Chapter 5

Microbial Nutrition and Culture

contd
Siti Sarah Jumali (ext 2123)
Room 3/14
sarah_jumali84@hotmail.com

Microbial Growth
Requirements

The Requirements for Growth

PHYSICAL REQUIREMENTS

CHEMICAL REQUIREMENTS
(NUTRITIONAL FACTORS)

Temperature
pH
Oxygen
Hydrostatic Pressure
Osmotic pressure

Carbon
Nitrogen, sulfur, and
phosphorous
Trace elements
Oxygen
Organic growth
factor

Physical Factors Required for Bacterial

Growth
1) pH

Optimum pH: the pH at which the microorganism

grows best (e.g. pH 7)
Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6
According to their tolerance for acidity/alkalinity,
bacteria are classified as:
Acidophiles (acid-loving): grow best at pH 0.1-5.4
Neutrophiles: grow best at pH 5.4 to 8.0
Alkaliphiles (base-loving): grow best at pH 7.0-11.5

2) Temperature

According to their growth temperature range, bacteria can be

classified as:
Psychrophiles
: grow best at 15-20oC
Psychrotrophs : grow between 0C and 2030C
Mesophiles
: grow best at 25-40oC
Thermophiles
: grow best at 50-60oC

Typical Growth Rates and Temperature

Minimum growth temperature: lowest temp which species can
grow
Optimum growth temperature: temp at which the species grow
best
Maximum growth temperature: highest temp at which grow is
possible

Food Preservation Temperatures

3) Oxygen

Aerobes: require oxygen to grow

Obligate aerobes: must have free oxygen for aerobic respiration (e.g.
Pseudomonas)
Anaerobes: do not require oxygen to grow
Obligate anaerobes: killed by free oxygen (e.g. Bacteroides)
Microaerophiles: grow best in presence of small amount of free
oxygen
Capnophiles: carbon-dioxide loving organisms that thrive under
conditions of low oxygen
Facultative anaerobes: carry on aerobic metabolism when oxygen is
present, but shift to anaerobic metabolism when oxygen is absent
Aerotolerant anaerobes: can survive in the presence of oxygen but do
not use it in their metabolism
Obligate: organism must have specified environmental condition
Facultative: organism is able to adjust to and tolerate environmental
condition, but can also live in other conditions

Patterns of Oxygen Use

4) Hydrostatic Pressure
Water in oceans and lakes exerts pressure exerted
by standing water, in proportion to its depth
Pressure doubles with every 10 meter increase in
depth
Barophiles: bacteria that live at high pressures,
but die if left in laboratory at standard
atmospheric pressure

5) Osmotic Pressure
Environments that contain dissolved substances
exert osmotic pressure, and pressure can exceed
that exerted by dissolved substances in cells
Hyperosmotic environments: cells lose water and
undergo plasmolysis (shrinking of cell)
Hypoosmotic environment: cells gain water and
swell and burst

Plasmolysis

Halophiles

Salt-loving organisms which require moderate to large

quantities of salt (sodium chloride)

Membrane transport systems actively transport sodium

ions out of cells and concentrate potassium ions inside

Why do halophiles require sodium?

1) Cells need sodium to maintain a high intracellular
potassium concentration for enzymatic function
2) Cells need sodium to maintain the integrity of their
cell walls

Responses to Salt

The Great Salt Lake in Utah

Chemical Requirement: Nutritional

Factors
1.
2.
3.
4.

Carbon sources
Nitrogen sources
Sulfur and phosphorus
Trace elements (e.g. copper, iron, zinc,
and cobalt)
5. Vitamins (e.g. folic acid, vitamin B-12,
vitamin K)

Chemical Requirements
Carbon
Structural organic molecules, energy source
Chemoheterotrophs use organic carbon sources
Autotrophs use CO2

Chemical Requirements
Nitrogen
In amino acids and proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3
A few bacteria use N2 in nitrogen fixation

Chemical Requirements
Sulfur
In amino acids, thiamine, and biotin
Most bacteria decompose proteins
Some bacteria use SO42 or H2S

Phosphorus
In DNA, RNA, ATP, and membranes
PO43 is a source of phosphorus

Chemical Requirements
Trace elements
Inorganic elements (mineral) required in small
amounts
Usually as enzyme cofactors
Ex: iron, molybdenum, zinc

