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Module 1 Microorganisms in Food and Spoilage

Introduction to Food Microbiology

1. Microbiology- The study of very small living organisms (Microorganisms or Microbes).


1.1. History of Microbiology
Historical milestones in microbiology
3.5 billion years ago-- Fossils of primitive microbes found dated.
3180 BC- earliest account of pestilence occurred in Egypt.
1900 BC- Bubonic plague (Greek army)
1500 BC- Ebers papyrus was discovered in a tomb in Thebes, Egypt containing
description of epidemic fever.
1122 BC- smallpox epidemic in China
790, 710 and 640 BC--- epidemics occurred in Rome
430 BC- Epidemics occurred in Greece
1493–appearance of French pox

Pioneers in the science of microbiology


Anton Van Leeuwenhoek- Discovered “animalcules” (live bacteria and protozoa). He is
known as the father of microbiology.
Louis Pasteur- Discovered the process of alcoholic fermentation, Pasteurization and
sterilization. He is known as the founder of microbiology.
Robert Koch- Developed methods of fixing, staining and photographing bacteria and also
developed methods of cultivating bacteria on solid media.
Joseph Lister- Aseptic techniques

1.2. Branches of microbiology


Bacteriology– the study of Bacteria
Phycology– the study of Algae
Mycology– the study of Fungi
Protozoology– the study of protozoa
Virology– the study of viruses
Immunology- the study of immune response
1.3. Application of microbiology
To study Indigenous microflora--- beneficial
To study Opportunistic pathogens—colonize or inhabit bodies.
To study Infectious disease and Microbial intoxication causing microbes

2. Micro organisms
Microorganisms are a heterogeneous group of several distinct classes of living beings. Based
on the difference in cellular organization and biochemistry, the kingdom protista has been
divided into two groups namely prokaryotes and eukaryotes. Bacteria and blue-green algae
are prokaryotes, while fungi (molds, yeast) ande other algae are eukaryotes.

PROKARYOTES EUKARYOTES
 Size: 1-5 μm  Size: 10-100 μm
 Genetic material is free in cytoplasm  Genetic material is in a membrane bound
 Histones (Complex proteins) are absent body
 Ribosome is 70S called nucleus
 Cell wall is made up of peptidoglycans  Histones are present
 Flagella is present  Ribosome is 80S
 Cilia is absent  No cell wall is present
 Pili is present  Flagella is absent
 Slime and capsule are present outside the cell  Pili is absent
wall  Cilia is present
 Example: Bacteria  A pellicle may be present around the cell wall
 Example: Fungi, Algae, Protozoa, Plants,
Animals
3. Bacteria- Morphology, Classification, Physiology, Growth, Nutrition &
Reproduction
3.1. Morphology
Size: The unit of measurement used in bacteriology is the micron (micrometer). Bacterial
cells are of the size of 1-5μm.
Shape: Bacterial cells may be rods (bacillus), sphere (coccus); curved (spirilla)
Cell structure: The outer layer or cell envelope consists of two components, a rigid cell wall
and beneath it a plasma membrane. The cell envelope encloses the cytoplasm, cytoplasmic
inclusions such as ribosomes and mesosomes, granules, vacuoles and the nuclear body.
(a) Slime layer and Capsule
Capsule is the outer most layer of the bacteria. It is a condensed well defined layer closely
surrounding the cell. They are usually polysaccharide, envelope the whole bacterium and
their production depends on growth conditions. They are secreted by the cell under harsh
conditions and are highly impermeable. When it forms a loose mesh work of fibrils extending
outward from the cell they are described as glycocalyx and when masses of polymer that
formed appear to be totally detached from the cell and if the cells are seen entrapped in it are
described as slime layer. The Capsule protects against complement and is antiphagocytic. The
Slime layer & glycocalyx helps in adherence of bacteria either to themselves forming
colonial masses or to surfaces in their environment and they resists phagocytosis and
desiccation of bacteria.
Cell wall/ Plasma membrane
Beneath the external structures is the cell wall. It is very rigid & gives shape to the cell. Its
main function is to prevent the cell from expanding & eventually bursting due to water
uptake. Cell Wall constitutes a significant portion of the dry weight of the cell and it is
essential for bacterial growth & division. Chemically the cell wall is composed of
peptidoglycan. Mucopeptide (peptidoglycan or murien) formed by N acetyl glucosamine & N
acetyl muramic acid alternating in chains, cross linked by peptide chains. Embedded in it are
polyalcohol called Teichoic acids. Some are linked to Lipids & called Lipoteichoic acid.
Lipotechoic acid link peptidoglycan to cytoplasmic membrane and the peptidoglycan gives
rigidity.
b) Cytoplasm
The cytoplasm is a colloidal system containing a variety of organic and inorganic solutes
containing about 70% Water. They are rich in ribosomes, DNA & fluid.
c) Single chromosome as genetic material called nucleoid (sometimes an additional DNA
called as plasmid may also be present
The nucleus is not distinct and has no nuclear membrane and the genetic material consist of
DNA. DNA is circular and haploid. They are highly coiled with intermixed polyamines &
support proteins. Plasmids are extra circular DNA.
d) Ribosome
They are the centers of protein synthesis. They are slightly smaller than the ribosomes of
eukaryotic cells.
e) Inclusions
The Inclusion bodies are aggregates of polymers produced when there is excess of nutrients
in the environment and they are the storage reserve for granules.
f) Flagella Flagella are long thread like helical filaments extending from cytoplasmic
membrane to exterior of the cell. It functions in cell motility. The parts of flagella are the
filament, hook and the basal body. Filament is external to cell wall and is connected to the
hook at cell surface, the hook & basal body are embedded in the cell envelope. Hook &
filament is composed of protein subunits called as flagellin.
The arrangement of flagella may be described as (i) Monotrichous – single flagella on one
side (ii) Lophotrichous – tuft of flagella on one side (iii) Amphitrichous – single or tuft on
both sides (iv) Peritrichous – surrounded by lateral flagella.

