preserving biological specimens, and in preparing vaccines.
Alkaline glutaraldehyde (Cidex®), acid glutaraldehyde (Sonacide®), and glutaraldehyde phenate solutions
(Sporocidin®) kill vegetative bacteria in 10-30 minutes and endospores in about 4 hours. A 10 hour
exposure to a 2% glutaraldehyde solution can be used for cold sterilization of materials. Ortho-
phthalaldehyde (OPA) is dialdehyde used as a high-level disinfectant for medical instruments.
9.Peroxygens: - Peroxygens are oxidizing agents that include hydrogen peroxide and per acetic acid.
Hydrogen peroxide is broken down into water and oxygen by the enzyme catalase in human cells and is
not that good of an antiseptic for open wounds but is useful for disinfecting inanimate objects. The high
concentrations of hydrogen peroxide overwhelm the catalase found in microbes. Per acetic acid is a
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disinfectant that kills microorganisms by oxidation and subsequent disruption of their cytoplasmic
membrane. It is widely used in healthcare, food processing, and water treatment.
10. Ethylene oxide gas: - Ethylene oxide is one of the very few chemicals that can be relied upon for
sterilization (after 4-12 hours exposure). Since it is explosive, it is usually mixed with inert gases such as
freon or carbon dioxide. Gaseous chemosterilizers, using ethylene oxide, are commonly used to sterilize
heat-sensitive items such as plastic syringes, petri plates, textiles, sutures, artificial heart valves, heart- lung
machines, and mattresses. Ethylene oxide has very high penetrating power and denatures microbial
proteins. Vapors are toxic to the skin, eyes, and mucous membranes and are also carcinogenic. Another
gas that is used as a sterilant is chlorine dioxide which denatures proteins in vegetative bacteria, bacterial
endospores, viruses, and fungi.
2. ANTIMICROBIAL CHEMOTHERAPEUTIC AGENTS: - Antimicrobial chemotherapy is the use
of chemicals to inhibit or kill microorganisms in or on the host. Chemotherapy is based on selective
toxicity. This means that the agent used must inhibit or kill the microorganism in question without
seriously harming the host. In order to be selectively toxic, a chemotherapeutic agent must interact with
some microbial function or microbial structure that is either not present or is substantially different from
that of the host. For example, in treating infections caused by prokaryotic bacteria, the agent may inhibit
peptidoglycan synthesis or alter bacterial (prokaryotic) ribosomes. Human cells do not contain
peptidoglycan and possess eukaryotic ribosomes. Therefore, the drug shows little if any effect on the host
(selective toxicity). Eukaryotic microorganisms, on the other hand, have structures and functions more
closely related to those of the host. As a result, the variety of agents selectively effective against eukaryotic
microorganisms such as fungi and protozoans is small when compared to the number available against
prokaryotes. Also keep in mind that viruses are not cells and, therefore, lack the structures and functions
altered by antibiotics so antibiotics are not effective against viruses. Based on their origin, there are 2
general classes of antimicrobial chemotherapeutic agents:-
1.Antibiotics: - substances produced as metabolic products of one microorganism which inhibit or kill
other microorganisms.
2.Antimicrobial chemotherapeutic chemicals: - chemicals synthesized in the laboratory which can be
used therapeutically on microorganisms.
Most of the major groups of antibiotics were discovered prior to 1955, and most antibiotic advances since
then have come about by modifying the older forms. In fact, only 3 major groups of microorganisms have
yielded useful antibiotics: the actinomycetes (filamentous, branching soil bacteria such as Streptomyces),
bacteria of the genus Bacillus, and the saprophytic molds Penicillium and Cephalosporium.
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Commonly used antimicrobial chemotherapeutic agents arranged according to their mode of
action:-
1. Antimicrobial agents that inhibit peptidoglycan synthesis. Inhibition of peptidoglycan synthesis in
actively-dividing bacteria results in osmotic lysis.
a. Penicillins (produced by the mold Penicillium)
There are several classes of penicillins:-
1.Natural penicillins are highly effective against Gram-positive bacteria (and a very few Gram-negative
bacteria) but are inactivated by the bacterial enzyme penicillinase. Examples include penicillin G, F, X, K,
O, and V.
2.Semisynthetic penicillins are effective against Gram-positive bacteria but are not inactivated by
penicillinase. Examples include methicillin, dicloxacillin, and nafcillin.
3.Semisynthetic broad-spectrum penicillins are effective against a variety of Gram-positive and Gram-
negative bacteria but are inactivated by penicillinase. Examples include ampicillin, carbenicillin, oxacillin,
azlocillin, mezlocillin, and piperacillin.
