Microbial Ecology

1
Chapter 6
Microbial Ecology
2
Microbial Ecology
To study the relations of organisms to one
another and to their physical surroundings (i.e.,
environment).

• Key Differences from clinical microbiology
• Communities of organisms, not isolated laboratory
  monocultures.
• Interaction with environment.

• Challenges
• Must work with low concentrations of nutrients
  and organisms
• Most clinical laboratory techniques do not work
  in the field.

• Objectives
• Identify organisms present (biodiversity) and
  their interactions predator-prey, syntrophy,
  symbiosis, etc.
• Identify new pathways (e.g., SO42- oxidation of
  CH4)
• Identify processes and measure rates, such as
  primary production, nitrification,
  methanogenesis, etc.
• Develop models for understanding and prediction.

3
A conventional Experiment illustrating
relationship between Microorganisms and their
environments Winogradsky column

1. Mud from the bottom of a lake or river is
   supplemented with cellulose (e.g. newspaper),
   sodium sulphate and calcium carbonate, then added
   to the lower one-third of the tube (30 cm tall
   and 5 cm diameter).
2. The rest of the tube is filled with water from
   the lake or river, and the tube is capped and
   placed near a window with supplementary strip
   lights.
3. What will happen with the mud?

4
Winogradsky column
The mud become stratified with different colors
and looks beautiful (right-handed figure)!
The different types of microorganism proliferate
and occupy distinct zones where the environmental
conditions favour their specific activities.
5
Winogradsky column
The Winogradsky column is a simple laboratory
demonstration - illustrates how different
microorganisms perform their interdependent
roles the activities of one organism enable
another to grow, and vice-versa. These columns
are complete, self-contained recycling systems,
driven only by energy from light!
6
If the column is treated with different
supplements as
Column 1 Column 2 Column 3 Column 4
No additions Same as Column 3, but placed in the dark. 10 g Sawdust5 g CaSO45 g CaCO310 mg NH4Cl5 mg K2HPO4 10 g Chitin5 g CaSO45 g CaCO310 mg NH4Cl5 mg K2HPO4
Column 5 Column 6 Column 7 Column 8
10 g Glucose5 g CaSO45 g CaCO310 mg NH4Cl5 mg K2HPO4 10 g Sawdust6 g FeSO45 g CaCO310 mg NH4Cl5 mg K2HPO4 Same as Column 3, but with only 1/4 the sediments. Freshwater5 g CaCO310 mg NH4Cl5 mg K2HPO4

7
                                              Column 1                                               Column 2                                               Column 3                                               Column 4
                                              Column 5                                               Column 6                                               Column 7                                               Column 8

8
Column 6
Column 7
9
Profiles of hydrogen sulfide (H2S) and methane
(CH4) versus depth
                                             Column 1                                                Column 3                                              Column 4
                                             Column 5                                                Column 7                                              Column 8
10
Column 1
Column 3
11
The importance of microorganisms
You could probably remove all higher organisms
without significantly altering the Earths
biogeochemistry or homeostatic(????) properties.
Microbial Systems responsible for decomposition
of dead organic material (detritus) under aerobic
and anaerobic conditions
Higher Trophic Levels Metabolically Simple
Animals
Microbial Systems
Detritus
Plants
As well as being consumed by higher trophic
levels, microbial systems also recycle many
inorganic nutrients N, P, S, Trace metals.
12
Deep Biosphere
Evidence is growing for the support of a
biosphere living up to 1 km below the earths
surface. Bacteria in this deep biosphere have
been found in even crystalline basalt rocks below
marine sediments, and their biomass may exceed
that above the surface. This biosphere is driven
by geogasses, and is similar to deep ocean vent
ecosystems. Could also exist on other planets,
e.g., Mars.
Chemistry of the Deep Biosphere
Hydrothermal Vent Tubeworms and bacterial
symbionts
13
Rumen Microbial Ecosystem
Complex anaerobic microbial system found in the
rumen
Feed (grasses or grain) Cellulose and Starch
Glucose
Fermentation
Lactate
Succinate
H2 CO2
Formate
Methanogens
Acetate Propionate Butyrate
CH4 CO2
Protein
Greenhouse gases
Digested
Reactions mediated by dozens of bacterial
species, including protozoan grazers such as
ciliates(???-????).
Similar systems found in termites
14
Chemical Potential Exploitation
H2S oxidation by NO3-
CH4 oxidation by SO42-
Anammox NH4 NO2- N2 2H2O
Boetius et al. 2000
Schulz et al. 1999 Thiomargarita namibiensis
Strous et al. 1999 Planctomycete
Observations consistent with systems maximizing
energy degradation and MEP
15
Other examples, Microbially-coupled Systems
Symbiosis and Endosymbiosis
Lichen Fungi Algae
Dinoflagellates (????) in flatworm(???)
Sulfur bacteria in Riftia (???)
Mycorrhizae (??)
16
Prokaryotic Differentiation
Cell-to-cell signaling compounds (such as N-acyl
homoserine lactones) allow bacterial species to
exhibit multicellular characteristics.
Ben-Jacob (1998) Paenibacillus dendritiformis
17
Other Important Microbial Processes

