Microbial Growth Flashcards
Growth
measured as an increase in the number of cells
Binary Fission
cell division following
enlargement of a cell to twice its minimum size
Generation Time
- time required for microbial cells to double in number
- Most bacteria have shorter generation times than eukaryotic microbes
- Generation time is dependent on growth medium and incubation conditions:
carbon source, pH, temperature, etc
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
Equation
Nt = No X 2^n
Nt is the final cell number
No is the initial cell number
n is the number of generations during the period of exponential growth
Growth Rate (k)
k = (Log Nt – Log N0) / 0.301(delta t)
N0 = number of cells at time1
Nt = number of cells at time2
Delta t = time2 - time1
Generation TIme (g)
g = 1 / k
- hr/gen -> min/gen (multiply by 60)
Clostridium perfringens
can double in numbers every 10 minutes under
optimal growth conditions (e.g. nice warm stew on a warming plate)
Escherichia coli
less than 30 min in a rich medium
Mycobacterium tuberculosis
cannot grow faster than one doubling every 24 h
Batch Culture
A closed-system microbial culture of fixed volume.
What are the four phases characterized by population of cells grown in a closed system?
- Lag phase
- Exponential Phase
- Stationary Phase
- Death Phase
Lag phase
Interval between inoculation of a culture and beginning of growth
Exponential Phase
Cells in this phase are typically in the healthiest state
Stationary Phase
- Cells metabolically active, but growth rate of population is zero
- Either an essential nutrient is used up, or waste product of the organism accumulates in the medium
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
Continuous Culture
An open system microbial culture of fixed volume
What is a Chemostat?
- Most common type of continuous culture device
- Both growth rate and population density of culture can be controlled independently and simultaneously
- Dilution rate: rate at which fresh medium is pumped in and spent medium is pumped out
- Concentration of a limiting nutrient controls the population size and the growth rate
Microbial Counts
- 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
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
Flow Cytometry
- Is an alternative method that can be used to count the total number of cells
- Uses laser beams, fluorescent dyes, and electronics.
Viable Cell Counts
- Measure only living cells
- Cells capable of growing to form a population
What are the two main ways to perm plate counts?
- Spread-plate method
- Pour-plate method
Spread-plate method
- Sample is pipetted onto surface of agar plate (0.1 ml or less)
- Sample is spread evenly over surface of agar using sterile glass spreader
- Incubation
- typical spread-plate results (Surface Colonies)
Pour-plate method
- Sample is pipetted into sterile plate
- Sterile medium is added and mixed well with inoculum
- Solidification & Incubation
- Typical pour-plate results (Surface Colonies & Subsurface Colonies)
The great plate anomaly
- Direct microscopic counts of natural samples reveal far more organisms than
those recoverable on plates - 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)
Why the great plate anomaly?
- 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
- Turbidity measurements are indirect, rapid, and useful counting methods
- Most often turbidity is measured with a spectrophotometer, and measurement is referred to as optical density (OD)
- Based on the fact that bacteria do behave like small particles and absorb and scatter light
- Only a portion of the incident light makes it to the photocell because particles (including cells) scatter light. The larger the number of particles, the greater the absorbance, the lower the light transmission to the photocell
- Caution: absorbance does not distinguish dead cells from living cells
Spectrophotometric Counts (Part 2)
- Turbidity measurements
- Quick and easy to perform
- Typically do not require destruction or significant disturbance of sample (some
spectrophotometers are specifically designed to use growth tubes as cuvette)
- To relate a direct cell count to a turbidity value, a standard curve must first be established to another counting method
- Viable cell counts
- Weight of biomass produced
- Measured as dry cell weight corresponding to a specific volume of cell
culture
- Measured as dry cell weight corresponding to a specific volume of cell
Spectrophotometric Counts (Part 3)
- Problems with optical density
- Has a finite linear range of measurement
- Only works if the cells are evenly distributed throughout the medium (no
clumps or biofilms) - Cuvette must not have scratches
- Culture may need to be diluted when the cells are at very high density
Other Counting Techniques
- Total mass of cells (dry cell weight)
- There are other spectrometric techniques to measure specific components of the cell
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
