CHAPTER 6 Microbial Nutrition and Growth

Objectives

1. Understand the various methods used by microbes for obtaining

carbon, energy and electrons
2. Learn about the benefits and dangers of using oxygen in
metabolism and how microbes can be classified by their ability to
use or tolerate oxygen
3. Learn about other chemical and environmental requirements of
microbes and how they can be classified by these needs.
4. Understand how microbes are cultured.
5. Learn how microbial populations grow in a culture medium.

Content

A. Patterns of metabolic pathways

a. Vary with sources of carbon, energy and electrons or hydrogen
atoms
b. -Troph: suffix referring to eating
c. Carbon
i. Autotrophs: source is CO2
ii. Heterotrophs: source is reduced organic compounds
d. Energy
i. Phototrophs: energy source is light
ii. Chemotrophs: energy source if from redox reactions
involving inorganic and organic compounds (aerobic
respiration, anaerobic respiration, fermentation)
e. Electrons or hydrogen ions
i. Organotrophs: from organic compounds, most
organisms
ii. Lithotrophs: from inorganic compounds; rarer
f. The organisms we’ll be studying are chemoheterotrophs as
humans
supply carbon, electrons and energy required for our pathogens
B. Oxygen requirements
a. Toxic forms of oxygen (free radicals)
i. Singlet oxygen: molecular O2 with energized electrons
ii. Superoxide radical (O2-) : superoxide dismutase
enzyme
required to change superoxide radical into hydrogen
peroxide
and oxygen; anaerobes die because they lack this
enzyme
iii. Peroxide ion (O22-): why hydrogen peroxide (H2O2) is
antimicrobial;
 - Catalase: H2O2 into H2O and O2
- Peroxidase: H2O2 into water and NAD+ (oxidized)
iv. Hydroxyl radical: most reactive of the four; enzymes
above
minimize production
b. Types of organisms and need for oxygen
i. Aerobes: use oxygen; have enzymes for detoxifying
ii. Facultative anaerobes: can use anaerobic
respiration or fermentation although prefer aerobic
respiration; Escherichia
iii. Aerotolerant anaerobes: don’t use but can tolerate
oxygen because they have some of the enzymes that
detoxify it (often peroxidases since they don’t make
more O2); Lactobacilli
iv. Microaerophiles: very limited ability to detoxify
oxygen; can tolerate smaller amounts than
aerotolerant anaerobes; Helicobacter pylori
v. Anaerobes: killed by oxygen because they can’t
detoxify it; Clostridia
vi. Obligate aerobes: algae, most fungi, protozoa and
many prokaryotes
vii. Facultative anaerobes: some yeast (fungi) and many
prokaryotes
viii. Other oxygen use patterns: many prokaryotes and a
few protozoa
c. Metabolic uses for oxygen
i. Structural component of organic compounds
(C,H,O,N)
ii. Final electron acceptor in aerobic respiration
pathway
iii. Part of bacteriocidal response in organisms with
lysosomes, neutrophils, macrophages, etc
iv. Photosynthesis waste product
C. Nitrogen requirements
a. Growth limiting nutrient
b. For use in metabolic reactions N must be in the form of
ammonium or
ammonia
c. Nitrogen fixation: rare but essential function; N2
(atmospheric gas)
 NH3
D. Other chemical requirements
a. May be limiting (phosphorus) and may be accumulated by
the
organism (inclusion bodies: sulfur)
b. Major elements: sulfur, magnesium, calcium, copper, iron,
 phosphorus, manganese
c. Trace elements: selenium, zinc, cobalt
d. Note that because of our similar metabolic pathways, in
general, a
(trace element), or micronutrient for a prokaryote is a
micronutrient for eukaryotes (check out your multivitamin
bottle)
e. Growth factors: some organisms need none (algae and
some bacteria = lithotropic photoautotrpohs), a few (many
prokaryotes synthesize their own vitamins), others require
standard vitamins and still others are fastidious requiring
many different and complex organic compounds
E. Temperature
a. Effects on
i. Shape of protein compounds
ii. Rigidity/fluidity of cell membranes & other lipid
structures
b. Minimum growth temperature  optimum growth
temperature  maximum growth temperature
decreibes the
temperature range of an organism
c. Psychrophiles: optimum is a low temperature
d. Mesophiles: medium temperatures; often our pathogens
e. Thermophiles: grow well at high to boiling temperatures;
include many Archaea and have been fruitful in providing
unique enzymes important in genetic engineering (stable at
high temperatures)
f. Cold temperatures do not kill organisms but do slow the
growth rate; extreme cold and drying (= lyophilization) may
even be used to preserve organisms in type culture
(standard strains) collections
F. pH
a. Effects on
i. Shape of protein and nucleic acid compounds
ii. Availablity for metabolic reactions
b. Neutrophiles
i. pH range of 6.5 to 7.5
ii. Our pathogenic organisms are of this type
iii. Organisms producing acid waste products in a limited
environment will eventually stop growing; has been
used in
food preservation (ex: sauerkraut & dill pickles)
c. Acidophiles
i. Obligate acidophiles: ex: chemoautotrophs that use
