Laboratory 2:

Bio 4905 Lab #2

  1. Bacterial Cell Growth (recapturing from your old Microbiology)

 

Growth is an orderly increase in the quantity of cellular components. In most bacteria, growth involves duplication of the bacterial chromosome, number of ribosomes , synthesis of new cell wall and plasma membrane,  and eventual partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission.
For unicellular organisms such as the bacteria, growth can be measured in terms of two different parameters: changes in cell mass and changes in cell numbers.

 

  1. Methods for Measurement of Cell Mass

 

    1. Methods for measurement of the cell mass involve both direct and indirect techniques.
      1. Direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation.
      2. Direct chemical measurement of some chemical component of the cells such as total N, total protein, or total DNA content.
      3. Indirect measurement of chemical activity such as rate of O2 production or consumption, CO2 production or consumption, etc.
    1. Turbidity measurements

Particulate objects such as bacteria scatter light in proportion to their numbers. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number. This method is simple and nondestructive. However, the sensitivity is limited to about 107 cells per ml for most bacteria, which requires proper dilution of cell suspension before measurements can be taken.

 

  1. The Bacterial Growth Curve and phase of cell growth

In the laboratory, under unlimited (unrestricted) conditions, the number of bacteria doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n (where n = the number of generations). This is called exponential growth. In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature where many growth restrict factors are present.

 

When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve

 

                 

    • Phase of Bacterial growth:     Four characteristic phases of the growth cycle are recognized.
      • Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass.

 

      • Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. During balanced growth, all components of cells are proportionally increased to the mass of cells. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population
      • Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space".

 

During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing.

      • Death Phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.).

Lab 2: Cell Culture and Measure Bacterial Growth Rate

    • Measure cell density from over night culture in LB medium (MC4100 in LB)

 

Dilute over night culture 10X with LB medium

  • 100ml (o/n culture) + 900ml LB medium

Measure o/n culture cell density with spectrometer at 600nm

  • Insert blank cuvette (1ml of LB medium) and press blank button on spectrometer
  • Insert test cuvette with sample and press sample

 

    • Cell culture in fresh LB medium (60ml)

Inoculate o/n culture to fresh LB medium (60ml in 250ml flask) to 0.05 OD600

  • Ex) o/n culture OD = 2.5,    then 2.5/0.05=50 

Need to dilute 50X to get 0.05 OD
60ml/50 = 1.2ml (noculate 1.2ml of o/n culture to fresh 60ml medium)

  • Calculate volume of o/n culture you need to start fresh culture (60ml LB)

o/n culture OD = ( _________ ) ,  then  __________    /0.05= ( ________ )
Need to dilute 50X to get 0.05 OD
60ml/ ( ________) =  ( _______ )ml

 

    • Measure cell growth: (work as 2 people/group)

Incubate inoculated culture at 37oC, 250 rpm shaker (start timer)

Measure cell density with spectrometer at 600nm

  • Measure every 10 min first 3 times

(you have to blank out with blank medium before measure your sample)

  • Then, measure every 30 minutes

*** when cell density reaches over 1.00 OD600, sample need to be diluted within 0.1 ~1.0 OD600

Ex) if OD600 without dilution is 1.2, need to dilute at least 2~3X with LB medium
       (if 2X dilution, 1 part sample and 1 part fresh LB medium)

Plot the cell growth on semi-log scale graph against time and cell density (OD600)
(x-axis time scale, y-axis cell density in logarithmic scale)

Predict cell growth on the semi-log scale plot

 

    •  Cell harvest and storage

 

  • Pipette out cell culture at OD 0.5,  1.0,  2.0,  3.0  for 3 OD equivalent volume

 Ex), OD= 0.5, 3/0.5= 6ml

  • Collect cells in 15ml Falcon-tube and centrifuge at 8,000rpm for 10 min
  • Discard supernatant
  • Add 1ml of 20mM Tris-HCl (pH 8.0) and vortex
  • Transfer to 1.6ml micro-centrifuge tube
  • Centrifuge at 10,000rpm for 5min
  • Discard supernatant and quick freeze in dry-ice to store at -80oC

 

    •  Growth rate calculation:

 

  • Based on the growth curve obtained on semi-log scale graph
  • Find an exponential phase of cell growth (liner portion of growth curve)
  • Find the cell doubling time (growth rate)
  • Generation time (G) is defined as the time (t) per generation (n = number of generations). Hence, G=t/n is the equation from which calculations of generation time derive.

 

 

 

 

 

 

 

 

 

 


This information is given as a guide to the student attending the Bio4905 laboratory as a means to review some of the information. It is not meant to replace the laboratory. No emphasis as to what will be required of the student is given in this text, indeed information that is given in the these transcripts may make little sense if the student has not first attended the relevant laboratory.

 


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