GENERAL MICROBIOLOGY

Chapter 5: Microbial
Growth and Reproduction
 FTEC 101
CHAPTER 5: MICROBIAL GROWTH & REPRODUCTION

OVERVIEW
Microbes are a varied group of organisms. Microbial growth is defined as a spike
in the amount of cells rather than the size of the cells. Microorganisms reproduce
several times during their development cycles, resulting in a substantial rise in
population size. To develop and live, various microorganisms (microbes) require
different conditions. These are physical or chemical features that characterize the
microbe's habitat. The physical factors include the temperature, pH, osmotic
pressure, radiation, moisture, hydrostatic pressure, and tonicity. Meanwhile, the
chemical factors includes the carbon and organic compounds, the nitrogen, sulfur,
phosphorus and other trace elements, and the oxygen.

Culturing bacteria is one of the latest practice of microbiologist in laboratory for

specific purpose. A culture is a group of microorganisms that has been cultured in
a laboratory. A culture medium, also known as a growth medium, is a liquid or gel
that aids in the development of microorganisms. There are several types of media
that may be used to grow various types of cells. Isolating a single variety of
bacteria from a source that contains numerous is an essential microbiology
technique. The streak plate approach is the most effective way to achieve this.
Other methods are selective media, differential media, enrichment media, and
etc.
LEARNING OBJECTIVES
At the end of this session, learners should be able to:

Identify the physical and chemical requirements and methods for the
growth and reproduction of microbes.

Share their insights on the importance of reproduction and growth

of microbes.

Apply knowledge on culturing bacteria and media culture.

PRE-ASSESSMENT
1. Which of the following elements influences the microorganism's reproductive
rate?
a. pH range
b. Nutrients
c. Oxygen
d. All of the above

2. Which of the following is the best definition of generation time in a bacterium?

a. The length of time it takes to reach the log phase
b. the length of time it takes for a population of cells to double
c. the time it takes to reach stationary phase
d. the length of time of the exponential phase.

3. What pH is best for the majority of bacterial growth?

a. pH 3
b. pH 5
c. pH 7
d. pH 12

4. What form of medium would you use if you wanted to select for a certain type
of bacteria?
a. Chemical Media
b. Selective Media
c. Differential Media
d. Nutrient broth

5. Which of the following media is an example of enrichment media?

a. Nutrient agar
b. Chocolate agar
c. Blood agar
d. All of the above
 INTRODUCTION
In the previous week, you learned the introduction of
microbial growth including the 4 phases, and the
process of measuring the number of microbes. As
part of learning outcome, you already compare and
contrast microbial growth and reproduction and its
4 phases. Now, we will move deeper on
understanding what are the physical and chemical
factors that may affect the microorganism growth
and development. Figure 1. Bacteria

RECALL
Figure 2. Microbes spiral shape
https://www.sciencenewsforstudents.org/article/genes-point-
to-how-some-bacteria-can-gobble-up-electricity
Reproduction of eukaryotes and microbes are
through asexual and sexual reproduction. The
creation of offspring is referred to as
reproduction. There are two types of
reproduction: sexual and asexual. When an
organism reproduces sexually, it integrates the
genetic information from each of its parents to
create a genetically distinct creature. One
parent duplicates itself to produce a genetically
identical child in asexual reproduction.

In fungi, unicellular
algae, and protozoa,
reproduction includes the
asexual process of mitosis,
which involves the
duplication of the nucleus,
and cytokinesis, or known
as the splitting of the cell. Figure 3. Reproduction process
https://edusaint.com/study/courses/class-10th-biology-lessons-based-on-ncert-
syllabus-free/lessons/how-do-organisms-reproduce-class-10th-notes-on-science/

Asexual reproduction occurs when two haploid nuclei join together to generate a
diploid cell with two sets of chromosomes. The offspring is subsequently subjected to a
series of alterations that result in a sexually generated offspring. Sexual reproduction
on the other hand has the benefit of mixing chromosomes to produce genetic variants
that asexual reproduction does not. Sexual reproduction, on the other hand, produces
fewer individuals than asexual reproduction.
 DISCUSSION

The Bacterial Growth

Microbes may thrive in a wide range of habitats because they are tiny, readily
disseminated, require only minute amounts of resources, and have a wide range of
dietary needs. The following are the factors and requirements for microbial growth.

