Microbial nutrition and growth 3

MODULE 3

MICROBIAL NUTRITION, CULTIVATION AND GROWTH

MICROBIAL NUTRITION
To obtain and construct new cellular components, organisms must have a supply
of raw materials or nutrients. Nutrients are substances used in biosynthesis and energy
production and therefore are required for microbial growth. The nutritional
requirements of microorganisms vary enormously among different species.

Growth Requirements for Microorganisms

A characteristic of microorganisms is their ability to grow and form a population
of organisms. One of the results of microbial metabolism is an increase in the size of the
cell. The many requirements for successful growth include those both chemical and
physical.

Two subgroups : physical and chemical

➢ Physical include: Temperature, pH and osmotic pressure.
➢ Chemical include: water, carbon, nitrogen, oxygen, hydrogen, sulfur,
phosphorous and trace elements.

Physical requirements

A. Temperature
• most microorganism grow well at the temperature favored by humans
• some bacteria capable of growing in an extreme temperature
• Classification of microorganisms:
1. psychrophiles – cold loving microorganism, some can grow at 0C but has
an optimum temperature of about 15C, they will not grow in warm temperature
at 25C.
2. psychrotrophs – cold loving organism but can grow at 20-30C but cannot
grow at about 40C, they are responsible for low temperature food spoilage, they
fairly grow well at refrigerator temperatures.
3. Mesophiles - moderate temperature loving microbes., with optimum growth
temperature of 25-40C, are the most common type of microbes. The optimum
temperature for many pathogenic bacteria is at 37C, that’s why all clinical
incubators are set at this temperature.

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4. Thermophiles –heat loving microbes, organism capable of growing at high

temperature, 50-60C, they cannot grow at temperature below 45C. They are
important organic compost piles.
5. Hyperthermophiles – microbes that can grow at 80C or even higher, such as
members of archaebacteria. Highest temperature in which bacteria can grow is
110C.
Incubation temperature:
• minimum growth temp-lowest temperature needed for the bacteria to
grow
• optimum growth temperature – in which species grows best
• maximum growth temp – highest temperature for the bacteria to grow
•
B. pH –refers to the alkalinity or acidity of a solution
• most bacteria grow best in a narrow pH range near neutrality, between pH 6.5 to
7.5.
• very few bacteria grow at acidic pH 4. This is why most foods are preserved from
spoilage by acid produced by bacterial fermentation.
• Acidophiles – are bacteria that can grow and tolerate acidity.
• Molds and yeast grow at slightly acidic environment with pH range of 5-6 ;
Ex: Saccharomyces cerevisiae
• Chemical buffers such as peptones, amino acid or phosphate are added to the
medium to neutralize the acid produced.

C. Osmotic pressure – related to the numbers of dissolved molecules or ions in a

solution ; Normally, the salt concentration of microbial cytoplasm is about 1 percent.
When the external environment also has a 1 percent salt concentration, then the
osmotic pressure is optimum
• microbes need a certain osmotic pressure to maintain integrity and get nutrients.
• most microorganisms obtained most of their nutrients from surrounding water.
High osmotic pressure has the tendency of removing water from a cell.
• When a microbe is in hypertonic solution, water will leave the cell and it will
cause shrinkage of the cell’s plasma membrane (plasmolysis).
• Growth of the cell is inhibited as the plasma membrane pulls away from the cell
wall, thus the addition of salt can be used to preserve foods.
• Halophiles – are microorganism that is adapted to high salt concentration that
they actually require them for growth ; They include diatoms and dinoflagellates,
two types of unicellular algae that lie at the base of oceanic food chains. There
are many other species of halophilic bacteria, fungi, protozoa, and algae.
• Obligate halophiles require high salt concentrations to survive

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• Facultative halophiles – require 2-5%

Chemical requirements

Nutrient is something that nourishes or promotes growth and repairs the natural
wastage of organic life. Nutrients may be divided into three general, often overlapping
categories: Those that supply energy, those that supply carbon, and those that supply
any and everything else. In this lecture we will consider nutrition from the point of view
of microorganisms, though many of the principles apply to macroorganisms equally
well.
A. Water - microbes require water for growth and are made of 80-90% water.

