Download Standard Growth Conditions and Measurement of Growth

Standard Growth Conditions
and Measurement of Growth
Binary fission in bacteria
Scanning electron
micrograph
Geometric progression
in the number of
bacteria in a
population resulting
from binary fission
Generation time 
length of time it takes
a single bacterium to
double
E. coli 25 minutes
Mycobacterium spp. 1-3
days.
Binary fission requires the addition of new
material at the growing sites of bacteria
Gram positive cells such as
this coccus, new material
is added at the division plane
Gram negative cells such as
this bacillus, new material is
added at the division plane and
also throughout the length of
the cell
Bacteria need to synthesize the macromolecules that allow for their
growth and reproduction.
Bacteria need to synthesize macromolecules
that allow for their growth and
reproduction— what are bacterial cells made
up of ?
Cells consist of WATER and MACROMOLECULES
Macromolecules are made up of smaller monomeric molecules
Small monomeric molecules are made up of atoms
Macromolecules of the bacterial cell
Proteins—The most abundant class of macromolecules and comprise
most of the structures of the cell as well as enzymes
amino acids—monomeric subunits of proteins
consists of carbon, hydrogen, oxygen, nitrogen, sulfur and
sometimes selenium
amino acids are covalently linked to form a peptide bond
H
H H
H
NH3
C
COOH
R
Where have you heard the
word tetra-peptide?
NH3
C
C N
R1 O
C
R2
Peptide bond
COOH
Macromolecules of the bacterial cell
(cont’d)
Polysaccharides—2nd most abundant of the bacteral macromolecules
sugars (monosaccharides)—monomeric units
consists of Carbon, Hydrogen and Oxygen atoms at a ratio
of 1:2:1
individual sugars are linked by a glycosidic bond
Polysaccharides form covalent linkages with other macromolecules
with proteins—glycoproteins
with lipids—glycolipids, lipopolysaccharides
Macromolecules of the bacterial cell
(cont’d)
Nucleic Acids
DNA—deoxyribonucleic acid
RNA—ribonucleic acid
Backbone of nucleic acids= polymer of phospho-ribose (RNA)
or phospho-deoxyribose (DNA)
The sugars are covalently attached to each other by phosphodiester bonds
Bases are attached to a carbon atom of the sugar moiety
cytosine, adenine, guanine (DNA/RNA); thymine (DNA)
uracil (RNA)
Comprised of the atoms Phosphorous, Carbon, Hydrogen, Oxygen
and Nitrogen
Macromolecules of the bacterial cell
(cont’d)
Lipids—made up of a long carbon chain—fatty acids—(14-20
carbons and one carboxylic acid group)
Saturated—Hydrogen atoms attached to most or all carbon
moieties.
Unsaturated—fewer Hydrogen atoms associated with carbons
Complex lipids are attached to simple sugars like phoshoglycerol
(ie phospholipids) or complex polysaccharides (LPS)
C, H, O, P
Bacterial Nutritional Requirements
NUTRITION::act of supplying microorganisms with the molecules
and atoms they require for the biosynthesis of small molecules
and macromolecules
Macronutrients: nutrients required in high amounts
Carbon, Nitrogen, Phosphorous, Sulfur
ALSO
Potassium, Magnesium, Calcium, Sodium
Micronutrients: nutrients required in small or even trace amounts
Chromium, Cobalt, Copper, Manganese, Molybdenum
Nickel, Selenium, Tungsten, Vanadium, Zinc, Iron
Growth Factors::organic compounds required in very small amounts
Vitamins, amino acids, purines and pyrimidines
Bacterial Nutritional Requirements
Autotroph: an organism capable of biosynthesizing all cell
material from CO2 as a sole carbon source
Heterotroph: an organism that requires carbon from preexisting
organic material
Photo--: solar energy converted to chemical energy
Chemo--: energy derived from chemical compounds
Catabolism: Act of breaking down complex molecular material
for energy or biosynthetic material
Anabolism: The act of biosynthesizing complex material from simpler
organic compounds
Culture media for artificial cultivation of bacteria in the lab
1. Complex (undefined)—enzymatic digests of milk protein
(casein), beef, yeast
2. Chemically defined—precise amounts of purified organic
and inorganic compounds are added to distilled water
Types of culture media
Fastidious
Brock, 10th edition
Other considerations with respect to
bacterial growth
1. pH—optimum pH of most organisms is 7.0
2. Water activity—most bacteria require a water activity
between 0.9 and 1.0
3. Osmolarity—The osmolarity of the bacterial cell cytoplasm
must be greater than that of its environment for cell growth—
turgor pressure
4. Oxygen—bacteria have a great variety of specifications
with respect to the amount of oxygen they require
5. Temperature—most organisms like 37oC
About oxygen
Aerobes—capable of growth at full oxygen tensions
Microaerophiles—can only grow when oxygen tensions are lower
than that found in air (soil, water bacteria)
Anaerobes—
obligate anaerobes—oxygen kills the organism
facultative anaerobes—prefer oxygen but can grow in its
absence
aerotolerant anaerobes—can grow in the presence of oxygen
but they don’t use it.
Quantifying bacteria
Direct microscopic cell count
Viable cell count (plate count or colony count)
Indirect measurement::microbial turbidity
Use of the hemocytometer or Petroff-Hausser
counting chamber (direct microscopic count)
Sample added to the surface of
the grid, the whole grid has 25
large squares, the total volume
that can be added is 0.02mm3
12 bacteria in one square
(assume 12 in each large
square) 12 X 25 = 300 bacteria
Total volume held is 0.02 mm3
300/.02 = 15000 or 1.5 X 104
Bacteria per mm3
1cm3 = 1000mm3 (10 x 10 x 10)
1.5 X 104 x 1000 = 1.5 X 107/cm3 or mL
Use of dilution and direct plating to measure
viable bacteria (viable cell count)
Use of the spectrophotometer to quantify
bacteria in a population
OD= Log Io/I
Io incident light
I unscattered light
Use of the spectrophotometer in measuring
the number of cells in a population
By using both spectrophotometry and dilutions
and plate counts one can
make a good correlation
between optical density
and cell population numbers
Stages of bacterial growth::typical growth
curve
Biphasic Growth Curve
2nd stationary phase
cells have exhausted
lactose
2nd log phase, cells prepare
enzymes required for the
catabolism of lactose
Premature stationary phase
cells have exhausted available
glucose