Microbial Growth:

Growth is an orderly increase in the quantity of cellular constituents. It depends upon the

ability of the cell to form new protoplasm from nutrients available in the environment.

Growth implies that all chemical components of the cell increase with the same speed and

after a certain time this leads to increase in cell number, which causes increase in size or

number of the individuals. Growth is normally performed batch-wise or continuously.

In any biological systems, growth can be defined as the orderly increase of all
chemical components. The amplification or multiplication of microorganisms such as
bacteria, algae, fungi, plankton and diatoms is called microbial growth. Increase of mass
might not really reflect growth because the cells could simply increasing their content of
storage products such as glycogen or poly-β-hydroxybutyrate. In an adequate medium to
which they become fully adapted, however, bacteria are in a state of balanced growth.
During a period of balanced growth, an increase of the biomass is accompanied by a
comparable increase of all other measurable properties of the population, e.g., protein,
RNA, DNA and intracellular water. In other words culture undergoing balanced growth
maintain a constant chemical composition.

Physical conditions required for microbial growth

The requirements for microbial growth can be divided into two main categories: physical and chemical.
Physical aspects include temperature, pH and osmotic pressure. Chemical requirements include sources of
carbon, nitrogen, sulphur, phosphorus, trace elements, oxygen, and organic growth factors.

Temperature

A particular microorganism will exhibit a range of temperature over which it can grow, defined by three
cardinal points in the same manner as pH. Considering the total span of temperature where liquid water
exists, the procaryotes may be subdivided into several subclasses on the basis of one or another of their
cardinal points for growth. For example, organisms with an optimum temperature near 37 degrees (the
body temperature of warm-blooded animals) are called mesophiles. Mesophilic organisms have important
uses in food preparation especially in cheese, yoghurt, in beer and wine making. Eg. Staphylococcus,
Pseudomonas, Actinotobacter

Organisms with an optimum T between about 45 degrees and 70 degrees are thermophiles. Eg.
Thermococcus litoralis, Pyrococcus furiosus.

Some Archaea with an optimum T of 80 degrees or higher and a maximum T as high as 115 degrees, are
now referred to as extreme thermophiles or hyperthermophiles. Eg. Methanopyrus, Aquifex, Pyrolobus
fumarii.

The cold-loving organisms are psychrophil a growth temperature optimum of 15oC or lower and cannot
grow in a climate beyond a maximum temperature of 200C. Eg. Chlamydomonas, Moritella,
Mathanococcoides burtonii, Polaromonas vacuolata. Psychrotrophs are the source of food storage in
refrigerators since they are invariably brought in from their mesophilic habitats and continue to grow in the
refrigerated environment where they spoil the food. Of course, they grow slower at 2 degrees than at 25
degrees.

pH

Microorganisms which grow at an optimum pH well below neutrality (7.0) are called acidophiles. Those
which grow best at neutral pH are called neutrophiles and those that grow best under alkaline conditions
are called alkaliphiles. Obligate acidophiles, such as some Thiobacillus species, actually require a low pH
for growth since their membranes dissolve and the cells lyse at neutrality. Several genera of Archaea,
including Sulfolobus and Thermoplasma, are obligate acidophiles. Among eukaryotes, many fungi are
acidophiles, but the champion of growth at low pH is the eucaryotic alga Cyanidium which can grow at a
pH of 0.

Most bacteria grow best in a narrow pH range near neutrality, between pH 6.5 and 7.5. Molds and
yeast will grow over a greater pH range than bacteria will, but the optimum pH of molds and yeasts is
generally below that of bacteria, usually about pH 5-6. When bacteria are cultured in the laboratory, they
often produce acids that eventually interfere with their own growth. To neutralize the acids and maintain
the proper pH, chemical buffers are included in the growth medium.

Water Availability

Water is the solvent in which the molecules of life are dissolved, and the availability of water is therefore a
critical factor that affects the growth of all cells. The availability of water for a cell depends upon its
presence in the atmosphere (relative humidity) or its presence in solution or a substance (water activity).
The water activity (Aw) of pure H2O is 1.0 (100% water). Water activity is affected by the presence of
solutes such as salts or sugars, that are dissolved in the water. The higher the solute concentration of a
substance, the lower is the water activity and vice-versa.

The only common solute in nature that occurs over a wide concentration range is salt [NaCl], and
some microorganisms are named based on their growth response to salt. Microorganisms that require some
NaCl for growth are halophiles. Eg. Halobacterium, Halococcus Mild halophiles require 1-6% salt,
moderate halophiles require 6-15% salt; extreme halophiles that require 15-30% NaCl for growth are
found among the archaea. Bacteria that are able to grow at moderate salt concentrations, even though they
grow best in the absence of NaCl, are called halotolerant. Although halophiles are "osmophiles" (and
halotolerant organisms are "osmotolerant") the term osmophiles is usually reserved for organisms that are
able to live in environments high in sugar. Organisms which live in dry environments (made dry by lack of
water) are called xerophiles.

Limiting water activities (Aw) for growth of certain procaryotes.

Organism Minimum Aw for growth

Caulobacter 1.00
Spirillum 1.00
Pseudomonas .91
Salmonella/E. coli .91
Lactobacillus .90
Bacillus .90
Staphylococcus .85
Halococcus .75

Chemical Requirements

Carbon

Carbon is the structural backbone of living matter; it is needed for all the organic compounds that make up
a living cell. Half the dry weight of a typical bacterial cell is carbon. Organisms able to use CO 2 as a
principal carbon source are termed as autotrophs; organisms dependent on an organic carbon source are
termed heterotrophs. By means of these criteria, four major nutritional categories can be distinguished:

Photoautotrophs use light as the energy source and CO2 as the principal carbon source. They include most
photosynthetic organisms: higher plants, algae and many photosynthetic bacteria.

Photoheterotrophs using light as the energy source and an organic compound as the principal carbon
source. This category includes certain of the purple and green bacteria.

Chemoautotrophs using a chemical energy source and CO2 as the principal carbon source. Energy is
obtained by the oxidation of reduced inorganic compounds, such as NH 4+, NO2-, H2, reduced form of
sulphur, CO and ferrous ion.

Chemoheterotrophs get most of their carbon from the source of their energy-organic materials such as
proteins, carbohydrates, and lipids. Chemoheterotrophs include all metazoan animals, protozoa, fungi, and
the great majority of bacteria.

Nitrogen, Sulphur and Phosphorus

Protein synthesis requires considerable amounts of nitrogen as well as some sulphur. The synthesis of DNA
and RNA also require nitrogen and some phosphorus, as does the synthesis of ATP, the molecule so
important for the storage and transfer of chemical energy within the cell. Nitrogen makes up about 14% of
the dry weight of a bacterial cell, and sulfur and phosphorus together constitute about another 4%.
Organisms use nitrogen primarily to form the amino group of the amino acids of proteins. Other bacteria
use nitrogen from ammonium ions which are already in the reduced form and are usually found in organic
cellular material. Still other bacteria are able to derive nitrogen from nitrates. Some important bacteria,
including many of the photosynthesizing cyanobacteria, use gaseous nitrogen directly from the atmosphere.
This process is called nitrogen fixation. Some organisms that can this method are free-living, mostly in the
soil, but others live cooperatively in symbiosis with the roots of legumes such as clover, soyabeans, alfalfa,
beans and peas. The nitrogen fixed in the symbiosis is used by both the plant and the bacterium.

Sulfur is used to synthesize sulfur-containing amino acids and vitamins such as thiamine and
biotin. Important natural sources of sulphur include the sulphate ion, hydrogen sulfide, and the sulphur
containing amino acids.

Phosphorus is essential for the synthesis of nucleic acids and the phospholipids of cell membranes.
Among other places, it is also found in the energy bonds of ATP. An important source of phosphorus is the
phosphate ion. Potassium, magnesium, and calcium are also elements that microorganisms require, often as
cofactors for enzymes.

