Lab Midterm 2 Study Guide

purpose of a viable plate count

Determine the number of living bacteria in a sample

why only living bacteria are counted

Only living cells can divide and form colonies

colony forming unit (CFU)

One viable cell or cell cluster capable of producing one colony

one colony does not always represent one bacterial cell

Cells may stick together, so multiple cells form one colony.

colonies develop only from viable bacteria

Dead cells cannot reproduce

plates containing 30–300 colonies

acceptable range

dilution plate used for CFU calculation

Each 1:10 dilution decreases concentration by a factor of 10.

serial dilutions

Reduce bacterial concentration so colonies become countable

Identify the total dilution of a sample

Example:

Tube	Total Dilution
1	10⁻¹
2	10⁻²
3	10⁻³
4	10⁻⁴

wrong dilution were plated

• Too concentrated → lawn or TNTC (too numerous to count)

• Too dilute → very few or no colonies

how dilution affects colony numbers

Greater dilution → fewer colonies.

all calculation steps

Example:

• 150 colonies

• 10⁻⁵ dilution

• 0.1 mL plated

CFU/mL = 150 ÷ (10⁻⁵ × 0.1)

= 150 ÷ 10⁻⁶

= 1.5 × 10⁸ CFU/mL

what OD600 measures

• Turbidity (cloudiness)

• Amount of light scattered at 600 nm

spectrophotometer

• Sends light through culture.

• More cells → more light scattered → higher OD.

OD600 indirect method

• Indirect count because it estimates cell number instead of counting cells

• Living cells, Dead cells , Cell debris

OD600 vs viable plate counts

OD may be higher because:

• Dead cells scatter light.

• Cell debris scatters light.

• Cell clumps count as one colony.

• Only living cells produce CFUs.

OD600 measures

living cells, dead cells, and cell debris

effects of Incubating plates right-side up

Water spreads colonies.

effects of Incubating upside down

Prevents condensation from dripping.

effects of Plating the wrong dilution

Counts inaccurate.

effects of Using too much inoculum

Lawn/TNTC.

effects of Using too little inoculum

Very few colonies.

purpose of a plaque assay

Count infectious bacteriophages.

plaque

Clear area where bacteria have been lysed.

plaque formation

comes from one infectious phage particle.

plaque originates from one infectious phage

• Adsorption (attachment)

• Infection

• Replication

• Lysis

• Release of new phages

adsorption

phage physically locks onto the bacterial cell wall

infection

phage tail sheath contracts, injecting its viral genetic material through the hollow core

replication

phage genome redirects the host bacterium's metabolic machinery

lysis

Phage-encoded enzymes are produced to degrade the bacterial cell wall and disrupt the cell membrane

release of new phages

Water rushes into the osmotically fragile bacterial cell, causing it to burst

soft agar

• allows bacterial growth

• allows limited phage movement

• produces localized plaques

Bacterial Lawn

• Phages require host bacteria.

• Without bacteria, no plaques form.

calculate original phage titer

Formula:

PFU/mL = Plaques ÷ (Dilution × Volume plated)

Example:

100 plaques

10⁻⁶ dilution

0.1 mL

PFU/mL

=100÷(10⁻⁶×0.1)

=100÷10⁻⁷

= 1 × 10⁹ PFU/mL

phages are not mixed with bacteria

No plaques.

phages incubate too long before plating

Fewer infectious phages.

wrong dilution is plated

Too many or too few plaques.

lytic cycle

Plaques demonstrate:

• Infection

• Replication

• Cell lysis

• Release of new phages

bacterial transformation

Uptake of foreign DNA (usually plasmids).

bacteria acquire plasmids

conjugation, transformation, transduction

transformation horizontal gene transfer

DNA moves between bacteria (not parent to offspring).

chemical competency

Cells treated to take up DNA.

calcium chloride

Makes membrane more permeable.

heat shock

Brief heat creates pores allowing plasmid entry.

