Earth Systems Science & Policy
Biotechnology Lab
an Introduction to Molecular Biology Laboratory Techniques
with Earth Systems applications
Henrik Kibak - Spring 2005

First Experimental Activity - Microbial Diversity of Elkhorn Slough (a National Estuarine Research Reserve)

This lab is inspired by Chapter 3 of "Molecular Microbiology Laboratory: A Writing-Intensive Course" by Walt Ream, Bruce Geller, Janine Trempy, and Katharine Field, of the Oregon State University Department of Microbiology, 2003 Academic Press.

Collecting and Isolating Pure Cultures of Bacteria from the vicinity of the Salt Ponds

Purifying Genomic DNA of Prokaryotes

PCR Amplification of 16S rRNA Genes

Purification of PCR Product

Subcloning for Sequencing

Collecting and Isolating Pure Cultures of Bacteria from the vicinity of the Salt Ponds

Day 1 - A trip to the Slough

• Select a "kit" for your trip to collect bacteria.  Your kit should include a Sharpie marking pen, sterile cotton swabs,

• Select one of each of the four types of agar plate and label them with your name and the correct label "HSdLB+amp," "Marine_dLB," "dLB" or "LSdLB." * Parafilm them tightly shut for the long trip to Elkhorn Slough Salt Ponds.

• Use your sterile cotton swabs to collect samples.  First collect from the colored dust/mud in one of the former salt ponds.  If you are swabbing from a dry surface, first wet the swab with sterile water or LB broth.

• When you have collected your sample on the cotton swab, paint it repeatedly along one-third of the side of the appropriate agar plate.  For example, use the High Salt with ampicillin plate for samples coming from the former salt pond.  Use the Marine plate if you soaked your swab in one of the microbial mats along the jetty.  Use the Low Salt plate if you are collecting from rain-washed soil. The dLB plate can be your default plate for use anywhere.  Record the locations where you collected in your lab books, preferably including Lat/Lon/Elev. GPS. Hopefully we will be able to photograph each of you as you collect your sample and supply the photo for your write-up.

• Secure the plates so that they do not open using tape or parafilm.

• Back in the lab we will finish streaking the plates using the sterile toothpicks:  Make three additional streaks, each with a fresh toothpick, by starting from the edge of the original swabbing on the plate, lightly drawing sort of a "Z" out from that first paint job.  Then, with a fresh toothpick, make a new "Z" out from the first "Z".  Finally make a last "Z" from the second, again using a fresh toothpick. If you only barely touch each "Z," this will create three "dilutions" of your original sample.

• Incubate the agar plates at 25° C for 24 hours, inspect.

Day 2 - Collecting descriptive results and subculturing

Remember when handling these cultures to treat them as potential pathogens.  It is entirely possible to isolate human pathogenic bacteria from Elkhorn Slough or almost any natural environment.  Follow instructions in the lab on gloves, protective eyewear, biohazard disposal, etc.  Most pathogens need to be at a certain titre to be able to beat your immune system, for example Vibrio cholerae needs to be at 10,000 cells per cubic centimeter of water or more to be infective when consumed... however, as you supply your isolates with nutrients they will quickly reach far greater numbers (an average colony on your plate will hold 10-50 million individual cells) and be in a position to overwhelm your immune response if they are pathogens.

• Write a brief description of the microorganisms growing on your plates. If you are able to photograph your plate, be sure to prepare labels that will show up on the image. Note the color, size, shape, and abundance of each class of colony on the plate.

• Choose two that are clearly isolated from any other colonies and use sterile toothpicks to streak each on fresh agar plates of the same type.  Use at least three toothpicks on each plate so that you can spread the colonies enough to produce a plate of isolated colonies, all of the same type. 

Skip Microscopy & Gram Stain Lab this year?

Day 3 - Use an isolated colony from each plate to inoculate LB broth.

• Label two sterile culture tubes with the sample source and your name.
• Pipette 3 mL of the correct LB broth into each tube and label the tube.
• Use sterile stick to inoculate a culture tube with an isolated colony from your plates.
• Incubate the cultures shaking overnight at 25° C. When the cultures have reached a reasonable optical density, they will be placed in the cold room.

Day 4 - Purify DNA from isolates

• Transfer 1.5 mL of your two best cultures to 1.5 mL microfuge tubes.
• Centrifuge your balanced tubes at maximum speed for 1 minute at room temperature.  Discard the supernatant safely in a biohazard container.
• Suspend the cells in 450 uL of sterile 25 mM Tris + 10 mM EDTA, pH 8, and centrifuge again.
• Follow the instructions from the Sigma GenElute Bacterial Genomic DNA purification kit.
• Determine the amount of template DNA and load an aliquot on an agarose gel.

Day 5 - PCR Amplification of 16S rRNA genes

We will be using ReadyMix Taq PCR Reaction Mix from Sigma.  The students add 25 uL ReadyMix to 25 uL of their template, our primers, and water, for a final volume of 50 uL.  The primer concentrations should be 10 uM and there should be about 100 ng of their template DNA.  We should also quantify the DNA yield from these different strains... see notes.

Day 6 - Subcloning of PCR Products

pGEM-T Easy Vector