Major elements, their sources and functions in bacterial cells.

Element % of dry weight Source Function

organic compounds
Carbon 50 Main constituent of cellular material
or CO2
H2O, organic
Constituent of cell material and cell water; O2 is electron
Oxygen 20 compounds, CO2,
acceptor in aerobic respiration
and O2
NH3, NO3, organic Constituent of amino acids, nucleic acids nucleotides,
Nitrogen 14
compounds, N2 and coenzymes
H2O, organic
Hydrogen 8 Main constituent of organic compounds and cell water
compounds, H2
inorganic Constituent of nucleic acids, nucleotides, phospholipids,
Phosphorus 3
phosphates (PO4) LPS, teichoic acids
SO4, H2S, So,
Constituent of cysteine, methionine, glutathione, several
Sulfur 1 organic sulfur
coenzymes
compounds
Main cellular inorganic cation and cofactor for certain
Potassium 1 Potassium salts
enzymes
Inorganic cellular cation, cofactor for certain enzymatic
Magnesium 0.5 Magnesium salts
reactions
Inorganic cellular cation, cofactor for certain enzymes
Calcium 0.5 Calcium salts
and a component of endospores
Iron 0.2 Iron salts Component of cytochromes and certain nonheme iron-
 proteins and a cofactor for some enzymatic reactions

Oxygen

Oxygen is a universal component of cells and is always provided in large amounts by H2O. However,
procaryotes display a wide range of responses to molecular oxygen O 2. Obligate aerobes require O2 for
growth; they use O2 as a final electron acceptor in aerobic respiration. Obligate anaerobes (occasionally
called aerophobes) do not need or use O2 as a nutrient. In fact, O2 is a toxic substance, which either kills or
inhibits their growth. Obligate anaerobic procaryotes may live by fermentation, anaerobic respiration,
bacterial photosynthesis, or the novel process of methanogenesis. Facultative anaerobes (or facultative
aerobes) are organisms that can switch between aerobic and anaerobic types of metabolism. Under
anaerobic conditions (no O2) they grow by fermentation or anaerobic respiration, but in the presence of O 2
they switch to aerobic respiration. Aerotolerant anaerobes are bacteria with an exclusively anaerobic
(fermentative) type of metabolism but they are insensitive to the presence of O2. They live by fermentation
alone whether or not O2 is present in their environment. Among microorganisms that are obligate aerobes,
some grow best at partial pressures of oxygen considerably below that (0.2 atm) present in air. They are
termed as microaerophilic.

Growth Factors

This simplified scheme for use of carbon, either organic carbon or CO 2, ignores the possibility that an
organism, whether it is an autotroph or a heterotroph, may require small amounts of certain organic
compounds for growth because they are essential substances that the organism is unable to synthesize from
available nutrients. Such compounds are called growth factors.

Growth factors are required in small amounts by cells because they fulfill specific roles in biosynthesis.
The need for a growth factor results from either a blocked or missing metabolic pathway in the cells.
Growth factors are organized into three categories.

1. purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA)

2. amino acids: required for the synthesis of proteins

3. vitamins: needed as coenzymes and functional groups of certain enzymes

Some bacteria (e.g. E. coli) do not require any growth factors: they can synthesize all essential purines,
pyrimidines, amino acids and vitamins, starting with their carbon source, as part of their own intermediary
metabolism. Certain other bacteria (e.g. Lactobacillus) require purines, pyrimidines, vitamins and several
amino acids in order to grow. These compounds must be added in advance to culture media that are used to
grow these bacteria. The growth factors are not metabolized directly as sources of carbon or energy, rather
they are assimilated by cells to fulfill their specific role in metabolism. Mutant strains of bacteria that
require some growth factor not needed by the wild type (parent) strain are referred to as auxotrophs. Thus,
a strain of E. coli that requires the amino acid tryptophan in order to grow would be called a tryptophan
auxotroph and would be designated E. coli trp-

Some vitamins that are frequently required by certain bacteria as growth factors are listed in Table . The
function(s) of these vitamins in essential enzymatic reactions gives a clue why, if the cell cannot make the
vitamin, it must be provided exogenously in order for growth to occur

Vitamin Coenzyme form Function

p-Aminobenzoic acid
- Precursor for the biosynthesis of folic acid
(PABA)
Transfer of one-carbon units and required for
Folic acid Tetrahydrofolate synthesis of thymine, purine bases, serine,
methionine and pantothenate
Biotin Biotin Biosynthetic reactions that require CO2 fixation
Lipoic acid Lipoamide Transfer of acyl groups in oxidation of keto acids
Mercaptoethane-
Coenzyme M CH4 production by methanogens
sulfonic acid
NAD (nicotinamide adenine
Nicotinic acid Electron carrier in dehydrogenation reactions
dinucleotide) and NADP
Coenzyme A and the Acyl Carrier Oxidation of keto acids and acyl group carriers in
Pantothenic acid
Protein (ACP) metabolism
Transamination, deamination, decarboxylation and
Pyridoxine (B6) Pyridoxal phosphate
racemation of amino acids
FMN (flavin mononucleotide) and
Riboflavin (B2) Oxidoreduction reactions
FAD (flavin adenine dinucleotide)
Decarboxylation of keto acids and transaminase
Thiamine (B1) Thiamine pyrophosphate (TPP)
reactions
Cobalamine coupled to adenine
Vitamin B12 Transfer of methyl groups
nucleoside
Vitamin K Quinones and napthoquinones Electron transport processes

Culture Media for the Growth of Microbes

For any bacterium to be propagated for any purpose it is necessary to provide the appropriate biochemical
and biophysical environment. The biochemical (nutritional) environment is made available as a culture
medium, and depending upon the special needs of particular bacteria (as well as particular investigators) a
large variety and types of culture media have been developed with different purposes and uses. Culture
media are employed in the isolation and maintenance of pure cultures of bacteria and are also used for
identification of bacteria according to their biochemical and physiological properties.

The manner in which bacteria are cultivated, and the purpose of culture media, varies widely. Liquid media
are used for growth of pure batch cultures, while solidified media are used widely for the isolation of pure
cultures, for estimating viable bacterial populations, and a variety of other purposes. The usual gelling
agent for solid or semisolid medium is agar, a hydrocolloid derived from red algae. Agar is used because of
its unique physical properties (it melts at 100oC and remains liquid until cooled to 40oC, the temperature at
which it gels) and because it cannot be metabolized by most bacteria. Hence as a medium component it is
relatively inert; it simply holds (gels) nutrients that are in aqueous solution.

Types of Culture Media

A nutrient material prepared for the growth of microorganisms in a laboratory is called a culture medium.
Culture media may be classified into several categories depending on their composition or use. A
chemically-defined (synthetic) medium is one in which the exact chemical composition is known.
Chemically-defined media are of value in studying the minimal nutritional requirements of
microorganisms, for enrichment cultures, and for a wide variety of physiological studies.

A complex (undefined) medium is one in which the exact chemical constitution of the medium is not
known. Complex media usually provide the full range of growth factors that may be required by an
organism so they may be more handily used to cultivate unknown bacteria or bacteria whose nutritional
requirement are complex (i.e., organisms that require a lot of growth factors, known or unknown). Complex
media are usually used for cultivation of bacterial pathogens and other fastidious bacteria.

Defined media are usually composed of pure biochemicals off the shelf; complex media usually contain
complex materials of biological origin such as blood or milk or yeast extract or beef extract, the exact
chemical composition of which is obviously undetermined. A defined medium is a minimal medium if it
provides only the exact nutrients (including any growth factors) needed by the organism for growth. The
use of defined minimal media requires the investigator to know the exact nutritional requirements of the
organisms in question.