g) Mesosomes They are vesicular, convoluted tubules formed by invagination of plasma


membrane into the cytoplasm. They are principal sites of respiratory enzymes
h) Pili: They are proteinaceous structures that extend from the cell membrane to external
environment. They are thinner, shorter and more numerous than flagella and they do not
function in motility. The pili help in conjugation.

Endospores (spores)
They are small, metabolically dormant cells with a thick, multi-layered coat,. They are highly
resistant to adverse environmental conditions and may survive desiccation, disinfectants or
boiling water for several hours. Spores are formed in response to limitations of nutrients by a
complex process (sporulation). Spores can remain dormant for long periods of time.
However, they are able to revert to actively-growing cells (i.e. germinate) relatively rapidly in
response to certain conditions such as the presence of specific sugars, amino acids or bile
salts. Spores have an important role in the epidemiology of certain human diseases.
3.2. Classification
Bacteria are classified on the basis of
(a) Gram staining (b) Cell Shape (c) Cell arrangements (d) Nutrition (e) Oxygen requirement
(f) Temperature requirement
(a) Gram staining: Gram-positive bacteria stain purple, whereas gram-negative bacteria
color pink or red by gram staining technique. The gram-positive cell wall consists of a single
20 to 80 nm thick homogeneous peptidoglycan or murein layer lying outside the plasma
membrane. In contrast, the gram-negative cell wall is quite complex. It has a 2 to 7 nm
peptidoglycan layer surrounded by a 7 to 8 nm thick outer membrane. Outer membrane is
found only in Gram-negative bacteria, it functions as an initial barrier to the environment and
is composed of lipopolysaccharide (LPS) and phospholipids. Because of the thicker
peptidoglycan layer, the walls of gram-positive cells are stronger than those of gram-negative
bacteria.

Figure: Schematic representations of cell envelopes of bacteria. (A) Gram-positive bacteria: SL:
surface layer proteins with protein subunits (1); CW: cell wall showing thick mucopeptide
backbone layers (2) covalently linked to peptides (4), wall teichoic acids (or teichouronic
acid); (3), lipoteichoic acids (anchored to cytoplasmic membrane; (5); CM: cytoplasmic
membrane with lipid bilayers containing phospholipids (7), glycolipids (6) and embedded
proteins (8). (B) Gram-negative bacteria: OM: outer membrane containing lipopolysaccharide
molecules, stabilized by divalent cations (1), phospholipids and proteins; MM, middle
membrane containing thin mucopeptide layers (4) covalently liked to peptides (3) and
lipoproteins (2); IM: inner membrane with phospholipid bilayers (5) and proteins (6).
Gram-positive Bacteria Gram-negative Bacteria
 Actinobacteria  Franciscella
Actinomyces  Klebsiella
Arthrobacter  Legionella
Bifidobacterium  Moraxella
Corynebacterium  Neisseria
Frankia  Pasteurella
Micrococcus  Salmonella
Micromonospora
 Serratia
Mycobacterium
 Shigella
Nocardia
Propionibacterium  Treponema
Streptomyces  Vibrio
 Bacilli, order Bacillales  Yersinia
Bacillus  Proteus
Listeria  Pseudomonas
Staphylococcus  Helicobacter
 Bacilli, order Lactobacillales  Hemophilus
Enterococcus  Campylobacter
Lactobacillus  Cyanobacteria
Lactococcus  Enterobacter
Leuconostoc  Erwinia
Pediococcus  Escherichia coli
Streptococcus  Acinetobacter
 Clostridia  Actinobacillus
Acetobacterium  Bordetella
Clostridium  Brucella
Eubacterium
Heliobacterium
Heliospirillum
Megasphaera
Pectinatus
Selenomonas
Zymophilus
Sporomusa
 Mollicutes
Mycoplasma
Spiroplasma
Ureaplasma
Erysipelothrix

