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I. Laboratory Culture of
Microorganisms
• 3.1 Cell Chemistry and Nutrition
• 3.2 Culture Media
• 3.3 Laboratory Culture
Microbial Nutrition
Categories
Energy source (Electron Donor) ED
Macronutrients
Micronutrients
Trace Elements
Terminal Electron Acceptor (TEA)
Energy Source
Organotroph- Organic Molecules
Lithotroph- Inorganic Molecules
Phototrophs- light
S + TEA------------- P (R/O) + Cell Mass + E
Macronutrients
C-Source
CO@-CO2-Autotroph
Organic-Heterotroph
N-Source
Organic-NH2
Inorganic NH3 or N03
P-Source
Organic-RNA
Inorganic H2P04
S-Source
Organic-Amino Acid(Cysteine)
Inorganic-SO4, S-2
3.1 Cell Chemistry and Nutrition
• Carbon
– Required by ALL cells
– Typical bacterial cell is ~50% carbon (by dry
weight)
– Major element in ALL classes of
macromolecules
– Heterotrophs use organic carbon
– Autotrophs use carbon dioxide (CO2)
3.1 Cell Chemistry and Nutrition
• Nitrogen
– Typical bacterial cell is ~13% nitrogen
(by dry weight)
– Key element in proteins, nucleic acids, and
many more cell constituents
3.1 Cell Chemistry and Nutrition
• Other macronutrients
– Phosphorus (P)
• Synthesis of nucleic acids and phospholipids
– Sulfur (S)
• Sulfur-containing amino acids (cysteine and methionine)
• Vitamins (e.g., thiamine, biotin, lipoic acid) and coenzyme
A
– Potassium (K)
• Required by enzymes for activity
3.1 Cell Chemistry and Nutrition
• Other macronutrients (cont'd)
– Magnesium (Mg)
• Stabilizes ribosomes, membranes, and nucleic acids
• Also required for many enzymes
– Calcium (Ca)
• Helps stabilize cell walls in microbes
• Plays key role in heat stability of endospores
– Sodium (Na)
• Required by some microbes (e.g., marine microbes)
3.1
• Iron
Cell Chemistry and Nutrition
– Key component of cytochromes and FeS proteins
involved in electron transport
• Growth factors
– Organic compounds required in small amounts by
certain organisms
• Examples: vitamins, amino acids, purines, pyrimidines
– Vitamins
• Most commonly required growth factors
Micronutrients
Cations- monovalent K+, Na+Di-valent Mg++. Ca++, Fe+2
cofactors/Enzymes
Anions- Cl-
Trace Elements
Co, Zn, Mo, Mn, Ni,Se
Vitamins, Yeast Extract
Culture Media
Difco, BBL
Liquid Media- Cell Numbers
Solid MediaAgar- Complex polysaccharide Sea Weed
1.5%
Melts 80-90
Solidifies 40-42 C
Inert Gel
3.2 Media and Laboratory
Culture
• For successful cultivation of a microbe, it is
important to know the nutritional
requirements and supply them in proper
form and proportions in a culture medium
Types of Culture Media
1. Synthetic or Defined Media
Chemically Defined
2. Complex Media/General Purpose
Chemical Composition not known
Biological or plant Extract
3. Selective Media
Selects one M.O. Over another
Bile salts Selects GM- over GM +
4. Differential Media
Distinguishes between a given group
of Bacteria
GP Media
Difco Nutrient Agar
Beef Extract
3g
Peptone
5g
Agar
Reagent Water
ml
1.5g
1000
3.2 Media and Laboratory
Culture
• Pure culture: culture containing only a
single kind of microbe
• Contaminants: unwanted organisms in a
culture
• Cells can be grown in liquid or solid culture
media
– Solid media are prepared by addition of a
gelling agent (agar or gelatin)
– When grown on solid media, cells form isolated
Enrichment Media
Environmental Tolerances
Oxygen
pH
Osmotic
Temperature
Pressure
Substrate
organic
inorganic
Km (Affinity)
Growth Rate
Effect of Oxygen
Obligate aerobes- Grow only in presence of Oxygen
Facultative Anaerobes- With or with out
Aerotolerant anaerobes- Ignore Oxygen
Obilgate Anaerobes- Die in Oxygen
Microaerophilis- 1-10% Oxygen
Toxic By Products
Superoxide (02- ): 02 + e-  02Hydrogen Peroxide: 02- + e- + H+ H2O2
Hydroxyl radical: H2O2 + e- + H+ -- H2O + 0H.
