Exam 2 Measuring microbial growth 9/21

Question 1

Cultivation independent methods

Answer 1

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).

Question 2

Metagenomics

Answer 2

DNA is isolated from an environmental sample and sequenced.

Metagenomic information must still be confirmed in cultured organisms.

Question 3

Bacterial Colony Morphology

Answer 3

Form
Elevation
Margin

Question 4

Unculturable bacteria

Answer 4

Microbial consortia

Question 5

Ecological Associations Among Microorganisms

Answer 5

Microbial association

• Symbiotic (Mutualism, Commensalism, and Parasitism)
• Non symbiotic (Synergism and Antagonism)

Question 6

Interrelationships Between Microbes and Humans

Answer 6

Human body is a rich habitat for symbiotic bacteria, fungi, and a few protozoa - normal microbial flora

Commensal, parasitic, and synergistic relationships

Question 7

Microbial Biofilms

Answer 7

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

Question 8

Quorum sensing

Answer 8

Communicate and cooperate in the formation and function of biofilms

Question 9

Division of bacterial cells occurs mainly through …

Answer 9

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

Question 10

Microbial growth occurs at two levels

Answer 10

Growth at a cellular level with increase in size, and increase in population

Question 11

Generation Time

Answer 11

Time it takes to get from step 1 to step 5 in binary fission

*DNA partitioning

Question 12

Rate of Population Growth

Answer 12

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

Question 13

How can we measure and count microbes?

Answer 13

Different methods exist, including:
- direct counts
- viable (living) cell counting
- turbidity (cloudiness) measurements
- flowcytometry

Question 14

Direct counts

Answer 14

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

Question 15

Using the Spread Plate and Pour Plate to determine cell count (viable)

Answer 15

Both may be used to determine the number of viable microorganisms in an original sample

Outcome measured as colony forming units (CFU)

Question 16

Pour Plate

Answer 16

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.

Question 17

What if the cells are already very diluted?

Answer 17

A filter apparatus can concentrate the cells.

Question 18

Turbidity

Answer 18

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.

Question 19

Methods of Analyzing Population Growth

Answer 19

Turbidity = Most simple.

Degree of cloudiness, turbidity, reflects the relative population size.

Question 20

Flow Cytometry

Answer 20

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