4.Semisynthetic broad-spectrum penicillins combined with beta lactamase inhibitors such as clavulanic
acid and sulbactam. Although the clavulanic acid and sulbactam have no antimicrobial action of their own,
they inhibits penicillinase thus protecting the penicillin from degradation. Examples include amoxicillin
plus clavulanic acid, ticarcillin plus clavulanic acid, and ampicillin plus sulbactam.
b. Cephalosporins (produced by the mold Cephalosporium)
Cephalosporins are effective against a variety of Gram-positive and Gram-negative bacteria and are
resistant to penicillinase (although some can be inactivated by other beta-lactamase enzymes similar to
penicillinase). Four "generations" of cephalosporins have been developed over the years in an attempt to
counter bacterial resistance.
1. First generation cephalosporins include cephalothin, cephapirin, and cephalexin.
2. Second generation cephalosporins include cefamandole, cefaclor, cefazolin, cefuroxime, and cefoxitin.
3.Third generation cephalosporins include cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone,
cefoperazone, ceftazidine, and moxalactam.
4. Fourth generation cephalosporins include cefepime and cefpirome.
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c.Carbapenems: - Carbapenems consist of a broad spectrum beta lactam antibiotic to inhibit
peptidoglycan synthesis combined with cilastatin sodium, an agent which prevents degradation of the
antibiotic in the kidneys. Examples include: imipenem, metropenem, ertapenem, and doripenem.
d.Monobactems: - Monobactems are broad spectrum beta lactam antibiotics resistant to beta lactamase.
An example is aztreonam.
e. Carbacephem: - A synthetic cephalosporins. An example is loracarbef.
f.Glycopeptides (produced by the bacterium Streptomyces): - Vancomycin and teichoplanin are
glycopeptides that are effective against Gram-positive bacteria.
f. Bacitracin (produced by the bacterium Bacillus):- Bacitracin is used topically against Gram-positive
bacteria.
h. Fosfomycin (Monurol)
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GROWTH CURVE
Growth: - Growth is defined as an increase in cellular constituents which leads to a rise in cell number.
As we are aware, microorganisms reproduce by binary fission or by budding. In order to study growth,
normally one follows the changes in the total population number. The cells copy their DNA almost
continuously and divide again and again by the process called binary fission. Binary fission which has
been described earlier as the form of asexual reproduction in single-celled organisms by which one cell
divides into two cells of the same size. Fortunately, few prokaryotic populations can sustain exponential
growth for long. Environments are usually limiting in resources such as food and space. Prokaryotes also
produce metabolic waste products that may eventually pollute the colony’s environment. Still, you can
understand why certain bacteria can make you sick so soon after infection or why food can spoil so
rapidly. Refrigeration retards food spoilage not because the cold kills the bacteria on food but because
most microorganisms reproduce very slowly at such low temperatures.
Growth Curve: - A growth curve is an empirical model of the evolution of a quantity over time. The
increase in the cell size and cell mass during the development of an organism is termed as growth. It is the
unique characteristics of all organisms. The organism must require certain basic parameters for their
energy generation and cellular biosynthesis. The growth of the organism is affected by both physical and
Nutritional factors. The increase in cell number or growth in population is studied by analyzing the growth
curve of a microbial culture. Bacteria can be grown or cultivated in a liquid medium in a closed system or
also called as batch culture. In this method, no fresh medium is added and hence with time, nutrient
concentration decreases and an increase in wastes is seen. As bacteria reproduce by binary fission, the
growth can be plotted as the logarithm of the number of viable cells verses the time of incubation. The
curve plotted shows four basic phases of growth; the lag, log, stationary, and death phase.
Lag phase: - When a microorganism is introduced into the fresh medium, it takes some time to adjust
with the new environment. This phase is termed as Lag phase, in which cellular metabolism is accelerated,
cells are increasing in size, but the bacteria are not able to replicate and therefore no increase in cell mass.
The length of the lag phase depends directly on the previous growth condition of the organism. When the
microorganism growing in a rich medium is inoculated into nutritionally poor medium, the organism will
take more time to adapt with the new environment. The organism will start synthesising the necessary
proteins, co-enzymes and vitamins needed for their growth and hence there will be a subsequent increase
in the lag phase. Similarly when an organism from a nutritionally poor medium is added to a nutritionally
rich medium, the organism can easily adapt to the environment, it can start the cell division without any
delay, and therefore will have less lag phase it may be absent.
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Exponential or Logarithmic (log) phase: - During this phase, the microorganisms are in a rapidly
growing and dividing state. Their metabolic activity increases and the organism begin the DNA replication
by binary fission at a constant rate. The growth medium is exploited at the maximal rate, the culture
reaches the maximum growth rate and the number of bacteria increases logarithmically (exponentially) and
finally the single cell divide into two, which replicate into four, eight, sixteen, thirty two and so on (That is
20
, 21
, 22
, 23
.........2n
, n is the number of generations) This will result in a balanced growth. The time taken
by the bacteria to double in number during a specified time period is known as the generation time. The
generation time tends to vary with different organisms. E.coli divides in every 20 minutes, hence its
generation time is 20 minutes, and for Staphylococcus aureus it is 30 minutes.
