Morphology of bacteria

Size of a Bacterial Cell:

There are great variations in size of bacteria. They measure from 0.5 µ to 1.5 µ but on an average each cell of
bacterium measures about 1.25 µ to 2 µ in diameter. Bacteria are so minute that a single drop of water may
contain about billions of bacteria.

Shape and Arrangement of Bacterial Cell:

Bacterial cells exhibit greater variation in their shape but usually four conventional shapes of cell have been
recognized (Fig. 12.1).

1. Cocci:
Simplest form of bacteria in which bacterium appears
like a spherical cell.
(i) Monococcus: When bacterium appears singly.
(ii) Diplococci: When they appear in pairs of cells.
(iii) Streptococci: When they appear in chain form.
(iv) Tetrad: Arranged in square of four.
(v) Sarcinae: When arranged in cuboidal or in different
geometrical or packet arrangement.
(vi) Staphylococci: Arranged in irregular clusters like
bunch of grapes.

2. Rod Shaped:
They are also called bacilli and are commonest in
microbial world.
They are of two kinds:
(i) Coccobacilli: Very short rods, occurring mostly
singly.
(ii) Long rods: Cylindrical shape, are known as Bacilli or
rods- occurring singly (Bacillus)
- in pairs (Diplobacilli)
- in chains (Streptobacilli)
- arranged by sides (Palisades)

3. Vibrios: They are curved rods or comma shaped,

their curvature is always less than a half turn.

4. Spiral bacteria: They are curved or spiral shaped

cells, their curvature exceeds that of a half turn. They
may be classified:
- Spirilla : one twist
- Spirochetes : multiple twisting

In addition there are:

• Star shaped cells (genus Stella)
• Square flat cells (halophilic archaebacteria)
• Triangular cells (Haloarcula)
• Branching mycelium – like filaments (Streptomyces)
Bacteria are mostly monomorphic (single shape), but some have many shapes which is known as
pleomorphic (Rhizobium and Corynebacterium)
 Ultra-Structure of Bacterial Cell
Examination of bacterial cells with electron microscope reveals various component structures. Some of these
are outside the cell membrane; others are internal to cell membrane (Figure).

Figure: Structure of bacterial cell

Structures Outside the Cell Membrane:

1. Capsule:
Some prokaryotic organisms secrete slimy or gumy materials (exopolymers) on their surface. A variety of
these structures consist of polysaccharides and a few consist proteins. The more general term glycocalyx is
also used.These layers may be thick, or thin, and rigid or flexible, depending on their chemical nature in
specific organism. The rigid layers are organized in a tight matrix that excludes Indian ink, this form is
referred to Capsule. If the substance is unorganized and only loosely attached to the cell wall it is referred as
Slime layer.
Functions of Capsule:
• The encapsulated strains of the bacterium are protected from phagocytosis and take part in virulence.
• It gives protection to the cell from desiccation under natural condition because outer polysaccharide
layer bounds a significant amount of water.
• It helps the bacteria to attach the surface of solid objects, in aqueous medium or to tissue surface of
animals and plants. Streptococcus mutans, a bacterium associated with the caries of teeth, is attached to
the dental surface with the help of glycocalyx, consists of water-insoluble polymer, glucan.

2. Flagella:
Most motile prokaryotes move by use of flagella thread like locomotor appendages, extending outwards
from the cell membrane and cell wall.
Bacterial flagella are slender, rigid structures, about 20 nm across and up-to 15 to 20 µ m long. Flagella are so
thin that they cannot be observed directly with bright field microscope hence can be observed by staining.
On the basis of arrangement of flagella, the bacteria are categorised into the following types (Fig. 2.16):
The number and arrangement of flagella on a cell are useful for identification and classification of bacteria.
(a) Monotrichous (Fig. 2.16B). Single flagellum at one pole of the cell, e.g., Vibrio cholerae.
(b) Amphitrichous (Fig. 2.16C). Each single flagellum is attached at both ends, e.g., Alkaligenes faecalis,
Nitrosomonas.
(c) Cephalotrichous (Fig. 2.16D). Two or more flagella at one end only, e.g., Pseudomonas fluorescens.
(d) Lophotrichous (Fig. 2.16E). A tuft of flagella at both ends, e.g., Spirillum volutans.
(e) Peritrichous (Fig. 2.16F). Numerous flagella are distributed all over the surface of the cell e.g., Bacillus
typhosus, Clostridium.
 Structure: flagellar apparatus is made up of three distinct regions:
(1) Filament- The outermost region is filament which is extended from the cell surface to tips.
(2) Basal body - consist of small central rods inserted into cell membrane
(3) Hook- A short curved segment that links the filament to basal bodies and acts as flexible coupling.

The filament is a hollow, rigid cylinder made up of protein subunits flagellin. Hook and basal bodies are quite
different from filaments. Basal body is more complex part of flagellum. In E. coli and most Gram-negative
bacteria, basal bodies bear 2 pairs of ring, outer pair (L and P ring) is situated at the level of outer membrane
and inner pair (S and M ring) is located near the level of cell membrane. The outer L and P ring associates
with lipopolysaccharides and peptidoglycan layer respectively. Inner M ring contacts the plasma membrane
while S ring lies just above attached to inner surface of peptidoglycon. Flageila of Gram-positive bacteria
have only lower S and M ring.
3. Fimbriae or Pili:
Some bacteria mostly (Gram negative bacilli) contain, non-flagellar, extremely fine appendages called
fimbrial or pile. The filament of pilus is straight and diameter is 7 nm. It is made up of pilin protein.
Pili are nonmotile but adhesive structure.
They enable the bacteria to stick firmly to other bacteria, to a surface or to some eukaryotic such as mould
plants, plants and animal cells including R.B.C and epithelial cells of elementary, respiratory and urinary
tracts.
Pilli help in conjugation (e.q. F-pili or Sex pili) of male bacteria for the exchange of genetic information, in the
attachment of pathogenic bacteria to their host cell.
4. Structure of Cell Membrane:
Cell membrane is a thin structure that completely surrounds the cell. This structure is a barrier separating the
inside of cell from environment. It is also highly selective barrier enabling the cell to concentrate a specific
metabolite and excrete waste material.
Mostly biological membrane is composed primarily of Phospholipids (about 20 to 30 percent) and proteins
(about 60 to 70 percent). The phospholipids form a bilayer in which most of the proteins are strongly held
(Integral proteins). Other proteins known as Peripheral proteins are only loosely attached and are present
on the periphery. The lipid matrix of membrane has fluidity, allowing the components to move around
laterally. (Fig. 12.5).
 Sterols are absent from membranes of all prokaryotic cells. A significant difference exists between the
phospholipids of eubacteria and those of archaebacteria.
Functions
(i) Transport:
(a) Active: involved in the active transport of selective nutrients. It is impermeable to ionised substances
and macromolecules.
(b) Passive: The passive transport of fat soluble micromolecular solutes takes place by diffusion.
(ii) Energy production: It is the site of electron flow in both respiration and photosynthesis leading to
phosphorylation and, therefore, the membrane is the site of carriers and enzymes in these reactions.
(iii) Polymer production: Cell membrane is the site of polymerising enzymes necessary for synthesis of cell
wall.
5. Cell Envelope of Prokaryote:
Bacteria can be divided into two major groups called Gram positive bacteria and Gram negative bacteria,
based on Gram stain. Gram positive bacteria and Gram negative bacteria differ in the appearance of cell wall.
The cell wall of Gram negative bacteria is multilayered structure and quite complex whereas Gram positive
bacteria contain primarily single type of molecule and is often much thicker (Fig. 12.7).