A selective medium is one which has a component(s) added to it which will inhibit or prevent the growth
of certain types or species of bacteria and/or promote the growth of desired species. For eg., bismuth sulfite
agar is one medium used to isolate the typhoid bacterium, the gram-negative Salmonella typhi from feces.
Bismuth sulfite inhibits gram-positive bacteria and most gram-negative intestinal bacteria (other than
S.typhi) as well. Dyes such as brilliant green selectively inhibit gram-positive bacteria, and this dye is the
basis oa a medium called brilliant green agar that is used to isolate the gram-negative Salmonella.
Differential medium make it easier to distinguish colonies of the desired organism from other colonies
growing on the same plate. Blood agar (which contain red blood cells) is a medium that microbiologists
often use to identify bacterial species that destroy red blood cells. These species, such as Streptococcus
pyogenes, the bacterium that cause strep throat, show a clear ring around their colonies where they have
lysed the surrounding blood cells

Sometimes selective and differential characteristics are combined in a single medium. Eg. selective,
differential medium for the isolation of Staphylococcus aureus, the most common bacterial pathogen of
humans, contains a very high concentration of salt (which the staph will tolerate) that inhibits most other
bacteria, mannitol as a source of fermentable sugar, and a pH indicator dye. From clinical specimens, only
staph will grow. S. aureus is differentiated from S. epidermidis (a nonpathogenic component of the normal
flora) on the basis of its ability to ferment mannitol. Mannitol-fermenting colonies (S. aureus) produce acid
which reacts with the indicator dye forming a colored halo around the colonies; mannitol non-fermenters
(S. epidermidis) use other non-fermentative substrates in the medium for growth and do not form a halo
around their colonies.

An enrichment medium contains some component that permits the growth of specific types or species of
bacteria, usually because they alone can utilize the component from their environment. An enrichment
medium for nonsymbiotic nitrogen-fixing bacteria omits a source of added nitrogen to the medium. The
medium is inoculated with a potential source of these bacteria (e.g. a soil sample) and incubated in the
atmosphere wherein the only source of nitrogen available is N2. A selective enrichment medium for growth
of the extreme halophile (Halococcus) contains nearly 25 percent salt [NaCl], which is required by the
extreme halophile and which inhibits the growth of all other procaryotes.

Enrichment Culture Techniques

The recognition that microorganisms in nature always exist in a mixture demanded that for the
study of various physiological types, pure cultures were essential. Realising this, S.Winogradsky
(1856. 1953) and M.W. Beijerinck (1851-1931) developed the technique of "enrichment culture".

Basically it uses the principle of natural selection. When a culture medium of a defined chemical
composition is inoculated with a mixed microbial population, only the organism to which the
environment is ideal will rapidly outgrow the others by repeated subculturing into new medium
of the same composition it is possible to isolate a pure culture.

Thus, by modifying the composition of the medium or the incubation conditions, it is possible to
Isolate any specific organism from a mixed population. Today, the enrichment culture method is
the most powerful tool available to microbiologists for the isolation of any type of
microorganism.

Methods of obtaining pure cultures of microorganisms

A culture that contains only one kind of microorganism is called a pure culture. The three most commonly
used methods for obtaining pure cultures of microorganisms are:
(i) Streak-plate
(ii) Pour-plate
(iii) Spread-plate
Other methods for obtaining pure cultures of microorganisms include: (a) use of enrichment media; (b) use
of differential and selective media; (c) use of media containing antibiotics; (d) selected techniques for the
cultivation of anaerobes.

Streak Plate method: This method offers a most practical method of obtaining discrete colonies
and pure cultures. It was originally developed by two bacteriologists, Loeffler and Gaffkey in the
laboratory of Robert Koch. In this method, a sterilized loop or transfer needle is dipped into a suitable
diluted suspension of organisms which is then streaked on the surface of an already solidified agar plate to
make a series of parallel, non-overlapping streaks. The aim of this exercise is to obtain colonies of
microorgnisms that are pure, i.e. growth derived from a single cell/spore.

Pour-plate method: The forerunner of the present pour- plate method was developed in the
laboratory of the bacteriologist, Robert Koch. In this technique, successive dilutions of the inoculum
(serially diluting the original specimen) are added into sterile Petri plates to which is poured melted and
cooled (42-45oC) agar medium and thoroughly mixed by rotating the plates which is then allowed to
solidify. After incubation, the plates are examined for the presence of individual colonies growing
throughout the medium. The pure colonies which are of different size, shape and colour may be
isolated/transferred into test tube culture media for making pure cultures.
Pour plates are also used as a means of determining the number of viable organisms in a
liquid such as water, milk, urine or broth culture as well as to determine the hemolytic activity of deep
colonies of some bacteria, such as the Streptococci by using an agar medium containing blood.

Spread-plate technique: The Spread-plate technique is used for the separation of a dilute, mixed
population of microorganisms so that individual colonies can be isolated. In this technique
microorganisms are spread over the solidified agar medium with a sterile L-shaped glass rod while the
 Petri dish is spun on a turntable. The theory behind this technique is that as the Petri dish spuns, at
some stage, single cells will be deposited with the bent glass rod on to the agar surface. Some of these
cells will be separated from each other by a distance sufficient to allow the colonies that develop to be
free each other.

Subculturing (or picking off) technique: After incubation has been completed in streak-
plate, pour-plate, or spread-plate techniques and appearance of the discrete, well-separated colonies
has been examined, the next step is to subculture some of the cells from one of the colonies to separate
agar plates or nutrient agar slants with a sterilized needle or loop for further examination and use. Each
of these new culture represents the growth of a single species and is called a pure or stock culture.
Subcluturing is used to describe the process of transferring of microorganisms from their parent growth
source to afresh one or from one medium to another. When the transfer is done from a solid medium
(agar) to a liquid medium (broth), the term picking off is used. This technique is also used routinely in
preparing and maintaining stock cultures, as well as in microbiological test procedures.

Broth cultures: Broth is a liquid formulation (medium) used for growing microorganisms in the
laboratory on a small scale or in an industrial fermentor on a large scale. The media termed broths,
milks, or nutrient solutions are made by dissolving various solutes in distilled water and later
sterilizing (autoclaving) it. Appropriate physical and chemical environments are required for growing
microorganisms in a broth. Temperature is the most vital requirement for growing cultures which is
supplied by an incubator in a laboratory. Many cultures also require oxygen, a certain amount of which
is absorbed into the surfaces of broth in test tubes placed in an undisturbed rack on the incubator shelf,
this condition is termed as static incubation. Roller drum apparatus in which cultures are placed at
an angle and rotated, are used to provide oxygen to broth cultures, a process used for aeration of broth
cultures. For larger volumes of broth, air is filter for aeration of broth cultures. For larger volumes of
broth, air is filter sterilized and pumped into large both-filled vessels termed as fermentors.
Growth of the microorganisms occurs throughout the container and may present a dispersed
cloudy or particulate appearance. Some microorganisms uniformly increase the turbidity (milkiness or
cloudiness) of the broth, due to increase in their numbers. Pellicle, a mat of cells, is formed by some
microorganisms which grow only at the surface of a static broth culture. Other microbial species may
settle to the bottom of the tune to form a sediment or button of cells that stick together. Filamentous
fungi form interwined mycelia (clumps) suspended within a broth.
The prime use of broth cultures is to study the characteristics of microorganisms and for the
production of various industrial products by the microorganisms, eg. Production of antibiotics, mass
production of baker’s yeasts.

Maintenance of Pure Cultures

Pure cultures of microorganisms are required for research as well as for other purposes. For this, it is
necessary to have a culture collection. The methods used for maintenance and preservation should
conserve all the characteristics of the organisms. Some of the simple methods of culture maintenance
and preservation are :

1. Transfer of Fresh Media: Microbial cultures can be maintained by periodic

transfer on fresh, sterile media in tubes. The frequency of transfer however varies with
the organisms. For example, a culture of E. coli needs to be transferred at monthly
intervals. After growth for 24 hours at 37°C, the slants can be stored at low
temperature for 20-30 days. To keep the cultures viable, it is necessary to use an
appropriate growth medium and a proper storage temperature. Many heterotrophic
bacteria can be maintained on a medium such as nutrient agar with transfers to fresh
medium after every 20-30 days.

