Microb Slides

Microbial
Ecology
Dr. Stacy
Stephenson-Clarke
stacy.stephenson02
@uwimona.edu.jm
MICR3213/BC31M:
Applied and
Environmental
Microbiology
Inspirational Quote of the Day
“We cannot fathom the marvelous complexity of an organic being; but

on the hypothesis here advanced this complexity is much increased.
Each living creature must be looked at as a microcosm--a little
universe, formed of a host of self-propagating organisms,
inconceivably minute and as numerous as the stars in heaven.”
― Charles Darwin
MICR3213
Timetable
MICR3213
Timetable
Course Outline
❑Microbial ecology
❑in situ measurement of microbial activity
❑Aquatic habitats: biomass distribution and oxygen relationships in lakes, rivers
and marine environments.
❑Biochemical oxygen demand and wastewater treatment: trickling filters,
activated sludge and anaerobic digesters.
❑Indicators of pollution. Soil as a microbial habitat: biodegradation of
xenobiotics, microbial remediation of polluted environments.
Course Outline (cont’d)
❑Deep subsurface microbiology.

❑Waterborne pathogens: their occurrence in nature, factors influencing
their presence in water supplies and means of control.
❑Industrial microbiology. Usefulness of microorganisms in
biotechnological applications and how the physiology of microbes are
related to their role in these processes
❑A practical section of 36 hours.
Course Assessments
❑Final Written Examination (2 hours): 60%
❑Course Work: 40%

▪ 2 In-course Tests 20%
▪ Tuesday Oct 19 at 12:00 noon
▪ Monday Dec 6 at 9:00 am
▪ Laboratory Reports 20%

Learning Objectives:
❑Understand the ecology of microorganisms at population, community and

ecosystem levels
❑Describe the approaches used to study microorganisms in their natural

environments and the limitations associated with each
❑Describe community function and dynamics at both the molecular and

the organismal level
Learning Objectives of
Course
❑Appreciate the (vast) genetic and physiological diversity of microbes, and

classify them based on their metabolic fuelling reactions
❑Understand how the specific environmental properties of soils, oceans and

biofilms affect microbial communities therein
❑Discuss how microbes are useful in biotechnological and environmental

applications such as sewage treatment, bioremediation, etc. and to relate
the physiology of microbes to their role in these processes
Microbial Ecology:
Further Reading
❑ See Madigan et al. (2020). Brock Biology of Microorganisms (16th
Ed). Person Education Limited
❑Chapters 19 and 20
Lecture Objectives
At the end of the lecture, students will be able to:
❑ Understand the ecology of microorganisms at

population, community and ecosystem levels
Microbial Ecology
❑ Study of inter-relationships between microorganisms and their
environments
ECOSYSTEM
COMMUNITY
GUILD
POPULATION
INDIVIDUAL
Microbial Ecology
Considerations
❑ Microbes and Ecosystem Niches

❑ Organization of Ecosystems
❑ Role of Microbes in Biogeochemical Cycling
❑ Microbial Environments and Microenvironments
General Ecological Concepts
❑Ecosystem
• The sum total of all organisms and abiotic factors in a
particular environment
❑Habitat
• Portion of an ecosystem where a community could reside
❑Population
• a group of microorganisms of the same species that reside in the
same place at the same time
• may be descendants of a single cell
❑Community
• a community consists of populations living in association with
other populations
Diversity of microbial species in an ecosystem is expressed in two ways

❑species richness: total number of different species present
❑species abundance: proportion of each species in an ecosystem
Microbial species richness and abundance are functions of the kinds
and amounts of nutrients available in each habitat
Ecosystem Service: Biogeochemistry and Nutrient Cycles
❑Guilds
• metabolically related microbial populations sets of guilds form
microbial communities that interact with macroorganisms and
abiotic factors in the ecosystem
❑Niche
• habitat shared by a guild
• supplies nutrients as well as conditions for growth
Microbial Ecology
Energetics and carbon
heterotroph
flow in microbial
metabolism
(e.g., NH4+, S, H2S, Fe2+)
autotroph
Objectives in Microbial Ecology
❑ Understanding the biodiversity of microorganisms in nature, and interactions in

communities
❑ Measurement of microbial activities in nature, and monitoring of effects on
ecosystems
❑ Activities commonly measured when studying microorganisms within an ecosystem:
❑ Primary production of organic matter (phototrophic, chemolithotrophic activity)
❑ CO2 + H2O + energy → new biomass
❑ Decomposition of organic matter (chemoorganotrophic/heterotrophic activity)
dead biomass → CO2 + H2O + energy
❑ Biogeochemical cycling of elements (C, O, N, P, S, Fe)
Microorganisms in Nature
❑Live in habitats suited to higher organisms, also in “extreme”

environments
❑ extremes in temperature, pH, pressure, salinity; anoxic habitats
❑ inanimate (soil, sediment, water, food) & animate habitats (on/in
animals, plants, insects)
❑ necessities for growth include available resources, suitable
physiochemical conditions
Psychrophiles, thermophiles,
hyperthermophiles: “extremophiles”
that live in habitats of extreme
temperature, including cold (e.g., deep
sea, Antarctica, the Arctic), or hot
habitats (e.g., compost piles, deep sea
hydrothermal vents)
Seawater evaporating ponds near San

Francisco Bay, used to harvest “solar”
salt. The red colour is due to pigments
of the extreme halophile
Halobacterium, an Archaeal species
that inhabits the ponds.
(Fig. 13.2(b), p. 423, Madigan & Martinko)

Microorganisms in nature
❑ Niche: the functional role of an organism
within an ecosystem; combined description
of the physical habitat, functional role, and
interactions of the microorganism occurring
at a given location
❑ Microenvironment: where a
microorganism lives, metabolizes within its
habitat
❑ physicochemical gradients
❑ spatial, temporal variability
Figure illustrates O2 contours within a soil

particle, measured by microelectrode. Each
zone could be considered a different
microenvironment
❑ Microcolonies in soil particles
❑ Very few microbes are free; most reside in microcolonies attached to soil
particles
❑ Soil aggregates can contain many different microenvironments supporting the

growth of several types of microbes
Environments and Microenvironments
❑Growth of microbes depends on resources and growth conditions

❑Differences in the type and quantity of resources and the physiochemical
conditions of a habitat define the niche for each microbe
❑realized niche or prime niche
• For each organism, there exists at least one niche in which that
organism is most successful
❑fundamental niche
• Full range of environmental conditions under which an organism can
exist
Resources and Conditions That Govern Microbial
Growth in Nature
Resources
Carbon (organic, CO2)
Nitrogen (organic, inorganic)
Other macronutrients (S, P, K, Mg)
Micronutrients (Fe, Mn, Co, Cu, Zn, Mn, Ni)
O2 and other electron acceptors (NO3−, SO42−, Fe3+)
Inorganic electron donors (H2, H2S, Fe2+, NH4+, NO2−)
Conditions
Temperature: cold→warm→hot
Water potential: dry→moist→wet
pH: 0→7→14
O2: oxic→microoxic→anoxic
Light: bright light→dim light→dark
Osmotic conditions: freshwater→marine→hypersaline
Nutrient levels and growth rates
❑ Microbial life in nature does not necessarily resemble microbial life
in lab culture
❑ Entry of nutrients into an ecosystem is often intermittent
❑ Feast-or-famine existence
❑ Adaptations
❑ Accumulate reserves in times of plenty
❑ High growth rate when growth possible; quiescence when growth is not possible
❑ Periods of extended exponential growth rare in nature
❑ Distribution of resources in nature is often non-uniform
❑ Competition for resources is likely
Microenvironments
❑Microenvironment
• The immediate environmental surroundings of a microbial cell or group of cells
• Soil particles contain many microenvironments
❑Physiochemical conditions in a microenvironment are subject to rapid change, both
spatially and temporally
❑Resources in natural environments are highly variable, and many microbes in nature
face a feast-or-famine existence
❑Growth rates of microbes in nature are usually well below maximum growth rates
defined in the laboratory
Environments and Microenvironments
❑Competition and cooperation occur between microbes in natural systems
❑Syntrophy
• microbes work together to carry out transformations that neither can
accomplish alone
• microbial partnerships are particularly important for anoxic carbon
cycling
• metabolic cooperation can also be seen in the activities of organisms
that carry out complementary metabolisms
General Ecological
Concepts: Symbiotic
Relationship
❑Many microbes establish relationships with other organisms (symbioses)

❑ Parasitism
❑ One member in the relationship is harmed, and the other benefits
❑ Mutualism
❑ Both species benefit
❑ Commensalism
❑ One species benefits, and the other is neither harmed nor helped
Examples of interactions between microbial populations
(i) Negative effect for (one or both) interacting populations:

* Competition – outcome depends on innate capabilities of nutrient uptake, metabolic rates
• “competitive exclusion” is one possible outcome
* Antagonism – specific inhibitor or metabolic product may impede growth/metabolism of others

• antibiotic or bacteriocin release, lactic acid production
(ii) Positive effect for (one or both) interacting populations:

* Cooperative interactions - interacting microbes must share same/nearby microenvironment
• Syntrophy – microorganisms together carry out transformation neither can conduct alone
(iii) Complementary metabolic interactions

• e.g., in nitrification: NH3 → NO2- (nitrosifying bacteria); NO2- → NO3- (nitrifiers)
• e.g., in S cycling: anaerobic sulfate reducing bacteria (SO42- → H2S) provide substrate for
microaerophilic sulfide-oxidizing bacteria (H2S → S0)
Surfaces and Biofilms
❑Surfaces are important microbial habitats
• Typically offering microbes greater access to nutrients and

protection from predation and physicochemical disturbances
• Nutrients adsorb to surfaces
• Attachment to a surface also offers cells a means to remain in a
favorable habitat, modify the habitat by their own activities,
and not be washed away
Biofilms
❑ assemblages of bacterial cells adhered to a surface and
enclosed in an adhesive matrix excreted by the cells
❑ The matrix is typically a mixture of polysaccharides.
❑ Biofilms trap nutrients for microbial growth and help prevent

detachment of cells in flowing systems.
❑ Biofilm formation is initiated by attachment of a cell to a

surface followed by expression of biofilm-specific genes.
❑ Genes encode proteins that synthesize intercellular

signaling molecules and initiate matrix formation.
Pseudomonas aeruginosa
❑ Biofilm producer
❑ Intracellular communication (quorum sensing) is critical in the
development and maintenance of a biofilm
❑ The major intracellular signaling molecules are acylated
homoserine lactones (AHL)
❑ Both intraspecies signaling and interspecies signaling likely occur in
biofilms
Biofilm: a community of microorganisms embedded in an organic
polymer matrix (extracellular polymeric substances, EPS), adhering to a
surface
❑ Surfaces are important
microbial habitats.
❑ Nutrients adsorb to surfaces and
microbial cells can attach to
surfaces
Time ❑ physicochemical gradients
within mature biofilm result in
a number of potential
microenvironments within a
small area
Biofilms: Heterogeneity
• Mature biofilm is a complex,
dynamic community of
microorganisms.
• Heterogeneity: differences in
metabolic activity and locations
of microbes.
• Interactions occur
among the attached
organisms.
• E.g. Exchanges take place
metabolically, DNA uptake and
communication.
Biofilms: Common in Nature
• Most microbes grow attached to surfaces (sessile) rather than free

floating (planktonic)
• Biofilm: attached microbes are members of complex, slime enclosed
communities.
• Biofilms are ubiquitous in nature in water.
• Can be formed on any conditioned surface.
Bacterial microcolonies developing on Natural biofilm on a leaf surface
a microscope slide immersed in a river ❑ cell colour indicates depth in biofilm: red (surface)
(phase contrast microscopy) → blue (18 μm deep)
(confocal laser scanning microscopy)
Biofilm developed on a stainless-steel pipe
❑ stained with DAPI (fluorescent; interacts with nucleic acids)
❑ note water channels through biofilm
Biofilm of iron-oxidizing prokaryotes on
rocks
Rio Tinto Spain
Evidence of a dental biofilm:

❑ left front tooth exposed to sucrose solution for 5 min while right served as
a control
❑ both then stained with iodine solution
❑ brown colouration results from reaction of iodine with extracellular
glucans (EPS) produced by the sucrose-supplied biofilm
Phototrophic Biofilms in Rivers and Streams
(a)Phototrophic biofilms colonizing the

rocky bottom of a stream flowing from
the Rhone Glaciers, Switzerland
(b)Cyanobacteria attached to the river

rocky in “tower-like” clusters with apical
oxygen bubbles
Thioploca Mats
(a, c) Filaments of the large sulfur-oxidizing

chemolithotroph Thioploca in the Bay of
Concepción off the Chilean coast.
(b) Thioploca form bundles of 10 to 20

filaments (trichomes) held together by a
gelatinous sheath
*Two species of Thioploca commonly

inhabit the same bundle: T. chileae and T.
araucae
Biofilms: Advantages to Microbes
❑ Bacteria form biofilms for several reasons:

❑ attachment to surface;
❑ nutrient trapping
❑ Self-defense
❑ Biofilms resist physical forces that sweep away unattached cells, phagocytosis
by immune system cells, and penetration of toxins (e.g., antibiotics), predators
❑ Allows cells to remain in a favorable niche (cooperative interactions
possible)
❑ Allows bacterial cells to live in close association with one another
Biofilms: Disadvantages to Microbes
❑highly competitive
❑localized biomass can be efficiently preyed upon,

infected by viruses
❑Can you think of any other?
Biofilms: Advantages to Humans
❑Exploitation of biofilms:
• slow sand filtration (water purification)
• microbial leaching of low-grade ores;
• Ferementation, vinegar production etc.
• Can you think of any other?
Biofilms: Disadvantages to Humans
❑Biofilms have been implicated in several medical and dental conditions.
• periodontal disease, kidney stones, tuberculosis, Legionnaires’ disease,
Staphylococcus infections, others
❑In industrial settings, biofilms can slow the flow of liquids through pipelines,
high microbial numbers in potable water distribution systems; accelerated
corrosion of pipelines and structural steelwork; increased drag on ship’s hull
❑Surface colonization and biodegradation of plastic alters the surface
chemistry, density, and sinking rates of MPs (Microplastics)
❑Few highly effective antibiofilm agents are available (https://www.future-
science.com/doi/pdfplus/10.4155/fmc.15.7#:~:text=These%20new%20antibiofilm%20age
nts%2C%20which,human%20medicine%20in%20the%20future.)
Microbial Mats
❑Microbial mats: very thick biofilms
❑ Built by phototrophic and/or
chemolithotrophic bacteria
❑ Photosynthetic mats contain filamentous
cyanobacteria
❑ Cyanobacterial mats are complete ecosystem
❑ Have existed for over 3.5 billion years
❑ Chemolithotrophic mats contain

filamentous sulfur-oxidizing bacteria
❑ Often found associated with hot springs, (Fig. 12.5, p. 334, Madigan & Martinko)
shallow marine basins
green:
cyanobacterial layers
(aerobic phototrophs)
~3 cm thick
orange:
layers of anoxygenic
phototrophic bacteria
A coring taken through a microbial mat from

a hot spring
Could you guess the microbes occupying the other layers?

