CH 7 microbial growth natural v lab

Question 1

CH 7 Nat vs Lab

Growth in the natural environment

Answer 1

Nutrients are typically a limiting factor - much less available with more competition
Oligotrophic environments:
- low nutrient levels
Surviving stravation
- starvation proteins
- spontaneous dormancy
- endospores and cysts

Question 2

CH 7 Nat vs Lab

Synthesis of starvation proteins

Answer 2

• Increase peptidoglycan crosslinking
• Bind and protect DNA
• Prevent protein denaturation
  Starved cells become more resistant to environmental stresses and pathogens may become more virulent

Question 3

CH 7 Nat vs Lab

Spontaneous dormancy in nutrient-rich conditions

Answer 3

Portion of the microbial population enters dormancy on purpose - never know when nutrients may run low
Resistant to other environmental stresses

Question 4

CH 7 Nat vs Lab

Biofilms

Answer 4

Organized community with multiple species encased in a protective slime layer. Cells attach to one another and to substrate. Generally one species’ waste is the others’ nutrients, and vice-versa. Ubiquitous in nature
Ex. dental plague, medical implants, urinary track infections, sinusitis, filters for sewage treatment, bioremediation of oil spills

Question 5

CH 7 Nat vs Lab

Formation of biofilms

Answer 5

Reversible attachment - putting down proteins, conditioning surface for next species
Irreversible attachment - secretion of slime layer; microbial growth
Detachment - detachment or sloughing of cells

Question 6

CH 7 Nat vs Lab

Quorum sensing

Answer 6

Chemical signaling to determine the number and type of cells in the surrounding environment - many things that bacteria do, they don’t like to do alone. - actions are effective only when a large number of cells are present.
utilization of nutrients, toxin production, bioluminescence
plasmid transfer–virulence

Question 7

CH 7 Nat vs Lab

Growth in the lab

Answer 7

Optimized conditions for growth (plenty of nutrients, no competition, temp, pH), so they grow better in the lab than in nature.
Unfortunately, we can only grow 2% of known bacteria in the lab.

Question 8

CH 7 Nat vs Lab

Culture media

Answer 8

All nutrients necessary for microbial growth.

Can be classified based on the physical state, chemical composition, and/ or function.

Question 9

CH 7 Nat vs Lab

Physical state of media

Answer 9

Liquid (broth)
Semisolid
- used to look at bacterial motility, oxygen requirements
Solid
- usu. agar, but can be gelatin to ID certain bacteria. Agar melts ~90C and solidifies ~45C

Question 10

CH 7 Nat vs Lab

Chemical composition of media

Answer 10

Defined

• all chemical compositions and ratios known Complex
• contain some ingredients of unknown composition and ratios
• Yeast extract, beef extract, peptone, tryptone

Question 11

CH 7 Nat vs Lab

Functions of media

Answer 11

Supportive: general bacterial growth
Enriched: fortified with extra nutrients (fastidious bacteria)
Selective: allows growth of some microorganisms while inhibiting others
Differential: distinguish among different groups of microorganisms

Question 12

CH 7 Nat vs Lab

Fastidious bacteria

Answer 12

Those that require specific needs for growth, either/ both environmental and nutrient factors.

Question 13

CH 7 Nat vs Lab

Blood agar

Answer 13

Can tell if the bacteria in the media are hemolytic or not - enriched and differential

Question 14

CH 7 Nat vs Lab

Chocolate agar

Answer 14

Blood agar where the erythrocytes have already been lysed - enriched, but not differential

Question 15

CH 7 Nat vs Lab

PEA

Answer 15

Phenylethanol Agar
Differential
Allows G+ to grow, but not G- because it is toxic to G-

Question 16

CH 7 Nat vs Lab

MacConkey Agar

Answer 16

Selective: G+ over G-
Differential: identifies bacteria that can ferment lactose (lactose positive)

Question 17

CH 7 Nat vs Lab

Eosin Methylene Blue

Answer 17

Selective

Differential: lactose positive or negative

Question 18

CH 7 Nat vs Lab

Mannitol Salt Agar

Answer 18

High salt concentration

Differential: mannitol fermentation

Question 19

CH 7 Nat vs Lab

Growing Anaerobic microorganisms

Answer 19

Anaerobic media

• can’t expose to oxygen, even when transferring between media
• reducing agents turn oxygen into water
• thioglycolate GasPak jar
  When working with obligate anaerobes, transfer between medias in a special hood with an inert gas

Question 20

CH 7 Nat vs Lab

Isolation of Pure Culture

Answer 20

Colony - population of bacteria arising from single cell
Three types:
- streak plate
- spread plate
- pour plate

Question 21

CH 7 Nat vs Lab

Enrichment cultures

Answer 21

Adjust conditions to ID microorganisms with a specific characteristic.
ex. metabolics, antibiotic resistant

Question 22

CH 7 Nat vs Lab

Colony morphology

Answer 22

Each species has its own unique morphology - dealing with form, elevation, and margin.

