MICROBIAL GROWTH

&
NUTRITION

Hera Nirwati
Dept. of Microbiology Fac.of Medicine UGM
Hera.nirwati@mail.ugm.ac.id
 Outline

• Microbial Reproduction
• Microbial Growth
• Culture Media
• Growth Measurement
Most bacteria reproduce by binary fission

Binary fission
produces genetically
identical daughter
cells
Generation time / doubling time
• Time required for a
bacterial cell to grow and
divide
• Dependent on chemical and
physical conditions

E. coli : 15 minutes
S. aureus : 30 minutes
M. tuberculosis: 15 hours
T. pallidum: 33 hours

Faster divisions times ➔

shorter incubation periode
A Skyrocketing Bacterial Population.
• The number of E. coli cells
progresses from 1 cell to 2 million
cells in a mere 7 hours.
• The J-shaped growth curve gets
steeper and steeper as the hours
pass.
• Only a depletion of food, build up
of waste, or some other limitation
will halt the progress of the curve.
 The Growth Curve for a Bacterial Population

A) During the lag phase, the population numbers remain stable as bacterial cells
prepare for division. (B) During the logarithmic (exponential growth) phase, the
numbers double with each generation time. Environmental factors later lead to
stationary phase (C), which involves a stabilizing population. (D) The decline
phase is the period during which cell death becomes substantial.
The Formation of a Bacterial
Spore by Bacillus subtilis

• A & B : When nutrient

conditions can support
growth and reproduction,
vegetative cells continue
through cycles of binary
fission.
• C-G: When nutrient
conditions become limiting
(e.g., carbon, nitrogen),
endospore formers, such
as B. subtilis, enter the
sporulation cycle shown
here.
 Optimal Microbial Growth is Dependent on
Several Physical Factors

• Growth of microbial population is sensitive to:

• Temperature
• Oxygen gas
• pH
 Temperature
• Temperature is one of the most important factors governing
growth
• Microorganism classified in 3 groups:
• psychrophiles (psychro = cold ➔cold loving microbes): 0-
20oC
• mesophiles (meso = middle ➔ moderate-temp- loving
microbes) 20-40oC
• thermophiles (thermo = heat ➔ heat-loving microbes) >
40oC ➔Hyper-thermophiles > 80oC
Microbial
-growth
 11

Classification of Microorganisms by Temperature

Requirements
 12

Oxygen and Microbial Growth

• Aerobes :
• Obligate aerobes : require oxygen to grow
• Facultative aerobes : can live with or without oxygen but
grow better with oxygen
• Microaerophiles : require reduced level of oxygen

• Anaerobes :
• Aerotolerant anaerobes : can tolerate oxygen but grow
better without oxygen.
• Obligate anaerobes : do not require oxygen ➔ inhibited or
killed if oxygen is present

A common way to test an organism’s oxygen sensitivity is to use

a thioglycollate broth, which binds free oxygen so that only fresh
oxygen entering at the top of the tube would be available
 The effect of oxygen on microbial growth

Each tube contains a thioglycollate broth into which was

inoculated a different bacterial species
 14

Hydrogen Ion Concentration (pH)

• The acidity or alkalinity of an environment can greatly affect
microbial growth.
• Most organisms grow best between pH 6-8, but some organisms
have evolved to grow best at low or high pH.
• Microorganisms regulate their internal pH over a wide range of
external pH values by pumping protons in or out of their cells.
• Acidophiles : organisms that grow best at low pH : optimum in pH:
1-4 ( e.g. Helicobacter pylori)
• Alkaliphiles : organismsa that grow best at high pH : optimum in
pH 8.5-11 (e.g. Vibrio cholera)
• Most of pathogenic bacteria are Neutrophiles: grow best in a
narrow range around neutral pH.

