BISC209/S12: Lab1

Lab 1 Introduction to the Microbiology Lab and to the Soil Bacterial Community 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. The human population, by and large, 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 has a barely conceivable number of members and 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 common 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/S12:ReadMe  Please watch these YouTube videos on lab safety at and 

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: [] You can download a pdf file of a greenhouse map and tour route here: []

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 S12; 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 <BR><BR>

With your teammates, go to the "tropical" greenhouse habitat you are to investigate. Before you take your soil sample, look around at this habitat and choose a location to sample. 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. <BR><BR>

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. <BR><BR>

TAKING YOUR SOIL SAMPLE:<BR> 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. <BR><BR>

To sample the soil, brush away any leaf debris or non-soil material that might end up in your sample. <BR> 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. <BR><BR>

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. <BR><BR>

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.<BR><BR>

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. <BR><BR>

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. <BR><BR>

BACK IN THE LAB<BR> Soil Preparation Sieve the fresh soil sample using the sterile sieve, beaker and pestle. <BR> Soil Extract Preparation<BR> 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 clean blender jar. Be careful not to contaminate the inside of the lid with your fingers when you place the lid on the jar. Blend at highest speed 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. This is your 1% soil extract. In making this extract, you have created a 10-2 or 1/100 dilution, which can be described in concentration units as 1% (wt/vol) since there is 1 gram/100ml. <BR>

After preparing this 1% soil extract, please transfer 1.0 ml of it to a new microfuge tube using a 1ml sterile pipette. Label the tube with your work-site letter using a piece of your team color tape. Make sure all the labeling is legible! Give this tube to your instructor. She will stain the soil microbes with a fluorescent DNA stain after you finish lab today, transfer all those microbes in a Poisson distribution to a small piece of filter paper, view and photograph them using fluorescence microscopy, and provide the photographs for you and your group next week to directly count discreet spots of fluorescence indicating individual microbial genomes. <BR>

PREPARING SOIL FOR CULTURE OF SPORE FORMING BACTERIA & FOR MEASURING THE DRY WEIGHT OF SOIL<BR> Weigh three 1 gram samples of your mixed and sieved soil into properly labeled aluminum weighing boats (label each with a piece of your team tape color on which you have identified the soil sample). You will find the aluminum weigh boats near the scale. It is important to use the aluminum boats and not the plastic ones so the soil can be oven dried. Leave the weighed samples on the metal tray marked with your lab section found beside the top-loading balances. These soil samples will be oven dried at at a low temperature (70°C) for you and returned to you for use in Lab 2. Drying the soil is part of the selection and enrichment for endospore forming bacteria.<BR>

WEIGH OUT SOIL FOR AN ENRICHMENT CULTURE TO BE STARTED TODAY:<BR> Weigh a 0.5gram soil sample onto a sheet of weighing paper or weighing boat. You will take it over to your bench to use later today in setting up an enrichment culture for a group (Azotobacter)of nitrogen fixing of soil bacteria that we seek.

Discard the rest of the sieved soil. Return it to the original 50 ml conical tube and dispose of it in the trashcan (NOT the autoclave bag). <BR><BR>

Asepsis and Aseptic Transfer
Asepsis<BR> 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<BR> Please watch the YouTube video on how to use the Bunsen burner

and the YouTube video on broth to broth Aspetic Transfer Technique .<BR><BR>

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. <BR><BR>

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. There is also a description of this protocol and other basic microbiological procedures in the Resources section of this wiki at BISC209/S12:Lab Basics<BR><BR>

Standard Plate Count of Soil Microorganisms on Dilute Nutrient Agar & on Nutrient Agar with 0.3% Starch one assay per sampling site (do this in groups of 4)<BR> 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. <BR> Gather the following materials to start your quantitation:<BR> 5 sterile 13 x 100 size sterile glass tubes with caps<BR> 1 sterile disposable plastic individually wrapped 1 ml pipets, <BR> 5 sterile dilute (1:10 diluted from full strength) nutrient agar plates,<BR> 5 sterile full strength nutrient agar plates with 2.5% starch 10 sterile plastic disposable spreaders <BR><BR> '''Setting Up a Standard Plate Count: <BR> 1. Label a set of 5 glass tubes 10-3, 10-4 ---through 10-7.