2. Over laying with Mineral oil: Many bacteria and fungi can be preserved by
covering the fresh growth in agar slants with sterile mineral oil. The oil must be above
the tip of the slanted surface. The method is very simple and cell viability is high
compared to frequent transfer and storage at low temperature. Mineral oil covered
cultures are stored at room temperature or preferably at 0-5°C; the latter, is desirable.
Some microorganisms have been preserved satisfactorily for more than 15-20 years by
this method

3. Lyophilization: It is the rapid dehydration of organisms while they are in a

frozen state. In this technique the culture is rapidly frozen at -70 oC and then
dehydrated by vaccum and the tubes containing freeze-dried cultures are sealed and
stored in dark at -4 oC in refrigerators.

4. Use of refrigerator or cold room storage: Live cultures on a culture

medium can be successfully stored in refrigerators or cold room maintained at 4 oC.
Generally the metabolic activities of the microorganisms will be greatly slowed down
at this temperature, but it is not low enough to stop metabolism completely. So, regular
subculturing is necessary for bacteria, it ranges from 2-3 weeks and for fungi from 3-4
months.

5. Liquid nitrogen method: Cultures are frozen in the presence of a protective

agent such as glycerol or dimethylsulfoxide in liquid nitrogen (- 196°C). Stabilizing
agent are added because that prevent the formation of ice crystals which may kill
frozen cells. This procedure has been successful with many organisms which cannot
be preserved by lyophilization.

6. Storage in sterile soil: Storage in Sterile Soil method is widely used for
preserving spore forming bacteria and fungi. Spore suspensions are added to sterile
 soil (sterilized for 2-3 hours at intervals of 1.2 days) and the mixture is dried at room
temperature and stored in a refrigerator. Bacterial cultures maintained by this
procedure have been remained for 70-80 years.

7. Storage in silica gel: Both bacteria and yeast can be 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 (paste) of
cells, mixed and stored at a low temperature. The basic principle in this technique is
quick dessication at low temperature, which allows the cells to remain viable for a
long period.
…………………………………………………………………………………………………………..

Actions of Microbial Control Agents

Alteration of Membrane Permeability

1. The susceptibility of the plasma membrane is due to its lipid and protein components.
2. Certain chemical control agents damage to the plasma membrane by altering its permeability

Damage to Proteins and Nucleic Acids

1. Some microbial control agents damage cellular proteins by breaking hydrogen bonds and covalent
bonds.
2. Other agents interfere with DNA and RNA replication and protein synthesis.

Physical Methods of Microbial Control

Heat
• Heat is frequently used to eliminate microorganisms. Heat appears to kill microorganisms by
denaturing their enzymes; the resultant changes to the three-dimensional shapes of these proteins
inactivate them.

• Moist heat kills microorganisms primarily by the coagulation of proteins, which is caused by
breaking of the hydrogen bonds that hold the proteins in their three-dimensional structure.
• Thermal death point (TDP) is the lowest temperature at which all the microbes in a liquid culture
will be killed in 10 minutes.

• Thermal death time (TDT) is the length of time required to kill all bacteria in a liquid culture at a
given temperature.
 • Decimal reduction time is the length of time in which 90% of a bacterial population which is
killed at a given temperature.

• Boiling (100OC) kills many vegetative cells and viruses within 10 minutes.

• Autoclaving is the most effective method of moist heat sterilization. The steam must directly
contact the material to be sterilized.

• In high-temperature short time (HTST) pasteurization, a high temperature is used for a short time
(72OC for 15 seconds) to destroy pathogens without altering the flavour of the food. Ultra high
temperature treatment (140OC for 3 seconds) is used to sterilize dairy products.
• Methods of dry heat sterilization include direct flaming, incineration, and hot-air sterilization. Dry
heat kills by oxidation.

• Different methods that produce the same effect (reduction in microbial growth) are called
equivalent treatments.

Filteration: Filteration is the passage of a liquid or gas through a filter with pores small enough to
retain microbes. Microbes can be removed from air by high-efficiency particulate air filters. Membrane
filters composed of nitrocellulose or cellulose acetate are commonly used to filter out bacteria, viruses,
and even large proteins.

Low temperatures: The effectiveness of low temperature depends on the particular microorganism
and the intensity of the application. Most microorganisms do not reproduce at ordinary refrigerator
temperatures (0-7oC). Many microbes survive at the subzero temperatures used to store foods.

High pressure: High pressure denatures proteins in vegetative cells.

Dessication: In the absence of water, microorganisms cannot grow but can remain viable. Viruses
and endospores can resist dessication.

Osmotic Pressure: Microorganisms in high concentrations of salts and sugars undergo plamolysis.
Molds and yeasts are more capable than bacteria of growing in materials with low moisture or high osmotic
pressure.

Radiation: The effects of radiation depend on its wavelength, intensity, and duration. Ionizing
radiation (gamma rays, X rays, and high-energy electron beams) has a high degree penetration and exerts
its effect primarily by ionizing water and forming highly reactive hydroxyl radicals. Ultraviolet radiation, a
form of non-ionizing radiation, has a low degree penetration and cause cell damage by making thymine
dimmers in DNA that interfere with DNA replication; the most effective germicidal wavelength is 260 nm.
Microwaves can kill microbes indirectly as material get hot.

Chemical Methods of Microbial Control

Chemicals agents are used on living tissue (as antiseptics) and on inanimate objects (as disinfectants). Few
chemical agents achieve sterility.

Principles and Evaluation of effective disinfection

Careful attention should be paid to the properties and concentration of the disinfectant to be used. The
presence of organic matter, degree of contact with microorganisms, and temperature should also be
considered.
In the use-dilution test, bacterial survival in the manufacturer’s recommemded dilution of a
disinfectant is determined. Viruses, endospore-forming bacteria, mycobacteria and fungi can also be used
in the use-dilution test. In the disk-diffusion method, a disk of filter paper is soaked with a chemical and
placed on an inoculated agar plate; a zone of inhibition indicated effectiveness.

Type of Disinfectants
Phenol and Phenolic
Phenolics exert their action by injuring plasma membranes. Bisphenols such as triclosan and
hexachlorophene are widely used in house hold products.

Biguanides
Chlorhexidine damages plasma membranes of vegetative cells.

Halogens
• Some halogens (iodine and chlorine) are used alone or as components of inorganic or organic
solutions.
• Iodine may combine with certain amino acids to inactivate enzymes and other cellular proteins
• Iodine is available as a tincture or as an iodophor
• The germicidal action of chlorine is based on the formation of hypochlorous acid when chlorine is
added to water

Alcohols
Alcohol exert their action by denaturing proteins and dissolving lipids. In tinctures, their enhance
effectiveness of other antimicrobial chemicals. Aqueous ethanol and isopropanol are used as disinfectants.
Heavy metals and their compounds
Silver, mercury, copper, and zinc are used as germicidals. They exert their antimicrobial action through
oligodynamic action. When heavy metal ions combine with sulfhydryl (-SH) groups, proteins are
denatured.

Surface-Active Agents
These agents decrease the surface tension among molecules of a liquid; soaps and detergents are examples.
Acid-anionic detergents are used to clean dairy equipment.

Chemical Food Preservatives

SO2, sorbic acid, benzoic acid, and propionic acid inhibit fungal metaboliam and are used as food
preservatives. Nitrate and nitrite salts prevent germination of Clostridium botulinum endospores in meats.

Antibiotics
Nisin and natamycin are antibiotics used to preserve foods, especially cheese.

