1.) Binary Fission - Bacterial reproduction occurs
through fission, a primitive form of cell
division that does not employ a spindle fiber
apparatus. [A spindle fiber apparatus made of protein filaments is responsible for
moving the chromosomes around during cell division (mitosis & meiosis) in most
eukaryotic cells. Bacteria do not have these
structures.] The bacterial cell doubles in
size and replicates its chromosome. Following
DNA replication, the two chromosomes attach to separate sites on the plasma membrane, and
the cell wall is laid down between them, producing two daughter cells.
2.) Budding - A few bacteria and some eukaryotes
(including yeasts) may also replicate by budding,
forming a bubble-like growth that enlarges and separates from the parent cell.
A.
Phases
of Growth - A
microbial lab culture typically passes through 4 distinct, sequential phases of growth
that form the standard bacterial growth curve: (Not all
growth phases occur in all cultures). See graph; be
able to draw & label.
1. Lag
Phase
- In the lag phase, the number of cells doesn't increase.
However, considerable metabolic activity is occurring as the cells prepare to grow. (This phase may not occur, if the
cells used to inoculate a new culture are in the log phase & provided conditions are
the same).
2. Log
Phase
(logarithmic or exponential phase) - cell numbers increase exponentially; during each
generation time, the number of cells in the population increases by a factor of two). The number of microbes in an exponentially
increasing population increases slowly at first, then extremely rapidly. Organisms in a tube of culture medium can maintain
log growth for only a limited time, as nutrients are used up, metabolic wastes accumulate,
microobes suffer from oxygen depletion.
3. Stationary
Phase
- The number of cells doesn't increase, but changes in cells occur: cell become smaller
and synthesize components to help them survive longer periods without growing (some may
even produce endospores); the signal to enter this phase may have to do with overcrowding
(accumulation of metabolic byproducts, depletion of nutrients, etc.).
4. Death
Phase
- In this phase, cells begin to die out. Death
occurs exponentially, but at a low rate. Death
occurs because cell have depleted intracellular ATP reserves. Not all cells necessarily die during this phase!
In the lab, cultures usually pass through
all growth phases - not in nature. In nature,
nutrients continuously enter the cell's environment at low concentrations, and populations
grow continually at a low but steady rate. The
growth rate is set by the concentration of the scarcest or limiting nutrient, not by the
accumulation of metabolic byproducts - in nature there is always some other microbe that
can use these metabolic byproducts for their own metabolism. In the lab, we have to continually replace the
media.
A. Indirect Measurements
(measure a property of the mass of cells and then ESTIMATE the number of microbes)
1. Turbidity
– Can hold tube up to the light and look for cloudiness as evidence of growth
(difficult to detect slight growth). A
spectrophotometer can measure how much light a solution of microbial cell transmits; the
greater the mass of cells in the culture, the greater its turbidity (cloudiness) and the
less light that will be transmitted. Disadvantages: Not sensitive in terms of numbers of bacterial
cells & not useful for detecting minor contamination.
2. Metabolic
Activity - 3
ways:
a. The
rate of formation of metabolic products, such as gases or acids, that a culture produces.
b. The
rate of utilization of a substrate, such as oxygen or glucose.
c. The
rate of reduction of certain dyes. Ex.
methylene blue becomes colorless when reduced.
B. Direct Measurements - Give
more accurate measurements of numbers of microbes.
1. Direct
Counts
- Coulter Counter - electronic counter; rapid & accurate only if bacterial cells are
the only particles present in the solution. [gives a total count - live & dead cells].
3. Plate
Count
– Bacterial colonies are viewed through the magnifying glass against a
colony-counting grid; called a Quebec colony counter (we have this in the lab). [gives a viable count]
4. Filtration
- A known volume of liquid or air is drawn through a membrane filter by vacuum. The pores in the filter are too small for
microbial cells to pass through. Then the
filter is placed on an appropriate solid medium and incubated. The number of colonies that develop is the number
of viable microbial cell in the volume of liquid that was filtered. This technique is
great for concentrating a sample, ex. a swimming pool, where small populations may go
undetected using some other methods. [gives a viable count]
III. Growth Factors
- Microbes
can exist in a great many environments because they are small, easily dispersed, need only
small quantities of nutrients, are diverse in their nutritional requirements.
A. Physical
Factors
1. pH
– bacteria
can classified as:
a.
acidophiles
(acid-loving) – grow best at a pH of 1
to 5.4; Ex. Lactobacilllus (ferments milk)
b.
neutrophiles
–
exist from pH to 5.4 to 8.5; most bacteria that cause human disease are in this category.
c.
alkaliphiles
(base loving) – exist from pH to 7.0 to 11.5; ex. Vibrio cholerae (causes cholera)
2. Temperature
– bacteria can be classified as:
a.
psychrophiles
(cold-loving)
15oC to 20oC; some can grow at 0oC.
b. mesophiles
- grow best between 25oC and 40 C; human
body temp is 37oC.
c.
thermophiles
(heat-loving)
– 50oC to 60oC; found in compost heaps and in boiling hot
springs.