Chlorophyll + hv------ Chlorophyll*
Chl* + 02------ ‘02
Growth
Binary Fission/Asexual Growth
Clones 1 cell=2 Cells
Cell Cycle=
G
G D
C=Chromosome Replication
G= distribution of cell synthesis
D=division
I. Bacterial Cell Division
•
•
•
•
5.1
5.2
5.3
5.4
Binary Fission
Fts Proteins and Cell Division
MreB and Cell Morphology
Peptidoglycan Biosynthesis
5.2 Fts Proteins and Cell Division
• Fts (filamentous temperature-sensitive) proteins
(Figure 5.2)
– Essential for cell division in all prokaryotes
– Interact to form the divisome (cell division
apparatus)
• FtsZ: forms ring around center of cell; related to tubulin
• ZipA: anchor that connects FtsZ ring to cytoplasmic
membrane
Outer membrane
FtsI
ZipA
Peptidoglycan
FtsA
ATP
FtsZ
ring
GTP
GDP +
Pi
FtsK
ADP +
Pi
Cytoplasmic
membrane
Divisome
complex
FtsZ ring
Cytoplasmic membrane
Figure 5.2
5.2 Fts Proteins and Cell Division
• DNA replicates before the FtsZ ring forms
(Figure 5.3)
• Location of FtsZ ring is facilitated by Min
proteins
– MinC, MinD, MinE
• FtsK protein mediates separation of
chromosomes to daughter cells
MinCD
Minutes
Cell wall
Cytoplasmic
membrane
Nucleoid
0
MinE
20
40
Divisome
complex
60
FtsZ ring
Septum
Nucleoid
80
MinE
Figure 5.3
FtsZ
Cell wall
Cytoplasmic
membrane
MreB
Sites of cell
wall synthesis
Figure 5.5a
II. Population Growth
• 5.5 Quantitative Aspects of Microbial
Growth
• 5.6 The Growth Cycle
• 5.7 Continuous Culture
5.5 Quantitative Aspects of
Microbial Growth
• Most bacteria have shorter generation times
than eukaryotic microbes
• Generation time is dependent on growth
medium and incubation conditions
Growth =Population Doubles/Unit Time
Generation Time=Time to double Population
Complex Series of Events
>2000 chemical reactions
1800 different Proteins
1 Complete Genomic DNA Replicated
1 Cytoplasmic membrane Synthesized
Short Time= E. coli= 20 minutes
5.6 The Growth Cycle
• Batch culture: a closed-system microbial culture
of fixed volume
• Typical growth curve for population of cells
grown in a closed system is characterized by
four phases (Figure 5.11):
– Lag phase
– Exponential phase
– Stationary phase
BATCH Culture: Growth curve
Batch Culture
Closed System
Nutrients Limited
Growth Conditions Optimize
pH
aeration
substrate
Incubate
Enumerate cells#/Unit Time
Exponential Growth Calculations
Log Phase
#/ T = Rise/Run= Kd
Geometric Progression of the # 2
2°21222324----2n
1
2
4
8
16----n # generations
Nt=No2n
Scientific Notation
LOG Transformation
Scientific Notation:
1000000000= 1.0 X 109
(Coefficient/Base 10/Exponent)
0.000000001= 1.0 X 10-9
LOG10= Coefficient=1.0 Then LOG10=Exponent
LOG10 2= 0.301(X 2.303=0.693)
e=ln=Natural Logs=ln 2 =0.693 (/2.303=0.301)
Multiplication/Division using LOGs
To Multiply Numbers Written in Exponential Notation
ADD the Exponents: (3X 104) X (2 X 103)=
(3X2) X 104 + 3= 6 X 107
To Divide, Divide the Coefficients and Subtract
the Exponents
3 X 104 = 3/2 X 104= 1.5 X 101
2 X 103
Logarithm
Power a base number is raised to produce a given number
Log10 0.00001= log10(1 X 105
= -5
Log101000000= log10(1 X 106)
= 6.0000
Exponential Growth Calculations
Log Phase
#/ T = Rise/Run= Kd
Geometric Progression of the # 2
2°21222324----2n
1
2
4
8
16----n # generations
Nt=No2n
Calculation of Growth Kinetics
Nt=No2n
gt= T/n
gt= Time for population to double
(Generation Time hr-1/division)
T= Time
n= # generations
gt= T/n; n = t/gt; t = n X gt
Log transformation of formula
Log10 NT= log10 No + (log10 2)(n)
Log10 NT/ log10 No = (log10 2)(n)
n = Log10
NT- log10 No = #
(log10 2)
0.301
Example of Growth rate Calculation
Nt=No2n
gt = 20 min
N0 = 1 Staphyloccus aureus/gram potato salad
Nt? After 4 hr?