Aldehydes
Aldehydes such as formaldehyde and glutaraldehyde exert their antimicrobial effect by in activating
proteins. They are among the most effective chemical disinfectants.

Peroxygens
Ozone, peroxide, and peracetic acid are used as antimicrobial agents. They exert their effect by oxidizing
molecules inside cells.

Growth Curve
When a few bacteria are inoculated into a liquid growth medium and the population is counted at intervals,
it is possible to plot a bacterial growth curve that shows the growth of cells over time.
There are four phases of growth: the lag, log, stationary and the death phase.
The Lag Phase: Little or no cell division and the cells are not dormant. The microbial population is
undergoing a period of intense metabolic activity involving, in particular, synthesis of enzymes and various
molecules.
The Log Phase: During this phase cellular reproduction is most active and the bacteria multiply at the
fastest rate. Microorganisms are particularly sensitive to adverse condition.
Stationary Phase: In this phase the growth rate slows, the number of microbial death balances the number
of new cells and the population stabilizes.
Death Phase: The number of death cells eventually exceeds the number of new cells formed and the
population enters the death phase.

Mathematical Expression of Growth

When a bacterial culture undergoing balanced growth, the rate of increase in bacteria at any
particular time is proportional to the number or mass of bacteria present at that time.
Rate of increase of cells=k (number or mass of cells)
K is called the growth rate constant
In mathematical terms:
dZ = kZ ………………….. (1)
dt

where Z is the amount of any cellular component/ml, t is the time, and k is the growth rate
constant

Upon integration of equation (1) yields

ln Z-ln Zo = k (t-to) ……………….. (2)

and on converting natural logarithms to logarithms to the base 10

2.303 log10Z - 2.303log10Zo= k (t-t0)

log10Z - log10Zo= k (t-t0)

2.303

K= (log10Z - log10Zo) 2.303 ………….. (3)

(t-t0)

where the values of Z and Zo correspond to the amount of any bacterial component of the culture
at times t and to respectively.

If the culture contains 104 cells/ml and 108 cells/ml at 4 hours later, the specific growth rate of the
culture is the

K= (8 - 4) 2.303 = 2.303/hours
4

How to calculate generation time

Doubling time or generation time defined as the time required for all components of the culture to
increase by a factor of 2 or time required for the cell population to double.
The relationship between g and k can be derived from equation 2.
Since if the time interval considered (t-t0) is equal to g, then Z will be twice Zo. Making these
substitutions, one obtain,

K= 0.693
g

where ln 2 is the natural log of 2 (determine this from your calculator) and g is the time in hours
taken from the population to double during the exponential phase of growth.

From example g= 0.693 = 0.3 hr. or 18 minutes

2.303

Example I: A bacterial culture in exponential growth contained 104 cells/ml at time zero and 108
cells/ml at time 4 h. What is the growth rate constant (k)?
From (1) k = (ln 108 - ln 104)/(4 - 0) = 2.3 h-1
What is the generation time?
From (3) g = ln 2/k = 0.693/2.3 = 0.30 h = 18 min

Example II: E. coli grew exponentially from 5 x 105 cells/ml to 3.5 x 107 cells/ml in 5 h. Its
generation time was 40 min.
Was there a lag phase?
From (1) and (3) (t2 - t1) = (ln N2 - ln N1)/k = g (ln N2 - ln N1)/ln 2 = 40 [ln (3.5 x 107) - ln (5 x
105)]/0.693 = 245 min
Total growth period = 5 h = 300 min
Lag = 300 - 245 = 55 min
Growth Yield:
The net amount of a bacterial culture is the difference between the cell mass used as an inoculum and the
cell mass present in the culture when it enters the stationary phase. When growth is limited by a particular
nutrient, there is a fixed linear relationship between the concentration of the limiting nutrient initially
present in the medium and the net growth. The mass of the cells produced per unit of limiting nutrient is
accordingly, a constant, the growth yield (Y). The value of Y can be calculated from single measurements
of total growth by the equation

Y=X-X0

Where X is the dry weight/ml of cells present when the culture enters the stationary phase, X0 is the dry
weight/ml of cells immediately after inoculation and C is the concentration of limiting nutrient.

Quantitative Measurement of Microbial Growth

Microbial growth to determine growth rates and generation times can be measured by different methods.
Since growth leads to increase both the number and the mass of the populations, either of the two may be
followed. It is necessary to make it clear that no single technique is always best; the most appropriate
approach depends upon the experimental situation.

Direct Measurement of cell numbers

1. Breed method

A known volume of microbial cell suspension (0.01 ml) is spread uniformly over a glass slide covering a
specific area (1 sq. cm). The smear is then fixed by heating, stained, examined under oil immersion lens,
and the cells are counted. Customarily, cells in a few microscopic fields are counted because it is not
possible to scan the entire area of smear. The counting of total number of cells is determined by calculating
the total number of microscopic fields per one square cm. area of the smear. The total number of cells can
be counted with the help of following calculations:
(a) Area of microscopic field= пr2
r(oil immersion lens)=0.08 mm
Area of microscopic field under the oil immersion lens
= пr2= 3.14x (0.08 mm)2 = 0.02 sq. mm

(b) Area of the smear one sq. cm. = 100 sq. mm.
Therefore the no. of microscopic fields= 100/0.02= 5000
(c) No. of cells / 1 sq. cm = Average no. of microbes per microscopic field x 5000 i.e. /0.01 ml of the
suspension.

2. Counting Chamber Technique

The number of cells in a population can be measured by taking direct microscopic count using Petroff-
Hausser counting chamber (for prokaryotic microorganisms) or hemo-cytometers (to larger eukaryotic
microorganisms). Prokaryotic microorganisms are more easily counted if they are stained or if phase I
contrast of florescence microscope is employed. These are specially designed slides that have chambers of
known depth with an etched grid on the chamber bottom. Each square on the grid has definite depth and
volume. Total number of microorganisms in a sample can be calculated by taking the count of number of
bacteria per unit area of grid and multiplying it by a conversion factor (depending on chamber volume and
sample dilution used). However, using this method dead cells are not distinguished from living cells and
also very small cells are usually missed.

Patroff-Hausser bacterial Count

1. Etched Grid 2. Cover Glass 3. Chamber Holding Bacteria

However, using this method dead cells are not distinguished from living cells and also very small cells are
usually missed.

3. Plate/Viable Count

The bacterial culture need not contain all living cells. There might be few dead as well. Only living
cells will form colony when grown in proper solid medium and under standard set or growth
conditions. This fact is used to estimate number of living or dead bacterial cells (viable count) in the
given culture. Estimates thus obtained are expressed as a colony forming unit (CFU).
Viable count technique is very much useful in the dairy industry and the food industry for quantitative
analysis of milk and spoilage of food products. For convenience, to obtain a colony count for bacteria
in milk, 1 ml of well mixed milk is placed in 99 ml of sterile dilute solution (may be water or nutrient
broth or saline solution).
 This results in dilution of 1: 100 or 1 × 10-2. To the petri dish containing pre solidified medium 1 ml of
1: 100 dilution is transferred and incubated at desired is repeated for the preparation of further dilution
as 1 : 1000 or 1 : 10, 0000 of bacteria per ml in original sample can be found by multiplying bacterial
colony count by the reciprocal of the dilution and of the volume used.
For Example, CFU = 50 for 1: 10, 000 if volume used is 1 ml then,
CFU = 50 × 10, 000 × 1
The plate count has certain disadvantages. If the suspension contains different microbial
species, then all of them may not grow on the medium used and under the specified conditions of
growth. Secondly, if the suspension is not homogeneous and contains aggregates of cells, the resulting
colony count will be lower than the actual number of organisms, since each aggregate will produce
only one colony. However, the most obvious advantage of the method is that it counts only living
organisms.

