Exam 2 Measuring microbial growth 9/21 Flashcards
Cultivation independent methods
DNA from unculturable bacteria can be amplified and sequenced by PCR.
Sequences can be used to produce fluorescent probes that will bind to complementary DNA (fluorescent in situ hybridization or FISH).
Metagenomics
DNA is isolated from an environmental sample and sequenced.
Metagenomic information must still be confirmed in cultured organisms.
Bacterial Colony Morphology
Form
Elevation
Margin
Unculturable bacteria
Microbial consortia
Ecological Associations Among Microorganisms
Microbial association
- Symbiotic (Mutualism, Commensalism, and Parasitism)
- Non symbiotic (Synergism and Antagonism)
Interrelationships Between Microbes and Humans
Human body is a rich habitat for symbiotic bacteria, fungi, and a few protozoa - normal microbial flora
Commensal, parasitic, and synergistic relationships
Microbial Biofilms
Biofilms result when organisms attach to a substrate by some form of extracellular matrix that binds them together in complex organized layers
Dominate the structure of most natural environments on earth
Quorum sensing
Communicate and cooperate in the formation and function of biofilms
Division of bacterial cells occurs mainly through …
Binary fission (transverse)
Parent cell enlarges, duplicates its chromosome, and forms a central transverse septum dividing the cell into two daughter cells
5 step process
Microbial growth occurs at two levels
Growth at a cellular level with increase in size, and increase in population
Generation Time
Time it takes to get from step 1 to step 5 in binary fission
*DNA partitioning
Rate of Population Growth
Time required for a complete fission cycle is called the generation, or doubling time
Each new fission cycle increases the population by a factor of 2 – exponential growth
Generation times vary from minutes to days
How can we measure and count microbes?
Different methods exist, including:
- direct counts
- viable (living) cell counting
- turbidity (cloudiness) measurements
- flowcytometry
Direct counts
Load known volume onto gridded slide.
Count under a light microscope.
- # of cells/volume
1 ml = 1000 µl
1 µl = 1000 µl
example: 9 cells in 1 µl
Direct count = 9 x 10^3
Using the Spread Plate and Pour Plate to determine cell count (viable)
Both may be used to determine the number of viable microorganisms in an original sample
Outcome measured as colony forming units (CFU)
Pour Plate
Original sample is diluted several times
Some of dilutions are mixed with warm agar and poured onto the plates
Isolated cells grow into colonies on the surface and within the medium. The isolated colonies can be counted or used to establish pure cultures.
What if the cells are already very diluted?
A filter apparatus can concentrate the cells.
Turbidity
A spectrophotometer sends light through a culture.
If the tube is cloudy, light won’t get through the tube and strike the unit’s sensor (high absorbance).
It can give a rough measure of cell density in the tube.
Methods of Analyzing Population Growth
Turbidity = Most simple.
Degree of cloudiness, turbidity, reflects the relative population size.
Flow Cytometry
microbial suspension forced through small orifice with a laser light beam
movement of microbe through orifice impacts electric current that flows through orifice
instances of disruption of current are counted
specific antibodies can be used to determine size and internal complexity
