Chapter 6
Microbial Growth
1
Growth vs. Reproduction
Growth: an orderly and permanent increase in the mass of
protoplasm of an organism or population.
"orderly" - a proportionate increase in all constituents
Reproduction: an increase in the numbers of individuals and can
be independent of growth
cells may grow without reproducing or may reproduce
without growth
2
Effects of Temperature
on Bacterial Growth
Temperature range for any organism :
minimum temperature: lowest where growth occurs
optimum temperature: where a species grows best
maximum temperature: highest where growth is possible
Psychrophiles: "cold-loving", grow at 0oC to 20oC, optimum
15oC
Mesophiles: "middle" loving, grow at 20oC to 45oC, optimum
20oC to 37oC
Thermophiles: "heat-loving”, grow at 37oC to 65oC
3
cold lovers
4
cold growers
5
6
pH and Bacterial Growth
For most organisms, optimum pH is generally between pH 6.5 pH 7.5
Acidophiles (acid loving) have optimums between pH 0 - 5.5
Basophiles: (basic loving) optimum from pH 8.5 to 11.5
organisms can change the culture media due to their own waste
products (generally acidic), may be toxic due to pH change
7
Osmotic Pressure & Bacterial Growth
Cells are 80-90% water
Isotonic solution (80-90% water): no net movement of water
Hypotonic: water will move into cell (cell rounds up and can
burst)
Hypertonic: water moves out of cell (cell shrinks, plasmolysis)
Some organisms can tolerate high osmotic pressure conditions
Halophiles: grow in high salt conditions (obligate halophile
requires high salt concentrations)
8
Chemical Requirements
for Bacterial Growth
9
Nutrients Required for Growth
Growth requires availability of macronutrients, trace elements,
and growth factors
Carbon: structural backbone of many molecules
Nitrogen, Sulfur, Phosphorus required items for production of
proteins as well as DNA/RNA (requires N & P)
Organic growth factors: vitamins, some amino acids, purines, pyrimidines
Trace elements – often needed for proper enzymatic functions
ANY of these nutrients can be limiting factors in growth
10
Oxygen Effects on Growth
organisms can produce more energy when growing in oxygen,
but toxic byproducts of oxygen can be fatal to cells (Table 6.1)
Toxic byproducts from use of O2
Superoxide Radical – extremely toxic to living cells. This
harmful molecule is neutralized by the enzyme superoxide
dismutase (SOD) which is produced by all organisms growing in
the presence of oxygen. SOD converts superoxide radical to
hydrogen peroxide & O2
note: hydrogen peroxide is still toxic, this will be dealt with shortly
11
Toxic Byproducts of O2
continued
Hydrogen Peroxide, toxic to cells can be neutralized two
different enzymes
Catalase
2 H2O2  2 H20 + O2
you can detect presence of this enzyme in an organism by
applying hydrogen peroxide to a culture (on solid media) and
observing presence of bubbles
Peroxidase H2O2 + 2 H+  2 H20
note: since this reaction doesn’t produce gaseous oxygen you can
not see bubbling when applying hydrogen peroxide.
12
Classification of Organisms
by O2 Requirements
Aerobes: require oxygen for growth
Facultative Anaerobes: can grow with or without oxygen
Anaerobes: cannot tolerate molecular oxygen
Microaerophiles: require oxygen, but can only grow at low
oxygen tension
13
14
Isolation of Pure Cultures
Most microorganisms in nature are found in mixed cultures.
Isolation of colonies is done using the streak plate technique.
Each isolated colony begins as a single bacterium.
A pure culture is a culture coming from an isolated colony on a
plate containing only one colony type.
