QUANTITATION OF

MICROORGANISMS
METHOD FOR ENUMERATION
 CELL NUMBER
Petroff-Hausser counting chamber
Hemocytometer
• Petroff-Hausser method is popular
for bacteria and offered in series of cell
depths. Hemocytometer is a specimen slide
which is used to determine the concentration
of cells in a liquid sample (to count blood
cells/WBC, RBC, ).
Petroff Hausser counting chamber
A slide with squares
• Area of square = 1/400 mm2
• cover slip rests at 1/50mm above the slide
volume : 1/400 x 1/50 = 1/20000 mm3

= ½ x 10 7 cm3

Average of 5 bacteria present then total is

5x 2x 10 7 cells/ml
Petroff Hausser counting chamber
 Electronic coulter counter

• The Coulter principle states that particles

pulled through an orifice, concurrent with an
electric current, produce a change in
impedance that is proportional to the volume
of the particle traversing the orifice
• The Coulter principle was named for its
inventor, Wallace H. Coulter
• By monitoring such pulses in electric current,
the number of particles for a given volume of
fluid can be counted. The size of the electric
current change is related to the size of the
particle, enabling a particle size distribution to
be measured, which can be correlated to
mobility, surface charge, and concentration of
the particles.
Coulter counter
Indirect methods to measure cell number

• Standard plate count method

Pour plate method
Spread plate method
Membrane filter techniques
By counting the colonies and arriving at number
of cells/ml of the sample
No of cell/ ml in pour plate
Membrane filter technique
 Procedure
• Collect the sample and make any necessary dilutions.
• Select the appropriate nutrient or culture medium.
Dispense the broth into a sterile Petri dish, evenly
saturating the absorbent pad.
• Flame the forceps, and remove the membrane from
the sterile package.
• Place the membrane filter into the funnel assembly.
• Flame the pouring lip of the sample container and
pour the sample into the funnel.
• Turn on the vacuum and allow the sample to draw
completely through the filter.
Rinse funnel with sterile buffered water. Turn on the vacuum and allow the
liquid to draw completely through the filter.
Flame the forceps and remove the membrane filter from the funnel.
Place the membrane filter into the prepared Petri dish.
Incubate at the proper temperature and for the appropriate time period.
 Measurement of cell mass
• Direct methods Indirect method
Dry weight method Turbidity method
Total nitrogen content
 Turbidity
• By using colorimeter or spectrophotometer
 Turbidity

• A widely used method for the determination of cell

number is a turbidometric measurement or light
scattering. This technique depends on the fact that as
the number of cells in a solution increases, the
solution becomes increasingly turbid (cloudy). The
solution looks turbid because light passing through it is
scattered by the microorganisms present and the
turbidity is proportional to the number of
microorganisms in the solution. The turbidity of a
culture can be measured using a photometer or a
spectrophotometer
• Both instruments measure the amount of transmitted
light, the light that makes it from the light source
through the sample to the detector.
• Absorbance increases in a linear fashion as the cell
number increases. When measuring growth of a culture
the term optical density (OD) is normally used to more
correctly represent the light scattering that is occurring.
• For most unicellular organisms changes in OD are
proportional to changes in cell number (within certain
limits) and therefore can be used as a method to follow
cell growth. If a precise cell number for a given OD is
desired, a standard curve can be generated, where
viable plate count or cell mass is plotted as a function of
OD.
 Direct methods
• Wet weight/ Dry weight method
 Total Nitrogen content
• MICRO KJELDAHL METHOD
• Two processes : Digestion and distillation
Bacterial cells are heated with tri acid mixture
Ammonia is liberated. Ammonia is distilled &
captured in boric acid solution. Titrate with
0.01N HCL Using methyl red- bromo cresol green
as indicator.
 Measuring biochemical activity
• The metabolic activity of microorganisms
taken as an index for measuring growth
• Eg : E. coli can grow in a medium supplied with
lactose as the sole carbon source, can convert
lactose into acid and Carbon dioxide.
• The amount of acid liberated can be
determined by biochemical methods.
• If the amount of acid production is more, it
can be concluded that more cells are present.
Environmental factors influencing growth

• Microorganisms interact with their

environment along more dimensions than pH,
temperature, and free oxygen levels, although
these factors require significant adaptations.
We also find microorganisms adapted to
varying levels of salinity, barometric pressure,
humidity, light and nutrient concentrations.
 Osmolarity

• All life depends on available water to grow.

Available moisture is measured as water
activity (aw), which is the ratio of the vapour
pressure of the medium of interest to the
vapour pressure of pure distilled water;
therefore, the aw of water is equal to 1.0.
• Bacteria require high aw (0.97–0.99), whereas
fungi can tolerate drier environments; for
example, the range of aw for growth
of Aspergillus spp. is 0.8–0.75.
• Most natural environments tend to have lower
solute concentrations than the cytoplasm of
most microorganisms. Rigid cell walls protect
the cells from bursting in a dilute environment
• Not much protection is available against
high osmotic pressure. In this case, water,
following its concentration gradient, flows out
of the cell. This results in plasmolysis (the
shrinking of the protoplasm away from the
intact cell wall) and cell death.
• Microorganisms called halophiles (“salt
loving”) actually require high salt
concentrations for growth. These organisms
are found in marine environments where salt
concentrations hover at 3.5%. Extreme
halophilic microorganisms, such as the red
alga Dunaliella salina and the archaeal
species Halobacterium, grow
in hypersaline lakes such as the Great Salt Lake
(3.5–8 times saltier than the ocean) and the
even saltier Dead Sea (10 times saltier than
the ocean).
How do osmotolerant organisms survive?
• Dunaliella spp. counter the tremendous osmotic pressure of
the environment with a high cytoplasmic concentration of
glycerol and by actively pumping out salt ions.
• Halobacterium spp. accumulate large concentrations of K+ and
other ions in their cytoplasm. Their proteins are designed for
high salt concentrations and lose activity at salt
concentrations below 1–2 M.
• staphylococci, micrococci, and corynebacteria that colonize
our skin tolerate salt in their environment.
 Light
• Photosynthetic bacteria (chlorobium, chromatium) depend
on sufficient light intensity at the wavelengths absorbed by
their pigments to grow and multiply. Energy from light is
captured by pigments and converted into chemical energy
that drives carbon fixation and other metabolic processes.
• Other microorganisms, such as the archaea of the
class Halobacteria, use light energy to drive their proton and
sodium pumps. . The light is absorbed by a pigment protein
complex called bacteriorhodopsin, which is similar to the eye
pigment rhodopsin.
• Photosynthetic bacteria are present not only in aquatic
environments but also in soil and in symbiosis with fungi in
lichens. The peculiar sight of pink snow, also called
watermelon snow, is caused by a microalga Chlamydomonas
nivalis, a green alga rich in a secondary red carotenoid
pigment (astaxanthin) which gives the pink hue to the snow
where the algae grows.