Download Chapter 6: Microbial Growth

Chapter 6:
Microbial Growth
1. Requirements for Growth
2. Culturing Microorganisms
3. Patterns of Microbial Growth
4. Measuring Microbial Growth
1. Requirements for Growth
Chapter Reading – pp. 167-174
Factors that affect Microbial Growth
Microbial growth depends on physical factors…
• temperature
• pH
• osmotic pressure
…and chemical factors
• availability of a useable carbon source
• useable sources of nitrogen, sulfur & phosphorus
• availability of trace elemental nutrients (Fe, Mg…)
• presence (or absence) of oxygen gas (O2)
1
Temperature & Microbial Growth
Growth rate
Thermophiles
Mesophiles
Hyperthermophiles
Psychrophiles
10
0
10
20
30
40 50 60 70
Temperature (°C)
80
90 100 110 120
Microorganisms can be grouped based on the temperature
range in which they can grow…
• each has an optimal temp. & minimum, maximum growth temps.
pH
Osmotic Pressure
• most microorganisms
grow best at pH levels
near neutral (6.5-7.5)
• few microorganisms
grow at the more
extreme pH levels
(below 4.0, above 10.0)
Hypertonic solutions can draw
water out of cells via osmosis:
• causes membrane to detach
from cell wall (plasmolysis)
• caused by high salt, sugar…
• inhibits bacterial growth
• microbial growth tends
to acidify the growth
medium, inhibiting
further growth
Oxygen (O2)
As we’ve learned, oxygen can promote growth
(via respiration in aerobes) or inhibit growth
(of obligate anaerobes).
Why is oxygen so toxic to some organisms?
• O2 is a reactive molecule that can result in the
formation of very forms of oxygen:
superoxide radical (most toxic!):
singlet oxygen:
O2-
O2 (“energized” O2)
hydroxyl radical:
peroxide anion:
OH
O22-
• aerobic organisms, unlike obligate anaerobes,
have enzymes to eliminate these dangerous radicals
e.g.
superoxide dismutase (SOD), catalase, peroxidase
2
Oxygen & Microbial Growth
Oxygen
concentration
High
Low
Loosefitting
cap
Obligate
aerobes
Obligate
anaerobes
Facultative
anaerobes
Aerotolerant
anaerobes
• thioglycollate medium produces an O2 gradient
• a given bacterial species will grow only in the
regions it can tolerate (e.g., anaerobes at bottom)
Chemical Factors for Growth
Source of Carbon
• autotrophs simply need access to CO2 to grow
• heterotrophs require an organic carbon source
• proteins, carbohydrates, lipids
**The carbon source a given organism can use depends
depends on its metabolic abilities (i.e., its enzymes!)**
Trace Elemental Nutrients
• all organisms need trace (small) amounts of
many so-called “mineral” elements:
iron (Fe), zinc (Zn), magnesium (Mg), calcium (Ca)…
• most are essential cofactors for various enzymes
Nitrogen, Sulfur & Phosphorus
• all organisms need access to nitrogen, sulfur &
phosphorus to make proteins, nucleic acids, vitamins
• some organisms require organic sources of these
elements, others are more flexible:
• e.g., nitrogen fixers are unique in being able to
obtain nitrogen from the atmosphere (N2), most
other organisms need Nitrogen in other forms
*One can effectively promote or inhibit the growth
of a microorganism of interest (or concern) by
controlling its physical & chemical environment!*
3
2. Culturing Microorganisms
Chapter Reading – pp. 175-182
Culture Medium
The culturing of microorganisms requires an
appropriate growth medium:
• material containing all nutrients required for
the desired organism to grow
• can be liquid or solid (i.e., solid agar)
• must initially be sterile (i.e., no live organisms)
• media can be sterilized by heat or by filtration
• growth should only occur following inoculation
of the medium with the desired organism
Defined vs Complex Medium
Defined medium has
a precisely known
chemical composition
• used for assessing
metabolic characteristics
Complex medium is rich in
nutrients though chemical
composition is not known
• used to sustain rapid growth
4
Selective & Differential Media
Selective media promote the growth of desired
organism(s), suppress growth of others:
• include something in the growth medium that
desired organism can tolerate, most other
organisms cannot (e.g., antibiotic, low pH, high salt)
• use defined media that sustain growth of desired
organism, not others (e.g., lactose as carbon source)
On differential media, microorganisms can be
distinguished based on appearance
• e.g., contain substances that change color due to
pH change, production of particular by-product
A
B
Selective medium
• compare A
(non-selective)
with B (selective)
C
D
Differential
medium
• C illustrates
differential growth
• D is differential
& selective
Culturing Obligate Anaerobes
• special chambers are used to remove and exclude any
oxygen (O2) that would otherwise kill such organisms
5
How to Obtain a Pure Culture
• quadrant streak to obtain isolated colonies
• inoculate an isolated colony (derived from a single original cell)
into liquid medium to obtain a pure culture
Plating Bacteria
2 basic methods:
1) mix 1 ml of culture
with molten agar
1
2
(not too hot, ~45-50o C.)
