The How of Microbial Ecology

The Principal
Methods used in the
Isolation
◤
of Indicator
Organisms from
Water
 ◤

Principal Methods

Membrane- Presence–absence
filtration Multiple-tube tests
method method
 Membrane
◤ Filtration
Technique
◤
Filtration

Filtration is an effective method for the

sterilization of heat-sensitive materials and it is
also utilized to sterilize different liquids and
gases (including air). It acts as a barrier and
removes the contaminating microorganisms
from the desired liquid or gas rather than
destroying them.
Filtration
◤

• A filter is made of millions of pores

which only allow the passage of liquid
or gas through them. These pores are
smaller than the microorganisms.
Hence it traps those particles which
are larger than the pores
 ◤
Filtration
There are present different types of filters that are used
for different purposes. Each of them are made of different
materials.
A filter can be made of

▪ Asbestos pad (Seitz filter),

▪ Diatomaceous earth(Berkefeld filter),
▪ Porcelain (Chamberland-Pasteur filter),
▪ Sintered glass disks(Sintered glass filter),
▪ Cellulose(Membrane filter),
▪ Borosilicate glass fiber(HEPA filter),
▪ mud(Candle filter).
 Membrane
◤ Filtration
Technique
 The Membrane filter is In biology laboratories,
also known as the membrane Filtration is
molecular or biological widely used to sterilize
filter. liquids.

Membrane
◤ Filtration
Technique The main purpose of the
Membrane filtration technique
is to ascertain the residence
The quality of water
and the quantity of or absence of a particular
microorganisms can coliform group that is usually
be determined by existing in wastewater and
using this membrane groundwater.
filtration method.
◤
Membrane filtration mechanism

• During membrane filtration, a part of fluid

termed as permeate (filtrate) moves
within the membrane, while other
components are expelled by the
membrane and clutched in the retentate
(concentrate) steam.
Membrane filtration Unit
◤
▪ Membrane filtration unit also known as the “Membrane filtration
assembly”.

Water
Sample

Funnel

Locking ring
Forcep
Membrane Filter

Vacuum pump Stainless base

Rubber stopper

Filter flask
◤

Membrane Filtration◤Technique
Advantages
▪ Quicker: quantitative results in
about 18 hours
▪ Less labour-intensive
▪ Requires less culture medium
▪ Requires less glassware
▪ Less sensitive
▪ Result obtained directly by colony
count (high precision)
▪ Readily adaptable for use in the
field
▪ No requirement for chemicals.
▪ Can remove 90–100% pathogens
from the water sample.
Disadvantages
▪ Not applicable to turbid waters
▪ There may be a risk of bacterial
abundance, as the water carries
numerous microorganisms.
▪ Glass filters are breakable and
can break quickly.
▪ The membrane filters can crack
easily.
▪ Only liquids are sterilized by this
method.
▪ Filters are costly to repair, mainly
nano-filters.
▪ Constitutional restrictions of
supplies used in filters alter the
effectiveness of this process such
as damage of glass filters, fracture
of the membrane filter, and
consumption of the filtrate by
Sietz filter.
▪ Require a high differential
pressure.
▪ Clogging can occur.
 Applications
Membrane Filtration◤Technique ▪ In industries and laboratories, it is used to sterilize the heat-labile
fluid materials.

▪ Most effective and acceptable method for filtration of drinking

water.

▪ In the pharmaceutical, cosmetics, electronics, and food and

beverage industries is is used to monitor the bacterial cells.

▪ Used in wastewater treatment.

▪ Used in cold sterilization of beverages and pharmaceuticals.

▪ Used for separation of milk fraction.

▪ Used to concentrating the proteins.

▪ Used for defeating skimmed milk and whey.

▪ Used for the partial demineralization of whey.

 Types of membrane filtration
◤
Microfiltration
It separates those particles that have
a size range of  0.1 to 10 μm.
Applications
In the water treatment plant, it is used
to separate pathogens such as the
protozoa Cryptosporidium and
Giardia lamblia, etc.

Ultrafiltration The pore size of an Ultrafiltration UF is used for the production of

membrane ranges from 0.1 μm to potable water. It removes the
0.01 μm. particulates and macromolecules
from raw water.

Nanofiltration The pore size of a Nanofiltration In fine chemistry and

membrane ranges from 0.001 μm to Pharmaceuticals industries, it is used
0.01 μm. The pore size of the for the recovery and management of
Nanofiltration membrane is smaller Non-thermal solvents, Room
than microfiltration and ultrafiltration. temperature solvent exchange.

Reverse osmosis (RO) The pore size of a Reverse osmosis it is used to purify the rainwater which
membrane ranges from 0.0001 μm to is collected from storm drains.
0.001 μm. It has the finest separation
membrane.
 Multiple-tube
◤
method
 It describes the statistical
estimation of total coliform density
in environmental water sources
such as fresh water, surface
water, ground water, etc. This test
can also be applied to wastewater
and effluent samples.

Multiple-tube
◤ method
The coliform group, as analyzed
for in this procedure, is defined as
all aerobic and facultative
anaerobic, gram-negative, non-
spore-forming, rodshaped
bacteria that ferment lactose with
gas formation within 48 hr at 35C.
Materials
◤

Flask
MacConkey broth
distilled water
Autoclave
Durham tube
Brilliant green lactose bile broth (BGLB)
Eosin methylene blue agar medium (EMB)
EMB medium
◤
◤
◤
 Advantages
Multiple-tube method
◤ ▪ Applicable to all types of water ▪ Slower: requires 48 houirs for
▪ May give better recovery of
stressed or damaged
organisms under some
circumstances.
Disadvantages
a negative or presumptive
positive result
▪ More labour-intensive
▪ Requires more culture
medium
▪ Requires more glassware
▪ More sensitive
▪ Result obtain indirectly by
statistical approximation (low
precision)
▪ Not readily adaptable for use
in the field
 Presence–
◤
absence tests
 The Presence Absence
(PA) test is a presumptive
detection for coliforms in
water

Presence–absence
tests
◤

this test is based on the

principle that coliforms and
other pollution indicator
organisms should not be
present in a 100 mL water
sample.
Materials
◤

▪ 20ml glass sampling bottle

▪ alcohol
▪ HACH PathoScreen Medium for 20ml
▪ 100ml glass bottle
▪ candle, lighter, alcohol
▪ UV lamp
▪ HACH Lauryl Typtose with Bromcresol Purple (LT/BCP) Broth with MUG
reagent for 100ml sample
◤
◤
 Advantages
Presence–absence◤tests ▪ No special skills are required
(anyone who can recognize
the species can do the
monitoring) and that the
monitoring requires very little
time.”
▪ Presence/absence sampling
can be a very fast method for
collecting information if the
only thing that is needed is
knowledge of whether or not
something was present
▪ .As such this method can
perform well for measuring or
understanding the distribution
of things across landscapes.
Disadvantages
▪  A significant limitation of this
method, however, is that it
does not provide information
on things like the abundance,
density, or condition of
individuals at the site. These
factors can be important
leading indicators of changes
that might take a long time to
be expressed through
changes in distribution.