Stationary phase: - As the bacterial population continues to grow, all the nutrients in the growth medium
are used up by the microorganism for their rapid multiplication. This result in the accumulation of waste
materials, toxic metabolites and inhibitory compounds such as antibiotics in the medium. This shifts the
conditions of the medium such as pH and temperature, thereby creating an unfavorable environment for
the bacterial growth. The reproduction rate will slow down, the cells undergoing division is equal to the
number of cell death, and finally bacterium stops its division completely. The cell number is not increased
and thus the growth rate is stabilized. If a cell taken from the stationary phase is introduced into a fresh
medium, the cell can easily move on the exponential phase and is able to perform its metabolic activities
as usual.
Decline or Death phase:- The depletion of nutrients and the subsequent accumulation of metabolic waste
products and other toxic materials in the media will facilitates the bacterium to move on to the Death
phase. During this, the bacterium completely loses its ability to reproduce. Individual bacteria begin to die
due to the unfavorable conditions and the death is rapid and at uniform rate. The number of dead cells
exceeds the number of live cells. Some organisms which can resist this condition can survive in the
environment by producing endospores.
Different phases of growth of a bacteria
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Significance of the Bacterial Growth Curve
1. The study of bacterial growth curves is important when aiming to utilize or inoculate known
numbers of the bacterial isolate, for example to enhance plant growth, increase biodegradation
of toxic organics, or produce antibiotics or other natural products at an industrial scale.
2. Knowledge of bacterial growth kinetics and bacterial numbers in a culture medium is important
from both a research and commercial point of view.
3. Growth kinetics is also useful for assessing whether particular strains of bacteria are adapted to
metabolize certain substrates, such as industrial waste or oil pollution.
4. Bacteria that are genetically engineered to clean up oil spills, for example, can be grown in the
presence of complex hydrocarbons to ensure that their growth would not be repressed by the
toxic effects of oil.
5. Similarly, the slope and shape of growth curves produced from bacteria grown with mixtures
of industrial waste products can inform scientists whether the bacteria can metabolize the
particular substance, and how many potential energy sources for the bacteria can be found in
the waste mixture
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Factors Influencing the Growth of Microorganisms in Food
Foods are mainly composed of biochemical compounds which are derived from plants and animals.
Carbohydrates, proteins and fats are the major constituents of food. In addition, minor constituents such as
minerals, vitamins, enzymes, acids, antioxidants, pigments, flavors are present. Foods are subject to
physical, chemical, and biological deterioration. The factors that affect microbial growth in foods, and
determine the nature of spoilage and any health risks can be cauterized in four groups. The major factors
affecting microbial growth in foods are as follows:-
1. Intrinsic Factors (Physico-chemical properties of the food itself)
2. Extrinsic Factors (conditions of the storage environment)
3. Implicit Factors (properties and interactions of the microorganisms present); and
4. Processing Factors.
Factors affecting the development of microbial associations in food
1 Intrinsic Factors 




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Nutrients
pH and buffering capacity
Redox potential
Water activity
Antimicrobial constituents
Antimicrobial structures
2 Environmental factors 


Relative humidity
Temperature
Gaseous atmosphere
3 Implicit factors 
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Specific growth rate
Mutualism
Antagonism
Commensalism
4 Processing factors 
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Slicing
Washing
Packing
Irradiation
Pasteurization
Extrinsic Factors:-
1. Relative Humidity: - Relative humidity and water activity are interrelated, thus relative humidity is
essentially a measure of the water activity of the gas phase. When food commodities having a
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low water activity are stored in an atmosphere of high relative humidity water will transfer from
the gas phase to the food.
It may take a very long time for the bulk of the commodity to increase in water activity,
but condensation may occur on surfaces giving rise to localized regions of high water activity. It is
in such regions that propagules which have remained viable, but unable to grow, may now
germinate and grow. Once microorganisms have started to grow and become physiologically
active they usually produce water as an end product of respiration. Thus they increase the water
activity of their own immediate environment so that eventually micro-organisms requiring a high
aw are able to grow and spoil a food which was initially considered to be microbiologically stable.
Such a situation can occur in grain silos or in tanks in which concentrates and syrups are stored.
Another problem in large-scale storage units such as grain silos occurs because the relative
humidity of air is very sensitive to temperature. If one side of a silo heats up during the day due to
exposure to the sun then the relative humidity on that side is reduced and there is a net migration
of water molecules from the cooler side to re-equilibrate the relative humidity. When that same
side cools down again the relative humidity increases and, although water molecules migrate back
again, the temporary increase in relative humidity may be sufficient to cause local condensation
onto the grain with a localized increase in aw sufficient to allow germination of fungal spores and
subsequent spoilage of the grain. This type of phenomenon can often account for localized caking
of grain which had apparently been stored at a ‘safe’ water content. The storage of fresh fruit and
vegetables requires very careful control of relative humidity. If it is too low then many vegetables
will lose water and become flaccid. If it is too high then condensation may occur and microbial
spoilage may be initiated.
