Lab 4: WQI and DNA Barcode Sample Preparation Flashcards
Why are nitrates important in water?
Nitrates are an important source of nitrogen necessary for plants and animals to synthesize amino acids and proteins.
How does nitrogen gas change into a form that is usable by plants and animals?
Through the nitrogen cycle. These conversions include industrial production of fertilizers, as well as natural processes, such as legume-plant nitrogen fixation, plant and animal decomposition, and animal waste.
What manmade sources can raise the concentration of nitrates?
Animal feedlots, runoff from fertilized fields, or treated municipal wastewater being returned to streams.
What can be caused by high nitrate concentrations?
Eutrophication. Unpleasant odor and taste of water, as well as reduced clarity, often accompany this process. Eventually, dead biomass accumulates in the bottom of the lake, where it decays and compounds the problem by recycling nutrients.
Also, high nitrate levels can cause blue-baby syndrome in which nitrate converts hemoglobin into a form that can no longer transport oxygen.
What units are nitrates measured in?
mg/L NO3–N
What do fecal coliform bacteria tests test for?
It tests for intestinal E. Coli and other coliform bacteria. (E. coli lives in the intestinal tracts of humans and animals)
What are coliform bacteria?
They are facultative anaerobes (can tolerate oxygen but can survive without it) that ferment lactose to produce gas.
How can fecal coliform be harmful?
Can cause typhoid fever, cholera, and dysentery
Why do we measure E. Coli?
Rather than testing for individual types of pathogens, water is tested for non-pathogenic intestinal bacteria, Escherichia coli in particular. As E. Coli is always found in the intestines and are not normally present in soil or water, its presence in water indicates that fecal material has contaminated the water supply. E. coli is a good sewage indicator because it is not normally present in water or soil, it is relatively easy to identify and survives a little longer in water than other enteric (from the intestines) bacteria. If they were hardier, they would make water purity tests too sensitive.
What is a presumptive test for testing fecal coliform?
You will not actually count or identify the actual bacteria; rather, you will infer from the amount of gas (usually CO2) produced how many E. coli are present in the water. This test also presumes that the gas is produced by E. coli as the gas could be produced by other coliform bacteria as well. Coliform bacteria are facultative anaerobes (can tolerate oxygen but can survive without it) that ferment lactose to produce gas.
How does E. coli get into the water?
Leaking septic or sewer systems, polluted runoff that has picked up animal waste en route to the stream, waterfowl in the stream, or wading cows.
What is DNA Barcoding?
The use of specific DNA sequences (DNA markers, genes) to identify organisms to species level. In this technique DNA sequence provides a unique way to identify the species, much like UPC Barcodes on products in stores that are used to uniquely identify the product.
Sequencing a short stretch of DNA. The DNA provides a unique way to identify the species. The specific characteristics of each species, and also for each individual within a species, is defined by their genomic DNA sequence. If the genomic sequence of each sample were known, then it would be possible to compare it with sequences from other organisms to determine if they are likely the same species or if they are different species.
What marker is used for animal samples?
The COI gene that codes for cytochrome c oxidase is used as a marker for animal samples. This is a highly conserved gene that is found in mitochondria and is important for respiration. Since all eukaryotes have mitochondria, this gene serves as a good DNA Barcode marker. It should generate around a 700 bp fragment.
What marker is used for plant samples?
The rbcL gene is found in the chloroplast genome and codes for ribulose-biphosphate carboxylase, commonly referred to as RuBisCo. This protein is involved in carbon fixation in one of the first steps in photosynthesis and is plant specific. The rbcL gene provides a DNA Barcode marker to specifically identify plants and enables scientists to distinguish the plant from other organisms that may be living in or on the plant. The primer set for the rbcL gene generates around a 600 bp fragment.
What marker is used for bacteria samples?
Since bacteria do not have mitochondria and chloroplasts, another gene is used as a DNA barcode marker. The 16S rRNA gene codes for the 16S ribosomal RNA that is part of the bacteria ribosome complex and the nucleotide sequence of this gene is highly conserved. In this course we will use a primer set for the 16S rRNA gene for the bacteria samples that should generate around a 1500 bp fragment.