Bacterial Cell Wall:

In the cell wall of bacteria, there is one rigid layer that is primarily responsible for strength of the wall. In
Gram negative bacteria addition layer is present outside this rigid layer and is known as Lipopolysaccharide
(LPS). The rigid layer of both Gram negative bacteria and Gram positive bacteria is very similar in chemical
composition and is called Peptidoglycan (or Murein).
A. Peptidoglycan: This layer is thin sheet composed of
the
he polysaccharide chain consisting of alternate
residues of the amino acid N-acetyl acetyl glucosamine
(NAG) and N-acetyl
acetyl muramic acid (NAM) linked
link in β-1,
4 glycosidie linkage and small number of amino acids
consisting of L-alanine, D-alanine,
alanine, D-Glutamic
D acid
and either L-lysine or meso-diaminopalmilic
diaminopalmilic acid acid.
Linear glycan (Polysaccharide) Chains connected through
throug
short tetrapeptide and pentaglycine
ycine bridge.
Tetra peptide is unusual
sual in that it contains D-amino
D acid,
which is rarely found in protein. Neighbouring
tetrapeptides are linked by pentaglycine bridge peptide,
each consisting of five glycine residues. Pentaglycine
bridge peptide links L-lysine
lysine of one tetrapeptide with the
terminal D-alanine
alanine of its neighbour. Because of extensive
cross linking the cell wall has a rigid structure frame work.
Peptidoglycan is present only in bacteria.

B. Teichoic Acids:
Gram positive bacteria have acidic polysaccharide called teichoic acids attached to their cell wall. The term
teichoic acid includes polymer of Glycerolphosphate
G or Ribitol phosphate residues.
es. These poly-alcohols
poly are
connected
ted by phosphate ester bond to NAM residues.
Teicohic acids are negatively charged and may function to effect passage of ions through the cell wall.
Certain Glycerol containing acids are bound to membrane lipids and are called lipo-teichoic
teichoic acid.
acid

Pseudopeptidoglycan and other Cell Walls of Archaebacteria:

Certain Archae contain cell walls constructed of a polysaccharide very similar to peptidoglycan.
peptidoglycan This material
is called Pseudo peptidoglycan (pseudomurein).
(pseudomurein). The backbone of Pseudopeptidoglycan is composed of
alternating repeats of N-acetyl glucosamine and N-acetyl-talosaminuric acid.
 Cell walls of other Archae lack both Peptidoglycan and Pseudopeptidoglycan and consist of Polysaccharide,
glycoprotein or protein.
The archaeal cell wall functions to prevent osmotic lysis and to define cell shape. Due to absence of
peptidoglycon in cell wall, all archae are naturally resistant to action of lysozyme.

C. Outer Membrane of Gram Negative Bacteria:

Gram negative bacteria outer membrane is covered by lipopolysaccharide.
The lipopolysaccharide consists of 3 regions- (a) Lipid A- complex polysaccharide covalently linked to fatty
acids, (b) Core polysaccharide and (c) O-polysaccharide.
The core polysaccharides consist of glucose, NAG and unusual sugars like ketodeoxyoctonate (KDO), seven
carbon sugar (heptose). Connected to the core is the O-polysaccharide which usually contains galactose,
Rhamnose, mannose (six carbon) and dideoxy sugar abequose. These sugars are connected in repeating
sequence (Fig 12.15) .

In outer membrane, the LPS associated with various proteins to form outer half of unit membrane sheet. A
lipoprotein complex is found on inner side of the outer membrane of Gram negative bacteria, lipoprotein is
small protein that functions as an anchor between the outer membrane and peptidoglycan.
Porins: These proteins are found in Outer membrane of Gram negative bacteria which makes it permeable to
small molecules and function as channels for the entrance and exit of hydrophilic low mol. wt. substance.

6. Periplasm:
Space between the outer surface of the cytoplasmic membrane and inner surface of the LPS containing outer
membrane is called Periplasm. It is gel like in consistency, because of the abundance of periplasmic protein.
Periplasm of gram negative bacteria several proteins including hydrolytic enzyme which functions in initial
degradation of food, binding protein which being the process of transporting substrate and chemoreceptors,
which are protein involved in chemotaxis.

Structure inside the bacterial cell:

1. Cytoplasm:
The cytoplasm is a colloidal system containing both organic and inorganic substances. It contains genetic
material, many ribosomes, few mesosomes, some inclusions or vacuoles. It is 80% water and 20% proteins.
2. Ribosome:
It is 70S type (S = sedimentation coefficient) and complex structure having two subunits- 50S and 30S. They
are the sites of protein synthesis.
3. Mesosomes (Chondroids):
These are convoluted multi-laminated localised infoldings of the cytoplasmic membrane into the cytoplasm
(Fig. 2.12). Their number is usually 2-4, but often found to be more in cells with high respiratory activity, e.g.,
Nitrosomonas. It serves to accommodate more spaces for respiration. In photosynthetic bacteria
(Rhodopseudomonas), they are the site of photosynthetic pigments.
4. Genetic Material of Bacteria:
The genetic material is present both in nucleoid and plasmid (Fig. 2.13).
The bacterial nuclear body is devoid of nuclear membrane, nucleolus and nuclear sap and is known as
Nucleoid. The nucleoid is composed of a double stranded circular DNA. When straightened the DNA
measures 1000 µm. It is devoid of any basic histone protein. The DNA is haploid and it undergoes semi-
conservative replication by simple fission and maintains genetic characteristics.

