EXAM 2-Chapter 7 part 2 Flashcards
Complex media
Know that it has all necessary macro and micronutrients but do not know specific concetrations
synthetic (defined) media
know everything in it and their concentrations
If you aren’t sure the nutrient requirements of your organism which media do you use?
synthetic
Components of complex media
peptones
extracts
agar
Peptones
Protein hydrolysates prepared by partial digestion of various protein sources
Peptones importance
break down into amino acids and supply macro and micronutrients
Peptone examples
gelatin
caseins
meats
Extracts
aqueous extracts usually of beefs or yeasts
Extract importance
supply macro and micronutrients
Agar
sulfated polysaccharide used to solidify media
usually extracted from red algae
Agar importance
melts at high temp- solidifies when cooled
most organisms cannot digest/degrade agar
functional media types
supportive/general purpose media
enriched media (supportive+)
Differential media
Selective media
Supportive/general purpose media
main function is to just grow your ordinary microorganisms
can grow a wide vareity of them
enriched media
general media plus some nutrients
What is enriched media used to grow?
Fastidious microorganisms
Fastidious microorganisms
Very picky and grow really slow on regular media
Differential
distinguishes between different groups based on their biological characteristics
Selective
favors the growth of some microorganisms and inhibits the growth of others
What type of media is blood agar?
enriched
functional- differential
Blood agar
Contains sheep red blood cells
differential: if lysis occurs a clear zone forms around the growth
What type of media is MacConkey agar>
complex
functional- differential and selective
MacConkey agar
differential- contains lactose and if fermentation occurs agar turns pink
selective- contains biosalts that allow growth of Gram-neg and inhibit Gram-positive bacteria
Why can Gram-negative bacteria grown on MacConkey agar?
Have an outer membrane that keeps bio salts out
pure culture
a plate with a single isolated culture
3 techniques for isolating colonies
Streak plate- T-streak
Spread plate
Pour plate
T streak method
spreading mixture of cells ON agar surface so that individual cells are separated from one another
involves bacteriological loop
Spread plate method
Make bacterial dilutions
pour 0.1mL on agar plate and spread with “hockey stick”
Bacteria grows ON surface
Pour plate method
make bacterial dilutions
put dilutions into molten agar and vortex to mix cells
Pour molten agar and bacteria mix into empty Petri dish
Bacteria grown ON and IN agar
Why cant we grow all microrganisms in lab?
we cannot replicate appropriate growth conditions
symbiotic relationships that we cannot recreate
How do we gain information about unculturable bacteria?
PCR
Fluorescent in situ Hybridization (FISH)
Metagenomics
PCR
DNA from unulturable bacteria can be amplified
FISH
sequences can be used to produce fluorescent probes that will bind to complementary DNA
Metagenomics
Take all DNA in a sample and get all genes in sequence
Limitations/drawbacks of metagenomics
results in a lot of info that must be analyzed
Results must still be confirmed in culture media
Microbial Consortia
Unculturable bacteria
organisms that will not grow on their own; require symbiotic relationship
Where are microbial consortials commonly found and give example?
interfaces between water and land
EX: biofilms
Bacterial colony morphology
Can tell us some things but not everything
cannot use morphology alone to determine microorganims
Differential colony morphology
the same colonies look different when grown in different conditions/on different media
Direct counts
see cells and count them
Two methods of direct counts
Direct count with Petroff-Hausen chamber
Flow cytrometry
Direct Count with Petroff Hausen chamber
uses a smaller aliquot to measure larger culture
petroff Hausen champer procedure
load knwon volume onto gridded side
count under a light microscope
petroff-haussen chamber limitations
Can lose accuracy
need to ensure aliquot is representative of entire culture- mixed well
Flow cytometry
microbial suspension is forced through a small orface with a laser beam
gives more accuracy
Flow cytometry procedure
one cell moves through light beam at a time and distrupts electrical current
When disruption happens the machine counts
Specific antibodies and flow cytometry
specific antibodies can be used to determine size and internal capacity
EX: set to recognize fluorescence with a live-dead stain or fluorescent probe
Limitations of Flow cytometry
very large and expensive
requires training
Visible counts
serial dilutions and measure as colony forming unites (CFU)
Visible counts process
create spread plate and pour plate
take dilutions at different times and spream 0.1mL on plate
COunt number of colonies on plate (avg) and divide by dilution (CFU/mL)
What is a reliable number of colonies for CFU?
20-400
What happens if starting culture is already diluted for CFU?
will not give a reliable count
must concentrate first
How to concentrate already diluted cultures?
filtering apparatus
cells get stuck on filter, transfer membrane filture onto growth medium and incubate
Turbidity
as culture gets more dense it gets cloudier and the absorbance increases
How to take turbidity measurements?
spectrophotometer sends light through culture
If tube is cloudy, light will not pass through and strike sensor
What does spectrophotometer find?
OD600
optican density at wavelength of 600
What is a CFU?
Colony forming unite
used to estimate the number of viable microbial cells in a sample
How to calculate CFU?
Count the number of colonies on the plate (average) and divide by the dilution
Gives CFU/mL
Indirect measurements of cell mass
Dry weight
Quantity of a particular cell constituent
Dry weight
putting cells on a balance and weighing it
How to get cells for dry weight
Take 5L of culture
Spin in multiple centrifuge tubes to separate pellet
Pour liquid and resuspend
Continue process multiple times
Limitations fo dry weight
time consuming
not very sensitive
can lose cells when pouring out liquid
Quantity of a particular cell constituent
using an assay of constituent
As constituent increases so are the sells
When can you use as a cell constituent?
protein
DNA
ATP
Chlorophyll
What are limitations of using cell constituents?
Must pick the right constituent
- cannot fluxuate randomly in cell
- needs to be expressed all the time