Thioploca mat: Filaments/sheaths of large sulfur-oxidizing chemolithotroph off Chilean coast
Fossing et al. 1995; Jørgensen and Gallardo 1999
Current trends in microbial
ecology
❑ Space exploration – microbes in extreme environments (hot springs,

thermal vents, lithosphere)
❑ Molecular techniques – diversity of microorganisms (Carl Woese), new

methods to assess presence or abundance of individual species in situ
❑ Realization that with pure culture/enrichment techniques, we know

somewhere between 1-10% of existing microbial species – lots to learn!
❑ Biology of climate change, global biogeochemistry

Recent Discovery
❑ In 2008, Prof. Gary Strobel ,Montana State University and students explored the
Patagonia rainforest they found an endophytic fungus inside the tissues of the
Ulmo tree. The fungus Gliocladium roseum liberates a number of volatile
compounds in the air, the mixture is similar to diesel fuel and can be produced
when grown in the lab with good yields on cellulose - dubbed mycodiesel
❑ Also, Prof. Scott Strobel and a group of Yale students in 2012 found in the
Amazon rainforest a fungus Pestialotiopsis microspora that degrades
polyurethane (plastic). The fungus is able to survive on polyurethane alone
under anaerobic conditions
History of Microbial Ecology
❑The term “microbial ecology” really wasn’t in common use until the late
1960s
❑Why?
❑Microbial ecology has its roots in microbiology, rather than ecology
❑The history of the field is largely a transition from laboratory pure cultures to
studying organisms in nature…
Louis Pasteur
(1822-1895)
❑ “basic” vs. “applied” science

❑ fermentation = biological process
carried out by microorganisms.
❑ Germ theory = foundation of
brewing of beer, wine-making, and
pasteurization.
❑ Nature of contagious diseases: potato
blight, silkworm diseases, and
anthrax.
❑ Immunization (anthrax, rabies)
❑ Public experiments!
"Imagination should give wings to our thoughts but we always need decisive
experimental proof, and when the moment comes to draw conclusions and to
interpret the gathered observations, imagination must be checked and
documented by the factual results of the experiment."
Robert Koch
(1843-1910)
❑ Discovered the Bacillus

strains that cause
cholera and anthrax
❑ Agar media for pure

cultures (earlier had tried
sliced boiled potatoes!)
❑ Pure culture
paradigm: isolate an
❑ Disease and medical organism and see what it
microbiology does
❑ Pure culture paradigm
Pure Culture Paradigm
❑ Extremely important conceptual development in microbiology (and in microbial ecology,
too)
❑ Remove organisms from complex communities
❑ Isolate key processes
❑ Obtain reproducible results
❑ This method is still used today
❑ Attitude of Koch’s time:

❑ “Work with impure cultures yields nothing but nonsense and Penicillium glaucum“ (Oscar
Brefield 1881)
Sir Alexander Fleming (1929), examining exactly such an impure culture (Staphylococcus
culture contaminated by Penicillium), led to the discovery of penicillin.
Agar petri dish zone of no bacterial

growth,
Staphylococcus due to penicillin
colonies produced
by fungus
Penicillium
contaminant
Interference competition!
classic ecological process
Sergei Winogradsky
(1856-1953)
❑ Isolated nitrifying bacteria
❑ Winogradsky column:
microbial communities develop
along a gradient of oxygen
tension; method still used
today
❑ Described oxidation of
hydrogen sulfide, sulfur,
ferrous iron
❑ …all leading to the concept of
chemoautotrophy – deriving
❑ Bacteria: central in element energy from chemical oxidation
of inorganic compounds and
transformations
carbon from CO2
❑ Founder of soil
microbiology
Martinus Beijerinck
(1851-1931)
“The way I approach

microbiology...can be concisely
stated as the study of microbial
ecology, i.e., of the relation
between environmental
conditions and the special forms
of life corresponding to them”
Founder of the Dutch Delft

School of Microbiology
Martinus Beijerinck (1851-1931)
❑ “a man of science does not marry”
❑ Isolated N fixers and S reducers
❑ ‘Microbial ubiquity’: all microorganisms are everywhere; conditions and
resources determine who flourishes
❑ Enrichment culture: growth medium tailored to suit particular metabolic
function
❑ With Winogradsky, recognized that microbes are the major players in
element transformations
❑ Led to field of global biogeochemistry
Albert Jan Kluyver
(1888-1956)
❑ Student of Beijerinck
❑ Microbial
physiology
❑ Comparative
approach
❑ Unifying metabolic
features among
microbes
❑ Leader of the Dutch

school after Beijerinck
Cornelius Bernardus
van Niel (1897-1985)
❑ Student of Kluyver, third in the
Dutch Delft School
❑ Isolated purple sulfur
bacteria
❑ Major contribution, chemistry of
photosynthesis:
❑ H2A + CO2 → CH2O + 2A + H2O
where A can be S or O
❑ Extended model green plants;
oxygen from water, not from CO2
❑ Also, chemistry of denitrification,
definition of prokaryote in 1961
(with R. Stanier)
❑ Taught lab course focusing on studying microbes from nature
(first course in microbial ecology?)
❑ Philosophy of hypothesis testing, falsification “moving from
clearly erroneous to more ‘correct’, but never immutable
conclusions”
Robert E. Hungate
(1908-2004)
❑ Student of van Niel
❑ Developed methods for

isolating anaerobes
❑ Devised culture methods that

select using natural substrates,
rather than guesses about what
organisms eat
❑ Microbiology of guts of rumen,

termites
❑ ASM president when

❑ AKA “Grampa Bob”
“Environmental Microbiology” and
“Microbial Ecology” formally
recognized
Other contributions
❑1960s: Ronald Atlas, Richard Bartha

❑ Studies of petroleum degradation
❑ Led to new field of bioremediation,
❑ Extended to many other pollutants: DDT, PCBs, mercury, selenium, industrial
solvents
❑1970s fuel-shortage:
❑ Shortage in N fertilizer
❑ Sparked interest in the biology of nitrogen fixers
Methods for Studying
Microbial Ecology
[Part 1]
MICR3213 [BC31M]: Applied and

Environmental Microbiology
Dr. Stacy A-M Stephenson-Clarke

stacy.stephenson02@uwimona.edu.jm
Learning Objectives – Part 1
❑ Explain why, to the best of our knowledge, most microorganisms resist culturing in the
laboratory
❑ Describe the use of enrichment cultures
❑ Explain why microbial communities are often investigated in situ
❑ Describe why, when, and how FISH and CARD-FISH are used
❑ Summarize how PCR is used to take a microbial census
❑ Explain why and how DGGE is used
❑ Describe the use of phylochips to assess microbial communities
❑ Differentiate metagenomics from metaproteomics
2 9/15/2021 Add a footer

❑ Explain how microbial ecologists measure community activity
❑ Describe why microelectrode measurements are important tools in the study of
microbial community activity
❑ Compare the application of traditional stable isotope analysis with stable isotope
probing
❑ Assess the advantages and disadvantages of measuring in situ mRNA abundance
❑ List the type of data that can be generated by MAR-FISH and compare this with
functional gene arrays
❑ Predict which techniques would be appropriate for assessing the quantity versus
diversity versus activity of a microbial community

Microbial Ecology Culturing Methods
• Microbial ecology is an interesting and different take on the standard
in-lab culturing methods.
• The idea takes into account the many microbes that cannot be
cultured—so how can we change what we’re doing to study them
when we can’t grow them?
• This section introduces a number of tools/methods for studying

microbes in their natural environments (in situ) and when they can’t
grow in the lab setting.
Methods in Microbial Ecology
• Understanding the biodiversity of microbes, and interactions in

communities
• Measurement of microbial activities, and monitoring of effects

on ecosystems
• There are many challenges associated with studying microbial

systems
Microbial Ecology
• Interdependent series of inquiries involving traditional, laboratory-based analyses,
metagenomics, and in situ biogeochemical assessments.
• Techniques to culture microbes (gold standard).
• Methods used to measure the activities in nature.
Methods in Microbial Ecology
• Culture-dependent methods
•Enrichment and isolation
•Culture methods
•Enrichment bias
•MPN
• Culture-independent methods
•Most accurately represent populations and communities
•Our focus
Culture-dependent methods: Enrichment
• Enrichment cultures
• Can prove the presence of an organism in a habitat
• Cannot prove that an organism does not inhabit an environment
• The ability to isolate an organism from an environment says nothing about its
ecological significance
• Enrichment bias
• Microorganisms cultured in the lab are frequently only minor components of the
microbial ecosystem
• Reason: the nutrients available in the lab culture are typically much higher than in nature
• Dilution of inoculum is performed to eliminate rapidly growing, but quantitatively insignificant,
“weed” species
Reasons Microbes May Be “Unculturable”
Potential Problem Example Method Designed to Overcome
Microbe is slow growing. Incubation times of weeks to months
Microbe is present in very low abundance. Extinction cultures, using many replicates
Different microbes in same habitat are Remove other microbes from the sample by physical methods
physiologically very similar. such as filtration or density-gradient centrifugation or use
OR extinction cultures.
Inhibition by other microbes in a mixed culture
Fastidious growth requirement Assess growth requirements of similar microbes, if known. Use
annotation of metagenomic sequences to infer nutritional
capabilities and requirements; grow in diffusion chambers that
allow influx of small molecules from natural environmental
samples without microbial contamination.
Cross-feeding or communication signals from Co-cultivate strains; use diffusion chambers; grow in
other microbes are needed. conditioned spent medium of “helper” microbe.
Triggers for growth or exit from a dormant state Add known growth triggers such as N-acetylmuramic acid.
are not present.
Three Reasons for Culture Discrepancy
❑ Viable but nonculturable (VBNC)
• Motility, dividing cells, grows in nature, or stains with
dyes for living cells.
❑ Great plate count anomaly (GPCA)

• Discrepancy between number of microbial
cells observed by microscopic examination
and number of colonies that can be
cultivated from the same natural sample.
❑ Not the right conditions for growth of

environmental microbes.
• Enrichment cultures.
©Dr. Rita B. Moyes

Culture-dependent methods: Isolation
❑Pure cultures contain a single kind of microorganism

• Can be obtained by streak plate, agar shake, or liquid dilution
❑Agar dilution tubes/plates are mixed cultures diluted in molten agar
• Useful for purifying anaerobic organisms
❑Axenic (pure) culture verified by

• Microscopy
• Observation of colony characteristics
• Tests of the culture for growth in other media
❑Laser tweezers (flow cytometry) useful for
• Isolating slow-growing bacteria from mixed cultures
Most Probable Number (MPN)
Technique for estimating
the number of microbes
in a natural sample.
• Samples include food or
water.
• Serial dilutions and
observation of growth.
• Microbe must be capable
of growth in laboratory.
Culture-dependent methods: Approaches to Microbial
Growth
• Use of unusual electron donors and acceptors.
• Prolonged time in culture.
• Unusual cell densities in nature (too low to appear turbid).
• Allowing bacteria to exchange secreted metabolites and signaling
molecules through filters or gels.
• Extinction culture technique.

• Dilution of natural sample to fewer than 10 cells.
• Incubation and screening for growth.
Culture-dependent methods: Microbial Growth—Flow
Cytometry
• Cells are tagged with a
fluorescent dye and injected into
a flowing stream of fluid.
• As the diameter of the stream

narrows, one cell at a time is
forced through a thin tube,
detected by a laser beam.
• Can detect population based on

cell size and morphology.
Culture-dependent methods: Isolation of Individual Cells
❑ Optical tweezers
• Laser beam used to drag microbe away from its neighbors if no
axenic (pure) culture.
❑ Isolation can be followed by analysis of organism’s DNA

for phylogenetic analysis or single cell genomic
sequencing.
Methods: Identity vs Function
• Culture-Independent Microscopic Analyses: Viability staining, Fluorescent Protein
Tags (e.g. GFP), FISH, CARD-FISH
• Culture-Independent Genetic Analyses of Microbial Communities: PCR, DGGE, T-

RFLP, ARISA, Microarrays, Metagenomics, Metatranscriptomics and
Metaproteomics
• Measuring Microbial Activities: Chemical assays, Radioisotopic methods (e.g. 14C,

35S ), Microsensors (e.g. microelectrodes), Stable isotopes (e.g. SIP, SIF)
• Linking Genes and Functions/Activity to Microorganisms: SIMS, NanoSIMS, Flow

Cytometry, MAR – FISH, DNA-SIP
Outline
I. Staining techniques: Culture-Independent Microscopic
Analyses of Microbial Communities
II. DNA-based techniques: Culture-Independent Genetic
Analyses of Microbial Communities
III. Measuring Microbial Activities
IV. Linking Genes and Functions to Microorganisms
❖ SEE BROCK Biology of Microorganisms 16th Ed – Chapter 19

❖ https://www.researchgate.net/publication/51905295_Culture-
independent_methods_for_studying_environmental_microorgan
isms_Methods_application_and_perspective
Staining Techniques: Examination of Microbial
Community Structure
❑ The most direct way to assess microbial community

structure is to directly observe communities in nature.
• Assessment can be done in situ using immersed slides.

• Assessment also by electron microscope grids placed in
locations of interest and then observed later.
I. Culture-Independent
Microscopic Analyses:
General Staining Methods
• Viability stains: enumerate and
differentiate between live and dead
cells
•Two dyes are used
•Stains based on integrity of cell membrane
•Green cells are live
•Red cells are dead
•Limitation: Can have issues with
nonspecific staining in environmental
samples
i. Culture-Independent Microscopic Analyses: General
Staining Methods
• Fluorescent staining using ', 6-diamidino-2-phenylindole (DAPI), acridine
orange (AO), or SYBR Green I (SYBR)
• DAPI-stained cells fluoresce bright blue (a)
• AO-stained cells fluoresce orange or greenish orange (b)
• SYBR-stained cells fluoresce green (c)
• Fluoresce under UV light and are used for the enumeration of microorganisms in environmental, food
and clinical samples
• Nonspecific and stain nucleic acids such as DNA (A-T rich regions)
• Limitation: Cannot differentiate between live and dead cells due to non-specific staining
I. Culture-Independent Microscopic Analyses: General
Staining Methods
• Fluorescent tags
•Fluorescent labelled antibodies
• Means of identifying or tracking microbes
•Fusion proteins (e.g. GFP gene) expressed in engineered cells
• Assess the effect of disturbances in microbial populations
I. Culture-Independent Microscopic Analyses: General
Staining Methods
• Green fluorescent protein can be genetically engineered into
cells to make them autofluorescent.
• Can be used to track live bacteria and bacterial processes such as infections
• Can act as a reporter gene to identify when a particular promoter is active
Limitation of Microscopy
• Inherent limitations exist for the use of microscopy as the sole research
tool.
• Staining methods do not accurately reveal the astounding diversity of

microorganisms, which may appear identical but are genetically distinct
• Thus, microscopic techniques are often coupled to or supplemented with

molecular-based tools that help reveal phylogenetic diversity.

