Transcript Lecture 7 & 8 Microbial Growth (Ch.6)

MICROBIAL GROWTH
Requirements for microbial
growth
• Physical
• Chemical
Physical
• Temperature
• pH
• Osmotic pressure
Temperature
• -10 to
C,
Psychrophiles
o
20
Temperature
• 0 to
C,
• Psychrotrophs
o
30
Temperature
• 10 to 50o C,
• Mesophiles
–Most
pathogens
Temperature
• 40 to
C,
thermophiles
o
• 65 to 110 C,
Extreme
thermophiles
o
70
Extreme
thermophiles
Thermophiles
Mesophiles
Psychrotrophs
Psychrophiles
-10 0 10 20 30 40 50 60 70 80 90 100 110
Degrees Centigrade
pH
• Most bacteria grow at pH
6.5 to 7.5
• Acidophiles (pH 1.0 - 5.5)
Lactobacillus acidophilus
pH
• Alkalophiles
(pH 8.5 - 11.5)
Bacillus alcalophilus
Osmotic Pressure (OP)
• Bacteria are better
adapted to low OP (i.e.
tap water)
• Plasmolysis occurs at
high OP
Osmotic Pressure (OP)
(cont.)
• Solutes (i.e. sugar and
salt) limit water
availability
Osmotolerant Organisms
(halophiles)
• Less tolerant
–Most Gram-negatives
• More tolerant
–Staphylococcus aureus
–Fungi
1. SEA ICE
cold-loving
microbes
(Psychrophiles)
Polaromonas
vacuolata
2. DEEP-SEA
VENT
heat-loving microbes
(Thermophiles and
Hyperthermophiles)
Methanopyrus
kandleri
3. SULFURIC
SPRING
acid-loving
microbes
(Acidophiles)
Sulfolobus
acidocaldarius
4. SALT
LAKE
salt-loving
microbes
(Halophiles)
Haloferax
volcanii
5. SODA
LAKE
alkali-loving
microbes
(Alkaliphiles)
Natronobacteri
um gregoryi
Chemical Requirements
• Carbon (all organic
compounds)
• Nitrogen (proteins)
Chemical Requirements
(cont.)
• Sulfur (proteins and
vitamins)
• Phosphorous (DNA,
RNA, ATP)
Trace Elements
• Needed in minute
amounts
-Iron
-Copper
-Molybdenum -Zinc
Trace Elements (cont.)
• Present in high enough
amounts in nature
• Enzymatic activity
–Cofactors
Organic growth factors
• Essential for growing
–Vitamins
–amino acids
–Purines and pyrimidines
Oxygen (O2)
• Electron acceptor during
aerobic respiration
• More efficient than other
acceptors (NO3, SO4, and
CO3)
Oxygen (O2)
• Highly reactive oxygen
radicals
–Singlet oxygen
–Superoxide free radicals
Neutralization of oxygen
radicals
• Superoxide Dismutase
(SOD)
• Catalase
Culture Medium
Forms an oxygen gradient
More Oxygen on top
O2
Obligate Aerobes
• Aerobic only
(O2)
O2
Mycobacterium tuberculosis
Facultative Anaerobes
• Primarily
aerobic
O2
• Alternatively
anaerobic
Escherichia coli
Obligate Anaerobes
• Killed by
oxygen
O2
Clostridium spp.
Aerotolerant Anaerobes
• Growth not
affected by
Oxygen
O2
Enterococcus faecalis
Microaerophiles
• Aerobic
• Killed by
O2
high (20%) O2
concentration
Campylobacter spp.
The Requirements for Growth:
Chemical Requirements
• Oxygen (O2)
Facultative anaerobe
Obligate aerobe
Aerotolerant aerobe
Obligate anaerobe
Microaerophilic
Bacterial Growth
Binary fission
• Asexual
reproduction
in which a
cell separates
into two cells
Resting cell
Cell elongation and DNA
replication
Cell wall and cell membrane
grow inward
Cell wall and Cell membrane
meet
Individual cells are formed
Generation or Doubling
Time
• Time required for a cell to
double in number
• Highly variable, 20
minutes to 24 hours
Generation Time (cont.)
