Chapter 6:

Microbial Growth
1. Requirements for Growth
2. Culturing Microorganisms
3. Patterns of Microbial Growth
4. Measuring Microbial Growth

1. Requirements for Growth

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)

Temperature & Microbial Growth

Growth rate

Thermophiles

10

Mesophiles

Hyperthermophiles

Psychrophiles

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
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)
microbial growth tends
to acidify the growth
medium, inhibiting
further growth

Osmotic Pressure
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

Oxygen (O2)
As weve 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

Oxygen & Microbial Growth

Oxygen
concentration
High

Low

Obligate
aerobes

Loosefitting
cap

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!*

2. Culturing Microorganisms

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

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

Selective medium
compare A
(non-selective)
with B (selective)

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

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

(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

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

60
Species A

40
30

30
20

10

10

2
1
Time (hours)

real living
organisms
reproduce
exponentially

40

20

Species B

50
Number of cells

50
Number of cells

exponential

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!

Microbial Growth Patterns

Microorganisms cannot undergo unlimited
growth, eventually the chemical and physical
environment in which theyre 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 WHATS 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**

4. Measuring Microbial Growth

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**

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!

Most Probable
Number
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

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

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