2. Label 5 destination plates of dilute nutrient agar and 5 destination plates of NA + starch. Include your name, date, lab section, soil sample code letter, type of medium, and the dilution (10-3 through 10-7) You want each dilution on each medium. 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 and cultured. However, today we are going to plate and culture all of our dilutions on both media.

3. Slightly dehydrate the medium on the plates. Clean the laminar flow hood with alcohol; turn on the fan; place all 10 plates in the hood; and position 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. (If you aren't very confident that you know how to use a micropipet properly or you want a review, ask your instructor for a quick tutorial before you start this step.) 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.

6. Using a new tip, transfer 100μL (0.1ml) of the 10-3 dilution to the tube labeled 10-4. Mix well by votexing or by putting a small piece of parafilm over the tube and inverting it several times. Mixing 0.1ml of the 10-3 dilution with 0.9ml of sterile water makes a 10-4 dilution.

7. Continue to transfer 100μL (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, pre-labeled culture 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 and the nutrient agar + starch plates. You should end with one plate of each medium inoculated with each dilution.

10. Allow the moisture to be absorbed into the agar before inverting the plates. Put a labeled piece of your team color tape around each set of plates (separate the two media). Incubate your cultures at room temperature (RT) until next week in a rack designated by your instructor.

11. DO NOT DISCARD your serial dilutions or your soil extract because you will use them again in one of the Enrichment/Selection Protocols that follow! <BR><BR>

Calculating the number of microorganisms per gram of soil<BR><BR>

Next week you will count the number of colonies on the plain nutrient agar plates, observe the variability in colonies on each plate and, select a couple of interesting colonies for isolation and further study. <BR><BR> If you divide the number of colonies you find on a culture plate, by the amount of inoculum (0.1ml) used times the dilution factor, you will obtain the number of cultivatable bacteria per gram of soil. Only cultures 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 you counted. Count number of visibly different bacterial colonies and covert to number of visibly different cultured colonies per gram wet soil.<BR><BR>

CFU/g (wt wet soil) = number colonies counted/(vol. of inoculum *dilution)<BR><BR>

Isolation & Study of Soil Bacteria from your habitat
Background<BR><BR> 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 the focus of our soil microbe isolations 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. For this reason we have plated our original soil extract on both on dilute nutrient agar (to reduce the richness of the medium and the growth rate of the fast growing organisms) and on full strength NA with starch. We will also use some specialized, defined media and culture conditions that will favor the groups we seek and discourage those we don't. <BR><BR> 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 bacteria by 16S rRNA gene sequencing analysis, a culture-independent 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. <BR><BR>

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. An example of selection of bacteria over other microorganisms in soil would be to add cycloheximine, a drug that prevents fungal growth but does not negatively affect bacteria. However, cycloheximine is also toxic to most eukaryotic cells (such as yours!), so we will not use in this project. One common selection approach is to provide a single carbon source if the microbes you desire are able to use this source for all of its carbon needs. <BR><BR>

Enrichment and selection procedures vary in the number of steps and amount of time required. Thus, unlike many of the lab protocols that everyone will accomplish at the same time, you will have to keep track of the progress of each of your isolations of different bacterial species because not all of your organisms will be ready for the next step at the same time. <BR><BR>

<BR> Many soil bacteria can fix nitrogen by reducing N2 to any or all of the other forms of nitrogen in the cycle, including nitrate, nitrite, and ammonia. Other soil bacteria can break down organic nitrogenous molecules to ammonia, nitrite, or nitrate to N2, allowing the cycle to run in both directions simultaneously and to recycle the raw materials. As a community, soil bacteria work co-operatively to provide these crucial metabolic raw materials for each other and for plants, animals, and other organisms that share their habitat.<BR><BR> In your soil sampling teams, divide up the following two primary enrichment cultures so that each soil sample is cultured in each medium.One of the cultures will start with seived soil and the other with the 1% soil extract that you prepared for your plate count. <BR><BR> Our goal is to isolate culturable bacteria that demonstrate your soil microbial community's ability to complete parts of the nitrogen cycle: We will select and enrich for nitrogen fixing bacteria (those that can use inorganic Nitrogen gas from the air in the creation of organic ammonium compounds). We will also select for bacteria that can break down organic ammonium compounds in order to use the nitrogen for other metabolic purposes. To find bacteria with these capabilities and to 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 and about the composition of the media you will use to select and enrich for them. We hope that, eventually, in the weeks to come, each student will be working with a unique subset of bacteria, including some with these basic characteristics.