• Nutrient cycling under aerobic and anaerobic
  conditions. Removal of excess nitrogen via
  nitrification and denitrification in eutrophic
  systems. NH4 ? NO3 ? N2
• Fixation of N2 gas into organic N, especially in
  root nodules via symbiosis with bacteria, such as
  Rhizobium.
• N2 ? Amino Acids
• Remediation of toxic substances (bioremediation
  or natural attenuation).

• Almost all biomass and processes in the oceans
  are dominated by microbes.
• Largest organism on Earth is a fungus (Armillaria
  ostoyae honey mushroom).
• Major sources and sinks for atmospheric trace
  greenhouse gasses (CH4, N2O).
• Cycling of iron (Fe3 ? Fe2), Manganese (Mn4 ?
  Mn2) and other metals.
• Cause of many diseases, especially in 3rd world
  countries.
• Very high species abundance, current estimate of
  107 species in 10 g of soil.

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Habitats and biogeochemistry
The world is not well-mixed, so that sources
and sinks of matter and energy produce gradients
and microenvironments. Microbial communities
develop as a function of resource availability.
Deep Lake
Gradients in Particles
Amount
Light
O2
Photic zone (aerobic)
Depth
Aphotic zone (aerobic)
O2
Anaerobic sediments
Radius
These gradient dominated environments (hot
springs, deep sea vents, hyper-saline, anoxic)
often can not be duplicated in the lab, so
research must be conducted in the field.
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Works in the lab, but not the field
Lab versus field measurements
Syntrophy Nutrient cycling
In Lab
B
1
3
C
In Field
2
A
D
Contained systems will often quickly diverge from
true system.
Finally, concentrations of compounds in natural
systems are often at the limit of detection! ?
New methods often produce new understandingParadi
gm Shifts
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How small are we talking?
Microbiology Requires a microscope to study ?
1 mm or less
Plankton Net movement dependent on flow field.
Nekton Move independent of flow field.
Phyto Autotrophic Zoo Heterotrophic

• Femtoplankton 0.02 - 0.2 mm
• Mostly viruses
• Picoplankton 0.2 - 2 mm
• Bacteria, cyanobacteria
• Nanoplankton 2 - 20 mm
• Flagellates, dinoflagellates
• Microplankton 20 - 200 mm
• Diatoms, ciliates.
• Mesoplankton gt 200 mm
• Zooplankton (copepods)

Surface area to volume

• Bacteria 0.2 mm - 1000 mm (1 mm)
• Typically 1 - 2 mm culture, or lt 1 mm natural
  environments.

Surface area 4 p r2 Volume 4/3 p r3 SA/V
3/r
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Prokaryotes and Eukaryotes

• Two general classes of cells
• Prokaryotes Most primitive cell. Lack internal
  organelles and nucleus. DNA is single molecule
  (no chromosomes) aggregated in the nucleoid,
  although plasmids can also be present.
  Reproduction occurs by simple division. Almost a
  synonym for bacteria.
• Eukaryotes Typically larger cells that contain a
  nucleus with DNA organized into chromosomes.
  Organelles, such as mitochondria or chloroplast,
  are usually present. Usually contain two copies
  of genes (diploid) and division occurs by
  mitosis. Example eukaryotes
• Algae Phototrophic, single cells (or colonies).
• Fungi Heterotrophic single (yeast) or
  multicellular (molds, mushrooms) with cell wall.
  
