BISC209/S11: Lab1

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Wellesley College-BISC 209 Microbiology -Spring 2011

Contents

Lab 1 Introduction to the Microbiology Lab and to the Soil Bacterial Communities Project

In this first lab you will learn:

  1. Who microbiologists are and what they do
  2. How to sample soil from a greenhouse habitat without contaminating it to start your semester long project
  3. To learn to work safely in a microbiology lab by practicing aseptic technique so that you don't contaminate yourself or your cultures
  4. To begin to become familiar with some of the basic equipment and procedures used in microbiological investigation, such as streaking for isolation on various types of media
  5. To use a lab notebook to record the progress of your experiments


Introduction To Microbiology

Welcome to the unseen world of microorganisms. For most of us, microbes are out of sight and out of mind and, largely, the human population would prefer it that way. Nevertheless, microbes have a major and continuing impact on us and on our planet; therefore, it behooves us to understand them better. By the end of this course, you will. Understanding the microbial world is a huge undertaking. A discipline that defines its scope as including all life forms (and some non-life forms like viruses and prions) that are invisible to the unaided human eye is a bit like saying we will study all humans and other animals shorter than some arbitrary height. Besides including a huge number of members, the diversity of such a group is overwhelming.

Where do we begin the study of microbiology? It's good to start with appreciating the power of these tiny, unseen life forms to thrive and spread without our permission or knowledge. It is also wise to recognize that, although a tiny fraction of the microbes in our world are disease causing, there are devastating infections caused by microbial pathogens. Although none of the microorganisms that we will knowingly work with this semester are commonly human pathogens, we require that you read and agree to certain rules for working in the microbiology lab that are designed to keep you from infecting yourself, your classmates, and the community. We will also begin to learn aseptic techniques that will reduce the chance of contaminating your cultures or the chance that your cultures will contaminate you.

Lab Safety

Please download, read, and sign your agreement to follow the Wellesley College Microbiology Lab Safety regulations: Media:Wellesley_College_Lab Safety.doc. These regulations can be found in this lab wiki at: BISC209/S11:ReadMe

Please watch these YouTube videos on lab safety at [1] and [2]

Introduction To the Tools and Techniques of Microbiology

Whether you are trying to keep a desired organism from being overgrown by a contaminant, or you are attempting to prevent contaminating your soil sample, yourself, your lab bench, or your lab partner with unwanted microorganisms; awareness of potential sources of contamination is critical. Your success in the lab depends on being open to learning and adopting the standard procedures used in microbiology. Today you will practice asepsis when you collect your soil sample and you will practice aseptic transfer technique when you begin to culture your soil sample bacteria.


Begin Soil Microbial Communities & Diversity Project

Today you will work with your table group to sample soil from a greenhouse habitat. You will begin to culture bacteria from that soil sample in order to characterize the bacterial soil community metabolic interactions. You will also begin to use traditional microbial tools and techniques to isolate a few bacteria from your soil community to physically characterize bacteria and to assess individual roles and relationships.


Link to the Wellesley College Margaret Ferguson Greenhouses: [[3]]
You can download a pdf file of a greenhouse map and tour route here: [[4]]

Soil Sampling

Gather your equipment before going to the Greenhouse to make notes and observations and to sample the soil from the environment you and your group will investigate. Wash your hands. Take off your lab coat and leave it in lab.
Take with you the following equipment for each sampling site (teams of 4):
your lab notebooks and pen;
1 JMC 18in steel soil corers (looks like a metal hollow rod with the T at the top)
4 copies of the green house map downloadable from:[| http://www.wellesley.edu/WCBG/Visit/WCBG_Greenhouses.pdf];
1 small stainless steel garden markers to designate where you take your soil samples. Label it: BISC209 Micro Course; date S11; TUES (or WED) LAB - Soil Sample Habitat______ A, or B, or C, etc.(Ask your instructor which habitat and letter code your group has been assigned)

