Tools and Techniques with methods used in microbial ecology study. general ,not specific. Designed to meet the authors syllabus so may not be enough

1. Tools and techniques of Experimental Microbial
Ecology
Prashant Dahal
BSc. Microbiology Final Year
Sunsari Technical College, Dharan-1

2. CONTENTS
• Introduction to Microbial Ecology
• Experimental Microbial Ecology
• Components of Microbial Ecology
• Tools and Techniques in Experimental;
1. Microbial Ecology of air
2. Microbial Ecology of water
3. Microbial Ecology of soil

3. Microbial Ecology
• Ecology: study of relationship between biotic and
abiotic and among biotic components
• Ecosystem : community of living organisms in
conjunction with the nonliving components of their
environment, interacting as a system
• Study of relationship of microbes with one
another and other biotic and abiotic components.
• Ubiquitous, air, water and soil; even deep level (2-3km),
↑temp.(2-300 C), ↓temp. (<-20), Salt, Anaerobic, Heavy
metal, desert, rocks, inorganic components, organic
matters, saprophytic, commensals, etc.

4. Experimental Microbial Ecology
• Experimental study of the microbial
diversity in a ecosystem, their roles and
interactions .
• It includes the use of tools and techniques
to enumerate and define microbiome of a
ecosystem.

5. Components of microbial ecology
Biotic
Microbes
Eukaryotes
(plants, animals,
human, insects, ….)
Abiotic
Soil
water
Air

6. Interaction of microbes
With biotic components With abiotic components
1. Mutualism
2. Syntrophism
3. Commensalism
4. Proto-cooperation
5. Predation
6. Parasitism
7. Competition
8. Ammensalism
1. Deterioration
2. Degradation
3. Decomposition
4. Mineralization
5. Assimilation
6. Support growth
7. Suppress growth

7. Tools of Experimental Microbial
Ecology
• Microscopy (SEM / TEM)
• FISH (Fluorescent in situ hybridization)
• PCR
• Denaturing gradient gel electrophoresis (DGGE)
• Spectrophotometry
• Gene sequencing
• Winogradasky column
• Culture
• Biochemical tests (METABOLIC STUDY)
• Chemical study of Ecosystem

8. Winogradasky column
• is a miniature, self-contained ecosystem which models ecological
conditions in varying ways
• nvented by the Russian bacteriologist Sergei N. Winogradsky in
1880
• is a column of pond mud and water mixed with a carbon source such
as newspaper (containing cellulose), blackened marshmallows or
egg-shells (containing calcium carbonate), and a sulfur source such
as gypsum (calcium sulfate) or egg yolk.
• Incubating the column in sunlight for months results in
an aerobic/anaerobic gradient as well as a sulfide gradient.

10. FISH (Fluorescent in situ hybridization)
• allows for the quantification and identification of
microorganisms in complex habitats
1. Fixation of cells with formaldehyde or ethanol
2. Permeabilization of the cell membranes to the probe
3. Hybridization of fluorescently labeled oligonucleotide
probes to ribosomes within target cells
4. Removal of excess probe by washing
5. Visualization and quantification using epifluorescent
microscopy or flow cytometry

12. TECHNIQUES IN SOIL MICROBIOLOGY
• Soil is the first and most impo. microbial Ecosystem
• Uses 2 approaches to enumerate and define soil microbiota;
1. Analytical approaches; analysis of chemical, bio-chemical
matters and diversity of microbiota in ecosystem.
2. Synecological approaches; study the interrelationship,
structure, development and distribution of ecosystem.

