Quantification of marine microbial population

ObjectivesStudents 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

• 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

• 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, freezer-blocks, 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

• 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.

Sample Preservation and Storage

• Maximum Holding Time (MHT) is the length of time a sample can be stored after collection and prior to analysis

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 non-sterile 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

• 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 103-106/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 103-

105/l• Yeast 10-8400/l• No data of fungi due to a

lack of method

• Many bacteria located on particulates

• Seawater bacteria occured in association with marine snow

• Some bacteria are free-living in the sea

• In the sediments

Where are thet located?

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