• Protozoa Single cells lacking rigid cell wall,
  heterotrophic.
• Metazoans Plants (phototrophic) and animals
  (heterotrophic) (cell differentiation).

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Five Kingdoms (old Phylogeny)

• Animalia
• Multicellular
• Organs
• No cell wall
• Ingests nutrition
• Nervous system
• Has locomotion

• Plantae
• Multicellular
• Cellulose cell walls
• Photosynthetic nutrition
• No nervous system
• No locomotion

• Fungi
• Most multicellular
• Chitin cell walls
• Absorbs food
• No nervous system
• No locomotion

• Monera
• Most unicellular, some colonial
• Cell walls of polysaccharides
• Absorbs food, chemosynthesizes or
  photosynthesizes
• No nervous system
• Locomotion in some

• Protista
• Most unicellular or simple multicellular
• Some have cell walls
• Absorbs food, ingests or photosynthesizes
• No nervous system
• Locomotion in some

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Phylogeny from DNA sequences

• Methodology
• Compare DNA sequences of different organisms
• Closer DNA sequences are to each other, closer
  the evolutionary distance separating the
  organisms.
• Use DNA of ribosomal RNA (rRNA), since all
  organisms have rRNA and rRNAs are evolutionarily
  stable.

Example
Evolutionary distance (base pairs)
A ? B 1 A ? C 4 A ? D 5 B ? C 3 B ? D 4 C ?
D 1
AACGTCGAAA (Organism A) AACCTCGAAA (Organism B)
AGGCTAGAAA (Organism C) AGGCTAGTAA (Organism D)
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Phylogenetic Tree (Three Domains)
Based on 16s or 18s rRNA sequences Carl R. Woese
Eukarya
Archaea
Multicellular
Bacteria
Animals
Extreme halophiles
Slime molds
Fungi
Green non-sulfur bacteria
Entamoebae
Methanobacterium
Plants
Mitochondrion
Thermoproteus
Ciliates
Gram bacteria
Thermoplasma
Methanococcus
Purple bacteria
Thermo- coccus
Pyrodictium
Chloroplast
Flagellates
Cyanobacteria
Trichomonads
Flavobacteria
Thermotoga
Microsporidia
Aquifex
Diplomonads

• 3 Kingdoms
• Hyperthermophiles-psychrophiles
• Halophiles- Methanogens
• Hyperthermophiles

• 15 Kingdoms

• Protists
• Fungi
• Plants
• Animals

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Endosymbiosis
Evolution of modern eukaryotic cells.
Animals
Modern eukaryote w/o mitochondria
Protozoa
Archaea (prokaryote)
Universal ancestor (prokaryote)
Primitive eukaryote
Nuclear line (prokaryote)
Bacteria (prokaryote)
Endosymbiosis with nonphototrophic cell
(primitive chloroplast)
Algae
Endosymbiosis with phototrophic cell (primitive
chloroplast)
Plants
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Evolution of life and atmospheric O2
0
Age of dinosaurs
Cambrian
Morphological evolution of metazoans
Cambrian explosion
Precambrian
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Origin of metazoans
1
10
O2 ( in atmosphere)
Origin of modern eukaryotes
Endosymbiosis
Development of ozone shield
Time before present (billions of years)
1
2
Oxygenated environment
0.1
Origin of oxygenic phototrophs (cyanobacteria)
Evolution of PS II
Archaea
Nuclear line
3
Bacteria
Microbial diversification
Origin of life
Anoxic
Chemical evolution
Photochemical synthesis of organic chemicals
4
Formation of the earth (4.5 109 years before
the present)
From Brock Biology of Microorganisms
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Redox Reactions and Energy Production
Gibbs Free Energy
A B ? C D
A ? B e - Oxidation C e - ? D- Reduction
Reduction-oxidation (redox) reactions
Aerobic Respiration Examples Organic CH2O
O2 ? CO2 H2O Inorganic NH4 2 O2 ? NO3-
H2O 2 H
Anaerobic Respiration Examples Organic 3
CH2O 2 SO42- ? 3 CO2 2 S 5 OH- H
Inorganic 3 H2 CO2 ? CH4 2 H2O
Fermentation Example C6H12O6 C6H12O6 ?
2 CO2 2 C2H6O
Autotrophy
H2S ? 2 H S 2 e - PS I H2O ? 2 H ½ O2
2 e - PS II
CO2 3 H 4 e - ? CH2O OH-
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Metabolic Classification of Life
Classification
Carbon Source
Energy Source