& pour in plate
• colonies grow IN
as well as ON agar
• some cells may be
harmed by higher temp.
2) spread small volume
of culture (0.1 ml) on
solid agar surface
• best method!
**Each colony starts with 1 CFU!**
3. Patterns of Microbial Growth
Chapter Reading – pp. 183-185
6
Bacterial Growth
• most bacteria divide by
binary fission ( a few by
budding)
• increase in cell numbers is
exponential
1 bacterium can become 1 billion in
just 30 generations!!!
Arithmetic vs Exponential Growth
70
70
arithmetic
60
Species A
40
30
30
20
10
10
2
1
Time (hours)
• real living
organisms
reproduce
exponentially
40
20
0
Species B
50
Number of cells
50
Number of cells
exponential
60
0
2
1
Time (hours)
Rates of Microbial Growth
The rate of microbial growth depends on the
generation time:
• the time for a microbial cell to divide
• depends on the type of microorganism
• also depends on the growth medium
***can be as short as 20 minutes
(E. coli) or >24 hr***
• a practical measure is the the time it
takes a microbial population to double
in size (doubling time)
• i.e., when every cell divides once!
7
Microbial Growth Patterns
Microorganisms cannot undergo unlimited
growth, eventually the chemical and physical
environment in which they’re growing will no
longer be able to sustain such numbers:
• sources of carbon, nitrogen, etc, get used up
• waste products accumulate, pH may change
Therefore, microbial growth tends to follow a
characteristic pattern:
Lag phase > Log phase > Stationary phase > Death phase
Phases of Microbial Growth
log phase growth is “linear”
(straight line) on a logarithmic plot
Lag phase: cells adjust to medium before dividing
Log phase: exponential growth
Stationary phase: growth = death (wastes, lack of nutrients)
Death phase: poor environment results in death > growth
Biofilms
It is estimated that the majority of bacteria in nature
live in biofilms, and that most bacterial diseases
are due to bacteria in a biofilm.
SO WHAT’S A BIOFILM?
• a gelatinous extracellular matrix (ECM) consisting
primarily of polysaccharides in the glycocalyces of
the bacteria in the biofilm
• forms on hard surfaces (rocks, teeth, prosthetics…)
• involves multiple bacterial species
• when sufficient bacterial numbers are present, a
signaling process called quorum sensing induces biofilm
**bacteria in biofilm are MUCH harder to get rid of than isolated bacteria**
8
4. Measuring Microbial Growth
Chapter Reading – pp. 186-190
How to Measure Microbial Growth?
There are a number of methods used to
count microorganisms and thus determine
the growth rate.
The method used depends on several things:
• the organism being analyzed
• how quickly one needs the result
• the degree of accuracy needed
• the nature of the sample being tested
Counting by Serial Dilution
1 ml original
culture
9 ml broth +
1 ml original
culture
1.0 ml
1:10
dilution
0.1 ml of each 0.1 ml
transferred to
a plate
1.0 ml
1:100
dilution
1.0 ml
1:1000
dilution
0.1 ml
0.1 ml
1.0 ml
1:10,000
dilution
1:100,000
dilution
0.1 ml
result
takes
~24 hr
Incubation
period
Too numerous to count
(TNTC)
TNTC
*
65 colonies
6 colonies
0 colonies
**ea colony starts w/1 CFU**
9
Counting by Filtration
Direct Microscopic
Counts
• place sample of culture
“counting chamber” slide
• count cells within grid and
calculate the cell density
• the volume covering each
grid or square is known so
the number of cells per unit
volume is easily determined
• gives immediate and
relatively accurate results!
• dilutions of test
sample are used to
inoculate sets of
differential media
# of tubes in
each group
with growth
is used for a
statistical
estimate of
the most
probable
number of
cells/100 ml
1.0 ml
1.0 ml
Most Probable
Number
Undiluted
1:10
1:100
Inoculate 1.0 ml into
each of 5 tubes
pH indicator
(phenol red)
added
Incubate
Results
4 tubes positive
2 tubes positive
1 tube positive
10
Spectrophotometry
One of the quickest, most
convenient methods to
determine cell density is
with a spectrophotometer.
• measures how much light is
transmitted through a liquid
culture sample
• more light blocked = greater
cell density (i.e., turbidity)
• % transmittance can be used
to calculate cell density
**Less precise, but gives
immediate results!**
Key Terms for Chapter 6
• psychrophile, mesophile, thermophile,
hyperthermophile
• superoxide dismutase, catalase, peroxidase
• defined, complex, selective & differential media
• binary fission, exponential growth, generation time
• Lag, Log, Stationary and Death phases
• biofilm, extracellular matrix, quorum sensing
• serial dilution, most probable #, spectrophotometry
Relevant Chapter Questions
MC: 1-7, 9, 10, 12, 15
FB: 1-10
11