For Each Each Sampling Site: Also Make up a kit in a gallon size ZipLock bag that contains:
(1) clean spatula or spoon,
(1) 50ml orange top sterile conical bottom plastic tubes labeled with your group's color code, initials, your lab section, the date, the greenhouse room that you plan to sample, and the code letter your instructor has given you to distinguish your soil sample from your classmates'.
(1) black Sharpie,
(several) paper towels
(4) pairs of disposable gloves in the appropriate size for you and your teammates


With your teammates, go to the greenhouse habitat you are to investigate. Before you take your soil sample, look around at this habitat and at your chosen location for sampling. Record your impressions as you discuss the habitat with your group. Does it seem cool, warm, or cold; dry, moist, or average in humidity? Is there abundant sun, shade, or is it mixed? How would you describe the variety of plants here? What are the largest, most abundant, most interesting or most typical plants? What else strikes you about this environment that you may want to add to your notes? Today you will draw a scaled "map" in your lab notebook to show where you sampled, containing enough detail so that someone else could easily locate the area even if the marker you will place here is removed.

Remember that you will get more variety of bacteria from soil near plants and their roots. The section of soil that contains plant roots is called the rhizosphere; if possible, you should take some of the rhizosphere. Avoid very wet soil and highly compacted soil but get close to a plant and record its name from the label on it. Do not choose an area where the plants are in pots.

TAKING YOUR SOIL SAMPLE:
When you and your teammates have agreed on where to take your soil sample, get your equipment laid out and ready. Place 2 or 3 paper towels down end to end in an open area near where you are going to sample and put on your gloves. The gloves are not sterile but be careful to avoid contaminating the exterior surface of the gloves with skin flora by touching your skin or anything other than the soil area you are going to sample.

To sample the soil, brush away any leaf debris or non-soil material that might end up in your sample.
Push the soil sampling device straight down, putting force with both of your hands until your corer is in about 15cm (6 inches). You may use your foot (if you can). It is best to twist the corer straight into the ground. Don't go deeper because we are not culturing in anaerobic conditions. You want to sample, primarily, aerobic or facultatively aerobic bacteria.

Empty the corer on the paper towels you made ready nearby. Knock the side of the corer and the soil should emerge as an intact cylinder of soil. If it doesn't, you may pull it out with your gloved hands. It doesn't matter if you don't get an intact cylinder. Your goal is to sample equally from this soil from the lower, middle, and upper areas of the core (avoiding the very top 30mm [1 inch] of surface soil), collecting enough soil to fill the 50ml tube.

Spoon (you may use the spatula you brought) the soil sample into the labeled 50ml conical tube. If you didn't get enough soil from your core to fill the tube, use your corer to take another sample adjacent to the first sampling site. When the 50ml tube has been filled, discard unused soil back into the sampled area and press down. Try to make the area look undisturbed. Mark the spot sampled by the team with your labeled garden marker.

Before you leave the greenhouse (after you have completed sampling and have removed your gloves), write down in your lab notebook the name of the plants that are located in or near your sampling area and mark on the map where you took the soil sample.

Return as quickly as you can to the lab with your labeled soil sample tube and all of your equipment. We have a lot to do today with these samples to start our analysis of the bacteria in this soil community.

BACK IN THE LAB
Soil Preparation Sieve the fresh soil sample using the sterile sieve, beaker and pestle.
Soil Extract Preparation
Weigh 1 gram of sieved soil using the top loading balance and add it to 100 mL of sterile water which you will find premeasured for you in a sterile 250 ml flask on your bench. Swirl to mix--- don't use the magnetic stirrer yet. Pour this soil suspension into a disinfected blender jar. Use gloves when you place the cap on the jar, first making sure that the rubber gasket is properly positioned and the seal at the bottom is tight (otherwise it will leak!). Blend for 3 pulses of 10 seconds on and 10 seconds off. Pour all of the suspension back into the 250 ml flask and add a sterile magnetic stir bar (on your bench). Place the flask on a magnetic stirrer at medium speed and mix for at least 15 min. Stop the stirring and let the soil settle until the larger particulate matter settles to the bottom. Not all visible particles need to settle, just the big stuff. Pour off about half of the supernatant (avoiding transferring the settled particles) into a new, labeled, sterile 50 ml conical tube. In making this extract, you have created a 10-2 or 1/100 dilution. This extract can also be described as a 1% (wt/vol) solution since there is 1 gram/100ml.