Plasmid: Bacterial cytoplasm may contain some extra-chromosomal genetic material which is called plasmid
or episomes. Lederberg (1952) termed as plasmid those extragenophoral genetic materials.
Plasmids are small ring-like double stranded DNA molecules which may contain about 100 genes. The
replication of plasmid seems self-controlled. They contain different non-essential characters. Based on host
properties, the plasmids are classified into different types.
These are:
(i) “F-factor” for fertility
(ii) “R-factor”—for antibiotic resistance
(iii) Tumor inducing plasmid (e.g., Agrobacterium tumifaciens)
(iv) Pathogenecity to mammals

5. Chromatophores:
These are the pigment- bearing structures, found in photosynthetic bacteria. They are found in different
forms such as membranes, vesicles, tubes, bundle tubes etc. or as thylakoids.
6. Inclusion bodies (Vacuoles):
Inclusion bodies may be found free in the cytoplasm or bound to membrane.
These are the sources of stored energy- starch or glycogen (polysaccharide) granules, lipid granules
(polyhydroxybutyrate), volutin (polyphosphate), nitrogen (cyanophycin granules) and sulphur granules.
7. Endospores:
Endospores are a survival mechanism. They are triggered to form during adverse environmental
conditions. Only one cell gives rise to one spore.
Endospores are resistant to: heat (withstand boiling for over one hour), desiccation, UV radiation and
chemical disinfectants. The resistance of these spores has serious consequence and some very
pathogenic bacteria have the ability of produce such spores.
 Classification of Bacteria

A. on the basis of mode of nutrition

B. on the basis of optimum temperature requirement for growth
C. on the basis of optimal pH for growth
D. on the basis of salt concentration
E. on the basis of gaseous requirement
F. on the basis of morphology
G. on the basis of gram staining
H. on the basis of flagella
I. on the basis of spore

A. Classification of bacteria on the basis of mode of nutrition

1. Phototrophs: Those bacteria which gain energy from light
Phototrophs are further divided into two groups on the basis of source of electron.
Photolithotrophs: these bacteria gain energy from light and uses reduced inorganic
compounds such as H2S as electron source. Eg. Chromatium okenii
Photoorganotrophs: these bacteria gain energy from light and uses organic
compounds such as succinate as electron source.
2. Chemotrophs: Those bacteria gain energy from chemical compounds
They cannot carry out photosynthesis
Chemotrophs are further divided into two groups on the basis of source of electron.
Chemolithotrophs: they gain energy from oxidation of chemical compound and
reduce inorganic compounds such as NH3 as electron source. Eg. Nitrosomonas
Chemoorganotrophs: they gain energy from chemical compounds and uses organic
compound such as glucose and amino acids as source of electron. eg. Pseudomonas
pseudoflava
3. Autotrophs: Those bacteria which uses carbon dioxide as sole source of carbon to prepare its
own food.
Autotrophs are divided into two types on the basis of energy utilized to assimilate carbon
dioxide. ex. Photoautotrophs and chemoautotrophs
Photoautotrophs: they utilized light to assimilate CO2. They are further divided into
two groups on the basis of electron sources. Ex. Photolithotropic autotrophs and
Photoorganotropic autotrophs
Chemoautotrophs: they utilize chemical energy for assimilation of CO2
4. Heterotrophs: Those bacteria which uses organic compound as carbon source
They lack the ability to fix CO2
Most of the human pathogenic bacteria are heterotropic in nature
Some heterotrophs are simple, because they have simple nutritional requirement.
However there are some bacteria that require special nutrients for their growth; known
as fastidious heterotrophs.

B. Classification of bacteria on the basis of optimum temperature of growth

1. Psychrophiles: Bacteria that can grow at 0°C or below but the optimum temperature of
growth is 15 °C or below and maximum temperature is 20°C are called psychrophiles
Psychrophiles have polyunsaturated fatty acids in their cell membrane which gives fluid
nature to the cell membrane even at lower temperature.
Examples: Vibrio psychroerythrus, Vibrio marinus, Polaromonas vaculata, Psychroflexus
2. Psychrotrophs (facultative psychrophiles): Those bacteria that can grow even at 0°C but
optimum temperature for growth is (20-30)°C
3. Mesophiles: Those bacteria that can grow best between (25-40)oC but optimum
temperature for growth is 37oC
Most of the human pathogens are mesophilic in nature
Examples: E.coli, Salmonella, Klebsiella, Staphylococci
4. Thermophiles: Those bacteria that can best grow above 45oC.
Thermophiles capable of growing in mesophilic range are called facultative thermophiles.
True thermophiles are called as Stenothermophiles, they are obligate thermophiles.
Thermophiles contain saturated fatty acids in their cell membrane so their cell membrane
does not become too fluid even at higher temperature.
Examples: Streptococcus thermophiles, Bacillus stearothermophilus, Thermus aquaticus,
5. Hypethermophiles: Those bacteria that have optimum temperature of growth above 80oC.
Mostly Archeabacteria are hyperthermophiles.
Monolayer cell membrane of Archeobacteria is more resistant to heat and they adopt to
grow in higher remperature.
Examples: Thermodesulfobacterium, Aquifex, Pyrolobus fumari, Thermotoga

C. Classification of bacteria on the basis of optimum pH of growth

1. Acidophiles: Those bacteria that grows best at acidic pH
The cytoplasm of these bacteria is acidic in nature.
Some acidopiles are thermophilic in nature, such bacteria are called Thermoacidophiles.
Examples: Thiobacillus thioxidans, Thiobacillus ferroxidans, Thermoplasma, Sulfolobus
2. Alkaliphiles: Those bacteria that grows best at alkaline pH; optimum pH of growth is 8.2
Example: Vibrio cholerae
3. Neutriphiles: Those bacteria that grows best at neutral pH (6.5-7.5)
Most of the bacteria grow at neutral pH
Example: E. coli

D. Classification of bacteria on the basis of salt requirement

1. Halophiles: Those bacteria that require high concentration of NaCl for growth.
Cell membrane of halophilic bacteria is made up of glycoprotein with high content of
negatively (-ve) charged glutamic acid and aspartic acids. So high concentration of Na+
ion concentration is required to shield the negative charge.
Example: Archeobacteria, Halobacterium, Halococcus
2. Halotolerant: Most of the bacteria do not require NaCl but can tolerate low concentration
of NaCl in growth media are called halotolerant.