Most bacteria grow between pH 6.5 and 7.5

Molds and yeasts grow between pH 5 and 6
 Osmotic Pressure
• Microorganisms obtain almost all
their nutrients in solution from the
surrounding water
• Solute concentration affect water
availability & osmotic pressure
• Cell wall structure- make them
relative change in osmotic pressure
• Extreme osmotic pressures can result
in death of bacteria

hypertonic environment will force water to diffuse out of a cell, it is said to

have high osmotic pressure or potential – Plasmolysis
 Classes of Microbes
Based on Physical Factors
 Types of Media
Physical state Chemical Functional
Composition type
1. Liquid 1. Synthetic 1. General
(chemically purpose
defined)
2. Semisolid 2. Enriched
3. Solid (can be 2. Non-synthetic 3. Selective
converted to liquid) (not chemically 4. Differential
defined)
4. Solid (cannot be 5. Specimen
converted to liquid) transport

Culture media contain the nutrients needed for optimal

microbial growth
 Functional Media
• General-purpose media are designed to grow as broad a
spectrum of microbes as possible. As a rule, they are non
synthetic and contain a mixture of nutrients that could support
the growth of pathogens and non pathogens alike.
• Examples include nutrient agar and broth, brain-heart infusion,
and trypticase soy agar (TSA).
• Enriched medium contains complex organic substances such as
blood, serum, haemoglobin, or special growth factors (specific
vitamins, amino acids) that certain species must have in order to
grow.
• Bacteria that require growth factors and complex nutrients are
termed fastidious
• Blood agar, which is made by adding sterile sheep, horse, or
rabbit blood to a sterile agar base is widely employed to grow
fastidious streptococci and other pathogens
 Selective Media

• Contains one or more agents that inhibit the growth of a

certain microbe and thereby encourages, or selects, a
specific microbe.
• Mannitol salt agar : selective for halophiles with 7% salt
(osmotic challenge) and differential for mannitol
fermenters: good for skin bacterial cultures.
• EMB Agar: kills gram positives with eosin and methylene
blue, selective for gram negatives. Differential for lactose
fermenters. Good for growing enterics.
• McConkey Agar: supresses gram positives with crystal
violet and bile salts; (also differential media)
The use of Mac Conkey agar as a selective and
differential medium
Bacterial colonies Fungal colonies

pH 7.3 pH 5.6

An example of the use of a selective medium

 Differential Media
• Distinguish between different species based on a
metabolic ability.
• Differential shows up as visible changes or variations
in colony size or color, in media color changes, or in
the formation of gas bubbles and precipitates.
• Example:
• Spirit Blue Agar to detect the digestion of fats by lipase
enzyme. Positive digestion (hydrolysis) is indicated by the
dark blue color that develops in the colonies.
• Blood agar for hemolysis (α,β,and γ hemolysis) etc
Beta-hemolysis
Alpha-hemolysis

No hemolysis
(gamme-hemolysis)

The Use Of Blood Agar as

a Differential Medium
 Durham tube
(inverted tube
to trap gas)

The use of
carbohydrate utilization
tubes as differential
Acid fermentation media
No fermentation with gas
 Transport media
• should fulfill the following criteria:
• temporary storage of specimens being transported to the
laboratory for cultivation.
• maintain the viability of all organisms in the specimen without
altering their concentration.
• contain only buffers and salt.
• lack of carbon, nitrogen, and organic growth factors so as to
prevent microbial multiplication.
• transport media used in the isolation of anaerobes must be free
of molecular oxygen.
• Example:
• Thioglycolate broth for strict anaerobes.
• Stuart transport medium - a non-nutrient soft agar gel
containing a reducing agent to prevent oxidation, and charcoal
to neutralize certain bacterial inhibitors
 Methods of culturing microorganism : The five I’s
Inoculation-Incubation – Isolation – Inspection - Identification
 Inoculation
• During inoculation, the sample is placed into a container of
sterile medium that provides microbes with the appropriate
nutrients to sustain growth
• Inoculation involves using a sterile tool to spread the sample
out on the surface of a solid medium or to introduced the
sample into a flask or tube

• Selection of media with

specialized functions can
improve later steps of isolation
and identification
• Some microbes may require a
live organism (animal, egg) as
the inoculation medium
 Incubation

• An incubator can be used to

adjust the proper growth
conditions of a sample

• Setting the optimum temperature and gas content

promotes multiplication of the microbes over a period of
hours, days and even weeks.
• Incubation produces a culture – the visible growth of the
microbe in the medium
 Bacterial Cultivation in Different Gas Environments.