I. Culture-Independent Microscopic Analyses :Molecular
Approaches to Staining
Fluorescent in situ hybridization (FISH)

• Fluorescently labeled oligonucleotides
(probes) specific for microbe of interest.
• Small subunit (SSU) rRNAs are commonly
used probes, specific to the microbe of
interest.
• When hybridized, probe fluoresces;
detected by epifluorescence microscopy.
• Video:
https://www.youtube.com/watch?v=b81DcJ
C1jAs
©Seana Davidson
I. Culture-Independent Microscopic Analyses: FisH
(Fluorescence in situ Hybridisation)
• Fluorescent Nucleic acid probe: DNA or RNA
complementary to a sequence in a target gene or
RNA
• Uses fluorescently labeled nucleotide probes to

label whole cells
• Typically rRNA sequence is used to design probes

specific to an organism
NB: The FisH method overlaps with
• Organisms identified based on their phylogeny culture-independent methods used
to link genes to microbial function
I. Culture-Independent Microscopic Analyses: FisH (Fluorescence
in situ Hybridisation)
• Fluorescing nucleotides complementary to rRNA

sequence
• Cells that possess the sequence become fluorescent
• FISH technology can also employ multiple
phylogenetic probes (colors specific to organism,
species, strains etc)
• Use different colored dyes to label different cells
different colors
❑ Advantage: Can be used for
quantitative analysis
❑ Can you think of any possible

• Application: Used in microbial ecology, food industry, disadvantage(s)?
and clinical diagnostics
I. Culture-Independent Microscopic
Analyses: FisH - Methodology
• To check whether a particular organism exists in a sample and
in what proportion:
• Find out the sequence of your organism (from database such as
NCBI)
• Make a probe (oligonucleotide) that is complementary to the
rRNA of the organism and label it with fluorescent probe.
• Fix your sample on a microscope slide and wash with labelled
probe.
• Look down a fluorescence microscope and see in which cells the
fluorescent probe has bound
I. Culture-Independent Microscopic Analyses: FISH
Methodology
I. Culture-Independent Microscopic Analyses: Usefulness of
FISH Method
Can identify a particular species, strain, or ecotype.
• Useful in clinical diagnostics and food microbiology.
Variation → CARD-FISH (Catalyzed reported deposition-FISH)

• Modification of FISH used to amplify the signal produced by microbe
cells in low numbers.
• Couple fluorescent probe with enzyme that makes lots of fluorescent
product when exposed to substrate.
I. Culture-Independent Microscopic Analyses: CARD-FISH
• Catalyzed reporter deposition FISH
• Used to measure gene expression in organisms in a natural sample
• Enhances the fluorescence signal for detection
• Useful in phylogenetic studies of prokaryotes that may be growing slowly (e.g.
microbes inhabiting open oceans of low temp. and nutrient conc.) with few
ribosomes and few mRNA
•Nucleic acid probe contains enzyme peroxidase conjugated instead of
fluorescent dye
•After hybridization, preparation treated with fluorescently labelled compound
(tyramide), which is substrate for peroxidase
•Substrate is then converted to reactive intermediate that covalently binds to
adjacent proteins
•Signal sufficiently amplified to be detected by fluorescence microscopy
I. Culture-Independent Microscopic Analyses:
CARD-FISH
https://www.arb-silva.de/fish-probes/fish-protocols/
I. Culture-Independent Microscopic Analyses: CARD-FISH
I. Culture-Independent Microscopic Analyses: BONCAT-
FISH
• A direct measure of translational activity
• Utilizes compounds are synthetic molecules that mimic natural metabolites
Bioorthogonal noncanonical amino acid tagging (BONCAT) combined with FISH

BONCAT - FisH
• Methionine analog (HPG) carrying alkyne groups is incorporated by the
cell in growing polypeptides.
• When treated with the fluorescent dye-labeled azide, the azide group
on the dye binds to the alkyne group on HPG to yield a fluorescent
protein - detected
Terms
• Bioorthogonal: refers to any chemical reaction that can occur inside of living
systems without interfering with native biochemical processes
• Noncanonical: deviation from naturally occuring pathway; Alternative pathways
• HPG = l-homopropargylglycine (methionine analog)
II. Culture-Independent Genetic Analyses of Microbial
Communities: Nucleic acid-based techniques
1) PCR Methods of Microbial Community Analysis
2) Microarrays for Analysis of Microbial Phylogenetic and Functional

Diversity
3) Environmental Genomics and Related Methods

Molecular Techniques
❑ DNA is extracted from soil, water, blood.
• Can then be used as template for amplification of specific genes by PCR or as
template for shotgun metagenomics.
❑ SSU rRNA analysis is used to identify community populations.

• DNA amplified by PCR directly from the environment.
• Protein-coding genes used to define phylotypes.
❑ Internal transcribed spacer region (ITS) between 16S and 23S rRNA
genes may also be used.
❑ PCR bias—certain nucleotide templates are more readily amplified

than others.
II. Culture-Independent Genetic Analyses of Microbial Communities:
1. PCR Methods of Microbial Community Analysis
• Specific genes can be used as a measure of diversity
• Total community DNA is isolated from the habitat, and that PCR amplifies
the target gene from all organisms containing the gene, which generates
many copies of each gene variant, referred to as a phylotype.
• Techniques used in molecular biodiversity studies

• DNA isolation and sequencing
• PCR
• Restriction enzyme digest
• Electrophoresis
• Molecular cloning
Steps in single-gene biodiversity analysis of a microbial community
Communities: 1. PCR-based Methods of Microbial Community
Analysis: Uses
As a preliminary step to amplify
As a stand-alone diagnostic
DNA for ARDRA, DGGE, SSCP or
tool
sequencing
PRIMERS
Universal or specific to a certain Specific ONLY to one organism or group
group of organisms of organisms
APPLICATIONS
Identifying novel taxa and Identifying the presence of known
community analysis organisms in the environment
1. PCR Methods of Microbial Community Analysis: Real Time PCR
• Purpose: to quantify the amount of template that is amplified by PCR (in real time)
• Can be used to quantify different species present in a community
• Need: Primers specific for the organisms you want to quantify
Molecular analysis
of microbial Native microbial
populations
Communities
Universal primers
Targeted analysis Extract DNA
Domain-specific primers
PCR
Kingdom-specific primers
Mixed 16S rRNA
Separate by cloning
into E. coli
Sequencing
Phylogenetic
identification
Communities: DNA Fingerprints - DGGE
❑ Denaturing Gradient Gel Electrophoresis (DGGE)
• Uses gradient of DNA denaturing agents to separate DNA

fragments.
• DNA that would form a single band on a non-gradient gel will
resolve into separate fragments (multiple bands).
• Popularity has declined in recent years due to poor gel-to-gel
reproducibility.
Communities: 1. PCR Methods of Microbial Community
Analysis: DGGE
• Denaturing gradient gel electrophoresis (DGGE) separates genes of
the same size based on differences in base sequence
• Denaturant is a mixture of urea and formamide
• Strands melt (denature) at different denaturant concentrations
Communities: 1. PCR Methods of Microbial Community
Analysis: DGGE methodology
• Once selective genes are amplified, PCR products are run out on an electrophoresis
gel; bands are cut out and then run on a DGGE gel.
• The sequences are the same size as they are all the same PCR produce. However, they
may differ in sequence.
• DGGE will separate several fragments of equal size based on sequence.
• Strands melt at different denaturant concentrations.
• Application:
•DGGE can be used to analyze mixed microbial samples from complex communities, such as sewage
and soil.
•Emphasizes value of interdisciplinary approaches to ecological and microbial molecular studies
DGGE analysis of 16S rRNA sequences
Step1 : PCR Amplification
Mixed Population DNAs PCR Primers Product
Separate on
+ Denaturing
16S rRNA Gene Gradient Gel
Step 2: Denaturing Gradient Gel Electrophoresis

Purified Bands for Sequence
Analysis
Mix A B C
Separation Based on
Differences in
Denaturant
Increasing
Nucleotide Sequence
(G+C content)
and Melting
Characteristics
DGGE study of temperature distribution of Octopus Spring
cyanobacterial mat 16S rRNA variants
ecotype
“Who is where?”
Communities: 1. PCR Methods of Microbial Community Analysis:
T-RFLP
• Terminal restriction fragment length polymorphism (T-RFLP):
• Target gene is amplified by PCR
• Restriction enzymes are used to cut the PCR products
• Use: Indicates biodiversity by generating DNA fingerprints of phylotypes based on # and
location of restriction sites in a specific length of DNA (e.g. 16S rRNA)
• METHODOLOGY:
• PCR with fluorescently labelled primers, so all PCR products have fluorescent terminal.
• Digest PCR products with restriction enzymes.
• Gel electrophoresis of digests
• Read results with a laser.
• The brighter the signal the more copies of DNA.
• Can identify individual species by checking T-RFLP pattern of pure culture.
T-RFLP (Terminal-Random Fragment Length Polymorphism)
• PCR with fluorescently labelled primers, so all
PCR products have fluorescent terminal.
• Digest PCR products with restriction enzymes.
• Run restriction digest on a gel and read with a
laser.
• The brighter the signal the more copies of DNA.
• Can identify individual species by checking T-
RFLP pattern of pure culture.
Brightness
• Video:
of Fluor.
https://www.youtube.com/watch?v=swQ_pm4c
R5o
Size of fragment
ARISA
• ARISA: automated ribosomal intergenic spacer analysis
• Related to T-RFLP; Uses DNA sequencing
• Use: Provides detailed info of phylotype diversity by analyzing the internal transcribed spacer (ITS) region,
which is length of DNA that separates 16S rRNA gene from 23S rRNA gene
• ITS region variable in length and base sequence in separate phylotypes. Hence diversity revealed
ARDRA
• ARDRA: Amplified Ribosomal DNA Restriction Analysis

• Amplify target rRNA gene of interest and clone into E. coli
• Amplify rRNA gene out of E.coli
• Digest DNA with restriction enzymes, and run restriction digest on an agarose gel
• Advantage: Can identify ARDRA types by sequencing or just make measure of

diversity by numbers of patterns
II. Culture-Independent Genetic Analyses of Microbial Communities: 1.
PCR Methods of Microbial Community Analysis: Quantitative
Measurement
• How many of each taxon are present????

• Terminal restriction fragment length polymorphism (T-RFLP)
• Fluorescent in situ hybridization (FisH)
• Real-time PCR
• DGGE and ARDRA can also give an idea of which organisms are most abundant.
• NB: true quantitation is virtually impossible.

• Disadvantage: PCR bias leads to unfair representation of quantity of each species present.
PCR Methods of Microbial Community Analysis: Next Gen. Seq.
❑Next-generation DNA sequencers such as MINion do not require a cloning

step
❑PCR products can be used directly for sequencing
❑Allows for deep sequence analysis and the detection of minor phylotypes
❑Results of PCR phylogenetic analyses shows that:

•Several phylogenetically distinct prokaryotes are present
•rRNA sequences differ from those of all known laboratory cultures
•Molecular methods conclude that fewer than 0.1% of bacteria have been
cultured and that enrichment bias is a real problem to culture-based
methods
2. Microarrays for Analysis of Microbial Phylogenetic and Functional
Diversity
• Microarrays are research tools with known short sequences

(oligonucleotides) attached to a slide.
• Total community DNA is then hybridized to the slide.
• If the community DNA contains the same DNA, it will bind to its complement on
the slide and light up.
• Advantage: Microarrays circumvent time-consuming steps of DGGE and T-RFLP
• Video: https://www.youtube.com/watch?v=0ATUjAxNf6U
Microarrays for Analysis of Microbial Phylogenetic and Functional
Diversity
• Phylochip: microarray that focuses on phylogenetic members of microbial
community
• Functional gene microarray (Geochip): microarray that focuses on genes of

biochemical significance
• Advantage: Encompasses many different metabolic pathways
Diversity: Phylochips
• Specialized microarrays used to access microbial diversity in natural
samples without nucleotide sequencing.
• Analysis of DNA hybridization reveals presence/absence of specific
genes in environment.
Diversity: Phylochips - Methodology
• Thousands of genes in the microbial community can be probed for at once
•Method: Affix oligonucleotide probe of targeted gene to the chip surface (glass,
plastic, silicon)
•Note position of each on the chip
• Isolate community DNA and amplify by PCR, fluorescently label 16S rRNA genes
•Add DNA to probe on the chip
•Fluorescence indicates hybridization to the probe and indicates presence of the
specific gene
Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity:
Phylochips
❑Advantages:
i.Do not require cloning and sequencing,
which saves time.
ii.fast and cost-effective tool to detect

specific microorganisms and study
gene expression
Diversity: Phylochips
• Disadvantages:
i. Expensive; price is not decreasing as it is with most sequencing
methods.
ii. May not be as specific as other methods, yielding false positives
by detecting sequences that are close but not exact.
iii.Though results are comparable, optimization of hybridization
conditions for a large number of probes may be challenging.
iv.Probe and array designing of probes and arrays time
consuming.
ITRC (Interstate Technology & Regulatory Council).
2013. Team. www.itrcweb.org.
ESD News and Events, 2008

Diversity: Geochips
Environmental Multi-Omics: Integration of Genomics, Transcriptomics,
Proteomics, and Metabolomics
❑A more complete understanding of how a microorganism functions

requires an integrated accounting of all central cellular processes.
• These include gene expression, functional knowledge of all gene products and
product activities, and all metabolites produced during growth.
❑Multi-omics : methods of genomic, transcriptomic, proteomic, and

metabolomic analysis required to unveil patterns of microbial diversity
that will ultimately lead to a more predictive understanding of microbial
community function and response to environmental change.
Communities: 3. Environmental Genomics and Related
Methods
• Environmental genomics (metagenomics)

• Method: DNA is cloned from microbial community and sequenced
• Detects as many genes as possible
• Yields picture of gene pool in environment
• Can detect genes that are not amplified by current PCR primers
• Powerful tool for assessing the phylogenetic and metabolic diversity of an environment
• Metatranscriptomics
• Analyzes community RNA
• Reveals genes in a community that are actually expressed
• Reveals level of gene expression
• Metaproteomics
• Measures the diversity and abundance of different proteins in a community
Methods: Metagenomics
❑ Metagenomics applied to diverse natural habitats

• Identification of new genes and gene products from uncultured
microbes.
• Assembly of whole or partial genomes.
• Comparisons of community gene content from microbial assemblages
of different origin.
Methods: Metagenomics
❑ Clone DNA fragments from environment to avoid PCR bias

• Predict biogeochemical conditions of a habitat based on its
metagenome.
❑ mRNA and cDNA

• Can identify previously unidentified genes.
• Shows active genes.
Methods: Metaproteomics and Metabolomics
❑ Metaproteomics
• Identify each protein present in a given microenvironment at the
time of sampling.
• Identifies the metabolic processes active at the time the community
was assessed.
❑ Metabolomics
• the comprehensive analysis of cellular and extracellular metabolites
of a microbial community
Communities: 3. Environmental Genomics and Related Methods:
Metaproteomics Procedure
❑ 2-D polyacrylamide gel electrophoresis

• Total peptide mass and amino acid sequence information is then used to
search databases.
❑ 2-D nano-liquid chromatography used when qualitative data

are most important
• Peptides are first separated by charge and then by hydrophobicity.
• Peptide masses determined by mass spectrophotometry.
• Tandem Mass Spectrophotometry is used to obtain amino acid sequence
information.
3. Environmental
Genomics and Related
Methods: Single gene vs
environmental genomic
approaches to Microbial
Community Analysis
3. Environmental Genomics and Related Methods: Multi-Omics
Methods Overview
III. Culture-Independent Genetic Analyses of Microbial
Communities: Nucleic acid-based approaches
❑ Two approaches for assessing the genetic diversity and structure of microbial
communities
• Whole community DNA analysis: characterizes all the genetic information

in the population
• Partial community DNA analysis: Particular targeted sequences are

amplified by PCR and studied in detail
III. Culture-Independent Genetic Analyses of Microbial
Communities: Nucleic acid-based approaches
❑ Whole community DNA analysis
• Guanine + Cytosine (GC) content

• Provides a coarse resolution of the different taxonomic groups present
• DNA reassociation/hybridization
• Rate of reassociation/hybridization depends on the similarity of sequences present
• Cross DNA hybridization (e.g., microarrays) – already covered

• A single array can contain thousands of DNA sequences
III. Culture-Independent Genetic Analyses of Microbial Communities:
Nucleic acid-based approaches
❑ Partial community DNA analysis
• Amplification of targeted DNA via PCR

• 16S rRNA gene analysis
• Denaturant gradient gel electrophoresis
(DGGE)
• Intergenic transcribed spacer (ITS) analysis
• Fluorescent in situ hybridization (FISH)
• Terminal restriction fragment length
polymorphism (tRFLP)
• Real-time PCR (RT-PCR)
Methods for
studying
microbial ecology
[Part 2]
Dr. Stacy A-M Stephenson-Clarke

stacy.stephenson02@uwimona.edu.jm
MICR3213 [BC31M]: Applied and
❑ Explain how microbial ecologists measure community activity
❑ Describe why microelectrode measurements are important tools in the study of
microbial community activity
❑ Compare the application of traditional stable isotope analysis with stable isotope
probing
❑ Assess the advantages and disadvantages of measuring in situ mRNA abundance
❑ List the type of data that can be generated by MAR-FISH and compare this with
functional gene arrays
❑ Predict which techniques would be appropriate for assessing the quantity versus
diversity versus activity of a microbial community

Outline
I. Experimental design and sampling
II. Staining techniques: Culture-Independent Microscopic Analyses of
Microbial Communities
III. DNA-based techniques: Culture-Independent Genetic Analyses of
Microbial Communities
IV. Measuring Microbial Activities
V. Linking Genes and Functions to Microorganisms
IV. Culture Independent Methods: Measuring
Microbial Activities in Nature
A.Chemical Assays, Radioisotopic Methods, and Microsensors
B. Stable Isotopes
C.Linking Genes and Functions to Specific Organisms: Stable Isotope Probing

and Single-Cell Genomics
D.Linking Genes and Functions to Specific Organisms: SIMS, Flow Cytometry,

and MAR-FISH
A. Measuring Microbial Activities in Nature: Chemical
Assays, Radioisotopes, and Microsensors
• In many studies, direct

chemical measurements are
sufficient
• Higher sensitivity can be
achieved with radioisotopes
• Proper killed cell controls must be
used
Assays
• Vast majority of microorganisms in nature have not been cultured

