Bacterial count methods

Jolly R Parikh
Bacterial growth

Bacterial count is required to measure bacterial

growth
Growth= an increase in the number of
cells, not an increase in size
Bacterial growth occurs by binary fission
• Generation time=time it takes for a cell to
divide and the population to double; (E. coli:
every 20 min)
Enumeration or counting bacteria
■ Goal:
■ It is often important to know not only what types of bacteria
are in a sample but also how many of them are present.
■ Food manufacturers are required by the FDA to monitor the
number and type of bacteria in their products.
■ Dairies monitor the number of bacteria present in milk after
pasteurization.
■ Water treatment plants monitor the effectiveness of their
sterilization process.
■ Biotechnology firms closely regulate bacterial growth as
they manipulate these organisms to produce useful
pharmaceutical products.
■ Beer and wine companies monitor the growth of yeast in
their distilling process.
■ Clinical laboratories monitor the growth rate of bacteria from
patients to determine their antimicrobial sensitivity.
Enumeration of Bacteria
■ Types of bacterial count
■ Viable: Count of living cells only
■ Total: Count of Living and dead cells
Viable Total

■ When you are concerned ■ When you need to know a

about only living and number of all organisms
metabolically active ■ EX) Microbial Ecology
organisms
■ EX) CLINICAL SAMPLES
Bacterial count methods

■ Both total count and viable count can be

determined by
■ Direct and
■ Indirect methods
■ Direct Count: It involves counting of bacteria
by direct visualization or by instrumentation ,
e.g. Staining and counting under microscope,
■ Indirect count: It estimates bacterial count
based on some indirect property
■ Spectroscopy: Estimate based on turbidity (or
transmittance of light)
■ Measuring metabolic activity of the bacteria
Direct Count Indirect Count

■ Advantage ■ Advantage
■ More accurate ■ Quicker to do
■ Disadvantage ■ Disadvantage
■ More time
■ Less accurate
■ Uses
■ Uses
■ To get accurate counts of
cells in clinical or ■ To get quick and dirty count
environmental samples in controlled circumstances
DIRECT COUNT METHODS

1. Plate method: Pour plate and spread plate

method
2. Membrane Filtration method
3. MPN estimation (MPN - most probable
number)
4. Direct counts on grid of slide using
microscopy
5. Electronic count method
6. Direct epifluorescence method [DEFT]
INDIRECT COUNT METHODS
1. Turbidimetric method
2. Measurement of metabolic activity of cells
Dry weight of cell mass
Direct chemical measurement of some cell
component such as total nitrogen, total protein, total
DNA content
3. Indirect measurement of chemical activity such as
rate of oxygen/ carbon dioxide
consumption/production
4. Micro calorimetry i.e. production of heat
5. Electrical conductivity
6. Bioluminescence
Viable count methods
■ Plate count methods
■ Membrane filtration method
■ DEFT
■ MPN
■ Bioluminescence
■ Estimation of metabolic activity due to
bacterial growth
■ Micro calorimetry
Total count methods
■ Microscopy
■ Electronic counting method
■ DEFT
■ Cell mass – dry weight or wet weight
■ Turbidity
Methods of bacterial growth and
their applications
Method Application Comments
Enumeration of Cannot distinguish
Direct microscopic
bacteria in milk or living from nonliving
count
cellular vaccines cells
Enumeration of
bacteria in milk, Very sensitive if
Viable cell count
foods, soil, water, plating conditions are
(colony counts)
laboratory cultures, optimal
etc.
Fast and non
Estimations of large
destructive, but
Turbidity numbers of bacteria
cannot detect cell
measurement in clear liquid media
densities less than
and broths
107 cells per ml
Methods of bacterial growth and
their applications
Method Application Comments
Measurement of total only practical
Measurement of total
cell yield from very application is in the
N or protein
dense cultures research laboratory
Measurement of Requires a fixed
Biochemical activity standard to relate
Microbiological
e.g. O2 uptake CO2 chemical activity to
assays
production, ATP cell mass and/or cell
production, etc. numbers
Measurement of dry
probably more
weight or wet weight
Measurement of total sensitive than total N
of cells or volume of
cell yield in cultures or total protein
cells after
measurements
centrifugation
Viable count methods : Plate
methods
Viable count methods
■ 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.
Plate count methods
■ 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.
Membrane filtration
● Membrane filters are very thin with a defined
pore size, e.g.
0.45 µm.
● Bacteria from a dilute sample are collected on
a filter; filter placed on agar plate, colonies
counted
Membrane filtration