TSA (P−)

Growth

TSA/Kan (P−)

No growth

TSA (P+)

Growth

TSA/Kan (P+)

Growth (transformed only)

kanamycin resistance gene

Plasmid contains:

• KanR gene

selectable marker

Allows transformed cells to survive.

plasmid omitted results

No growth on Kan plate.

heat shock omitted results

Little/no transformation.

wrong plate used results

Cannot determine transformation.

kanamycin omitted results

Everyone grows.

contamination occurred results

Unexpected growth.

calculate TE

Formula:

TE = Transformants ÷ μg DNA

Higher TE = More successful transformation.

antibiotic resistance

transformation spreads it

occurs when bacteria mutate and evolve to survive the drugs designed to kill them

virulence genes

transformation spreads it

specific genetic sequences in pathogens that enable them to cause disease

spread of plasmids

transformation spreads it

small, circular, extrachromosomal DNA molecules that replicate independently of chromosomal DNA

psychrophiles

Cold-loving

mesophiles

• Moderate (20–45°C)

• Human pathogens grow best at 37°C.

thermophiles

Hot

hyperthermophiles

Extremely hot

temperature-dependent pigment production

changes with temperature because enzyme activity changes

pH

Scale:

• 0–14

Each pH unit = 10× difference.

Example:

• pH 4 is 10× more acidic than pH 5.

• pH 4 is 100× more acidic than pH 6.

halophile

Requires high salt.

High salt inhibits many bacteria by causing plasmolysis.

halotolerant

Can tolerate salt.

plasmolysis

Water leaves cell → growth stops.

MSA

Selective:

• 7.5% NaCl

Differential:

• Mannitol + phenol red

Yellow:

• Mannitol fermented → acid produced.

Pink/red:

• No fermentation.

obligate aerobe

Top only.

obligate anaerobe

Bottom only.

facultative anaerobe

Everywhere, mostly top.

aerotolerant anaerobe

Even throughout.

microaerophile

Thin band just below surface.

Resazurin

Pink = oxygen present.

Top is pink because oxygen diffuses from air.

Anaerobe Jar

Removes oxygen chemically.

Needed for obligate anaerobes because oxygen kills them.

salt tolerance media

MSA

mannitol fermentation media

MSA color

oxygen requirements media

Fluid thioglycollate medium (FTM)

Kirby-Bauer Test

Purpose:

• Determine bacterial susceptibility to antibiotics.

Provides:

• Susceptible (S)

• Intermediate (I)

• Resistant (R)

In vitro:

In Vitro vs. In Vivo

Lab results may differ because of:

• Immune system

• Drug absorption

• Infection location

• Biofilms

• Drug metabolism

Standardization

• Mueller-Hinton agar depth

• Inoculum density

• Incubation time

• Temperature

• Disk potency

• Ensures accurate, comparable results.

Mueller-Hinton agar depth Standardization

standardized (4 mm) to ensure antibiotics diffuse at a consistent rate

inoculum density Standardization

standardized to ensure every test begins with the same bacterial concentration

incubation time Standardization

so bacteria have enough time to grow and antibiotics have enough time to diffuse consistently through the agar

incubation temperature Standardization

standardized to ensure consistent bacterial growth and antibiotic diffusion

disk potency Standardization

exact amount of antibiotic contained in each antibiotic disk used in the Kirby-Bauer test

Zone of Inhibition

Measure:

• Diameter across the entire clear zone (mm).

Larger zone does not always mean susceptible because interpretation depends on CLSI breakpoints.

Breakpoints

Use CLSI chart:

• S = Susceptible

• I = Intermediate

• R = Resistant

Susceptible

microorganism cannot grow and thrive in the presence of a specific antimicrobial drug

Intermediate

classifies a microorganism's response to a drug as uncertain

Resistant

the ability of a microorganism to survive and grow in the presence of an antimicrobial drug that would normally kill or inhibit them

MIC

Definition:

• Lowest antibiotic concentration preventing visible bacterial growth.

Determined from broth microdilution by finding the first clear well.

Lower MIC = More effective antibiotic.

No antibiotic control

• Confirms bacteria can grow.

Expected:

• Turbid.

If clear:

• Experiment failed.

No bacteria control

• Confirms broth is sterile.

Expected:

• Clear.

If cloudy:

• Contamination.

Acidophiles

Acid

Neutrophiles

Around pH 7

Alkaliphiles

Basic

In vitro

Performed in the laboratory

Kirby-Bauer

Qualitative
Measures zone diameter
Faster and cheaper
Reports S/I/R