Photoautotrophs
Light(Phototrophs)
PS I anaerobic, H2SPS II aerobic, H2O
Photoheterotrophs
CO2(Autotrophs)
Inorganic(Chemolithotrophs)
Chemolithoautotrophs
Aerobic (majority)Anaerobic (few)
Chemical(Chemo- lithotrophs)
Chemolithoheterotrophs (or mixotrophs)
Organic(Heterotrophs)
Organic(Chemoorganotrophs)
Chemoorgano-heterotrophs
Aerobic respirationAnaerobic respirationFeremtat
ion
Chemo-organoautotrophs
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Course Overview
Lab and Lecture Topics 1. Introduction Backgroun
d information and setup of Winogradsky columns.
2. Bacterial Abundance Techniques to determine
bacterial densities (plate and direct counts).
3. Bacterial Production Measurement of bacterial
growth rates using 14C. 4. Enzyme
Assays Technique to measure activity of
extracellular enzymes. 5. Chemolithotrophy Use
Winogradsky columns to study microbial
biogeochemical diversity. 6. Bacterial
Grazers Microbial loop concept and determination
of the bacterial consumption rate by predators.
7. Bacteria-Phyto. Comp. Examine competition
between bacteria and phytoplankton for inorganic
nutrients. 8. PCR Technique Polymerase chain
reaction.
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Winogradsky Column

• Sediment supplements
• CaCO3
• CaSO4
• Carbon source

O2
H2S
Cyanobacteria Algae
Water
Sulfur bacteria
Purple nonsulfur bacteria

• All five major metabolic groups will develop
• Sulfate reduction
• S oxidation
• Fermentation
• Photosynthesis PS II
• Photosynthesis PS I
• Methanogenesis
• Nitrification
• Denitrification?

Purple S bacteria
Green S bacteria
Sediment
Desulfovibrio
Clostridium
Wear shoes that can get muddy for next
Thursdays Lab
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Bacterial Abundance

• Objective
• Measure bacterial numbers and mass per unit
  volume.
• Note, we are not concerned with identification
  here.
• Why do we want to know abundance?
• Allows determination of biomass pool size.
• Provides crude estimate of element fluxes.
• Helps to characterize dynamics of ecosystem.
• Challenges with natural samples
• Low concentrations
• Methods
• Dry and weigh (not with natural samples).
• Plate (or viable) count (Today).
• Direct count. (Thursday).

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Why do we want to measure bacterial concentration?
E.g., Bacterial concentration is 100 cells ml-1
or 100 fg C ml-1

• Estimate bacterial pool size
• Ocean 109 cells l-1
• 20 fg C cell-1 (20 ? 10-15 g C cell-1)
• 1.37 ? 1021 l oceans-1
• Crude estimate of element fluxes (x bacterial
  biomass)
• Growth rate G ?x ? specific growth rate
• Uptake rate U ?x/? ? growth efficiency
• Typical ? 1 d-1 ? 0.2
• Ecosystem dynamics

27 Gt C oceans-1
R
Conc.
Time
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How is bacterial concentration measured?

• Laboratory cultures
• Measure optical density and cell dry weight
• Problems
• High cell densities required.
• Must be only cells (i.e., no debris or detritus)
• High predator abundance would also skew results.
• Technique does not work in the field!
• Dilution Plates
• Grow single cells on Petri plate until colonies
  are visible, then count colonies.
• Must use serial dilution so that colonies are in
  countable range.
• This method has a major problem. What is it?
  (Akin to growing fish in chicken soup)
• Direct Counts
• Use microscope to directly count bacteria.
• Problem Bacteria in natural environments are
  very small and difficult to see and distinguish
  from detritus using standard light microscopy.