E. Classification of bacteria on the basis of gaseous requirement

1. Obligate aerobes: Those bacteria that require oxygen and cannot grow in the absence of O2.
These bacteria carryout only oxidative type of metabolism.
Examples; Mycobacterium, Bacillus
2. Facultative anaerobes: Those bacteria that do not require O2 but can use it if available.
Growth of these bacteria become better in presence of O2
These bacteria carryout both oxidative and fermentative type of metabolism
Examples: coli, Klebsiella, Salmonella
3. Aerotolerant anaerobes: Those bacteria do not require O2 for growth but can tolerate the
presence of O2.
Growth of these bacteria is not affected by the presence of O2.
These bacteria have only fermentative type of metabolism.
Example: lactobacillus
4. Microaerophiles: Those bacteria that do not require O2 for growth but can tolerate low
concentration of O2.
At atmospheric level of Oxygen growth of these bacteria is inhibited.
These bacteria only have oxidative type of metabolism
Example: Campylobacter
5. Obligate anaerobes: Those bacteria that can grow only in absence of Oxygen.
Oxygen is harmful to obligate anaerobes
These bacteria have only fermentative type of metabolism
Examples: Peptococcus, Peptostreptococcus, Slostridium, Methanococcus
6. Capnophiles: Those bacteria that require carbon dioxide for growth.
They are CO2 loving organism
Most of the microaerophiles are capnophilic in nature.
Example: Campylobacter, Helicobacter pylori, Brucella abortus

F. Classification of bacteria on the basis of Morphology

1. Coccus:
These bacteria are spherical or oval in shape
On the basis of arrangement, cocci are further classified as-
i) Diplococcus: coccus in pair. Eg, Neissseria gonorrhoae, Pneumococcus
ii) Streptococcus: coccus in chain. Eg. Streptococcus salivarius
iii) Staphylococcus: coccus in bunch. Eg. Staphylococcus aureus
iv) Tetrad: coccus in group of four.
v) Sarcina: coccus in cubical arrangement of cell. Eg. Sporosarcina
2. Bacilli:
These are rod shaped bacteria
On the basis of arrangement, bacilli are further classified as-
i) Coccobacilli: Eg. Brucella
ii) Streptobacilli: chain of rod shape bacteria: Eg. Bacillus subtilis,
iii) Comma shaped: Eg. Vibrio cholarae
iv) Chinese letter shaped: Corynebacterium dephtherae
3. Mycoplasma
They are cell wall lacking bacteria
Also known as PPLO (Pleuropneumonia like organism)
Mycoplasma pneumoniae
4. Spirochaetes: They are spiral shaped bacteria. Ex. Spirochaetes
5. Rickettsiae and Chlamydiae; They are obligate intracellular parasites resemble more closely
to viruses than bacteria
6. Actinomycetes: They have filamentous or branching structure
They resemble more closely to Fungi than bacteria. Example: Streptomyces

G. Classification of bacteria on the basis of Gram staining

1. Gram positive bacteria:
cell wall of these bacteria is composed of peptidoglycan layer only.
Eg. Staphylococcus, Streptococcus, Micrococcus
2. Gram negative bacteria:
cell wall of these bacteria is composed of Peptidoglycan and outer membrane.
Eg. E. coli, Salmonella
H. Classification of bacteria on the basis of Flagella
1. Monotrichous bacteria: bacteria having single flagella in one end of cell. eg. Vibrio cholera,
Pseudomonas aerogenosa
2. Amphitrichous bacteria: bacteria having flagella at both ends of cell. eg. Alkaligenes faecalis
3. Lophotrichous bacteria: bacteria having bundle of flagella in one end of cell.
eg. Pseudomanas fluroscence
4. Peritrichous bacteria: bacteria having flagella all over the cell surface. Eg. E.coli,
Salmonella, Klebsiella
5. Atrichous bacteria: bacteria without flagella. Eg. Shigella

I. Classification of bacteria on the basis of Spore

1. Spore forming bacteria: Those bacteria that produce spore during unfavourable condition.
These are further divided into two groups:
i) Endospore forming bacteria: Spore produced within the bacterial cell. Bacillus, Clostridium,
Sporosarcina etc
ii) Exospore forming bacteria: Spore produced outside the cell. Methylosinus
2. Non spore forming bacteria: those bacteria which do not produce spore. E. coli, Salmonella

Classification of bacteria in Bergey’s Manual of Determinative Bacteriology

1. Gram positive eubacteria that have cell walls: Gram positive rods
Gram positive cocci
2. Gram negative eubacteria that have cell walls: Gram negative rods
Gram negative cocci
3. Cell wall-less eubacteria: Mycoplasma
4. Pseudomurein: Archaebacteria
 Culture media

Culture media contains nutrients and physical growth parameters necessary for microbial growth.
All microorganisms cannot grow in a single culture medium and in fact many can’t grow in any
known culture medium.
Organisms that cannot grow in artificial culture medium are known as obligate parasites.
Mycobacterium leprae, rickettsias, Chlamydias, and Treponema pallidum are obligate parasites.
Bacterial culture media can be distinguished on the basis of composition, consistency and
purpose.

Classification of culture on the basis of consistency-

1. Solid medium- Solid medium contains agar at a concentration of 1.5-2.0% which is an inert
solidifying agent. Solid medium has physical structure and allows bacteria to grow in physically
informative or useful ways (e.g. as colonies or in streaks). Solid medium is useful for isolating
bacteria or for determining the colony characteristics of the isolate.
2. Semisolid media- They are prepared with agar at concentrations of 0.5% or less. They have soft
custard like consistency and are useful for the cultivation of microaerophilic bacteria or for
determination of bacterial motility.
3. Liquid (Broth) medium- These media contains specific amounts of nutrients but don’t have agar.
Broth medium serves various purposes such as propagation of large number of organisms,
fermentation studies, and various other tests. e.g. sugar fermentation tests, MR-VP broth.

Classification of culture media based on the basis of composition-

1. Synthetic or chemically defined medium- A chemically defined medium is one prepared from
purified ingredients and therefore whose exact composition is known. Synthetic medium may be
simple or complex depending up on the supplement incorporated in it.
2. Non synthetic or chemically undefined medium- Non-synthetic medium contains at least one
component that is neither purified nor completely characterized nor even completely consistent
from batch to batch. Often these are partially digested proteins from various organism sources.
Nutrient broth, for example, is derived from cultures of yeasts. A simple non-synthetic medium is
capable of meeting the nutrient requirements of organisms requiring relatively few growth factors
where as complex non-synthetic medium support the growth of more fastidious microorganisms.

