Studied by 2 people
Why are bacterial plate counts important?
it can help us know how fast bacteria grow
Why do we serially dilute bacteria before we count them?
So that we can count the number of colonies on the plate
Diluent
a fluid used to dilute the concentrated sample (like water)
Aliquot
a smaller volume withdrawn from a total sample volume
Dilution factor
fraction by which your original sample concentration is diluted
What does standard plate count tell you?
tells us how many bacteria are in a sample
Which plates count as valid for counting (Approximately how many colonies should these plates have)?
there should be 30-300 colonies in the plate
Why would plates with too many colonies be considered invalid?
overcrowding can lead to two colonies looking like one
Why would plates with too little colonies be considered invalid?
could be a sampling error
TNTC
too numerous to count
TFTC
too few to count
Why is it useful to perform growth characteristic tests for bacteria?
useful for isolating, identifying, and/or describing microbes; microbes have different requirements that they like to grow
Osmosis
movement of water across a semi permeable membrane from a lower solute concentration to a higher solute concentration
Solute
substance dissolved in a solution
How does water move relative to a solute?
water moves to equalize solute
Osmotic pressure
pressure exerted by the flow of water into the compartment w/ a higher solute concentration through a semi permeable membrane separating two solutions w/ different concentrations
Hypertonic
higher solute concentration outside the cell; water moves out of the cell
What can this type of environment (hypertonic) do to an organism that does not thrive in a hypertonic environment?
Cause the cells/organisms to shrink
Hypotonic
lower solute concentration outside the cell; water moves into the cell
What can this type of environment (hypotonic) do to an organism that does not thrive in a hypotonic environment?
Cause the cell to burst (osmotic lysis)
Isotonic
equal solute concentrations
Halophiles
salt lovers; can tolerate hypertonic environment
Strict aerobes
can’t grow w/o O2, requires O2 to grow
Strict (obligate) anaerobes
can’t survive in the presence of O2
Facultative anaerobes
can grow in the presence or absence of O2
Aerotolerant anaerobes
can’t use O2 to grow, but can tolerate it
Microaerophilic bacteria
grow best in high CO2/low O2 environment
What does thioglycolate media do? How does it do this?
binds to O2, which creates an O2 gradient because of presence of resazurin
What color does the resazurin turn in the presence of oxygen? In the presence of no oxygen?
In presence of oxygen, resazurin turns pink; colorless when no oxygen
What reaction occurs in the Brewer anaerobic jar?
Removes oxygen from the environment
What color does the methylene blue indicator strip turn in the presence of oxygen?
In the presence of no oxygen? Methylene blue is blue with oxygen; white when no oxygen is present
In what pH do bacteria grow best?
6.5 to 7.5
Acidophiles
prefer acidic conditions
Alkaliphiles
prefer alkaline conditions
Why is it important to know which pH and temperature bacteria grow best in? (hint: enzymes)
improper pH will fold the proteins of the cell differently, which will impair their function
Minimum growth temperature
lowest temp in which species will grow; minimal growth rate
Maximum growth temperature
highest temp in which species will grow; minimal growth rate
Optimum growth temperature
temp at which species have the highest growth rate
Psychrophilic bacteria
grows best between 0-5 degrees C (deep sea bacteria, Arctic regions, food spoilage microbes)
Mesophilic bacteria
grows best between 25-40 degrees C (human gut flora)
Thermophiles
grows best between 45-65 degrees C (hot springs bacteria)
Diffusible pigment
colony bleeds into agar
Non-diffusible pigment
stays/confined in the colony
Do pigments change in response to environmental changes?
Species experiencing larger temperature increases over time displayed reductions in pigmentation, while those experiencing increases in aridity displayed increases in pigmentation.
Why is it important to perform physiological/biochemical tests on bacteria?
It allows us to identify an unknown organism
citrate test: What kind of broth/slant is used?
Simmon’s Citrate Slant
citrate test: What indicates a positive result? (color, appearance, etc.)
blue
citrate test: What indicates a negative result (color, appearance, etc.)
green
citrate test: What does a positive test tell you about the bacteria? (what the bacteria uses as an energy source, what enzymes it uses, what characteristics the bacteria has, etc.)
citritase/citrate demolase; breaks down citrate for energy source
citrate test: Why does the slant/broth change colors/appearance? (know the products of the reaction, if a change in pH is what makes the slant change, etc.)
pH turns alkaline
lactose fermentation test: What kind of broth/slant is used?
BCP lactose broth
lactose fermentation test: What indicates a positive result? (color, appearance, etc.)
yellow
lactose fermentation test: What indicates a negative result (color, appearance, etc.)
purple
lactose fermentation test: What does a positive test tell you about the bacteria? (what the bacteria uses as an energy source, what enzymes it uses, what characteristics the bacteria has, etc.)
no enzyme; bacteria ferments lactose
lactose fermentation test: Why does the slant/broth change colors/appearance? (know the products of the reaction, if a change in pH is what makes the slant change, etc.)
acids/alcohols produced; BCP
hydrogen sulfide test: What kind of broth/slant is used?