PREPARING SOIL FOR CULTURE OF SPORE FORMING BACTERIA & FOR MEASURING THE DRY WEIGHT OF SOIL
Weigh three 1 gram samples of your mixed and sieved soil into properly labeled weighing boats (label each with a piece of your team tape color on which you have identified the soil sample). Leave these on the tray marked with your lab section found beside the top-loading balances. These will be dried for you and returned to you for use in Lab 2 for a Streptomyces enrichment.

WEIGH OUT SOIL FOR FUTURE GENOMIC DNA ISOLATION
To start the culture independent bacterial identification (sequencing 16s rRNA genes), each student will need a 0.25 gram sample of sieved soil from your sample. Weigh out these 4 aliquots using the top loading balance and weighing paper that you are careful not to contaminate. Fold up the edges of the weighing paper to use as a crude funnel and pour one 0.25g soil sample for each teammate into separate, sterile microfuge tubes. Label them with your initials, date, and sample code on a piece of your team color tape. Give these tubes to your instructor to freeze. She will return them to you when we start our genomic DNA extraction next week.

WEIGH OUT SOIL FOR ENRICHMENT CULTURES TO BE STARTED TODAY:
Weigh two different 0.5gram soil samples onto different sheets of weighing paper or weighing boats. You will take these over to your bench to use later today in setting up two enrichment cultures for specific groups of soil bacteria we seek.


Save the rest of the sieved soil. Return it to the original 50 ml conical tube, make sure it is labeled properly, and give it to your instructor to refrigerate for you.

Asepsis and Aseptic Transfer

Asepsis
Microbiologist must constantly be aware of the ubiquity of microbes on every surface and in the air. We must follow set procedures designed to avoid the inadvertent contamination of microbes from the environment into our samples and avoid contaminating ourselves with our cultures. These procedures are called aseptic technique. You will need to learn them well and follow them rigorously at all times throughout this semester, beginning today.

Since your skin is covered with a thick coating of bacteria and eukaryotic microorganisms and it is estimated that every cubic meter of room air contains at least 106 fungal spores or other microbes, you must be aware that touching anything sterile with your hands or any part of your body immediately negates its sterility. If you maintain sterility successfully but you leave sterile samples or equipment uncovered for any longer than absolutely necessary, you have also increased the likelihood that that equipment or sample contains microbes from the air rather than exclusively from the source you desire. When we gather our equipment today and take a soil sample for culture, be aware of the potential sources of contamination and minimize the risk by avoiding touching the part of your equipment that will come in contact with your sample. Work quickly so that your sample is exposed to air or other potential contaminants for as short a time as possible. It is impossible to avoid all sources of contamination but following aseptic technqiues will minimize the risk

Aseptic Transfer
Please watch the YouTube video on how to use the Bunsen burner [5]

and the YouTube video on broth to broth Aspetic Transfer Technique [6].

Manipulation of the many tubes, plates and transfer tools that you will use in each lab requires patience and practice. Mastery is vital to success in the microbiology laboratory. By the end of the semester you will become proficient at many of the transfer methods, you will know when and what to sterilize, and you will be able to keep pre-sterilized tools sterile.

Activity: Plate Count : Assessing the Number of Culturable Microbes In Your Soil

To perform a plate count of your soil sample microorganisms you must first dilute the soil serially so that the colony numbers will be manageable and so that you are more likely to have a countable number of cells. If you don't remember how to make a serial dilution, here is a link to a helpful animation for making dilutions http://www.wellesley.edu/Biology/Concepts/Html/serialdilutions.html.