B. Carbon- structural backbone of living matter ; Carbon can be obtained from

organic materials in the environment, or it may be derived from carbon dioxide ;
• Autotrophs is using CO2 as their starting compound, they are known
ecologically as the producers ; Indeed, autotrophs are "self-feeders."
• Heterotroph or organotroph -An organism which uses organic compounds as
its principle source of carbon ; Heterotrophs feed on others and obtain their
carbon compounds by consuming other organisms ; Heterotrophs are
thus consumers, decomposers, scavengers, predators, herbivores, etc.
• Photoautotroph - An organism whose energy source is light and whose
principle carbon source is carbon dioxide ; Photoautotrophs include plants,
algae, cyanobacteria, as well as some photosynthesizing bacteria.
• Photoheterotroph - An organism whose energy source is light and which uses
organic compounds as its principle source of carbon ; These organisms are
unable to convert CO2 to sugar nor produce O2.
• Chemoautotroph - -An organism whose energy sources are electron donating
compounds and whose principal carbon source is carbon dioxide.
Chemoautotroph constitutes the producers at the deep sea vents, i.e., they
extract energy from inorganic compounds dissolved in sea water which are
vented at these locations.
• Chemoheterotroph - An organism whose energy source is electron donating
compounds and which uses organic compounds as its principal source of carbon.
; Humans are an example.
• Medically relevant microorganisms are almost always chemoheterotrophs. This is
because pathogens tend to derive both their energy and their carbon from
organic compounds obtained from their hosts such as human bodies.

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• In some cases, some organisms must have an environment rich in carbon dioxide.
Organisms such as these are said to be capnophilic.

C. Oxygen - is used by aerobic bacteria during the process of cellular respiration as

a final electron acceptor.
• For aerobic organisms, oxygen is an absolute requirement for their growth
for energy-yielding properties. Certain microorganisms can grow in the
absence of oxygen and are described as anaerobic (Clostridium species);
facultative anaerobes, obligate anaerobes, aerotolerant anaerobes,
microaerophiles ;
• Facultative anaerobes – species that grow in the presence or absence of
oxygen ;
Ex: E. coli (Enterics) and most importantly yeast.
• Obligate anaerobes cannot use molecular oxygen or do not tolerate the
presence of oxygen and most are harmed by it ; Species of Bacteriodes,
Fusobacterium, Clostridium, Methanococcus are examples of obligate
anaerobes. Harmful oxygen includes H2O2, Superoxide (O2-) and hydroxyl
radical (OH.) ; Many obligate anaerobes don’t have the enzymes to deal with
these reactive oxygen species (ROS).
• Aerotolerant anaerobes cannot use oxygen but tolerate it well, they can
grow well even with or without oxygen ; Enterococcus faecalis
• Microaerophilic are species of bacteria that can grow in low concentrations
of oxygen (2-10%) such as Campylobacter

D. Nitrogen– needed for synthesis of protein and other cellular material(DNA and
RNA) ; Bacteria that obtain nitrogen directly from the atmosphere are called
nitrogen-fixing bacteria. They include species of Rhizobium and Azotobacter,
both found in the soil.
E. Sulfur – use in protein synthesis
F. Phosphorus - is an essential element for nucleic acid synthesis and for the
construction of
phospholipids.
G. Trace elements : copper, iron, molybdenum, and zinc - These elements often
are used for the synthesis of enzymes
H. Organic growth factors such as vitamins may also be required by certain
bacteria. Amino acids, purines, and pyrimidines should also be available. Some
but not all organisms are able to synthesize all 20 amino acids directly from

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intermediates of carbohydrate metabolism ; Escherichia coli is an example of an

organism which can synthesize all 20 amino acids.

Fastidious bacteria - means to have complex nutrient requirements.

Examples of fastidious genera include: Neisseria, Mycoplasma, Mycobacterium
A fastidious microorganism is one whose nutrient requirements include a significant
number of organic growth factors.

MICROBIAL CULTIVATION/ CULTURE MEDIA

When microorganisms are cultivated in the laboratory, a growth environment

called a medium is used. The medium may be purely chemical (a chemically defined
medium), or it may contain organic materials, or it may consist of living organisms such
as fertilized eggs. Microorganisms growing in or on such a medium form a culture. A
culture is considered a pure culture if only one type of organism is present and a mixed
culture if populations of different organisms are present. When first used, the culture
medium should be sterile, meaning that no form of life is present before inoculation
with the microorganism.