A. Physical Factors
Temperature. The most essential element
influencing microorganism growth and
survival is temperature. Microbes can be
harmed by high temperatures because
enzymes, transport carriers, and other
proteins are denaturized. Extreme
temperatures disrupt microbial membranes.
At a very low temperature, however, it
solidifies, preventing the enzymes from
working correctly.
Bacteria have a minimum, optimal, and maximum growth temperature, and may
be categorized into three classes depending on this temperature:

1. Psychrophiles. The word "psychrophiles"

was fisrt use by S. Schmidt-Nelson. This
bacteria are called as "Extremophilic"
organisms that are capable of growth in cold
temperature. Their ideal growing temperature
is between -5 and 15 degrees Celsius. They
are most commonly found in the Arctic and
Antarctic regions, as well as in glacier-fed
streams. Examples are: Oscillatoria,
Chlamydomonas nivales, Methanogenium and etc.
Figure 6. Oscillatoria Bacteria

2. Mesophiles. This is the type of bacterium

that thrives in a temperate environment. Their
ideal growing temperature is between 25 and
45 degrees Celsius. The majority of bacteria,
including ordinary soil bacteria and bacteria
that dwell in and on the body, are mesophilic.
Examples are:Streptococcus , Escherichia Coli and etc.

Figure 7. E. Coli

 Mesophiles are microorganisms that thrive at temperatures in the

middle of the range, such as Bacteria, Fungi, and even Archaea.

3. Thermophiles-heat-loving. Microorganisms
known as "thermophiles" have been isolated from a
variety of marine and terrestrial geothermally
heated habitats, including shallow terrestrial hot
springs, hydrothermal vent systems, volcanic island
sediment, and deep sea hydrothermal vents, with
optimal growth temperatures ranging from 60 to
108 degrees Celsius. Example: Thermus aquaticus,
Geogemma barossii and etc.
Figure 8. Thermus aquaticus

4. Hyperthermophiles. Bacteria that thrive at extremely high temperatures. Their

ideal growing temperature is between 70 and 110 degrees Celsius. They are often
Archaea members that thrive around hydrothermal vents at vast depths in the
ocean. Archaea is home to a large number of hyperthermophiles. Example:
Pyrolobus fumarii (an archaeon that can survive at 113 degrees Celsius in Atlantic
hydrothermal vents), Pyrococcus furiosus (an archaeon that can survive at 100
degrees Celsius), Methanococcus jannaschii and etc.

pH Level
pH stands for negative logarithm of hydrogen
ions, and a dramatic shift in pH can disrupt
bacteria's plasma membrane. The following are
the bacteria that live in a particular pH range.

Acidophiles (acid-loving) – grow best at a pH

of 1 to 5.4; Ex. Lactobacilllus (ferments milk)

Neutrophiles – exist from pH to 5.4 to 8.5; most

bacteria that cause human disease are in this
category; Ex. Pseudomonos aerunginosa

Alkaliphiles (base loving) – exist from pH to 7.0

to 11.5; ex. Vibrio cholerae (causes cholera) and
Thermococcus alcaliphilus.
 Most bacteria grow best in a pH range of 6.5 to 7.0, however others
thrive in extremely acidic environments and can even withstand pH
levels as low as 1.0.