gt= 0.333 hr-1/division
2n = ?
212
Log transformation
(12)(log 2)
(12)(.301)= 3.612
Antilog 3.612 = 4092 cells
Alternate way to calculate Growth Kinetics
#/ T = Rise/Run= Kd
Nt=Noet
= logNt-Log No Page 143
(log 2) (t)
 = 8.0-7.69 (Nt = 1 X 108; No = 5 X 107)
(.301)(t=2hr)
 = 0.5 div/hr-1
gt? = 1/ = 1/0.5 = 2 hr
gt = t/n = 2hr/n=1 = 2 hr
 (k), gt, t, n
One can calculate different organisms growing
under different conditions
What do we do with this Information?
1. Optimize culture conditions!
2. Test for “+” or “-” effects of an experimental
Treatment!
Anaerobic jars
Anaerobic glove box
Methods of Enumeration
?
(k), gt, t, n
Direct Methods
Direct Microscopic Methods
D
Counting chambers
i
fixed Film
r
e
Particle Sizer
c
t
Indirect Methods (Viable Cell Counts)
Plate Counting Techniques
Pour Plate
Spread Plate
Most Probable Number (MPN)
Membrane Filtration (MF)
Turbidity (absorbance)
Ridges that support coverslip
To calculate number
per milliliter of sample:
12 cells X 25 large squares X 50 X 103
Coverslip
Number/mm2 (3 X 102)
Sample added here. Care must be
taken not to allow overflow; space
between coverslip and slide is 0.02 mm
1 mm). Whole grid has 25 large
( 50
squares, a total area of 1 mm2 and
a total volume of 0.02 mm3.
Microscopic observation; all cells are
counted in large square (16 small squares):
12 cells. (In practice, several large squares
are counted and the numbers averaged.)
Number/mm3 (1.5 X 104)
Number/cm3 (ml) (1.5 × 107)
Figure 5.15
5.8 Microscopic Counts
• Limitations of microscopic counts
–
–
–
–
–
–
–
Cannot distinguish between live and dead cells without special stains
Small cells can be overlooked
Precision is difficult to achieve
Phase-contrast microscope required if a stain is not used
Cell suspensions of low density (<106 cells/ml) hard to count
Motile cells need to immobilized
Debris in sample can be mistaken for cells
5.9 Viable Counts
• Viable cell counts (plate counts):
measurement of living, reproducing
population
– Two main ways to perform plate counts:
• Spread-plate method (Figure 5.16)
• Pour-plate method
• To obtain the appropriate colony number,
the sample to be counted should always be
diluted (Figure 5.17)
Counting the number of viable cells by
serial dilution and plate count
Figure 6.5 (Part 1) Concentration of cells by
membrane filtration
Concentration of cells by
membrane filtration
Concentration of cells by membrane filtration
Relationship between
light absorbency and cell mass
Relationship between nutrient
concentration and total cell mass
Continuous culture in a chemostat
Steady-state relationship between substrate concentration
and output of bacterial mass
Response of bacterial growth
to oxygen availability