Bacterial Growth
Lecture Outline:
1. A pure culture: A population of cells derived from a single
cell. The study of bacteria involves the study of a population of
cells rather than individual cells. In nature most bacteria are found
living with other organisms, i.e., as a mixed culture, and not as
pure cultures.
2. How are pure cultures obtained:
- Problems- need to establish conditions for growth of the
bacterium you want to pure culture, and organism of interest may
be a minority in population and/or dependent on another organisms
for growth. Not all bacteria observed in nature have been pure
cultured.
- technique- Streak plate technique developed by Robert
Koch. Employs a solidified medium. Rational of procedure is to
physically separate the various cells from one another by
spreading them over solid surface, immobilize them, and then allow
sufficient time so that each cell grows until it produces a colony
of cells, a clone, that is visible to the eye (1 X 106
cells). To a liquid medium, agar, an extract from a marine algae,
is added to a concentration of 1.5% (W/V). To sterilize medium,
render it free of all living organisms, and melt agar, agar
containing medium is subjected to autoclaving (heat under
pressure). Following autoclaving medium is cooled, and poured into
a container either a petri dish (a two part glass/plastic dish)
and/or test tube. Substances that can not withstand
temperature/pressure of autoclave are sterilized by other
means.
3. Nutritional requirements: In the simplest of terms, all
organisms must have (1) a source of energy, (2) a source of carbon,
and (3) certain essential elements. The major chemical
elements in terms of amounts required for growth are carbon,
hydrogen, oxygen, phosphate, nitrogen, sulfur, magnesium, and iron.
In addition other elements, called trace elements, are needed
in smaller amounts (manganese, zinc, cobalt, molydenum, nickel, and
copper) and/or vitamins (biotin, B12, niacin). These elements can
exist in many different chemical forms, and the ability to use these
different compounds for growth defines the nutritional
properties/characteristics of the bacterium.
4. The major groups of
organisms based on their carbon and energy requirements
5. Types of media used to culture bacteria:
6. Bacterial cell division: An asexual process Fig. 4.1
- process- Binary fission (one cell divides into two). Bacterial
growth is exponential or logarithmic. Mitosis and meisois does not
occur in bacteria.
- generation time or doubling time- the time it take a cell or
culture to double.
- nutritional, chemical, and physical factors influence growth
(see below).
7. Methods to monitor
growth: Methods to monitor growth measure an increase in cell
numbers. (WILL NOT COVER IN SUMMER QUARTER)
8. Phases of growth in a batch or closed system (a finite amount
of nutrients are provided, and as bacteria grow conditions change):
Cells are dividing asynchronously. Fig. 4.6
- lag- cell metabolism occurring, but cells not dividing. Cells
are adjusting to medium and growth conditions imposed.
- exponential- cells are dividing as rapidly as possible. It is
during this time that the generation time of culture can be
determined. Generation time at its minimum.
- stationary- no increase in cell numbers, and may be due to no
cells dividing or for every cell that divides, an equal number of
cells die. Cells enter stationary phase because have either
depleted nutrients and/or environment is no longer conducive to
growth (toxic products have accumulated in medium).
- death- cell death is occurring. What two bacterial species
would not exhibit a death phase and why?
9. Factors that influence growth:
- medium- generation time shorter in a complex medium versus a
minimal medium. Why?
- temperature- Fig. 4.9
- psychrophiles- cold loving bacteria (-5 to
20oC).
- Psychrotrophs- 20-30oC, but can grow well at
lowerer temperatures.
- mesophiles- mild temperature growing bacteria
(25-45oC).
- thermophiles- hot loving bacteria (45 to
70oC).
- Hyperthermophiles- 70-110oC
- oxygen requirement/sensitivity- Table 4.3
- obligate (strict) aerobes- have an absolute requirement for
oxygen.
- obligate (strict) anaerobes- killed by oxygen.
- facultative anaerobes- can grow in the presence/absence of
oxygen, but grow better with oxygen.
- aerotolerant- can grow in presence/absence of oxygen, but
derive no benefit from oxygen (indifferent).
- microaerophilic- need reduced amounts of oxygen (2 to 10%).
Higher concentrations toxic.