Osmotic Pressure
Water produces
osmotic pressure on the
semi-permeable membrane
(plasma membrane) that
surrounds the cell. In
reaction to an uneven
distribution of dissolved
solutes in the environment,
water flows across the
plasma membrane. Water
Activity = Vapor Pressure of
Solution/ Vapor Pressure of
Pure Water
Classification of Microorganism based on
osmotic pressure
Osmotolerant. Are those microorganisms which
can grow at relatively high salt concentration.
Example: Aeromonas spp., Staphylococcus spp.,
and etc.
Halophiles. Grow in the presence of salt at
concentration above 0.2 to 0.6. Ex.
Halobacterium.
Figure 10. Halophiles

Moisture. Another physical factors Tonicity. The use of salt as a preservative

include the moisture of the in curing meats and the use of sugar in
surroundings. nly the spores of sport- creating jellies is based on the concept
forming bacteria can exist in a that a hypertonic environment kills or
dormant state in a dry environment. inhibits microbiological development.
Oceans are home to halophiles (salt-
Hydrostatic pressure. Some bacteria lovers).
can only live in high hydrostatic
pressure conditions (ex. ocean Radiation. UV and gamma radiation have
valleys in excess of 7000 meters); the potential to induce DNA alterations
the high pressure is required to and even kill microbes. Some bacteria
preserve their enzymes in the have enzyme systems that are capable of
appropriate 3-D structure; without it, repairing mutations.
the enzymes lose their shape and
denature, and the cell dies.
 B. Chemical Factors

Various microorganisms have variable nutritional needs, as well as different

amounts of each nutrient. A macronutrient is a nutrient that organisms require in larger
concentrations, whereas a micronutrient is a chemical that an organism requires in tiny
amounts to survive. A trace element is a nutrient that is required in minute quantities.
Some trace elements are also available in the environment at extremely low quantities,
whereas macronutrients and micronutrients are more plentiful and simpler for
microorganisms to access. All microorganisms require certain ingredients in order to
grow.
Oxygen Requirements

Aerotolerant. This are Strict or obligate aerobes. Lack of

bacteria that do not use
oxygen, it does not hurt
them. Example, Lactobacillus
Microaerophiles. This are
bacteria that has aLow
oxygen levels and high
carbon dioxide levels, for
example Campylobacter,
oxygen kills the bacteria; ex.
Pserdomonas.
Facultative anaerobes. Can shift
their metabolism (anaerobic if
oxygen is absent or aerobic if
oxygen is present); ex. E. coli,
Staphylococcus
Strict or obligate anaerobes. Oxygen
kills the bacteria; ex. Clostridium
tetani.

Nutritional (Biochemical) Factors

Every microorganism may be classified according to how it obtains sustenance.

Microbes use two types of nutrient transport: passive, which requires no energy, and
active, which requires energy. An autotroph synthesizes some nutrients using substances
it uptakes, whereas a heterotroph receives all of its nutrients from its environment. A
chemoautotroph is a living creature that gets its energy from chemical interactions
with specific organic or inorganic substances, such as sulfur compounds. A
photoautotroph converts carbon dioxide into sugar using light energy.

Nitrogen - Amino acids and nucleotides Sulfur – needed for amino acids,
are required; some organisms can synthesis coenzymes.
all 20 amino acids, while others must have
some in their medium.

Carbon – carbon containing compounds Vitamins – Many bacteria produce

are needed as an energy source (ex.
glucose) and for building blocks.
Phosphorus – ATP, phospholipids,
nucleotides all require it.
their own, but some require the
presence of a medium; germs
residing in the human gut produce
and vitamin K, which is essential for blood
clotting, as well as several B vitamins,
so helping their host.
 CULTURING BACTERIA
Bacterial culture is a technique for growing
bacteria in or on a culture medium under
carefully regulated laboratory conditions.
Depending on the target bacterial species, the
exact circumstances necessary for optimum
replication will vary.

How to culture bacteria?

