CH 7 microbial growth natural v lab Flashcards
CH 7 Nat vs Lab
Growth in the natural environment
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
CH 7 Nat vs Lab
Synthesis of starvation proteins
- Increase peptidoglycan crosslinking
- Bind and protect DNA
- Prevent protein denaturation
Starved cells become more resistant to environmental stresses and pathogens may become more virulent
CH 7 Nat vs Lab
Spontaneous dormancy in nutrient-rich conditions
Portion of the microbial population enters dormancy on purpose - never know when nutrients may run low
Resistant to other environmental stresses
CH 7 Nat vs Lab
Biofilms
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
CH 7 Nat vs Lab
Formation of biofilms
Reversible attachment - putting down proteins, conditioning surface for next species
Irreversible attachment - secretion of slime layer; microbial growth
Detachment - detachment or sloughing of cells
CH 7 Nat vs Lab
Quorum sensing
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
CH 7 Nat vs Lab
Growth in the lab
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.
CH 7 Nat vs Lab
Culture media
All nutrients necessary for microbial growth.
Can be classified based on the physical state, chemical composition, and/ or function.
CH 7 Nat vs Lab
Physical state of media
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
CH 7 Nat vs Lab
Chemical composition of media
Defined
- all chemical compositions and ratios known Complex
- contain some ingredients of unknown composition and ratios
- Yeast extract, beef extract, peptone, tryptone
CH 7 Nat vs Lab
Functions of media
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
CH 7 Nat vs Lab
Fastidious bacteria
Those that require specific needs for growth, either/ both environmental and nutrient factors.
CH 7 Nat vs Lab
Blood agar
Can tell if the bacteria in the media are hemolytic or not - enriched and differential
CH 7 Nat vs Lab
Chocolate agar
Blood agar where the erythrocytes have already been lysed - enriched, but not differential
CH 7 Nat vs Lab
PEA
Phenylethanol Agar
Differential
Allows G+ to grow, but not G- because it is toxic to G-
CH 7 Nat vs Lab
MacConkey Agar
Selective: G+ over G-
Differential: identifies bacteria that can ferment lactose (lactose positive)
CH 7 Nat vs Lab
Eosin Methylene Blue
Selective
Differential: lactose positive or negative
CH 7 Nat vs Lab
Mannitol Salt Agar
High salt concentration
Differential: mannitol fermentation
CH 7 Nat vs Lab
Growing Anaerobic microorganisms
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
CH 7 Nat vs Lab
Isolation of Pure Culture
Colony - population of bacteria arising from single cell Three types: - streak plate - spread plate - pour plate
CH 7 Nat vs Lab
Enrichment cultures
Adjust conditions to ID microorganisms with a specific characteristic.
ex. metabolics, antibiotic resistant
CH 7 Nat vs Lab
Colony morphology
Each species has its own unique morphology - dealing with form, elevation, and margin.
CH 7 Nat vs Lab
Characteristics looked for in identifying bacteria on morphology
Size
Texture of surface
Overall texture (butyrous, brittle, viscous)
Optical characteristics (opaque, translucent, dull, iridescent)
Pigment (most don’t produce color unless stressed)
CH 7 Nat vs Lab
Types Cultures
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
CH 7 Nat vs Lab
Growth Curve for Batch cultures
Lag phase > exponential phase > stationary phase > death and senescence phase
CH 7 Nat vs Lab
Lag phase of growth curve
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
CH 7 Nat vs Lab
Exponential phase of growth curve
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)
CH 7 Nat vs Lab
Stationary phase of growth curve
Some live and some dead cells
Nutrient availability decreasing and wastes building up
Critical population level = too dense (10 9 cells/ mL)
CH 7 Nat vs Lab
Senescence and death
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)
CH 7 Nat vs Lab
Math of Growth
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
CH 7 Nat vs Lab
Why is the math important?
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.
CH 7 Nat vs Lab
Measuring growth
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 *****
CH 7 Nat vs Lab
Direct cell counts
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)
CH 7 Nat vs Lab
Petroff-Hausser counting chamber
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
CH 7 Nat vs Lab
Coulter Counters
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
CH 7 Nat vs Lab
Fluorescence and direct cell counts
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.
CH 7 Nat vs Lab
Viable cell count
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.
CH 7 Nat vs Lab
Biomass measurement
Dry weight determination (filter out of liquid culture)
Time consuming and not very sensitive/ accurate
CH 7 Nat vs Lab
Spectrophotometry
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
CH 7 Nat vs Lab
Detecting cell products
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
CH 7 Nat vs Lab
Continuous Culture System
Constantly adding nutrients and taking out wastes so the stationary phase is never reached.
Two types: chemostats and turbidostats
CH 7 Nat vs Lab
Chemostats
- 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
CH 7 Nat vs Lab
Turbidostats
- 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
