MICROBIAL GROWTH Chapter 4, pp 83-107
I. Growth requirements (Table 4-1, p87)
A) physical requirements
1) temperature - generally the higher the temp, the faster the
growth
a)Psychrophiles = 0 to 20oC (Fig 4-4, p
86)
- more double bonds in fatty acids of membranes
b) Mesophiles = 25 to 40oC (includes most pathogens)
c) Thermophiles = 40 to 80oC
some can grow at 90 - 100oC, membranes rich in saturated fatty
acids
2) pH - usually between pH 5 - 8, acidophiles = low pH (2 - 3)
Buffers maintain proper pH of growth medium
3) Osmotic pressure = results from solute concentration
(hypertonic and hypotonic)
High solute concentration may be lethal due to plasmolysis (Fig
4-5, p 89)
obligate halophiles = require 3 - 6 molar salt
Halobacterium, Halococcus (Dead Sea)
B) Chemical requirements (see Table 4-3, p90)
1) Carbon - about 50% dry weight of cell, all life needs a carbon
source.
Many cells (including ours) use glucose, however, bacteria are
diverse in
this aspect
2) Nitrogen - about 14% dry weight of cell; inorganic sources =
NH3, NO3, NO2, N2; organic sources = amino acids, urea
a) Nitrogen Fixation
N2 ---> NH3
biologically, only done by some bacteria and Cyanobacteria,
inhibited by oxygen.
Rhizobium = symbiotic with plants produces
leghemoglobin; heme is bacterial
in origin, globin is of plant origin.
b) NH3 = most efficient N source
3) Sulfur most microbes use SO4
SO4 --->
---> H2S
H2S + serine ---> cysteine + H2O
4) Phosphorus = inorganic phosphate used by most
5) Oxygen - (Table 4-2, p 88); Reactive forms of oxygen may be be
toxic
a) obligate aerobes = requires oxygen
b) microaerophiles = require O2 at lower concentrations
c) obligate anaerobes = unable to use oxygen and it is toxic to
them
d) facultative anaerobes = can grow with or without O2 (and use
it)
e) aerotolerant anaerobes = can grow in presence of O2 but
don’t use it
Oxygen Toxicity
toxic forms include: H2O2, O2-, OH-
Obligate anaerobes (such as Clostridium) lack enzymes
such as superoxide dismutase and catalase to deal with toxic
forms of oxygen and therefore cannot survive in the presence of
oxygen (Table 4-2, p 88 shows enzymes involved)
6) Mineral metabolism: includes Mg, Ca, Mn, Zn, Fe; minerals are
cofactors for enzymes, also involved in electron transfer
(oxidation-reduction)
Fe importance:
- upon infection, Fe in body is “tied up”
- E. coli has 13 genes involved in iron uptake and use
7) Organic growth factors (some bacteria are able to make all
from a simple carbon source.
a) vitamins (coenzymes)
b) amino acids
c) purines and pyrimidines
- some microbes release extracellular proteases to generate amino
acids, e.g. Clostridium perfringens (gas gangrene)
In culture, the above requirements must be satisfied by the
culture media used.
II. Bacterial cell cycle - generally cell divides by binary
fission.
A) general events: Fig 4-3, p 86
1) growth, DNA replication (DNA replication occupies entire cell
cycle, in E. coli replication takes 40 min, however, since a cell
may divide every 20’ there must be multiple replication
forks to replicate DNA
2) cell wall and membrane begins to grow in (septum formation)
3) division - cells may or may not actually separate
B) Generation time
1) Mathematics of bacterial growth = exponential growth for
binary fission
2) time for cell cycle - for most bacteria is 1 - 3 hrs; E.
coli under optimal
conditions may divide every 20’
C) Culturing bacteria: may be done in batch cultures or
continuous cultures
1) batch cultures = closed system (like a petri dish or test
tube)
growth curve has 4 phases (Fig 4-17, p 102):
a) lag phase - cells acclimate; have zero growth rate, RNA and
protein synthesis; have lag in cell division.
b) log phase - cell division is constant and maximum, growth rate
constants may be determined, have “balanced” growth
c) stationary phase, have zero growth rate,
“unbalanced” growth
d) death phase, have negative growth rate, cell lysis
Note: length of lag time (and other phases) can vary depending on
conditions.
2)Continuous cultures = consists of an open system which
maintains cells in log phase (page 103)
3) Biofilms (page 104)
D) Determining cell number (Table 4-7, p97)- direct methods
1) colony count or plate count, here may use slurries or
suspensions (food, soil), but sample is heated, generates viable
counts.
a) spread plate method
b) pour plate method Fig 4-13, p98
2) filtraion methods = used where bacterial counts are low such
as in water
viable counts
3) Most probable number = a statistical method to determine
viable counts Fig 4-15, p 100
4) direct microscopic counts = cells counted on an etched slide
(Petroff - Hausser cell counter) results in total counts, viable
and non-viable Fig 4-11, p 96
E) Indirect methods
1) turbidity = density of cell suspension - usually done with
photometers, spectrophotometers; may measure absorbance (optical
density) = Abs, or may measure transmittance = %T; as cell number
(density) increases, Abs. increases and %T decreases Fig 4-16,
p 101
2) Cell mass - can determine dry weight or wet weight
3) Chemical methods = measure chemical components such as RNA,
DNA, protein, N, C
4) Metabolic measurements such as oxygen uptake, moles of acid
produced
III. Media
Solid media usually contains agar = plates, slants, deeps; vs.
liquid broths.
A) Basic types of media
1) Complex (undefined) = enriched; contains extracts (meat,
plant, or yeast digests)
ie: nutrient broth, nutrient agar (Tables 4-5 and 4-6, p93)
2) Synthetic (defined) = all components and their amounts are
known
B) Special media = includes enrichment cultures, selective media,
differential media
1) enrichment: enhances growth of one type of organism (Fig 4-10,
p95)
2) selective media: inhibits growth of unwanted microbes
3) differential media: indicates or differentiates colonies
Fig, 4-7, p94
IV. Culture techniques
A) Pure cultures
1) streak plate Fig 4-2, p85
2) pour plate Fig 4-13, p98