(b) & (c) Cell shape and arrangements


Shape: Bacterial cells may be rods (bacillus), sphere (coccus); curved (spirilla)
Arrangements: i) Diplococcus- coccus in pairs ii) Streptococcus- coccus in chains iii) Tetrad-
coccus in squares iv) coccus in cubes v)Staphylococcus- coccus in random planes vi)
Streptobacillus- bacillus in chains vii) vibrio- comma shaped xi) spirillum- thick spiral x)
spirochete- thin spiral
(d) Nutrition
Bacteria are classified into two main groups according to the type of compounds that they can
utilise as a carbon source:
Autotrophs: utilise inorganic carbon from carbon dioxide and nitrogen from ammonia,
nitrites and nitrates; they are of minor medical importance.
Heterotrophs: require organic compounds as their major source of carbon and energy; they
include most bacteria of medical importance.
(e) Oxygen requirement
(i) Aerobes- Bacteria that require oxygen for growth
(ii) Anaerobes- bacteria that don’t require oxygen for growth
(iii) Obligate aerobes- bacteria that grow only in presence of oxygen
(iv) Facultative anaerobes- bacteria that can grow with lack of oxygen or in absence of
oxygen
(v) Obligate anaerobes-bacteria that grow strictly in absence of oxygen
(f) Temperature requirement
(i) Thermophiles- bacteria that grow at a temperature of 60-80 ℃
(ii) Mesophiles- bacteria that grow at a temperature of 20-40 ℃
(iii) Psychrophiles- bacteria that grow best at temperatures below 20°C
3.3. Physiology, Growth and Nutrition
For bacteria, growth is increase in size or increase in number. Bacterial cells undergo cell
division, binary fission, septum formation or constriction to multiply. Under suitable
conditions (nutrients, temperature and atmosphere) a bacterial cell will increase in size and
then divide by binary fission into two identical cells. These two cells are able to grow and
divide at the same rate as the parent cell, provided that conditions including nutrient supply
remain stable. Most bacteria of medical importance require carbon, nitrogen, water, inorganic
salts and a source of energy for growth. They have various gaseous, temperature and pH
requirements, and can utilise a range of carbon, nitrogen and energy sources. Temperature
requirements: refer above thermophiles etc. Gaseous requirements: refer above aerobes etc
Energy sources: refer autotrophs and heterotrophs above pH requirements: Most pathogenic
bacteria grow best at a slightly alkaline pH (pH 7.2–7.6).
3.4. Reproduction
Binary fission: Most bacteria reproduce by binary fission. During binary fission, the single
DNA molecule replicates and both copies attach to the cell membrane. The cell membrane
begins to grow between the two DNA molecules. A cell wall then forms between the two
DNA molecules dividing the original cell into two identical daughter cells.
Sexual Reproduction
Bacterial Recombination: In order to incorporate some genetic variation, bacteria use a
process called recombination. Recombination is the process in which one or more nucleic
acids molecules are rearranged or combined to produce a new nucleotide sequence. Usually
genetic material from two parents is combined to produce a recombinant chromosome with a
new, different genotype. Bacterial recombination can be accomplished by three ways i)
Conjugation ii) Transformation iii) Transduction
i) Conjugation: Some bacteria are capable of transferring pieces of their genes to other
bacteria that they come in contact with. During conjugation, one bacterium connects itself to
another through a protein tube structure called a pilus. Genes are transferred from one
bacterium to the other through this tube.
ii) Transformation is the uptake by a cell of a naked DNA molecule or fragment from the
medium and the incorporation of this molecule into the recipient chromosome in a heritable
form. In natural transformation the DNA comes from a donor bacterium. The process is
random, and any portion of a genome may be transferred between bacteria.
iii) Transduction is the exchange of bacterial DNA through bacteriophages. It is of two types
(a) Generalised transduction (b) Specialised transduction
4. Yeasts
Yeasts are unicellular eukaryotes belonging to kingdom fungi.
4.1. Size: yeast cells are about 3-4 μm in diameter, although some grow upto 40 μm.
4.2. Shape & Morphology: Cell shape is ovoid or spheroid. Colonies of yeast are moist,
slimy, mealy, whitish, and also grow as a film scum. Yeast cell lacks flagella and other organ
of locomotion. The various shaped cells, with regard to the mode of their production and the
place on the mother cell, where they are formed, are named by special terms.
4.3. Cell structure
The Cell wall is thin and chitinous. The protoplasm is surrounded by cell membrane which
contains all the usual cell organelles like ribosomes, mitochondria, ER, nucleus and other
granules. Vacuole is single, large and centrally located.

4.4. Physiology, growth, and nutrition


Yeasts are chemoautotrophs and grow anaerobically (known as obligate anaerobes). They
grow in neutral or slightly acidic pH environment. They grow in varied temperature (-2 to
45℃).

4.5. Reproduction
Asexual reproduction
Yeasts generally reproduce by Asexual method such as Budding. It Occurs during abundant
supply of nutrition. The protoplasm bulges out the cell wall, the bulge grows and finally walls
off. Bud grows as a tube like projection. Replicated material divides between the cells
(mother and daughter cells).
Sexual reproduction
Under high stress conditions such as nutrient starvation, haploid cells (imperfect asexual
stage) die and diploid cells (sexual stage) undergo sporulation and enter sexual reproduction
by meiosis, producing haploid spores, which later conjugate and reform the diploid. Sexual
reproduction is carried out by true yeasts (ascomycotina) which produce ascospores. The
yeast cell serves as ascus. The usual number of spores/ ascus is a characteristic of yeast. The
ascospores may differ in color, smoothness, roughness of wall and their shape (round, oval,
bean or sickle shaped). False yeasts produce no ascospores or other sexual spores and are
known as fungi imperfectii. Cells of some yeasts become chlamydospores by the formation of
a thick wall around cell e.g. conidia.

4.6 Classification
Yeasts are classified on the basis
a) Ascospores produced: True yeasts produce ascospores and false yeasts do not produce
ascospores
b) Appearance of ascospores: Yeasts are also classified on the basis of size, shape and color
of ascospores produced
c) Appearance of vegetative cells- according to the size and shape of cells
d) Method of asexual reproduction
e) Growth as a film or throughout
f) Color of growth
g) Appearance of growth
Mycelium: when thread like growth is produced
Pseudomycelium: when cell associations are present where cells cling together in chains
h) Physiological characteristics- source of nutrition (carbon or nitrogen), Vitamin
requirement, oxidative or fermentative, or by activity (lipolysis, urease activity, acid
production).

