Biology Lab Quiz #3

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a smaller volume withdrawn from a total sample volume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

1. inhibiting cell wall synthesis

2. inhibiting protein synthesis

3. alteration of cell membranes

4. inhibition of nucleic acid synthesis

5. 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