Aseptic Transfer of Soil or Soil Extract to Begin Enrichment for Specific Bacterial Groups from a Mixed Population
<font size=+1>Activity: Begin Enrichment/Selection of Nitrogen Cycling Bacteria from Soil</font size=+1><BR> I. Follow the ENRICHMENT directions described below to begin the search for Nitrogen extracting bacteria. The full protocol for the complete isolation to pure culture is found in the section: Finding Nitrogen Cycling Bacteria using Azotobacter and Simmons Citrate media in the Protocol section Culture Media. We will only set up the initial enrichment/selection culture today, described below. <BR><BR>

Protocols:<BR> Nitrogen Fixing Bacterial Enrichment/Selection<BR> Each soil sampling group (not each student) should set up both of these two enrichment/selections:<BR>
 * 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.)<BR>
 * 2) Mix well but do not invert because, if you do, the contents will spill.<BR>
 * 3) Label the flask using a piece of your team color tape with your soil sample code, initials, lab section, date, medium name, and N2 fixer enrichment.
 * 4) Place the flask in your closed bench cabinet so the culture will incubate in the dark at RT for one week. <BR>

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. <BR><BR>

Ammonium Using Bacteria Enrichment/Selection<BR> <BR><BR> <font size=+1>What's Next? </font size=+1><BR> 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. <BR><BR>
 * 1) Obtain and label three plates of sterile Simmons Citrate medium with your soil sample code letter, your initials, lab section, date, name of medium, and a soil extract dilution (10-2, 10-3, or 10-4---note that it would be equally correct to label these dilutions 1%, 0.1%, and 0.01%).
 * 2) Using your P200 and a sterile tip, gently swirl to mix your 1% soil extract and inoculate 100μL of it onto the center of a plate of Simmons Citrate medium.
 * 3) Use a sterile disposable spreader to spread the inoculum evenly all over the surface of the sterile medium.
 * 4) Use different sterile tips to inoculate 100μL of the 10-4 and 10-5 dilutions onto separate  plates of Simmons Citrate Medium
 * 5) Use clean sterile disposable spreaders to spread the inoculum evenly over the surface of the other dilutions
 * 6) When the inoculum has absorbed into the medium, invert the plates, and incubate them at room temperature in your team's rack for a week.

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. <BR><BR>

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.<BR><BR>

GENERAL CLEAN UP INSTRUCTIONS
1. Culture plates, stocks, etc. that you are not finished with should be identifiable with your team color tape and well labeled. 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.<BR><BR>

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 non contaminated and empty disposable glass tubes can be discarded in the glass disposal box.<BR><BR>

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.<BR><BR>

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

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

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. <BR><BR>

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

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

12. Wash your hands.<BR><BR>

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 and organizing a lab notebook in the Guidelines for Maintaining A Good Lab Notebook in the BISC209/S12:Resources section of this wiki. 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 goals and approaches to our investigation, it may be wise to rethink the usual notebook organization of an exclusively time-course description of each lab period. Since this is a semester long project with many experiments that answer different experimental questions, it will be helpful to you to section your lab notebook in 4 parts: Suggested Sections (1-4) & Subsections:<ul> <li>1. Soil sampling & Making a soil extract <li>2. Enumeration: <ul> <li>a. Culture-dependent plate count <li>b. Culture-independent direct count by fluorescent stain</ul> <li>3. Community level analyses of diversity and community behavior (co-operation &/or competition)<ul> <li>a. Functional metabolic diversity by Carbon Source Utilization Testing <li>b. Exo-enzyme testing</ul> <li>4. Example level analyses (on bacterial isolates) of diversity and community behavior (co-operation and/or competition)<ul> <li>a. Enrichment/Selection of Bacteria of Specific Groups to Pure Culture<ul> <li>     i.Nitrogen Fixing Bacteria (Azotobacter medium) <li>     ii. Ammonium Using Bacteria (Simmons Citrate Medium) <li>     iii. Endospore Forming Bacteria (from dried soil and GYE medium) <li>     iv. Other interesting Bacteria (from dilute NA or NA +starch)</ul> <li>b. Other Tests</ul> </ul>

