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What is the "quickie" formula for Fahrenheit to Celsius?
- quickie: F= (C x 2)+30
- Actual: F=(C x 9/5) + 30
What is the purpose of this graph?
this graph shows the different temperatures of different types of microorganisms
What are Psychrophiles?
- Grow best at 10C
- are found in oceans, polar regions, and Alps
- *note, most fridges run at 4c
What are Psychrotrophs?
- Grow best at 20C
- Are widespread in water, dirt, and plants
What are Mesophiles?
- Grow best about 40C
- *these are most of the mammalian (37C) and the avian pathogens as well as normal microbiota
What are Thermophiles?
- Grow best at 60C
- found in soil-sporeformers
- also used in laundry detergents and mushroom soil
What are Hyperthermophiles?
- Grow best at 95C
- Found in hot springs
- Used for DNA Fingerprinting
How does temperature effect microbial growth?
After a certain point, the enzymes denature irreversibly and the MO dies, thats why the growing part is a good slant and after it hits the peak temperature it dies quickly
What is the optimal pH for most clinically-significant bacteria as well as most fungi?
- Most clinically significant bacteria: 7
- Most Fungi: 5
Give a brief overview on how to measure pH
- Each number is 10x more acidic or basic than the next.
- ex: pH9 is 10x more basic than pH8 and 100X more basic than pH7
What are pH buffers and why does media have it? Why does MSA lack pH buffers?
pH buffer is a molecule that can either give off or suck up H+
to maintain the current pH
- MO end products, like for those that use carbs, produce acid and can change the pH (MOs that use proteins produce alkaline, making it more basic).
- -This can stop growth! pH buffers prevent this
- MSA lacks pH buffers because it contains manitol and peptone which change color if the MO gives off an acid or a base. This helps us to ID the MO and is useful
- -basic: pink
Why are media formulated to have low osmotic pressure?
low osmotic pressure=low solute concentration
low osmotic pressure creates a hypotonic environment which has low nutrients, but grows pretty good
if you add more nutrients, it raises the osmotic pressure, making it hypertonic and the cell shrinks!
What are the uses and sources of the 4 elements needed in large amounts for microbial growth?
Elements: Carbon, Nitrogen, Sulfur, and Phosphates
Carbon: builds "backbone" of all biological molecules-various sugars, peptone (partially hydrolyzed protein)
Nitrogen: essential component of proteins (-NH2 (amino) groups) and nucleotides (nitrogenous bases: ATCGU)-peptone
Sulfur: essential component of certain a.a.s. (cysteine, methionine), and acetyl CoA-peptone
Phosphates: essential component of nucleotides, including ATP-phosphate salts
What are trace elements and how are they added to media?
trace elements are metal ions that are required for the function of many proteins
they are added to the media simply as contaminants, not actually as notable quanitites
What are growth factors and how are they added to media?
- Growth factors are small organic molecules the microbe cant biosynthesize such as vitamins, a.a.s
- -they are added to the media like an ingredient in a recipe
- -different MO need different growth factors
What is beneficial about O2 and when can it be harmful?
is a final e-
acceptor in aerobic respiration
- BUT in any oxic environment there will be a small but significant about of O2-
- -a "super oxide free radical"
- -free radical are potent oxidizers of DNA and protein molecules
- In ordxer for cells to survive in an oxic enviorment, they need protective enzymes
- -Superoxide Dismutase (SOD)
O + O2
Having no living cells, spores, or viruses
Define Culture Medium
a solid or liquid substance used to grow a microorganism
a container of medium with MO growing in or on it
What are the special properties of agar that make it useful for solidifying media?