• Termed viable but not culturable (VBNC)
• Primary source of information for these microorganisms is their
biomolecules;
Lipids, proteins and DNA/RNA
Assays
Phospholipid fatty acids (PLFA)
❑ Lipids
• Extract, concentrate, structural analysis
❑ Quantitative
❑ Insight into
• viable biomass, community composition,
nutritional-physiological status, evidence for
metabolic activity
Assays - PLFA
• Designated based on:

• The total number of C atoms
• Degree of unsaturation (double bonds)
• Position of the double bonds
• Branching patterns
• Examples:
• 16:0 = 16 carbons, no double bond
• 18:25 = 18 carbons, 2 double bonds at the 5th position from the aliphatic end
• i15:0 = 15 carbons, no double bond with iso-branching
• a15:0 = 15 carbons, no double bond with ante iso-branching
Assays - PLFA
• Some ecologically important patterns have been recognized:

• Ratios of i15:0 and a15:0 PLFA to 16:0 PLFA is a useful index of the
proportion of bacteria and eukarya in the community, [iso – methyl
group on second-to-last C; ante iso- methyl group on third-to-last C]
• Also ratios of trans- and cis- isomers of saturated to unsaturated
fatty acids may indicate physiological conditions of organisms or
environmental stress.
• Proportion of diglycerides – a function of cell death/lysis/action of

phospholipases
Assays - PLFA
• Some fatty acids are biomarkers
• Bacteria = i14:0, i15:0, a15:0, 18:17c,
cy19:0
• Algae = 20:53, 18:33
• Fungi = 18:26, AMF 16:15*
• Actinomycetes = 10Me17:0, 10Me18:0
• Sulfate reducers = i17:1, 10Me16:0
• Methanotrophs = 16:18c, 18:18c
• Single most quantitative, comprehensive insight into in-situ microbial

community
• Can indicate micro-niches of populations
A. Measuring Microbial Activities in Nature: Chemical Assays,
Radioisotopic Methods, Microsensors, and Nanosensors
• In many studies, direct chemical measurements are sufficient
• Lactate, SO42−, and H2S can all be measured with high sensitivity by
chemical assay
• Higher sensitivity can be achieved with radioisotopes
• Proper killed cell controls must be used to separate the chemical action
of microbes from that of abiotic processes
• In some activity measurements, it is useful to inhibit or encourage the
activities of certain organisms
• Acetylene as an inhibitor or alternative substrate
A. Measuring Microbial Activities in Nature: Microbial Activity
Measurements
A. Measuring Microbial Activities in Nature: The Acetylene Reduction
Assay of Nitrogenase Activity in Nitrogen-Fixing Bacteria
A. Measuring Microbial Activities in Nature: Microsensors
• A microelectrode is a conductor through which electric current is passed,

between a metallic part and a nonmetallic part of an electrical circuit
• Small glass electrodes with the tip sizes ranging from 2 to 100 μm in
diameter
• Electrochemical reactions due to the presence of a substrate change the

current – a substance is detected
• Can measure pH, oxygen, N2O, CO2, H2,H2S, and others

A. Measuring Microbial Activities in Nature: Microsensors
• Microsensors
•Can measure a wide range of activity
•pH, oxygen, CO2, and others can be measured
•Small glass electrodes, quite fragile
•Electrodes are carefully inserted into the habitat (e.g., microbial
mats)
• Measurements taken every 50–100 mm
Use of Microelectrodes: Microbial Mats
Babauta et al.(2014). Front. Microbiol.

Seawater
Denitrification Nitrification
O2 NO3–
Oxic sediment
and DRNA
Anoxic sediment
A. Measuring Microbial Activities in Nature: Stable Isotopes
and Stable Isotope Probing
❑Stable isotopes: nonradioactive isotopes of an element
❑used to study microbial transformations in nature
1H 2H
12C 13C 14N 15N
32S
33S
34S
36S
B. Measuring Microbial Activities in Nature: Stable Isotopes
Element Isotopes Abundance

Hydrogen 1H, 2H 1H = 99.985%
2H = 0.015%
Carbon 12C, 13C 12C = 98.89%

13C = 1.11%
Nitrogen 14N, 15N 14N = 99.633%

15N = 0.366%
Oxygen 16O, 17O, 18O 16O = 99.759%

17O = 0.037%
18O = 0.204%
Sulfur 32S, 33S, 34S, 36S 32S = 95.00%

33S = 0.76%
34S = 4.22%
36S = 0.014%
❑ Stable isotope ratios (e.g.,13C/12C) are measured using a mass spectrometer.
Three masses of CO2 (44/45/46)

are measured to determine the
amount of 13C and 12C in a sample
12C+16O+16O = 44
13C+16O+16O = 45
12C+18O+16O = 46
B. Measuring Microbial Activities in Nature: Stable
Isotopes
❑ Mass spectrometers can measure very small differences in isotope ratios.
• 13C/12C = 0.011225
• 13C/12C = 0.011071
• 13C/12C = 0.010918
• Raw ratios are converted into

delta (d) values:
13C/12C - 13C/12Cstandard
sample
d13Csample = x 1000
13C/12C
standard
• Not radioactive but metabolized differentially by microorganisms

• Enzymes typically favour the lighter isotope
• Two methods:
• stable isotope fractionation (SIF) and
• stable isotope probing (SIP)
Enzyme substrates Fixed carbon
Enzyme that
fixes CO2
12CO
2
12C
organic
13CO 13C
2 organic
B. Measuring Microbial Activities in Nature: Stable
Isotope Fractionation
• Isotope fractionation
• Carbon and sulfur are commonly used

• Lighter isotope is incorporated
preferentially over heavy isotope
• Indicative of biotic processes
• Isotopic composition reveals its past
biology (e.g., carbon in plants and
petroleum)
• The activity of sulfate-reducing

bacteria is easy to recognize from their
fractionation of sulfur in sulfides
B. Linking Genes and Functions to Specific Organisms: Stable
Isotope Probing
• Stable isotope probing (SIP): links specific metabolic activity to diversity using a stable
isotope
• Microorganisms metabolizing stable isotope (e.g., 13C) substrate will incorporate it into their DNA
• DNA with 13C can then be used to identify the organisms that metabolized the 13C
• SIP of RNA also possible

B. Linking Genes and Functions to Specific
Organisms: Stable Isotope Probing
B. Linking Genes and Functions to Specific
Organisms: Stable Isotope Probing
• Principles:
▪ Incorporation of 13C-labeled substrate into cellular biomarkers (e.g.
nucleic acids);
▪ Separation of labelled from unlabeled nucleic acids by density
gradient centrifugation;
▪ Molecular identification of active populations carrying labelled
nucleic acid
• Advantage: Allows the identification of active microorganisms without

the use of radioactive isotopes.
B. Linking Genes and Functions to Specific Organisms:
Stable Isotope Probing
• Disadvantages:
▪ Possible biases caused by the
incubation with the isotope
▪ Cycling of the stable isotope
within the microbial
community.
C. Linking Genes and Functions to Specific Organisms:
SIMS
• Analysis of cells by secondary ion mass spectrophotometry (SIMS)
• Detection of ions released from sample placed under focused high energy primary
ion beam
• High-energy ion beam impacts a sample
• Secondary ions are released (sputtering)
• Mass spectrometry of secondary ions
• Data generated from mass spectrometer reveals elemental and

isotopic composition of released materials (secondary ion)
• FISH-SIMS – traces incorporation of different elements or isotopes into

individual cells of specific cell populations (previously labelled with FisH
probes)
SIMS
• NanoSIMS
• SIMS devices that yield information on single cells
• Uses multiple detectors to provide simultaneous analysis of ions
• Allows for a two-dimensional image of the distribution of specific ions on the sample surface
• reveals info on single cells using O2 beam (generates positive secondary ions to analyse metals (e.g.
Fe, Mg) and Cs+ beam (generates negative secondary ions for analysis of cellular elements e.g. C, N, P,
S, O, H and halogens)
Bi = Bismuth
Cs = Caesium
ChiuHuang et al. (2014). ASME. J. Nanotechnol. Eng. Med. 5(2):021002-021002-5

Fluorescence and NanoSIMS images of a microbial consortium consisting of filamentous
cyanobacteria (Anabaena sp. strain SSM-00) and alphaproteobacteria (Rhizobium sp.
strain WH2K) attached to heterocysts
Behrens et al.( 2008) Appl. Environ. Microbiol. 74:10 3143-3150

C. Linking Genes and Functions to Specific Organisms: Linking
Functions to Specific Organisms
• Raman Microspectroscopy
• Be used to characterize the molecular and

isotopic composition of single cells by
nondestructive illumination with
monochromatic light generated by a laser
• When combined with confocal microscopy,

Raman spectrometers can determine the
elemental composition and appearance of
a single microbial cell
• Confocal microscopy - increases optical

resolution and contrast by use of a spatial
pinhole to block out-of-focus light when
forming an image
C. Linking Genes and Cellular properties to Individual
Cells: Flow Cytometry
• Flow cytometry and multiparametric analysis
• Natural communities contain large populations
• Flow cytometer examines specific cell parameters
very fast as they pass through a detector
• Cell size
• Cell shape
• Fluorescence
• Parameters can be combined and analyzed
(multiparametric analysis)
• Example: resolved two populations of marine
cyanobacterium (Prochlorococcus and Synechococcus)
based on differences in size and fluorescence in the
late 1980s
• Video:
https://www.youtube.com/watch?v=mcnFTjcmykE
C. Linking Genes and Functions to Individual Cells: MAR-FisH
• Radioisotopes are used as measures of microbial activity in a

microscopic technique called microautoradiography (MAR)
• Radioisotopes can also be used with FISH
• microautoradiography FISH (MAR-FISH)

• combines phylogeny with activity of cells
• Simultaneous assessment of metabolic activity and phylogenetic
identity of microbes of interest at the level of a single cell in
complex microbial communities.
MAR-FISH
• Radioisotopes are used as measures of
microbial activity in a microscopic
technique called microautoradiography
(MAR)
• Radioisotopes can also be used with

FisH
• Microautoradiography FISH (MAR-FISH)
• Combines phylogeny with activity of cells )
MAR-FISH
❑FISH used in combination with
microautoradiography
• Assesses both activity and identity

• Identifies organisms metabolizing
radiolabelled substance
• Other techniques plus FISH-
• ISRT-FISH (in situ reverse transcriptase PCR/FISH)
used to examine gene expression
• CARD-FisH
Cottrell, M. (2016). Website accessed at

https://www.ceoe.udel.edu/our-
people/profiles/mattcott
FISH-MAR: Methodology
❑ When left in the dark radioactive decay of incorporated substance exposes silver grains in
the emulsion.
❑ Appear as black dots within and around the cells.
C. Linking Genes and Functions to Individual Cells:
MAR-FisH
Access the text alternative for these images

©Cleber Ouverney
O'Donnell et al. (2007). Nature Reviews
Microbiology
10/13/2014
Biodegradation and
Bioremediation
Paul D. Brown, PhD

Email: paul.brown@uwimona.edu.jm
MICR3213: Applied & Environmental Microbiology
What organisms may do as part of

their metabolism
Biodegradation, Bioremediation,
Biocatalysis
What we call it when

we exploit some
microbial activity for
our (industrial) use What we call it when we
exploit some biodegradation
activity for “clean-up”
purposes
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Oil Spills
• Exxon Valdez accident

spilled 11,000,000
gallons of crude oil in
Prince William Sound,
Alaska on March 24,
1989
Exxon Valdez
• Tanker accidents contribute ~5% of total oil entering the seas each
year
• There were 16,000 accidents in 1988 - spilling four times the
amount of the Exxon Valdez oil spill into US waters
• About 35% of oil entering the seas each year comes from normal
ballasting and washing operations of tankers and 36% comes from
urban and industrial run-off
Alaskan beach contaminated by Exxon Valdez

spill (1989)
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Terminology
Biodegradation can be defined as the
biologically catalyzed reduction in complexity
of chemicals.
Complete
Mineralization leads to the conversion C, N,
P, S and other elements in the original
compound to inorganic products (i.e., CO2,
NH4, SO4, etc.)
Partial
Biotransformation - changing of a
compound to another reasonably stable
molecule; Often simpler compound,
sometimes it is more complex (e.g.,
polymerization)
Biodegradation
Principle of microbial infallibility (M. Alexander, 1965)
The empirical observation that no natural organic
compound is totally resistant to biodegradation
provided that environmental conditions are
favourable
Xenobiotic (completely synthetic) chemicals not
included!
Factors influencing biodegradability, rate of
biodegradation include:
pH, temperature, organic matter content
Presence/absence of required microbial consortium
Bioavailability of target compound
Compound degradability may depend on:
Elemental composition
Structure of basic repeating units (in a polymer)
Linkage between units
Degree of branching in the molecule
Arrangement and types of substituents
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Relative biodegradability of
glucose polymers
CH2OH CH2OH CH2OH

O O O starch, glycogen
α 1→4 linkages
O O O O • easy to degrade
CH2OH CH2OH
O cellulose
O O O
β 1→4 linkages
• not readily degraded
O
O O
CH2OH
Relative biodegradability of
chlorinated compounds
2,4,5-T: also a herbicide
• More recalcitrant
2,4-D: a herbicide
Substance Time for 75-100%

2,4-D (2,4-dichlorophenoxyacetic acid) ~ 4 weeks
2,4,5-T (2,4,5-trichlorophenoxyacetic acid) ~20 weeks
• Presence of additional –Cl on C-5 increases persistence of

herbicide 2,4,5-T compared to related herbicide 2,4-D
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Hydrocarbon biodegradation
•Oil-degrading bacteria congregate at the oil-water

interface but not within the droplet itself
•In general, only dissolved hydrocarbon
molecules are available to the degraders
• Hydrocarbon degraders are ubiquitous in environment
• Most biodegradation is aerobic because O2 is a direct reactant →
oxygenases
• Methane: a special case
• Degraded by specialized C1 microorganisms (methanotrophs)
methane
monooxygenase
CH4 CH3OH CH2O HCOO- CO2
O2 H2O
• Some generalizations:
• Aliphatics:
• Up to C4 (i.e., methane, …, butane) – gases
• Up to C9 – can be toxic; biodegradable; relatively volatile
• Often lost from spill
• ~C10 - ~C24 – rapidly, readily biodegraded
• Larger than C24, or branching in molecule
• decreases biodegradation
• Unsaturated compounds (i.e., alkynes, alkenes) more rapidly
degraded than saturated (alkanes)
• Alkanes (most readily degraded) → aromatics → alicyclics (least)
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H3C-CH2-CH2-CH2-CH2-CH3 Hexane: an aliphatic hydrocarbon
Benzene: an aromatic ring hydrocarbon
Cyclohexane: an alicyclic ring hydrocarbon

(also aliphatic)
H2C=CH2 Ethene (or ethylene), an alkene
H3C-CH3 Ethane, an alkane
Oxidation of n-octane (C8 aliphatic):
Alkane
Alcohol
- OH
β-oxidation of fatty acids:
→ 2C units (acetyl CoA)
→ feed into central
Aldehyde, metabolism (TCA cycle)
ketone +
- CHO reducing power (FADH,
NADH)
→ used to generate ATP, or
directly in biosynthesis
Acid
- COOH
β-oxidation pathway
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Oxidation of aromatic hydrocarbons:

• Monoaromatics, e.g., benzene, toluene, ethylbenzene, xylenes (BTEX)
• Polyaromatic hydrocarbons (PAHs), e.g., naphthalene
benzene p-xylene naphthalene anthracene
CH3
CH3
Common themes in aerobic biodegradation:

• Oxygenases (di- or mono-)
• Initial steps lead to catechols, protocatechuate (aromatic rings
with adjacent –OHs)
• Subsequent ring cleavage between hydroxyl groups (ortho
fission), beside the hydroxyl groups (meta fission)
• Eventual formation of compounds that enter central metabolic
pathways (TCA cycle)
• “Funnelling” of many different aromatic compounds (including
substituted aromatics) through a few key intermediates (e.g.,
catechols) to common pathways
General Metabolic Redox Model in

