Measurement of microbial growth

MEASUREMENT OF MICROBIAL
GROWTH
Dr. Kalavati Prajapati

MEASUREMENT OF MICROBIAL GROWTH
A. Measurement of Cell count
B. Measurement of Viable cell
C. Measurement of Cell Mass
D. Measurement of Cell constituents

A. MEASUREMENT OF CELL COUNT:
 To determine microbial numbers is through
direct counting.
 Using a counting chamber is easy, inexpensive,
 and relatively quick; it also gives information
about the size and morphology of
microorganisms.
 Petroff-Hausser counting chambers can be used
for counting procaryotes
 Hemocytometers can be used for both
procaryotes and eucaryotes.

1. The Petroff-Hausser Counting Chamber.(Direct microscopic count)
400 to500 magnification.
The average number of bacteria
in these squares is used to
calculate the concentration of cells
in the original sample.
Since there are 25 squares
covering an area of 1 mm2,
the total number of bacteria in 1
mm2 of the chamber is
(number/square) (25 squares).
The chamber is 0.02 mm deep and
therefore,
bacteria/mm3 = (bacteria/square)
(25squares) (50).

2.ELECTRONIC COUNTERS(COULTER COUNTER)
 Larger microorganisms such as protozoa, algae, and
nonfilamentous yeasts can be directly counted
 An electrical current flows through the hole, and
electrodes placed on both sides of the orifice
measure its electrical resistance. Every time a
microbial cell passes through the orifice, electrical
resistance increases (or the conductivity drops) and
the cell is counted.
 Limitation: counts of all cells, whether alive or dead.
It is not as useful in counting bacteria because of
interference by small debris particles, the formation
of filaments, and other problems.

Culture sample
Capillary tube
Light beam
Photodetector
Digital Readout
Electronoic Cell Counter

3.DRY SLIDE/ DIRECT MICROSCOPIC COUNT
 Preapare a smear of bacterial culture to study
 Stained with suitable dye
 Used to quick checkof milk as it enters the
dairy to determine the level of contamination
before pasteurization.

1. The Membrane Filtration Procedure.
Usually the counts obtained with this approach are much higher than those from
culture techniques because some of the bacteria are dead.
 Sample is first filtered through a black polycarbonate membrane filter to
provide a good background for observing fluorescent objects. The bacteria then
are stained with a fluorescent dye such as acridine orange or DAPI and observed
microscopically.
 Acridine orange–stained microorganisms glow orange or green and are easily
counted with an epifluorescence microscope
B.Measurement of Viable cell

Filtration
DIRECT MEASUREMENTS OF MICROBIAL GROWTH
Figure 6.17a, b

2.SPREAD-PLATE AND POUR-PLATE
TECHNIQUES(VIABLE CELL COUNT )
 Plating techniques are simple, sensitive, and widely
used for viable counts of bacteria and other
microorganisms in samples of food, water, and soil.
 it is not possible to be absolutely certain that each
colony arose from an individual cell, the results are
often expressed in terms of colony forming units
(CFU) rather than the number of microorganisms.
 Limitations:
 Suitability of medium
 Suitability of incubations conditions.
 Hot agar used in the pour-plate technique may
injure or kill sensitive cells.

 Plate Counts: Perform serial dilutions of a
sample
Figure 6.15, top portion

 Inoculate Petri
plates from
serial dilutions

 After incubation, count colonies on plates that
have 25-250 colonies (CFUs)
PLATE COUNT

Multiple tube
MPN test
Count
positive
tubes and
compare to
statistical
MPN table.
3. DIRECT MEASUREMENTS OF MICROBIAL GROWTH (MPN)

B. MEASUREMENT OF CELL MASS
1. Dry Weight Estimation (Gravimetric method):
 The most direct approach is the determination of
microbial dry weight.
 Cells growing in liquid medium are collected by
centrifugation, washed, dried in an oven, and
weighed.
 This is an especially useful technique for
measuring the growth of fungi.
 It is time consuming, however, and not very
sensitive. Because bacteria weigh so little, it may
be necessary to centrifuge several hundred
milliliters of culture to collect a sufficient
quantity (mg dry wt /ml).