- sensitivity to toxic forms of
oxygen
- water availability: water must not only be present, but
available to cell. In high sugar/salt environment water leaves
cells. How do cells that live in low water availability
environment (high salt/sugar) deal with their environments? What
are some of the properties of Staphylococcus aureus that
contribute to its virulence?
- pH: As a group bacteria
grow of the pH range of 2-11. How is Helicobacteria pylori
able to grow in the highly acid environment of the
stomach.
Learning
Objectives:
- What is meant by a pure culture?
- What is the basic principle behind the streak plate
technique?
- How are organisms grouped by their carbon and energy
requirements?
- How does a minimal medium differ from a complex medium? How
does a selective medium differ from an enrichment medium? and what
are you trying to accomplish by growing bacteria on a differential
medium?
- Describe the process of asexual reproduction.
- What is meant by the generation time of a culture, and how is
it determined?
- What are the advantages and disadvantages of the various ways
to monitor bacterial growth.
- Describe the growth pattern of bacteria in a "closed"
system
- How are bacteria grouped according to their temperature and
oxygen requirements (sensitivity)?
- What accounts for "resistance" or sensitivity of organisms to
toxic forms of oxygen?
Classification
of Bacteria According to Their Source of Energy and
Carbon
|
Name
|
Energy Source
|
Carbon Source
|
Photoautotrophs
|
Light
|
C02
|
Photoheterotrophs
|
Light
|
Various Organic Compounds
|
Chemolithotrophs
|
Inorganic compounds such as H2,
NH3, N02-,
Fe2+, H2S
|
C02
|
Chemoheterotrophs
|
Organic
|
Organic compounds
|
Methods to Determine
Bacterial Growth:
A. Determination of cell number:
1. Total cell count methods:
a. Direct microscopic count- See fig. 4.5 pg. 91
Advantages- quick and easy
Problems- can not distinguish be live and dead cells, can
not detect less than 106 bacteria/ml.
b. Coulter count (electronic count):
Advantages- very quick and easy
Disadvantages- same as above, count dust and debris, and
apparatus very expensive.
2. Viable cell count method: See fig. 4.7 pg. 93
advantages- can count as few as 1 bacterium/ml, only
count live cells
disadvantages- requires time for growth, may need to make
dilution's of preparation and make dilution calculations
(examples), error due to
growth of bacteria in clumps and chains.
B. Determination of cell mass-
1. dry weight determination:
advantages- only way to determine growth of
filamentous bacteria.
disadvantages- cumbersome, not very accurate, If cell
numbers important must relate weight to total cell count
number.
advantages- rapid and easy
disadvantages- does not give you cell numbers or increase in
mass (must correlate to one of above), can not distinguish
between live and dead cells, and must work within certain
absorbency (more than 107 and less than
108).
C. Determination of cell constituents- measure increase in a
specific cell material, i.e., DNA, RNA, Protein, or etc.
Dilution
Problems:
(no. of bacteria/ml in original sample = no. of colonies on plate
X 1/total dilution X 1/ volume sample plated)
1. You are interested in determining the number of bacteria in
saliva. You spit into a tube, and then do four 1:10
dilution's. From the last dilution tube you plate 1.0 ml onto an
appropriate medium, and observe 100 colonies on the agar surface
after overnight growth. How many bacteria are present in the original
sample?
2. A friend of yours tells you that there should be no bacteria in
hamburger meat, and having had micro you say not true. To show
him/her you do the following: You take 1 gram of meat and blend it in
100 ml of sterile water. You then do the following dilution: 1:10,
1:100, 1:10, and a 1:100. You then take 0.1ml from the last dilution,
and plate onto an appropriate medium, and find that after 18 hours of
growth that there are 125 colonies on the plate. How many bacteria
were present in the original sample, per ml of blended material and
per gram of hamburger meat?
Enzyme Content
of Bacteria With Different Requirements (sensitivity) for
Oxygen
|
Name
|
Enzyme Content for O2 detoxification
|
Strict aerobe
|
catalase
H2O2 ---> H2O +
O2
superoxide dismutase
O2- + 2H+ -->
O2 + H2O2 -->
H2O + O2
|
Facultative anaerobe
|
catalase and Superoxide dismutase
|
Strict anaerobe
|
neither catalase nor superoxide dismutase
|
Microaerophile
|
small amounts of catalase and superoxide
dismutase
|
Aerotolerant
|
superoxide dismutase
|
6/14/09