The Growth of Bacterial Populations (page 2)
(This chapter has 4 pages)
© Kenneth Todar, PhD
Methods for Measurement of Cell Numbers
Measuring techniques involve direct counts, visually or
instrumentally,
and indirect viable cell counts.
1. Direct microscopic counts are possible using special
slides
known as counting chambers. Dead cells cannot be distinguished from
living
ones. Only dense suspensions can be counted (>107
cells
per ml), but samples can be concentrated by centrifugation or
filtration
to increase sensitivity.
A variation of the direct microscopic count has been used to observe
and measure growth of bacteria in natural environments. In order to
detect
and prove that thermophilic bacteria were growing in boiling hot
springs,
T.D. Brock immersed microscope slides in the springs and withdrew them
periodically for microscopic observation. The bacteria in the boiling
water
attached to the glass slides naturally and grew as microcolonies on the
surface.
2. Electronic counting chambers count numbers and measure
size
distribution of cells. For cells the size of bacteria the suspending
medium
must be very clean. Such electronic devices are more often used to
count
eucaryotic cells such as blood cells.
3. Indirect viable cell counts, also called plate counts,
involve plating out (spreading) a sample of a culture on a nutrient
agar
surface. The sample or cell suspension can be diluted in a nontoxic
diluent
(e.g. water or saline) before plating. If plated on a suitable
medium,
each viable unit grows and forms a colony. Each colony that can be
counted
is called a colony forming unit (cfu) and the number of cfu's
is
related to the viable number of bacteria in the sample.
Advantages of the technique are its sensitivity (theoretically, a
single
cell can be detected), and it allows for inspection and positive
identification
of the organism counted. Disadvantages are (1) only living cells
develop
colonies that are counted; (2) clumps or chains of cells develop into a
single colony; (3) colonies develop only from those organisms for which
the cultural conditions are suitable for growth. The latter makes the
technique
virtually useless to characterize or count the total number
of
bacteria in complex microbial ecosystems such as soil or the animal
rumen or gastrointestinal tract. Genetic probes can be used to
demonstrate
the diversity and relative abundance of procaryotes in such an
environment,
but many species identified by genetic techniques have so far proven
unculturable.
Table 1. Some Methods used
to
measure bacterial growth
Method |
Application |
Comments |
Direct microscopic count |
Enumeration of bacteria in milk or cellular vaccines |
Cannot distinguish living from nonliving cells |
Viable cell count (colony counts) |
Enumeration of bacteria in milk, foods, soil, water,
laboratory cultures,
etc. |
Very sensitive if plating conditions are optimal |
Turbidity measurement |
Estimations of large numbers of bacteria in clear liquid
media and
broths |
Fast and nondestructive, but cannot detect cell densities
less than
107 cells per ml |
Measurement of total N or protein |
Measurement of total cell yield from very dense cultures |
only practical application is in the research laboratory |
Measurement of Biochemical activity e.g. O2 uptake CO2
production,
ATP production, etc. |
Microbiological assays |
Requires a fixed standard to relate chemical activity to cell
mass
and/or cell numbers |
Measurement of dry weight or wet weight of cells or volume of
cells
after centrifugation |
Measurement of total cell yield in cultures |
probably more sensitive than total N or total protein
measurements |

Figure 2. Bacterial colonies
growing on a plate of nutrient agar. Hans Knoll Institute. Jena,
Germany.
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