2. Temperature: - Microbial growth can occur over a temperature range from about — 8°C up to
100o
C at atmospheric pressure. The most important requirement is that water should be present in
the liquid state and thus available to support growth. No single organism is capable of growth over
the whole of this range; bacteria are normally limited to a temperature span of around 35°C and
moulds rather less, about 30°C. A graph showing the variation of growth rate with temperature
illustrates several important features of this relationship (Figure no. 1). Firstly, each organism
exhibits a minimum, optimum and maximum temperature at which growth can occur.
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Figure no. 1 :- Effects of temperature on growth
These are known as cardinal temperatures and are, to a large extent, characteristic of an organism,
although they are influenced by other environmental factors such as nutrient availability, pH and
aw. Micro-organisms can be classified into several physiological groups based on their cardinal
temperatures. This is a useful, if rather arbitrary, convention, since the distribution of micro-
organisms through the growth temperature range is continuous. To take account of this and the
effect of other factors, it is more appropriate to define cardinal temperatures as ranges rather than
single values (Table no. 1).
Table no. 1 : - Cardinal Temperatures for Microbial Growth
In food microbiology mesophilic and psychrotrophic organisms are generally of greatest
importance. Mesophiles, with temperature optima around 37°C, are frequently of human or animal
origin and include many of the more common foodborne pathogens such as Salmonella,
Staphylococcus aureus and Clostridium perfringens. As a rule mesophiles grow more quickly at
their optima than psychrotrophs and so spoilage of perishable products stored in the mesophilic
growth range is more rapid than spoilage under chill conditions. Because of the different groups
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of organisms involved, it can also be different in character. Among the organisms capable of
growth at low temperatures, two groups can be distinguished: the true or strict psychrophiles
(‘cold-loving’) have optima of 12 — 15°C and will not grow above about 20°C. As a result of this
sensitivity to quite moderate temperatures, psychrophiles are largely confined to Polar Regions
and the marine environment. Psychrotrophs or facultative psychrophiles will grow down to the
same low temperatures as strict psychrophiles but have higher optimum and maximum growth
temperatures. This tolerance of a wider range of temperature means that psychrotrophs are found
in a more diverse range of habitats and consequently are of greater importance in the spoilage of
chilled foods. Thermophiles are generally of far less importance in food microbiology, although
thermophilic spore formers such as certain Bacillus and Clostridium species do pose problems in
a restricted number of situations. Another feature evident from Figure no.1 is that the curve is
asymmetric – growth declines more rapidly above the optimum temperature than below it. As the
temperature is decreased from the optimum the growth rate slows, partly as a result of the slowing
of enzymatic reactions within the cell. If this were the complete explanation however, then the
change in growth rate with temperature below the optimum might be expected to follow the
Arrhenius Law which describes the relationship between the rate of a chemical reaction and the
temperature. The fact that this is not observed in practice is, on reflection, hardly surprising since
microbial growth results from the activity of a network of interacting and interrelating reactions
and represents a far higher order of complexity than simple individual reactions. A most important
contribution to the slowing and eventual cessation of microbial growth at low temperatures is now
considered to be changes in membrane structure that affect the uptake and supply of nutrients to
enzyme systems within the cell. It has been shown that many micro-organisms respond to growth
at lower temperatures by increasing the amount of unsaturated fatty acids in their membrane lipids
and that psychrotrophs generally have higher levels of unsaturated fatty acids than mesophiles.
Increasing the degree of unsaturation in a fatty acid decreases its melting point so that membranes
containing higher levels of unsaturated fatty acid will remain fluid and hence functional at lower
temperatures. As the temperature increases above the optimum, the growth rate declines much
more sharply as a result of the irreversible denaturation of proteins and the thermal breakdown of
the cell’s plasma membrane. At temperatures above the maximum for growth, these changes are
sufficient to kill the organism – the rate at which this occurs increasing with increasing
temperature.
3. Gaseous Atmosphere: - Oxygen comprises 21% of the earth’s atmosphere and is themost
important gas in contact with food under normal circumstances. Its presence and its influence on
redox potential are important determinants of the microbial associations that develop and their
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rate of growth. Since under redox potential, this will be confined to the microbiological effects of
other gases commonly encountered in food processing. The inhibitory effect of carbon dioxide
(CO2) on microbial growth is applied in modified-atmosphere packing of food and is an
advantageous consequence of its use at elevated pressures (hyperbaric) in carbonated mineral
waters and soft drinks. Carbon dioxide is not uniform in its effect on micro-organisms. Moulds
and oxidative Gram-negative bacteria are most sensitive and the Gram positive bacteria,
particularly the lactobacilli, tend to be most resistant. Some yeasts such as Brettanomyces spp.
also show considerable -tolerance of high CO2 levels and dominate the spoilage microflora of
carbonated beverages. Growth inhibition is usually greater under aerobic conditions than
anaerobic and the inhibitory effect increases with decrease of temperature, presumably due to the
increased solubility of CO2 at lower temperatures. Some micro-organisms are killed by prolonged
exposure to CO2 but usually its effect is bacteriostatic. The mechanism of CO2 inhibition is a
combination of several processes whose precise individual contributions are yet to be determined.