PIA slant
hydrogen sulfide test: What indicates a positive result? (color, appearance, etc.)
black
hydrogen sulfide test: What indicates a negative result (color, appearance, etc.)
yellow
hydrogen sulfide test: What does a positive test tell you about the bacteria? (what the bacteria uses as an energy source, what enzymes it uses, what characteristics the bacteria has, etc.)
break down sulfur in AA; cysteine (S-S) desulfurase
hydrogen sulfide test: Why does the slant/broth change colors/appearance? (know the products of the reaction, if a change in pH is what makes the slant change, etc.)
produces hydrogen sulfide; reacts w/ ferric/iron —> black precipitate
indole production test: What kind of broth/slant is used?
tryptone broth
indole production test: What indicates a positive result? (color, appearance, etc.)
red ring
indole production test: What indicates a negative result (color, appearance, etc.)
yellow ring
indole production test: What does a positive test tell you about the bacteria? (what the bacteria uses as an energy source, what enzymes it uses, what characteristics the bacteria has, etc.)
break down tryptone; tryptophanase
indole production test: Why does the slant/broth change colors/appearance? (know the products of the reaction, if a change in pH is what makes the slant change, etc.)
Kovac’s reagent detects presence of indole, has tryptophanase
methyl red test: What kind of broth/slant is used?
MRVP broth
methyl red test: What indicates a positive result? (color, appearance, etc.)
red gradient
methyl red test: What indicates a negative result (color, appearance, etc.)
yellow
methyl red test: What does a positive test tell you about the bacteria? (what the bacteria uses as an energy source, what enzymes it uses, what characteristics the bacteria has, etc.)
formic hydrogenylase; ferments glucose; presence of acids
methyl red test: Why does the slant/broth change colors/appearance? (know the products of the reaction, if a change in pH is what makes the slant change, etc.)
methyl red: pH indicator —> acids/alcohols are produced and that is what changes the color
motility test: What kind of broth/slant is used?
semi-solid agar
motility test: What indicates a positive result? (color, appearance, etc.)
turbidity
motility test: What indicates a negative result (color, appearance, etc.)
bacteria contained in the stab site
motility test: What does a positive test tell you about the bacteria? (what the bacteria uses as an energy source, what enzymes it uses, what characteristics the bacteria has, etc.)
motile/non-motile; appearance of flagella
motility test: Why does the slant/broth change colors/appearance? (know the products of the reaction, if a change in pH is what makes the slant change, etc.)
semi solid agar allows motile bacteria to flow out from the stab site
urease test: What kind of broth/slant is used?
urea slant/broth
urease test: What indicates a positive result? (color, appearance, etc.)
pink
urease test: What indicates a negative result (color, appearance, etc.)
orange
urease test: What does a positive test tell you about the bacteria? (what the bacteria uses as an energy source, what enzymes it uses, what characteristics the bacteria has, etc.)
urease; breaking down urea
urease test: Why does the slant/broth change colors/appearance? (know the products of the reaction, if a change in pH is what makes the slant change, etc.)
alkaline —> pH indicator
Why is it important to make sure caps are loose before incubating?
to allow a free exchange of air, which leads to growth
What is a Durham tube and what does it tell you?
The Durham tube is used to detect gas produced as a byproduct of fermentation. As gas forms, a bubble will appear in the tube.
Does a Durham tube tell you what kind of gas is produced?
no
tests requiring extra step to see a color change & what are the names of these reagents
indole production test - Kovac’s reagent
methyl red test - methyl red
What is the purpose of using chemical agents such as antiseptics and disinfectants?
Used to control microbial growth
Antiseptics
used on living tissues; controls infections by killing bacteria, virus, & fungi
Disinfectants
used on inanimate objects; toxic for living organisms; controls infections by killing bacteria, virus, & fungi
Bactericidal agent
completely kills bacteria (antibiotics, antiseptics, disinfectants)
What are some bactericidal processes?
Autoclaving, boiling, radiation, UV light
Bacteriostatic agent
temporarily inhibits further growth of bacteria
What are some bacteriostatic processes?
refrigeration (depending on the bacteria)
Can a bactericide also be bacteriostatic?
yes
From the results we obtained in lab, what disinfectants and antiseptics have the largest ZOIs? Which have the smallest?
antibiotic
natural antimicrobial agents produced by microbes; safe enough to be ingested
Why is an antibiotic sensitivity test performed?
used to estimate the effectiveness of known antibiotics vs certain bacteria; helps to understand which antibiotic(s) will be most effective in combatting bacterium in question
Are antibiotics bactericidal or bacteriostatic?
they can be both; bacteriostatics at high concentration can be bactericidal & vice versa
5 ways antibiotics achieve antimicrobial effect
inhibiting cell wall synthesis
inhibiting protein synthesis
alteration of cell membranes
inhibition of nucleic acid synthesis
interfering w/ metabolic pathways
broad spectrum
effective against a wide variety of different bacteria
narrow spectrum
effective against a particular group of bacteria
minimum inhibitory concentration (MIC)
lowest concentration of antibiotic needed to kill/inhibit bacteria in vitro; measured in mm & compared to standard table
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