Transcript Document

Quantification of marine
microbial population
Objectives
Students will be able to:
• explain the significance of marine microbial for human life
• describe techniques used to get microbial samples from marine
sediment and water
• explain important key that should be considered for sampling
microbe
• Explain method to approch marine microbe quantification
Key concept
• Marine microbes exist in huge numbers and form a major
component of biomass on Earth.
• Although there is a wide range of sizes, most marine
microbes are exceptionally small.
• A wide range of physical and chemical conditions provide
diverse specialized habitats.
• Microbes are major components of plankton and marine
snow.
• Microbes are important in sediment formation and there is
abundant life below the seafloor.
• Microbes colonize the surfaces of inanimate (non-living)
objects and other living organisms by the formation of
biofilms.
Water sampling - microbes
• Sterile technique:
– Containers must be
sterilized by autoclaving
or with gas used to kill
microbes
– Take care not to
contaminate the container
– Water samplers should be
swabbed with 70 %
alcohol
Sampling from water
• The best is to collect samples directly into the
appropriate bottle or jar
• use of an intermediate container should be
avoided, but if have to 
• intermediate containers have to be
prewashed (for example syringes and filters)
or flushed with existing site water before
being used for the final collection of samples
if possible.
• If sampling using a pole with a large clamp (or
other suitable device) to hold the sampling
(see Figure 3.2), the rod may becomes
contaminated  wash it promptly,
• make sure the washings cannot contaminate
any samples or any material about to be
sampled (for example by disposing of
washings downstream of the sampling site).
Sequence of Sampling Matrices
• Project deals with multimedia and/or multiple parameters use
following sequence:
– Collect from least to most contaminated sampling locations
– If sediment and water is being collected, collect water first to
minimize effects from suspended bed materials
– For shallow streams, start downstream and work upstream to
minimize sediment effects due to sampling disturbances
– If sampling at different depths, collect surface samples first and
then proceed deeper
Sample Amount
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Minimum sample required depends on the concentration of the analytes present
Should take enough for all analyses and additional for any QA/QC work required
Heterogeneous samples generally require larger amounts to be representative of sample
variations
Taking too much sample can lead to problems with storage and transportation
Sample Preservation and Storage
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Purpose – minimize physical, chemical and biological changes
3 approaches:
– Refrigeration
– Use of proper sample container
– Addition of preserving chemicals (formalin or glutaraldehyde)
Proper Sample containers
• For microbiological analysis,
strong, thick-walled, glass sample
bottles with a minimum capacity
of 300 ml should be used.
• They should have screw caps of a
type that will maintain an
effective seal, even after they
have been sterilised many times
in an autoclave.
• Some technicians fasten a Kraft
paper cover over the bottle caps
before autoclaving to protect
them from contamination during
handling.
• Alternatively, plastic or
aluminium sleeves may be used.
• The neck of the bottle should not
be plugged with cotton wool. To
prepare sample bottles,
• they should be washed with a
non-ionic detergent and rinsed at
least three times (five is better)
with distilled or deionised water
before autoclaving.
• New bottles require the same
preparation. If distilled or
deionised water is not available,
clean chlorine-free water may be
used.
Sample carrier boxes
• keep samples at suitably
low temperatures, by
adding block or crushed
ice, dry ice, freezerblocks, or other similar
substance, or are
refrigerated by a power
source
• then transported in
cleaned/ uncontaminated
insulated carrier boxes
(coolers).
• Dry ice (solid carbon
dioxide) is used where
samples must be frozen
immediately after
collection.
• When storing chilled or
frozen samples in coolers,
note that the coolers can
be a source of sample
contamination under
some circumstances. 
be sure to clean it
thoroughly before use
Sample Preservation and Storage
•
Maximum Holding Time (MHT) is the length of time a sample can be stored after
collection and prior to analysis
• On arrival at the laboratory,
samples for bacteriological
analysis should be placed in a
refrigerator and analysis should
be started within 2 hours.
• Any samples arriving more than
24hours after they were
collected, or arriving unchilled
more than 2 hours after they
were collected, should be
discarded.
• Analysis of such samples is
unlikely to reflect the
bacteriological condition of the
water at the time of sampling.
Selection of Sampling Equipment
Surface Water and Wastewater Sampling
• Grab sampler, weighted bottle sampler, Kemmerer bottle
Water sample collection – grab
samples
Grab samples for fecal
coliforms are taken with
sterile containers
Selection of Sampling Equipment
Groundwater Sampling
• Collected from wells using a bailer or by pumps (peristaltic and bladder)
• Samples do not come into contact with mechanical components of the pump
Nansen bottle  for nonsterile water sample
Niskin sampler for sterile
water sample
Selection of Sampling Equipment
Soil Sampling
• Soil depth and whether or not each soil horizon is necessary to sample are main
considerations
• Scoops and trowels, tube sampler, augers, split spoon sampler (drilling)
Selection of Sampling Equipment
Sediment Sampling
• Dredges (Ekman dredge,
Peterson dredge,
Ponar dredge)
• Core samplers (Livingstone,
Kullenberg, and
Mackereth)
sample labeling
• An unlabeled sample
may as well just be
dumped down the
drain.
• Use good labels not
masking tape, etc. Poor
labels often fall off when
frozen samples are
thawed.
• Use permanent markers
NOT ball point pens,
pencils in a pinch
Where are they live?
• The largest population
present in the uppermost
layer of water
• Upperlayer of sediments
• Location that contain high
organic matter
• Seasonal influence
coiciding with changed with
temperature, tides, etc
• The greater population in
the shore
Heteregeneous
distribution
Regular sampling
is necessary
Factor affecting fluctuation in
microbial population
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Temperature
Complex nutritional
Physico-chemical condition in ecology
Interaction with other microorganism, ex L
grazing rates by zooplankton app. 106 cell/ml or
33-50% elimination
Number of marine microbial
• Number of bacteria in
the marine environmen
t reach between 103106/ml
• Max 108 in upper layer
• Number of amoeba
reach 1,2-1,3x103/ml
• Ciliated 0-23/ml
• Dinoflagellates 103-107/l
• Flagellated 3-2400/l
• Phytoflagellates 103105/l
• Yeast 10-8400/l
• No data of fungi due to
a lack of method
Where are thet located?
• Many bacteria located
on particulates
• Seawater bacteria
occured in association
with marine snow
• Some bacteria are freeliving in the sea
• In the sediments
Approch to estimation of bacteria
population
• It is difficult to ascertain which method provide the
most meaningful data
• Direct method : present of dormant bacteria/non
culturable cells, clumping, dead cell and uninformly
shaped particles
• The viable count only reach 0,1 % of organisms
observed
• Approch :
– microcospy of viable cell using nalidixic acid
– Transmission electron microscopy
– Spread technique
• Thank You