34
Dilution Plates
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
9 ml
10-1
10-2
10-3
10-4
10-5
10-6
Statistically relevant colony density 30 - 300
Technique largely used for isolation or water
testing, such as coliform test.
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Dilution Plate Calculations
N Number of colonies on plate VS Volume
pipetted onto Petri plate. D Dilution factor
for test tube plated out. ? Concentration of
cells in original sample (cells ml-1)
Example N 33 VS 100 ml D 10-4
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Fecal Coliform Counts
The abundance of fecal coliform bacteria are used
as an indicator of fecal contamination of both
drinking water and recreational water (i.e.,
swimming, shellfishing). Fecal coliform bacteria
inhabit the intestinal tracks of animals. While
the indicator bacteria are typically not
pathogens, they indicate that the water has
become contaminated with fecal material, either
by human or other animals. Although it would be
better to assay for pathogens directly (such as
hepatitis), it is too difficult to culture these
organism quickly and reliably.

• Basic method
• Aseptically collect and filter water onto sterile
  filter.
• Place filter on sterile pad that contains medium
  for the culturing of fecal coliform bacteria
  (contains eosin-methylene blue dye)
• Incubate filter at 37ºC
• Count colonies to determine colonies/100 ml water
  
• EPA requirements (cfu/100ml)
• Drinking water None
• Shellfishing ? 14
• Swimming ? 200

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Some Drinking Water Pathogens

• Viruses
• Hepatitis
• Bacteria
• Cholera (Vibrio cholera)
• typhoid fever (Salmonella typhi)
• Fecal bacteria (often Escherichia coli)
• Protists
• Cryptosporidia
• Giardia

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Direct Bacterial Counts

• Challenges with Direct Count Method
• Natural samples contain low concentrations of
  bacteria (106 cells ml-1)
• Must concentrate bacteria
• Bacteria are small (0.2 - 1 mm) so difficult to
  see and differentiate from detritus using
  microscope with normal or phase contrast lighting
  techniques.
• Must stain with fluorescent dye and use
  epifluorescence microscopy.
• Procedure outline
• Incubate water sample with fluorescent dye.
• Concentrate sample onto 0.2 mm filter.
• Place filter on slide, and count bacteria in grid
  
• Calculate bacterial numbers.

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Epifluorescence Microscopy

• Fluorescence
• Compound is excited at a particular wavelength
  of light (usually in the UV)
• Compound then emits light at a different
  wavelength.
• Advantage contrast is extremely high, which
  allows detection of weak light.
• Dyes used
• Acridine orange (AO)
• DAPI (46-diamidino-2-phenylindole)
• Mechanisms
• AO fluoresces when bound to DNA or RNA. Cells
  appear orange.
• DAPI fluoresces when bound to DNA and is more
  specific. Cells appear blue.

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Epifluorescence Details
Light source
Excitation filter
Eyepiece
Beam splitter, Emission filter
Objective
Sample
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Slide Preparation for DAPI
Drop of immersion oil
Cover slip
Drop of immersion oil
Filter, bacteria side up!
Drop of immersion oil
Microscope Slide

• Notes
• Place filter so that bacteria are on the top
  side.
• Use small drops of immersion oil
• Cover slips stick together. If you have more than
  one, you will not be able to focus well.
• Label slide.

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Cell Density Calculations

• Known or measured
• Volume of sample filtered VS
• Area of filter occupied by sample AF
• Area of grid in field of view AG
• Average number of cells grid-1 N
• Cell Concentration
• Cell Conc r

Whole filter
Filter wetted by sample
AF pRF2
RF
AG
What is the main assumption in this calculation?
43

• SES Methods in Microbial Ecology
• Fall 2005
•  
• Problem Set 1 (Due 15 Sept 05)
•  
• Answers to the questions below should be short,
  usually just one sentence or one word, unless
  otherwise requested. Each question is worth 10
  pts unless otherwise indicated. Partial credit is
  given only if you show your calculations.
•  
•  
• 1)           Explain how microbes that are strict
  anaerobes (that is, they perish in the presence
  of oxygen) can survive and flourish in
  environments where the bulk phase is replete with
  oxygen, such as in soils and the oceans.
•  
• 2)           What reactions do bacteria use to
  ameliorate eutrophication?
•  
• 3)           Primary productivity (CO2 fixation
  rate) in aquatic systems is often measured by
  placing water in an airtight glass bottle and
  measuring the increase in oxygen over a several
  hour incubation period. Why may this approach
  fail in very shallow aquatic environments?
•  
• 4)           A) In DNA-based phylogeny why is it
  important to choose a gene that does not exhibit
  rapid mutation rates? B) Which prokaryote domain
  has a close relation to Eukarya?
•  
• 5)           A) List four macromolecule
  constituents of living cells. B) What are
  micronutrients? C) What is a guild?
•  
• 6)           We do not find chemoorganoautotrophs
  in nature. Why not?

