Microbio Lab: practical 1
what happens in the death phase of bacterial growth?
# dying cells> # new cells produced; lots of turbidity due to presence of living and dead cells, cell particles and metabolites
what is a - and + result for a motility (movement) in the SIMs test?
++= very motile= entire tube is turbid += microbe is motile= some growth away from the stab -= microbe is non-motile= only growth in stab line
what is a - and + result for a Sulfide (production of hydrogen sulfide H2S) in the SIMs test?
+= H2S production/ sulfur present= black precipitte -= No H2S = no precipitate
describe 3 things about flagellar motility
- "run" and "tumble" - whip-like in eukaryotes - some use cilia that move in unision
what is a - and + result for a fermentation test?
- = no fermentation = red += fermentation occuring= orange OR yellow and CO2 bubbles
what is the viable counting method?
- a single colony or colony forming unit came from a single cell - serial dilutions are set up and plated using a spread plate technique; count # of colonies - only counts living cells
what are the 4 types of microbial motility?
- corkscrew & bending motion - flagellar - twitching/gliding movement - brownian motion
what is the direct counting method
- requires a microscope an d counting chamber, count the number of cell sin each portion of the grid on the counting chamber - both living and dead cells are counted
describe 3 things about brownian motion
- result of water molecules moving around and hitting the bacteria - usually bacteria lacking appendages (non-motile) placed in a wet environment - NOT MOTILITY
what is fluorescent microscopy?
- visualize specimens that can fluoresce either naturally or after treatment (fluorochromes) - used in combination with specific antibodies to provide specificity
what is dark microscopy?
-dark field, light objects, -used to view live, unstained organisms that do not stain well
what is phase contrast microscopy?
-high-contrast image of transparent specimens to view living cells (unstained/stained) - staining is not needed - useful for dehydrated structures within cells (spores, lipid bodies, PHB structures)
what is bright microscopy?
-light field, dark object, - stains are used to increase the contrast between the object and the background -staining usually kills cells
what uses twitching/gliding movement?
-pili - on moist surfaces
what are the 6 things to do to properly store a microscope?
1. DO NOT leave slide on stage 2. 100x objective should be cleaned of immersion oil (lens paper/lens cleaning solution) 3. The stage should be as far down as possible (far from the objective lens) 4. Lowest objective lens (4x, 10x) in position 5. Wrap the power cord up 6. Put it back into the cabinet
what are the 4 characteristics of the spectroscopic method?
1. fastest counting method, uses a spectrophotometer to quantify the turbidity of the culture 2. spec measures the amount of light transmitted through the sample 3. turbidity vales are presented as absorbance of light at a particular wavelength (optical density) 4. inversely related: as turbidity increases, transmission of light decreases= lots of cells present
what are the 3 characteristics of scanning electron microscopy (SEM)?
1. specimen is coated with electron-dense material (heavy metal) to kill the cells, the electrons bounce off the sample and produce the image 2. SEM images are 3DD 3. Living cells require chemical fixation to preserve and stabilize structure
what are characteristics of both Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)?
1. specimens are viewed in a vacuum and produce useful magnification up to 250,000x 2. view dead cells or specimens
what are the characteristics of Transmission Electron Microscopy (TEM)?
1. the specimen is fixed in resin and must be thin enough for electrons to pass through
what does the eyepiece/ocular lens do?
10x magnification
countable colonies fall between
30-300 colonies per plate TFTC-> 30-300 -> TNTC
what is aseptic technique?
a method used to prevent contamination with microorganisms (flaming top of tube, heating inoculating loop, never leave plates/tubes uncovered, inoculate plates lid side down)
what happens in the lag phase of bacterial growth?
adaptation - microbes are synthesizing enzymes, metabolites, and other nutrients
why is heat fixing important for staining?
adheres cells to slide so they don't get washed away with dye - strengthens cell for staining - speeds up evaporation
what does the objective lens do?
adjust the strength of the magnification (4x-100x)
what is transmission electron microscopy (TEM)?
allows for cross sections (internal structures) of microorganisms to be viewed
what is Scanning Electron Microscopy (SEM)?
allows the surface of the specimen to be viewed
what are the 4 types of microscopy?
