20-Feb-18

Chapter 6
HOW CELLS GROW

Amit Jain, Ph.D.

Asst. Professor,
Dept. of Chemical Engg.,
BITS Pilani – Pilani Campus

Presentation Outline
• Introduction
• Batch Growth:
– Quantifying Cell Concentration
• Determining Cell Number Density
• Determining Cell Mass Concentration

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Introduction
• Microbes response to physiochemical
environment:
– Biosynthesis and product formation (secondary
response)
– Growth (primary response)
• Replication
• Cell size
substrate + cells  extracellular products + more
cells

• Autocatalytic: the rate of growth is directly related to

cell concentration.

Introduction
• The rate of microbial growth is characterized by
the net specific growth rate,

• Replacing ‘X’ with ‘N’ gives rate in terms of cell

number concentration and µ would be µR.

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Batch Growth
• Initial charge of medium in a vessel.
Quantifying Cell Concentration:
• Essential for determining Kinetics and
Stoichiometry of microbial growth.
• Methods of quantification:
– Direct
• Infeasible due to suspended solids and other interfering
compounds.
– Indirect
• Cell mass concentration
• Cell number density / concentration.

Determining Cell Concentration

1. Cell Number Concentration
a) Hemocytometer or Petroff-Hausser Slide
- a grid based method
b) Petri Dish
- Viable (capable of reproduction) cell counts expressed in terms of
CFU.
c) Electronic Particle Counter
- the no. of pulses is measure of no. of particles.
- height of pulse is a measure of cell size.
d) Nephelometry
- measures the scattered light intensity using phototube.

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Hemocytometer

Cell Staining Method

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Cell Staining Reagents

Determining Cell Concentration

1. Cell Number Concentration
a) Hemocytometer or Petroff-Hausser Slide
- a grid based method

b) Petri Dish
- Viable (capable of reproduction) cell counts
expressed in terms of CFU.
c) Electronic Particle Counter
- the no. of pulses is measure
of no. of particles.
- height of pulse is a measure
of cell size.
d) Nephelometry
- measures the scattered
light intensity using phototube.

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Petri Dish: Streak Plate Method

Petri Dish: Streak Plate Method

https://microbeonline.com/streak-plate-method-principle-purpose-procedure-results/

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Determining Cell Concentration

1. Cell Number Concentration
a) Hemocytometer or Petroff-Hausser Slide
- a grid based method
b) Petri Dish
- Viable (capable of reproduction) cell counts expressed in terms of
CFU.
c) Electronic Particle Counter
- the no. of pulses is measure of no. of
particles.
- height of pulse is a measure of cell size.
d) Nephelometry
- measures the scattered light intensity using phototube.
- the intensity of scattered light is proportional to cell concentration.

Electronic Particle Counter

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Determining Cell Concentration

1. Cell Number Concentration
a) Hemocytometer or Petroff-Hausser Slide
- a grid based method
b) Petri Dish
- Viable (capable of reproduction) cell counts expressed in terms of
CFU.
c) Electronic Particle Counter
- the no. of pulses is measure of no. of particles.
- height of pulse is a measure of cell size.
d) Nephelometry
- measures the scattered light intensity using
phototube.
- the intensity of scattered light is proportional to cell
concentration.

Determining Cell Concentration

(cont.)
2. Cell mass concentration
a) direct methods
→ dry weight (filtration or centrifugation)
– Samples of culture broth are centrifuged or
filtered and washed with a buffer solution or
water.
– The washed wet cell mass is then dried at
80oC for 24 hrs.
– Dry cell weight is then measured.

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Determining Cell Concentration

(cont.)
→ packed cell volume (centrifugation)
→ optical density (light scattering, 600-700 nm)
– Use a wavelength that minimizes absorption
by medium components,
– “blanking ” against medium,
– Use of calibration curve ( to relate the
absorbance with cell dry weight)

Determining Cell Concentration (cont.)

• The ATP content of a typical bacterial cell is 1 mg ATP/g

dry-weight cell, approx. , measured by photometers or
scintillation counter.

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Thank You!

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