– Used for samples with low microbial

concentration
– A measured volume (usually 1 to 100 ml)
of sample is filtered through a membrane
filter (typically with a 0.45 μm pore size)
– The filter is placed on a nutrient agar
medium and incubated
– Colonies grow on the filter and can be
counted
Membrane filtration
Direct Epifluorescence filtration
technique
■ It has been found that if bacteria are stained
with acridine orange and examined under
fluorescent microscope, viable cells fluoresce
with an orange hue. This observation has
been adapted for determination of bacterial
content.
Direct Epifluorescence filtration
technique [DEFT]
■ This method consists of filtering the liquid to be tested
through a membrane filter, staining the filter with
acridine orange and examining the filter under a
fluorescent microscope.
■ The organisms may be counted making this
technique quantitative
■ This method is used to determine microbial
contamination in IV fluids and is able to detect
organisms at a level of 25/ml.
■ DEFT presents problems if fluid to be examined is
viscous. However it may be overcome by dilution.
Microcalorimetry
■ This method depends on the fact that bacteria like all
living organisms produce heat when they metabolize.
Due to the small amount of heat produced, specially
sensitive calorimetric devices are required. Hence the
name microcalorimetry
■ The specimen to be evaluated is diluted with a
nutrient medium and if microorganisms are present
and can metabolize, heat is produced and can be
measured
■ Different organisms produce different heat outputs
and this may provide a means of identification
Electrical conductivity
■ When organisms grow in a liquid media their
metabolic products can create a change in
the conductivity of the media. Colony
numbers of 10/ml are required to produce a
measurable conductivity change.
■ The actual changes can be measured by
changes in impedance [resistance to
alternating current] or change in electrical
capacity
Electrical conductivity
■ Limitations:
■ If the level of initial concentrate of the product
is low, incubation of a sample in a suitable
broth will be necessary to increase the
organism to near 10/ml. This will add to the
time of the test
■ It is difficult to detect non fermenting
organisms whose metabolic activity produces
little change in media conductivity.
MOST PROBABLE NUMBER
■ Statistical method based on probability theory
■ In this method Multiple serial dilutions are performed
to reach a point of extinction [dilution level at which
no cells are deposited]
■ The greater the number of bacteria in a sample, the
more the dilution required to a point where no
bacteria are left to grow in the tubes in a dilution
series
■ MPN method is most suitable when microbes
counted will not grow on solid media.
■ The MPN is only a statement that there is 95%
chance that the bacterial population falls within a
certain range and that MPN is statistically the most
probable number.
MOST PROBABLE NUMBER
■ In the MPN dilution series
1. There are three sets of tubes and five tubes
in each set
2. Each tube in the first set receives 10ml of
inoculum
3. Each tube in the second set receives 1ml of
inoculum
4. Each tube in the third set receives 0.1ml of
inoculum
MOST PROBABLE NUMBER
The pattern to tubes that show growth
(brown) and those that do not (orange)
are compared with a statistical table to
calculate MPN
MOST PROBABLE NUMBER
Volume of Tubes of nutrient Number of
inoculums for medium positive tubes in
each set of 5 a set
tubes
10 ml 5 5

1ml 5 4

0.1ml 5 1
MOST PROBABLE NUMBER
■ There are enough bacteria in the first set of
five tubes that tubes show growth and are
recorded as positive
■ In the second set which received 1/10th
amount of inoculum, 4 tubes are positive
■ In the third set which received 1/100th amount
of inoculum only 1 tube is positive
■ Hence the pattern is 5 4 1.
■ Refer in statistical table to obtain total no of
microrganisms
 Bioluminescence
■ Certain insects and two or three genera of bacteria
possess the ability to emit light. This property is
utilized in quality control and research
■ Light generation depends on the oxidation of a
substance known as luciferin.
■ This is a fatty acid aldehyde known as dodecanal.
■ An enzyme called luciferase extracted form fire flies
catalyzes this reaction.
■ This reaction also requires ATP.
■ Thus measurement of light emission measures ATP
Bioluminescence
The method of ATP bioluminescence is derived
from the reaction that occurs naturally in the
firefly.
1. Firefly luciferase catalyzes the production of
light from luciferin in the presence of ATP,
Mg+2 and molecular oxygen.
2. The intensity of emitted light measured by
the luminometer indicates the amount of
extracted ATP.
Bioluminescence
■ The determination of adenosine triphosphate ( ATP)
with a bioluminescence assay may solve the
problems encountered in the detection of viable
bacteria.
■ The main function of the assay is to quantitate ATP,
the important compound in metabolism that is found
within all living cells.
■ The assay is based on the reaction between the
luciferase (enzyme), luciferin (substrate), and ATP.
Light is emitted during the reaction and can be
measured quantitatively and correlated with the ATP
quantity extracted from bacteria.
Bioluminescence
■ The detection of bacteria by this method
depends on the fact that they like all living
organisms contain ATP.
■ There arises a potential problem when
determining bacterial count of food, clinical
material and water. Elaborate techniques are
required to eliminate non bacterial ATP from
the samples.
Microscopic count
■ Direct microscopic counts are performed by
spreading a measured volume of sample over a
known area of a slide, counting representative
microscopic fields, and relating the averages back to
the appropriate volume-area factors.
■ Specially constructed counting chambers, such as
the Petroff-Hauser and Levy counting chambers,
simplify the direct counting procedure because they
are made with depressions in which a known
volume overlies an area that is ruled into squares.
■ The ability to count a defined area and convert the
numbers observed directly to volume makes the
direct enumeration procedure relatively easy.
Microscopic cell counts

