Direct methods of measurement of microbial growth includes various methods of enumeration of both viable and non viable cell also includes growth curve. Helpful for UG and PG programs of microbiology

2. Measurement of Bacterial Growth
 The bacterial cell cycle involves the formation of new cells
through the replication of DNA and partitioning of cellular
components into two daughter cells.
 Bacterial growth is the asexual reproduction
of bacterium into two daughter cells, in a process
called binary fission.
 The resulting daughter cells are genetically identical to the
original cell.
 Both daughter cells from the division do not necessarily
survive.
 The bacterial population undergoes exponential growth.

4. Generation Time
• In prokaryotes (Bacteria and Archaea), the generation time is
also called the doubling time and is defined as the time it
takes for the population to double through one round of binary
fission.
• Bacterial doubling times vary enormously.
• Whereas Escherichia coli can double in as little as 20 minutes
under optimal growth conditions in the laboratory, bacteria of
the same species may need several days to double in
especially harsh environments.
• For any number of starting cells, the formula is adapted as
follows:
Nn is the number of cells at any generation n,
N0 is the initial number of cells, and
n is the number of generations.

5. The Growth Curve

6. PURPOSE OF MEASUREMENT OF
MICROBIAL GROWTH
 The number of bacteria in a clinical sample serves as an
indication of the extent of an infection.
 Quality control of drinking water,
 Quality control of food,
 Quality control of medication, and
 Quality control of even cosmetics relies on estimates of
bacterial counts
 To detect contamination and prevent the spread of disease.

7. BACTERIAL CELL COUNT
 Estimating the number of bacterial cells in a sample, is
known as a bacterial count.
 Two major approaches are used to measure cell number.
1. DIRECT METHODS
2. INDIRECT METHODS
 The direct methods involve counting cells, whereas the
indirect methods depend on the measurement of cell
presence or activity without actually counting individual
cells.
 Both direct and indirect methods have advantages and
disadvantages for specific applications.

8. BACTERIAL CELL COUNT
 Estimating the number of bacterial cells in a sample, is
known as a bacterial count.
 Two major approaches are used to measure cell number.
1. DIRECT METHODS
2. INDIRECT METHODS
 The direct methods involve counting cells, whereas the
indirect methods depend on the measurement of cell
presence or activity without actually counting individual
cells.
 Both direct and indirect methods have advantages and
disadvantages for specific applications.

9. DIRECT METHODS
Direct microscopic count
Electronic counter
Standard plate count
Membrane filtration
MPN
INDIRECT METHODS
Turbidity
Metabolicactivity
Dryweight
Automatedmicrobial
identificationsystem.

10. DIRECT METHODS OF MEASURING MICROBIAL GROWTH
Direct Microscopic Count Using a Petroff Hausser slide:
• A Petroff-Hausser chamber( Depth 0.02 mm) is a special slide
designed for counting the bacterial
cells in a measured volume of a sample.
• A grid is etched on the slide to facilitate
precision in counting.
• It can be used for counting procaryotes.
• It is similar to a Hemocytometer used to
count red blood cells.
• Hemocytometers ( Depth 0.1 mm) can be
used for both procaryotes and
eucaryotes.

11. • Stain is added to visualize bacteria. Procaryotes are
more easily counted in these chambers if they are
stained
• Newly developed fluorescence staining techniques
make it possible to distinguish viable and dead
bacteria.
• These viability stains (or live stains) bind to
nucleic acids, but the primary and secondary stains
differ in their ability to cross the cytoplasmic
membrane.
• The primary stain, fluorescence green, can
penetrate intact cytoplasmic membranes, staining
both live and dead cells.
• The secondary stain, which fluorescence red, can
stain a cell only if the cytoplasmic membrane is
considerably damaged.
• Thus, live cells fluorescence green because they only absorb the green stain,
whereas dead cells appear red because the red stain displaces the green stain on
their nucleic acids (Figure).