sulfur as
 the acceptor of excess hydrogen ions to produce
sulfuric acid
(H2S)
ii. Acid-tolerant organisms:
- May manipulate environment: Helicobacter pylori
secretes
bicarbonate and urease (urea  ammonia which is
basic)
d. Alkalinophiles
i. Yeasts: disruption of normal acid-tolerant bacteria
permits
overgrowth of yeasts in more alkaline vaginal
environment
ii. Increases in alkalinity of soil and water by natural
changes in
the environment encourage growth of the cholera
bacterium
Vibrio cholerae
G. Osmotic pressures
a. Cell wall protects in normal hypotonic environmnet
b. Obligate halophiles: extremophiles
c. Facultative halophiles: Staphylococcus aureus can tolerate
the higher
salt concentrations found on human skin
H. Ecological associations
a. Antagonistic: role of natural antibiotics and organic acids in
competition
b. Synergistic:
i. Symbiotic: each organism benefits
ii. Biofilms: complex bacterial communitites that allow
better
attachment to and control of their local environment
iii. Quorum sensing: ability of bacteria to respond to
density of
nearby bacteria and change expression of genes to
form
biofilms
iv. Ex: dental plaque
I. Culturing microbes: must supply microbe with needed nutrients and
environmental requirements (temperature, pH, etc)
a. Inoculum: microbes placed into the growth environment
b. Culture: the growth of (a culture) or the act of (to culture)
growing
microbes
c. Note that the anatomy of a colony can be used to help
identify
 organisms (pre-microscope & metabolic tests was once one
of the
main ways): pge 178: shape of entire colony, margin of
colony,
shape, size, texture, pigmentation and optical property
d. Getting pure cultures
i. Aseptic technique: methods used to minimize
contamination (unwanted microbial growth)
ii. Streak plate: smearing fewer and fewer microbes
until
isolated colonies are produced
iii. Pour plate: the microbes in serial dilutions are
poured into
individual petri dishes; if dilutions are accurate, this
can also
give an estimate of the number of microbes in the
original
sample
iv. Both of the methods used to get pure cultures rely on
media
solidified by agar; it’s advantages are:
- Can’t be digested by most microbes
- Liquid at a temperature that doesn’t damage
most
nutrients but can be sterilized
- Solid at temperatures useful for culturing
microbes
e. Types of culture media
i. Defined media: the exact chemical composition is
known
ii. Complex media: contains digests of heart, brain,
blood,
yeast, milk, etc; the exact composition is not defined
iii. Selective media: substances are present that
encourage
growth of the desired microbe, or inhibit the growth of
undesired ones; ex: MacConkey agar
iv. Differential media: appearance of the growing
colonies are
not the same; ex: presence and type of hemolysis in
blood
agar plates, carbohydrate utilization broths and
ability to
ferment lactose in Macconkey agar
iv. Anaerobic media
- Stab cultures into deeps
 - Reducing media: thioglycollate combines with
oxygen
v. Transport media
- Maintain ratios of organisms so overgrowth by
robust
ones doesn’t occur
- Support survival of delicate pathogens outside of
the body
- Keep anaerobes alive
J. Growth of microbial populations
a. Binary fission: duplication of the chromosome followed by
equal
division of the cytoplasm between the daughter cells
b. Logarithmic or exponential growth: number of cells in the
population = 2 n, where n is the number of generations
c. Generation time
1. Time from one cell division event to the next, under
optimal
conditions
2. Can have consequences in pathogenicity and lab culture
3. E. coli and S. aureus = 20 minutes
4. Mycobacterium = 10 days
d. Phases on microbial growth
1. Lag phase: adjustment to new medium
2. Log phase
i. Rapid constant log growth
ii. Most pathogenic in this phase
iii. Physiological traits usually expressed best
(staining
reactions, etc)
iv. Can be maintained indefinitely if in a chemostat
that
replaces nutrients and removes wastes
3. Stationary phase: rate of growth = rate of death
4. Death phase
i. Death exceeds growth
ii. If microbes can form resistant stages (cysts,
endospores,etc) these will be made in this phase
e. Measuring microbial growth
1. Direct methods
i. Viable plate counts: serial dilutions are plated out
until
colonies on one plate can be counted
ii. Membrane filtration: known volume of sample is
filtered
 through a membrane and the membrane is placed
on
a suitable culture medium
iii. Direct counting on a grid or electronically with a
Coulter
Counter
iv. Most probable number (MPN): number of
samples showing growth at each dilution will give
results that can be used to estimate density of
original culture
2. Indirect methods
i. Metabolism: rate of usage or production of
measurable
compound; can measure living microbes
ii. Dry weight: limited to measuring dead microbes
iii. Turbidity: low tech (reading newspaper print) to high
tech
methods (spectrophotometer)