GROWTH, ISOLATION, CULTIVATION,

& CONTROL/DESTRUCTION OF
MICROORGANISMS

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Bacterial Growth Growth of Microorganisms (Bacteria)

BACTERIAL GROWTH
▪ In multicellular organisms:
growth is manifested by an
increase in size and mass.
▪ In unicellular microorganisms
(e.g. bacteria): growth is usually
defined as the increase in
number and mass.
▪ Most bacteria and archaea are
haploid and reproduce via binary
fission.
Phases of Bacterial Growth Growth of Microorganisms (Bacteria)

Binary Fission in Prokaryotes

1. In the initial step of binary fission, the DNA
replicates or copies its chromosome.
2. Next, each copy of the DNA is attached to a
separate portion in the cell.
3. In the next step, the cell elongates to
approximately twice its average cell length and
since the DNA is attached to different locations of
the cell, they separate from one another.
4. At this point, a pinch is formed at the center,
creating a septum that serves as a separation wall
between the 2 compartments of the cell.
5. Finally, the cell splits into new daughter cells,
each having a copy of the DNA
 Factors Affecting Growth Growth of Microorganisms (Bacteria)

FACTORS AFFECTING
GROWTH
a) Nutrients – carbon, nitrogen + other
macro- and micronutrients
b) Temperature – psychrophiles,
mesophiles, thermophiles,
hyperthermophiles
c) Moisture – availability of water
(water activity, Aw)
d) Aeration – different oxygen
requirements
e) pH - expression of acidity or
alkalinity

https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.04%3A_Temperature_and_Microbial_Growth
Phases of Bacterial Growth Growth of Microorganisms (Bacteria)

Phases of Bacterial Growth: The Growth Curve

During cell division, a cell reproduces to

form 2 cells so that for each generation, both
the cell number and cell mass doubles.

https://microbiologynotes.org/bacterial-growth-curve-definition-phases-and-measurement/
Measurement of Growth Growth of Microorganisms (Bacteria)

MEASUREMENT OF GROWTH:
Standard Plate Count (SPC) ✓ In this method, viable cells are
counted, or those cells that can
divide and reproduce.
✓ It is called standard plate count
because agar plates are needed
for this method.
✓ Assumption: one viable cell will
give rise to one colony that will
grow on an agar plate = no. of
colonies reflects the cell no.

Advantage: can determine # of living cells

Disadvantage: underestimation of cell population
https://pharmacyscope.com/methods-of-isolation-of-pure-culture/
Serial Dilution Growth of Microorganisms (Bacteria)

SERIAL DILUTION

• A turbid bacterial broth

contains millions of bacteria
thus, when it is plated it will
only produce a lawn of
bacterial growth.
• Serial dilution is done initially
to “thin out” or decrease the
concentration of cells so that A dilution is prepared by adding a specific volume or weight of
distinct colonies can be sample to a volume of sterile diluent such as water, saline
water or phosphate buffer to dilute or make the bacterial
counted after plating. suspension less concentrated than the original sample.
Series of tubes become less concentrated.

https://chemistnotes.com/biochemistry/isolation-of-bacteria/
Plating Methods Growth of Microorganisms (Bacteria)
PLATING METHODS
After serial dilution, plating technique can be done
either by pour-plate or spread-plate method:
a. In pour-plate method, 0.1 to 1 milliliter of culture
is pipetted to a sterile petri plate. Molten agar
medium at around 50 degrees Celsius is poured onto
the plate and is mixed gently before the agar
solidifies.
b. In spread-plate method, 0.1 milliliter of culture is
pipetted on the surface of an agar plate. The aliquot
will be spread on the surface of the agar plate using
a sterile glass spreader or L-rod.
 Plating Methods Growth of Microorganisms (Bacteria)
An example of agar plates with colonies
for a standard plate count.
https://microbenotes.com/plate-count-agar-pca/
• For both methods, the number
of colonies that grow on the
plates should not be too many
or too few.
• If colonies are too many,
colonies may fuse with one
another leading to erroneous
counts. If colonies are too few,
the statistical significance will
be low.
Valid no. of colonies in the
plate count method: 25 to 250 colonies
Computation Growth of Microorganisms (Bacteria)

To compute for the colony-forming units or CFU per ml, the product of the
average number of colonies and dilution factor is divided by the volume
plated in ml. DF or dilution factor is equal to 1 divided by the dilution.
Sample Computation Growth of Microorganisms (Bacteria)

SAMPLE COMPUTATION
Definition of Terms Isolation & Cultivation of Microorganisms

PURE CULTURE
(AXENIC CULTURE)
• a culture which contains a single
species of microorganism
• a population of cells arising from a
single cell
• to study a single type of
microorganism
• to characterize an individual
species
Definition of Terms Isolation & Cultivation of Microorganisms
Isolation Techniques Isolation & Cultivation of Microorganisms

STEPS IN PREPARING PURE CULTURES

• Isolation
• Transfer desired colony to a slant or stab
• Verify the purity
• Microscopy
• Re-streak on agar medium
• Physiological and biochemical test
• Make stock cultures
Definition of Terms Isolation & Cultivation of Microorganisms

CULTIVATION
-increasing the population of
microorganisms by providing
their nutritional and physical
requirements
NUTRIENTS
- extracellular substances which
provide the cell with materials for
building protoplasm and for energy
generation

CULTURE MEDIUM
- any nutrient material for growth
and cultivation of microorganisms in
the laboratory
 Culture Media Isolation & Cultivation of Microorganisms