• Escherichia coli
–20 minutes
• Mycobacterium tuberculosis
–12 hours
Escherichia coli’s
generation time 8
16
32
4
2
1
0
20
40
60 80 100 120 140
Minutes
Logarithmic growth
Number of bacteria
Bacterial Growth Curve
Lag phase
Time
Number of bacteria
Bacterial Growth Curve
Log
Phase
Time
Number of bacteria
Bacterial Growth Curve
Stationary
Phase
Time
Number of bacteria
Bacterial Growth Curve
Death
Phase
Time
Measurement of Microbial
Growth
• Plate counts
• Pour plates
Direct Measurements of
Microbial Growth
• Plate Counts: Perform serial
dilutions of a sample
Figure 6.15, top portion
Serial Dilutions
• Transfer of part of a
sample through a series of
specific volumes of
diluent
Serial Dilutions (cont.)
• Reduce high numbers of
microorganisms gradually
(i.e. form 100,000,000 to
10)
Membrane filtration
Sterile
cup
Vacuum
Membrane
0.45 mm
Membrane filtration
Culture medium
Direct Measurements of
Microbial Growth
• Filtration
Figure 6.17a, b
Most Probable Number
(MPN)
• Statistical estimation of
the number of organisms
present in a sample
Most Probable Number
(MPN) (cont.)
Water samples Positives
0.1 ml
1/5
1 ml
2/5
10 ml
4/5
CULTURE MEDIA
Nutritional types of
Microorganisms (energy)
• Phototrophs (light)
• Chemotrophs (organic or
inorganic compounds)
Nutritional types of
Microorganisms
(Carbon source) (Cont.)
• Autotrophs (CO2)
• Heterotrophs (organic
carbon)
Culture Medium
• Material prepared in the
laboratory for the growth
of microorganisms
Culture
• Microorganisms that
grow in or on a culture
medium
Streak Plate for obtaining
a pure culture
Figure 6.10a, b
Sterile
• Absence of life
Chemically Defined Media
• Exact chemical
composition is known
• Mostly autotrophic
organisms
Chemically Defined Media
(cont.)
• Ammonium sulfate 0.5 g
• Potassium phosphate 0.2
• Calcium chloride
0.04
• Magnesium sulfate 0.04
• Water
1 liter
Complex Media
• Exact composition is
unknown
• Slight differences from
batch to batch
• Extracts from meats,
milk, vegetables and yeast
Complex Media
Composition
• Soy peptone
5.0 g
• Beef extract
3.0
• Sodium chloride 8.0
• Agar
15.0
• Water
1 liter
Agar
• Complex polysaccharide
• Solidifying agent
• Extracted from red algae
Deserts using agar
Agar-agar – jello made out of seaweed, much firmer than the kind
you get in hospitals.
Agar used in the lab
Robert Koch and Agar
(History and Microscopy lecture)
• He invented new methods –
• of cultivating pure cultures of bacteria
on solid media such as potato, and on
agar kept in the special kind of flat
dish invented by his colleague Petri,
which is still in common use
• (He also developed new methods of
staining bacteria which made them
more easily visible and helped to
identify them)
Anaerobic Growth Media
(broth)
• Sodium thioglycolate
scavenges O2 by
combining with it
Growth Media
Sodium
thioglycolate
O2 O2
O 2 O2
O2 O2
Clostridium spp.
Aerobic
Anaerobic
Capnophiles require high
• Candle jar CO2
• CO2-packet
Figure 6.7
Anaerobic Growth Media
(solid)
• Anaerobic atmospheres
(without O2) are obtained
using special jars
Anaerobic Growth Media
(solid)
CO2
H2
Pouch with
CO2 , H2 and
Na borohydride
Anaerobic jar
Anaerobic Culture Methods
• Anaerobic
chamber
Figure 6.6
Fastidious Organisms
• Do not grow on known
media
• Difficult to grow
Fastidious Organisms
(cont.)
• Mycobacterium leprae
–Animals
• Treponema pallidum
• Rickettsias and
Chlamydias
Selective Media
• Suppresses the growth of
unwanted bacteria,
without affecting the
target organism
Selective Media (cont.)
• Bismuth sulfite agar
–Salmonella typhi
• Mannitol salt agar
–Staphylococcus aureus
Selective Media
• Suppress
unwanted
microbes and
encourage
desired
microbes.
Figure 6.9b, c
Differential Media
• The target organism
forms distinctive
colonies, usually of
different color or
appearance
Differential Media (cont.)
Blood agar
Streptococcus pyogenes
Differential Media
• Make it easy to distinguish
colonies of different
microbes.
Figure 6.9a
Differential-Selective
Media
Mannitol-salt
agar
Staphylococcus aureus
Staphylococcus epidermidis
Chromogenic medium
Enrichment Media (broth)
• Selective medium that
favors the growth of the
target organism when
present in low numbers