MEASUREMENT OF MICROBIAL GROWTH
MEASUREMENT OF BACTERIAL GROWTH
Growth of micro-organisms can be quantitatively measured by
various techniques.
1. DETERMINATION OF CELL NUMBER:-
A] TOTAL COUNT /DIRECT METHODS:
B] VIABLE COUNT / INDIRECT METHODS:
2. DETERMINATION OF CELL MASS:
A] DIRECT METHOD:
B] INDIRECT METHOD:
1. DETERMINATION OF CELL NUMBER:
A] TOTAL COUNT OR DIRECT METHODS:
In this method the total
count of micro-organisms in any given suspension indicates the total number of cells
which includes both living and dead. Dead cells cannot be distinguished from living
cells by this method.
The following methods are used for the determination of total
count.
DIRECT MICROSCOPIC
COUNT: - This method is also called as “breed method”. These methods are possible using special slides
known as counting chamber. Only dense suspension can be counted (>107 cells
per ml) but sample can be concentrated by centrifugation or filtration to
increase sensitivity.
A known volume of cell
suspension (0.01 ml) is spread uniformly over a glass slide within a specific area
(1 sq. cm). The smear is then fixed, stained, examined under the oil immersion
lens and the cells counted. It is impossible to examine entire area (1 sq. cm),
therefore practically only a few microscopic areas are observed. Several
microscopic fields are counted and an average is taken. The total cells per
square cm are then calculated by determining the number of microscopic fields
per square cm.
COUNTING CHAMBER
METHOD: - In this method the total cells (both living and
dead) of liquid sample are counted easily and accurately by using a special
microscope glass slide which is called as Petroff - Hausser counting chamber or
Haemocytometer.
This method is useful
for counting both prokaryotes (Haemocytometer) and eukaryotes (Petroff -
Hausser counting chamber).
In the haemocytometer
or counting chamber method, a minute drop of the culture is placed in a tiny,
shallow, rectangular glass slide, called “Neubar’s” slide, in this chamber a
grid is marked on the surface of the glass slide with squares of known area.
The whole grid has 25 large squares, a total area of 1 mm2 and a total volume
of 0.02 mm3 (1/50 mm).
A suspension of
unstained bacteria can be counted in the chamber, using a phase contrast microscope
and calculate the count of number of bacteria per unit area of grid &
multiply it by conversion factor (depending on chamber volume and sample
dilution used).
PROPORTIONAL COUNT
METHOD: - In this method a standard suspension of particles
(plastic beads, number of particles/volume is known) is mixed with an equal
amount of cell suspension. This mixed suspension is spread on the slide, fixed
and stained. The particles and cells in the microscopic field are counted. An
average count of the particles and the cell is taken from the number of fields.
E.g.
Suppose an average count of 10 particles and 50 cells per field is obtained. If
the number of particles in 1 ml of standard suspension is 25,000.Then the
number of cells/ml of suspension is:
50/10 x
25,000 = 1,25,000 cells/ml.
ELECTRONIC COUNTER
METHOD: - In this method an electronic instrument, such as
Coulter counter can be used for the direct enumeration of cells in a
suspension. In this technique, the bacterial suspension is passed through a
capillary tube or orifice. The diameter of this tube is so small that it allows
only one cell to pass at a time. The instrument can count thousands of cells in
a few seconds.
Advantages: -
1.
Direct counting methods are rapid and
simple.
2.
The morphology of cells can also be observed
when they are counted under the microscope.
Disadvantages: -
1.
The major disadvantage of this method is
that it gives the total cell count which includes both viable and non-viable
cells.
2.
Accuracy also declines with very dense
and very dilute suspensions because of clumping and statistical errors,
respectively.
3.
The Coulter counter counts even dust
particles. Hence, the suspension must be absolutely free of any foreign
particles.
B] VIABLE COUNT / INDIRET METHODS: -
In this method the viable count of micro-organisms in suitable growth medium
indicates the number of viable cells or spores. The viable cells under growth
medium multiply and that each cell or spore forms a colony. Each colony that can be counted is called as
colony forming unit (CFU), and the number of CFU’s is related to the number of
bacteria in the sample.
