Microbial Growth and Measurement Methods

Turbidity

Cloudiness in broth caused by bacterial cells scattering light.

Transmittance vs. Absorbance (OD)

Transmittance measures light passing through; Absorbance (OD) measures light blocked/scattered.

Can optical density (OD) distinguish living from dead cells?

No, because intact dead cells still scatter light and contribute to turbidity.

Membrane Filtration Method

Traps bacteria on a filter with tiny pores, then incubates filter on medium to count CFUs.

Colony-Forming Unit (CFU)

A single cell or group of cells that multiplies into one visible colony.

Countable range for viable plate counts

30 to 300 colonies per plate (or 25 to 300).

Why choose a plate with 30-300 colonies?

Too many colonies overlap; too few provide an unreliable statistical estimate.

Direct Microscopic Cell Count

Counts cells directly on a grid (hemocytometer/Petroff-Hausser chamber) without incubation.

Direct Methods of Measuring Microbial Growth

Microscopic count, plate count, membrane filtration, Coulter counter, flow cytometry, MPN.

Indirect Methods of Measuring Microbial Growth

Turbidity, metabolic activity, and dry weight.

Bacteriuria

The presence of bacteria in urine.

Closed System (Batch Culture)

A culture system where fresh nutrients are not added and wastes are not removed.

Lag Phase

Cells adjust to environment, make enzymes, and repair components without increasing in number.

Log (Exponential) Phase

Cells divide at a constant, maximum rate; population doubles at regular intervals.

When does sporulation begin in endospore formers?

Late log phase, in response to nutrient limitation.

Stationary Phase

Rate of cell division equals rate of cell death; living population stays constant.

Death (Decline) Phase

Dying cells outnumber new cells due to toxic waste build-up and nutrient depletion.

Binary Fission

Asexual reproduction where one bacterial cell divides into two identical daughter cells.

Septum in Binary Fission

The dividing wall that forms between two daughter cells before separation.

Generation Time

The time required for a cell to divide or a population to double.

Formula variables: N₀, n, Nₜ

N₀ = initial cells, n = number of generations (doublings), Nₜ = final cells (Nₜ = N₀ × 2ⁿ).

Primary goal of culture preservation methods

Slow down cell metabolism to prevent nutrient exhaustion and waste build-up.

Lyophilization

Freeze-drying culture under an intense vacuum for long-term storage.

Deep-Freezing (-80°C)

Preserves cells frozen in liquid media containing glycerol as a cryoprotectant.

Obligate Aerobe

Requires oxygen to grow.

Obligate Anaerobe

Cannot grow in the presence of oxygen; oxygen is toxic.

Facultative Anaerobe

Grows with or without oxygen; grows better with oxygen.

GasPak Anaerobic Jar

Enclosed vessel that uses chemical sachets to create an oxygen-free environment.

Thioglycolate Medium

A reducing medium that chemically removes dissolved oxygen from the broth.

Resazurin

Oxidations-reduction indicator: pink in presence of oxygen, colorless without oxygen.

Stab Culture

Inoculation deep into solid agar to protect cells from surface oxygen exposure.

Enriched Media

Contains extra nutrients or growth factors to support fastidious organisms.

Selective Media

Inhibits growth of unwanted microbes while permitting desired ones to grow.

Differential Media

Distinguishes between different microbial types based on metabolic reactions.

Mannitol Salt Agar (MSA)

Selective (7.5% salt) and differential (mannitol fermentation; yellow = positive).

Staphylococcus aureus vs. S. epidermidis on MSA

S. aureus grows yellow (ferments mannitol); S. epidermidis grows red (does not ferment mannitol).

Chocolate Agar

Enriched, non-selective medium supplying growth factors (heme, NAD) for fastidious species.

Why can't Chlamydia and Rickettsia grow on nutrient agar?

They are obligate intracellular parasites that require living host cells to reproduce.

Unculturable in vitro model organisms

Mycobacterium leprae (grown in armadillos) and Treponema pallidum (grown in rabbits).