5. Molds
5.1. Cell Structure
The protoplasm is surrounded by cell membrane which contains all the usual cell organelles
like ribosomes, mitochondria, ER, nucleus and other granules. Vacuole is single, large and
centrally located.

5.2. Morphology
Molds are microscopic, multicellular, and composed of long branching filaments called
hyphae. The whole mass of these hyphae is known as mycelium. The mycellium of molds
may appear fuzzy, white or colored. They display two distinct morphological stages: the
vegetative and reproductive. The vegetative body of molds (hyphae and mycelium) is called
thallus. Hyphae have a tubular structure, and those tubes can be divided by cross walls called
septa. Non-septate hyphae are coenocytic, so one continuous hyphal segment may contain
multitudes of nuclei. Then there are monokaryotic hyphae, when one nucleus is in one hyphal
segment or the special dikaryotic hyphae when two nuclei are in one segment.

5.3. Physiology, growth, and nutrition


Molds grow at a moisture content of 14-15% and are heterotrophs.
Temperature requirement: i) mesophiles, ii) thermophiles, iii) psychrophiles (refer above)
Most of the molds are mesophiles.
Oxygen requirement: molds are aerobic in nature
pH: molds grow at a pH of 2-8.5 but acidic pH favors growth.

5.4. Reproduction
Asexual reproduction: it involves spores which are light, small, and resistant to drying.
Three types of spores are produced.
a) Arthropores/oidia: Arthrospores are hyphal segments that are formed by the fragmentation
of hyphae.
b) Conidiospores: are formed at the tips of hyphae andare known as microconidia if they are
small and unicellular. And macroconidia if they are large and contain more than one spore.
c) Sponrangiosphores: are spherical and produced in sac-like structures containing sporangia.
Sexual reproduction:
It involves the four kinds of spores
(i) Oospores: Oomycetes produce gametangia. The oogonium contains haploid oocytes
produced by meioses. Beside the oogonium, antheridia develop and produce haploid nuclei
via meioses. These nuclei migrate to oogonia across a fertilization tube developed by the
oogonium to fertilize an oocyte. Their fusion produces diploid oospores that germinate and
produce coenocytic non-septate hyphae with many nuclei.
(ii) Zygospores: do not occur inside any kind of enclosing structure, but are produced by the
direct fusion of two hyphal protrusions from neighbouring filaments.
(iii) Ascospores: are produced within spherical to cylindrical cells called asci. Usually the
asci are produced within some kind of enclosing structure and thus are not found exposed on
the hyphae.
(iv) Basidiospores: are always produced externally on a structure called a basidium. Basidia
come in a variety of forms, but those commonly encountered on moulds will be club-shaped
and bear four or eight spores on sharp projections at the apex.
5.5. Classification
Molds are classified on the basis of the spores they produce into oomycetes (oospores)
zygomycetes (zygospores) ascomycetes (ascospores).

6. Virus
Viruses are not classified into any of the five kingdoms. They are not cellular and are
dependent on host cells for their replication. Viruses are classified in two different ways:
(i) According to their structure - genetic (DNA or RNA?) and physical (shape etc) – this
scheme is favoured by scientists doing fundamental work.
(ii) According to the type of disease they cause, this scheme is favored by medical workers
who need to correlate given viruses with given diseases.
There are five stages in virus replication:
(i) Attachment- specific attachment of the virus to the host cell, it attaches because it binds
with specific molecules on the cell wall surface of the bacterium, the same principle of
specific recognition of a binding or attachment site is found with viruses that infect animal
and other cells that do not have a cell wall, and in that case it is molecules projecting from the
target cell membrane that act as recognition sites so that the virus will attach to the cell.
(ii) Penetration - the virus (or in some cases - just its genome) enters the cell. When a cell has
a cell wall (bacteria, fungi, plant cells, algal cells) the usual strategy is for insertion of just the
genome of the virus into the cell cytoplasm, cell walls are rigid and difficult for entire virus
particles to penetrate. In animal cells, the entire virus enters.
(iii) Synthesis - viral genes are transcribed and then direct the host cell membrane to make
components of the virus. In many cases the first step is the destruction of the host cell
genome, followed by transcription and translation of viral nucleic acid that results in
generation of virus components.
(iv) Maturation - the viral components assemble into complete virus copies, this is a
thermodynamic process, it is not directly (as far as I can gather) controlled, it is just that there
is a thermodynamically favourable tendency for the different virus particle components to
associate in the correct way to form a functioning virus particle.
(v) Release - the new viruses are released, which may or may not kill (lyse – this word means
to burst in this context) the host cell. In some cases, as with cells that have cell walls, the
virus has to direct lytic processes that lyse or burst the cell by first damaging the cell wall. In
other cases, as with animal cells that lack a cell wall, there may be an immediate release of
virus particles that bursts and kills the host cell, or the virus can escape from the host cell by
forming an envelope derived from the host cell membrane, and this can be done in a way
(essentially exocytosis) that does not cause host cell destruction, at least not immediately, so
that many viruses leave the cell in this fashion.
Figure: Photograph of microbial morphology. (A) Molds: Conidial head of Penicillium sp.
showing conidiophore (stalk) and conidia. (B) Yeasts: Saccharomyces cerevisiae, some
carrying buds. (C) Rod-shaped bacteria: Bacillus sp., single and chain. (D) Spherical-shaped
bacteria: Streptococcus sp., chain. (E) Spherical-shaped bacteria: tetrads. (F) Bacillus cells
carrying spores, center and off-center. (G) Clostridium cells, some carrying terminal spore
(drumstick appearance). (H) Motile rodshaped bacterium (Clostridium sp.) showing
peretrichous flagella.
Pure Culture Techniques and Maintenance of Cultures

