CHAPTER 6 - Microbial Growth
Note: These notes are outlines of my lectures and they in no way represent all the material covered in class!!
Physical requirements for the growth of microorganisms
WHAT FACTORS AFFECT GROWTH?
Physical factors - temp, pH, water, oxygen, pressure
Nutrients - Cell constituents and energy sources
I. PHYSICAL FACTORS:
1. Temperature:
How does temperature affect optimal growth?
minimum, optimum, maximum temperatures for growth
Minimum - no growth below this temperature
enzymes don't function
transport ceases
cells dormant not dead
dangers of freezing and thawing
Optimum - closer to maximum than minimum (5 to 10 C below max)
Maximum - Bacterium A grows at temperatures up to 40°C. What might be happening to the cell at
41°C?
Categories based on optimum temperatures:
psychrophiles <20 C
high levels of unsaturated fatty acids --> remain fluid at low temperature
mesophile 20-40 C
thermophile >40 C and < 65 C
extreme thermophiles > 65 C
hot water heaters, hot springs, vents
E.g. Pyrodictum, min 82 opt 105 max 110 C
extremes have lipids, proteins adaptation
PCR - Thermus aquaticus
2. pH: usually narrow range of growth most don't grow below pH 4 and above pH 9 internal pH always near
neutrality may produce end products that change local pH/need buffers acidophiles - pH 1 - 5.5
require hydrogen ions for membrane integrity
do not survive at neutrality
prevent uptake into cytoplasm and pump out
neutrophiles - pH 5.5 - pH 8.5
alkalophiles - pH 8.5 - 11.5
pump in protons
generate ammonia (+) by ammino acid degradation
Bacteria min pH opt pH max pH
Thiobacillus 1.0 2-2.8 4-6
E. coli 4.4 6-7 9.0
C. sporogenes 5.4 6-7.6 9.0
P. aeruginosa 5.6 6.6-7 8.0
Nitrobacter 6.6 6.6-8.6 10.0
3. Classification based on water activity/osmotic pressure (remember, low water activity = high
osmotic pressure)
halophiles- require sodium >9%; extremes may live in 30%
marine salt about 3%
cell wall and membrane fall apart w/o sodium
nonhalophile - 0 to 1.5%
What happens when water activity is high (cell hypertonic to environment)?
Water enters cell and stretches to cell wall
What happens when water activity is low (cell hypotonic to environment)?
Water leaves cell; dehyration
How do microbes adapt to low water activity?
Microbes can change their internal osmotic environment
E. coli in GI tract
produce or import compounds that increase internal solutes
compatible solutes - do not harm host
ions, sugars, amino acids
4. Oxygen:
How does oxygen affect optimal growth?
aerobes - require oxygen
obligate/strict anaerobes - killed by oxygen
aerotolerant anaerobes - does not require oxygen but not killed
facultative anaerobe - grows in presence or absence of oxygen with different metabolic
strategies
microaerophiles - require reduced levels of oxygen
Why is oxygen toxic to some microbes?
metabolic byproducts all organisms produce are toxic to cells
hydrogen peroxide
superoxide
present in WBC to kill bacteria
aerobes have catalase and/or peroxidase and superoxide dismutase (SOD)
catalase: 2H202 -> 2H20 + 02
peroxidase: H202 + 2H+ -> 2H20
SOD: 202- + 2H+ -> 02 + H202
strict anaerobes lack these enzymes
microaerophiles either reduced amounts of these enzymes or oxygen-sensitive forms of enzymes
aerotolerant have these enzymes but not those used in aerobic metabolism
Toxic forms of oxygen are broken down by several enzymes; one of these is catalase. This
enzyme breaks down hydrogen peroxide to form water and molecular oxygen. Our tissues as
well as many microorganisms are catalase positive - thats why the bubbles when you pour
peroxide on a cut.
Why would our tissues have peroxide in them?
Some theories of cancer hypothesize that oxidants (like toxic forms of oxygen) damage DNA
and thus cause the mutations which in turn cause cancer. These theories predict that antioxidants
such as beta-carotene, vitamin E and vitamin C would thus have anti-mutation activity.
Organic growth factors
commonly called vitamins, these are organic substances required for the growth of an organism but
which the organism can not synthesize.