Buffer
To neutralize acids and maintain proper pH
Peptones and amino acids or phosphate salts
may act as buffers

Organic Growth Factors

Organic compounds obtained directly from the
environment
Ex: Vitamins, amino acids, purines, and
pyrimidines

Preparation of Culture Media

Culture medium: Nutrients prepared for
microbial growth
Sterile: No living microbes
Inoculum: Introduction of microbes into
medium
Culture: Microbes growing in/on culture
medium

Agar
Complex polysaccharide
Used as solidifying agent for culture media in
Petri plates, slants, and deeps
Generally not metabolized by microbes
Liquefies at 100C
Solidifies at ~40C

Types of Culture Media

Natural Media: In nature, many species of
microorganisms grow together in oceans, lakes, and soil
and on living or dead organic matter
Synthetic medium: A medium prepared in the
laboratory from material of precise or reasonably welldefined composition
Complex medium: contains reasonably familiar
material but varies slightly in chemical composition
from batch to batch (e.g. peptone, a product of enzyme
digestion of proteins)

Types of Culture Media

Type of Media

Purpose

Chemically
Defined

Growth of chemoheterotrophs and

photoautotrophs: microbiological assays

Complex

Growth of most chemoheterotrophic organisms

Reducing

Growth of obligate anO2

Selective

Suppresion of unwanted microbes; encouraging

desired microbes

Differential

Differentiation of colonies of desired microbes

from others

Enrichment

Similar to selective media but designed to increase

numbers of desired microbes to detectable levels.

Culture Media
Chemically defined media: Exact chemical
composition is known
Complex media: Extracts and digests of
yeasts, meat, or plants
Nutrient broth
Nutrient agar

Selective, Differential, and Enrichment Media

Selective medium: encourages growth of some
organisms but suppresses growth of others
(e.g. antibiotics)
Differential medium: contains a constituent that
causes an observable change (e.g. MacConkey agar)
Enrichment medium: contains special nutrients that
allow growth of a particular organism that might not
otherwise be present in sufficient numbers to allow it
to be isolated and identified

Selective Media
Suppress unwanted microbes and encourage desired
microbes
Ex: Sabourauds Dextrose Agar: used to isolate fungi, has
a pH of 5.6, outgrow most of bacteria

Differential Media
Make it easy to

distinguish colonies of
different microbes.
Ex: Blood Agar: bacteria
that can lysed blood
cells causing a clear
areas around the
colonies.

Three species of Candida can be differentiated in mixed

culture when grown on CHROMagar Candida plates

Identification of urinary tract pathogens with

differential media (CHROMagar)

Enrichment Media
Encourages growth of desired microbe
Assume a soil sample contains a few phenol-degrading
bacteria and thousands of other bacteria
Inoculate phenol-containing culture medium with the
soil, and incubate
Transfer 1 ml to another flask of the phenol medium,
and incubate
Transfer 1 ml to another flask of the phenol medium,
and incubate
Only phenol-metabolizing bacteria will be growing

Culturing Bacteria

Culturing of bacteria in the laboratory

presents two problems:
1. A pure culture of a single species is needed
to study an organisms characteristics
2. A medium must be found that will support
growth of the desired organism

Obtaining Pure Cultures

Pure culture: a culture that contains only a
single species or strain of organism
A colony is a population of cells arising from a
single cell or spore or from a group of attached
cells
A colony is often called a colony-forming unit
(CFU)
The streak plate method is used to isolate pure
cultures

The Streak Plate Method uses agar plates to

prepare pure cultures

The Streak Plate Method

Figure 6.11

Anaerobic Culture Methods

Reducing media
Contain chemicals (thioglycolate or oxyrase) that
combine O2
Heated to drive off O2

Anaerobic Jar

Figure 6.6

To culture obligate
anaerobes, all molecular
oxygen must be removed
and kept out of medium.
Agar plates are incubated
in sealed jars containing
chemical substances that
remove oxygen and
generate carbon dioxide
or water

Anaerobic Transfer

An Anaerobic Chamber

Figure 6.7

Capnophiles
Microbes that require high CO2 conditions
CO2 packet
Candle jar

Preserved Cultures

1.
2.
3.