Many preservation techniques have been used to preserve microbial cultures. The objective
of preservation methods is to maintain the viability and genetic stability of the culture. The
techniques that have been developed and used can be divided into three categories:
All preservation methods follow an essentially similar protocol with distinct stages:
 Culture purity check
 Preparation of the ampoules (labelling, sterilizing)
 Growth of the culture
 Suspension of the cells in preservation medium
 Dispensing of cell suspension into ampoules
 Preservation (by method of choice)
 Ampoule stock storage
 Update ampoule stock records
 Ampoule recovery and testing (viability, purity, genetic stability)
Regular Subculture: Periodic transfer on fresh, sterile media can maintain microbial culture.
The culture preserved in this way is maintained by alternate cycles of active growth and
storage periods obtained by series of subcultures. Sub culturing can lead to change of
characteristics, i.e., characteristics may be lost, reduced, or intensified.
Paraffin Method: In this method sterile liquid paraffin is poured over the slant culture of
microbes and stored upright at room temperature. The layer of paraffin prevents dehydration
of the medium and ensures anaerobic conditions. It slows the metabolic activity by reduced
growth through reduced oxygen tension.
Storage in Soil: Soil storage involves inoculation of 1 ml of spore suspension into soil (that
has been autoclaved twice) and incubating at room temperature for 5-10days. This initial
growth period allows the fungus to use the available moisture and gradually to become
dormant. The bottles are then stored at refrigerator. Spraying few soil particles on a suitable
medium retrieves culture.
Storage in Silica Gel: The basic principle in this technique is quick desiccation at low
temperature, which allows the cell to remain viable for a long period. Cells are stored in silica
gel powder at low temperature for a period of 1-2 years. In this method, finely powdered,
heat sterilized and cooled silica powder is mixed with a thick suspension of cells and stored at
low temperature.
Storage at Refrigerator or Cold Room Storage Live cultures on a culture medium can be
successfully stored in refrigerators or cold rooms, when the temperature is maintained at 4ºC.
This method cannot be used for a very long time because toxic products get accumulated
which can kill the microbes.
Storage by Freezing: Thick bacterial suspensions can be frozen at a temperature of - 30ºC.
Metabolic rates are reduced by lowering the temperature and in the extreme case of storage in
liquid nitrogen at - 196ºC, are considered to be reduced to nil. Cultures can be preserved very
effectively if frozen in the presence of a cryoprotectant, which reduces damage from ice
crystals. Glycerol or dimethylsulphoxide (DMSO) are commonly used as cryoprotectants.
The simplest way to preserve a culture is to add 15% (v/v) glycerol to the culture and then to
store it at -20ºC or -80ºC in a freezer.
Storage by Freeze Drying: Freeze-drying is the most widely used technique for maintaining
bacterial cultures. Freeze drying is also called lyophilization. Freeze-drying or lyophilization
whereby the culture or serum is dried rapidly in vacuum from the frozen state largely avoids
the problem. The material is frozen by a suitable method and then dried by sublimation of the
ice. Freeze- drying is a multistage process; it begins with freezing, a temporary stop to
metabolic activity, then continues with the removal of water without thawing (sublimation),
and ends with a dried product.
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.

1. Physical methods
1.1 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.
(a) High Temperature
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.
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. For example: Autoclaving, Boiling water
Dry heat
Dry heat kills microorganisms through a process of protein oxidation rather than protein
coagulation. Examples of dry heat include: Hot air sterilization, Incineration, Pasteurization
b) 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.

1.2 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.

1.3. Osmotic Pressure


Microorganisms, in their natural environments, are constantly faced with alterations in
osmotic pressure. Water tends to flow through semipermeable membranes. 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.

1.4. Radiation
Ultraviolet Radiation
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.
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.
1.5 Chemical methods
Disinfectants, Antiseptics and Sanitizers
Disinfection is the elimination of microorganisms from inanimate objects or surfaces,
whereas decontamination is the treatment of an object or inanimate surface to make it safe to
handle. Examples: Phenol and phenol derivatives; Soaps and detergents; Alcohols; Acids and
alkalies; Heavy metals; Chlorine; Iodine and iodophores; Aldehydes; Ethylene oxide gas
(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.
Microorganisms Important In Food Microbiology

Food serves as growth medium for different kinds of microorganisms. Microorganisms enter
into food and grow as contaminants or intended additions. Growth of microorganisms in food
may spoil food quality or improve depending on the types of microorganisms and the changes
they bring about. Food is assessed for their quality in terms of physical, chemical, sensory
and microbiological characteristics. Microbiological characteristics are assessed in terms of
the microorganisms present in food, their characters, ability to change the quality, their
influence on health of consumer. It is necessary for food microbiologists to become
acquainted with the microorganisms important in food at least to the extent that will enable
them to identify the main types with their characteristics. Important microorganisms
associated with food are molds, yeasts, bacteria, viruses and some protozoa parasites.
Knowledge of general characters and primary identification methods is necessary for the
people working with food microbiology.