What is the difference between a defined and an undefined medium?
WHAT FACTORS AFFECT GROWTH?
Nutrients - Cell constituents and energy sources
Physical factors - temp, pH, water, oxygen, pressure
I. Nutrients:
ELEMENT CELL FUNCTION
C backbone of organic cell components, energy
H water, organic components, pH, hydrogen bonds, re-dox
O water, organic components, respiration
N amino acids, nucleotides, coenzymes, ATP
S amino acids, coenzymes, enzymes
P nucleic acids, phospholipids, coenzymes, ATP
Fe cytochromes, enzymes
Na, K, Ca, Cl, Mg, Mn Trace elements: transport, ionic balance, cofactors (e- donor/acceptors)
II. CLASSIFICATION OF ORGANISMS BASED ON carbon, energy, and electron sources
Chemotrophs - Derives energy from chemicals
Phototrophs - Derives energy from sunlight
Autotrophs - Use carbon dioxide for carbon
Heterotrophs - Use organic substrates for carbon
Lithotrophs - Inorganic compounds for electrons
Bacterial Growth
Bacteria grow by binary fission. Starting with one organism how many organisms would you
have after 1,2,3,4,5,6,7,8,9,10,11,12 generations.
If you started with 10 organisms, how many would you have after each of the above
generations?
What is microbial growth?
Increase in cell numbers
HOW DO BACTERIA REPRODUCE?
Binary fission
Budding
Fragmentation
What occurs during binary fission?
DNA duplication
DNA repication
single origin of replication, bidirectional
theta intermediate
Separation of DNA and cytoplasmic contents
Cross wall formation
Generation time = doubling time
time required for a cell to divide or a population to double
1 to 2 or 100 to 200 or 1 million to 2 million
Most common bacteria have a generation time 30-60 min under opt. conditions.
Most common pathogens in the body, about 5-10 hours.
Mycobacterium tuberculosis (in lab) 12 hrs
Clostridium botulinum (in lab) 0.58
E. coli (in lab) 0.30
E. coli (in mouse) 20 hrs
What happens when you inoculate a single bacterium from slant stored in refrigerator into
nutritional medium and incubate?
Why doesn't it grow immediately?
What determines how fast it grows?
Why does it stop growing?
Phases in bacterial growth in batch culture (see above picture)
Lag phase - synthesis of new components or repair
Log phase - reproduction at maximum rate (shortest generation time)
exponential growth 1->2->4->8->16->32->64 Bf = Bi x 2n
Log Bf = log Bi + .3t/dt
Stationary phase - no net increase, balance between cell division, cell "death",
maintenance Nutrients vs toxins synthesis of storage materials
sporulation, survival genes
Death phase - do they really die? some autolysis
small subpopulation persists; accumulation of toxins
Mathematics of growth- generation time
Growth equation:
n = log10Nf - log10 Ni
0.301
Where n = number of generations, Nf = final conc. of cell (e.g. 109/ml),
Ni = initial conc. of cells (e.g. 103/ml), and 0.301 is factor to convert log2 to log10.
Example: measure culture at 9 a.m.: No = 10,000 cells/ml. Then, measure culture at 3 p.m.: Nf = 100,000
cells/ml. Calculate n = (5 - 4)/0.3 = 1/0.3 = 3.33 generations.
Generation time: Total time = 6 hours = 360 minutes/3.33 generations = 108 minutes/generation
Conclude: generation time = 108 minutes
Note: be able to calculate g.t. Pay attention to units!
Graphical measurement of growth
· Plotting # of cells vs. time gives a curved line.
· Plotting log # of cells vs. time gives a straight line --- easier to interpolate, use.
· Plotting # of cells vs time on semilog paper also gives a straight line --- easiest way in
practice to work with growth measurements.
· Note: often what is plotted on the Y-axis of semilog paper is not # of cells, but something
more easily measurable, such as Absorbance (see below).
.
CULTURE MEDIA
A culture medium is any material prepared for growth of an organism in a laboratory setting. Microbes
that can be cultured on a petri-plate or in a test-tube containing media are said to grow under in vitro
conditions ("within-glass".)