To avoid risk of contamination and to reduce

mutation rate, stock culture organisms should be
kept in a preserved culture, a culture in which
organisms are maintained in a dormant state
Lyophilization (freeze-drying): Frozen (54 to 72C)
and dehydrated in a vacuum
Deep Freezing: 50 to 95C
Refrigeration
Reference culture (type culture): a preserved culture
that maintains the organisms with characteristics as
originally defined

CHAPTER 6:
MICROBIAL GROWTH

Growth and Cell Division

Microbial growth is defined as the increase in the
number of cells, which occurs by cell division
Binary fission (equal cell division): A cell duplicates its
components and divides into two cells
Septum: A partition that grows between two daughter
cells and they separate at this location
Budding (unequal cell division): A small, new cell
develops from surface of exisiting cell and subsequently
separates from parent cell

Binary Fission

Binary Fission

Thin section of the bacterium Staphylococcus,

undergoing binary fission

Budding in Yeast

Phases of Growth

Consider a population of organisms

introduced into a fresh, nutrient medium

Such organisms display four major phases of

growth in batch culture:
1.
2.
3.
4.

The lag phase

The logarithmic phase
The stationary phase
The death phase

The Lag Phase

Organisms do not increase significantly in number
They are metabolically active
Grow in size, synthesize enzymes, and incorporate
molecules from medium
Produce large quantities of energy in the form of
ATP

The Log Phase

Organisms have adapted to a growth medium
Growth occurs at an exponential (log) rate
The organisms divide at their most rapid rate
a regular, genetically determined interval
(generation time)

Synchronous growth: A
hypothetical situation in
which the number of
cells in a culture would
increase in a stair-step
pattern, dividing
together at the same
rate
Nonsynchronous growth:
A natural situation in
which an actual culture
has cell dividing at one
rate and other cells
dividing at a slightly
slower rate

Stationary Phase
1) Cell division decreases to a point that new cells
are produced at same rate as old cell die.
2) The number of live cells stays constant.

Decline (Death) Phase

1) Condition in the medium become less and less
supportive of cell division
2) Cell lose their ability to divide and thus die
3) Number of live cells decreases at a logarithmic
rate

Microbes growing continuously in a chemostat

Measuring Microbial Growth

Direct Methods

Indirect Methods

Turbidity
Metabolic activity
Dry weight

Plate counts
Filtration
MPN
Direct microscopic
count

Direct Methods
1) Plate Count

Must perform
- Serial Dilutions
- Pour plate or Spread Plate Method

often reported as colony-forming units (CFU)

Serial Dilution

Plate Counts

Figure 6.17

Plate Counts
After incubation, count colonies on plates that
have
25250 colonies (CFUs)

Figure 6.16

2) Counting Bacteria by Membrane Filtration

3) The Most Probable Number (MPN) Method

Method to estimate number
of cells
Multiple tube MPN test
Count positive tubes

Compare with a statistical

table.

4) Direct Microscopic Counts

Another way to measure bacterial growth by:
1) Petroff-Hausser counting chamber
2) Colony counting chamber
In Petroff-Hausser counting chamber, bacterial suspension
is introduced onto chamber with a calibrated pipette
Microorganisms are counted in specific calibrated areas
Number per unit volume is calculated using an appropriate
formula

The Petroff-Hausser Counting Chamber

Direct Microscopic Count

Counting colonies using a bacterial colony counter

Bacterial colonies viewed through the magnifying

glass against a colony-counting grid

Countable number of colonies

(30 to 300 per plate)

Which of these plates would be the

correct one to count? Why?

Indirect Methods
1) Turbidity
Practical way of monitoring bacterial
growth.
Measure turbidity using spectrophotometer

A Spectrophotometer: This instrument can be used to measure bacterial growth by

measuring the amount of light that passes through a suspension of cells

Turbidity

Turbidity

The less light transmitted, the more bacteria in sample.

2) Metabolic Activity
Assuming the amount of a certain metabolic
product, ex acids, CO2 produced = direct
propotion of no of bacteria present
3) Dry Weight
For filamentous bacteria and molds
Apply filtration of amount of broth culture
on filter paper and dried in a dessicator
Weight of dried culture = direct propotion
of no of bacteria present

Biosafety Levels
1: No special precautions
2: Lab coat, gloves, eye protection
3: Biosafety cabinets to prevent airborne
transmission
4: Sealed, negative pressure
Exhaust air is filtered twice

Biosafety Level 4 (BSL-4) Laboratory

Questions?