Chapter 3: Determination of

Microorganisms in the Environment:

Microbial Analysis Methods

Environmental Microbiology (EnEg21012// By : Dr. Tsedekech G/mesekel// Year:2023/24

 Outline
Introduction
Culture-Dependent Methods
Plating method
Membrane Filter Method
Multiple Tubes Fermentation Method

Culture-Independent Methods
PCR based methods
Non PCR based Methods
 Introduction
 Microbial analysis: the study of microorganisms and their
characteristics, including their composition, diversity, and function
within a given sample or environment.

 Methods used in microbial analysis

 Culture-Dependent Methods: involve the cultivation of microorganisms
on specific growth media

 Culture-Independent Methods: methods bypass the need for cultivation

of microorganisms.
 Culture-Dependent Methods
General Media Used for Culturing Bacteria

 The major nutrient components of microbiological media

include:
 A source of carbon for incorporation in biomass, such as glucose for
heterotrophic bacteria or CO2 for autotrophic bacteria.

 Nitrogen, which is needed for growth and commonly supplied as

ammonia, proteins, amino acids, peptones, or extracts from plants or
meat.
 Culture-Dependent Methods

Buffers to maintain a suitable pH.

Growth factors such as defined trace minerals or metals.
Many media also contain selective components that favor the
growth of specific organisms while inhibiting the growth of non
target organisms

 For solid media used in plating, agar is the most commonly used
solidifying agent.
 Culture-Dependent Methods
1. Plating Method
 After serial dilutions, the sample is added to petri dishes containing a
growth medium consisting of agar mixed with selected nutrients. Two
types of platting method
A. Spread plate method, a 0.1 - ml aliquot of selected dilutions of the
sample is uniformly spread on top of the solid agar with the aid of a
sterile glass rod
B. Pour plate method, 1 - ml aliquots of appropriate sample dilutions
are mixed with molten agar (45°C) in a petri dish and allowed to
solidify
Dilution and Spread Plating Technique
Dilution and Pour Plating Technique
 Culture-Dependent Methods

 The spread plate technique is advantageous in that it allows

colonies to develop on the surface of the agar.
making it easier to distinguish different microorganisms on the
basis of morphology.
facilitates further isolation of the colonies.

 After plating, the samples are incubated under specified

conditions, allowing the bacteria to multiply into macroscopic,
isolated colonies known as colony-forming units (CFUs)
 Culture-Dependent Methods
 Main criticism: only a small fraction of the total population, as
observed microscopically, can be cultured on laboratory media
2. Membrane Filter Methods
 Widely used technique in microbiology for the enumeration and
isolation of microorganisms in liquid samples.

 It is particularly useful for water and food samples, where the

microorganisms are present in relatively low concentrations.
 Culture-Dependent Methods
 Preparation of the filtration system:
 The filtration apparatus consists of a filter holder, a membrane filter (usually
made of cellulose ester or nitrocellulose), and a vacuum source.

 The filter holder is sterilized before use.

 Sample filtration:
 The liquid sample is passed through the membrane filter using the vacuum. The
filter acts as a physical barrier, capturing the microorganisms present in the
sample while allowing the liquid to pass through.

 The size of the filter pores is selected based on the target microorganisms.
 Culture-Dependent Methods
 Transfer and incubation:
 After filtration, the membrane filter is carefully transferred onto a suitable
(selective) growth medium, e.g. m-FC agar for fecal Coliform .

 The filter is placed on the agar surface with the captured microorganisms facing
up.

 Incubation:
 The agar plates with the filters are incubated under appropriate conditions
(temperature (35oC total coliform, 44.5oC fecal coliform), humidity, and
duration) to allow the microorganisms to grow and form colonies.
 Culture-Dependent Methods
 Colony counting and analysis:
 After incubation, the colonies that have developed on the membrane
filter are counted and recorded.

 The number of colonies corresponds to the number of microorganisms in

the original sample. The colonies can be further characterized or
subjected to additional tests if necessary.
 Culture-Dependent Methods

 Fecal coliforms results

in blue colonies, and
non fecal coliform
colonies are gray to
cream colored
Membrane Filter Technique.
(a) Total Coliform test: Total coliform on a membrane filter (M-Endo MF broth medium).
Notice the dark red to purple colonies with a metallic sheen.
(b) Fecal Coliform Test: Fecal coliform on a membrane filter (M-FC broth medium).
Notice the blue-colored colonies
 Hints and Precautions

 The broth media should be freshly prepared on the day of the exercise.

 Water should not be used that is high in turbidity or contains a lot of

algae.

 Coliform density is always expressed in terms of a 100-ml water

sample.

 If the water sample will not be tested immediately, store it in the

refrigerator to prevent extra microbial growth.
 Culture-Dependent Methods
3. Multiple Tube Fermentation Methods (MTF)
 Rely on the ability of different microorganisms to ferment specific
substrates and produce characteristic metabolic end products.
 Principle of MTF
 Inoculating multiple tubes or vials containing different selective or differential media
with a sample suspected to contain a particular group of microorganisms.
 The tubes are then incubated under specific conditions to promote the growth and
metabolic activity of the target microorganisms.
 The presence or absence of specific metabolic end products is observed as an indicator
of the fermentation capabilities of the microorganisms.
 Culture-Dependent Methods

 Application in environmental microbiology

 MTF methods help identify microorganisms involved in the
degradation of various environmental pollutants.

 For example, the BTEX (benzene, toluene, ethylbenzene, and xylene)

degradation test employs MTF methods to determine the ability of
microorganisms to ferment these compounds.
 Culture-Dependent Methods
Steps in MTF
 Serial dilution to extinction

 Inoculate multiple tubes (5 or 10) of media with across the increasing series
of dilutions

 Incubate
 35oC or

 44.5oC

 Count positive growth tubes

 Use Most-Probable-Number (MPN) table to estimate density

 Culture-Dependent Methods

 MPN (Most probable number): is a method where a series of

dilutions is made to determine the highest dilution still yielding
growth.