Standard Plate Count of Soil Microorganisms done in teams (one assay per soil extract)
The Soil Extract you prepared is now at a 1:100 dilution (1 gram/100 ml). This could also be called a 1% (w/v)suspension.
Gather the following materials to start your quantitation:
5 sterile 13 x 100 size sterile glass tubes with caps
6 sterile disposable plastic individually wrapped 1 ml pipets,
5 sterile dilute nutrient agar plates,
5 sterile plastic disposable spreaders

Setting Up a Standard Plate Count:
1. Label 5 tubes 10-3, 10-4 etc. through 10-7.

2. Label 5 destination plates of nutrient agar with dilution and identifying information. Because your goal is to obtain 30-300 well isolated colonies on a plate, generally only the 10-4 through 10-7 dilutions are plated. However, we are going to plate all of our dilutions.

3. Slightly dehydrate the medium on each plate by cleaning the laminar flow hood with alcohol, turning on the fan, placing the plates in the hood, and positioning the covers so they are slightly ajar for 10 minutes or until the medium surface shows no visible moisture.

4. Use a sterile 1ml disposable pipet and your blue Pipetman to pipet 0.9 ml of sterile water into the 5 tubes labeled in step 1. (You may use the same sterile 1ml pipet for all of them.)

5. Using your P200 micropipet and autoclaved tips, transfer 100 microliters (0.1ml) of your soil extract (1:100 dilution) to the tube labeled 10-3, mix well by vortexing. (If you don't know how to use a micropipet properly or you want a review, ask your instructor for a quick tutorial.)

6. Using a new tip, transfer 100μL (0.1ml) of the 10-3 dilution to the tube labeled 10-4. Mix well. Mixing 0.1ml of the 10-3 dilution with 0.9ml of sterile water makes a 10-4 dilution.

7. Continue to transfer 0.1ml aliquots (after mixing well) from each dilution to the next tube of 0.9ml water until you have carried the dilution to 10-7. Use a new tip for each transfer.

8. Starting with the most dilute extract and a new tip, transfer 100μL (0.1ml) of it and dispense it to the center of the appropriate, labeled plate. Use a sterile plastic disposable spreader to gently push the dispensed sample two or three times clockwise around the dish, and then several times counterclockwise. Make sure all of the surface area of the plate has been inoculated. Don't press too hard as force will cause the microorganisms to collect at the edge of the spreader, resulting in uneven distribution.

9. Repeat step 8 to inoculate the rest of your prelabeled dilute nutrient agar plates.

10. Allow the moisture to be absorbed into the agar before inverting the plates. Put a labeled piece of your team color tape around the set. Incubating your set of standard plate count plates at room temperature (RT) until next week in a rack designated by your instructor.


Calculating the number of microorganisms per gram of soil

Next week you will count the number of colonies on these plates, observe the variability in colonies on each plate and, perhaps, select one or more of these colonies for isolation and further study.

If you divide the number of colonies you find, by the amount of diluent used times the dilution factor, you will obtain the number of cultivatable bacteria per gram of soil. Only plates with between 30 and 300 colonies per plate give accurate calculations. Record the number of microbes/gm wet soil in your lab notebook. Take photos of the plate your counted. Count number of visibly different bacterial colonies and covert to number of visibly different cultured colonies per gram wet soil.

CFU/g (wt wet soil) = number counted on plate/(diluent plated*dilution of plate counted)

Isolation & Study of Soil Bacteria from your habitat

Background

There are large numbers of both beneficial and non-beneficial bacteria in soil. Often their roles are not well understood. The main antibiotic producing genera of soil microbes include the Bacillus, Cephalosporium, Penicillium, and Streptomyces. Nitrogen cycling bacteria (such as Azotobacteria, Berjerinckia, Cyanobacteria, Rhizobium, Frankia, Azospirillum, Clostridium, some Klebsiella), and/or sulfur utilizing bacteria (such as Disulfovibrio), methylotrophic bacteria, as well as other types of recyclers are equally important to the soil microbial community & to the ecosystem of which they are a crucial part. Since we are limiting our focus to bacteria, we will choose media that encourage growth of specific types of bacteria and/or discourage growth of fungi and other eukaryotic microorganisms. A wide variety of growth media and incubation conditions can be used to isolate bacteria from soil. In general, we can favor the growth of certain groups over others by altering the composition (e.g. pH, osmolarity) and/or nutrients available. Some bacteria will grow so fast on rich media (nutrient agar, TSA, etc.) that they will mask other slower growing genera. We will try a variety of defined media that will favor slower growing bacterial genera.

