Lab 4 - WQI and DNA Barcode Sample Preparation

Nitrates

Concentration of nitrate ions in water
Important source of nitrogen for plants and animals to synthesize amino acids and proteins
mg/L NO3-N unit
Usually 0.1-4 mg/L
Below 1.0 mg/L for healthy bodies of water
Can lead to eutrophication

Factors that lead to high nitrates concentration

Animal feedlots
Runoff from fertilized fields
Treated wastewater

Eutrophication

Process by which nutrients like phosphorous and nitrogen become highly concentrated leading to increased growth of organisms such as algae or cyanobacteria

Fecal coliform bacteria test

Determines whether or not there is E. coli present in water
Presence of E. coli implies existence of pathogens, diseases, viruses, and parasites as well as fecal contamination
Presumptive test
CFU/100ml unit

E. coli

Types of bacteria that live in the intestinal tracts of humans

Factors that lead to high E. coli levels

Leaking septic or sewer systems
Polluted runoff with animal waste
Waterfowl
Wading cows

Steps to fecal coliform presumptive test

Water sample is added to lactose in a tube
Coliform bacteria will ferment lactose and generate CO2 into the Durham tubes
Tubes are incubated at 44.5C to kill any other bacteria
Number of positive tubes is compared to the table to determine the Most Probable Number of bacteria in the sample

Negative fecal coliform result

Absence of gas in Durham tube

Doubtful fecal coliform result

Less than 10% of the Durham tube is filled with gas

Positive fecal coliform result

More than 10% of the Durham tube is filled with gas

Steps to measure nitrate concentration

Soak Nitrate ISE in the high standard solution for 30 minutes
Calibrate
Collect nitrate concentration data
Properly store Nitrate ISE

Steps to soak nitrate ISE in high standard solution

Attach the probe clamp to the support stand
Insert the nitrate ISE to the probe clamp
Raise clamp to allow room for the rinsate beaker
Rinse tip with water and wipe
Uncap the high standard
Insert the probe into the high standard so that the white dot is submerged
Let soak for 30 minutes

Steps to calibrate nitrate ISE

Plug ISE sensor into Channel 1

Go to sensors

Select calibrate

Select nitrate ISE

Select calibrate now

Wait for live voltage reading to stabilize

Enter 100 for value 1

Press keep

Remove nitrate ISE from high standard by raising probe clamp

Swap out high standard for rinsate beaker

Rinse tip with water and wipe

Swap out rinsate beaker for low standard

Lower probe clamp into low standard

Wait for live voltage reading to stabilize

Enter 1 for value 2

Press keep

Press ok to finish calibration

Steps to collect nitrate concentration data

Rinse tip with water and wipe

Place tip into sample water

Leave probe submerged for 1 minute

Begin data collection for 10 seconds

Stop data collection

Go to analyze

Go to statistics

Record mean

Steps to properly store nitrate ISE

Rinse tip with water and wipe
Insert probe into long term storage bottle (do not touch sponge)
Tighten the lid

Colony forming units assay

Determines the number of colony forming units per unit volume of a water sample
Uses spot plates and agar gel

Reading results of colony forming units assay

Look at the growth on the spots and enter the number of colonies observed
Some colonial batches may be too dense to count (CC)
Different tubes have different dilutions
Tubes with higher concentrations (lower dilutions) will be harder to count since they have more CFU per unit volume

CFU/ml of a sample

(Average # CFUs) (Total dilution factor) / Volume plated (ml)

DNA barcoding

Genome sequencing used to differentiate species with indistinguishable morphological characteristics

Conserved region of DNA

A nucleotide sequence with little to no variation across a species

DNA barcoding preparation

Extraction of DNA
Amplification by PCR
Visualization via agarose gel electrophoresis or Sanger method
Analysis

COI gene

Barcode to identify animal samples
Codes for cytochrome c oxidase
Important for respiration
700 bp fragment

rcbL gene

Barcode to identify plants
Important for carbon fixation in photosynthesis
600 bp fragment

16S rRNA

Barcode to identify bacteria
Codes for 16S ribosomal RNA
1500 bp fragment

Agar

A solid matrix of agarose with added nutrients to provide a growth medium for bacteria

Direct amplification

PCR without DNA extraction due to the heat in PCR lysing cells to release DNA
Used in DNA barcoding for bacteria which lack membrane bound organelles