Classification of Bacterial Culture Media based on the basis of purpose-

Many special purpose media are needed to facilitate recognition, enumeration, and isolation of
certain types of bacteria. To meet these needs, numerous media are available.
1. General purpose media/ Basic media- Basal media are basically simple media that supports
most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar are considered as
basal medium. These media are generally used for the primary isolation of microorganisms. ex.
Nutrient Agar Medium.
2. Enriched medium (Added growth factors)- Addition of extra nutrients in the form of blood,
serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to
grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum
slope etc are few of the enriched media. Blood agar is prepared by adding 5-10% (by volume)
blood to a blood agar base. Chocolate agar is also known as heated blood agar or lysed blood
agar.
 3. Selective and enrichment media are designed to inhibit unwanted commensal or
contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective
media are agar based, enrichment media are liquid in consistency. Both these media serve the
same purpose. Any agar media can be made selective by addition of certain inhibitory agents that
don’t affect the pathogen of interest. Various approaches to make a medium selective include
addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these.
a. Selective medium - Selective medium is designed to suppress the growth of some
microorganisms while allowing the growth of others. Selective medium are agar based (solid)
medium so that individual colonies may be isolated.
Examples of selective media include:
1. Thayer Martin Agar used to recover N.gonorrhoeae. It contains antibiotics; vancomycin,
colistin and nystatin.
2. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus. It contain 10% NaCl.
3. MacConkey’s Agar used for Enterobacteriaceae members. It contains bile salt that inhibits
most gram positive bacteria.
4. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating
malachite green.
5. Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of dye
brilliant green.
6. Selective media such as TCBS Agar used for isolating V.cholerae from fecal specimens have
elevated pH (8.5-8.6), which inhibits most other bacteria.
b. Enrichment culture medium - Enrichment medium is used to increase the relative
concentration of certain microorganisms in the culture prior to plating on solid selective medium.
Unlike selective media, enrichment culture is typically used as broth medium. Selenite F broth,
tetrathionate broth and alkaline peptone water (APW) are used to recover pathogens from fecal
specimens.
4. Differential/ indicator medium: differential appearance- Certain media are designed in such a
way that different bacteria can be recognized on the basis of their colony colour. Various
approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that
utilize them appear as differently coloured colonies. Such media are called differential media or
indicator media. Differential media allow the growth of more than one microorganism of interest
but with morphologically distinguishable colonies.
Examples of differential media include:
1. Mannitol salts agar (mannitol fermentation = yellow)
2. Blood agar (differentiates between bacteria exhibiting various kinds of hemolysis i.e. α, β and γ
hemolysis)
3. Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter produces
pale or colorless colonies)
4. TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)
5. Transport media- Clinical specimens must be transported to the laboratory immediately after
collection to prevent overgrowth of contaminating organisms or commensals. This can be
achieved by using transport media. Such media prevent drying (desiccation) of specimen,
maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of
these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to
neutralize inhibitory factors.
• Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to
transport feces from suspected cholera patients.
• Pike’s medium is used to transport streptococci from throat specimens.
6. Anaerobic media- Anaerobic bacteria need special media for growth because they need low
oxygen content, reduced oxidation–reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such
media may also have to be reduced by physical or chemical means. Boiling the medium serves to
expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05%
cysteine or red hot iron filings can render a medium reduced. Before use the medium must be
boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin.
Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in
the medium. Under reduced condition, methylene blue is colorless.
7. Assay media- These media are used for the assay of vitamins, amino acids and antibiotics. E.g.
antibiotic assay media are used for determining antibiotic potency by the microbiological assay
technique.

Methods of Preservation of Microbial Cultures

Agar Slant Cultures (Refrigeration):

All microbiology laboratories preserve micro-organisms on agar slant. Pure cultures can be
successfully stored at 0-4°C either in refrigerators or in cold-rooms. This method is applied for
short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities
of the microorganisms are greatly slowed down but not stopped.
Thus their growth continues slowly, nutrients are utilized and waste products released in medium.
This results in, finally, the death of the microbes after sometime. These cultures are periodically
transferred to fresh media. Time intervals at which the transfers are made, varies with the origin
and condition of growth.
Agar Slant Culture Covered with Oil (Parafin Method):
This is a simple and most economical method of preserving bacteria and fungi where they remain
viable for several years at room temperature. The layer of paraffin prevents dehydration of the
medium and by ensuring an aerobic condition, the microorganism remain in dormant state.
Therefore, the culture is preserved for several years.
The agar slants are inoculated and incubated until good growth appears. They are then covered
with sterile mineral oil to a depth of 1 cm above the tip of slant surface. Transfers are made by
removing a loop full of the growth, touching the loop to the glass surface to drain off excess oil,
inoculating a fresh medium and then preserving the initial stock culture.
Saline Suspension:
Sodium chloride in high concentration is frequently an inhibitor of bacterial growth. Bacteria are
suspended in 1% salt solution in screw cap tubes to prevent evaporation. The tubes are stored at
room temperature. Whenever needed the transfer is made on agar slant.
Preservation at Very Low Temperature (Cryopreservation):
Cryopreservation (i.e., freezing in liquid nitrogen at-196°C) helps survival of pure cultures for long
storage times. In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen
at -196°C in the presence of stabilizing agents such as glycerol, that prevent the formation of ice
crystals and promote cell survival. The organisms are suspended in nutrient broth containing 15%
glycerol. The suspension is frozen and stored at -15°C to -30°C in sealed ampoules. The frozen
cultures are kept in liquid nitrogen refrigerator.
Preservation by Drying in Vacuum:
The organisms are dried over calcium chloride in vacuum and are stored in the refrigerator.
Preservation by Freeze Drying (Lyophilization):
In this process the microbial suspension is placed in small vials. A thin film is frozen over the inside
surface of the vial by rotating it in mixture of dry ice (solid carbon dioxide) and alcohol, or acetone
at a temperature of −78oC .The vials are immediately connected to a high vacuum line. This dries
the organism while still frozen. Finally, the ampules are sealed off in a vacuum with small flame.
To revive microbial cultures it is merely necessary to break open the vial aseptically, add a suitable
stale medium, and after incubation make further transfers. The process permits the maintenance
of longer number of culture without variation in characteristics of the culture and greatly reduces
the danger of contamination.
 CULTURE TECHNIQUES
Pure Culture: Population of cells arising from a single bacterial cell, to study characteristics in detail.
Pure Culture Technique- Developed by Robert Koch. In natural habitat i.e., clinical sample, bacteria of interest
usually grow in complex mixed population. It is required to separate the bacteria of interest from the mixed
population, as pure culture to study the characteristics of the bacteria in detail. Generally, initial incubation of
clinical sample is done using liquid broth i.e., Nutrient Broth for growing the bacteria or increasing the number of
bacteria in the given clinical sample, in laboratory using artificial culture media.

A. Cultivation of Aerobic Bacteria

1. Inoculation of Nutrient Broth : The clinical samples should be added to the nutrient broth aseptically to avoid
contamination and incubate the nutrient broth for 24 Hrs at 37oC.
In nutrient broth after incubation:
Turbidity : Indicates the growth of bacteria
Transparency: Indicates no growth of bacteria.
2. Streak Plate Method:
To obtain a pure culture , it is required to obtain separate , single bacterial colony. To attain this, well known
‘Quadrant streaking method’ can be used.
The microbial mixture is seeded on the edge of an agar plate with inoculating loop and than streaked out over the
surface.
After seeding the culture, first sector is streaked originating from seeding area. After first sector is streaked,
inoculum loop is sterilized and second sector is streaked using inoculum from the first sector. Similarly, Third sector
is streaked.
In this way, the number of bacteria in seeding area is diluted in first sector, second sector.
Eventually, very few cells remain on the loop in third or fourth sector and single cells dropped from inoculation loop
will develop into separate colonies.
Separate single colony is picked up, streaked on fresh plate to get a pure culture.