 The MPN method evaluates detectable growth by observing

changes in turbidity, bubble or color due to metabolic activity.
The accuracy increases with the number of replicates examined.
Results are expressed as MPN units per 100 ml.
 Culture-Dependent Methods
 Based on the results one can estimate the number of viable microorganisms
in a sample using a statistical procedure.
 The statistical estimate of average number of cells per tube is based on
Poisson distribution (named after French mathematician Siméon Denis
Poisson.)
1. The average cell per tube can be calculated from the probability of
(frequency) of 0 cells per tube, i.e from the frequency of negative tubes in a
series of replicates giving both positive and negative results. In this cases,
P(0)=e-m
Where, m = the average number of cells per volume of sample used
P(0)= the fraction of negative tubes
Culture-Dependent Methods
 Culture-Dependent Methods
2. Using tables – statistical estimate of MPN for different dilutions and 95%
confident (Poisson distribution)
 Tables are used when three and more serial sample volumes (dilutions)
are conducted.
 Table are prepared for different volumes (10,1and 0.1 ) or dilutions (1,
0.1 and 0.01).
 Culture-Dependent Methods

Corrections on MPN estimate

If lower or higher serial dilutions are used , MPN values obtained from table must be
adjusted accordingly.

Example: Table is prepared for (10, 1 and 0.1 ml) and the sample is analyzed for
higher dilution (1m, 0.1ml and 0.01ml) the result (MPN) Value must be
multiplied by 10. Or

For some table when the dilution/sample is taken for lower one (100ml, 10ml,
and 1ml) the result MPN from table must be corrected by dividing with 10.
 Culture-Dependent Methods
 In situation where more than three test dilutions have been run; the
following rules are applied to select the three dilutions to be used in
determination of MPN value from table :

 Choose the highest dilution that gives positive results in all five portions
tested and the two next higher dilutions.
 Culture-Dependent Methods
Typical application of MPN method is the estimation of the number of coliforms in a sample of
drinking water,

MPN test is completed in three steps : -

1. Presumptive test (Total coliform)

Lauryl lactose brose

2. Confirmed test (Faecal coli forms )

mFC agar (membrane fecal coliform agar)

or m-Endo agar (membrane Endo agar).

3. Completed test (E.coli)

Eosin methylene blue agar

 Culture-Independent Methods

 Molecular method: the common approach is to target a small subunit

of the ribosomal rRNA (16S rRNA gene in bacteria and archaea, while
the 18S rRNA gene is used in eukaryotes).
I. PCR (polymerize chain reaction) based (clone library method,
genetic fingerprinting, DNA microarrays, or by a combination of
these techniques.)
II. Non PCR based (staining and florescent in-situ hybridization
(FISH))
 Culture-Independent Methods
 PCR (polymerase chain reaction) – is a technique used to amplify/
produce a copy of target DNA.
Template (sample DNA)
Primer (specific for certain genome)
Nucleotides
Polymerase

 Thermo-cycler (instrument)
 Culture-Independent Methods

 There are 3 steps in a PCR amplification cycle:

i. template denaturation;
ii. primer annealing; and
iii. primer extension.
 All 3 steps occur at different but defined temperatures and time
intervals
 These repeating cycles are performed in an automated, self-
contained, Thermo-cycler
 Culture-Independent Methods

 Non PCR based

1. Staining is technique used in microscopy to enhance contrast in the
microscopic image in which, stains and dyes are frequently used for
viewing, often with the aid of different microscopes. (specifically used for
identification of bacteria)

 Bacteria have nearly the same refractive index as water, therefore, when
they are observed under a microscope they are opaque or nearly invisible
 Culture-Independent Methods
Staining process
Fixation –aims to preserve the shape of the cells or tissue involved as much
as possible.
Heat fixation is used to kill, adhere, and makes them permeable so they
will accept stains.

 stain/dye
A stain is a substance that adheres to a cell, giving the cell colour.
The presence of colour gives the cells significant contrast so they are
much more visible.
 Culture-Independent Methods
Types of stain
 Basic dye stain: stains bacteria
 Acidic dye stain: stains background

 stains are basically made of dissolved organic salts: Consists of +ve and –ve
ions
One of these ions is coloured is called chromospheres
The uncoloured one is called counter ion

Basic / stain: when the +ve ion is the chromophore (colorant)

 this stains the bacteria : because bacterial cell is negatively charged
 Culture-Independent Methods

Acidic dye /stain: when the –ve ion is chromophore.

Different stains have different affinities for different organisms, or

different parts of organisms.

used to differentiate different types of organisms or to view specific parts

of organisms.

 Different types of staining methods are used to make the cells and their
internal structures more visible under the light microscope.
Culture-Independent Methods
 Culture-Independent Methods
 Fluorescent in situ hybridization (FISH)
 is a molecular biology technique used to detect and localize specific
nucleic acid sequences within cells or tissues.
 It combines the principles of nucleic acid hybridization and
fluorescence microscopy, allowing researchers to visualize the presence
and location of target DNA or RNA sequences.
 In this technique, the full set of chromosomes from an individual is
affixed to a glass slide and then exposed to a ―probe‖—a small piece of
purified DNA tagged with a fluorescent dye
 Culture-Independent Methods

 The fluorescently labeled probe finds and then binds to its matching
sequence within the set of chromosomes.

 With the use of a special microscope, the chromosome and sub-

chromosomal location where the fluorescent probe bound can be seen.
Culture-Independent Methods