Culture media is a nutrient material prepared for the growth of microorganisms

in the laboratory. For the cultivation of bacteria, a commonly used medium is nutrient
broth, a liquid containing proteins, salts, and growth enhancers that will support many
bacteria. To solidify the medium, an agent such as agar is added. Agar is a
polysaccharide that adds no nutrients to a medium, but merely solidifies it. The medium
that results is nutrient agar. Important properties: microbes cannot degrade it, liquefies
at 100oC and solidifies at 40oC.

Some bacteria can grow well on just about any culture medium, others require
special media, others still cannot grow on any non living medium. Most fungi require
extra carbohydrate and an acidic environment for optimal growth. The medium
employed for these organisms is potato dextrose agar, also known as Sabouraud
dextrose agar. For protozoa, liquid media are generally required, and for rickettsiae and
viruses, living tissue cells must be provided for best cultivation. For anaerobic
microorganisms, the atmosphere must be oxygen free.

• Inoculum – refers to microbes that are introduce into a culture media

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• Culture – refers to the microbes that grow and multiply in or on a culture medium.
• Liquid medium – prepare, dispense in tubes and then autoclave
• Solid medium – prepare, sterilize/autoclave, then dispense. There is a possibility of
contamination.
• Cotton plugs is used to cover test tubes to allow oxygen to enter the tube to support
aerobic bacteria.

Synthetic or defined media - Chemically defined medium is one in which the exact
chemical composition is known.
• Used for laboratory experimental work, research or culturing autotrophic bacteria.
• Simple media for growing cyanobacteria and eukaryotic algae contains carbon
dioxide containing compound as source of carbon, nitrate or ammonia as
nitrogen source, sulfate, phosphate and a variety of minerals.

Complex media is made up of nutrients from extracts of yeasts, meat, plants or digests
of protein.
• Very useful in culturing many different microorganism since it has sufficient
nutritional requirements.
• Contain components like peptones, meat extract, and yeast extract. Beef extract
contains amino acids, peptides, nucleotides, organic acids, vitamins, and minerals.
• Yeast extract is an excellent source of B vitamins as well as nitrogen and carbon
compounds.
• Examples: nutrient broth , nutrient agar, trypticase soy broth, and MacConkey agar.
• Solid media- 1.5-2% agar
• Semi-solid – 0.5% agar

Classification of culture media according to its used

1. Simple culture media – such as nutrient agar or broth, support non fastidious
bacteria.
2. Enrichment culture media – supports the growth of fastidious microbes, added
ingredient increase the number of growing bacteria. Examples : Brain heart
Infusions, BAP,CAP ; – Enrichment media is usually a liquid media and provides
nutrients and environmental conditions that favor the growth of a particular
microbe. When attempting to isolate Salmonella species from fecal samples, for
instance, it is helpful to place a sample of the material in an enriched medium to
encourage Salmonella species to multiply before the isolation techniques begin.

3. Selective culture media

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• Favor the growth of particular microorganisms ; used to suppress the

growth of unwanted bacteria and encourage the growth of desired
microbes.
• Bile salts or dyes like basic fuchsin and crystal violet favor the growth of
gram-negative bacteria by inhibiting the growth of gram positive.
• sodium azide, 7.5% NaCl, Colistin nalidixic acid are added which favors
Gram (+) bacteria
• McConkey – allows gram negative bacteria to grow.
• Manitol Salt Agar – allows gram positive bacteria to grow and halophilic
microbes such as Staph. Aureus;
• Sabourad’s dextrose agar which has a pH of 5.6 is used to isolate fungi
because of pH.

4. Differential culture media

• Media that distinguish between different groups of bacteria and even
permit tentative identification of microorganisms based on their biological
characteristics ; media make it easier to distinguish between colonies of
the desired organism from other colonies.
• Blood agar is both a differential medium and an enriched one. It
distinguishes between hemolytic and non hemolytic bacteria. (produce
clear zone around their colonies because of red blood cell destruction.
• Streptococcus pyogenes the bacteria causes lysis of the blood cells in
blood agar and makes a ring around the cells. Differentiates hemolytic
pattern of microorganisms as to :
Alpha-hemolytic ----with greenish zone of hemolysis
Beta-hemolytic ---with clear zone of hemolysis
Gamma-hemolytic or non hemolytic ---no zone of inhibition

• McConkey – differentiate lactose (pink-E.coli) from non lactose fermenters

(colorless – pseudomonas/ Shigella/ Salmonella) ;
• Certain media are both selective and differential. For instance, McConkey
agar differentiates lactose-fermenting bacteria from non lactose-
fermenting bacteria while inhibiting the growth of Gram-positive bacteria.