15. • Cells are counted and multiplied by a factor to obtain
concentration.
 The bacteria in several of the central squares are counted,
usually at x400 to x500 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).

16. Number of per (ml) = Number of cells counted X dilution (if used)
X 50,000*
[The factor of 50,000 is used in order to determine the cell count for 1 ml: 1 ml = 1000 mm3
*50,000 = 50 (cell depth is 1/50) X 1000 (1000 cubic mm = 1 milliliter)
 The chamber is 0.02 mm deep and therefore,
 bacteria/mm3 (bacteria/square)(25 squares)(50)
 The number of bacteria per cm3 is 103 times this value.
 For example, suppose the average count per square is 14 bacteria:
bacteria/cm3 = (14 bacteria) (25 squares)(50)(103)
(103) = 1000
= 17,500,000
OR
= 1.75 X 107.

17. bacteria/cm3 = (28 bacteria) (25 squares)(50)(103)
= 35,000,000
OR
= 3.5 X 107.
(103) = 1000

19. Advantages: -
 No incubation time
required
 Easy to perform
 Inexpensive
 Gives information about
size and morphology
 Cannot always
distinguish between live
and dead bacteria.
 Non viable cells are also
counted
 Motile bacteria are
difficult to count.
 Requires a high
Concentration of
bacteria (10 million/ml)
Disadvantages: -

20. CLEANING THE COUNTING CHAMBERS
To clean the counting chamber: After completing the count,
remove the cover glass and clean the counting chamber
with water or a mild cleaning solution (10% solution of
bleach).
Dry the counting chamber with a soft cloth or wipe, or rinse
with acetone.

21. Measurement of microbial growth-CELL NUMBER
Coulter counter- electronic method
 Larger microorganisms such as protozoa, algae, and non
filamentous yeasts can be directly counted with electronic
counters such as the Coulter Counter.
 The microbial suspension is forced through a small hole or
orifice.
 Every time a microbial cell passes through the orifice,
electrical resistance increases (or the conductivity drops)
and the cell is counted.
 The Coulter Counter gives accurate results with larger cells
and is extensively used in hospital laboratories to count red
and white blood cells.

22.  In Electronic instrument as Coulter counter
Microbial suspension is forced through small hole or
orifice or capillary tube
 The diameter of this tube is so microscopic
that allows only one cell to pass at a time.
 Can count thousands of cells in a few
seconds.
 Movement of microbe through orifice/capillary tube
impacts electric current that flows through orifice

23.  A glass tube with a small opening is immersed in an
electrolyte solution.
 A first electrode is suspended in the glass tube.
 A second electrode is located outside of the tube.
 As cells are drawn through the small aperture in the glass
tube, they briefly change the resistance measured
between the two electrodes and the change is recorded
by an electronic sensor (Figure); each resistance change
represents a cell.

24. WORKING OF A COULTER COUNTER

27. STANDARD PLATE COUNT
 One method of measuring bacterial growth is the standard
plate count.
 This technique relies on the fact that under proper
conditions, only a living bacterium will divide and form a
visible colony on an agar plate.
 These are referred to as viable counting methods because
they count only those cells that are alive and able to
reproduce.
 Plating techniques are simple, sensitive, and widely used
for viable counts of bacteria and other microorganisms in
samples of food, water, and soil.

28.  The results are usually expressed as colony-forming units
per milliliter (CFU/mL) rather than cells per milliliter
because more than one cell may have landed on the same
spot to give rise to a single colony.
 Several plating methods can be used to determine the
number of viable microbes in a sample.
 Two commonly used procedures are
1. The spread-plate technique and
2. The pour-plate technique.
Although the final inoculation procedure differs between
these two methods, they both start with a serial dilution of
the culture.