3.Pour Plate Method

The sample is diluted several times to reduce the microbial count sufficiently to obtain separate colonies. When
plating , small volumes of several diluted samples are mixed with molten agar that has been cooled to 45OC before
pouring into sterile plates.
Bacteria and fungi are not killed at 45OC for a short exposure. After solidification of agar each bacterial cell is fixed
in a place and form an individual colony.
Like the spread plate, pour plate can also be used to determine the number of cells in a population. Plates
containing 30-300 clonies are counted. The total number of colonies equals the number of viable bacterial
cells.The count of bacterial cells should be multiplied by dilution factor to work. Can also yield isolated colonies.
Surface colonies can be used to prepare the pure culture.

4.Spread Plate/Lawn Culture

In this method, small volume of dilute microbial mixture containing around 30-300 bacterial cells is transferred to
the centre of agar plate and with the help of spreader, spread evenly over the surface of agar medium. The
dispersed cells develops into a single colony.
Numbers of colonies are equal to the number of viable organisms in the sample.
This type of method is used in Antibiotic Sensitivity /Drug Sensitivity Testing.
Spread plate method can be used to count microbial population.

5.Slant and stab culture

Slants of agar in a test tube are generally used for maintaining the bacteria in pure culture. The bacterial colonies
are streaked on the surface of slants

B. Cultivation of Anaerobic Bacteria

Main Principle: reduce the O2 content of culture medium and remove any oxygen already present inside the
system or in the medium .
Oxygen is ubiquitous in the air so special methods are needed to culture anaerobic microorganisms. A number of
procedure are available for reducing the O2 content of cultures; some simple but suitable mainly for less sensitive
organisms, others more complex but necessary for growth of strict anaerobes.
Bottles or tubes filled completely to the top with culture medium and provided with tightly fitting stopper.
Suitable for organisms not too sensitive to small amounts of oxygen.
Addition of a reducing agent that reacts with oxygen and reduces it to water e.g., Thioglycolate in thioglycolate
broth. After thioglycolate reacts with oxygen throughout the tube, oxygen can penetrate only near the top of
the tube where the medium contacts air.
 Obligate aerobes grow only at the top of such
su tubes.
Facultative organisms grow throughout the tube but best near the top.
Microaerophiles grow near the top but not right at the top.
Anaerobes grow only near the bottom of the tube, where oxygen cannot penetrate.
A redox indicator dye called resazur
resazurin
in is added to the medium because the dye changes color in the presence
of oxygen and thereby indicates the degree of penetration of oxygen into the medium.
Strict anaerobes, such as methanogenic bacteria can be killed by even a brief exposure to O2. In these cases, a
culture medium is first boiled to render it oxygen free, and then a reducing agent such as H2S is added and the
mixture is sealed under an oxygen
oxygen- free gas. All manipulations are carried out under a tiny jet of oxygen free
hydrogen or nitrogen
en gas that is directed into the culture vessel when it is open, thus driving out any O2 that
might enter. For extensive research on anaerobes, special boxes fitted with gloves, called anaerobic glove
boxes, permit work with open cultures in completely ano
anoxic atmospheres.

Stringent anaerobes can be grown only by taking special precautions to exclude all atmospheric oxygen from the
medium. Such an environment can be established by using one of the following methods:
1. Pre-reduced media
During preparation, the culture medium is boiled for several minutes to drive off most of the dissolved
oxygen. A reducing agent e.g., cysteine, is added to further lower the oxygen content. Oxygen free N2 is
bubbled through the medium to keep it anaerobic. The medium is then dispensed into tubes which are
being flushed with oxygen – free nitrogen, stoppered tightly, and sterilized by autoclaving.
autoclaving Such tubes
are continuously flushed with oxygen free CO2 by means of a cannula, restoppered, and incubated.
2. Anaerobic Chambers
This refers to a plastic anaerobic glove box that contains an atm atmosphere of H2, CO2, and N2. Culture
media are placed within the chamber by means of an air lock which can be evacuated and refilled with
N2. Any oxygen in the media is slowly removed by reaction with hydrogren, forming water; this reaction
is aided by a palladium
lladium catalyst. After being rendered oxygen free, the media are inoculated within the
chamber (by means of the glove ports) and incubated (also within the chamber).
3. Anaerobic Jar- GasPak system (Fig 16.20)
Anaerobic jar is a heavy- walled jar with a gas tight seal within which tubes, plates, or other containers to
be incubated are placed along with H2 and CO2 generating system (GasPak system). After the jar is
sealed oxygen present in the atmosphere inside jar and dissolved in the culture medium, is gradually
used up through reaction with the hydrogen in the presence of catalyst. The air in the jar is replaced with
a mixture of H2 and CO2, thus leading to anoxic conditions.
 Physical and Chemical Control of Micro-organisms

Control of microorganisms is essential in order to prevent the transmission of diseases and

infection, stop decomposition and spoilage, and prevent unwanted microbial contamination.
Primary targets of microbial control are microorganisms capable of causing infection or
spoilage.
Microorganisms are controlled by means of physical methods and chemical agents.
Choice of a method for controlling microorganisms is based on the following factors:
1. The intensity and nature of a physical agent used
2. The concentration and kind of a chemical agent used
3. The length of exposure to the agent
4. The temperature at which the agent is used
5. The number of microorganisms present
6. The organism itself
7. The nature of the material bearing the microorganism.

Controlling Microorganisms
The methods of microbial control used result in four possible outcomes
- sterilization (kills all microbial life)
- disinfection (kills vegetative microbes)
- decontamination / sanitization (cleans and reduces microbes on nonliving surfaces)
- antisepsis / degermation (reduces microbes on living tissues)

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. Bacterial
endospores have traditionally been considered the most resistant microbial entities. The goal
of any sterilization process is the destruction of bacterial endospores any process that kills
endospores will invariably kill other microbial forms.

Modes of Action of Antimicrobial or Sterilization Agents

Cellular targets of physical and chemical agents:
1. The cell wall - cell wall becomes fragile and cell lyses
-ex. some antimicrobial drugs, detergents, and alcohol
2. The cell membrane - loses integrity; ex. detergent, surfactants
3. Cellular synthetic processes (DNA, RNA) - prevention of replication, transcription.
- ex. some antimicrobial drugs, radiation, formaldehyde, ethylene oxide
4. Proteins -interfere at ribosomes to prevent translation, disrupt or denature proteins.
- ex. alcohols, phenols, acids, heat

Thermal Death Measurements

• Thermal death time (TDT)- shortest length of time required to kill all test microbes at a
specified temperature.
• Thermal death point (TDP)- lowest temperature required to kill all microbes in a sample
in 10 minutes.
• Decimal Reduction Time (DRT)- Minutes to kill 90% of a population at a given
temperature.
 I. Physical Methods in Microbial Control
Following physical methods may be used to control of microorganisms
a. Temperature- High and Low
b. Osmotic Pressure
c. Dessication
d. Radiation
e. Filteration

A. 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 (killing) action, since they denature
microbial enzymes and other proteins. Temperature is a very common and effective way of
controlling microorganisms.