In bacterial culture methods, bacteria must be provided with nutrients in the

culture media in order to be cultivated successfully. There are a variety of
formulations available to meet the nutritional requirements of different bacterial
species. Also, the sort of medium you use will be determined by the culture's goal.
When trying to bulk up a pure culture and get the bacterial cells in good shape,
rich, nutrient, or complete medium might be beneficial. Minimal media, on the
other hand, will only provide the minimal necessary for survival and may be used
to control which pathways in the bacteria are activated.

Sample agar in laboratory
What is media?
Media is a tool use to cultivate a
bacteria, it can classified as defined or
undefined media. In the defined media,
all of the nutrients is given, however the
undefined media tend to have a complex
mixtures of nutrients and other
compounds that are unknown like the
yeast extract. Petri-dish is a two-part,
glass or plastic-covered container that
contains culture medium.

Agar Pure Culture

Agar, is a polysaccharide extracted
from marine algae, is used to solidify a
A pure (or axenic) culture is a
specific nutrient solution. It is not easily
population of cells or multicellular
degraded by a bacteria.
creatures that grows without the
presence of other species or types of
Classification of Culture Media organisms. A pure culture can come
1. Consistency from a single cell or organism, in which
2. Nutritional component case the cells are genetic clones.
3. Functional Use
 CLASSIFICATION BASED ON CONSISTENCY

Liquid media. These can
be placed in test tubes,
bottles, or flasks. It's also
known as a broth culture,
example is the nutritious
broth. Liquid medium will
also dilute waste
products as they develop,
allowing them to be
distributed throughout
the culture.
Solid media. An agar Semi-solid media. Such
plate is a Petri dish media are fairly soft
that contains a growth and are useful in
medium (typically agar demonstrating bacterial
plus nutrients) used to motility and separation
culture microorganisms. motile from non-motile
2% of agar is added. strains. Example the
Agar is the most Hugh and Leifson's
commonly used oxidation fermentation.
solidifying agent.

CLASSIFICATION BASED ON NUTRITIONAL COMPONENTS

Simple Media Complex Media Synthetic Media

Simple media
such as
A defined
medium
Complex media are
peptone water, nutrient (also known as a
those media that are
agar can support most chemically defined
other than basal media.
non-fastidious bacteria. medium or synthetic
They include unique
Basal media, also known media) is one in which
chemicals that promote
as general-purpose all of the chemicals
the development of
media, are simple media used are known and
microbes. These unique
that allow most non- there is no yeast,
substances, such as yeast
fastidious bacteria to animal, or plant tissue
extracts or casein
thrive. Basal media present. A chemically
hydrolysate, are made up
include peptone water, defined media is a
of a complex blend of
nutrient broth, and culture medium for
molecules in unknown
nutrient agar (NA). microorganisms or
proportions.
animal cells.
 CLASSIFICATION BASED ON FUNCTIONAL USE FOR
APPLICATION
Enriched Media. Adding blood, serum,
or egg to the medium enriches this
media. Blood agar and Lowenstein-
Jensen media are two examples of
enriched media. In blood agar media,
it contains streptococci proliferate.
Selective Media. These media
promote the growth of a certain
bacteria by preventing the growth of
undesirable bacteria while permitting
the growth of beneficial bacteria.
MacConkey agar, Lowenstein-Jensen
media, and tellurite media are among
examples (Tellurite inhibits the growth
of most of the throat organisms except
diphtheria bacilli). Antibiotics might be
used.
Transport Media. When specie-men
can't be cultivated right away, these
mediums are employed. Cary-Blair
medium, Amies medium, Stuart medium
are some examples.
Storage media. The bacteria are
stored in this media for a long time.
Egg saline medium, chalk cooked beef
broth are two examples.
Differential media. include substances
that enable groups of microorganisms
to be visually discriminated by the look
of the colony or surrounding media,
generally due to a biochemical
difference between the two groups.
One sort of differential media is blood
agar, which allows bacteria to be
identified based on the type of
hemolysis they cause (see below).
Indicator Media. In this medium, it use
an indicator. A specific organism is the
source of the indication, such as blood,
neutral red, or tellurite, changes. Blood
agar and MacConkey agar are two
examples of indicator agars media.

