Chapter 5
Microbial Nutrition and Culture
cont’d
Siti Sarah Jumali (ext 2123)
Room 3/14
sarah_jumali84@hotmail.com
Microbial Growth
Requirements
The Requirements for Growth
PHYSICAL
REQUIREMENTS
– Temperature
– pH
– Oxygen
– Hydrostatic Pressure
– Osmotic pressure
CHEMICAL REQUIREMENTS
(NUTRITIONAL FACTORS)
– 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 0°C and 20–30°C
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 100°C
• Solidifies at ~40°C
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: Sabouraud’s 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 organism’s 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 –72°C)
and dehydrated in a vacuum
Deep Freezing: –50° to –95°C
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
25–250 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?