1. High Temperature
High temperature may be applied as either moist heat or dry heat.

Moist heat
Moist heat has high penetration in cells and therefore more effective than dry heat for
killing microorganisms.
i) Sterilization by moist heat is done by Autoclaving.
Autoclave is an apparatus which employs steam under pressure. During autoclaving, the
materials to be sterilized are placed under 15 pounds per square inch (psi) of pressure in a
pressure-cooker type of apparatus fitted with heating element immersed in water. 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.
iii) Pasteurization
Pasteurization is the mild heating of milk and other materials to kill particular spoilage
organisms or pathogens (Mycobacterium tuberculosis, Salmonella typhi, etc.). It does not,
however, kill all organisms.
Milk is usually pasteurized by –
Heating to 71.6°C (160°F) for at least 15 seconds (Flash method)
Heating to 62.9°C (145°F) for 30 minutes (Holding method)
Ultra-high-temperature: 140°C for <1 sec

Dry Heat
Dry heat method use higher temperatures than moist heat. Dry heat kills microorganisms
through a process of protein oxidation rather than protein coagulation. Examples of dry
heat include:
i) 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.
ii) Incineration
Incinerators are used to destroy disposable or expendable materials by burning. We also
sterilize our inoculating loops by incineration.

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.

B. Osmotic Pressure
Hypotonic and isotonic environments are not usually harmful to microorganisms. However,
hypertonic environment, results into plasmolysis of the microbial cell and its growth is
inhibited.
The canning of jams or preservation 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/degradation of the food.

C. Desiccation
Desiccation removes water from cells which leads to metabolic inhibition. 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.
It is not an effective microbial control – many cells retain ability to grow when water is
reintroduced.

D. Radiation
• Non-ionizing radiation (UV rays)
• Ionizing radiation (X rays, gamma rays, electron beams)

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.
UV lights are frequently used to reduce the microbial populations in hospital operating rooms and
sinks 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.
2. Ionizing Radiations
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.

E. 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. Filters can also be
used to remove microorganisms from water and air for microbiological testing.
For sterilization three types of filters are used:

Membrane filters: These are thin filters do not absorb liquids during filtration. The
which are made of cellulose. They can be disadvantage is that they are very brittle and
employed for online sterilization during break easily.
injection by placing the membrane between
the syringe and needle. They are highly
efficient to sterilize liquid and solvents.
The disadvantage is there are chances of
rupture of membrane leading to improper
sterilization.
Seitz filters: These are made of asbestos or
other material. They are pad like and thicker
than membrane filters. They do not rupture
during filtration. But the solution might get
absorbed by the filter pad itself. An
alternative type of filter is sintered glass
filters. These are made of glass and hence

Candle filters: These are made of clay like diatomaceous mud. This special mud has minute
pores made of algae. The filters have many tiny lengthy pores. The microbes get stuck
during their travel through the pore in the candle.
 II. Chemical Agents in Microbial Control
Chemical agent includes Disinfectants, Antiseptics, Sterilants, Degermers and Preservatives.
Desirable qualities of chemical control agent:
– rapid action in low concentration
– solubility in water or alcohol, stable
– broad spectrum, low toxicity
– penetrating
– noncorrosive and nonstaining
– affordable and readily available

The germicides are evaluated in terms of their effectiveness in destroying microbes -

i. High level germicides - kill endospores and are used as Sterilants.
ii. Intermediate level germicides - kill fungal, but not bacterial spores, resistant pathogens
and viruses
iii. Low level germicides - eliminate only vegetative bacteria, vegetative fungal cells and
some viruses.

Chemical Disinfectant Groups

a. Ethylene oxide (gaseous) f. Acids/Alkalis
b. Aldehydes g. Heavy metals
c. Halogen-Based Biocides h. Oxidants
d. Alcohols i. Surfactants
e. Phenolics

Ethylene oxide- It is a highly reactive gas (C2H4O), flammable, toxic, and a strong mucosal
irritant. Ethylene oxide can be used for sterilization at low temperatures (after 4-12 hours
exposure). Ethylene oxide has very high penetrating power and denatures microbial
proteins. Gaseous chemosterilizers are commonly used to sterilize heat-sensitive items
such as plastic syringes, petri plates, textiles, sutures, artificial heart valves, heart-lung
machines, and mattresses.
A drawback is that this gas cannot kill dried microorganisms and requires a relative humidity
level of 40–90% in the sterilizing chamber. Vapors are toxic to the skin, eyes, and mucous
membranes and are also carcinogenic. Since it is explosive, it is usually mixed with inert
gases such as freon or carbon dioxide.
Another gas that is used as a sterilant is chlorine dioxide

Aldehydes-
Formaldehyde (HCHO) is the most important aldehyde which is used for gas sterilization.
This substance is used to disinfect surfaces and objects in 0.5–5% solutions. Formaldehyde is
a water-soluble gas. Formalin is a 35% solution of this gas in water. Formaldehyde is a
broad-spectrum germicide for bacteria, fungi, and viruses. At higher concentrations, spores
are killed as well. Its biocidal action is through alkylation of carboxyl, hydroxyl and sulfhydryl
groups on proteins and therefore causing protein denaturation. Similarly affect the ring
nitrogen atoms of purine bases. It is used in embalming, preserving biological specimens,
and in preparing vaccines.
Formaldehyde’s drawbacks are reduction in efficacy at refrigeration temperature, its
pungent, irritating odor, and several safety concerns. Formaldehyde irritates mucosa. Skin
contact may result in inflammations. Also, Formaldehyde is presently considered to be a
carcinogen or a cancer-suspect agent according to several regulatory agencies.

Paraformaldehyde is a solid polymer of formaldehyde. Paraformaldehyde generates

formaldehyde gas when it is depolymerized by heating to 232 to 246°C (450 to 475°F); the
depolymerized material reacts with the moisture in the air to form formaldehyde gas. This
process is used for the decontamination of large spaced and laminar-flow biological safety
cabinets when maintenance work or filter changes require access to the sealed portion of
the cabinet.
Glutaraldehyde is a colorless liquid and has the sharp, pungent odor typical of all aldehydes.
Two percent (2% v/v) solution of glutaraldehyde exhibits very good activity against
vegetative bacteria, spores and viruses. It is ten times more effective than formaldehyde
and less toxic. However, it must be limited and controlled because of its toxic properties and
hazards. A 10 hour exposure to a 2% glutaraldehyde solution can be used for cold
sterilization of materials like clinical instruments.