Microorganisms
Reduced chemical; Terminal e- Acceptor
Electron donor
e-
Oxidation to Reduction to
yield energy; can provide e- “sink”
be multiple steps (costs energy)
Oxidized Product Reduced Product
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Primary degradation
Aerobic Respiration Anaerobic respiration
Benzene O2 SO42-
Benzene
e- e-
Energy Coupled *Energy Coupled
production reduction production reduction
CO2 CO2 H2 S
H2 O
*Less net energy derived than with O2
Aerobic biodegradation of monoaromatic

compounds
Ortho
cleavage
succinic acid
acetyl CoA
(central
metabolism)
Meta
cleavage
central
metabolism
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Anaerobic biodegradation of aromatic compounds
• Ring reduction followed by ring cleavage to form fatty acid

• β-oxidation to acetyl-CoA units
• Added oxygen is from H2O not O2
• BTEX compounds may be slowly biodegraded by similar
pathway in absence of oxygen
Biotransformation of metals from oxidized

(soluble and toxic) to reduced (insoluble,
less toxic)
Organic C Oxidized
or inorganic metal (e.g.,
(eg., H2S) U6+ or Cr6+)
Energy e Reductive
-
production dechlorination
CO2 or oxidized Reduced metal

product (e.g., (e.g., U4+ or
SO42-) Cr3+)
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Co-metabolism
Mycobacterium vaccae
Oxidation of primary substrate
propane cometabolic reaction

½O2 cyclohexane
propane
monooxygenase
propanol cyclohexanol
Pseudomonas
cyclohexanone
propanone
energy +
production CO2 + biomass
of energy, biomass
Metabolic transformation of a substance while a

second substance serves as primary E or C source
Biodegradation of xenobiotics
• Examples include pesticides, polychlorinated biphenyls

(PCBs), munitions, dyes, chlorinated solvents
• Losses from environmental compartments may be due to
abiotic + biotic reactions
• Volatilization
• Leaching
• Spontaneous chemical decomposition
• Biological reactions
• Cometabolic reactions can be important
• Products may sometimes be more complex/more harmful
than parent compound
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Reductive dechlorination of chlorinated solvents

tetrachloroethene (PCE) and trichloroethene
(TCE)
Cl H
C=C trans-1,2-DCE
H Cl
Cl H H H H H H H
Cl Cl
C=C C=C C=C C=C
C=C
Cl Cl Cl Cl H Cl H H
Cl Cl
PCE TCE cis-1,2-DCE VC Ethene
carcinogen? carcinogen? carcinogen, relatively
mutagen innocuous
• PCE, TCE widely used as degreasers, dry cleaning fluid
• common contaminants of groundwater
• Each dechlorination step requires electrons + H+, Cl- is released
• cis-DCE is main DCE intermediate in biological reduction
• This reductive dechlorination occurs in anoxic, highly reduced
environments
• Microorganisms that conduct this most efficiently are able to
halorespire (chlorinated compound = terminal electron
acceptor in respiration)
Biodegradation of 2,4,5-T by
Burkholderia cepacia
• O2 required
• Note the following
• –Cl replacement by -OH
• Substituted catechol
formation; subsequent
ring fission
• 2,4,5-T is used as a
growth substrate;
succinate, acetate feed
into central metabolic
pathways
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Plastics
• Classically, polymers
of polyethylene,
polypropylene, PVC
• Examples of very
recalcitrant
xenobiotic
compounds
• Accumulate
(landfills)
• Consequent interest in developing biodegradable plastics

• Photodegradable polymers
• Starch-linked polymers
• Plastics based on microbial storage polymers (poly-
β-hydroxyalkanaotes, PHAs)
• Poly-β-hydroxybutyrate (PHB) is one you should know
bacterial cells
with inclusions of
PHB
12
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Poly-β-hydroxyalkanoate (PHA)
• Manipulation of growth conditions and/or genetic modifications of

PHA-producing bacteria have been used to produce PHAs with
different melting points, flexibility, tensile strength
• A copolymer of two PHAs produced by strain of Ralstonia eutrophus
been used in European “bioplastic” shampoo bottles
Two advantages to “natural” plastics:

• More readily biodegraded
• Technology is not petroleum-based
An example of biocatalysis
• Naphthalene dioxygenase catalyzes the first step of
aerobic naphthalene degradation
• The enzyme has a broad substrate range, and much potential
for use in stereospecific biocatalysis
Naphthalene
Indole
NH
Naphthalene
OH dioxygenase
OH OH
OH
NH
further bacterial metabolism

spontaneous reactions
CO2 + H2O + new cells

Indigo
(blue jean dye)
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Microbial corrosion
Bacterial or microbiologically-
induced corrosion (MIC)
 Sulfate-oxidisers
 Thiobacillus thiooxidans, T. thioparus, and T.
concretivorus
 Acidothiobacillus (H2SO4), Ferrobacillus (FeOx
and Fe(OH)x)
 Sulfate-reducers
 Desulfovibrio and Desulfotomaculum
 Require reducing environment (H2S)
 Form metal sulfides/H2S
 Major problem in stagnant
(fresh/salt) water systems
 Can form concentration cells
 Enhance galvanic corrosion
 Selective/localized leaching;
leads to brittleness
Microbial corrosion
Bacterial or microbiologically-
induced corrosion (MIC)
 Can affect metals, plastics, concrete
 Hydrocarbon utilizing microorganisms
 HUM bugs
 Mostly Cladosporium resinae and P.
aeruginosa
 Commonly present in jet fuel
 Live in the water-fuel interface of the
water droplets, form dark
black/brown/green, gel-like mats
 Cause microbial corrosion to Rusticle from HMS Titanic
plastic and rubber parts of the
aircraft fuel system by
consuming them
 (Continuous) use of biocides
and mechanical cleaning can
reduce MIC
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Microbial corrosion
 MIC can also occur in air
Corrosion of concrete
 Sulfur-oxidizing bacteria (primarily Thiobacillus
thiooxidans)
 Sewage systems, wastewater treatment facilities,
cooling towers and concrete/cement paste used to
immobilize radioactive and heavy metal wastes
 Oxidize various sulfur compounds (H2S) to produce
H2SO4
 Reacts with free Ca(OH)2 to form gypsum (CaSO4 ·
2H2O), which produces a corroding layer that
penetrates into the concrete
 Gypsum crystals can react with calcium aluminate to
produce ettringite (3CaO · Al2O3 · 3CaSO4 · 32H2O),
which increases the internal pressure, and leads to the
formation of cracks
 Provide additional sites for acid penetration
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Corrosion of concrete
SEM photomicrographs of cement surfaces after 30

days of exposure to a sterile medium (A) and to a
semicontinuous culture of H. neapolitanus (B).
Bars = 100 μm.
Bioremediation
 The quality of life on Earth is linked inextricably to
the overall quality of the environment
 The contamination of soil and water with organic
and inorganic pollutants is of increasing concern
and the subject of legislation.
 These pollutants include complex organic
compounds, heavy metals, and natural products
such as oils and are derived from industrial
processing, deliberate release, and accidental
release.
 Bioremediation is defined as the process
whereby organic wastes are biologically
degraded under controlled conditions to an
innocuous state, or to levels below
concentration limits established by regulatory
authorities.
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Economic Realities
 US contaminated groundwater cleanup costs
estimated in excess of $1.7 trillion using
“conventional” treatments
 As of 1995, bioremediation had been

employed at over 400 cleanup sites with costs
averaging 80-90% lower than landfill disposal
or incineration.
 US bioremediation revenues are increasing by

~ 10-15% per year. In 2000 bioremediation
spending in US exceeded 0.5 billion dollars.
 Environmental regulations have driven

bioremediation.
Bioremediation
 Bioremediation uses naturally occurring
bacteria and fungi or plants to degrade
or detoxify substances hazardous to
human health and/or the environment.
 The microorganisms may be indigenous
to a contaminated area and stimulated
in activity (biostimulation) or they
may be isolated from elsewhere and
brought to the contaminated site
(bioaugmentation).
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Major Issues in Bioremediation

 Regulatory acceptance and peer-reviewed reports
of controlled studies
 Bioremediation of recalcitrant compounds in

complex environments.
 Traditional kinetic approaches to predictions are

flawed.
 How clean is clean enough?
 How do you know bioremediation is happening?
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Benefits of Bioremediation
 In many cases the clean-up of contaminated sites has been
carried out using physical and chemical methods-
immobilization, removal (dig and dump), thermal, and
solvent treatments.
 Bioremediation is often cheaper, require less energy
input than the chemical and physical options, and can deal
with lower concentrations of contaminants more
effectively, although the process may take longer.
 Several approaches for bioremediation in both soil and
water:
 Rely in the indigenous microbial population.
 Biostimulation: Encourage the indigenous population.
 Bioaugmentation: addition of adapted inoculants.
 Addition of genetically modified micro-organisms (‘bag-
o-bugs’).
 Phytoremediation
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Bioremediation Technologies:
Typical interaction with other types of treatments
Specific, commonly used approaches

land farming
soil piling
in situ stimulation
bioreactor technology
marine oil spill cleanup
Largely ineffective approaches

bag-o-bugs
“casual” additions of bacterial cultures
Often added organisms

turn out to be very
expensive fertilizer for
existing organisms.
Rarely are nutrient

additions tested
separately from the
bacteria. Clients just
assume the bacteria were
responsible for
degradation.
20
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Land farming bioremediation:

tilling for aeration
Large scale soil pile

bioremediation
21
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Pump and treat/biostimulation
22
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Multi component treatment

systems
Constructed wetland technology
23
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Generalized Central Metabolism
Universal Metabolic Precursors
24
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Introduction to
Sampling of
Environmental Media
Paul D Brown, PhD

MICR3213: Applied and Environmental Microbiology
Sampling Considerations
What we want:
• Fast
• Sensitive
• Specific
• Easy to Perform
• Reliable (Accurate/Precise)
• Compatible with Downstream Detection
What do we have???
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The Challenge of Environmental

Sampling for Pathogens
• Variation in microbe type and distribution

• Low microbe numbers: need to concentrate
them
• Non-random distribution and physical state
of microbes of interest: aggregated,
particle-associated, embedded, etc.
• Volume considerations
• Environmental factors may inhibit or
interfere with downstream detection
• Separate them from interfering and excess
other material
Detection of Pathogens
in The Environment
• Three main steps:
• (1) recovery and concentration,
• (2) purification and separation, and
• (3) assay and characterization.
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Pathogen Detection
Techniques
Targets:
• ATP
• Nucleic Acid
– PCR methods
– Microarray methods (fluorimetric,
electrochemical)
• Protein/Lipid
– Immunological methods
– Mass Spectrometry methods
• Whole Organism
– Microscopy
– Culture
Water Concentration
• Distribution of pathogens in water

necessitates sampling of large volumes
of water (1-1000s of litres)
• Filtration is typically used for
concentration
• Several formats utilized:
– Membrane filter, pleated capsule, cartridge,
hollow fibre
• Several types of media
– cellulose ester, fiberglass, nylon,
polycarbonate, diatomaceous earth
(diatoms), polypropylene, cotton,
polysulfone, polyacrylonitrile, polyether
sulfone
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Filters to Recover and Concentrate Microbes from Liquids
Types of Filtration
• Size Exclusion/
Retention
• Adsorption/Elution
4
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Surface Sampling
• Current Methods (5-90% recoveries, generally
poorly characterized)
– Swabs (better for gram negatives?)
• Cotton / Dacron / Calcium Alginate (may inhibit PCR
and be toxic to cell culture) / Sponge (Polyurethane and
Cellulose)
– Swipes/Wipes
• Cotton / Nitrocellulose membranes / Polyester bonded
cloth / Velvet or Velveteen
– Vacuum Filtration
• Hepa bag vac / Wet Vac
– Rinse/Elute
– Contact Plates and Paddles (RODAC) (better for gram
positives?)
• New Methods
– Adhesive Strips and Paddles
– Scraping/Aspiration
Yamaguchi, et al. 2003; Cloud, et al. 2002; Lemmen, et al, 2001; Poletti, 1999;
Craythorn, et al. 1980; Osterblad, et al. 2003; Taku, et al. 2003
5
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Aerosol Sampling
• Impactor
– Anderson single and multistage sampler
– Slit sampler
– Rotary arm sampler
• Impinger
– AGI sampler
– Biosampler (SKC) sampler
• Filters
– IOM/Button filter sampler
– Foam plug filter sampler
• Centrifugal
– Cyclone sampler
– Centrifugal sampler
• Precipitators
– Electrostatic precipitator
– Condensation trap
• Hybrid
Impactors
6
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Impingers
Filters
7
10/22/2014
Large Volume Aerosol

Samplers
• Biocapture BT 550 (Mesosystems)
– Rotary arm impactor, liquid collection
– 150L/min (~15 min)
• Bioguardian (Innovatek)
– Wet-walled multi cyclone,
w/centrifugal impactor for removal of
large particles
– 100-1000L/min (1 min-12 hours)
• Spincon (Sceptor)
– Centrifugal wet concentrator,
w/cyclonic pre-separation
– 450L/min (5 min-6 hours)
Aerosol Samplers
8
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Separation and Purification

Methods
• Purification, separation and

concentration of target microbes in
primary sample or sample concentrate
– Separate target microbes from other

particles and from solutes
– Reduce sample size (further

concentrate)
Separation/Purification
Methods
• Variety of physical, chemical and
immunochemical methods:
– Sedimentation and flotation (primarily
parasites)
– Precipitation (viruses)
– Filtration (all classes)
– Immunomagnetic separation or IMS (all
classes)
– Flow cytometry (bacteria and parasites); an
analysis, too
9
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Secondary Concentration and

Purification
• PEG (polyethylene glycol)
• Organic Flocculation
• IMS (Immunomagnetic separation)
• Ligand capture
• BEaDs (Biodetection Enabling Device)
• Capillary Electrophoresis
• Microfluidics
• Nucleic Acid Extraction
• Spin Column Chromatography
• Floatation
• Sedimentation
• Enrichment
10
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Aeromicrobiology
Paul D. Brown, PhD

paul.brown@uwimona.edu.jm
MICR3213: Applied & Environmental Microbiology
Aerobiology
 The interdisciplinary science that deals
with the movement and dispersal of
bioaerosols
 The movement of bioaerosols is
generally passive and is greatly
influenced by the environment
 The survival of viable bioaerosols is also
dependent on the environmental
conditions
1
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Bioaerosols
 Biological agents carried in the air as
large molecules, volatile compounds,
single particles, or clusters of particles
that are living or were released from a
living organism
 Particles sizes – typically 0.5m to 100
m
 Capable of eliciting diseases that may
be infectious, allergic, or toxigenic with
the conditions being acute or chronic
2
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3
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Aerosols and Respiratory

Deposition
 Aerosols >5 microns in diameter are removed in the

upper respiratory tract, especially the nose.
 Particles are brought to the pharynx by mucociliary
activity of the upper respiratory epithelial mucosa,
where they are expectorated or swallowed.
 Swallowed particles containing enteric microbes can
initiate enteric infections.
4
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Aerosols and Respiratory

Deposition
 Particles <5 microns in diameter, esp. 1-3 microns
diameter, penetrate to the lower respiratory tract
 Can be deposited in the bronchioles, alveolar ducts and
alveoli.
 Deposition efficiency in lower respiratory tract is ~50% for
particles 1-2 microns diameter.
 Particles <0.5 microns dia. can also be deposited in the lower
respiratory tract, especially particles <0.25 microns dia.
 Particles deposited in the lower respiratory tract can be
phagocytized by respiratory (alveolar) macrophages
 can be destroyed or
 carried to the ciliary escalator, where they are transported
upward to the pharynx.
Hydroscopicity and Aerosol