One factor often identified is the effect of CO2 on pH. Carbon dioxide dissolves in water to
produce carbonic acid which partially dissociates into bicarbonate anions and protons. Carbonic
acid is a weak dibasic acid (pKa 6.37 and 10.25); in an un-buffered solution it can produce an
appreciable drop in pH, distilled water in equilibrium with the CO2 in the normal atmosphere will
have a pH of about 5, but the effect will be less pronounced in buffered food media so that
equilibration of milk with 1 atmosphere pCO2 decreased the pH from 6.6 to 6.0. Probably of more
importance than its effect on the growth menstruum is the ability of CO2 to act in the same way as
weak organic acids, penetrating the plasma membrane and acidifying the cell’s interior. Other
contributory factors are thought to include changes in the physical properties of the plasma
membrane adversely affecting solute transport; inhibition of key enzymes, particularly those
involving carboxylation/decarboxylation reactions in which CO2 is a reactant; and reaction with
protein amino groups causing changes in their properties and activity.
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Intrinsic Factors:-
1. Nutrient Content: - Like us, micro-organisms can use foods as a source of nutrients and energy.
From them, they derive the chemical elements that constitute microbial biomass, those molecules
essential for growth that the organism cannot synthesize, and a substrate that can be used as an
energy source. The widespread use of food products such as meat digests (peptone and tryptone),
meat infusions, tomato juice, malt extract, sugar and starch in microbiological media bears
eloquent testimony to their suitability for this purpose. The inability of an organism to utilize a
major component of a food material will limit its growth and put it at a competitive disadvantage
compared with those that can. Thus, the ability to synthesize amylolytic (starch degrading)
enzymes will favour the growth of an organism on cereals and other farinaceous products. The
addition of fruits containing sucrose and other sugars to yoghurt increases the range of
carbohydrates ‘available and allows the development of a more diverse spoilage microflora of
yeasts. The concentration of key nutrients can, to some extent, determine the rate of microbial
growth. The relationship between the two, known as the Monod equation, is mathematically
identical to the Michaelis - Menten equation of enzyme kinetics, reflecting the dependence of
microbial growth on rate-limiting enzyme reactions:-
Where:-
µ is the specific growth rate;
µm the maximum specific growth rate;
S the concentration of limiting nutrient; and
Ks the saturation constant.
When S>>Ks, a micro-organism will grow at a rate approaching its maximum, but as S fallsto
values approaching Ks, so too will the growth rate. Values for Ks have been measured
experimentally for a range of organisms and nutrients; generally they are extremely low, of the
order of 10-5
M for carbon and energy sources, suggesting that in most cases, nutrient scarcity is
unlikely to be rate-limiting. Exceptions occur in some foods, particularly highly structured ones
where local microenvironments may be deficient in essential nutrients, or where nutrient limitation
is used as a defence against microbial infection, for example the white of the hen’s egg.
2. pH and Buffering Capacity:- pH is equal to the negative logarithm of the hydrogen ion activity.
The acidity and alkalinity of an environment affects growth and metabolism of microorganisms as
the activity and stability of macromolecules, enzymes and nutrient transport is influenced by pH.
Generally, bacteria grow fast at pH 6-8. But bacteria that produce acids have optimum pH
between pH 5 and 6 (Ex: Lactobacillus and Acetic acid bacteria). Yeast grows best at pH 4.5-6.0
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and Fungi at 3.5 – 4.0. In low pH foods (Ex. Fruits), spoilage is mainly by yeasts and fungi than
bacteria. Fishes with pH around neutrality (6.5-7.5) favour bacterial growth and spoil rapidly than
meat (pH: 5.5 – 6.5). Ability of low pH to restrict microbial growth has been employed as a
method of food preservation (Ex: use of acetic and lactic acid). Buffering capacity refers to the
ability of foods to withstand pH changes. Microorganisms have ability to change pH of the
surrounding environment to their optimal level by their metabolic activity. Decorboxylation of
aminoacids releases amines which increases surrounding pH. Deamination of aminoacids by
enzyme deaminases release organic acids causing decease in pH. Thus, protein rich foods like fish
and meat have better buffering capacity than carbohydrate rich foods.
In general, bacteria grow fastest in the pH range 6.0 - 8.0, yeasts 4.5 - 6.0 and
filamentous fungi 3.5 - 4.0. As with all generalizations there are exceptions, particularly among
those bacteria that produce quantities of acids as a result of their energy-yielding metabolism.
Examples important in food microbiology are the lactobacilli and acetic acid bacteria with optima
usually between pH 5.0 and 6.0. Most foods are at least slightly acidic, since materials with an
alkaline pH generally have a rather unpleasant taste (Table no. 2). Egg white, where the pH
increases to around 9.2 as CO2 is lost from the egg after laying, is a commonplace exception to
this. A somewhat more esoteric example, which many would take as convincing evidence of the
inedibility of alkaline foods, is fermented shark, produced in Greenland, which has a pH of 10 -
12.