Anaerobic growth media and method

• Must use reducing media that contain chemicals like sodium thioglycolate that
combine with oxygen to or remove it.
• Labs may have special incubators for anaerobes and capnophiles (microbes that
grow better with increased CO2)

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• If you need to grow an anaerobe on a plate a anaerobic jar.candle jar may be used
• Pouch containing NaHCO3 , Naborohydrate,
• N. miningitidis and N. gonorrheae/Streptococcus pyogenes

Special culture technique

• Some organisms cannot be cultured easily on laboratory media.
– Example: Mycobacterium leprae cultured in armadillos.

ISOLATION METHODS

• One is the streak plate method, in which a sample of mixed bacteria is streaked
several times along one edge of a Petri dish containing a medium such as
nutrient agar. A loop is flamed and then touched to the first area to retrieve a
sample of bacteria. This sample is then streaked several times in the second area
of the medium. The loop is then reflamed, touched to the second area, and
streaked once again in the third area. The process can be repeated in a fourth
and fifth area if desired. During incubation, the bacteria will multiply rapidly and
form colonies.
• Second isolation method is the pour plate method. In this method, a sample of
bacteria is diluted in several tubes of melted medium such as nutrient agar. After
dilution, the melted agar is poured into separate Petri dishes and allowed to
harden. Since the bacteria have been diluted in the various tubes, the plates will
contain various dilutions of bacteria, and where the bacteria are most diluted,
they will form isolated colonies

MICROBIAL GROWTH
Reproduction patterns: During their growth cycles, microorganisms undergo
reproduction many times, causing the numbers in the population to increase
dramatically.
In fungi, unicellular algae, and protozoa, reproduction involves a duplication of
the nucleus through the asexual process of mitosis and a splitting of the cell in
cytokinesis. Reproduction can also occur by a sexual process in which haploid nuclei
unite to form a diploid cell having two sets of chromosomes. Various changes then
follow to yield a sexually produced offspring. Sexual reproduction has the advantage of
mixing chromosomes to obtain genetic variations not possible with asexual
reproduction.
Bacteria reproduce by the asexual process of binary fission. In this process, the
chromosomal DNA duplicates, after which the bacterial membrane and cell wall grow

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inward to meet one another and divide the cell in two. The two cells separate and the
process is completed.
One of the remarkable attributes of bacteria is the relatively short generation
time, the time required for a microbial population to double in numbers. The generation
time varies among bacteria and often ranges between 30 minutes and three hours.
Certain bacteria have very brief generation times. Escherichia coli, for example, had a
generation time of about 20 minutes when it is dividing under optimal conditions. After
20 generations a single cell of E. coli would be over 1,000,000 ; 30 generations = 10
hours = 1,000,000,000
• Difficult to graph such high numbers that is why logarithmic scales are
used, bacterial growth curve
• Logrithmic representations are much easier to graph and is necessary for proper
understanding of microbial populations.

The growth curve. The growth of a bacterial population can be expressed in various
phases of a growth curve. The logarithms of the actual numbers in the population are
plotted in the growth curve along the side axis, and the time is plotted at the base. Four
phases of growth are recognized in the growth curve.
a. Lag phase - the population remains at the same number as the bacteria become
accustomed to their new environment. Metabolic activity is taking place including DNA
and enzyme synthesis, and new cells are being produced to offset those that are dying.
b. Log phase - or exponential growth phase-bacterial growth occurs at its optimal
level and the population doubles rapidly. This phase is represented by a straight line,
and the population is at its metabolic peak. Research experiments are often performed
at this time ; Generation time equals a constant minimum rate that is why you see a
straight line ; Microbes are at their most sensitive here many antimicrobial agents work
best during this phase by interfering with growth
c. Stationary phase - reproduction of bacterial cells is offset by their death, and the
population reaches a plateau. The reasons for bacterial death include the accumulation
of waste, the lack of nutrients, and the unfavorable environmental conditions that may
have developed. If the conditions are not altered, the population will enter its decline,
or death phase.
d. Decline phase or death phase or logarithmic decline - The bacteria die off rapidly,
the curve turns downward, and the last cell in the population soon dies.

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A growth curve of a bacterial population showing the four major phases of the curve.

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CULTURE MEDIA

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