Deposition in the Respiratory
Tract
When inhaled, aerosol particles derived from aqueous fluids
pick up moisture (water) while traveling in the respiratory
passageways, thereby increasing in size.
 Increased size changes deposition site
H2O H2O H2O
5
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Respiratory Deposition -
Impaction
 Each time airflow changes due to a bifurcation in the
airways, suspended particles tend to travel along their
original path due to inertia and may impact on an airway
surface.
 This mechanism is highly dependent on aerodynamic
diameter, since the stopping distance for very small
particles is quite low.
 Occurs mostly for larger particles that are very close to
airway walls, near the first airway bifurcations.
 Therefore, deposition by impaction is greatest in the
bronchial region.
 Impaction accounts for the majority of particle deposition on
a mass basis.
Sedimentation
 Settling out of particles in the smaller

airways of the bronchioles and alveoli,
where air flow is low and airway
dimensions are small.
 Rate of sedimentation is dependent on the
terminal settling velocity of the particles
 Sedimentation plays a greater role in the
deposition of particles with larger
aerodynamic diameters.
 Hygroscopic particles may grow in size as
they pass through the warm, humid air
passages, thus increasing the probability of
deposition by sedimentation.
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Interception
 Occurs when a particle contacts an airway surface due
to its physical size or shape.
 Unlike impaction, particles that are deposited by
interception do not deviate from their air streamlines.
 Most likely to occur in small airways or when the air
streamline is close to an airway wall.
 Interception is most significant for fibers, which easily
contact airway surfaces do to their length.
 Furthermore, fibers have small aerodynamic
diameters relative to their size, so they can often
reach the smallest airways.
Respiratory Deposition - Diffusion
 Primary mechanism for particles <0.5 microns in diameter

 Governed by geometric rather than aerodynamic size
 Net transport of particles from a region of higher to lower
concentration due to Brownian motion.
 Brownian motion: random wiggling motion of a particle
due to the constant bombardment of air molecules.
 Occurs mostly when particles have just entered the
nasopharynx
 Also most likely to occur in the smaller airways of the
pulmonary (alveolar) region, where air flow is low.
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Concentrations in Air
 Concentrations of indoor bacteria range
from <100-1500 cfu/m3; avg 0-500
 heated dwellings lower
 Avg concentrations of outdoor bacteria
200-1000
 Avg concentrations of indoor Fungi
range from 10-500
 Avg concentrations of Outdoor Fungi
range from 100-1000
Microbial IAQ
 Outdoor spores enter readily
 Many fungi can also amplify indoors –
anytime moisture is available fungi can
grow on many indoor substrates
 Penicillium, Aspergillus, and
Cladosporium are most common
indoors
 Many can form mycotoxins – Aspergillus
spp and Stachybotrys
8
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Important Airborne Pathogens
9
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Airborne Plant Pathogens
10
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11
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12
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13
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Environmental factors that

influence indoor fungal
contamination
 Outdoor concentration and type
 Type and rate of ventilation
 Activity levels
 Indoor moisture levels
 Modern building materials
Indoor air sample
14
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15
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Typical 80/20 recirculation/ventilation consistent with ASHRAE 62-89
Dry Air Spora
16
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Diurnal Rhythm of
Cladosporium
Hourly Cladosporium Levels

Tulsa May 24, 1999
30,000
25,000
20,000
Spores/m 3
15,000
10,000
5,000
0
12:00 2:00 4:00 6:00 8:00 10:00 12:00 2:00 PM 4:00 PM 6:00 PM 8:00 PM 10:00
AM AM AM AM AM AM PM PM
17
10/13/2014
Cladosporium spores peak hourly

concentration of >120,000 spores/m3
during a spore plume
140,000
Cladosporium ,
120,000 30 September 1998
Tulsa
100,000
3
Spores/m
80,000
60,000
40,000
20,000
0
0:00 2:00 4:00 6:00 8:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00
Basidiospore Rhythm
May of 1998
 Need for moisture 2500
confines spore 2000
release to periods
spores/m 3
1500
of high humidity
1000
500
 Typically peak 0
levels in pre-dawn
2:00 AM
4:00 AM
6:00 AM
8:00 AM
10:00 AM
12:00 AM
12:00 PM
2:00 PM
4:00 PM
6:00 PM
8:00 PM
10:00 PM
hours and low

levels in afternoon
18
10/13/2014
Airborne Fungal Spore

Concentrations in Tulsa 2002
60,000
2002
50,000
40,000
Spores/m3
30,000
20,000
10,000
0
J F M A M J J A S O N D
23 Taxa identified: Cladosporium, Ascospores,

Basidiospores, and Alternaria Comprised 90% of Total
Cladosporium Ascospores
45000 12000
40000
10000
35000
30000 8000
spores/m3
25000
spores/m
20000 6000
15000
4000
10000
5000 2000
0
0
1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1
1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1
Basidiospores Alternaria
5000 2500
4500
4000 2000
3500
spores/m 3
spores/m 3
3000 1500
2500
2000 1000
1500
1000 500
500
0 0
1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1 1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1
19
10/13/2014
Spore plumes show the

influence of environmental
conditions
Total Spore Concentration and Wind Speed
Hectorville, OK - Sept 21, 1998
200000 7
180000
6
160000
Total Spore Concentrations
140000 5
Wind Speed (m/s)

(spores/m^3)
120000
4
100000
3
80000
60000 2
40000
1
20000
0 0
12:00 2:00 4:00 6:00 8:00 10:00 12:00 2:00 4:00 6:00 8:00 10:00
a.m. a.m. a.m. a.m. a.m. a.m. p.m. p.m. p.m. p.m. p.m. p.m.
Time
20
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21
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Sources of Aerosols
Point Sources
 Personal (i.e. sneezes, vomitus, etc)
 Natural
 Anthropogenic
 Agricultural
 Intentional
 Industrial
22
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23
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24
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25
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What are other sources?

 Home IAQ
 Occupational Sources
 Healthcare setting
 Food processing
 Other
 Outdoor Sources
26
10/13/2014
Factors Affecting
Survival of Pathogens
in Bioaerosols
Factors Affecting Bioaerosol

Stability
 Temperature
 Humidity
 Composition (moisture, salts, protein,
etc.)
 (Sunlight)
 (Wind)
27
10/13/2014
Stability of Airborne
Microbes
 Relative Humidity
 Temperature
 Oxygen
 Open Air Factors (e.g. Ozone, olefins..)
 Radiation (e.g. UV, γ-rays, X-rays)
 Suspending media (salts, proteins,
sugars)
 (Air Movement- e.g. wind shear)
28
10/13/2014
Major Losses of Viability

 Maillard Reactions (dessication)
 Membrane phase Changes
 Cross linking
 Free radical damage (hydroperoxide
and hydroxyl)
 Resulting in
 Phospholipid crystalization
 Change in permeability barrier function
 Energy production capability
 Temp. associated death mechanism
Mobility of Aerosols
29
10/13/2014
Transport of aerosols
 Gravitational Settling
 Stokes Law: v= pd2g/18n (cm/sec)
v= velocity, p= particle density, d=
particle diameter, g= acceleration due to
gravity, n=viscosity of air
Assumes spherical shape
 For particles >1 micron diffusion due to
gravitational settling is dominant
 For particles <0.5 micron, V→→0
Movement/Transport in Air
 Advection
 Mechanical Diffusion
 Molecular Diffusion
30
10/13/2014
Movement/Transport in Air
 Advection (a transport mechanism of a
substance or conserved property by a fluid due
to the fluid's bulk motion. Ex. the transport of
pollutants or silt in a river by bulk water flow
downstream)
 Mechanical Diffusion (arise from physical

obstruction of the flow by any barrier; when
barrier encountered, the particle must move
laterally; Fick’s law; involves Reynold’s number)
 Molecular Diffusion (caused by random

molecular motion due to thermal kinetic energy
of the aerosol)
31
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32
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Deposition
 Surface impaction
 Essentially a collision between particles and
surfaces
 Not necessarily permanent
 Rainfall
 Electrostatic
 Turbulent Diffusion
 Thermal gradients
 Electromagnetic radiation
33
10/13/2014
Deposition
Deposition
34
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Deposition
Deposition
35
10/13/2014
Deposition
 Molecular Diffusion
 Tend to be downwardly directed by
gravity
 Random movements
 Highly influenced by wind
 Increases with wind strength
36
10/13/2014
Brownian Motion
 Bioaerosols are
subject to
Brownian motion
as described by
Einstein’s equation
 X=root mean
square particle
displacement, cm
 t= time, s
 r=particle radius
37
Risk Assessment &
Risk Management
Paul Brown, PhD
MICR3213: Applied and Environmental Microbiology
Risk Assessment
US Airways Magazine, October 1991
1
Risk Management
US Airways Magazine, October 1991
Risk Assessment/Risk Management
 Risk Identification
– Adverse events?
 Risk Estimation
– Probability of adverse event?
 Risk Management
– Control measures?
2
Risk (Definitions)
 “Possibility of loss, injury, disease, or death.”

Webster's Medical Desk Dictionary (1986)
 “The probability that exposure to a hazard will

lead to a negative consequence.” David Ropeik,
George Gray (2002)
 “To risk living is to risk dying.” Anonymous
Risk Assessment
 The emergent science based on toxicology,

epidemiology and statistics that utilizes
qualitative and quantitative hazard analysis to
provide the public with a reasonable estimate
of probability of harm.
 “Not a scalpel, but a crude tool that allows

you to make estimates.” Peter Preuss, US EPA
3
Risk Assessment
 Difficult process
(expertise of many fields needed)
 Involves uncertainty
 Range provided (not a specific number)
 Estimates for society (individual risk may
vary)
 “Reasonable worst-case estimate” (better to
overestimate than underestimate risk)
 Costs and benefits of proposed actions
helpful
4 Steps in Risk Assessment (Jeff

Wheelwright, 1996)
 1) Identify health hazard

 2) Quantify hazard
 3) Exposure assessment (from source to at
risk person)
 4) Determine probability of disease (based
on exposure estimate and potency of agent)
4
Biohazard Epidemiology
 Incidence of Hepatitis among Danish

clinical chemistry workers 7X higher
than general population (Skinholj, 1974)
 Risk of acquiring TB 5X greater among

medical lab workers in England than
general population (Harrington &
Shannon, 1976)
Hierarchy of Controls
 Anticipation
 Recognition
 Evaluation
 Control
– substitution
– administrative
– engineering
– work practices
– personal protective clothing
– facility features
5
Biohazard Risk Assessment
 Qualitative exercise
(inexact)
 General guidelines
to assess/control risk:
– agent in use, volumes, concentration
– proposed practices/procedures
– proposed location
– training, experience, health status of
worker
Biohazard Risk Assessment

 Use to determine appropriate combination of
containment
– lab practices
– safety equipment
– facility design
 Primary Containment
– protects handlers and those in immediate vicinity
 Secondary Containment
– protects environment and those outside the lab
6
Biohazard Risk Assessment Pathway
 Principal Investigator (initiates risk review)
 Biosafety Officer (assists PI)
 Institutional Biosafety Committee (must
review and approve PI’s submission)
 Assistance through
– published listings, guidelines (U.S. and abroad)
– other experts at host institution, local public health
– other institutions working with same agents
– Government entities (CDC, NIH, USDA, FDA, etc.)
Risk Assessment Pathway

 Principal Investigator initiates process
– Qualitative process
– Agent
• Virulence, pathogenicity, communicability,
environmental stability, dose, route of
exposure, availability of therapy
– Use Risk Group Lists
– Consider proposed procedures
• Operations, quantity (volume/concentration),
generation of aerosols, sharps, animals
7
Routes of Exposure to
Infectious Agents
 Inhalation of aerosols
 Through intact or non-intact skin (needlestick,
injury (broken glass), animal bites or
scratches, vector (mosquito, tick,
parasite), eczema, dermatitis
 Mucous membranes of eyes, nose or mouth
 Ingestion (mouth pipetting)
 Contact (indirect transfer from hands or
contaminated surfaces)
8
Infectious Agents are Classified by
Level of Hazard
4 Agent Risk Group Classifications
RG1 RG2 RG3 RG4

Low individual Moderate High individual risk
risk individual risk
No risk to High risk to

Low risk to community community
community
Risk Groups (RG)
 RG1
• Not infectious to healthy adults
• e.g. E. coli K12 strains, B. subtilis, S. cerevisiae
 RG2
• Infectious agents of varying severity, treatment
usually available, predominantly bloodborne,
ingestion, and mucous membrane routes of
exposure
• e.g. Salmonella, Shigella, Vibrio, Plasmodium,
Hepatitis B Virus, Cryptococcus neoformans
– Both RG1/RG2 can be used in a basic lab
• containment equipment to contain aerosols
9
Risk Groups (RG)
 RG3
• potential to cause serious or lethal disease,
airborne route of exposure (and others),
treatment generally not available, lower
infectious dose.
• Containment Lab 2 doors off general corridor,
dedicated air handler, controlled airflow, all
work contained
• e.g. TB, Vesicular Stomatitis Virus, Yellow
Fever Virus, Coxiella burneti, Francisella
tularensis
Risk Groups (RG)
 RG4
• Dangerous, exotic agents with high risk to
individual and community. Aerosol
transmission along with all other routes. Very
low infectious dose, high mortality rates.
• Building within building approach for research
purposes.
• e.g. Ebola virus, Marburg virus, Junin, Lassa,
Machupo, Sabia, Equine Morbillivirus (Hendra-
like viruses), Tick-Borne Encephalitis Viruses
10
 Principal Investigator responsible for

completing initial risk assessment
– Start with risk group for parent organism
– Consider the proposed procedures
– Identify Risk Management Procedures
• Facility design elements
• Safety or containment equipment
• Work practices
Laboratory Safety
Containment Levels
4 Laboratory Biosafety Levels
BSL1 BSL2 BSL3 BSL4

Basic laboratory, Containment lab, 2 Maximum containment
confine aerosols in door separation from lab, building w/in building,
biosafety cabinet if general traffic, all features isolated, pos.
needed negative air flow, pressure suits, glove box
alarms type isolation
11
Hybrid Biosafety Level
 BSL2/BSL3 (BL2+)
– Creutzfeld Jacob
– HIV
– High risk clinical specimens
 BSL3-Enhanced (HEPA filtered exhaust
Lab)
– Yellow Fever, Rift Valley Fever Virus, VEE
– Rickettsia rickettsii
Unknown Specimens
 Facility Evaluation (highest level of

protection available)
 “B.A.R.E”
– Block All Routes
of Exposure
12
Containment achieved with:
 Good microbiological
practices
 Safety Equipment
 Facility Design

 Institutional Biosafety Committee
verifies and approves PI Risk
Assessment
– Review of written risk assessment
– Verification of personnel training and
experience
• Biosafety courses
• Hands-on experience/proficiency
• Safety record
– Inspection of facility and work practices
– Formal approval of protocol
13
Find Assigned Risk Group for:
 Brucella canis  Rabies virus
 Chlamydia trachomatis  HIV/SIV
– diagnostic work – research scale
– high concentrations  Vesicular Stomatitis
virus
 Francisella tularensis
– lab adapted strains
– diagnostic/clinical work
– Isolates from livestock
– cell culture experiments
 rDNA, Insertion of
 Coccidioides immitis oncogene into human
– clinical specimens cells
– cultures  Vesicular Stomatitis
 Prions virus-NJ with HIV gp 120
– human prions  Botulinum toxin
– animal prions
Risk Assessment & Risk

Management
 Prior Planning Prevents Poor

Performance
14
Risk Assessment & Risk
Management
 Pathogen (Agent)
 Procedures (Protocol)
 Personnel
 Protective Equipment
 Place (Proposed lab facility)
P-1: Pathogen
 Should this agent be used in this

experiment? On this campus?
 Note: concentration or amplification in

lab may present greater hazard than in
nature.
15
PATHOGEN
 Agent Classification (Prior LAI’s)

 Source of agent
 Routes of Exposure
 Infectious Dose (LD50’s for toxins)
 Pathogenicity
 Virulence
 Antibiotic resistance
Infectious Dose
 Agent  Dose
– Ebola virus – 1
– TB – 1 - 10
– Tularemia – 10
– Anthrax – >1300?????
– Cholera – 10^8
– Salmonella typhi – 10^5
– E. coli – 10^8
– Shigella – 10^9
16
PATHOGEN
 Quantity/Concentration
 Incidence in the Community
 Immunization/Treatment
 Communicability
 Presence of Vectors
 Environmental Concerns (stability)
 Data from animal experiments
 Clinical specimens
Immunizations
– Vaccinia
– Tetanus
– Meningococcal Immunization
– Typhoid
– Botulinum
– Hepatitis B virus, Hepatitis A virus
– Yellow Fever, EEE
– Rabies
17
rDNA Molecules
 Classification of parent agent