■ Calibrated “Petroff-Hausser counting

chamber,” similar to hemacytometer, can be
used
■ Generally very difficult for bacteria since cells
tend to move in and out of counting field
■ Can be useful for organisms that can’t be
cultured
■ Special stains (e.g. serological stains or stains
for viable cells) can be used for specific
purposes
Microscopic cell counts

■ Direct counting procedures are rapid but have

the disadvantage that they do not discriminate
between living and dead cells. This method is
used to assess the sanitation level of a food
product
Microscopic cell counts
Microscopic cell counts
Microscopic cell counts

Cell density is determined by:

1) Counting the number of bacteria in one small square
2) Multiplying by the dilution factor of the sample
Dilution factor of 107
Number of bacteria in square = 5
5 X 107 = 50,000,000 bacteria per ml
Coulter Counter : Electronic count
method
■ Coulter-counter: single-file cells detected by
change in electric current
Flow cytometry
■ This technique together with coulter counter technique depends
on a simple device.
■ A potential difference is maintained in a circuit which includes a
tube with a small orifice submerged in a conducting fluid.
■ If a liquid containing particulate matter, bacteria or blood cells is
passed down the tube, when a particle passes through the
orifice a change in resistance in the circuit occurs and the
change maybe recorded by detection or printout devices.
■ Both the number of particles and their size maybe determined.
Disadvantages:
Both the living and dead particles may be counted
Orifice may become clogged during use.
Turbidimetric methods
■ A quick and efficient method of estimating the
number of bacteria in a liquid medium is to
measure the turbidity or cloudiness of a
culture and translate this measurement into
cell numbers.
■ This method of enumeration is fast and is
usually preferred when a large number of
cultures are to be counted.
■ Although measuring turbidity is much faster
than the standard plate count, the
measurements must be correlated initially
with cell number
Turbidimetric methods
■ This is achieved by determining the turbidity of
different concentrations of a given species of
microorganism in a particular medium and then
utilizing the standard plate count to determine the
number of viable organisms per milliliter of sample.
■ A standard curve can then be drawn in which a
specific turbidity or optical density reading is matched
to a specific number of viable organisms.
■ Subsequently, only turbidity needs to be measured.
The number of viable organisms may be read directly
from the standard curve, without necessitating
time-consuming standard counts.
Turbidimetric methods
■ Based on the diffraction or “scattering” of light
by bacteria in a broth culture
■ Light scattering is measured as optical
absorbance in a spectrophotometer
■ Optical absorbance is directly proportional to
the concentration of bacteria in the suspension
Indirect counting method
■ Quick and convenient, shows relative change
in the number of bacteria, useful for
determining growth (increase in numbers).
■ Does NOT distinguish between live and dead
cells.
■
Turbidimetric methods
More about Spectrophotometry
Direct measurement of microbial
biomass
■ Cell mass is determined directly by weighing
whole cells; biomass can be correlated with
cell numbers by reference to a standard
curve. Wet weight or dry weight of bacteria
may be used for estimation of cell numbers.
■ Mass determination
■ Cells are removed from a broth culture by
centrifugation and weighed to determine the
“wet mass.”
■ The cells can be dried out and weighed to
determine the “dry mass.”
Indirect Measurement of Microbial
Biomass
■ Microbial biomass is estimated by measuring
relatively constant biochemical components of
microbial cells, such as protein, ATP,
lipopolysaccharides, peptidoglycan, and
chlorophyll; biomass can also be indirectly
estimated by measured turbidity that can then
be correlated with cell numbers by reference
to a standard curve..
Indirect Measurement of Microbial
Biomass
■ Various procedures based on the detection of specific
microbial macromolecules or metabolic products can
be used to estimate numbers of microorganisms. For
example, peptidoglycan can be quantified, and
because this biochemical occurs exclusively in the
cell wall of bacteria, the concentration of
peptidoglycan can be used to estimate bacterial
numbers.
■ Such biochemical approaches for determining
bacterial numbers depend on the development of
analytical chemical procedures for quantifying the
particular biochemical and determining what
proportion of bacterial cell is composed of the specific
biochemical constituent
Exponential growth
■ Balanced growth”
■ Numbers of bacteria are doubling at regular
intervals.
■ All components of bacteria are increasing in
amount at the same rate
■ 2x as many bacteria = 2x as much protein, 2x as
much peptidgolycan, 2x as much LPS, etc.
■ During exponential growth, bacteria are not
limited for any nutrients, i.e. they are not short
of anything
Exponential growth
In this example, total biomass increases exponentially over time.