Molds
Molds are important in food because they can grow even in conditions in which many
bacteria cannot grow, such as low pH, low water activity (aw), and high osmotic pressure.
Many types of molds are found in foods. Growth of molds on food as fuzzy or cottony
appearance is the common observation for every one and moldy or ‘mildewed’ food are
considered as unfit to eat. Molds are concerned in spoilage of many kinds of food, but some
special molds are useful in manufacture of certain food or ingredients of food. Molds are used
in making Oriental food, such as soy sauce, tempeh, and ripening of some kinds of cheese.
Many are used to produce food additives and enzymes. Some of the most common genera of
molds found in food are listed below,
a) Aspergillus: It is widely distributed and contains many species important in food.
Members have septate hyphae and produce black-colored asexual spores on conidia.
Many are xerophilic (able to grow in low Aw) and can grow in grains, causing
spoilage. They are also involved in spoilage of foods such as jams, cured ham, nuts,
and fruits and vegetables (rot). Some species or strains produce mycotoxins (e.g.,
Aspergillus flavus produces aflatoxin). Many species or strains are also used in food
and food additive processing. Asp. oryzae is used to hydrolyze starch by α-amylase in
the production of sake. Asp. niger is used to process citric acid from sucrose and to
produce enzymes such as β-galactosidase.
b) Alternaria: Members are septate and form dark-colored spores on conidia. They
cause rot in tomatoes and rancid flavor in dairy products. Some species or strains
produce mycotoxins. Species: Alternaria tenuis.
c) Fusarium: Many types are associated with rot in citrus fruits, potatoes, and grains.
They form cottony growth and produce septate, sickle-shaped conidia. Species:
Fusarium solani.
d) Geotrichum: Members are septate and form rectangular arthrospores. They grow,
forming a yeastlike cottony, creamy colony. They establish easily in equipment and
often grow on dairy products (dairy mold). Species: Geotrichum candidum.
e) Mucor: It is widely distributed. Members have nonseptate hyphae and produce
sporangiophores. They produce cottony colonies. Some species are used in food
fermentation and as a source of enzymes. They cause spoilage of vegetables. Species:
Mucor rouxii.
f) Penicillium: It is widely distributed and contains many species. Members have
septate hyphae and form conidiophores on a blue-green, brushlike conidia head. Some
species are used in food production, such as Penicillium roquefortii and Pen.
camembertii in cheese. Many species cause fungal rot in fruits and vegetables. They
also cause spoilage of grains, breads, and meat. Some strains produce mycotoxins
(e.g., Ochratoxin A).
g) Rhizopus: Hyphae are aseptate and form sporangiophores in sporangium. They cause
spoilage of many fruits and vegetables. Rhizopus stolonifer is the common black
bread mold.
Yeasts
Yeasts are important in food because of their ability to cause spoilage. Many are also used in
food bioprocessing. Some are used to produce food additives. Most yeasts are useful in food
microbiology as fermenting organisms. Art of making breads, cakes, wines, beers and other
fermented food making use of yeasts is oldest process of food preparation. Metabolic
characters of yeasts to utilize sugars and produce variety of products in fermented food make
different food products with difference in quality and nutritive value. Generally yeasts are
more osmo-tolarant than molds and bacteria and therefore used in food fermentations.
Variety of yeast genera are usually found on fruits and bring about spoilage of fruit products.
Many yeasts are capable of attacking the sugars found in fruits and bring about fermentation
with production of alcohol and carbon dioxide. Contamination and unwanted growth of
yeasts in food leads to spoilage and reduction in shelf-life.
Several important genera are briefly described below,
a) Saccharomyces: Cells are round, oval, or elongated. It is the most important genus
and contains heterogenous groups. Saccharomyces cerevisiae variants are used in
baking for leavening bread and in alcoholic fermentation. They also cause spoilage of
food, producing alcohol and CO2.
b) Pichia: Cells are oval to cylindrical and form pellicles in beer, wine, and brine to
cause spoilage. Some are also used in oriental food fermentation. Species: Pichia
membranaefaciens.
c) Rhodotorula: They are pigment-forming yeasts and can cause discoloration of food
such as meat, fish, and sauerkraut. Species: Rhodotorula glutinis.
d) Torulopsis: Cells are spherical to oval. They cause spoilage of milk because they can
ferment lactose (e.g., Torulopsis versatilis). They also spoil fruit juice concentrates
and acid foods.
e) Candida: Many species spoil foods with high acid, salt, and sugar and form pellicles
on the surface of liquids. Some can cause rancidity in butter and dairy products (e.g.,
Candida lipolyticum).
f) Zygosaccharomyces: Cause spoilage of high-acid foods, such as sauces, ketchups,
pickles, mustards, mayonnaise, salad dressings, especially those with less acid and
less salt and sugar (e.g., Zygosaccharomyces bailii).