The vast majority of bacteria in soil (90-99%) will not grow on your plates at all. We will only find the ‘culturable’ bacteria that like the growth conditions you choose. The more types of media and growth conditions you use, the greater the variety and number of bacteria that you will find. Although most soils contain a rich and unbelievably diverse community of microorganisms, we will focus part of our investigation on isolating just a few types of bacteria that contribute to this unseen world. To get a sense of the true diversity of the bacterial community in your habitat, in the other parts of this three part project, we will isolate genomic soil DNA and id a sample of both culturable and unculturable bacteria by 16S rRNA gene sequencing analysis. We will also use soil extracts to test the culturable part of the whole microbial community for co-operative and competitive roles in fulfilling the communities' metabolic needs and those of the larger ecosystem.

Aseptic Transfer of Soil or Soil Extract to Begin Enrichment for Specific Bacterial Groups from a Mixed Population

The formulation of enrichment media supplies specific nutrients that encourage the growth of bacteria types that grow too slowly or not at all in media missing these nutrients. Often enrichment media is also selective media. Selective media is selective because it contains one or more ingredient(s) that inhibits the growth of competitor microbes (such as cycloheximine, a drug that prevents fungal growth but does not affect bacteria negatively). In some cases growth on enrichment media will take up to 2 weeks. Thus, you will have to keep track of the progress of your isolation of different bacterial species as not all of your organisms will be ready for the same steps at the same time.

Activity: Begin Enrichment of Nitrogen Cycling and Methylotrophic Bacteria from Soil
In your soil sampling teams, divide up the following two enrichments so that each soil sample is cultured in each medium.
Our goal is to isolate culturable bacteria that belong to two important groups of soil bacteria: Azotobacter, among the nitrogen cyclers; and Hyphomicrobia, among the methylotrophs. To find them and separate them from the dizzying number of competitor microorganisms present in 1 gram of your soil sample is a challenge that will take time, effort, and a lot of knowledge about the metabolic and physical structure of these groups of bacteria. To start you will use enrichment media described in the Culture Media in the Protocol section of this wiki. We will attempt to find culturable bacteria from each of these groups from each soil sample, if possible. We hope that, eventually, in the weeks to come, each student will be working with a unique subset of bacteria with these basic characteristics.

I.Follow the ENRICHMENT directions described below to begin the search for Hyphomicrobia. The full protocol is found in the section: Finding Denitrifying Methylotrophs (Hyphomicrobia) Bacteria using Denitrifying Methyloptrophs Medium (DMMM) with Methanol in the Protocol section Culture Media. We will only set up the initial enrichment culture today, described below.

Protocol:

  1. Inoculate 0.5g of soil into a small screw cap tube containing Denitrifying Methylotroph (Hyphomicrobium) Medium with methanol(DMMM). Label the tube with tape in your team color including your initials, lab section, date, soil sample identifier, medium name (DMMM)and Hyphomicrobia enrichment. Put on gloves. Take precautions when handling this medium as the methanol in it is highly toxic. Go over to the laminar flow hood where you will find a bottle of liquid DMMM medium. Using a P1000 and sterile tips, add more liquid DMMM medium until the tube is completely full. Screw on the cap tightly leaving no air interface. The goal is to create an anaerobic environment to facilitate the growth of anaerobic Hyphomicrobia bacteria. The environment will become anaerobic after the aerobic bacteria in the soil sample have used up all the oxygen.
  2. Incubate at 30°C for several days to 1 week in the 30°C room in the rack set up for your lab section.
  3. Watch the culture for the development of turbidity.
  4. When the solution begins to look turbid, slowly loosen the cap while watching for the appearance of bubbles rising in the liquid. The bubbles appear because of decreased pressure from loosening the cap and indicate the production of N2 gas by these denitrifying bacteria that use methanol as their carbon source.

