Lecture 10 - bacterial growth Flashcards
What is binary fission?
Method of bacteria division in which one parent cell splits into 2 equivalent daughter cells
When can a colony of bacteria be visually detected?
When the colony has around 10 thousand cells
Bacterial growth curve in a closed system
- Lag phase
- Log phase
- Stationary phase
- Death phase
Lag phase of bacterial growth curve
Bacteria are preparing their cell machinery for growth
Log phase of bacterial growth curve
Growth approximates an exponential curve (straight line on a logarithmic scale)
Stationary phase of bacterial growth curve
Resources run out. Cells stop growing, shut down growth machinery, and turn on stress responses to retain viability.
Death phase of bacterial growth curve
Cells die with a “half life” that approximates a negative exponential curve
What can happen to the environment as bacteria grow?
Bacteria can release waste product into the environment that change the conditions
Equation for bacterial growth by binary fission
Nt = N0 x 2^(t/td); td is the doubling/generation time of the population
What percentage of recognized bacteria can be cultured using routine lab growth media and conditions?
0.1-1%
Methods of isolation of individual bacteria/bacterial clones
- Streaking
- Serial dilution and plating
What is streaking?
Bacteria are streaked across the surface of an agar plate to spread the cells out into individuals. Inoculation loop is heat sterilized between streaks
Serial dilution and plating
Bacterial suspension is serial diluted and samples spread onto agar
How are bacterial cells counted?
- viable bacteria can be counted as colony forming units (cfu) using serial dilution and plating
- indirect counting by turbidity or optical density of broth cultures
- direct counting using Petroff-Hausser counting chambers
What are Petroff-Hausser counting chambers?
Specialized microscope slides with chambers of defined volue and a inscribed grid
Downside to direct counting using a Petroff-Hausser counting chamber?
Can’t tell if cells are alive or dead
Method to differentiate dead and alive bacteria?
Live/dead dye: acts like DAPI and gets into spaces in DNA
- if cell is alive, the stain won’t penetrate the cell and fluoresce
Types of growth media
- complex media
- synthetic media
- enriched media
- selective media
- differential media
Complex media
Nutrient rich media. Contents are poorly defined/not standardized
Synthetic media
Precisely defined growth media. (aka defined media)
Enriched media
Complex media to which specific blood components are added
Why are blood components added to enriched media?
Pathogenic species often need host materials in blood to grow
Selective media
Favors the growth of one organism over another
What is one way to make selective media?
Including an antibiotic
Differential media
Exploits differences between two species that grow equally well
What is LB broth?
Lysogeny broth
- most common rich media, good for E. coli growth and most gram negative and gram positive bacteria
What makes azotobacter medium (a defined medium) unique?
Doesn’t provide nitrogen because azotobacter is able to fix nitrogen from the air
What makes defined media for sulfur oxidizers unique?
Doesn’t provide a carbon source because it is for bacteria that use sulfur for its energy supply
What is MacConkey’s Agar?
Differential media that has lactose and pH sensitive dye
- bacteria that metabolize lactose will make the agar red from environment acidification
- bacteria that don’t metabolize lactose will have yellow agar
What bacteria does MacConkey’s agar suppress the growth of?
Gram + bacteria. Media contains bile acids that act as detergent to disrupt the membranes
Alternate methods of growing bacteria in culture
- Growth in chip with multiple chambers for bacteria that grow in soil
- Continuous culture
- Chemostat
How does chip growth of bacteria help with finding new antimicrobial compounds?
Overlay a gel covered in the target bacteria. If there is no growth over a particular bacteria colony from the chip chambers, the colony contains bacteria that are releasing a potential antibiotic.
What is continuous culture?
All cells in a population achieve a steady state.
- allows the study of bacterial physiology
What is a chemostat?
Device that ensures logarithmic growth of bacteria by constantly adding and removing equal amounts of culture media.
- brings in new nutrients and removes waste
What is a biofilm?
Surface-attached community of bacteria.
- can be single or multi-species
- can be on organic or inorganic surfaces
Why are biofilms of medical significance in surgery?
Biofilms can form on medical implants if there is contamination during placement. Very hard to get rid of biofilms with antibiotics.
Describe the process of biofilm formation and propagation
- Flagellar bacteria move to a new region and attach to surface as a monolayer
- Cells stick to their surface and stop expressing flagella to form microcolonies
- Exopolysaccharide is produced to form the “glue” that holds the bacteria together
- Biofilm towers form –> mature biofilm
- Towers dissolve and allow bacterial dispersal
What do bacteria use to stick to surfaces?
Pili, fimbriae, and adhesins
What are pili and fimbriae? What is the difference between the two?
- straight filaments of pilin protein monomers
- pili are much longer than fimbriae
- fimbriae are only expressed for adhesion, pili can also be used for conjugation
What makes pili an fimbriae adhesive?
Tip of the filaments have an adhesive molecule (ex: Pap or Fim) at the tip specific to a particular target molecule
Pap vs Fim
Pap: pyelonephritis associated pilus
Fim: binds mannose on outer surface of most cells
How do bacteria use Pap and Fim differently?
Uses Fim to climb up the urinary tract and resist the flow of urine. Uses Pap to adhere in the kidney.
Peritrichous cells
Have flagella randomly distributed around the cell
Lophotrichous cells
Have flagella localized to cell poles
Monotrichous cells
Have a single flagellum
How to peritrichous cells control their movement?
Clockwise vs counterclockwise rotation of flagella
- clockwise spreads out the flagella –> tumbling
- counterclockwise causes flagella to bundle together and act as one flagellum to steer cell in one direction
Composition of flagella
- filament is composed of a flagellin polymer
- filament is rotated by a motor driven by proton motive force
Is the flagellar motor the same in Gram - and Gram + cells?
No, there are different layers of rings that make up the motor depending on the membranes present in the cell