Bacteria
Bacteria have spectrum of metabolic reactions and produce variety of products using a wide
range of substrates. This character is very useful in preparing several food of microbial
fermentation and fermented milk products. They produce several enzymes like amylases,
proteases, lipases, etc. which are important in food industry. Bacteria have become part of
every household food preparations and maintaining good health. Lactic acid bacteria like
Lactobacillus sp are good probiotic candidates for prevention of several infectious
gastroenteritis diseases and are part of natural flora of human body. Several bacteria are the
causative agents of human diseases. Some of the important human pathogens are
Staphylococcus aureus, Streptococcus pyogenes, Clostridium botulinum, Salmonella typhi,
Shigella dysenteriae. These are also important food contaminants and agents of food borne
diseases and food poisoning. It is necessary to know about characters of such bacteria for
their growth, metabolic products and the diseases they cause. Important bacteria of food
fermentations like Lactobacillus acidophilus, L. lactis, Acetobacter sp are grown as starter
cultures and used for large scale production of fermented food products.
Lactobacillus: Rod-shaped cells that vary widely in shape and size, some are very long
whereas others are coccobacilli, appear in single or in small and large chains; facultative
anaerobes; most species are nonmotile; mesophiles (but some are psychrotrophs); can be
homo- or heterolactic fermentors. Found in plant sources, milk, meat, and feces. Many are
used in food bioprocessing (Lactobacillus delbrueckii subsp. bulgaricus, Lab. helveticus,
Lab. plantarum) and some are used as probiotics (Lab. acidophilus, Lab. reuteri, Lab. casei
subsp. casei).
Coxiella: Gram-negative; nonmotile; very small cells (0.2 x 0.5 mm); grow on host cells.
Relatively resistant to high temperature (killed by pasteurization). Coxiella burnetii causes
infection in cattle and has been implicated with Q fever in humans (especially on consuming
unpasteurized milk).
Listeria: Short rods (0.5 x 1 mm); occur singly or in short chains; motile; facultative
anaerobes; can grow at 1oC; cells removed by bactrifugation. The species are widely
distributed in the environment and have been isolated from different types of foods. Some
Listeria monocytogenes strains are important foodborne pathogens.
Staphylococcus: Spherical cells (0.5 to 1 mm); occur singly, in pairs, or clusters; nonmotile;
mesophiles; facultative anaerobes; grow in 10% NaCl. Staphylococcus aureus strains are
frequently involved in foodborne diseases. Sta. carnosus is used for processing some
fermented sausages. Main habitat is skin of humans, animals, and birds.
Lactococcus: Ovoid elongated cells (0.5 to 1.0 mm); occur in pairs or short chains;
nonmotile; facultative anaerobes; mesophiles, but can grow at 10oC; produce lactic acid.
Used to produce many bioprocessed foods, especially fermented dairy foods. Species:
Lactococcus lactis subsp. lactis and subsp. cremoris; present in raw milk and plants and
several strains produce bacteriocins, some with a relatively wide host range against Gram-
positive bacteria and have potential as food biopreservatives.
Leuconostoc: Spherical or lenticular cells; occur in pairs or chains; nonmotile; facultative
anaerobes; heterolactic fermentators; mesophiles, but some species and strains can grow at or
below 3oC. Some are used in food fermentation. Psychrotrophic strains are associated with
spoilage (gas formation) of vacuum-packaged refrigerated foods. Found in plants, meat, and
milk. Species: Leuconostoc mesenteroides subsp. mesenteroides, Leu. lactis, Leu. carnosum.
Leu. Mesenteroides subsp. dextranicum produces dextran while growing in sucrose. Several
strains produce bacteriocins, some with a wide spectrum against Gram-positive bacteria, and
these have potential as food biopreservatives.
Clostridium: Rod-shaped cells that vary widely in size and shape; motile or nonmotile;
anaerobes (some species extremely sensitive to oxygen); mesophiles or psychrotrophic; form
endospores (oval or spherical) usually at one end of the cell, some species sporulate poorly,
spores are heat resistant. Found in soil, marine sediments, sewage, decaying vegetation, and
animal and plant products. Some are pathogens and important in food (Clostridium
botulinum, Clo. perfringens) and others are important in food spoilage (Clo. tyrobutyricum,
Clo. saccharolyticum, Clo. laramie). Some species are used as sources of enzymes to
hydrolyze carbohydrates and proteins in food processing.