3. Moisture
– only the spores of sport-forming bacteria can exist in a dormant state in a dry
environment.
4. Hydrostatic
pressure – pressure
exerted by standing water (ex. lakes, oceans, etc.); some bacteria can only survive in
high hydrostatic pressure environments (ex. ocean valleys in excess of 7000 meters); the
high pressure is necessary to keep their enzymes in the proper 3-D shape; without it, the
enzymes lose their shape and denature and the cell dies.
5. Tonicity
(hypotonic,
hypertonic, isotonic) – The use of salt as a preservative in curing meats and the use
of sugar in making jellies is based on the fact that a hypertonic environment kills or
inhibits microbial growth. Halophiles (salt lovers) inhabit the oceans.
6. Radiation
– UV rays and gamma rays can cause
mutations in DNA and even kill microorganisms. Some
bacteria have enzyme systems that can repair some mutations.
B. Oxygen
Requirements
1. strict
or obligate anaerobes – oxygen
kills the bacteria; ex. Clostridium tetani
2. strict or obligate aerobes – lack
of oxygen kills the bacteria; ex. Pserdomonas
3. facultative anaerobes – can
shift their metabolism (anaerobic if oxygen is absent or aerobic if oxygen is present); ex. E. coli,
Staphylococcus
4. aerotolerant
– the
bacteria don’t use oxygen, but oxygen
doesn’t harm them; ex. Lactobacillus
5. microaerophiles
– like
low oxygen concentrations and higher carbon dioxide concentrations; ex. Campylobacter
C. Nutritional
(Biochemical) Factors – Nutrients
needed by microorganisms include:
¨ Carbon
– carbon containing compounds are needed as an energy source (ex. glucose) and for
building blocks.
¨ Nitrogen
- needed for amino acids and nucleotides; some can synthesize all 20 amino acids; others
have to have some provided in their medium.
¨ Sulfur
– needed for amino acids, coenzymes,
¨ Phosphorus
–
needed for ATP, phospholipids, and nucleotides
¨ Vitamins
– a vitamin is an organic substance that an organism requires in small amounts and
that is typically used as a coenzyme; many bacteria make their own, but some are required
in the medium; microbes living in the human intestine manufacture vitamin K, needed for
blood clotting, and some of the B vitamins, thus benefiting their host.
¨ Certain
trace elements
– ex. copper, iron, zinc, sodium, chloride, potassium, calcium, etc.; often serve as
cofactors in enzymatic reactions.
A. Methods
of Obtaining Pure Cultures (a
culture that contains only 1 species of organism)
1. The
Streak Plate Method – Bacteria
are picked up on a sterile wire loop, and the wire is moved lightly along the agar
surface, depositing streaks of bacteria on the surface.
The loop is flamed and a few bacteria are picked up from the region already
deposited and streaked onto a new region. Fewer
and fewer bacteria are deposited as the streaking continues, and the loop is flamed after
each streaking. Individual organisms
(individual cells) are deposited in the region streaked last. After the plate is incubated at a suitable growth
temperature for the organism, small colonies (each derived from a single bacterial cell)
appear. The loop is used to pick up a portion
of an isolated colony and transfer it to another medium for study. The use of aseptic technique assures that the new
medium will contain organisms of a single species. We’ll
do this in lab.
1. Synthetic
medium – prepared
in the lab from materials of precise or reasonably well-defined composition.
2. Complex
medium –
contains certain reasonably familiar materials but varies slightly in chemical composition
from batch to batch (contains extracts from beef, yeasts, blood); ex. nutrient agar,
nutrient broth
1. Selective
– one
that encourages the growth of some bacteria but suppresses the growth of others.
2. Differential
–
has an ingredient that causes an observable change in the medium when a particular
biochemical reaction occurs (ex. a color or pH change).
1. Candle
jars – the
inoculated tube or plate is placed in a jar; a candle is lit before the jar is sealed; the
burning candle uses the oxygen in the jar and adds carbon dioxide to it; when the carbon
dioxide extinguishes the flame, condition are optimum for the growth of microorganisms
that require small amounts of carbon dioxide (ex. Neisseria
gonorrhoeae)
2. Thioglycollate
medium
– oxygen-binding agent added to the medium to prevent oxygen from exerting toxic
effects on anaerobes; media is usually dispensed in sealed screw-cap tubes.
3. Anaerobic
Chamber (Brewer Jar) – A
catalyst is added to a reservoir in the lid of the jar.
Water is added to the gas-pak. Water
is converted into hydrogen gas and carbon dioxide. The
hydrogen gas can then bind with any oxygen in the jar to form water. A methylene blue test strip is included in the
jar to ensure that anaerobic conditions are reached.
When oxidized (oxygen is present) the strip is blue; when reduced (no oxygen), the
strip is clear.
H2O -----------> CO2 +
H2
H2
+ O2 ----------->
H2O