Table no.2:- Approximate pH ranges of some common food commodities
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The acidity of a product can have important implications for its microbial ecology, and the rate
and character of its spoilage. For example, plant products classed as vegetables generally have a
moderately acid pH and soft-rot producing bacteria such as Erwinia carotovora and
pseudomonad sp. play a significant role in their spoilage. In fruits, however, a lower pH prevents
bacterial growth and spoilage is dominated by yeasts and moulds. As a rule, fish spoil more
rapidly than meat under chill conditions. The pH of post-rigor mammalian muscle, around 5.6, is
lower than that of fish (6.2 - 6.5) and this contributes to the longer storage life of meat. The pH-
sensitive genus Shewanella (formerly Alteromonas) plays a significant role in fish spoilage but
has not been reported in normal meat (pH < 6.0). Those fish that have a naturally low pH such as
halibut (pH ≈ 5.6) have better keeping qualities than other fish. The ability of low pH to restrict
microbial growth has been deliberately employed since the earliest times in the preservation of
foods with acetic and lactic acids. With the exception of those soft drinks that contain phosphoric
acid, most foods owe their acidity to the presence of weak organic acids.
3. Redox Potential (also known as redox potential, oxidation / reduction potential, ORP, pe, ε,
or Eh):- An oxidation-reduction (redox) reaction occurs as the result of a transfer of electrons
between atoms or molecules. It measured in millivolts or volts. This is represented in its most
general form to include the many redox reactions which also involve protons and have the overall
effect of transferring hydrogen atoms.
Where, n is the number of electrons, e, transferred
In living cells an ordered sequence of both electron and hydrogen transfer reactions is an essential
feature of the electron transport chain and energy generation by oxidative phosphorylation. The
redox potential we measure in a food is the result of the following factors:-
 Redox couples present.
 Ratio of oxidant to reductant
 pH.
 Poising capacity.
 Availability of oxygen (physical state, packing).
 Microbial activity Redox Potential The redox potential in food is the result of several
factors
Redox potential exerts an important elective effect on the microflora of a food. Individual
microorganisms are conveniently classified into one of several physiological groups on the
basis of the redox range over which they can grow and their response to oxygen.
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Obligate or strict aerobes are those organisms that are respiratory, generating most of their
energy from oxidative phosphorylation using oxygen as the terminal electron acceptor in the
process. Obligate anaerobes tend only to grow at low or negative redox potentials and often
require oxygen to be absent. Obligate anaerobes, such as Clostridia, are of great importance in
food microbiology. They have the potential to grow wherever conditions are anaerobic such
as deep in meat tissues and stews, in vacuum packs and canned foods causing spoilage and, in
the case of C. botulinum, the major public health concern: botulism. Aero-tolerant anaerobes
are incapable of aerobic respiration, but can nevertheless grow in the presence of air. Many
lactic acid bacteria fall into this category; they can only generate energy by fermentation and
lack both catalase and superoxide dismutase, but are able to grow in the presence of oxygen
because they have a mechanism for destroying superoxide based on the accumulation of
millimolar concentrations of manganese.
Factors influencing O/R potential of a food
 The characteristic of O/R potential of the original food.
 Poising capacity – (Resistance to change in potential of food)
 Oxygen tension of the storage atmosphere of food
 Access that the atmosphere has to the food
 Microbial activity
Eh requirement of microorganisms
 Aerobic microorganisms require oxidized condition (+ Eh) for growth. Ex. Bacillus sp
 Anaerobes require reduced condition (-Eh). Ex. Clostridium sp
 Microaerophils are aerobes growing at slightly reduced condition. Ex. Lactobacillus,
Campylobacter.
 Facultative anaerobes have capacity to grow both under reduced and oxidized
condition. Eg. Yeasts.
 Plant foods have positive Eh (fruits, vegetables) and spoilage is mainly caused by
aerobes (bacteria and molds).
 Solid meat and fish have negative Eh (-200 mv), and minced meat positive Eh (+200
mv).
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Microorganisms and Eh of food
 Microorganisms affect Eh of food during growth. Aerobes reduce the Eh of
environment due to oxygen utilization. Growth medium becomes poorer in
oxidizing and richer in reducing substances.
 Microorganisms reduce Eh by releasing metabolites. Hydrogen sulphide released
by anaerobic microorganisms reacts with oxygen and creates reduced condition
 Presence or absence of appropriate quantity of oxidizing/ reducing agents in the
medium influences growth and activity of all microorganism
4. Water Activity or Moisture content (aw):- Moisture content of the food affects microorganisms in
foods, and the microbial types present in foods depends on the amount of water available. Water
requirement for microorganisms is described in terms of water activity (aw) in the environment
and is defined as the ratio of the water vapor pressure of food substrate to the vapor pressure of
pure water at same temperature.
aw = P/Po
P = vapour pressure of water in substrate
Po = vapour pressure of solvent (pure water)
aw is related to relative humidity (RH)
RH = 100 x aw
Water activity of solutes and requirements of certain microorganisms
aw of pure water: 1.0
NaCl solution (22 %): 0.86
Saturated NaCl solution: 0.75
 Bacteria generally require higher value of aw than fungi
 G – ve bacteria require higher aw than G +ve bacteria
 Most spoilage bacteria do not grow at aw below 0.91
 Spoilage molds grow at aw of 0.80
 Halophilic bacteria grow at aw of 0.75
 Xerophilic and osmophilic yeasts grow at of 0.61
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 Microorganisms like halophiles, osmophiles and xerophiles grow better at reduced aw.