bright dark phase contrast fluorescent
what is the CFU/mL equation?
cfu/mL = (# of colonies * dilution factor) / volume of culture plated (mL)
the prefix strepto means
chain
the prefix staphyl means
cluster
what are the 3 types of bacterial morphology
coccus (sphere) bacillus (rod) spiral (spirillym, spirochetes) also [ vibrio: comma shaped]
what does the stage control knobs do?
control left to right, back and forth movement of the stage
what does the condenser light source & diaphragm do?
controls the intensity and position of the light illuminating the specimen
what is the purpose of safranin in gram staining?
counter staining causes gram - cells to turn pink
define magnification
describes the enlargement of an object under the microscope
define resolution
describes the sharpness and detail of the image
what does the MR/VP in the indole- Methyl red- vogues proskauer citrate (IMViC) test for?
determines which fermentation pathway is used -> mixed acid (4:1 acidic:neutral) or butanediol pathway (1:1)
what are the 3 methods used to count colonies?
direct counting method, viable counting method spectroscopic method
what is the purpose of ethanol in gram staining?
disrupts the outer membrane of gram - cells, allowing the crystal violet complex to leave - destaining
Magnification is compounded by the use of two lenses: the _ and the _
eyepiece and objective lens
what does the condenser do?
focuses light
what is the purpose of iodine in gram staining?
forms a large complex with the crystal violet dye - complex becomes trapped in the gram +, causing it to take a purple color
what is the purpose of crystal violet in gram staining?
initial stain, turns cells purple
why do we stain bacteria?
it allows us to visualize them and differentiate between cell types (Gram + vs gram -)
what are the phases of bacterial growth in order?
lag, exponential, stationary, death
what happens in the logarithmic/exponential phase of bacterial growth?
microbial population doubles; cell metabolism is the most active; cells are more susceptible to antimicrobial agents that target protein, DNA, and cell wall synthesis
why is aseptic technique important for staining?
minimizes contamination to ensure accurate results
what does the fine focus do?
moves stage similar to coarse focus, but in smaller increments
what does the coarse focus do?
moves the stage closer to or further from the objective lens
define total magnification
ocular lens magnification X objective lens magnification
describe heat fixing
pass the slide with a sample on it through a flame a few times, but do not hold it in the flame; we need all the liquid to evaporate
how does the sugar fermentation test work?
phenyl red as pH indicator: red -> yellow if fermentation occurs (acidic byproducts are produced and pH is lowered) small tube (Durham tube) that captures gas produced (CO2) by the microbes)
what does a gram - microbe look like?
pink, bacillus shaped (rod); inner and outer membrane with a thin cell wall with a low amount of peptidoglycan
what does a gram + microbe look like?
purple, coccus shaped only inner membrane wit a thicker cell wall with a high amount of peptidoglycan
what does the motility (movement) in the SIMs test test for?
semi- solid media used in test allows for identification of motile bacteria
what usually used corkscrew & bending motion?
spirochetes
what does the indole (hydrolysis of tyrptophan) in the SIMs test test for?
test if the microbe contains tryptophanase to breakdown tryptophan and use it as carbon and nitrogen source
what does the Sulfide (production of hydrogen sulfide H2S) in the SIMs test test for?
test microbe's ability to reduce sulfate to sulfide by hydrolysis reaction - most useful for determining the presence/absence of enteric bacteria
what does the sugar fermentation test test for?
test to see if microbe metabolize sugar (glucose, sucrose, lactose) to produce acid and gas as byproducts
why would we perform a gram stain?
to differentiate gram - and gram + bacteria
what is the purpose of water in gram staining?
to rinse off excess stain without losing the fixed culture
how does the indole (hydrolysis of tyrptophan) in the SIMs test work?
tryptophan hydrolysis -> indole, pyruvic acid, ammonia
what does the iris (or diaphragm) do?
varies the intensity and size of the cone of light
what is a - and + result for a indole (hydrolysis of tyrptophan) in the SIMs test?
visualized by Kovac's reagent += indole present= red line - = indole not present= yellow line
what happens in the stationary phase of bacterial growth?
when cells run out of essential nutrients; inhibited by their own waste products, or lack physical space ( # new cells = # dying cells)
what does the stage do?
where slides are placed
