MICB 211: LECTURE 6: MEASUREMENT OF BACTERIAL GROWTH

 Can be done in two ways:

i) Measuring changes in cell mass/turbidity
ii) Measuring changes in cell numbers/cell counts.

A. Measurement of cell mass

 During growth, all cellular components increase in direct proportion to the
increase in cell numbers
 Thus, instead of measuring cell numbers, the quantity of cellular components can
be measured
 Methods of measurement can be direct and indirect.

Direct methods
1. Physical measurements
Involve measurement of:-
 Dry weight
o Cells growing in a liquid medium are collected by
centrifugation, washed, dried in the oven, and weighed
 Wet weight
o Cells are collected by centrifugation and weighed without
drying
 Not accurate/not sensitive
o Bacteria weigh so little and it may be necessary to centrifuge
several hundreds of cultures to collect sufficient quantities that
can be weighed.
 Time-consuming.

2. Chemical measurements
Involve the measurement of chemical components of cells such as:-

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 o Total N
o Total protein
o Total DNA
o Chlorophyll -to measure cyanobacterial populations
o Quantity of ATP –to estimate the amount of living microbial mass
An increase in bacterial population is reflected by an increase in the concentration of
cellular components

Indirect methods
1. Measurement of chemical/metabolic activities
 The metabolic activities of living cells can be
o Anabolic - synthesis
o Catabolic – breakdown of substances
 During growth, various metabolic activities occur
 oxygen consumption,
 acid and gas production,
 nutrient utilization,
 change in pH
 waste production and accumulation
 Activities such as
o Rate of O2 production or consumption
o Rate of CO2 production or consumption
o Rate of acid production

2. Optical methods/turbidity measurements

 Employs the spectrophotometer to determine the amount of light
scattered by a suspension of cells.
o The turbidity or optical density of a suspension of cells is directly
related to cell mass or cell numbers.

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  The more the cells present in the suspension, the more the
light that is blocked from passing through the suspension
and the greater the turbidity
 The less the amount of light that is scattered by the
suspension
 Turbidity readings can therefore be used as a substitute for total or viable
counting methods

 Advantages
o They are quick and easy to perform
o Can be done without destroying or disturbing cultures of
microorganisms
 Drawbacks
o A standard curve must first be prepared that relates cell numbers,
dry weight or protein content to turbidity.
o Different organisms differ in size and shape and equal cell numbers
of two organisms may yield different turbidity measurements
o Some bacteria do not grow uniformly
 Can grow clumped to one another
 Form films on sides of growth tubes
o In such instances, optical density may be
inaccurate as a measure of total cell mass
o The cultures can be shaken and stirred to mix
cells well and prevent the formation of
aggregates/clumps

B. Measurement of cell numbers

Direct methods
 Are used to count bacterial numbers without culturing them
 Are easy to perform and the size and morphology of cells can be studied

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1. Microscopic counts
 Microscopes to enumerate the numbers of cells present in a culture or
sample
 Microscopic counts can be done on:
o Samples dried/fixed on slides
 For dried samples, staining is usually done to increase the
contrast between cells and their backgrounds
o Samples in liquid form
 Specially designed counting chambers or microscope slides
with grids/squares of known areas are used
 The special microscope slides are called cytometers
 The number of cells per unit area of the grids can be counted
under the microscope
 The number of cells per ml of sample/suspension can then
be determined
 Disadvantages
o Is difficult to use when cells are motile.
o Dead cells cannot be distinguished from living ones.
o Only dense suspensions can be counted
 Samples can be concentrated by centrifugation or filtration
to increase sensitivity.
o Precision is difficult to achieve/unreliable
 Some cells can easily be missed
 Debris in the sample can easily be mistaken for cells
 Advantages
o Sensitivity is increased by staining cells.
o Is a simple, quick, and easy way of estimating microbial cell
numbers

2. Electronic counting chambers/coulter counters

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  Are automatic counters that electronically scan a culture for cells
 Can also measure the size distribution of cells
 Commonly used to count, sort, size, and identify cells in suspension
 Mostly used in hospitals
 to count and identify blood cells and cancer cells.
o Advantage:
 It is quick
 the size of cells can be recorded.
o Disadvantage:
 it is expensive
 dead cells and other particles may be counted.

Indirect methods
1. Viable cell counts/cultural methods/plate counts
 Only viable cells are counted
 A viable cell is a cell that is living and can divide to form an
offspring
 It is assumed that each viable cell can grow and divide to yield a
visible colony
 Thus colony numbers are used to reflect cell numbers

 Determination of viable cell counts:

o Determination of the number of cells in a sample that can grow and
form colonies on a suitable agar medium is done
o Involve plating out (spreading) a sample of a culture on a nutrient
agar surface.
o Each viable cell grows and forms a colony that can be counted.
o The number of colonies is reported as CFUs
o The number of CFUs is related to the viable number of bacteria in
the sample.

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  Disadvantages
o If the concentration of bacteria is high, the colonies will grow
clumped into each other and the plates will be uncountable.
 The sample or cell suspension can be diluted before plating.
o Colonies develop only from those organisms which can grow in the
culture
 Some organisms are viable but un-culturable (VBNC)
o If samples are dilute, plates prepared with the samples will too few
colonies to be counted.
 The concentration of bacteria can be increased by
filtering the sample before plating.
 Advantages:
o Best determines the number of viable cells in a sample
o Its sensitivity (only living cells develop colonies)
 As few as one viable cell per sample plated out can be
detected
 It allows for studies on colonies of the organism counted.

2. Membrane filtration technique.

 The bacteria are retained on the surface of a membrane filter
and then transferred to a culture medium to grow and
subsequently be counted.
 Useful for samples with low bacterial concentrations.
o Advantage
 Only viable cells are counted.

o Disadvantage
 time-consuming

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  not all bacteria are culturable

3. The most probable number (MPN) method

 It is a statistical estimation of the number of cells.
 Can be used for microbes that grow in a liquid medium
 A dilution series is prepared and plated as in the other methods
 The number of cells per plate is counted
 The combination of numbers of cells per plate is used to get the MPN of cells in
the initial sample by referring to the MPN table.
 Used for microbes that won't grow on solid media

Reading assignment: Understand the process/steps involved in the MPN method

 Advantage:
o Growth is in liquid media so other characteristics of bacteria can be
studied
o Can be used even for turbid samples as opposed to membrane filtration
where membranes can clog
 Disadvantage
o A statistical estimation – not accurate
o Process is long and tedious

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Assignment: Some Methods used to measure bacterial growth

Method Application Comments

Enumeration of bacteria in milk or Cannot distinguish living from nonliving

Direct microscopic count
cellular vaccines cells

Enumeration of bacteria in milk, foods, Very sensitive if plating conditions are

Viable cell count (colony counts)
soil, water, laboratory cultures, etc. optimal

Estimations of large numbers of

Fast and nondestructive, but cannot detect
Turbidity measurement bacteria in clear liquid media and
cell densities less than 107 cells per ml
broths

Measurement of total cell yield from only practical application is in the research
Measurement of total N or protein
very dense cultures laboratory

Measurement of Biochemical activity e.g. Requires a fixed standard to relate

O2 uptake CO2 production, ATP Microbiological assays chemical activity to cell mass and/or cell
production, etc. numbers

Measurement of dry weight or wet weight of Measurement of total cell yield in probably more sensitive than total N or
cells or volume of cells after centrifugation cultures total protein measurements