Lecture #4 Microbial Growth

What is Binary fission?

Binary fission is a type of growth (acts like a photocopier)

Binary fission: cell division following enlargement of a cell to twice its minimum size.

• asexual

• Minimal mutation

• Reproduces species

Growth: an increase in the number of cells

What is generation time?

Generation time (g): The time required for microbial cells to double in number. The generation time in bacteria is typically shorter than the generation times in eukaryotic microbes.

In Eukaryotic cell division… each daughter cells receives a chromosome and enough copies of all other cell constituents to exist as an independent cell.

• growth, replication of DNA and separation via mitosis are separated into interphase and mitosis

In prokaryotic cell division… growth in cell size, chromosome replication and even septum formation typically occur simultaneously

• mitosis does NOT occur

g= 1/k

What is generation time dependent on

The time varies based on whether the species growing is prokaryotic or eukaryotic, and is dependent on growth medium and incubation conditions

Ex: carbon source, pH, temperature

What is exponential growth?

Exponential growth: Growth of a microbial population in which cell numbers double at a constant and specific time interval

A relationship exists between the initial number of cells present in a

culture and the number present after a period of exponential growth:

N t = No x 2n

• Nt is the final cell number

• N0 is the initial cell number

• n is the number of generations during the period of exponential growth

the increase in cell number is initially slow but increases at an ever faster rate following an exponential curve

• Only when plotting on a log scale can one appreciate that the cells are doubling at a constant rate

Growth rate

Exponential growth: When growth is unlimited. It generates a curve whose slope increases continuously

Growth rate (k): is the rate of increase in population number or biomass. For each organism there is a specific growth rate that is the fastest growth rate in the best growth medium at optimal temperature

• Since bacteria and archaea grow by binary fission, the growth rate is

expressed as the number of doublings per hour

• The specific growth rate (k) can be calculated using the formula: Where:

k = (Log Nt – Log N0) / 0.301 delta t

Examples of growth rate

Clostridium perfringens: can double in numbers every 10 minutes under optimal growth conditions (e.g. nice warm stew on a warming plate)

Esherichia coli: less than 30 min in a rich medium

Mycobacterium tuberculosis: cannot grow faster than one doubling every 24 h

Growth curve patterns

Can study a growth curve by observing a batch culture. A typical growth curve for population of cells grown in a closed system is characterized by 4 phases.

Batch culture: a closed cycle microbial culture of fixed volume

1. Lag phase: Interval between inoculation of a culture and beginning of growth

• nutrient rich, waste poor

• essentially familiarizing, looking around to see what food it got and preparing to build enzymes

2. Exponential phase: cells int his phase are typically in the healthiest state

3. Stationary phase: cells are metabolic alt active but the growth rate of the population is zero (either an essential nutrient is used up or waste product accumulates in the medium)

• nutrient poor waste rich

Death phase: If incubation continues after cells reach stationary phase, the cells will eventually die

• Not all bacteria die, some bacteria form spores/cysts or dormant stages that allow a significant proportion of cells to survive for a long time

What is a continuous culture?

Continuous culture: an open-system microbial culture of fixed volume, supports indefinite growth (essentially comes back everyday to clean)

Chemostat: Most common type of continuous culture device. Allows for both growth rate and population density of culture to be controlled independently and simultaneously

• Concentration of a limiting nutrient controls the population size and the growth rate

Dilution rate: rate at which fresh medium is pumped in and spent medium is pumped out

What is a microbial count

Microbial count: Microbial cells can be enumerated by direct microscopic observations using a Petroff-Hausser counting chamber • Each square corresponds to a calibrated volume

• Results can be unreliable (exaggerated, can’t differentiate btwn sleeping and dead cells)

Limitations of microscopic counts

• Cannot distinguish between live and dead cells without special stains

• Small cells can be overlooked

• Precision is difficult to achieve (need a lot of counts)

• Phase-contrast microscope required if a stain is not used

• Cell suspensions of low density (<106 cells/ml) hard to count

• Motile cells need to immobilized

• Debris in sample can be mistaken for cells

• Cells may move (Brownian motion), some form clumps Based on random distribution and dispersal of the cells

What is Flow cytometry?

Flow Cytometry: is an alternative method that can be used to count the total number of cells

• Uses laser beams, fluorescent dyes, and electronics

What is a viable cell count and what are the methods used to obtain it?

Viable cell counts: Measure only living cells (Cells capable of growing to form a population)

• Two main ways to perform plate counts

1. Spread-plate method

2. Pour-plate method

Issues with viable cell counts

• Requires lots of preparation (dilution tubes, agar plates), and incubation time (overnight or more) to get the measurements for a single culture

• Plate counts can be highly unreliable when used to assess total cell numbers of natural samples (Ex: soil and water)

• Selective culture media and growth conditions target only particular species

• A single medium will never grow every microbe

• Can only count the types of bacteria that can grow in the medium you selected to use

What could be responsible for the great plate anomaly?

The great plate anomaly: Direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates

• anomaly= inconsistency

Modern genomic techniques suggest that only 1-10% of microbial diversity is culturable from most environmental samples (including the diversity of organisms in our own microbiomes)

• Microscopic methods count dead cells, whereas viable methods do not

• Different organisms may have vastly different requirements for growth

• We do not know the specific requirements for all organisms

Spectrophotometric Counts

Spectrophotometry measures turbidity (the cloudiness of a bacterial culture), to estimate cell concentration indirectly and rapidly. The measurement, known as optical density (OD), relies on how bacterial cells absorb and scatter light: as cell numbers increase, light transmission decreases and absorbance increases.

While this method is quick, easy, and non-destructive, it cannot distinguish living from dead cells. To relate turbidity readings to actual cell numbers, a standard curve must be established using another counting method (e.g., viable cell counts or dry weight measurements).

Problems with spectrophotometric counts: optical density had a finite linear range of measurement and only works if the cells are evenly distributed throughout the medium, the cuvette must also not have scratches and may need to be diluted when cells are at high density

What are the other counting techniques

Total mass of cells (dry cell weight): a specific aliquot (volume) cells are concentrated, washed to remove media components, concentrated and dried.

• There are other spectrometric techniques to measure specific components of the cell: protein, DNA etc. which are proportional to

the whole mass of cells