Question 23

CH 7 Nat vs Lab

Characteristics looked for in identifying bacteria on morphology

Answer 23

Size
Texture of surface
Overall texture (butyrous, brittle, viscous)
Optical characteristics (opaque, translucent, dull, iridescent)
Pigment (most don’t produce color unless stressed)

Question 24

CH 7 Nat vs Lab

Types Cultures

Answer 24

Batch System

• closed system with fixed nutrients Continuous System
• constantly adding new media/ nutrients and removing waste
• used in industry to produce proteins and other molecular components

Question 25

# CH 7 Nat vs Lab Growth Curve for Batch cultures

Answer 25

Lag phase > exponential phase > stationary phase > death and senescence phase

Question 26

# CH 7 Nat vs Lab Lag phase of growth curve

Answer 26

Subculturing bacteria from one  media to another - takes some time for them to start actively growing again. No increase in cell number - synthesizing cellular components

Question 27

# CH 7 Nat vs Lab Exponential phase of growth curve

Answer 27

Max rate of growth Doubling at regular intervals (generation time) Balanced growth biochemical assays done at this time b/c the populations are uniform (physiologically the same)

Question 28

# CH 7 Nat vs Lab Stationary phase of growth curve

Answer 28

Some live and some dead cells Nutrient availability decreasing and wastes building up Critical population level = too dense (10 9 cells/ mL)

Question 29

# CH 7 Nat vs Lab Senescence and death

Answer 29

Irreversibly lose the ability to reproduce - viable but unable to grow Apoptosis - some cells commit suicide to release nutrients for the other cells to keep living (through quorum sensing)

Question 30

# CH 7 Nat vs Lab Math of Growth

Answer 30

``` g = generation time (cell # to double) N t = N o x 2 n N t is the cell # at time t N o is the initial cell # n is the # of generations at time t ```

Question 31

# CH 7 Nat vs Lab Why is the math important?

Answer 31

Microbes in exponential phase are more susceptible to antimicrobial agents. Exponential phase is favored because of uniform growth - major targets of antibiotics are proteins, cell walls, and DNA replication that occur during the exponential phase. During senescence is when we see the production of starvation proteins.

Question 32

# CH 7 Nat vs Lab Measuring growth

Answer 32

``` Direct cell counts Viable cell counts Biomass measurements Detecting specific products from cells ***** be able to ID which is best to use in a given situation ***** ```

Question 33

# CH 7 Nat vs Lab Direct cell counts

Answer 33

Petroff-Hausser counting chamber Advantages: * quick, inexpensive, info about size and morphology Disadvantages: * labor intensive (tedious), culture must be densely populated, can't distinguish dead from live cells (total cell count, not viable count)

Question 34

# CH 7 Nat vs Lab Petroff-Hausser counting chamber

Answer 34

Slide holds a known sample volume Grid pattern assists in counting Average number of bacteria per square is used to calculate concentration of the origin sample

Question 35

# CH 7 Nat vs Lab Coulter Counters

Answer 35

Cells are counted electronically - pass through opening in single file Advantages: * quick, not labor intensive Disadvantages: * debris interferes with counting, works better for larger cells, can't differentiate between dead and live cells

Question 36

# CH 7 Nat vs Lab Fluorescence and direct cell counts

Answer 36

Reagents used to distinguish between living and dead cells. Can be used with coulter counters as well for more accurate results. Often has higher concentration estimates than viable cell counts.

Question 37

# CH 7 Nat vs Lab Viable cell count

Answer 37

Examines the number of colony forming units. Filter plate methods to check water samples - place filter onto plate of agar and count number of colonies that grow. Advantages: * living cells, if using types of media you can begin to differentiate between types of bacteria Uses serial dilution, pour plate method, and then spread plate method.

Question 38

# CH 7 Nat vs Lab Biomass measurement

Answer 38

Dry weight determination (filter out of liquid culture) | Time consuming and not very sensitive/ accurate

Question 39

# CH 7 Nat vs Lab Spectrophotometry

Answer 39

Turbidity of culture increases as cell number increase Measure absorbance/ scattering of light passing through the culture - proportional to the number of cells in a culture. Advantages: quick and easy Disadvantages: doesn't distinguish living and dead cells

Question 40

# CH 7 Nat vs Lab Detecting cell products

Answer 40

Measure amounts of cellular products (ex. proteins or nitrogen levels) Concentration of product has to be constant from one cell to another Many industrial uses

Question 41

# CH 7 Nat vs Lab Continuous Culture System

Answer 41

Constantly adding nutrients and taking out wastes so the stationary phase is never reached. Two types: chemostats and turbidostats

Question 42

# CH 7 Nat vs Lab Chemostats

Answer 42

* fresh media is fed into system at fixed rate media has a limiting nutrient that regulates growth rate * all cells are in same stage of growth * maintain stage of growth for long periods of time shut down if contaminated or if toxin from bacteria froms on the walls of apparaturs

Question 43

# CH 7 Nat vs Lab Turbidostats

Answer 43

* maintain constant cell density * measure absorbance of the culture * flow rate of media is determined by turbidity (cloudiness) of the culture * media contains nutrients in excess, can allow maximal growth rate of cells useful for photosynthetic microbes, can flush out some cells as add new nutrients