THINK ABOUT IT!

1. What is the main difference of a selective and an enriching culture?

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2. Distinguish those media that are complex and those that are chemically
defined.
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 Bacterial Growth Characteristics
Bacteria can have distinct growth patterns even on general-
purpose growth medium. Bacteria develop as colonies on agar
plates, which are clusters of cells. Each colony is formed by a
single or a few bacteria. Individual cells are too tiny to see,
however large groups of cells may be seen. Colonies come in a
variety of shapes, sizes, heights, and colors. One type of
information that microbiologists can use to identify unknown
bacteria is colony features. At the conclusion of the lab, several
examples of growth characteristics on various types of growth
media are shown.

GENERAL PROCEDURE OF INCUBATING MEDIA

1. In the heat of a Bunsen burner, sterilize an inoculating loop or

needle. For efficient sterilization, the section of the loop or
needle that will come into contact with the stock culture or
growing media must turn bright orange. Place the loop at the
peak of the inner blue cone of flame for the fastest sterilization
—this is where the Bunsen burner's temperature is the highest.
After the loop has been suitably heated, remove it from the
flame; leaving it in the flame for too long will cause it to shatter.
2. Cool the inoculating loop on agar that
does not contain any bacteria if you are
choosing a colony from a plate colonies.
3. Take a tiny bit of bacteria and put it in
your mouth (you do not need much).
Remove the lid of the tube (do not place
the cap down on the table) and burn the
tube's lip if you're inoculating a tube of
broth or an agar slant.

Hold the tube at an angle during the

operation to limit the chance of particles
entering the orifice. Transfer bacteria to
the growing media by inserting the loop
into the tube. Only the sterilized portion
of the loop should touch the tube or enter
the growing liquid.
4. Before reinstalling the cap,
light/flame the test tube's lip.
5. Re-sterilize the inoculation loop.
 METHODS OF OBTAINING PURE CULTURE
STREAKING FOR SINGLE COLONIES

Bacteria thrive in communities made up of many different bacterial species

outside of the laboratory. If you need to identify the many species of bacteria in
environmental or medical samples, you'll need a technique to isolate them and
create clean cultures. A pure culture includes only one type of bacteria, but a
mixed culture may contain a variety of microorganisms. The streak plate method
shows how you'll separate distinct species of bacteria in a combination using a
streak plate.

STEP-BY-STEP PROCEDURE
https://www.youtube.com/watch?v=_1KP9zOtjXk
https://www.youtube.com/watch?v=fND5I_A7wNM
Watch the videos and observe the process of culturing the bacteria.

Observations:
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INOCULATING A PLATE FROM A BROTH CULTURE

1.Sterilize the inoculating loop. 5 . Flame the lip of the tube

2 . Remove the cap from tube. Do
NOT put the cap of the tube
down on the lab bench—hold it
in your hand.
3 . Flame the lip of the tube.
4 . Place sterile portion of
inoculating loop into broth, then
remove.
6 . Replace the cap.
7 . Gently streak the surface of an
agar plate with the inoculating
loop.
8 . Sterilize the inoculating loop
 COMMON MEDIA IN ROUTINE USE