PLATE COUNT TECHNIQUE:
-: The plate count method relies on bacteria growing a
colony on a nutrient medium. The colony becomes visible to the naked eye and
the number of colonies on a plate can be counted. To be effective, the dilution
of the original sample must be arranged so that on average between 30 and 300
colonies of the target bacteria are grown. Fewer than 30 colonies makes the
interpretation statistically unsound and greater than 300 colonies often
results in overlapping colonies and imprecision in the count.
In this method, a
measured amount of diluted bacterial suspension is introduced into a Petri plate,
after which the agar medium (liquid form 45°C) is added. Immediately, mix the agar medium with the inoculums
by rotating the plate. After the solidification of medium, the plates are
incubated at 37°C for 24 hours in an inverted position. A plate having 30 to
300 colonies is selected for counting the number of microorganisms.
MEMBRANE FILTERS
COUNT: - This method work as same principle as plate count
technique only difference is as in this method a diluted suspension of
micro-organisms is filtered through a Millipore filter. The microbes are
retained on the filter disc & this disc is placed in a culture medium in a
Petri plate. The plates are incubated & the colonies are counted on the
filter disc.
2. DETERMINATION OF CELL MASS: - In this method the weight or mass of the
cells is estimated as an indicator of increased growth.
A] DIRECT METHOD: -
DRY WEIGHT
MEASUREMENT:- This is
a simple and direct method of measuring the cell mass. The cell mass of a very dense cell suspension can be
determined by this technique. In this technique, the culture suspension is centrifuged and the microorganisms are
removed from the medium by filtration and the microorganisms on filters are
washed to remove all foreign particles, and dried in desiccators by putting in
weighing bottle (previously weighed). The dried microbial content is then
weighed accurately. This technique is especially useful for measuring the
growth of micro fungi like molds.
MEASUREMENT
OF CELL NITROGEN: - A major chemical constituents of
microbial cell is proteins, as the micro-organism grow; there is an increase in
the protein concentration (i.e. nitrogen concentration) in the cell. Thus, cell
mass can be subjected to quantitative chemical analysis methods to determine
total nitrogen that can be correlated with growth. The cells are obtained by
centrifugation as mention in dry weight measurement. This method is useful for dense cell
suspension for determining the effect of nutrients or antimetabolites upon the
protein synthesis of growing culture.
B] INDIRECT METHOD: -
TURBIDIMETRIC METHOD: - A most widely used method
for measuring cell mass is by observing the light scattering capacity of the
sample. Turbidometry is based on the fact that microbial cells scatter light
striking them. Since the microbial cells in a population are of roughly
constant size, the amount of scattering is directly proportional to the biomass
of cells present and indirectly related to cell number.
A
suspension of unicellular organisms is placed in a colorimeter or
spectrophotometer & light pass through it. This instruments work on Beer
and Lambert law i:e light absorbance is directly proportional to turbidity of
medium . One visible characteristic of growing bacterial culture is the
increase in cloudiness of the medium (turbidity). When the concentration of
bacteria reaches about 10 million cells (107) per ml, the
medium appears slightly cloudy or turbid.
Further increase in
concentration results in greater turbidity. When a beam of light is passed
through a turbid culture, the amount of light transmitted is measured. Greater
the turbidity, lesser would be the transmission of light through medium.
The absorbance is measured
in terms of optical density. Measurement
of optical density does not give value of cell numbers or cell mass but the
cell number can be calculated by plotting calibration curve which indicates the
direct relationship of optical density and mass. Cell number of an unknown sample
can be determined by taking the optical density and comparing it with the
corresponding value on the standard curve.
3. DETERMINATION OF
CELL ACTIVITY: -
MEASUREMENT OF
BIOCHEMICAL ACTIVITY: - Measurement
of a specific chemical change by metabolic activities of microorganisms can be
correlated with the microbial growth. Cell metabolic activity results in to
formation of any specific metabolite.
E.g. lactic acid, H2S, Co2, enzymes etc. The measurement of these products forms the principle of measurement of cell activity. The amount of acid produced to the magnitude of cell suspension.
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