(A) A candle jar, in which microaerophilic bacterial species grow in an

atmosphere where the oxygen is reduced by the burning candle.
(B) An anaerobic jar, in which hydrogen is released from a generator and then
combines with oxygen through a palladium catalyst to form water and
create an anaerobic environment
 Isolation
• The end result of inoculation and
incubation is isolation of the
microbe in macroscopic form.
• The isolated microbes take the
form of separate colonies (discrete
mounds of cells) on solid media, or
turbidity in broths.

• Further isolation, also known as sub culturing, involves taking

a tiny bit of growth and inoculating an additional culture of it.
• This is one way to make a pure culture that contains only a
single species of microbe.
Streak plate of isolation
Pour plate method of isolation
Inspection
• The cultures are observed macroscopically for
obvious growth characteristic (colour, texture, size)
that could be useful in analyzing the specimen
contents.
• Slides are made to assess microscopic details such
as cell shape, size and motility.
• Staining techniques may be used to gather specific
information on microscopic morphology.
Characteristic of
bacterial colonies
 Identification
• A major outcome is to pinpoint an isolate down to the level of species
• Summaries of accumulated data are used to develop profiles of the microbe
or microbes isolated from the sample.
• Information can include relevant characteristics already taken during
inspection or additional tests that further describe and differentiate the
nature of microbe isolated
• Other types of specialized tests include biochemical tests to determine
metabolic activities specific to microbe, immunology test and genetic
analysis.
Bacterial Identification
 Estimating Bacterial Numbers
• Direct Measures
• Plate counts of viable bacterial forming colonies
• Counting low viable bacterial numbers by filtration
• Counting viable bacteria with Most Probable Number
• Counting bacteria per ml in direct microscopy
• Indirect Measures
• Turbidity/Absorbance with a spectrophotometer
• Metabolic activity tracking conversion of colored molecules
• Dry weight by weighing a set volume and knowing weight of
one cell
Plate Assays: Spread Plate or Pour Plate Methods
• After incubation, count colonies on plates that have 30-
300 colonies (CFUs)

The dilution in a
particular tube =
ml of fluid added
to tube/total
volume after
addition; e.g.
1ml/(9ml + 1ml) =
1/10 = 10-2
Use of membrane
filtration to
estimate microbial
population-
overview
 1.0 ml 1.0 ml

Undiluted 1:10 1:100

Inoculate 1.0 ml into

each of 5 tubes

Phenol red, pH
color indicator, The most
added
probable
Incubate number (MPN)
method for
Results
estimating
microbial
4 tubes positive 2 tubes positive 1 tube positive numbers
 Direct Measurements of Microbial Growth

• Multiple tube
MPN test
• Count positive
tubes and
compare to
statistical MPN
table
• Produces a range
of concentrations
Direct Measurements of Microbial Growth
Temperature Growth Ranges and Food Safety
Synthetic media
• Media whose compositions are chemically defined
• Such media contain pure organic and inorganic
compounds that vary little from one source to another
and have a molecular content specified by means of an
exact formula

Complex / non-synthetic media

• at least one ingredient that is not chemically definable—
not a simple, pure compound and not representable by an
exact chemical formula
• these substances are extracts of animals, plants, or yeasts,
including such materials as ground-up cells, tissues, and
secretions e.g. blood, serum, meat extracts, milk, yeast
extract, soybean digests, and peptone
Four Basic groups of organism
Slant

Butt