4. Coulter Counter: Coulter counter is an electronic used to count number of microbes

preferably protozoa microalgae and yeasts. In this method, the sample of microbes is forced through a
small orifice (small hole). On the both sides of the orifice, electrodes are present measure the electric
resistance or conductivity when electric current is passed through the orifice. Every time a
microorganism passes through the orifice, electrical resistance increases or the conductivity drops and
the cell is counted. The Coulter counter gives accurate results with larger cells. The precaution to be
taken in this method is that the suspension of samples should be free of any cell debris or other
extraneous matter. The major disadvantages of these methods is that it gives a total cell count which
includes both viable and non viable cells.

Coulter Counter

1. Electrode 2. Flow of Suspending Fluid 3. Bacterial Cell 4. Orifice

5. Electrode 6. Particle Location 7. Measurement of Voltage

5. Membrane-filter technique
Microbial cell numbers are frequently determined using special membrane filters possessing millipores
small enough to trap bacteria. In this technique a water sample containing microbial cells are passed
 through the filter. The filter is then placed on solid agar medium or on a pad soaked with nutrient broth
(liquid medium) and incubated until each cell develops into a separate colony. Membranes with
different pore sizes are used to trap different microorganisms. Incubation times for membranes also
vary with medium and the microorganism. A colony count gives the number of microorganisms in the
filtered sample, and specific media can be used to select for specific microorganisms. This technique is
especially useful in analysing aquatic samples.

Indirect Measurement of Cell mass:

1. Dry weight Technique
The cell mass of a very dense cell suspension can be determined by this technique. In this technique,
the microorganisms are removed from the medium by filtration and the microorganisms on filters are
washed to remove all extraneous matter, and dried in dessicator by putting in weighing bottle
(previously weighted). The dried microbial content is then weighted accurately. This technique is
especially useful for measuring the growth of microfungi. It is time consuming and not very sensitive.
Since bacteria weigh so little, it becomes necessary to centrifuge several hundred millions of culture to
find out a sufficient quantity to weigh.

2. Measurement of nitrogen content

As the microbes (bacteria) grow, there is an increase in the protein concentration (i.e. nitrogen
concentration) in the cell. Thus, cell mass can be subjected to quantitative chemical analysis methods
to determine total nitrogen that can be correlated with growth. This method is useful in determining the
effect of nutrients or antimetabolites upon the protein synthesis of growing culture.

3. Measurement of Turbidity
A most widely used technique of measuring cell mass is by observing the light-scattering capacity of
the sample. A suspension of unicellular organisms is placed in a colorimeter or spectrophotometer, and
the light is passed through it. The amount of the light absorbed or scattered is proportional to the mass
of the cells in the path of light. When cells are growing exponentially, increase in cell mass is directly
related to cell number. One visible characteristic of growing bacterial culture is the increase in
cloudiness of the medium (turbidity). When the concentration of bacteria reaches about 10 million
cells (107) per ml, the medium appears slightly cloudy or turbid. Further increase in concentration
results in greater turbidity. When a beam of light is passed through a turbid culture, the amount of light
transmitted is measured, Greater the turbidity, lesser would be the transmission of light through
medium. Thus, light will be transmitted in inverse proportion to the number of bacteria.

Continuous cultures of microorganisms

Microbial populations can be maintained in a state of exponential growth over a long period of time by
using a system of continuous culture.

The growth chamber is connected to a reservoir of sterile medium. Once the growth has been
initiated, fresh medium is continuously supplied from the reservoir. As the rate of utilization of nutrients
will increase, the culture density increases until the depletion of one nutrient begins to limit the growth rate.

As long as the growth rate exceeds the rate of loss through the siphon overflow, cell density will
increase, the limiting nutrient in the growth vessel continue to decrease and a consequence the growth rate
will decrease until the rate of increase of cells through growth will equal to the rate of loss of cells through
the overflow.

Simplified diagram of a continuous culture system

Under the steady state operation of a continuous culture device, the rate of the addition of the nutrient from
reservoir must equal the rate at which it is utilized by the culture together that lost through the overflow.

(substrate added from reservoir)= (substrate used for growth)+ (substrate lost through overflow)

or DCr = dc/dt + DC

where Cr is the concentration of the limiting nutrient in the reservoir and dc/dt is the rate of loss of
nutrients through overflow and C is the concentration of the limiting nutrient utilized by the culture.

Substitute dX/dt = KX
 And dc/DX=1/Y

We have

DCr = KX/Y + DC

In a steady state, D=K

So, X=Y (Cr – C)

This equation yield relation between cell concentration (X) and the concentration of the limiting nutrient
(C) in the growth vessel.

Cell number and the concentration of the limiting nutrient change little at low dilution rate. As the
dilution rate approaches Kmax (growth rate at saturating concentration of nutrient), cell drops rapidly to zero
and the conc. of limiting approaches its conc. in the reservoir (Cr).

Two Common Major Types of Continuous Culture Systems:

Chemostats: The chemostat is a continuous system cultivator in which the medium is thoroughly mixed
to obtain maximium homogeneity. The fresh medium flows into the culture vessel from a reservoir of
sterile medium at a defined and constant rate. The volume in the culture vessel is kept constant by a device
that allows the culture medium, together with accumulated waste products and older and dead cells, to
leave the culture vessel at the same rate. The level of the growth is controlled by maintaining a fixed,
limiting concentration of a particular nutrient in the medium. The remaining constituents essential for the
growth of the selected organisms are added in the medium in excess of requirement. The growth limiting
nutrient is added into the medium at a concentration below that required for maximum growth in a batch
culture, i.e., a closed vessel.

In a chemostat, indefinite growth at any constant rate can be maintained.

The rate of loss of cells through the overflow ca be expresses as

dM/dt=F/V=DM
Where flow rate F is measured in culture volumes V per hour. The expression F/VM is called the dilution
rate D. In a continous culture, KM=DM
K=D

Under such conditions growth of the culture linear rather than exponential. Since the amount of
mass per unit volume (growth rate) remains constant.
Turbidostat: The turbidostat is an another continuous culture apparatus In a turbidostat the system
includes an optical-sensing device which measures the absorbancy of the culture density (turbidity) in the
growth vessel. Changes in turbidity retard (or increase) passage of light through the culture. These changes
activate mechanism that control the flow of nutrient into, and the flow of waste out, of the main culture
vessel.

Chemostat and turbidostat are usually operated at different dilution rates. The dilution rate is the
ratio of inflowing amount of nutrient medium per hour to the volume of the culture. In the chemostat,
maximum stability is attained within a range of dilution rates over which cell density changes only slightly
with changes in dilution rates, i.e. at low dilution rates. In contrast, in the turbidostat, maximum sensitivity
and stability are achieved at high dilution rates, within a range over which culture density changes with
dilution rate.

The dialysis technique is another device which maintains the culture in the logarithmic phase for a
slightly longer period. In this device fresh nutrients are always available to the culture, and waste products
are continually removed.

Continuous culture systems offer valuable advantages. They provide a constant source of cells in
the logarithmic phase of growth for the study of physiological and genetics of the organisms. Secondly,
these systems allow the cells to be grown continuously in limiting concentration of the nutrient. Such
growth gives valuable information on the catabolism of the limiting substrate. This is very important in
getting rid of a common industrial waste product or a poisonous pollutant.

Synchronous culture of microorganisms

Synchronous culture are composed of populations of cells that are at the same stage of their life cycle. All
the cells in the culture will divide at the same rate, will grow for a generation time, and all will divide again
at the same time. Thus the entire population is kept uniform with respect to growth and division. It is not
possible to analyse a single bacterial cell to obtain the information about growth behaviour. Synchronous
culture provides the entire cell crop in the same stage of growth.

