Oxygen Requirements & Enumeration Techniques (8/20/2026)

Oxidative stress

an excess of unstable molecules such as peroxides and free radicals which damage cells

Peroxides

act as oxidizing agents, which can strip electrons off the molecules

Free radicals

• attacks double bonds and causes permanent DNA damage

• Highly Reactive

• have unpair electrons

Catalase

An enzyme that breaks down H2O2 (hydrogen peroxide; usually used to counteract products of cellular respiration

Superoxide Dismutase

enzyme that breaks down superoxide radial into o2 and h2o2

Obligate Aerobes

Only aerobic growth; oxygen required

Facultative Anaerobes

Both aerobic and anaerobic growth; however increase growth with oxygen

Obligate Anaerobes

only anaerobic growth; dies in the presence of oxygen

Aerotolerant anaerobes

only anaerobic growth, but continues in the presence of o2.

Microaerophiles

Only aerobic growth, but oxygen required in low concentration

How does the fluid thioglycolate medium differentiate?

Oxygen gradient (differentiates aerotolerance).

Active reducing agent in FTM

Sodium Thoglycollate (lowers oxidation / reduction potential)

Indicator in FTM

Resazurin; turns red in presense of O2

Brewer’s Jar/ Gas Pak

These are airtight laboratory containers used to grow anaerobic bacteria

Gas Pak

A chemical packet called a sachet is placed inside the jar that releases hydrogen and carbon dioxide; O2 is turned into water and CO2 is building up.

Candle jar; why do capnophiles love it

high CO2 levels and lower oxygen levels created by burning of candle

(microaerophiles like)

capnophiles

CO2 loving

air exposure disk shows

• oxygen tolerance

  • obligate aerobe

  • facultative anaerobe

  • aerotolerant anaerobe

• growth outcome

  • if no growth = obligate anaerobe

Gas pak pouch shows

Whether an organism can grow in the absence of oxygen.

Why is fluid thiglycollate medium more efficient

oxygen gradient; allows all 5 category identification

What stress reducing enzymes would an obligate aerobe have?

Catalase + superoxide dismutase detoxify harmful reactive oxygen species produced during aerobic respiration.

What stress reducing enzymes would an obligate anaerobe have?

They lack these oxidative stress enzymes; they cannot neutralize toxic byproduct, which harms and kills the cell.

e. coli

facultative anaerobe

Clostridium sporogenes

obligate anaerobe

Alcaligenes Faecalis

Obligate Aerobe

Direct microscopic counts

using microscopes to count small sample area in a slide (cell/ml)

Turbidometric methods

uses light and the absorbance of light to estimate densities if cultures or to count particles (cell/ml)

Coulter Counter

Electronic counter that measures and counts particles

that pass through micro-channel.

Culture methods

Using serial dilution to create countable cultures that determine the concentration of an original sample

Enumeration

How many microbes are in a sample

Types of Direct Microscopic counts

• Cell count method

• Epifluorescence microscope- uses a special stain; cells can be counted by fluorescence.

• Coulter counter

Turbidometric

Spectrophotometer: measures turbidity by light.

standard plate count

Measures the number of live, growing bacteria in a sample

• the pour plate method

• the spread plate method

Why do serial dilution?

To reduce the concentration of a substance to a usable or measurable level.

Countable

30-300

Formula

OCD= CFU/FDF

(# of colonies on each plate)

(Final dilution factor)

examples of enumeration in the world

• food, agriculture, environmental laboratories

• can be used to test water for microbes

• Can be used to detect disease casing microbes in animals or humans

How can we use this data to enumerate an unknown sample concentration of the same sample

construct a standard curve/ calibration curve.