Bacterial Growth, Culture, and Cell Count

Essential Nutrients

Those that must be supplied

Macronutrients

Required in large quantities (C, H, O, N, P, S)

Micronutrients

Required in small quantities

• Trace elements necessary for enzyme function (Co, Cu, Mn, Zn, Mo, Ni)

Carbon in Life

• Heterotrophs: use preformed organic molecules. (release CO2)

• Autotrophs: fix CO2 and assemble into organic molecules (mainly sugars)

  • Phototrophy: Uses light energy

  • Lithotrophy: energy from oxidation of minerals

All the Trophs

Carbon Source for Biomass

• Auto, Hetero

Energy Source:

• Photo, Chemo

Electron Source:

• Litho, Organo

Chemolithoautotroph: Produces energy from oxidizing inorganic molecules (Fe, S, N). Energy is used to fix CO2.

The Nitrogen Cycle

Facilitate Nitrogen into our ecosystem (Some organisms are unable to use atmospheric nitrogen due to the triple bond between the two N atoms).

1. Nitrogen Fixing Bacteria: Covert Nitrogen gas to NH4+

2. Nitrification: Nitrifies oxidize NH4+ and create NO3- (Nitrate) ions

3. Denitrification: Organisms use nitrate and other inorganic forms of N as terminal electron acceptor in ETC — reduce nitrate to N2

Root Nodules In Nitrogen Cycle

• Light Hemoglobin appears red

• Promote plants output and growth

• Rhizobia secrete Nod factors that bind to receptors in membranes of host plant root hair cells.

The Growth Cycle

• Most bacteria divides by binary fission: where one parent spilts into two equal daughter cells

• Some divide asymmetrically by budding

• All must replicate and segregate the genome prior to splitting into 2 distinct cells.

Three Phases of Bacterial Cell Cycle

1. Period of growth after the cell is born

2. Chromosome replication and partitioning

3. Cytokinesis

Exponential Growth

• The growth rate, or rate of increase in cell numbers or biomass, is proportional to the population size at given time.

• It is called “exponential” because it generates an exponential curve. Never lasts indefinitely.

• If a cell divides by binary fission, the number of cells is proportional to 2n (n=number of generations).

Batch Culture

A liquid medium within a closed system.

• Bacterial growth curves can be observed when microorganisms are cultivating in batch culture.

• Has 5 distinct phases: Lag Phase, Exponential Phase, Stationary Phase, Death Phase, Long-term Stationary Phase

Lag Phase

Cell synthesizing new components necessary for reproduction.

• Varies in length dependent upon the organism

• Some cases, can be very short or even absent

Exponential Phase

• Also called Log Phase

• Has more living than dead cells

• Rate of growth and division is constant and maximal

• Population is most uniform in terms of chemical and physical properties during this phase

• Stops because of resources. Nutrients is down.

Stationary Phase

• Closed system population growth eventually ceases, total number of viable cells remains constant

• Active cells stop reproducing and is balanced with death rate

• Remain metabolically active

• Possible reasons for stationary phase: Nutrient limitation, limited oxygen availability, toxic waste accumulation, critical population density reached

Death Phase

• Cells are dying at a constant rate

• More dead cells than living cells

• Nutrient deprivation and buildup of toxic waste

Continuous Culture

All cells in a population achieve a steady state, which allows detailed study of bacterial physiology.

Chemostat

Ensures logarithmic growth by constantly adding and removing equal amounts of culture media. (Ex: GI Tract)

Turbidostat

• Regulates flow rate of media through vessel to maintain predetermined turbidity or cell density.

• Dilution rate varies

• Contains all nutrients in excess

• Operates best at high dilution rates

Generation Time

The time it takes for a population to double.

G=t/n

• G= Generation Time

• T= Time interval in hours or minutes

• N= Initial number of cells

Binary Fission Generation Time

Nt= No TIMES 2n

• Nt= the final cell number

• No= the original cell number

• n= number of generations

• 2 bc of the division of daughter cells

Culture

Pure Culture= In lab. Only 0.1%.

Culture Media

• Liquid or Broth: Useful for studying the growth characteristics of a pure culture

• Solid (gelled with Agar): Useful to separate mixed cultures

Types of Media

Complex Media: Nutrient rich but poorly defined

Synthetic Media: Precisely defined

Enriched Media: Complex media to which specific blood components are added

• Fastidious- requiring blood

Selective Media: Favor the growth of one organism over another

Differential Media: Exploit differences between two species that grow equally well.

MacConkey Media: Inhibits some organism but always will show differences in organism (with lactose) based upon different capabilities of the organisms. Red= Acid that is a by product from sugar

Isolating Pure Colonies Techniques

1. Dilution Streaking: Dragging a loop across the surface of an agar plate. Flame the Loop!!

2. Spread Plate: Serial dilution are performed on a liquid culture. A small amount of each dilution is then plated.

Measurements of Microbial Growth

• Can measure changes in number of cells in a population

• Can measure changes in mass of population

Direct Measurements of Cell Numbers

• Counting chambers

• On membrane filter

• Flow Cytometry: computer counts cells.

• Electronic Counters- the Coulter counter

Counting Chambers

• Squaring off areas to make it easier to count

• Useful for counting both eukaryotes and prokaryotes

• Gives information about the size and morphology of microorganisms

• Cannot distinguish living from dead cells

Membrane Filters

• Cells filtered through special membrane that provides dark background observing cells.

• Cells are stained with fluorescent dyes

• Useful for counting bacteria

• With certain dyes, can distinguish living from dead cells.

Viable Cell Count

Count replication colony forming cells.

• Pour Plate Method: Uses malted/melted agar

  • Dilutions are performed on a liquid culture

  • A small amount of each dilution is then plated

  • A plate with 30-300 colony forming units (CFU) is counted

Optical Density (Spectrophotometer)

• The fastest way to measure cell density

• Measures everything in broth (live and dead)