USES OF CULTURE MEDIUM

✓ for growth and maintenance of
microbial cultures
✓ to favor the production of particular
compounds
✓ to study microbial action on some
https://microbeonline.com/types-of-bacteriological-culture-medium/
constituents of the medium
Culture Media Isolation & Cultivation of Microorganisms

Nutrient materials for growing and cultivating microorganisms

Must contain essential elements needed by the microorganisms
C, H, O, N, P, S – basic elements in the cell for biosynthesis
and energy production
Ingredients vary from one type of media to another.
pH is properly adjusted since different microorganisms have
different pH requirements
Culture Media Isolation & Cultivation of Microorganisms

Almost all solid media has agar as its component

Solidifying agent with no nutritive value
Very few microorganisms can degrade agar
Melts at 100°C, solidifies at 40°C

Image from:
https://www.indiamart.com/proddetail/agar-agar-
powder-bacteriological-grade-18746755488.html
Types of Culture Media Isolation & Cultivation of Microorganisms

1. Based on composition

• Synthetic – all components are chemically defined

• Complex – not all components are chemically
defined
Types of Culture Media Isolation & Cultivation of Microorganisms

2. Based on consistency

Image from: https://www.jlindquist.com/generalmicro/dfnewthiopage.html

• Liquid (Broth) – no solidifying agent

• Semi-solid – 0.5%-0.7% solidifying agent
• Solid – 1.5%-2.0% solidifying agent
Types of Culture Media Isolation & Cultivation of Microorganisms

3. Based on purpose/use

a. General Purpose - can support most or almost all types of species e.g. Nutrient Agar (NA) ,
Potato Dextrose Agar (PDA)

b. Differential - distinguishes one type of bacteria from another; with special reagents like pH
indicators or dyes e.g. Eosin Methylene Blue Agar (EMBA)

c. Selective - allows the growth of a specific type of microorganism only; with selective agents
(ex. salts, dyes, antibiotics, etc.) e.g. Bacillus Cereus Agar (BCA)

d. Enrichment - used to increase the number of microorganisms with unusual physiological

characteristics; with special nutrients (ex. blood) e.g. Blood Agar

e. Assay - of prescribed composition used for assay of vitamins, amino acids and antibiotics; used
to determine qualitative/quantitative production of such a compound by an organism
Types of Culture Media Isolation & Cultivation of Microorganisms

3. Based on purpose/use

Image from: https://orbitbiotech.com/mannitol-salt-agar/

Image from: https://orbitbiotech.com/blood-agar-%CE%B1-hemolysis-%CE%B2-hemolysis-%CE%B3- Image from: https://microbiologyinfo.com/citrate-

Image from: https://microbiologie-clinique.com/nutrient-broth.html hemolysis-alpha-prime-wide-zone-alpha-hemolysis-composition-procedure-usus/ utilization-test-principle-media-procedure-and-result/
Media Preparation Isolation & Cultivation of Microorganisms

Guide for the Determination of Total Volume

Media Preparation Isolation & Cultivation of Microorganisms

Example: Prepare 10 plates (maximum vol.), 10 big stabs, and 10 small

slants of Nutrient Agar (NA). How much NA (in ml) should be
prepared?

10 plates x 20ml/plate = 200ml

10 big stabs x 10ml/big stab = 100ml
10 small slants x 3ml/small slant = 30ml

TOTAL VOL. TO BE PREPARED: 330ml NA

Media Preparation Isolation & Cultivation of Microorganisms

How much of each component (in ml)

should be prepared to make 330ml NA?
Isolation Techniques Isolation & Cultivation of Microorganisms

ASEPTIC TRANSFER/TECHNIQUE
Isolation Techniques Isolation & Cultivation of Microorganisms

ASEPTIC TRANSFER/TECHNIQUE

https://youtu.be/i6b-V02KFwY
Isolation Techniques Isolation & Cultivation of Microorganisms
Isolation Techniques Isolation & Cultivation of Microorganisms

STREAK PLATING
- The inoculum is streaked over the agar
surface in such a way that it “thins out” the
bacteria.
- Some individual bacterial cells are
separated and well-spaced from each other.
As the original sample is diluted by
streaking it over successive quadrants, the
number of organisms decreases.
- Usually, by the third or fourth quadrant,
only a few organisms are transferred which
will give discrete colony forming units
(CFUs).
- Surface colonies formed
Isolation Techniques Isolation & Cultivation of Microorganisms

https://www.youtube.com/watch?v=hHk0dRuNr_U
Isolation Techniques Isolation & Cultivation of Microorganisms

SPREAD AND POUR PLATING

Culture Preservation Isolation & Cultivation of Microorganisms

CULTURE PRESERVATION

Objective
• to retain the viability of the stock culture for a
long period of time while maintaining its purity
Culture Preservation Isolation & Cultivation of Microorganisms

CULTURE PRESERVATION
Culture Preservation Isolation & Cultivation of Microorganisms

CULTURE PRESERVATION
Culture Preservation Isolation & Cultivation of Microorganisms

CULTURE PRESERVATION
Culture Preservation Isolation & Cultivation of Microorganisms

CULTURE PRESERVATION
Importance Control and Destruction of Microorganisms

Why control growth of microorganisms?

Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Agents Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms

https://www.worldhealth.net/news/one-step-towards-
daily-use-deep-uv-light-source-sterilization-and-
disinfection/
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms
Methods Control and Destruction of Microorganisms