Our semester long investigation explores abundance and richness in a soil microbial community and community behavior among the microorganisms in that community. <BR> Our lines of investigation are 3 fold: 1) Abundance (how many microbes are in our soil comunity?)<BR> We will answer that with two different experiments: <BR> a) a culture-dependent colony plate count (that you set up in Lab 1 and will get data and complete in LAB 2), and<BR> b) a culture-independent direct count of fluorescent stained microbial genomes that your instructor started today and will provide data for you to complete for homework and discuss in LAB 2. <BR> 2) Richness (is the soil community diverse and, if so, in what ways?) and<BR> 3) Community Behavior(how do the microbes living in this unbelievably crowded soil community, co-operate and/or compete to find and maintain a niche and to keep the soil resources from being depleted?<BR> In order to provide evidence to answer this broad, complex question, we will study a)a few example bacterial community members. We will try to find out who they are and what they can do to survive in the community and, perhaps, to help others survive. You and your partners will isolate as many as feasible different looking bacteria of specific types to pure culture. Once you have isolated them and grown them in pure culture, we will attempt to identify them by 16s rRNA gene sequencing and test them for certain behaviors or metabolic characteristics that provide evidence for community co-operative or competitive behavior and/or for diversity (richness) of your soil community. 2)Simultaneously, you will make and use soil extracts to perform tests on the whole microbial community to gather evidence for community bunctional metabolic diversity and for co-operative and competitive behavior within the community.<BR><BR>

You must keep our experimental questions and goals in mind as you perform your experiments and you must keep all of your work well organized or it will be difficult to make sense of your findings or to write your Materials and Methods and Results sections for your final paper. There is a more complete suggested organization scheme for your notebook available in the downloadable document [[Media:Labnotebookguide_209_12(3).doc]]. There is also a sample notebook available for you to look at done by a previous student in the course so that you see how she kept a good, well organized notebook. For the present, divide your lab notebook into the 4 suggested main sections and subsections, leaving lots of empty space between parts. Your notebook will be checked in a few weeks to see how your are doing and it will be collected and graded for a few points (not many, don't worry!) 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: [] You can download a pdf file of a greenhouse map and tour route here: []<BR><BR>

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. <BR><BR>

Make sure you have read all of the introductory information on the wiki about the project on the project home page BISC209/S12:Project, reviewed what was accomplished in LAB 1, and read LAB 2 carefully before coming to lab next time. Organize your lab notebook using the instructions in the previous section and in the Resources section of the wiki. Prepare for Lab 2 by making brief flow diagrams of the work you will do in the appropriate section of your notebook. Note that when you are to repeat something that you have already described in your notebook (like making a soil extract or collecting a new soil sample), you need NOT repeat that information. Instead, add some notes to the previous description explaining when you repeated the procedure and if you plan to or did anything differently this time. These additions are one reason it is wise to leave lots of empty space around every section and subsections of your notebook entries. Note that it will save you a lot of time in lab if you prepare for lab by finding and including the recipes (ingredients and concentrations) for all media and reagents stocks that you will use in any protocol. If this information is in your notebook in the section where you describe the use of this media or reagent, it will save you a lot of time when you write the M&M section for your paper. Sometimes this information is in the LAB work description in the wiki and other times you will have to hunt for it in the Protocol section of the wiki or it may not be in the wiki at all and you will have to ask your instructor in lab to write the information on the board for you to copy. <BR><BR>

Graded Assignment: Bring to Lab 2, a Discussion with References(references formatted in the NAME/YEAR style of the journal Cell) of how the enrichment/selection/differentiation culture techniques and media you will use this semester will separate and differentiate microbes in the soil community. Be sure to read the directions for this assignment found at: Assignment: Enrichment/Selection/Differentiation of culturable bacteria of specific groups. Please complete this assignment individually and turn in an electronic copy of it to your drop-box in the lab Sakai site before you come to lab. In addition, be sure to bring a hard copy to class. You will work with your teammates in lab to complete an exercise that should improve your understanding of general purpose, selective, enrichment, and differential media and of culture based tools that use physical and metabolic characteristics of different microorganisms to isolate them from each other. Your discussion assignment should be useful in this group activity. Notice that, unlike the Lecture part of this course, we are not using the ASSIGNMENTS tool in Sakai for graded work submissions, but the more informal DROP-BOX tool.

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