Agar Agar is a polysaccaride derived from kelp-you can buy it in the store or you can buy it in a brown powder form, but it is very expensive
very few microbes can metabolize it
- it melts at 95C, but stays liquid until it cools down to 42C, providing plenty of time to inncoluate it or add nutrients
- -can add live thermophiles, mesophiles, and even add RBCs to it
What is the difference between undefined/complex media and defined media?
we know the exact proportions of everything in the media
- Undefined/complex media: we do not know the exact proportions of nutrients in every ingredient (such as when you add beef extract)
- -much more common in medical labs
What makes a media either liquid, semi-solid, or solid?
The amount of agar added to the media will determine whether it is a solid or a liquid
What is the function of an anaerobic jar?
An anaerobic jar contains no O2 (once the O2 is vacuumed out) and is used for incubating anaerobic microorganisms. They jar contains paper which is blue in the presence of O2 and white in it's absence. This helps us tell if there is O2 present in the jar
What is the purpose of selective and/or differential media? How does this apply to EMB, PEA, and Blood Agar?
- Selective media is chemically composed to inhibit the growth of certain MO
- -chemical added retard the growth of certain MO
- Differential Media helps you ID the MO that is growing
- -chemicals added to differentiate between type of MO growing
- EMB is both selective and differential
- -it is selective for gram negative bacteria and is differential for those that produce lactose (like between salmonella and shigella)
selective for gram positive MO
is neither selective or differential
What is the theory behind a streak plate? How do you do a streak plate? What is it's application?
get isolated colonies
- -to check for culture purity
- -make pure cultures
- -assess colony morphology (clue for identity)
How can bacterial cultures be preserved by deep freezing (-80C)?
- 1. Grow a dense broth culture
- 2. cetrifuge, decant supernatant, leaving cell pellet
- 3. add very cold glycerol to cell pellet and store in -80C freezer
This is a good way to store a LOT of cultures for a LONG period of time
What is binary fission? Describe the process
- Binary fission: how microorganisms populate
What is doubling time?
- Amount of time it takes for the population of an MO to double
- -this time is constant
What are the doubling times for the following:
S. Aureus-30 min
M. Tuberculosis-15 hours
T. Pallidum-33 hours
M. Leprae-13 days
How does binary fission lead to logarithmic growth?
- it is a constant doubling, leading to logarithmic growth during the log phase
- -exponential growth
What is the classic bacterial growth curve?
- This curve shows all four phases of microbial growth
- -the bottom shows the length of time while the left side shows the # of live cells
- -log is used because the numbers become large quickly
- *it is important to remember that cells tend to die at a slower rate than pictured here
What is happening in each phase of the bacterial growth curve? Why?
- Lag phase: cells are synthesizing catabolic enzymes and transport proteins
- -no growth in numbers occuring
- Log phase: population doubles at constant intervals
- -abundant nutrients and proper tools thanks to lag phase
- stationary phase: division stops- nutrients are depleted and wastes are abundant
- -about 1 billion/mL
cellls die and the population halves at a constant rate
What is the process for doing a serial dilution? How would you do a plate count from this?
- The process of a serial dilution includes taking 1 mL from the original sample and adding it to 9mL of a sterile solution. This series is repeated with several test tubes
- -the reasoning is to get a countable amount in one of the tubes
- -these are plated with agar plates and then the most reasonable number is counted
- #of coliforms/mL in tube
- #of coliforms/100mL
What is CFU?
Colony forming Unit
- each produces 1 colony
- -great for plate counts
Describe the filtration technique
- A filtrate system is used with a filter
- -Large amount of sample is placed into the funnel and passes through a membrane filter
- (MO now on filter)
- -the liquid flows into a container where there is a vaccuum pump that draws the liquid from the funnel
The filter is then removed and placed in a nutrient plate for the MO to grow
What types of samples are best taken by a serial dilution/plate count and what samples are best taken by filtration?
- Serial dilutions are used for small very accurately measured amounts of sample on a plate
- -assumed to have a large amount of bacteria in small amount of sample
- Filtration is used for a large very accurately measure amounts of a sample onto a plate
- -may contain very few bacteria in a large sample