II.Each group will also follow the ENRICHMENT directions described below to begin the search for Azotobacter nitrogen cycling bacteria). The full protocol is found in the section Finding Nitrogen Cyclying Bacteria: Azotobacter in the Protocol section Culture Media. We will only set up the initial enrichment today (described as follows).

Protocol:

  1. Inoculate 0.5 g of soil into 25 ml of liquid Azotobacter medium. (The medium is already aliquoted for you in small flasks with cotton plugs or a loose cover.)
  2. Mix well but do not invert as the contents will spill.
  3. Label the flask using a piece of your team color tape with your initials, lab section, date, medium name, and Azotobacter enrichment.
  4. Place the flask in your closed bench cabinet so the culture will incubate in the dark at RT for one week.

NEXT LAB: (7 days later) examine the air-liquid interface in your flasks and look for a slimy growth. The slimy growth may only be on the sides of the flask or it may extend across the liquid surface (a pellicle). Once you find slimy growth you may move on to the next step in the isolation process and the original culture flask can be autoclaved.

You are beginning an investigative project that is, increasingly, uniquely your own. Although the culture-independent molecular techniques will be done by all of you at the same time, a lot of the work that you will do each week on characterizing your culturable bacteria depends on the unique properties and metabolic capabilities of the bacteria you choose to isolate. The goal is to identify and characterize a diverse population of bacteria from a defined habitat. You will present your findings in a poster presentation to the class at the end of the semester.

Come to the lab and check on your cultures often over the next few days. Make observations about the number, size, color and shape of the various colonies that appear and draw and photograph the growth you observe. Keep a careful record in your lab notebook.

These enrichment, isolation, and identification protocols vary in the length of time between steps. We can't make this become regular, once-a-week work to fit our lab schedule. You will need to be highly organized and to remember when you need to come to lab to do the next part of the isolation or characterization tests. Fortunately, much of what you will need to do outside of lab time is not time consuming. Usually, it will amount to taking a well-isolated colony and subculturing it onto new media (a few minutes of work); however, your lab instructor can't keep track of all the isolations in progress and remind you that it is time for the next step. The success of this project depends on your organizational skills and your commitment to time-sensitive attention to the task at hand. It is your responsibility to check your cultures often and subculture or move them to your lab section's designated rack in the walk-in cold room to halt growth before the isolated colonies we seek become a mess of overgrown lawn growth on your plates and the isolation must be started all over.

GENERAL CLEAN UP INSTRUCTIONS

1. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of labeled team color tape around the whole stack.

2. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save provided stock cultures.

3. Remove tape from all liquid cultures in glass tubes and place the glass tubes in racks by the sink near the instructor's table. Do not discard the contents of the tubes. Place the non-disposable caps for these tubes in the wire basket provided in the clean-up area near the sink.

4. Glass slides or empty disposable glass tubes can be discarded in the glass disposal box.

5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.

6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 110x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.

7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.

8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.

9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.

10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.

11. Take off your lab coat and store it in the blue cabinet with your microscope.

12. Wash your hands.

13. See you next time!

Keeping A Good Lab Notebook For This Microbiology Project This Semester

Your lab notebook should be a useful source of information needed when your write the Materials and Methods section of your final paper. There is good advice about keeping a lab notebook in the Guidelines for Maintaining A Good Lab Notebook in the BISC209/S11:Resources section of this wike. You will also find there a downloadable .doc file of organization suggestions and specific do's and don'ts that you should print out and paste in your lab notebook. Since this is a project that has multiple approaches to our overall investigation, it would be wise to rethink the usual notebook organization of a time-course description of each lab period. You should organize your notebook hierarchially after describing the goals of the project and the soil sample collection.