Microorganisms cannot grow below aw 0.60, and in such situations spoilage of food is not
microbiological but due to chemical reactions (Ex: oxidation).
Relationship between aw, temperature and nutrition
 Growth of microorganisms decreases with lowering of aw
 The range of aw at which the growth is greatest occurs at optimum temperature for
growth
 The presence of nutrients increases the range of aw over which the organisms can
survive
5. Antimicrobial Barriers and Constituents: - An antimicrobial is a substance that either kills or
inhibits the growth of microorganisms such as bacteria, fungi, or protozoan's. All foods have one
or the other mechanism to prevent or limit potentially damaging effects by microorganisms
through protective physical barriers to infection (Ex. skin, shell, and husk) and antimicrobial
components. Natural covering of some foods provide excellent protection against entry and
subsequent damage by spoilage microorganisms. These include outer covering of fruits, outer
shell of egg, skin covering of fish and meats. The outer covering is usually composed of
macromolecules and these are resistant to degradation and create inhospitable environment for
microorganisms due to low aw and shortage of readily available nutrients. The antimicrobial
substances such as short chain fatty acids in animal skin and essential oils in plant surfaces help to
prevent entry of microorganisms. Physical damage to outer barrier allows microbial invasion and
cause spoilage. Some foods are resistant to attack by microorganisms and remain stable due to the
presence of naturally occurring substances which have antimicrobial property. Many plant species
possess essential oils which are antimicrobial. As part of the natural protection against
microorganisms, milk has several non-immunological proteins which inhibit the growth and
metabolism of many microorganisms including the following most common:-
 lactoperoxidase
 lactoferrin
 lysozyme
 xanthin
Effect of Pressure on Foods: - Pressure-cooking is an old but reliable cooking method that makes quick
work of tough cuts of meats, dense vegetables, hard beans and more. A pressure cooker works by trapping
steam inside the sealed pot. This causes the atmospheric pressure to rise, which increases the
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boiling temperature of water. So, instead of cooking food at 212°F (if you are at sea level), you cook it at
250°F, resulting in dramatically faster cooking times without harsh boiling.
Effects on food:-
 Pressure cooking can reduce heat-sensitive nutrients (e.g., vitamin C, folate) and bioactive
phytonutrients, such as betacarotene, glucosinolates (helpful compounds found in cruciferous
vegetables) and omega-3 fatty acids, that are beneficial for human health. But so do other
cooking methods—and generally to more or less the same extent.
 With vegetables and fruits, the heat-sensitive nutrients (e.g., vitamin C, folate and bioactive
phytonutrients) are generally most susceptible to degradation during pressure cooking.
Consuming the cooking water can help restore some of these losses.
 In the case of grains and legumes, although the vitamins and heat-sensitive vitamins and
phytonutrients are vulnerable to deterioration, the net result of pressure-cooking is a positive
nutritional gain—from the increased digestibility of the macronutrients (protein, fiber and
starch) and the increased bioavailability of the essential minerals.
 Pressure-cooked meat-based dishes show a significant reduction in unsaturated fat contents,
but it appears that iron is not lost.
 In addition to making foods like grains and legumes more digestible, pressure cooking does
not create any of the unhealthy chemicals associated with baking and grilling methods.
 High heat damages some nutrients, such as polyunsaturated fats and certain vitamins, and can
cause the formation of unhealthy chemicals as found in grilling and baking.
RADIATION: - Electromagnetic radiation is a form of energy that propagates as both electrical and
magnetic waves traveling in packets of energy called photons. In the context of sterilization, ionizing
radiation is a type of short wavelength, high intensity radiation that is used to destroy all microorganisms
during sterilization. The forms of ionizing radiation used for sterilization are known as gamma irradiation,
electron irradiation and x-ray irradiation.
Irradiation: - the process of applying radiation to matter.
Food irradiation (application of irradiation as ionizing energy to foods): - Food irradiation is a cold,
non-chemical process that exposes food to ionizing radiation that can penetrate food to kill, or prevent
reproduction of microorganisms, insects and pests. Insects require a lot less irradiation than bacteria and
viruses. Many different types of food preservation alter the taste and appearance of food.
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Sources of radiation used in food irradiation
1. Gamma Rays are emitted from radioactive forms of the element cobalt (Co60) or of the element
cesium (Cs137). Gamma radiation is used routinely to sterilize medical, dental and household
products and is also used for the treatment of cancer.