 Toxins
 Antibiotic resistance genes
 Altered host range or tropism
 Replication competency
 Integration into host genome
 Toxicity, allergenicity, other
P-2: Personnel
 Are the proposed researchers capable

of safely conducting these experiments?
18
PERSONNEL
 Host Immunity
 neoplastic disease/infection
immunosuppressive therapy
age, race, sex, pregnancy
surgery (splenectomy, gastrectomy)
diabetes, lupus
 Reproductive age
 Contraindications for therapy
PERSONNEL
 Medical Surveillance
 prophylactic immunizations
 serum storage
 post-exposure prophylaxis/treatment
 screening
19
PERSONNEL
 aware of hazards
 prior documented work experience
 microbiological proficiency (observed)
 comfort/choice
PERSONNEL
 Safety Attitude
 Those who have fewer accidents:
adhere to safety regulations
respect infectious agents
defensive work habits
able to recognize potential hazards
 Women
 Older employees (age 45-64)
20
PERSONNEL
This image cannot currently be display ed.
 Safety Attitude
 Those who have more accidents:
low opinion of safety
take excessive risks
work too fast
less aware of risks
 Men
 Younger employees (age 17-24)
P-3: Protective Equipment
 Has the Principal Investigator selected

the appropriate combination of personal
protective clothing and safety
equipment for the safe conduct of
research?
21
Protective Equipment
 Personal Protective Equipment

(clothing)
 Containment Equipment
– Biological safety cabinets
– Safety centrifuges
– Sealed sonicators, blenders, homogenizers
– Sealed tubes, transport carriers
– Safe sharps, needleboxes, medical waste
bags, tongs, forceps, etc.
PERSONAL PROTECTIVE
EQUIPMENT
 Protect: skin
clothing
mucous membranes
respiratory system
 Use: gloves (double, kevlar)
lab coats, solid-front gowns
sleeve covers
full face protection
respiratory protection
22
PERSONAL PROTECTIVE
EQUIPMENT
 Disposable
 Decontamination
 Dedicated to area
 Donning/Doffing
– Compromised (wet/contaminated/torn)
– Respiratory Protection Program
P-4: Place (Facility Design)
 Does this research group have (or have

access to) a laboratory with the
requisite containment features this
work?
23
PLACE – FACILITY DESIGN
 Restricted access/Door sign

 Easily cleanable
 Hand washing sinks (near exit door)
 Eye wash
 Autoclave
 Vacuum system protection
 Biosafety Cabinet
PLACE – FACILITY DESIGN
 Anteroom
 Negative pressure gradient
 Airflow monitor
 Air changes per hour (10-15)
 Sealed penetrations, coved flooring
 Facility alarms/interlocks
 Communication outside the lab
24
P-5: Procedures
 Has the Principal Investigator outlined

all of the proposed steps in the
protocol?
 Has the lab outlined sufficient protective
work practices to minimize the risk to
those working and those outside the
lab?
PROCEDURES
 Develop standard written practices

(SOP’s) for handling pathogens
 Job Safety Analysis (JSA)
– identify each task
– describe all steps
– hazard assessment at each step
– incorporate safety
 Focus on containing aerosol generating
procedures and equipment
25
Aerosols
– Procedures that impart energy into a

microbial suspension are a potential source
of aerosol (Chatigny, 1974)
– Many common lab procedures and accidents

have capability of releasing aerosols
– homogenization, sonication, blending, mixing, grinding,
shaking, vortexing, spills, opening vials, pipetting,
animals excreting agent, opening vials under pressure,
etc.
Viable Particles Recovered from Air

(Chatigny, 1974)
 Procedure  # Particles/ft3 of air

– sonic oscillator – 6
– mixing w/ pipette – 7
– overflow from mixer – 9
– opening lyophilized vial – 135
– top removed after – 1500
blending
– dropping flask of culture – 1551
– dropping lyophilized – 4839
culture
26
Procedures - Sharps Hazards
 Syringe/Needle
– adjusting volume
– withdrawal from stopper
– separation from syringe
– leaking syringe
– leakage from injection site
– inappropriate disposal
– poor work practices
Procedures - Sharps Precautions
 Syringe/Needle
– use needle-locking syringes
– cover with disinfectant soaked gauze
– animal restraints
– cleanse inoculation site
– safe needle practices
– immediate collection/disposal
27
Procedures - Sharps Precautions
 Needle/syringe
– removal of needle from syringe (hemostat)
– no recapping, bending, breaking, etc.
– immediate disposal of intact needle/syringe
– location of needlebox (vicinity, height)
– replacement of needleboxes
– eliminate/minimize use/safe sharp devices
– avoid glass Pasteur pipettes
Procedures presenting risk
 Microbiological loop
– streaking plates
– spreading material on slides
– cooling loop in media
– heating loop in an open flame
28
Precautions in bacteriology
 Microbiological loop
– smooth plates
– disposable plastic loops
– well formed loops with short staff
– glass spreaders
– electric (walled) micro-incinerators
– work within a biosafety cabinet
Procedures with general risk
 Pipetting
– mouth pipetting
– glass Pasteur pipettes
– blow-out pipettes
– mixing suspensions
– spill of droplets onto hard surfaces
 Eating, drinking, smoking, applying
cosmetics
29
PROCEDURES
 Pipetting
– no mouth pipetting
– disposable plastic pipettes
– mark to mark pipettes
– collect within biosafety cabinet
– work over disinfectant-wet pad
 Restrict consumption of food or
beverage to well defined break areas
PROCEDURES
 Centrifugation
– broken/leaking tubes
– microfuge tubes (snap caps)
– (flawed/overfilled)
 Protective Measures
– check O-rings on rotors (use O-ring tubes)
– safety cups/sealed rotors
– load/unload in a biosafety cabinet
30
Risk Assessment Example
 Hantavirus Protocol
– Application of 5 P’s
– Hierarchy of controls
• Pathogen
• Personnel
• Place
• Procedures
• Protective Equipment
31
11/8/2018
ENVIRONMENTAL
DISINFECTION AND
SANITATION
Paul D Brown, PhD
MICR3213: Applied and

Kofi Annan
Former UN Secretary-General
“We shall not finally defeat AIDS,

tuberculosis, malaria, or any of the
other infectious diseases that
plague the developing world until
we have also won the battle for
safe drinking-water, sanitation and
basic health care.”
1
11/8/2018
Global Burden of Disease Attributable

to Selected Major Risk Factors
Underweight Tobacco
Unsafe sex Alcohol
burden (within region)
Overweight
Percent of total
5% - Water, sanitation and

hygiene (5.5%)
Indoor air (3.7 %)
Physical
Zinc deficiency inactivity
Tobacco
Alcohol
1% -Overweight Occupational risks
Lead Ambient Unsafe sex
Water, sanitation
Ambient air air
Lead and hygiene
Developing countries Developed countries
(high mortality)
More recent estimate even
higher!
Global Burden of Poor Water,

Sanitation and Hygiene (WSH)
 1.1 billion people (~17% of the
population) lack access to improved
water
 tap water in house/yard from public
distribution systems,
 protected wells & springs
 public stand posts/pipe
 rain water collection
 2.6 billion (42% of population) lack

access to basic sanitation
 sewerage, on-site septic waste treatment
system, latrine
2
11/8/2018
Global Burden of Poor Water,

Sanitation and Hygiene (WSH)
 1.8 million people die every year

from diarrheal diseases (including
cholera)
 90% are children under 5, mostly in
developing countries.
 80% of the population without access
to drinking-water are rural dwellers,
but future populations will be mainly
urban
 Peri-urban slums are among the most
underserved and unsanitary places
on earth!
The Older Conventional View:
Lack of WSH = Disease and Poverty

• Inadequate water supply • Time, financial cost
• Unsafe water resources • Disease burden
• Inequitable access • Health care costs
POVERTY
3
11/8/2018
The Newer Optimistic View!
WSH = An Engine for Development

and Productivity
• Improved water supply • Time, financial savings
• Safe water resources • Averted disease costs
• Universal access • Healthy populations
Development
Millennium Development Goals

•Goal 1 Eradicate extreme poverty and hunger
•Goal 2 Achieve universal primary education
•Goal 3 Promote gender equality and empower women
•Goal 4 Reduce child mortality
•Goal 5 Improve maternal health
•Goal 6 Combat HIV/AIDS, malaria, and other diseases
•Goal 7 Ensure environmental sustainability
• Target 9: Integrate the principles of sustainable
development into country policies … reverse loss of
environmental resources.
• Target 10: Halve by 2015 the proportion of people
without sustainable access to safe drinking water
and basic sanitation
• Target 11: improve the lives of at least 100 million slum
dwellers
•Goal 8 Develop a global partnership for development
4
11/8/2018
The “F”s of WSH
 Feces
 Fingers
 Flies
 Fields/Food
 Fluids
 Fomites
Water Treatment
Other important aspects
 Air pollution
 Solid waste
management
 Vectors &
vector-borne
diseases
 Disasters and
emergencies
 Climate
change health
effects
5
11/8/2018
Human Sanitation:
Fundamental but Often Lacking
 Excreta
management
and disposal
 Hygiene
behaviors
 Handwashing
 Safe water
Sanitation: Our Biggest Failure

 Our sanitation
systems don’t work
well and result in
pathogen release
 Whether community
or on-site, they all Roman latrine
fail or have serious
deficiencies
 Sanitation is one of Latrine
the biggest
technological gaps
we have globally
 Pathogens go
everywhere as a
result
VIP latrine
6
11/8/2018
Inferior/Absent Community
Wastewater Treatment Systems
Rx.
No Rx. Rx. Often Absent!
Untreated/poorly treated wastewater is discharged to

land or natural waters
Water, Disease and Health

 Water-borne
 Exposure mainly by ingestion of contaminated
water
 Primarily enteric diseases transmitted by the
fecal-oral route
 Water-washed
 Exposure is reduced by water use for personal
and domestic hygiene: washing (clothes,
floors, other household chores), bathing &
other personal hygiene
 Water contact and water vector-borne
 Exposure by skin contact with contaminated
water
 Ex: schistosomiasis
 Exposure to water habitat "insect vector"
diseases
7
11/8/2018
Issues in Water and

Health
 Quality
 Quantity
 Access
 Habitat and Ecology
 Resources and Management
 Economics
 Behavior and Beliefs
 Enabling Environment and Policies
COMPONENTS OF
ENVIRONMENTAL SANITATION
 Water sanitation
 Food and milk sanitation
 Excreta disposal
 Sewage disposal
 Refuse disposal
 Vector and vermin control
 Housing
 Air sanitation
8
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WATER SANITATION
WATER SANITATION
9
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WATER
SANITATION
WATER SANITATION
10
11/8/2018
WATER SANITATION
WATER SANITATION
11
11/8/2018
WATER SANITATION
Water analysis assess the following

properties:
 Physical
 Chemical
 Radiological
 Biological
 Bacteriological
WATER SANITATION
 Public water supply must be-

Safe
Reasonably soft
Plentiful
Cheap
12
11/8/2018
WATER SANITATION
 Household treatment of water

 Boiling, i.e., beyond 2 minutes
 Chlorination- 1-5 ppm
 Iodine treatment- 10 drops per
gallon (about 3 drops/L)
 Filtration
 Aeration
BACTERIOLOGICAL EXAMINATION
OF WATER SAMPLES
Population Max. Interval Min # of

served between samples/per
sampling pop’n/month
Up to 20,000 One month One sample/
5000
20,001-50,000 Two weeks One sample/
5,000
50,001- 100,000 Four days One sample/
10,000
More than One day One sample/
100,000 10,000
13
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WATER SANITATION
-CHEMICAL QUALITY
CHEMICAL CONCENTRATION [mg/L]
Arsenic 0.2
Barium 1.0
Cadmium 0.01
Chromium 0.05
Cyanide 0.01
Lead 0.1
Selenium 0.05
Silver 0.05
WATER SANITATION
-CHEMICAL QUALITY
14
11/8/2018
WATER SANITATION
-CHEMICAL QUALITY
WATER REQUIREMENTS
1. Minimum Demand Per Person Per Day
 2 L for drinking
 10 L for food preparation and cooking
 15 L for bathing
 15 L for laundry
 10 L for sanitation and hygiene
2.Hospitals And Clinics

 Out-patient: 5 L / patient/ day.
 In-patient: 40-60 L/ patient/ day
 Feeding centers: 20-30 L/person/day
15
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WATER REQUIREMENTS
WATER REQUIREMENTS
16
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WATER REQUIREMENTS IN
EMERGENCY SITUATIONS
17
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WATER REQUIREMENTS IN
WATER REQUIREMENTS…IN
QUALITY CONTROL
 To preserve public health, a large amount of
reasonably safe water is preferred over a small
amount of purified water.
 Bacteriological, biological, chemical, physical and
radiological quality of water must be deemed safe.
 There must be no fecal coliforms per 100 ml at the
point of delivery
 People drink water from a protected or treated
source in preference to other readily available
water sources.
 Steps must be taken to minimize post delivery
contamination
 No negative health effect should be detected.
18
11/8/2018
WATER REQUIREMENTS…IN
DECONTAMINATION AND DISINFECTION
 Water purifier: 2tabs/person/day
 HTH [high test hypochlorite]: stock soln: 1L/20
families/5 days
 Shock disinfection: 50-100 ppm of 70% available
chlorine
OTHERS REQUIREMENTS
 Drinking water: one container of 10 L per family
 Communal water storage tank: 10 L per person
/day. Volume of tank must be good for two days
 Shallow well: for toilet flushing and cleaning
only
FOOD AND MILK SANITATION

 The GOLDEN RULE of food
sanitation is:
“Keep it cold or hot, and keep it
covered”
3 ENEMIES OF FOOD STORAGE:

High (ambient) temperature
High humidity
Contamination by strong odors
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FOOD SANITATION:
FOOD BORNE DISEASES
FOOD POISONING
OR
INTOXICATION
BACTERIAL PLANT OR CHEMICAL

-Staphylococcus ANIMAL •DDT, Lead,
-Streptococcus •Mushroom • Mercury, Cadmium
-Cl. botulinum •Mussels
•Fish
•Herbs
MILK SANITATION
 STERILIZATION- The application of high

temperature for the purpose of
destroying all types of microorganisms.
 PASTEURIZATION- The application of

heat to milk for the purpose of
destroying pathogenic microorganisms
with minimum injury to the substance
20
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MILK SANITATION
 TYPES OF PASTEURIZATION:
 HOLDING OR VAT PASTEURIZATION:
142—143 F FOR 30 MINS.
 HIGH TEMPERATURE, SHORT TIME
[HTST]- 160-162 F FOR 15 MINS.
 FLASHPASTEURIZATION- 190 F FOR FEW
SECONDS.
MILK SANITATION
21
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22
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23
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Sanitation & Disease (Contd.)