Synchronous culture of bacteria can be obtained by a number of techniques. In the first approach,
a synchronous population of cells can be sorted out according to the age or size by physical separation of
cells. In methods of second type, a culture is induced by manipulating the physical environment or the
chemical composition of the medium to obtain a synchronously dividing population.

The most widely used method for obtaining synchronous cultures is the Helmstetter-Cummings
technique. A population of cells is passed through a membrane filter of pore size small enough to trap
bacteria in the filter. The filter is then inverted, and fresh nutrient is allowed to flow through it. After
loosely associated bacteria are washed from the filter, the only bacterial cells in the effluent stream of the
medium are those which arise through division. If the sample of this stream is collected over a short period
of time, all the cells in this sample are newly formed, and are therefore of the same age and will divide
synchronously. The method has one disadvantage, that the population size is very small and changes in
cellular constituents, therefore cannot be measured.

Helmstetter-Cummings technique

Diauxic growth

Key features:
  In a medium containing two carbon sources, bacteria such display a growth curve which
is called diauxic. J. Monod, about 35 years ago, first observed this phenomenon when be
grew E. coli in a medium containing glucose and lactose
 Microbes have a choice of carbon source in the medium
 Initially the growth curve is the same as a standard growth curve
 The stationary phase is in fact another lag phase
 In this second lag phase new enzymes are being synthesised by the bacteria to utilise the
secondary carbon source
 The second lag phase is then followed by a second exponential phase followed by the
stationary phase and death phase

Arithmetic growth expression

Arithmetic growth refers to the situation where a population increases by a constant number of persons
(or other objects) in each period being analysed.

In certain abnormal situations, the kinetics of bacterial growth may become arithmetic rather than
exponential. Under these circumstances, the rate of increase is a constant, i.e..,

dN/dt=C

where C is constant; and on integration:

N-N0= C(t-to)

The number of cells (N), rather than the logarithm of the number of cells, is a linear function of time (t)

An arithmetic sequence, also called an arithmetic progression, is a sequence that begins with a fixed
number, a , and then each subsequent term is found by adding a constant value, d, called the common
difference.

General Form: a, a + d, a + 2d, a + 3d + . . .

Geometric growth expression:

Geometric growth refers to the situation where successive changes in a population differ by a constant ratio
(as distinct from a constant amount for arithmetic change). Geometric growth where the population is
measured at fixed discrete time intervals whereas the exponential growth is where the population is
measured at any point.

A population may change in size over a discrete time interval as a result of four factors; birth, death,
immigration or emigration. If we consider a closed population where there is no immigration or emigration
we can see that the change in population size over a time interval (ΔN/Δt) is equal to the number of
birth(B) less than the number of death (D) during the same time interval as shown in following expression:

ΔN/Δt=B-D

The change in population size as well as the number of births(B) and death (d) are related to the size of the
population, N, and the rates per capita (i.e. rates/individual) are determined by dividing through the
population size N, at the start of Δt to obtain the following:

ΔN/Δt = B-D

N N

However, the birth rate (B/N) minus the death rate (D/N) is equal to the per capita, or per individual, rate
on increase Rm and so above equation can be written as

ΔN/Δt = Rm

In mathematics, a geometric progression, also known as a geometric sequence, is a sequence of numbers

where each term after the first is found by multiplying the previous one by a fixed non-zero number called
the common ratio. For example, the sequence 2, 6, 18, 54, ... is a geometric progression with common ratio
3. Similarly 10, 5, 2.5, 1.25, ... is a geometric sequence with common ratio 1/2. The sum of the terms of a
geometric progression is known as a geometric series.

Thus, the general form of a geometric sequence is

a, ar, ar2, ar3, ar4……

and that of a geometric series is

a+ ar+ar2+ar3+ar4…

where r ≠ 0 is the common ratio and a is a scale factor, equal to the sequence's start value.
The nth term of a geometric sequence with intial value a and common ratio r is given by:

an=ar n-1

Another kind of model, logistic growth, is a generalized form of geometric growth.

Example of Geometric Growth

o The number of Microorganisms in a culture dish will grow exponentially until an

essential nutrient is exhausted. Typically the first organism splits into two daughter
organisms, who then each split to form four, who split to form 16, and so on.
o A virus ([for example [SARS]], West Nile or smallpox) typically will spread
exponentially at first, if no artificial immunization is available. Each infected person can
infect multiple new people.
o Human population, if the number of births and deaths per person per year were to remain
at current levels (but also see logistic growth).
o Many responses of living beings to stimuli, including human perception, are logarithmic
responses, which are the inverse of exponential responses; the loudness and frequency of
sound are perceived logarithmically, even with very faint stimulus, within the limits of
perception. This is the reason that exponentially increasing the brightness of visual
stimuli is perceived by humans as a linear increase, rather than an exponential increase.
This has survival value. Generally it is important for the organisms to respond to stimuli
in a wide range of levels, from very low levels, to very high levels, while the accuracy of
the estimation of differences at high levels of stimulus is much less important for survival

Sterilization
Sterilization refers to any process that effectively kills or eliminates transmissible agents (such as fungi,
bacteria, viruses, and spore forms etc.) from a surface, equipment, foods, medications, or biological culture
medium. Sterilization does not, however, remove prions. Sterilization can be achieved through application
of heat, chemicals, irradiation, high pressure or filtration.

There are two types of sterilization: physical and chemical.

Physical sterilization includes:

• heat sterilization
• radiation sterilization

Chemical sterilization includes:

• ethylene oxide
• ozone
• chlorine bleach
• glutaraldehyde
• formaldehyde
• hydrogen peroxide
• peracetic acid

Heat Sterilization

Steam sterilization

A widely-used method for heat sterilization is the autoclave. Autoclaves commonly use steam heated to
121 °C or 134 °C. To achieve sterility, a holding time of at least 15 minutes at 121 °C or 3 minutes at 134
°C is required. Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial
spores, which can be quite resistant. It will not necessarily eliminate all prions. For prion elimination,
various recommendations state 121–132°C (270 °F) for 60 minutes or 134 °C (273 °F) for at least 18
minutes.

Cooking and canning are the most common applications of heat sterilization. Boiling water kills
the vegetative stage of all common microbes. Roasting meat until it is well done typically completely
sterilizes the surface. The common methods of cooking food do not sterilize food - they simply reduce the
number of disease-causing micro-organisms to a level that is not dangerous for people with normal
digestive and immune systems. Pressure cooking is analogous to autoclaving and when performed correctly
renders food sterile.

Flaming: A variation on flaming is to dip the object in 70% ethanol (or a higher concentration) and
merely touch the object briefly to the Bunsen burner flame, but not hold it in the gas flame. The ethanol
will ignite and burn off in a few seconds. 70% ethanol kills many, but not all, bacteria and viruses, and has
the advantage that it leaves less residue than a gas flame. This method works well for the glass "hockey
stick"-shaped bacteria spreaders.

Incineration: It is a waste treatment technology that involves the combustion of organic materials and/or
substances. Incineration and other high temperature waste treatment systems are described as "thermal
treatment". Incineration of waste materials converts the waste into incinerator bottom ash, flue gases,
particulates, and heat, which can in turn be used to generate electric power. The flue gases are cleaned of
pollutants before they are dispersed in the atmosphere. Incineration will also burn any organism to ash. It is
used to sanitize medical and other biohazardous waste before it is discarded with non-hazardous waste.

Boiling in water for fifteen minutes will kill most vegetative bacteria and viruses, but boiling is ineffective
against prions and many bacterial and fungal spores; therefore boiling is unsuitable for sterilization.
However, since boiling does kill most vegetative microbes and viruses, it is useful for reducing viable
levels if no better method is available.

Tindalization /Tyndallization: named after John Tyndall is a lengthy process designed to reduce the level
of activity of sporulating bacteria that are left by a simple boiling water method. The process involves
boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, boiling,
cooling, incubating for a day, boiling, cooling, incubating for a day, and finally boiling again. The three
incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to
form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. This is
effective because many spores are stimulated to grow by the heat shock. The procedure only works for
media that can support bacterial growth - it will not sterilize plain water. Tindalization/tyndallization is
ineffective against prions.