It was not until the era of Robert Koch and his coworkers that Agar was introduced as a a common
medium for bacterial growth. Agar is a complex polysaccharide derived from a marine sea weed. Few
bacteria possess enzymes capable of digesting agar and therefore it is useful as a solidifying agent and
for isolating microbes in pure culture. Prior to the advent of agar, gelatin was used as a growth
medium. Unfortunately, many bacteria possess enzymes that liquify gelatin and therefore this medium
is not useful for isolating pure cultures. However, gelatin liquefaction is one among a series of
biochemical tests that helps differentiate species of bacteria.
What is a PURE CULTURE?
A pure culture represents a single species (clonal in nature) of microorganisms
A clone is a genetically identical population of microbes that have descended from a single
parent cell
Colonies are visible clones that have grown on solid media and represent millions of bacterial
cells
Distinctive characteristics of colonies should be noted such as:
pigmentation
odor
elevation
margin (border of the colony)
consistency, such as mucoid, irridescence, filamentous, etc.
Media vary in their chemical composition. In turn, the composition of the media determines
microbial growth and the type of microbes that will grow.
a) Chemically defined media: exact chemical composition is known. Such media is often
commercially prepared.
b) Selective media. Contain chemicals which encourage growth of certain types of microbes
but inhibits the growth of others.
c) Differential media allows different microbes to be distinguished on the basis of various
biochemical reactions. Fermentation reactions involving the catabolism of various sugars are
particularly useful biochemical tests
Note: Many media are both selective and differential, such as MacConkey (Mac) agar and
Mannitol Salt agar (MSA).
d) Enrichment media contains a rich supply of nutrients to encourage the encourage
growth of microorganisms. A commonly used enrichment medium is blood agar. This
medium is also differential and it permits detection of differnt patterns of hemolysis.
Measurement of growth
a) Total Cell count
· Petroff-Hausser chamber slide -- needs large conc. (107 cells/ml minimum).
· Coulter Counter (for larger microbes; fungi, yeasts, protozoa, etc.) --- uses electrical charge
difference in passing through small hole. Not so useful with bacteria, get errors due to
clumping, debris, unable to differentiate bewteen live and dead cells, etc.
b) Viable count
CFU (colony forming units) assay
1. carry out dilution series
2. plate known volumes on plates
3. count only plates with 30 - 300 colonies (best statistical accuracy)
4. extrapolate to undiluted cell conc.
Measures colony forming units (CFU), may or may not be same as number of cells --- accurate, but
requires time for incubation.
Two ways to carry out viable count:
1. Spread plate: bacteria are spread on the surface of agar using some sterile spreading
device. Advantages: if properly carried out, all colonies should be easily counted.
Disadvantages: takes some time, not always reliable in inexperienced hands, cells with low
tolerance to oxygen will not grow. If "spreaders" are present may overgrow plate surface.
2. Pour plate: bacteria are mixed with melted agar and cooled; colonies grow throughout the
agar. Advantages: almost fail-proof technique, colonies well separated. Can allow growth
of organisms with lower oxygen tolerance in agar. Disadvantages: colonies variable size,
harder to see similarity in colony morphology between those on surface and in agar.
Counting may be more difficult. Heat may kill some cells before agar cools and gels.
c) Light techniques
Often, can estimate cell numbers accurately by measuring visible turbidity. Light scattered is
proportional to number of cells. This only works above cell densities of 107 in pure cultures. Eyeball
method. This is not a precise measurement, but shoud allow estimation within an order of magnitude.
• no turbidity means less than 107 cells/ml
• Slight turbidity = 107 - 108 cells/ml
• high turbidity= 108 - 109 cells/ml
• Very. high turbidity = greater than 109 cells/ml (cultures rarely get as high as 1010 cells/ml)
Absorbance (usually at wavelengths around 400-600 mn). Accurate measure of cells when
concentration not too high. Easy and quick to measure (can sample in less than a minute).
d) Batch vs. Continuous culture methods
· Batch method: put small inoculum of pure culture into sterile medium, let grow. Common lab
procedure, but not typical of many real environments.
• Continuous culture (see text section 6.11): use chemostat or turbidostat. Trickle fresh
medium into culture at slow but steady rate, displace = volume of culture as overflow.
• Cells remain in exponential (but suboptimal) state, growing at known rate. Good simulation for
study of many natural environments.