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MEASUREMENT OF MICROBIAL GROWTH OR

ENUMERATION OF MICROORGANISMS
Enumeration of microorganisms is especially important in dairy microbiology, food
microbiology, and water microbiology.

• 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.
• Pharmaceutical industry 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.
• To determine growth rates and generation times of bacteria.

There are many ways to measure microbial growth:

1. Direct Measurement of microbial growth (cell number method)


• Standard Plate Counts
• Filtration
• Most Probable Number (MPN) Method
• Direct Microscopic Counts
2. Indirect Measurements
• Turbidity
• Metabolic Activity
• Dry Weight

1. Direct Measurement of microbial growth

(a) STANDARD PLATE COUNT METHOD or VIABLE COUNT:

The most frequently used method of measuring bacterial populations is the plate count. Commonly
used for enumeration of bacteria in a wide variety of samples including milk, soil and other food.

Two main ways to perform plate counts


 Spread-plate method
 Pour-plate method

Pour Plate method:

 Either 1.0 ml or 0.1 ml of dilutions of the bacterial suspension is introduced into a Petri dish.
 The nutrient medium, in which the agar is kept liquid by holding it in a water bath at about
50°C, is poured over the sample, which is then mixed into the medium by gentle agitation of
the plate.
 When the agar solidifies, the plate is incubated .
 With the pour plate technique, colonies will grow within the nutrient agar (from cells
suspended in the nutrient medium as the agar solidifies) as well as on the surface of the agar
plate.
Drawback: Some relatively heat-sensitive microorganisms may be damaged by the melted agar and
will therefore be unable to form colonies.

Spread plate method :


 A 0.1 ml inoculum is added to the surface of a solidified agar medium.
 The inoculum is then spread uniformly over the surface of the medium with a specially
shaped, sterilized glass or metal rod.
 When a plate count is performed, it is important that only a limited number of colonies
develop in the plate.
 When too many colonies are present, some cells are overcrowded and do not develop; these
conditions cause inaccuracies in the count (Too numerous to count, TNTC)

The U.S. Food and Drug Administration convention is to count only plates with 25 to 250 colonies,
but many microbiologists prefer plates with 30 to 300 colonies. (less than 30 are Too few to count,
TFTC).

Serial dilution: To ensure that colony counts will be within this range, the original sample is
diluted several times in a process called serial dilution.
• The colonies growing on the plate are considered to have started from one viable bacterial
unit. Because bacteria are usually not found as individuals, the colony may have started from
a single cell or a group of cells.
• Hence, The results are reported as colony forming units (CFU’s) /ml of sample.

CFU per ml of sample = number of colonies


x dilution factor
amount of sample plated

Limitations:
• Plate counts show lower no. of cells than direct microscopic count as media may not support
certain type of organisms.
• Only aerobic organisms are counted.
• Volumes greater than 0.1ml are avoided in spread plate as excess liquid doesnot soak in the
media and may cause the colonies to coalesce as they form.
• Sometimes hard to distinguish between bacteria and food particles.
• Organisms which cannot tolerate 45-50oC temperature are difficult to grow in pour plating
method.
• Others include plating inconsistencies, such as inaccurate pipetting of a liquid sample, a
nonuniform sample (for example, a sample containing cell clumps), insufficient mixing, and
other factors.
• Hence, if accurate counts are to be obtained, great care and consistency must be taken in
sample preparation and plating, and replicate plates of key dilutions must be prepared.

2. Direct microscopic count of total cell number


This method is commonly used to count number of bacteria in liquid food especially milk.

Petroff-Hauser Counting Chamber is used.


 A measured volume of a bacterial suspension is placed within a defined area on a microscope
slide.
 These specially designed slides have chambers of known depth with an etched grid on the
chamber bottom.
 Prokaryotes are more easily counted in these chambers if they are stained, or when a phase
contrast is used.
 The number of microorganisms in a sample can be calculated by taking into account the
chamber’s volume and any sample dilutions required.
 The bacteria in several of the central squares are counted. 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)(25 squares) x (dilution if any)

Volume (1x 0.02mm3)


Advantages: Microscopic determination of bacterial numbers through direct counting is easy,
inexpensive, and relatively quick method;
It also gives information about the size and morphology of microorganisms

Limitations of direct microscopic count:


• Dead cells and living cells are all counted
• Small cells are difficult to count
• Precision is difficult to achieve
• Require phase contrast microscope when the sample is not stained.
• Need high cell concentration to be seen in microscopic field( > 106cells/ml) because such a
small volume is sampled.
• Motile cells must be immobilized before counting

3. Filtration
When the quantity of bacteria is very small, as in lakes or relatively pure streams, bacteria can be
counted by filtration method.
• In this technique, at least 100 ml of water are passed through a thin membrane filter
(Membrane filters are very thin with a defined pore size, e.g. 0.45 µm) whose pores are too
small to allow bacteria to pass.
• Thus, the bacteria are filtered out and retained on the surface of the filter.
• This filter is then transferred to a Petri dish containing a pad soaked in liquid nutrient
medium, where colonies arise from the bacteria on the filter's surface.
• This method is applied frequently to detection and enumeration of coliform bacteria, which
are indicators of fecal contamination of food or water.

4. MPN Method:
It is commonly used when the growth of bacteria in a liquid differential medium (lactose broth) is
used to identify the microbes (such as coliform bacteria, which selectively ferment lactose to acid, in
water testing).
 A series of serial dilutions of water sample is made.
 Transfer 1 mL sample from each of the serial dilutions to five fermentation test tubes
containing lactose medium and inverted gas collection tube.
 Incubate tubes for 24 hours at 35 ° C in a water bath
 Accumulation of gas (CO2) in inverted gas tubes is considered a +ve reaction (No gas –ve
reaction)
 After incubation, the pattern of positive and negative tubes is noted, and
 A standardized MPN Table is consulted to determine the most probable number of
organisms (causing the positive results) per unit volume of the original sample.
Estimating Bacterial Numbers by Indirect Methods
1. Turbidity
Estimating turbidity is a practical way of monitoring bacterial growth. As bacteria multiply in a
liquid medium, the medium becomes turbid, or cloudy with cells. A suspension of cells looks cloudy
(turbid) to the eye because cells scatter light passing through the suspension. The more cells that are
present, the more light is scattered, and hence the more turbid the suspension.
The instrument used to measure turbidity is a spectrophotometer (or colorimeter). It passes light
through a cell suspension and measures the unscattered light that emerges.
As bacterial numbers increase, less light will reach the detector. The unit of turbidity is called optical
density.
Commonly used wavelengths for bacterial turbidity measurements include 480 nm, 540 nm, 600 nm
and 660 nm.
Relating OD to Cell Numbers
For unicellular organisms, OD is proportional to cell number. Turbidity readings can therefore be
used as a substitute for total or viable counting methods. However, before this can be done, a
standard curve must be prepared that relates cell number (microscopic or viable count) to turbidity.

Advantages :
• Quick and easy to perform
• Typically do not require destruction or significant disturbance of sample

Limitations:
 Does NOT distinguish between live and dead cells.
 Sometimes problematic (e.g., microbes that form clumps or biofilms in liquid
medium)
 About 10 million to 100 million cells per milliliter are needed to make a suspension
turbid enough to be read on a spectrophotometer. Therefore, turbidity is not a useful measure
of cell number in liquids having small numbers of bacteria.
2. Dry weight:

During exponential growth, all cellular components increase in proportion to the increase in cell
numbers. Thus, instead of measuring changes in cell number over time, one could instead measure
the increase in dry weight of a culture as a barometer of growth. The cell mass of a very dense cell
suspension can be determined by this technique. 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 filamentous bacteria or molds.
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.

Automated methods
Coulter counter: Larger microorganisms such as protists and yeasts can be directly counted with
electronic counters such as the Coulter Counter. The microbial suspension is forced through a small
hole or orifice in the Coulter Counter. 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.
The Coulter Counter gives accurate results with larger cells and is extensively used in hospital
laboratories to count red and white blood cells.
Limitation: It is not commonly used in counting bacteria because of interference by small debris
particles or the formation of filaments.

Flow cytometer
Can differentiates between live and dead cells. Commercially available fluorescent dyes which can
stain live and dead cells differently are now available, making it possible to count directly the
number of live and dead microorganisms in a sample.
(certain dyes can penetrate only impermeable memb. of dead cells and some fluorescing dyes
penetrates all cells, viable or not.)

When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher
energy state, emit light energy at higher wavelengths. Commonly used dyes include propidium
iodide, phycoerythrin, and fluorescein. The light signals are detected by photomultiplier tubes and
digitized for computer analysis.