Dry heat: Can be used to sterilize items, but as the heat takes much longer to be transferred to the
organism, both the time and the temperature must usually be increased, unless forced ventilation of the hot
air is used. The standard setting for a hot air oven is at least two hours at 160 °C (320 °F). A rapid method
heats air to 190 °C (374 °F) for 6 minutes for unwrapped objects and 12 minutes for wrapped objects. Dry
heat has the advantage that it can be used on powders and other heat-stable items that are adversely affected
by steam (for instance, it does not cause rusting of steel objects).

Prions can be inactivated by immersion in sodium hydroxide (NaOH 0.09N) for two hours plus
one hour autoclaving (121 °C/250 °F). Several investigators have shown complete (>7.4 logs) inactivation
with this combined treatment. However, sodium hydroxide may corrode surgical instruments, especially at
the elevated temperatures of the autoclave.

Chemical sterilization
Chemicals are also used for sterilization. Although heating provides the most reliable way to rid objects of
all transmissible agents, it is not always appropriate, because it will damage heat-sensitive materials such as
biological materials, fiber optics, electronics, and many plastics.

Ethylene Oxide

Ethylene oxide gas is commonly used to sterilize objects such as plastics, optics and electrics. Ethylene
oxide treatment is generally carried out between 30 °C and 60 °C with relative humidity above 30% and a
gas concentration between 200 and 800 mg/L for at least three hours. EtO can kill all known viruses,
bacteria and fungi, including bacterial spores and is satisfactory for most medical materials, even with
repeated use. However it is highly flammable, and requires a longer time to sterilize than any heat
treatment. The process also requires a period of post-sterilization aeration to remove toxic residues.
Ethylene oxide is the most common sterilization method, used for over 70% of total sterilizations, and for
50% of all disposable medical devices.

Spore testing

Bacillus subtilis, a very resistant organism, is used as a rapid biological indicator for EO sterilizers. If
sterilization fails, incubation at 37 °C causes a fluorescent change within four hours, which is read by an
auto-reader. After 96 hours, a visible color change occurs. Fluorescence is emitted if a particular (EO
resistant) enzyme is present, which means that spores are still active. The color change indicates a pH shift
due to bacterial metabolism. The rapid results mean that the objects treated can be quarantined until the test
results are available.

Ozone

Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for surfaces. It has the
benefit of being able to oxidize most organic matter. On the other hand, it is a toxic and unstable gas that
must be produced on-site, so it is not practical to use in many settings.

Bleach

Chlorine bleach is another accepted liquid sterilizing agent. Household bleach consists of 5.25% sodium
hypochlorite. It is usually diluted to 1/10 immediately before use; however to kill Mycobacterium
tuberculosis it should be diluted only 1/5, and 1/2.5 (1 part bleach and 1.5 parts water) to inactivate prions.
The dilution factor must take into account the volume of any liquid waste that it is being used to sterilize.
Bleach will kill many organisms immediately, but for full sterilization it should be allowed to react for 20
minutes. Bleach will kill many, but not all spores. It is highly corrosive and may corrode even stainless
steel surgical instruments.
Glutaraldehyde and Formaldehyde

Glutaraldehyde and formaldehyde solutions (also used as fixatives) are accepted liquid sterilizing agents,
provided that the immersion time is sufficiently long. To kill all spores in a clear liquid can take up to 12
hours with glutaraldehyde and even longer with formaldehyde. The presence of solid particles may
lengthen the required period or render the treatment ineffective. Sterilization of blocks of tissue can take
much longer, due to the time required for the fixative to penetrate. Glutaraldehyde and formaldehyde are
volatile, and toxic by both skin contact and inhalation. Glutaraldehyde has a short shelf life (<2 weeks), and
is expensive. Formaldehyde is less expensive and has a much longer shelf life if some methanol is added to
inhibit polymerization to paraformaldehyde, but is much more volatile. Formaldehyde is also used as a
gaseous sterilizing agent; in this case, it is prepared on-site by depolymerization of solid paraformaldehyde.
Many vaccines, such as the original Salk polio vaccine, are sterilized with formaldehyde.

Phthaladehyde

Ortho-phthalaldehyde (OPA) is a chemical sterilizing agent that received Food and Drug Administration
(FDA) clearance in late 1999. Typically used in a 0.55% solution, OPA shows better myco-bactericidal
activity than glutaraldehyde. It also is effective against glutaraldehyde-resistant spores. OPA has superior
stability, is less volatile, and does not irritate skin or eyes, and it acts more quickly than glutaraldehyde. On
the other hand, it is more expensive, and will stain proteins (including skin) gray in color.

Hydrogen Peroxide

Hydrogen peroxide is another chemical sterilizing agent. It is relatively non-toxic once diluted to low
concentrations (although a dangerous oxidizer at high concentrations), and leaves no residue.

Low Temperature Plasma sterilization chambers use hydrogen peroxide vapor to sterilize heat-
sensitive equipment such as rigid endoscopes. A recent model can sterilize most hospital loads in as little as
20 minutes. The Sterrad has limitations with processing certain materials such as paper/linens and long thin
lumens. Paper products cannot be sterilized in the Sterrad system because of a process called cellulostics, in
which the hydrogen peroxide would be completely absorbed by the paper product.

Hydrogen peroxide and Formic acid

Hydrogen peroxide and formic acid are mixed as needed in the Endoclens device for sterilization of
endoscopes. This device has two independent asynchronous bays, and cleans (in warm detergent with
pulsed air), sterilizes and dries endoscopes automatically in 30 minutes. Studies with synthetic soil with
bacterial spores showed the effectiveness of this device.
Dry sterilization process

Dry sterilization process (DSP) uses hydrogen peroxide at a concentration of 30-35% under low pressure
conditions. This process achieves bacterial reduction of 10-6...10-8. The complete process cycle time is just 6
seconds, and the surface temperature is increased only 10-15 °C (18 to 27 °F). Originally designed for the
sterilization of plastic bottles in the beverage industry, because of the high germ reduction and the slight
temperature increase the dry sterilization process is also useful for medical and pharmaceutical
applications.

Peracetic acid

Peracetic acid (0.2%) is used to sterilize instruments in the Steris system.

Radiation sterilization

Methods exist to sterilize using radiation such as electron beams, X-rays, gamma rays, or subatomic
particles.

• Gamma rays are very penetrating and are commonly used for sterilization of disposable medical
equipment, such as syringes, needles, cannulas and IV sets. Gamma radiation requires bulky
shielding for the safety of the operators; they also require storage of a radioisotope (usually
Cobalt-60), which continuously emits gamma rays (it cannot be turned off, and therefore always
presents a hazard in the area of the facility).
• Electron beam processing is also commonly used for medical device sterilization. Electron beams
use an on-off technology and provide a much higher dosing rate than gamma or x-rays. Due to the
higher dose rate, less exposure time is needed and thereby any potential degradation to polymers is
reduced. A limitation is that electron beams are less penetrating than either gamma or x-rays.
• X-rays are less penetrating than gamma rays and tend to require longer exposure times, but require
less shielding, and are generated by an X-ray machine that can be turned off for servicing and
when not in use.
• Ultraviolet light irradiation (UV, from a germicidal lamp) is useful only for sterilization of
surfaces and some transparent objects. Many objects that are transparent to visible light absorb
UV. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between
uses, but is ineffective in shaded areas, including areas under dirt (which may become
polymerized after prolonged irradiation, so that it is very difficult to remove). It also damages
many plastics, such as polystyrene foam.
• Subatomic particles may be more or less penetrating, and may be generated by a radioisotope or a
device, depending upon the type of particle.