For convenience, bacteria important in foods have been arbitrarily divided into several
groups on the basis of similarities in certain characteristics. This grouping does not have any
taxonomic significance. Some of these groups and their importance in foods are listed here.
(a) Lactic Acid Bacteria
They are bacteria that produce relatively large quantities of lactic acid from carbohydrates.
Species mainly from genera Lactococcus, Leuconostoc, Pediococcus, Lactobacillus, and
Streptococcus thermophilus are included in this group.
(b) Acetic Acid Bacteria
They are bacteria that produce acetic acid, such as Acetobacter aceti.
(c) Propionic Acid Bacteria
They are bacteria that produce propionic acid and are used in dairy fermentation. Species
such as Propionibacterium freudenreichii are included in this group.
(d) Butyric Acid Bacteria
They are bacteria that produce butyric acid in relatively large amounts. Some Clostridium
spp. such as Clostridium butyricum are included in this group.
(e) Proteolytic Bacteria
They are bacteria that can hydrolyze proteins because they produce extracellular proteinases.
Species in genera Micrococcus, Staphylococcus, Bacillus, Clostridium, Pseudomonas,
Alteromonas, Flavobacterium, Alcaligenes, some in Enterobacteriaceae, and Brevibacterium
are included in this group.
(f) Lipolytic Bacteria
They are bacteria that are able to hydrolyze triglycerides because they produce extracellular
lipases. Species in genera Micrococcus, Staphylococcus, Pseudomonas, Alteromonas, and
Flavobacterium are included in this group.
(g) Saccharolytic Bacteria
They are bacteria that are able to hydrolyze complex carbohydrates. Species in genera
Bacillus, Clostridium, Aeromonas, Pseudomonas, and Enterobacter are included in this
group.
(h) Thermophilic Bacteria
They are bacteria that are able to grow at 50oC and above. Species from genera Bacillus,
Clostridium, Pediococcus, Streptococcus, and Lactobacillus are included in this group.
(i) Psychrotrophic Bacteria
They are bacteria that are able to grow at refrigerated temperature (≤ 5oC). Some species
from Pseudomonas, Alteromonas, Alcaligenes, Flavobacterium, Serratia, Bacillus,
Clostridium, Lactobacillus, Leuconostoc, Carnobacterium, Brochothrix, Listeria, Yersinia,
and Aeromonas are included in this group.
(j) Thermoduric Bacteria
They are bacteria that are able to survive pasteurization temperature treatment. Some species
from Micrococcus, Enterococcus, Lactobacillus, Pediococcus, Bacillus (spores), and
Clostridium (spores) are included in this group.
(k) Halotolerant Bacteria
They are bacteria that are able to survive high salt concentrations (≥ 10%). Some species
from Bacillus, Micrococcus, Staphylococcus, Pediococcus, Vibrio, and Corynebacterium are
included in this group.
(l) Aciduric Bacteria
They are bacteria that are able to survive at low pH (<4.0). Some species from Lactobacillus,
Pediococcus, Lactococcus, Enterococcus, and Streptococcus are included in this group.
(m) Osmophilic Bacteria
They are bacteria that can grow at a relatively higher osmotic environment than that needed
for other bacteria. Some species from genera Staphylococcus, Leuconostoc, and Lactobacillus
are included in this group. They are much less osmophilic than yeasts and molds.
(n) Gas-Producing Bacteria
They are bacteria that produce gas (CO2, H2, H2S) during metabolism of nutrients. Species
from genera Leuconostoc, Lactobacillus, Propionibacterium, Escherichia, Enterobacter,
Clostridium, and Desulfotomaculum are included in this group.
(o) Slime Producers
They are bacteria that produce slime because they synthesise polysaccharides. Some species
or strains from Xanthomonas, Leuconostoc, Alcaligenes, Enterobacter, Lactococcus, and
Lactobacillus are included in this group.
(p) Spore Formers
They are bacteria having the ability to produce spores. Species from Bacillus, Clostridium,
and Desulfotomaculum are included in this group. They are further divided into aerobic
sporeformers, anaerobic sporeformers, flat sour sporeformers, thermophilic sporeformers, and
sulfide-producing sporeformers.
(q) Aerobes
They are bacteria that require oxygen for growth and multiplication. Species from
Pseudomonas, Bacillus, and Flavobacterium are included in this group.
(r) Anaerobes
They are bacteria that cannot grow in the presence of oxygen. Species from Clostridium are
included in this group.
(s) Facultative Anaerobes
They are bacteria that are able to grow in both the presence and absence of oxygen.
Lactobacillus, Pediococcus, Leuconostoc, enteric pathogens, and some species of Bacillus,
Serratia, and coliforms are included in this group.
(t) Coliforms
Species from Escherichia, Enterobacter, Citrobacter, and Klebsiella are included in this
group. They are used as an index of sanitation.
(u) Fecal Coliforms
Mainly Escherichia coli is included in this group. They are also used as an index of
sanitation.
(v) Enteric Pathogens
Pathogenic Salmonella, Shigella, Campylobacter, Yersinia, Escherichia, Vibrio, Listeria,
hepatitis A, and others that can cause gastrointestinal infection are included in this group.

Protozoa
These organisms may be free living in marine and fresh water and in soil or they can be
symbionts in or on living hosts. Some protozoa are pathogenic to higher animals, like
Entamoeba histolytica, Plasmodium sp. Trepanosoma sp., Trichomonas sp. Some of these are
obligate parasites like Entamoeba and cause food or waterborne diseases frequently. Under
developed countries face the challenge of diseases like amoebiasis, malaria, sleeping sickness
caused by Entamoeba, Plasmodium, Trepanosoma. Diverse forms of protozoa are classified
based on the structural features of cells as seen under high magnification microscopes. The
general groups of protozoa are flagellates, amoebas, sporozoa and ciliates. Protozoa are
observed under light compound microscopes and identified based on the morphology and
ecological characteristics.

Viruses
Viruses are considered as borderline between living and non-living. They behave as non-
living in the absence of suitable host cell and as living in suitable host cell. Being obligate
parasites, viruses do not grow on culture media as do bacteria and fungi and therefore do not
grow in food. Since they do not replicate in food, their numbers may be expected to be low
relative to bacteria and therefore extraction and concentration methods are necessary for their
study in food. Viruses are important in food for three reasons. Some are able to cause enteric
disease, and thus, if present in a food, can cause foodborne diseases. Hepatitis A and
Norwalk-like viruses have been implicated in foodborne outbreaks. Several other enteric
viruses, such as poliovirus, echo virus, and Coxsackie virus, can cause foodborne diseases. In
some countries where the level of sanitation is not very high, they can contaminate foods and
cause disease. Some bacterial viruses (bacteriophages) are used to identify some pathogens
(Salmonella spp., Staphylococcus aureus strains) on the basis of the sensitivity of the cells to
a series of bacteriophages at appropriate dilutions. Some bacteriophages are used to transfer
genetic traits in some bacterial species or strains by a process called transduction (e.g., in
Escherichia coli or Lactococcus lactis). Laboratory identification of viruses in food is not
much practiced. Most of the food borne viral diseases are caused by the presence of viruses
as contaminants in food and their consumption. Viral gastroenteritis is the common disease
caused in people consuming food and water contaminated by viruses. Some of the common
food borne viral pathogens are picornaviruses (polio viruses), reoviruses (rotaviruses),
hepatitis A virus, parvoviruses (human gastrointestinal viruses), papovaviruses (human BK
and JC viruses) and adenoviruses (human adenoviruses). It is necessary to detect and
eliminate viruses contaminated in food for safety, particularly in meat and meat products.