44
  Carbon Source Energy Source
a) CO2 Light
b) C6H12O6 C6H12O6 ? 2 CO2 2 C2H6O
c) (CH2O)n H2S ½ O2 ? S H2O
d) C2H6O Light
e) CO2 NH4 2 O2 ? NO3- H2O 2 H

•  
• 8)           A) How much faster (or slower) will
  1 L of bacteria 0.1 mm in diameter grow compared
  to 1 L of bacteria 1.0 mm in diameter? B) Will a
  single 1.0 mm diameter bacteria grow faster than
  a single 0.1 mm diameter bacteria? Explain.
•  
• 9)           A) Which are more closely related
  humans and trees or bacteria and archaea? B) Why
  are prokaryotes only one kingdom (Monera) in the
  older phylogeny tree?
•  
• 10)       What evidence supports the
  endosymbiotic theory?

45
SES Methods in Microbial Ecology, Fall
2005 Problem Set 2 (Due 22 Sept 05)   To get
partial credit, please show all your
calculations. If you give only a number answer,
and it's incorrect, you will get zero on that
question.   1)     Plate count lab A) Report the
number of colonies counted on each dilution
plate. From the statistically appropriate plates,
calculate the concentration of bacteria in the
original sample. Specify which plate count is
being used for the calculation. B) Report the
colonies counted on the fecal coliform plates,
the volume of sample water filtered, the location
of where the sample was collected and calculate
the number of fecal coliforms per 100 ml of
sample.   2)     DAPI count lab Report the
average number of bacteria per grid counted under
the microscope using the DAPI stain. From this
number, calculate the bacterial concentration in
the original sample. Specify the volume of sample
you stained/filtered. Note, the size of the grid
in the microscope is 10 mm x 10 mm.   3)     Why
doesnt detritus fluoresce just like the bacteria
when stained with DAPI?   4)     A) What are two
disadvantages of using Acridine Orange for direct
bacterial counts? B) What cellular
macromolecule(s) does Acridine Orange bind
to?   5)     Why are bacterial numbers measured
by plate counts lower than bacterial numbers
measured by direct counts?   6)     A) What
organism caused the largest outbreak of a
waterborne disease in US history? B) Does the
fecal coliform count measure the concentration of
pathogenic organisms in a water sample? Explain
your answer.   7)     A) Assuming a bacterial
concentration of 1.4 ? 106 cells ml-1 (this
notation is the same as cells/ml) and that a cell
has 20 fg C (f is femto, 10-15), what is the
carbon concentration associated with the
bacterial biomass? Please give your answer in
mmol C l-1. B) If we assume that the bacteria
were growing at 1 d-1, and had a growth
efficiency of 20, what would the rate (in mmol C
l-1 d-1) of CO2 production by bacteria
be?   8)     A) Mechanistically, it would be
possible to stain bacteria with DAPI, then put a
drop of the stained bacteria on a microscope
slide and count them. What is the filtration step
for in the DAPI method? B) Why do we run a blank
along with the sample in the DAPI
counts?   9)     Why do we use fluorescence and
not phase contrast to count bacteria in natural
samples?   10) A) What benefit does immersion
oil provide in light microscopy? B) What is the
approximate size of the smallest object that can
be resolved using light microscopy?
46
Signature Lipid Profiling
Methylation
Liberate fatty acids and ether lipids
Sample


Lipid extraction
COOH
O
OH
O
O
Gas chromatograph
HO
Separate fatty acids and ether lipids
O
The identification of fatty acid and ether lipid
components of cell membranes can be used to
identify micro-organisms and trace these strains
in leaching environments.
identify and quantify fatty acids and ether lipids
Chromatogram