Nutrient Agar. It is solid at 37°C. 2.5% agar is added in nutrient

broth. It is heated at 100°C to melt the agar and then cooled.
Peptone Water. Peptone 1% and sodium chloride 0.5%. It is used as
base for sugar media and to test indole formation.
Blood Agar. Most commonly used medium. 5- 10% defibrinated
sheep or horse blood is added to melted agar at 45-50°C. Blood
acts as an enrichment material and also as an indicator.
Chocolate Agar or Heated Blood agar: Prepared by heating
blood agar. It is used for culture of pneumococcus, gonococcus,
meningo- coccus and Haemophilus. Heating the blood inactivates
inhibitor of growths.
MacConkey Agar. Most commonly used for enterobac-teriaceae. It
contains agar, peptone, sodium chloride, bile salt, lactose and
neutral red. It is a selective and indicator medium :
Mueller Hinton Agar. Disc diffusion sensitivity tests for antimicrobial
drugs should be carried out on this media as per WHO
recommendation to promote reproducibility and comparability of
results.
Hiss's Serum Water Medium. This medium is used to study the
fermentation reactions of bacteria which can not grow in peptone
water sugar media, e.g. pneumococcus, Neisseria,
Corynebacterium.
Lowenstein-Jensen Medium. It is used to culture tubercle bacilli. It
contains egg, malachite green and glycerol. (1) Egg is an
enrichment material which stimulates the growth of tubercle bacilli,
(2) Malachite green inhibits growth of organisms other than
mycobacteria, (3) Glycerol promotes the growth of Mycobacterium
tuberculosis but not Mycobacterium bovis.
Dubos Medium. This liquid medium is used for tubercle bacilli. In this
medium drug sensitivity of tubercle bacilli can be carried out. It
contains 'tween 80', bovine serum albumin, casein hydrolysate,
asparagin and salts. Tween 80 causes dispersed growth and bovine
albumin causes rapid growth.
Loeffler Serum. Serum is used for enrichment. Diphtheria bacilli
grow in this medium in 6 hours when the secondary bacteria do not
grow. It is used for rapid diagnosis of diphtheria and to
demonstrate volutin granules. It contains sheep, ox or horse serum.
Tellurite Blood Agar. It is used as a selective medium for isolation of
Cotynebacterium diphtheriae
IMPORTANCE OF CULTURE MEDIA IN MICROBIAL GROWTH &
REPRODUCTION.

Most microbiological experiments require culture media in order to create,

develop and count microbial cells, and nurture and select microorganisms. The
likelihood of obtaining accurate, reproducible, and repeatable
microbiological test findings is diminished without high-quality medium.
Given the world's diversity of microorganisms, it's evident that we still don't
know a lot about how they grow. Continuing to learn more about microbial
development will allow humans to coexist securely with microorganisms in our
communities while also taking use of their particular characteristics.
Also, culture media help biologist to diagnose infections
Through culture media manipulation of the genome of bacterial strains may be
desired for a variety of reasons, including trying to understand fundamental
biology, attenuating it when making vaccine strains, overproducing proteins,
and creating a reference strain with a detectable marker, to mention a few.
This allows microbiologist to look at how bacterial populations evolve over
time, which can help with treatment, vaccine, and diagnostic design and
updates, as well as look at transmission events, which can help with public
health policy and guidance.
In order to combat a bacterial infection, you must normally be able to
cultivate it as well. It may be required to cultivate strains in order to
comprehend their genomes, amplify their genes, or alter them during the
production of vaccines
Probiotics and starter cultures are two types of bacteria that are used in the
production of many foods.
Probiotics are commonly cultured for their health benefits, which are often
achieved through our gut microbiome. While probiotics can contain a variety
of bacteria, Lactobacillus and Bifidobacterium are popular culture options.
Some bacteria are beneficial in the food production process, some can also
be a contaminant and cause serious foodborne illnesses. In this problem,
culture media can be a good practice to detect such foods that can cause
harm.
COMMON PROBLEMS IN BACTERIAL CULTURE

Contamination Overgrowth of Antibiotic Incorrect Non-culturable

some treatment growth and slow-
species in agar prior to conditions growing
sampling organisms
REFERENCES

https://www.technologynetworks.com/immunology/articles/an-introduction-to-
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https://www.austincc.edu/rohde/CHP7a.htm
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961714/
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forms-reproduction-bacteria/
https://sitn.hms.harvard.edu/flash/2020/how-microbes-grow/