32. COLONY COUNTER

34. LIMITATIONS STANDARD PLATE COUNT
 The samples should yield between 30 and 300 colonies for
best results.
 Of course the counts will also be low if the agar medium
employed cannot support growth of all the viable
microorganisms present.
 The hot agar used in the pour-plate technique may injure or
kill sensitive cells; thus spread plates sometimes give
higher counts than pour plates.
 Several problems, however, can lead to inaccurate counts.
 Low counts will result if clumps of cells are not broken up
and the microorganisms well dispersed.

35.  Because 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.
 Furthermore, samples of bacteria that grow in clusters or
chains are difficult to disperse and a single colony may
represent several cells.
 Some cells are described as viable but nonculturable and
will not form colonies on solid media.
 For all these reasons, the viable plate count is considered a
low estimate of the actual number of live cells.
 These limitations do not detract from the usefulness of the
method, which provides estimates of live bacterial
numbers.

36. MEMBRANE FILTER
A diluted suspension of microorganism/Cells is filtered
through special membrane that provides dark background
for observing cells
Cells are retained on filter
The disc is placed in a culture media in a petri plate
Incubated at ideal growth factors
Useful for counting bacteria
With certain dyes, can distinguish living from dead cells
Membranes with different pore sizes are used to trap
different microorganisms.
Incubation times for membranes also vary with the
medium and microorganism.

38. Colonies on Membrane Filters
Membrane-filtered samples grown on a variety of media.
(a) Standard nutrient media for a total bacterial count. An indicator colors colonies
red for easy counting.
(b) Fecal coliform medium for detecting fecal coliforms that form blue colonies.
(c) m-Endo agar for detecting E. coli and other coliforms that produce colonies with
a green sheen.
(d)Wort agar for the culture of yeasts and molds.

39. MOST PROBABLE NUMBER (MPN)
 When samples contain too few organisms to give reliable
measures of population size by the standard plate count
method, as in food and water sanitation studies, or when
organisms will not grow on agar, the most probable
number (MPN) is used.
 With this method, the technician observes the sample,
estimates the number of cells in it, and makes a series of
progressively greater dilutions.
 As the dilution factor increases, a point will be reached at
which some tubes will contain a single organism and
others, none.

40.  A typical MPN test consists of five tubes of each of three
volumes (using 10, 1, and 0.1 ml) of a dilution.
 Those that contain an organism will display growth by
producing gas bubbles and/or by becoming cloudy when
incubated.
 The most probable number test is used to determine the
bacterial density in a water sample by serial dilution in
multiple tubes using fermentation technique.
 The number of organisms in the original culture is
estimated from a most probable number table.

41.  One of the most useful applications of the MPN method is
in testing water purity.
 It is used to examine the portability of water.
 It indicates the number of bacteria which are present in the
given sample.
 Used mainly to measure bacteria that will not grow on
solid medium.
 Dilute a sample repeatedly and inoculate several broth
tubes for each dilution point.
 Count the number of positive tubes in each set.
 Statistical method: Determines 95% probability that a
bacterial population falls within a certain range

47. REFERENCES
PRESCOTT, HARLEY AND KLIENS MICROBIOLOGY
7TH Edition
Joanne M.Willey,Hofstra University.Linda M. Sherwood,Montana State University.
Christopher J.Woolverton,Kent State University
Published by McGraw-Hill
MICROBIOLOGY PRINCIPLES AND EXPLORATION
9TH Edition
JACQELYN G. BLACK, LAURA J. BLACK
Published byTheWileyPLUS Advantage
THE IMPRINT 2014 Edition Biochemistry scanner
MBH – 104 MICROBIALAND BIOCHEMICALTECHNIQUES
PROF. BALASUBRAMANIAN SATHYAMURTHY
A TEXTBOOK OF MICROBIOLOGY
Dr. R.C.Dubey , Dr. D.K. Maheshwari
Published by S.Chand publishers

48. Internet References
https://microbialgrowth101.weebly.com/direct-methods-of-measuring.html
www.google images .com
www.google scholars.com
www.biologydiscussion.com
www.biolibretexts.com