2.TURBIDOMETRIC AND SPECTROPHOTOMETRIC
METHOD METHOD:
Determination of microbial mass
by measurement of light
absorption.
As the population and turbidity
increase, more light is scattered
and the absorbance reading given
by the spectrophotometer
increases.
Advantage: More rapid, sensitive
techniques depend on the fact
that microbial cells scatter light
striking them. Because 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

C. MEASUREMENT OF CELL CONSTITUENTS:
1.Determination of N-contents.
 Major constituents is protein in cell.
 One can also measure a bacterial population or
cell crop in terms of bacterial nitrogen.
 From total cell dry weight 14% is N.
 For the N measurement first harvest the cells
then wash them with free of medium and
perform a quantitative chemical analysis of
N.(mg N/ ml).
 It is only applicable for concentrated
population.

2.Measurement of a specific chemical change produced on a
constituent of the medium ( Biochemical activity).
 Glucose fermentation in medium.
 The cell concentration is measured from acid produced by
culture.(miliequivalent acid produced/ ml culture).
 Other biochemical reactions also used for measurement of
growth at particular phase.

3. ATP BIOLUMINOMETRY
ATP is rapidly lost from dead cells. It is useful to determine
viable cell.
 This techniques utilize enzyme-substrate complex.
 Luciferase enzyme obtained from firefly Photinus pyralis
converts Luciferin to oxyluciferin by bioluminometer.
 ATP+O2+luciferin Luciferase
Oxyluciferin+AMP+CO2+PPi+
Photon of light (562 nm)
 Very rapid and sensitive under optimum conditions as little
as 10 -15 mol of ATP can be detected.

4. Online estimation
 It provide real time estimation of biomass, and
minimizes the requirements for repeated sampling
and off-line estimation.
 O.D. based or capacitance based probe,
 Carbon dioxide evolution estimation,
 Oxygen utilization estimation

1. Which of the following is used to grow bacterial cultures
continuously?
a) Chemostat
b) Coulter Counter
c) Hemostat
d) Petroff-Hausser chamber
2. Which of the following techniques would be best to use to
determine the number of living cells in a culture?
a) Viable plate count
b) Coulter counter
c) Microscopic count
d) Most Probable Number
MULTIPLE CHOICE QUESTIONS

3.Which instrument is used for counting procaryotes
a) Chemostat
b) Coulter Counter
c) Hemostat
d) Petroff-Hausser chamber
4. Which enzyme is used in ATP bioluminometry method
a) Luciferase
b) Peroxidase
c) Oxy luciferase
d) None of these

5.Electronic counter method is not highly used because…
a) Counts all cells
b) Count dead cell debris
c) Counts alive or dead.
d) All of these
6.Which techniquie ids used for viable count of bacterial
cells
a) Membrane filtration
b) Plate method
c) a and b
d) None of these

7. All of the following are drawbacks to the use of
spectrophotometry in estimating microbial growth EXCEPT:
a) It cannot distinguish between live and dead cells.
b) It can not be used if there are less than one million cells per
milliliter.
c) It will underestimate the true number of cells if they are not
uniformly in suspension.
d) It kills the organisms as they are being counted.
8. Full form of CFUs
a) Colony formal unit
b) Colony forming unit
c) Counting forming Unit
d) None of these

9. Turbididmetric measurements of bacterial population
size are usually made with_________
a) Peter hoff hauser chamber
b) Spectrophotometer
c) Centrifuge
d) Dry slide technique
10. ___________are usedf to maintain culture in
exponential phase in industrial ferementation.
a) Chemostate
b) Turbidostate
c) Diauxic growth
d) a and b

REFERENCES
 Prescott, L.M., Harley, J.P., Klein, D.A. (2002). Microbiology. Fifth Edition.
Wm. C. Brown Pub. Dubuque, Iowa. pp. 112-125.
 Pelczar, M.J., Chan, E.C.S., Krieg, N.R.(1993) Introduction to Microbiology .
Fifth Edition. Tata Mc-Graw – Hill Edition.pp-119-132
 Ingraham, J.L. and Ingraham C. A(2008) Introduction to Microbiology. Third
Edition
 Modi H.A. (2014) A handbook of Elementary Microbiology .Shanti Prakashan.
pp-203-216

Measurement of microbial growth

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Measurement of microbial growth