Halogen-Based Biocides- Chlorine, iodine, and derivatives of these halogens are suitable for use as
disinfectants. Chlorine and iodine show a generalized microbicidal effect and also kill spores.
Chlorine Compounds
Chlorine compounds are good disinfectants. They have a broad spectrum of antimicrobial
activity and are inexpensive and fast acting. Hypochlorites, the most widely used of the
chlorine disinfectants, are available in liquid (e.g., Sodium hypochlorite), household bleach
and solid (e.g., calcium hypochlorite, sodium dichloroisocyanurate) forms. Household bleach
has an available chlorine content of 5.25%, or 52,500 ppm. Because of its oxidizing power, it
loses potency quickly and should be made fresh and used within the same day it is
prepared.
There are two potential occupational exposure hazards when using hypochlorite solutions.
The first is the production of the carcinogen bis-chloromethyl ether when hypochlorite
solutions come in contact with formaldehyde. The second is the rapid production of chlorine
gas when hypochlorite solutions are mixed with an acid. Care must also be exercised in
using chlorine-based disinfectants which can corrode or damage metal, rubber, and other
susceptible surfaces. Bleached articles should never be autoclaved without reducing the
bleach with sodium thiosulfate or sodium bisulfate.
Chloramine T which is prepared from sodium hypochlorite and p-toluenesulfonamide is a
more stable, odorless, less corrosive form of chlorine but has decreased biocidal activity in
comparison to 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.
Chlorine gas reacts with water to form hypochlorite ions, which denature microbial
enzymes. Chlorine is used in the chlorination of drinking water, swimming pools, and
sewage.
Iodophors
Iodophors are used both as antiseptics and disinfectants. An iodophor is a combination of
iodine and a solubilizing agent or carrier; the resulting complex provides a sustained-release
reservoir of iodine and releases small amounts of free iodine in aqueous solution. Antiseptic
iodophors are not suitable for use as hard-surface disinfectants because they contain
significantly less free iodine than do those formulated as disinfectants.
The most important iodine preparations are the solutions of iodine and potassium iodide in
alcohol (tincture of iodine) used commonly as a topical antiseptic to disinfect skin and small
wounds. They are generally effective against vegetative bacteria, Mycobacterium
tuberculosis, fungi, some viruses, and some endospores.
Wescodyne®, Betadine®, Povidone-Iodine and other iodophors are commercially available
Iodine-based disinfectants, which give good control when the manufacturer’s instructions
for formulation and application are followed. Both bleach and iodophors should be made
up in cold water in order to prevent breakdown of the disinfectant.

Alcohols-
The types of alcohol used in disinfection are ethanol (80%), propanol (60%), and isopropanol
(70%). Ethyl and isopropyl alcohols are the two most widely used alcohols for their biocidal
activity. These alcohols are effective against lipid-containing viruses and a broad spectrum
of bacterial species, but ineffective against spore-forming bacteria.
Alcohols work through the disruption of cellular membranes, solubilization of lipids, and
denaturation of proteins by acting directly on S-H functional groups. They evaporate rapidly,
which makes extended contact times difficult to achieve unless the items are immersed.
They are used to clean instruments and wipe down interior of Biological Safety Cabinets and
surgical and hygienic disinfection of the skin and hands bottles, etc

Phenol and Phenolics-

Phenol (5-10%) was the first disinfectant commonly used. However, because of its toxicity
and odor, phenol derivatives are now generally used. Lister was the first to use phenol
(carbolic acid) in medical applications. Phenols denature proteins.
Phenolics are phenol (carbolic acid) derivatives in which phenol is substituted with organic
groups and/or halogens (alkylated, arylated, and halogenated phenols). They retain more
activity in the presence of organic material than other disinfectants. Cresols,
hexachlorophene, alkyl- and chloro derivatives and diphenyls are more active than phenol
itself. Available commercial products are in Lysol®, O-syl®, Staphene®, and Amphyl®.
Orthophenylphenol is the agent of Lysol. Triclosan is a chlorine-containing phenolic
antiseptic very common in antimicrobial soaps and other products.
These agents kill most bacteria, most fungi, and some viruses, but are usually ineffective
against endospores. They alter membrane permeability and denature proteins.

Acids/Alkalis-
Strong mineral acids and alkalis have disinfectant properties depending on the extent of
their dissociation in solution. In general acids are better disinfectants than alkalis. Mode of
action is attributed to an increase of H+ and OH– species in solutions which interfere with
certain microbial functions, however the total effect is not only dependent on pH alone.
Weak organic acids are more potent than inorganic acids since they disrupt secondary and
tertiary conformation of enzymes and structural proteins.
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).
Heavy Metals- Heavy metals, such as mercury, silver, and copper, denature proteins. They
attack on protein sulfhydryl groups and disrupting the enzyme functions.
Soluble salts of mercury, mercuric chloride and mercurous chloride are efficient
bactericidal agents and are not effective against endospores. Silver nitrate (1%) is
sometimes put in the eyes of newborns to prevent gonococcal infections. Copper sulfate is
used to combat fungal diseases of plants and is also a common algicide. Selinium sulfide
kills fungi and their spores.

Oxidants- This group includes ozone, hydrogen peroxide, potassium permanganate, and
peracetic acid. Their relevant chemical activity is based on the splitting off of oxygen. Most
are used as mild antiseptics to disinfect mucosa, skin, or wounds.

Surfactants- These substances (also known as surface-active agents or detergents) include

anionic, cationic, amphoteric, and nonionic detergent compounds, of which the cationic and
amphoteric types are the most effective. The bactericidal effect of these substances is only
moderate. They have no effect at all on tuberculosis bacteria, pores, or non-encapsulated
viruses. Their efficacy is good against Gram-positive bacteria, but less against Gram-negative
rods. Their advantages include low toxicity levels, lack of odor, good skin tolerance, and a
cleaning effect.
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. They are also
inactivated by soaps and organic materials like excreta. Cationic detergents include the
quaternary ammonium compounds such as zephiran, diaprene and phemerol. Quaternary
ammonium compounds are generally odorless, colorless, nonirritating, and deodorizing.
They have detergent action and they are good disinfectants. The mode of action of these
compounds is through inactivation of energy producing enzymes, denaturation of essential
cell proteins, and disruption of the cell membrane. Many of these compounds are better
used in water baths, incubators, and other applications where halide or phenolic residues
are not desired. 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 soap contains added antiseptics to increase antimicrobial activity.