The three main sections should reflect our different investigative approaches:
1.Culture-Independent Analysis: Community phylogenetic relationship analysis by rRNA sequencing
2.Community Culture-Dependent Analyses :Functional soil community profiling
3.Culture-Dependent Isolates Analysis: Enrichment, selection and isolation procedures and tests on cultured isolates

If you want to prepare your notebook for the whole project, you could first organize by those 3 lines and then organize specific protocols under each of these sub-sections:

  1. Culture-Independent Identification of Bacteria in a Soil Community by 16s rRNA Gene Sequencing of whole soil genomic DNA(notice goal is before tool)
    1. Isolation of genomic DNA from soil
    2. Amplification of 16s rDNA by polymerase chain reaction. This protocol should include the adjustment of DNA concentration, the pcr product "cleanup" and the assessment of success by agarose gel electrophoresis). The final goal of this protocol is to end up with lots of copies of double-stranded DNA amplified from the 16s rRNA gene template found in as many as possible of the bacteria in your soil community.
    3. Separating the 16s rRNA genes of bacteria in a soil community. This protocol should in include cloning the 16s rDNA into a plasmid vector; transforming chemically competenet E. coli with the clones; selecting for transformants with a plasmid containing a soil community bacteria's 16s rRNA gene; preparing glycerol stocks of successful, cultured transformants; DNA sequencing of 16s rRNA genes from clones (name the sequencing method, facility and location we will use)
  2. Culture-Dependent Soil Microbial Community Community Physiological Profiling
    1. Carbon source utilization: average metabolic response (AMR) and community metabolic diversity (CMD)
    2. Exoenzymes: Cellulase, Amylase, Phosphate Solubilization
  3. Culture-Dependent Assessment of Functional Roles and Relationships in Selected and Isolated Bacteria from a Soil Community
    1. Isolation of Methylotropic Denitifiying Bacteria to Pure Culture
      1. Physical Characteristics of Isolates: Gram stain, Special Stains, Motility
      2. Metabolic Characteristics (Roles in the community): Enzymes, Nitrogen Cycling Assessment
    2. Isolation of Azotobacter Bacteria to Pure Culture
      1. Physical Characteristics of Isolates: Gram stain, Special Stains, Motility
      2. Metabolic Characteristics (Roles in the community): Enzymes, Nitrogen Cycling Assessment
    3. Antibiotic Production Testing
    4. Quorum Sensing Assessment
    5. Mutualistic and Antagonistic Interaction Assessement



We offer these organizational hints in the hope that they will help you have all the information you need in a clear and organized format when it is time to write your final paper. Having a complete and accurate record of your work and your results always makes writing about your findings a significantly less ornerous task. Your notebook will be collected and graded at the end of the semester.

Assignment

Revisit the greenhouse (hours and more information found at: | http://www.wellesley.edu/WCBG/Visit/info.html ) using the downloadable greenhouse map to guide you.
Link to the Wellesley College Margaret Ferguson Greenhouses: [[7]]
You can download a pdf file of a greenhouse map and tour route here: [[8]]

Improve the notes you took during the soil sampling visit, paying special attention to the plants around your sample site and how this habitat is different from others in the Wellesley College Greenhouses.

Make sure you have read all of LAB 2 carefully before coming to lab next time. In your lab notebook (not to turn in), organize, outline or make flow diagrams of all the procedures that you will perform, starting a clear, hierachial schema of where those procedures fit in the different approaches to our soil communities project. You can get a broad sense of the progress of the different aspects of our project from the overview and the schematics found on the Introduction to the Project page at BISC209/S11:Project1. Make sure you read and understand what we are doing and know how each protocol fits into our overall goal of getting a sense of the abundance, diversity, co-operative/competitive functional and phylogenetic relationships among the bacteria in a soil community.

Graded Assignment: Turn in at the beginning of Lab 2, a Discussion with References of how the enrichment culture techniques and media you will use will select soil bacteria of the specific groups we seek and differentiate them from other microbes in the community. Be sure to read the directions for this assignment found at: Assignment: Enrichment for culturable bacteria of specific groups.

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11

Lab 12
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