Transmission of Excreted Pathogens depends
on
 Excreted Load (Number of pathogens excreted
in the environment is termed as “excreted
load”)
 Changes in number of pathogens in the
Environment
Changes in number of pathogens during
environmental transmission are governed by
three key properties:
 Latency (i.e., how long it takes for the pathogens
to become ineffective)
 Persistence (i.e., how long pathogens survive in
the environment)
 Multiplication (i.e., ability of pathogens to
multiply)
Sanitation & Disease (contd)

Category Transmission Examples Transmission
Features of infection Focus
1. Non-Bacterial Non-latent Hepatitis A Personal

Fecal- Oral High infectivity &E Domestic
Diseas Low to medium Diarrhea
persistence
es Giardiasis
Unable to multiply
No intermediate host
2. Bacterial Non-latent Medium to Cholera Personal
Fecal-Oral low infectivity Medium Typhoid Domestic
Diseas to high persistence Water
Able to multiply
es Crops
No intermediate
host
3. Latent Hookworm Domestic
Geohelminthiases High persistence Ascariasis Field
Unable to multiply Crops
Very high
infectivity
4. Excreta- Infections 1-3 Water
related insect intermediate host transmitted
vector diseases mechanically
by flies and
cockroaches
24
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“Fecal-Oral” Transmission Routes of
Diseases
INSECTS
25
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Definition of Sanitation
System
o A sanitation system involves all arrangements
necessary to store, collect, process and
deliver human waste or other forms of waste
back to nature in a safe manner.
o Sanitation system with respect to human

waste management may be considered to have
the following functions
o Excretion and storage
o Collection and Transportation
o Process/ Treatment
o Disposal/ Recycle
Classification of Sanitation
System
(1)On site Sanitation System: When the wastes
are disposed of at or close to the point of generation
Example: Pit latrines (rural), septic tank system

(urban).
Conventional Simple Pit Latrine
26
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Classification of Sanitation System

(Contd.)
(1) On site Sanitation System (Contd.):
Basic Principle:
•Liquid infiltrates into the soil (infiltration capacity of soil
and location of ground water table are important issues).
•Solids are retained (confined) and anaerobically
digested. Solids have to be removed periodically, or a
new pit has to be dug at regular intervals.
Features:
•Designed to dispose of human waste only.
•Wastewater from other sources (kitchen, washing,
bathing) has to disposed separately.
•Suitable for sparsely settled rural areas with low
population density, and low water
consumption.
•Not feasible in areas with: (a) high population density (b)
high water consumption; (c) low infiltration capacity of soil
(d) high groundwater table.
System (Contd.)
(2) Off-Site Sanitation System: When the wastes are
collected and transported to somewhere else for treatment
disposal.
Examples: Conventional sewage system; small bore sewerage
system (SBS); Bucket latrines
Conventional Sewerage
System
27
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System (Contd.)
(2) Off-Site Sanitation System (Contd.):
Basic Principle:
• Basic elements of off-site sanitation system include
collection, transportation, treatment, disposal and/ or reuse.
•The waste is collected either through house sewers or
manually using buckets or vaults, transported either by
sewer system, cart or truck to a suitable distant place
where it is treated prior to disposal or reuse.
System (Contd.)
(2) Off-Site Sanitation System (Contd.):
Features:
•Collection and transportation of waste through a sewer
reticulation system requires that the waste be diluted by
water.
•Hence piped water supply is essential where this system is
to be applied.
•Most satisfactory system of waste disposal, provided
sufficient funds are available for its construction and
maintenance.
•Because of high cost, preferable to introduce gradually;
where possible existing sanitation system (e.g., septic tank
system) should be upgraded and improved (e.g., SBS
system utilizing existing septic system). In this system,
the costs can be significantly reduced because of smaller
sewer size and lower gradients.
28
11/8/2018
System (Contd.)
Sanitation system may be further classified into:
(a) Dry Sanitation System: No water is used for the

dilution of waste. Applied in areas with no piped water
supply.
Example: Pit latrines (rural, on-site), Bucket Latrines
(Urban, off-site).
(b) Wet Sanitation System: Waste is diluted with flushes

of water (to carry it away from the point of generation).
Example: Septic Tank System (on-site), Conventional
Sewerage System (off-site).
System (Contd.)
Sanitation system may be further classified into:
(i) Permeable System: Allows infiltration of liquid

portion of waste into the soil, causing potential
pollution of groundwater.
Example: Pit latrines.
(ii) Confined System: Does not allow infiltration of

Liquid portion of waste into the soil.
Example: Septic tank (not septic tank system, which
also includes a soakage pit
29
11/8/2018
Suitability of Sanitation System

Most important factor affecting suitability of
sanitation system:
 Level of Water Supply:
 Pit latrines would not be appropriate with
piped water supply
 Water borne system (e.g., conventional
sewerage system) is not feasible with bucket
carried or hand pump water supply
 Population Density
 On site system are more appropriate for
low density rural settings, and low density
urban areas
 Off-site systems are suitable for high density
urban centers.
Suitability of Sanitation
System
Appropriate sanitation
Various Sanitation systems based on water consumption level

and population density (Source: Veenstra et. Al., 1997).
30
11/8/2018
Rural Sanitation Options
• Different types of sanitation options

are in use in rural and low-income
urban areas, not all of which are
hygienic.
• A wide range of on-site sanitation

technologies exist that are low cost
and can be selected for use in
different hydrological, socio-
economic, and cultural conditions.
Typical Sanitation Options:

Rural/Urban Slums
31
11/8/2018

Rural/Urban Slums

Rural/Urban Slums
32
11/8/2018
Rural Sanitation Options
What are the criteria for

acceptable sanitation
options?
Hygienic Latrine
• A “hygienic latrine” is defined as a sanitation

facility which effectively breaks the cycle of
disease transmission.
• A hygienic latrine would include all of the
following:
• Confinement of waste (feces),
• Sealing of the passage between the squat hole
and the pit to effectively block pathways for flies
and other insect vectors, thereby breaking the
cycle of disease transmission, and
• Venting out of foul gases generated in the pit
through a properly positioned vent pipe to keep
latrine odor free and encourage its continual use
33
11/8/2018
Hygiene or Sanitary Latrine
• The most fundamental health objectives of

sanitation must be achieved through proper
design, installation, and use of a sanitary
or hygiene latrine.
• There is no universal design of a
sanitary/hygiene latrine that could be
used for all socio-economic and hydro-
geological conditions.
• Therefore, a wide range of sanitary/
hygienic latrine technologies should be
available to suit different conditions.
• Most technological options are:
• Pit latrines
• Pour flush latrines (often both types
are referred to as “pit latrines”)
Pit Latrines
• A pit is simply a hole in the ground that
receives human waste. Urine and other
liquids soak into the ground and solid
materials are retained and decomposed in
the pit.
• All forms of pit latrines are not fully
sanitary/hygienic. With slight modifications
in design and with some interventions,
conventional pit latrines could be improved
to be hygienic.
• The major types of pit latrines are:
• Simple or Home-made Pit Latrines
• Ventilated Improved Pit (VIP) Latrines
• Reed Odorless Earth Closet
Zaki Uddin Ahmad, Lecturer, Department of CE, UITS
34
11/8/2018
Prevention of
Groundwater pollution
Typical Pit Construction
Liquid infiltration only Perforated Concrete Liquid infiltration

through the bottom Rings would
increase pit life by 4-5
times
Typical Pit in
Typical Pit in loose/ unconsolidated soil relatively stale soil
Typical Dimension of Each Ring:

Diameter: ~ 3 feet/ 1 m
Height: 1 feet/ 0.3 m
Wall Thickness: 1.25 to 1.50 inch
Depth of Pit:
5 to 6 ring (i.e., 1.5 to 1.8 m) are most common (manually dug pit)
35
11/8/2018
How people see their city
River &
Environs
City
Peri- Ward
domestic
Home
(street,
school,
work-
place)
An environmental view
Home
Peri-
domestic (street,school,
workplace)
Ward
City
Central Treatment
Works
Collectors
Street
Sewers
House
Connections
36
11/8/2018
A public health view
Street Sewer River &

Sewer Mains Environs
City
Peri- Ward
Interceptor/ domestic
Collector Home
House
Connection
Treatment
Plant/Outfall
EXCRETA DISPOSAL
 METHODS :
1. WITH WATER CARRIAGE

2. WITHOUT WATER CARRIAGE
37
11/8/2018
EXCRETA DISPOSAL
1. WITHOUT WATER CARRIAGE

 CAT-HOLE
 STRADDLE TRENCH
 SANITARY PIT PRIVY
 BORED-HOLE
 CHEMICAL TOILET
 PAIL SYSTEM
 OVERHUNG LATRINE -”POUR-FLUSH”
EXCRETA DISPOSAL
2. WITH WATER CARRIAGE

-WATER SEALED
SEPTIC TOILET/AQUA PRIVY
- IMHOFF TANK SYSTEM
38
11/8/2018
EXCRETA DISPOSAL
 CHARACTERISTICS OF ADEQUATE EXCRETA

DISPOSAL FACILITIES FOR RURAL AREAS.
 Simple, cheap and easy to
construct
 Easy to maintain
 Affords easy protection against
the elements and provide desired
privacy
 Acceptable to the users
REFUSE/WASTE DISPOSAL
 REFUSE IS A GENERAL TERM APPLIED TO SOLID AND SEMI

SOLID WASTE MATERIALS OTHER THAN HUMAN EXCRETA
39
11/8/2018
REFUSE DISPOSAL
 PUBLIC HEALTH REASONS FOR

PROPER DISPOSAL OF WASTES
 Breeding place for insects and rats
 Gives out foul smell
 “Eye sore”
 Fire hazard
REFUSE DISPOSAL
 TYPES OF REFUSE
 GARBAGE: LEFT-OVER VEGETABLES, ANIMAL AND FISH
MATERIAL FROM KITCHENS AND FOOD ESTABLISHMENTS.
 RUBBISH: WASTE MATERIAL SUCH AS BOTTLES, BROKEN
GLASS, TIN CANS, WASTE PAPERS, DISCARDED
PORCELAINWARE, PIECES OF METAL, WRAPPING PAPERS
ETC.
40
11/8/2018
REFUSE DISPOSAL
 TYPE OF REFUSE:.. Con’t..

 ASHES: LEFT-OVER FROM BURNING OF WOOD AND COAL.
 DEAD ANIMALS/ CARCASSES
 STABLE MANURE
 STREET SWEEPING: DUST, MANURE, LEAVES, CIGARETTE
BUTTS, WASTE PAPER AND OTHER MATERIALS THAT ARE
SWEPT FROM THE STREETS
REFUSE DISPOSAL
 TYPES OF REFUSE ..con’t..

 NIGHT SOIL: HUMAN WASTE WRAPPED AND THROWN INTO
SIDEWALKS AND STREETS
 YARD CUTTINGS: LEAVES, BRANCHES, GRASS
41
11/8/2018
REFUSE DISPOSAL
 CHARACTERISTICS OF CONTAINERS
 SMALL ENOUGH TO BE EASILY CARRIED
 SUFFICIENT IN NUMBER
 PROVIDED WITH TIGHT-FITTING COVERS
 MADE OF STURDY MATERIAL
 STEADY
 PLACED IN AN ACCESSIBLE LOCATION
REFUSE DISPOSAL
 COMMUNITY REFUSE DISPOSAL METHODS:

 DUMPING ON LAND
 SANITARY LANDFILL
 COMPOSTING
 INCINERATION
 REDUCTION AND SALVAGE
42
11/8/2018
REFUSE DISPOSAL
 REFUSE DISPOSAL METHODS FOR HOUSEHOLDS

 BURIAL
 BURNING
 FEEDING TO ANIMALS
 COMPOSTING
 GRINDING AND DISPOSAL TO SEWER
REFUSE DISPOSAL
 REFUSE COLLECTION
 Frequent collection of refuse, specially
garbage, is necessary for good sanitation
 A longer interval between collection
creates problem of storage and foul odor
for the homeowner
 It is necessary to cover the refuse in the
vehicles during transportation to final
disposal sites to prevent flies, minimize
odors or remove traveling “eye sores”.
43
11/8/2018
REFUSE DISPOSAL
 REFUSE COLLECTION
 It is important to have adequate and
properly maintained collection carts, trucks
and other vehicles to eliminate collection
delays and complaints from residents.
 The route to the final disposal should be as
direct as possible from the point of origin.
It should preferably not pass busy streets.
 It is preferable to have collection done at
night
VERMIN CONTROL
[RODENT AND INSECTS]
 TYPES
 PHYSICAL OR MECHANICAL
 CHEMICAL
 BIOLOGICAL
 ENVIRONMENTAL
 EDUCATIONAL
44
11/8/2018
HOUSING SANITATION
 CHARACTERISTICS OF AN ACCEPTABLE
HOUSE
 ADEQUATE SPACE: AT LEAST 50 SQ.FT. per
PERSON FOR BEDROOM
 ADEQUATE LIGHTING: AT LEAST 100 FT.
CANDLES (100 lumens) FOR READING
 ADEQUATE WATER SUPPLY: 15-20 GALLONS
(56.8 – 75.7 L) PER CAPITA PER DAY
…..CONT….
HOUSING SANITATION
 CHARACTERISTICS OF AN ACCEPTABLE
HOUSE…[cont]…
 NOISE:SHOULD NOT BE MORE THAN 30
DECIBELS
 ADEQUATE HEAT AND VENTILATION
 EQUIPPED WITH SANITARY TOILET, FOOD
STORAGE AND PROPER REFUSE DISPOSAL
45
11/8/2018
…IN EMERGENCY SITUATIONS

 OTHER SANITARY REQUIREMENTS:
 LATRINE
 __ONE /FAMILY
 ----MIN. 1 SEAT/20 PERSONS
 --- 50 METERS AWAY FROM HOUSES
- WASTE DISPOSAL
- ONE COMMUNAL PIT/500 PERSONS [2X5X2 M]
 SOAP
 250G/PERSON/MO
…CONT…
…IN EMERGENCY
SITUATIONS..
 OTHER REQUIREMENTS…
cont…
 SHELTER
 Individual: 4 sq.m./Person
 Collective:30 sq,m,/person
[including shelter, sanitation
services, community activities,
warehousing, access etc]
46

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Microbial

“We cannot fathom the marvelous complexity of an organic being; but

❑Deep subsurface microbiology.

❑Final Written Examination (2 hours): 60%

❑Course Work: 40%

▪ Laboratory Reports 20%

❑Understand the ecology of microorganisms at population, community and

❑Describe the approaches used to study microorganisms in their natural

❑Describe community function and dynamics at both the molecular and

❑Appreciate the (vast) genetic and physiological diversity of microbes, and

❑Understand how the specific environmental properties of soils, oceans and

❑Discuss how microbes are useful in biotechnological and environmental

At the end of the lecture, students will be able to:

❑ Understand the ecology of microorganisms at

❑ Microbes and Ecosystem Niches

Diversity of microbial species in an ecosystem is expressed in two ways

(e.g., NH4+, S, H2S, Fe2+)

❑ Understanding the biodiversity of microorganisms in nature, and interactions in

❑Live in habitats suited to higher organisms, also in “extreme”

Seawater evaporating ponds near San

(Fig. 13.2(b), p. 423, Madigan & Martinko)

Figure illustrates O2 contours within a soil

❑ Soil aggregates can contain many different microenvironments supporting the

❑Growth of microbes depends on resources and growth conditions

❑Competition and cooperation occur between microbes in natural systems

❑Many microbes establish relationships with other organisms (symbioses)

(i) Negative effect for (one or both) interacting populations:

* Antagonism – specific inhibitor or metabolic product may impede growth/metabolism of others

(ii) Positive effect for (one or both) interacting populations:

(iii) Complementary metabolic interactions

❑Surfaces are important microbial habitats

• Typically offering microbes greater access to nutrients and

❑ The matrix is typically a mixture of polysaccharides.

❑ Biofilms trap nutrients for microbial growth and help prevent

❑ Biofilm formation is initiated by attachment of a cell to a

❑ Genes encode proteins that synthesize intercellular

• Most microbes grow attached to surfaces (sessile) rather than free

Evidence of a dental biofilm:

(a)Phototrophic biofilms colonizing the

(b)Cyanobacteria attached to the river

(a, c) Filaments of the large sulfur-oxidizing

(b) Thioploca form bundles of 10 to 20

*Two species of Thioploca commonly

❑ Bacteria form biofilms for several reasons:

❑localized biomass can be efficiently preyed upon,

❑ Chemolithotrophic mats contain

A coring taken through a microbial mat from

Could you guess the microbes occupying the other layers?

❑ Space exploration – microbes in extreme environments (hot springs,

❑ Molecular techniques – diversity of microorganisms (Carl Woese), new

❑ Realization that with pure culture/enrichment techniques, we know

❑ Biology of climate change, global biogeochemistry

❑Microbial ecology has its roots in microbiology, rather than ecology

❑ “basic” vs. “applied” science

❑ Discovered the Bacillus

❑ Agar media for pure

❑ Attitude of Koch’s time:

Agar petri dish zone of no bacterial

“The way I approach

Founder of the Dutch Delft

❑ Leader of the Dutch

❑ Student of van Niel

❑ Developed methods for