13. • Several techniques are used by soil microbiologists;
1. Determination of form and arrangement of
microbes in soil
2. Isolation and characterization of soil microbes
3. Detection of microbial activity in soil
4. Determination of microbial biomass

14. Determination of form and arrangement of
microbes in soil
1. Microscopic examination of stained soil
(uncultured)
2. Fluorescent antibody techniques (FIA, FISH)
3. Hemocytometer approach
4. Contact slide method
5. Soil enrichment method
6. Non-selective Culture

16. Isolation and characterization of soil
microbes
• Soil serial dilution
• Inoculation in suitable media (selective)
and incubation
• Biochemical study

19. Detection of microbial activity in soil
1. Study growth curve to determine growth
rate
2. Study of enzymes produced
3. Rate and type of respiration
4. Product accumulation rate
5. Mycelial extension
6. Study of substrate utilization capacity
7. Study rate of substrate disappearance and
metabolite accumulation

21. Determination of Microbial biomass
1. Direct counting
2. Plate count method (CFU / g)
3. MPN method
4. Determining without counting;
a. Soil fumigation methods
b. Determination of ATP contents in soil
c. Determination of cell wall components

22. CFU/gram =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠
𝑣𝑜𝑙 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 ×𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
Eg: if 23 colonies from 1 ml sample inoculation
from 10−6 dilution plate, then,
𝐶𝐹𝑈/𝑔 =
23
1 × 10−6
= 23 × 106
𝑐𝑓𝑢/𝑔𝑚

23. TECHNIQUES IN AIR MICROBIOLOGY`
• Air is not the medium for growth and proliferation of microbes, it
only acts as temporary reservoir or transporter from several source
that makes microbes to enter the air current and remain there due to
air flow via. entrapped in water drops, aerosols or dusts or also in
spore form.
• So, no study of metabolites, substrate degradation
• Only focuses on microbial load, diversity and roles

24. 1. Direct microscopy;
2. Isolation and characterization
3. Determination of microbial biomass

25. Direct microscopy
1. Air is passed through filter or sticky surface (gravity
slide method is widely used, slide coated with
petroleum jelly or silicone grease – held horizontally
for limited time; microbes traps in the jell)
2. Staining of the material with trapped organism and
microscopy
3. Or, direct microscopy without staining

26. Isolation and Characterization
Two methods are commonly used for this purpose:
1. Solid impingement method (settle plate
method)
2. Liquid impingement method (passes air through
sterile water or liquid medium, and culture the
broth or transfer to solid medium)

28. Determination of microbial biomass
• Plate Count Method and calculation of CFU/𝑚3
• 𝑁 = 5𝑎 × 10−4(𝑏𝑡)−1
• N= CFU/𝑚3
• a = no. of colonies per petri plates
• b = surface area of the plate
• c = time of exposure in minutes

29. Techniques in WaterMicrobiology
• in general study is of 2 types; marine and fresh water
• Water is a good medium for growth as well as good
disseminator
• Coliforms (both FCB & TCB ) are indicator of water
being contaminated (only for pathological
significance // drinking water quality)
• But, in studying water microbiome, it is not followed

30. Techniques used
1. Determination of form and arrangement of
microbes in water
2. Isolation and characterization of water microbes
3. Detection of microbial activity in water
4. Determination of microbial biomass

31. Determination of form and arrangement
1. Direct microscopy
2. Antibody testing
3. Hemocytometer approach
4. Molecular approach

32. Isolation and Characterization
1. Serial dilution and culture (specific)
2. Direct inoculation
3. Membrane Filtration

36. Detection of microbial activity
1. Study growth curve to determine growth rate
2. Study of enzymes produced
3. Rate and type of respiration
4. Study of substrate utilization capacity
5. Study rate of substrate disappearance and metabolite
accumulation
6. Radioisotope cycling studies

37. Determination of microbial biomass
1. Direct counting
2. PC method (CFU/ml)
3. MPN method
4. Membrane Filtration Method (MF)
5. Determination of ATP content
6. Determination of cell wall components
7. Turbidity measurement method

38. MPN
• method used to estimate the concentration of viable microorganisms
in a sample by means of replicate liquid broth growth in ten-fold
dilutions.
• commonly used in estimating microbial populations in soils, waters,
agricultural products
• MPN test is performed in 3 steps
• Presumptive test
• Confirmatory test
• Completed test

40. THANK YOU !!