2. X-rays are produced by reflecting a high energy stream of electrons off a target substance (usually
one of the heavy metals) into food. X-rays are also widely used in medicine and industry to
produce images of internal structures.
3. Electron beam (or e-beam) is similar to x-rays and is a stream of high-energy electrons propelled
from an electron accelerator into food.
Dose and dose rate
 Ionizing energy processes create enough of an absorbed dose to destroy microbes.
 Unit of absorbed dose in food is kGy (kilograys).
 Dose can be divided into three categories: 1.Radicidation 2.Radurization 3.Raddapperization
“Low” doses <1 kGy (Radicidation)
 Controls insects in grains and fruits
 Inhibit sprouting in tubers
 Delay the ripening of some fruits/vegetables
 Reduce the problems of parasites in products of animal origin. (e.g: Trichinella spiralis in pork).
“Medium” dose (1~10 kGy) (Radurization)
 Control Salmonella, Shigella, Campylobacter, Yersinia, Listeria and E.coli in meat poultry and
fish.
 Delay mold growth on strawberries and other fruits “High” dose (>than 10kGy)
(Radapperization)
 Kill microorganisms and insects in spices
 Commercially sterilize foods, destroying all microorganisms of public health concern (i.e, special
diets for people with weakened immune systems)
To make the food become radioactive, it will require a lot of energy; 15MeV. Foods are actually
naturally radioactive. Due to natural presence of Ca, P, K, and S elements in the food.
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Effects of irradiation on microorganisms
Indirect effects: - Due to formation of the free radicals during radiolysis of water molecules. →Free
radicals are highly reactive - form stable products. → Combine with one another or oxygen molecules
–oxidizing agents. → can damage bacterial cell components. → unstable free radicals react with
bacterial cell membranes to change or damage their structure- bacterial death.
Direct effects: - Ionizing radiation kills microbes by damaging biomolecules of their cells. →
Incoming photon hit electrons in the atoms of microbes or food molecules. → during the collision,
photon’s energy is transferred to the electron changing the photon’s direction. → Electron free to
collide with neighboring electron. →This cause chemical bonds breakage interrupts normal cell
metabolism and division
Effect of food irradiation on food quality
 The food molecules are made of water, lipids, proteins, carbohydrates and vitamins.
 Radiation energy generates a degradative reaction when it interacts with food → radiolysis.
Products of radiolysis is known as radiolytic products.
 Irradiation cause changes to food molecules particularly at high doses.
 Sterilization levels causes nutrient loss and desirables effects.
WHY SHOULD WE IRRADIATE FOODS?
 To decrease the growing food-borne illness rate.
 The control of ripening, sprouting and insect damage.
 Along with other preserving technologies there are advantages and disadvantages.
ADVANTAGES OF IRRADIATION OF FOOD
Pathogen Reduction
 Generate short-lived and transient radicals (e.g. the hydroxyl radical, the hydrogen atom and
solvated electrons).
 That in turn damages the bonds in the DNA molecules, causing disruption in the genetic makeup
of microbes beyond its ability to repair.
 The target organism ceases all the processes of maturation and reproduction.
Shelf Life Extension
 Low doses of radiation (up to 1 kGy) can prolong the shelf-life of many fruits and vegetables.
 For example: Irradiating strawberries extends their refrigerated shelf-life to up to three weeks
without decay or shrinkage, versus three to five days for untreated berries.
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 Moreover irradiation also causes delay in sprouting and ripening adding to shelf-life
Insect Disinfestation
 Irradiation of spices, herbs, and dry vegetable seasonings.
 It is an alternative to the use of chemicals or fumigants, such as ethylene oxide and methyl
bromide.
 Moreover unlike chemicals, irradiation does not leave any residuals that can lead to reinfestation.
Sterilization
 Irradiation can also be used to sterilize food, which can then be stored for years without
refrigeration.
 Sterilized food are used in hospitals for patients with severely impaired immune systems, such as
patients with AIDS or undergoing chemotherapy.
 National Aeronautics and Space Administration (NASA) astronauts eat meat that has been
sterilized by irradiation to avoid getting foodborne illnesses when they fly in space.
Irradiation of Meat and Poultry
 Treating raw meat and poultry at slaughter plant with high doses of irradiation can cause
elimination of E. coli, Salmonella, and Campylobacter.
 Irradiation of animal feeds could also prevent the spread of Salmonella and other pathogens to
livestock through feeds.
DISADVANTAGES OF FOOD IRRADIATION
 Irradiated foods may form chemical products called “radiolytic products”
 Irradiation cannot be used with all foods. It can causes undesirable flavor and texture changes.
 Food irradiation can destroy bacterial spores but is not effective against viruses
 Unknown long-term effects on human health
 Increased consumer cost- Irradiated meats cost approximately 3 to 5 cents more a pound than non-
irradiated meat.
 Prices of irradiated foods are expected to decrease
 Food irradiation reduces the nutritional content of foods
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Introduction and growth of microorganisms in food

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