15
Other culture techniques
Spread plate – a diluted liquid inoculum is spread over surface
of a plate (fig 6.16 part b) – another isolation technique
Pour plate – a diluted liquid inoculum is mixed with melted
nutrient agar and colonies are allowed to grow – another isolation
technique
Broths - propagation (rapid growth) of cultures
Slants – maintenance of cultures
16
Types of Media
Culture media: nutrient material prepared for the growth of
microorganisms in the laboratory (must initially be sterile)
Synthetic (or Defined) Medium: made of known amounts of
chemicals
example: E. coli minimal salts medium
Complex medium: some ingredients are of unknown
composition or amount (extracts of plants, yeast or meat)
 Nutrient broth
 Tryptic soy broth
17
Solid, Semi-Solid, Liquid?
Solid media: a synthetic or complex medium can be solidified
using agar.
Agar: from red algae, sulfated polymer of D-galactose, 3,6 anhydro-Lgalactose, and D-glucuronic acid, is not degraded by most microorganisms.
Agar melts in boiling water, solidifies when cooled to about 40oC, does not
melt again until 80 - 90oC
Solid media: 1.5% agar, give surface for growing bacteria (often
used to maintain cultures)
Semi-solid media: usually 0.5% agar, used for motility studies
Liquid media: no agar, used for propagation of cultures
18
General or Specific Media?
General medium - provides basic needs of organisms
Selective medium - encourages the growth of certain organisms
while discouraging the growth of others. examples: crystal violet
or basic fuschin dyes selective for Gram- (against G+)
MacConkey, EMB
Differential medium - distinguishes between different groups of
bacteria medium contains constituents which cause an observable
change (color or pH change)
example: MacConkey contains lactose and neutral red, lactose
fermenters appear pink; Blood agar - hemolysis
19
Cultivation Of Anaerobes
Special media/conditions necessary may include :
reducing media (chemicals to remove oxygen, ex. sodium
thioglycollate)
non-oxygen atmosphere (candle jar or anaerobic jar)
Enrichment medium: provide conditions which will only allow
bacteria with a specific desired trait to survive and grow
(particular carbon source, growth conditions, etc.).
example: Sabouraud’s dextrose agar (fungi)
20
Microbial Growth
Bacterial division: binary fission, two identical cells, each with
one full chromosome
Generation time: the time required for cells to divide (and thus
double the population), can be as short as 20 minutes or longer
than a day
Calculation of generation time (use logarithmic plot), fig. 6.13
and 6.14
21
Microbial Growth Curve
Sufficient media to
support cells present
Excess media
22
Insufficient media
to support life.
Metabolic waste buildup
Measuring Growth
Direct methods:
1. Plate counts (serial dilutions) increase in number of bacteria
with time (viable count), each colony represents one bacterium
2. Filtration – known volume of bacterial culture collected on a
filter disk then the disk is placed in media and allowed to grow
(fig 6.17 p 177) each colony represents one bacterium in original
sample
3. Most probable number (MPN): statistical method
4. Direct microscopic count: Petroff-Hausser counter
23
24
25
explanation of MPN
Most Probable Number Method uses statistics to provide an
estimate of number of bacteria in a given sample.
In the book’s example you inoculate three sets of five tubes
Our original sample has 100ml total volume.
Question, how many bacteria does this contain?
Series 1 tubes each get 10 ml of the original sample.
Series 2 tubes each get 1 ml of original sample.
Series 3 tubes each get 0.1 ml of original sample
26
How many tubes in each series had
bacterial growth? (indicating presence of
at least one bacterium in our inoculation)
Series 1 = all five tubes have growth
Series 2 = three tubes
Series 3 = only one tube
Interpretation of results.
5 – 3 – 1 (find in table)
95% of the time, a sample giving this pattern
will have at least 40 bacteria in original
sample possibly as many as 300. The average
number of bacteria in a sample with this
pattern is 110
27
28
Measuring Growth
Indirect methods:
1. increase in turbidity with time
(cloudier media = more bacteria)
2. metabolic products – measurable metabolic products assumed
to increase as numbers of bacteria increase
3. dry weight – bacteria separated by centrifuge and then weighed
29
30
STOP
31