Lecture 11: Factors That Affect Bacterial Growth Flashcards
(24 cards)
Determing cell population
What is direct counting?
what are some difficulties?
- directly measures cells or colonies themselves
- counts both viable (live) and nonviable (dead) cells
- difficult to count motile cells (with phagelum) so they are often killed with chloroform (CHCl3)
How can you differentiate between viable and nonviable cells?
You can dye the cells with methylene blue which will be uptake by dead cells and push out (not stained) by live cells.
Determining Cell Populations
What are the three mechanisms of direct counting?
- spread plate counting - put diluted culture onto solid medium in a petri dish, spread the culture around and incubate –> count
- pour plate method - pour sample directly onto petru dish, then pour molten agar, incubate –> will have subsurface and surface colonies
- petroff-hause counting chamber - have small amount of sample under cover slip and individually count cells on grid
Determining cell populations
What is indirect counting?
- using other means, like turbidity to count (usually with liquid medium)
- can measuer turbidity with spectrophotometer (OD) or klett photomer (klett units)
- turbidity is directly proportional with cell count (viable and nonviable)
turbidity - cloudiness/haziness of media
what do spectrophotometers and klett photomers do?
They will direct light into the culture in the liquid medium and measure how much light is absorbed.
* need to have standard curve to determine estimated amount
* compare turbidities of the same species (not different)
Why are serial dilutions done?
In the context of the class, this is done to dilute the culture so that there is a lower concentration to count. If this is not done then there is too much growth and it becomes much harder to count individual cells.
How are serial dilutions done?
The culture will be put in a tube and diluted by broth. Each dilution will be decreasing in concentration (e.g., 1/10, 1/100, 1/1000), therefore decreasing in cells. Once you have a dilution that is countable then you can count and multiple the number according to the dilution factor.
How can plate counting errors occur?
- unsuitable culture conditions (e.g., inappropriate medium, inappropriate oxygen concentration)
- procedural errors (e.g., inaccurate transfers or dilutions)
- cell clumping - colonies will come from one cell, but clumping can affect the colony
What factors affect microbial growth?
- temperature
- pH
- water availability
- oxygen concentration
What are the cardinal temperatures of growth?
They describe the relative enzymatic or protein activity levels that promote growth
* maximum - when it starts to denature
* optimum - optimal temp for enzyme to function
* minimum - minimal temp to grow
What are the categories of cells based on their growth abilities at various temperatures?
- psycholophile (grow in freezing temperatures)
- mesophile (medium temp, room temp)
- psychotolerant - can grow at freezing temp but prefer 20-40 C (mesophile)
- thermophile (near to our body temp)
- hyperthermophile (boiling temp, hyper hot water; sub class of thermophile)
What are the adaptations that enable growth under these temperatures for psychrophily?
name them
- enzymes active in cold (more alpha helices, more polar side chains, fewer weak interactions)
- more unsaturated lipids (double bonds so can’t hold as tightly together)
- cryoprotective mechanisms (resisting ice crystallizations)
want more flexbilibility
What are the adaptations that enable growth under these temperatures for thermophily?
- amino acids subsitutions in key places to increase stability at high temps
- more ionic bonds
- denser hydrophobic protein cores
- cytoplasmic solutes
- more saturated fatty acids in bacteria
- lipid monolayers in archaea
overall more rigid and strong interactions
What is the difference between acidophiles and alkaliphiles?
Acidophiles like low pH (high [H+]) while alkaliphiles like high pH (low [H+]).
What is an example of an acidophile and an alkaliphiles?
Acidophiles: picrophilus oshimae has a pH optimum at 0.7 and lyses when pH is above 4.
Alkaliphiles: Bacillus firmus grows in pH up to 11 and uses Na+ gradient to drive transport and locomotion
What are the qualities related to pH that affect growth?
list them
Concentration of H+ since this is important for driving transportation and locomotion. However, organisms that live in alkaline (low H+) conditions have adpated to use sodium to drive this.
What is Taq polymerase and why has it been of use in biotechnology?
It is a thermostable polymerase that has been use in molecular biology techniques like PCR for replicating DNA.
What is water activity?
water activity: water available for a microbe
high wa = lots of available
low wa = little water
Microbes & Osmotic Pressures
What important adaptation enables growth at high osmolarity?
They will use compatible solutes which are compatoble with living conditions of cells found in the cytoplasm, creating a high solute concentration in the inside. This is so that water is not just moving out of the cell.
What are examples of compatible solutes?
compounds that bring water in
- amino acid-type: glycine betaine, ectoine, proline
- carbohydrate-type: sucrose, trehalose
- alcohol-type: glycerol, mannitol
they are highly soluble compounds and have a lot of places to bind water
What is the relationship that various microbes have with respect to osmotic concentrations?
- Nonhalophile - grow best in reduced water concentration; higher salt concentration
- Halotolerant - tolerate higher salt contration
- Halophile - flourish in high salt conditions
- Extreme halophile - need very high salt conditions to grow
What are the classes of relationships that microbes have with oxygen?
5 total
- obligate aerobes - need O2 for growth
- obligate anaerobes - O2 is toxic and inhibits growth
- facultative anaerobes/aerobes - can switch between aerobic and anaerobic respiration
- microaerophiles - need some O2 but a lot is toxic
- aerotolerant anaerobes - anaerobic metabolism only, but can grow in presence of O2
- note that on tube diagram the red is oxic zone and yellow is anoxic zone
Microbe & Oxygen - classes
Why do microbes have a relationship with oxygen?
Could be necessary or toxic for metabolism and growth
What are oxygen’s toxicity methods used to control oxygen concentrations during laboratory growth?
Superoxide (O2-): O2 gets aditional e-, can be destroyed by superoxide dismutase/reductase and combo of superoxide dismutase + catalase
Hydrogen Peroxide (H2O2): destroyed by catalse and peroxidase
Hydroxyl Radical (OHdot): missing electron
start with O2 and when you add an electron you get the toxic forms above in order, then the fourth electron added makes water
