BISC209/S11: Lab1
Lab 1 Introduction to the Microbiology Lab and to the Soil Bacterial Communities Project
In this first lab you will learn:
- Who microbiologists are and what they do
- How to sample soil from a greenhouse habitat to start your semester long project without contaminating it
- To learn to work safely in a microbiology lab by practicing aseptic technique so that you don't contaminate yourself or your cultures
- 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
- 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 4 ft. Besides including a huge number of members, the diversity of such a group is overwhelming.
Where do we begin in 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 today 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
If possible, 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.
Asepsis and Aseptic Transfer
Asepsis
Although the microbial world is largely out-of-sight and out-of-mind, microbiologist must constantly be aware of the ubiquity of microbes on every surface and in the air. We must always 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 [3]
and the YouTube video on broth to broth Aspetic Transfer Technique [4].
Aseptic transfer technique is important to prevent contamination of your cultures, yourself, others, and the laboratory. Manipulation of the many tubes, plates and transfer tools requires patience and practice and its 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.
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: [[5]]
You can download a pdf file of a greenhouse map and tour route here: [[6]]
Habitat 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 team of 4:
2 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:[[7]];
2 small stainless steel garden markers to designate where you take your soil samples. Label them: BISC209 Micro Course S11 TUES (or WED) LAB - Soil Sample (A, B, etc.) Taken Here for Microbial Flora analysis- DATE)
lab notebooks and pen;
For Each Pair or Each Sampling Site in a Habitat: Make up a kit in a gallon size ZipLock bag that contains:
(1) clean spatulas or spoons,
(2) 50ml orange top sterile conical bottom plastic tubes labeled with your group's color code, initials, your lab section, the date, and the greenhouse room that you plan to sample- if there are to be two samples taken from this habitat (one per pair), distinguish your soil sample from the other taken at this site as A or B;
a black Sharpie,
several paper towels
(2) pairs of disposable gloves in the appropriate size for you and your partner
1 small sandwich bag (for collecting a few leaves from plants in your sampling area)
With your partner and your other team members, go to the greenhouse habitat you are to investigate. Before you and your partner take your soil sample (1 sample per pair -two samples per habitat), 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; this is the area that you should sample. You will take one sample per pair in slightly different locations. Avoid very wet soil and highly compacted soil that will be difficult to sample but get close to a plant and record it's name from the label on it.
In the tropical house choose somewhere near a plant growing in the center circle rather than from the periphery where most of the plants are in pots.
TAKING YOUR SOIL SAMPLE:
When you and your partners have agreed on where each pair should take a 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 it 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 for, 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 two 50ml tubes from each sampling site.
Spoon or use the spatula you brought to get the soil sample into the labeled 50ml conical tubes. If you didn't get enough soil from your core to fill both tubes, use your corer to take another sample adjacent to the first sampling site. After both 50ml tubes have 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 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 tubes 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
In the lab, you will mix your soil sample by sieving. Wear gloves and avoid contaminating your soil with skin flora or other lab environment organisms. There will be a piece of screening material and a large beaker at your bench. Wearing fresh gloves (with an uncontaminated finger surface), place the screen over the beaker and have your partner secure it with an elastic band, avoiding contaminating the center surface of the screen with human skin flora. Pour the soil from the tubes onto the screen in small aliquots. Your goal is to sieve out the large non-soil materials. This can be expedited by pushing gently on the soil with uncontamined gloved fingers. The mixed, seived soil will be your SOIL source for weighing aliquots for dry soil preparation and for genomic testing, for inoculating some enrichment media to culture specific desired groups of bacteria, and for making a soil extract (described below).
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. The instructor will dry these and return them to you for use in Lab 2 for a second 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 an additional 0.25 gram sample of sieved soil from your sample. Weigh out an aliquot using the top loading balance and wieghing paper that you are careful not to contaminate. Fold up the edges of the weighing paper to use as a crude funnel and pour the 0.25g of soil into a special PowerBead tube from the MoBIO PowerSoil DNA isolation kit (these tube will be at the instructor's bench). Make sure you use the right tube and that your soil isn't too wet. (See the Wet Soil option of the Soil DNA isolation protocol below.) Each student will do a genomic DNA soil isolation, but not today. Label this tube with a piece of your team color tape containing your name, lab section, soil sample location and today's date. Give this tube to your instructor to freeze. She will return it to you when we start our genomic DNA extraction.
SOIL EXTRACT preparation
Weigh 1 gram of soil and add it to 100 ml of sterile water in a sterile 250 ml flask with magnetic stirrer (on your bench). This will become your soil extract. Place the flask on a magnetic stirrer at medium speed for at least 15 min. After you stop the stirring, 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 some of the supernatant, avoiding transfering the settled particulate matter, into a new labeled sterile 50 ml conical tube. This supernatant will be your SOIL EXTRACT and this extract will be used for some of the culturable bacterial enrichment protocols you will start today in Part C. Leave the rest of your soil extract in the flask for the time being. You will discard it at the end of the lab period.
Save the rest of the soil you collected. You will use soil, rather than soil extract,in several of your culture enrichment protocols.
The most time efficient way to organize your work is to leave your soil extract stirring on the magnetic stirrer while you start the culture of specific groups of bacteria from soil rather than soil extract. It doesn't matter if your extract stirs longer than 15 minutes.
Activity: Plate Count of Soil Bacteria: Assessing the Number of Culturable Bacteria In Your Soil
To perform a standard plate count of your soil sample bacteria you must first serially dilute the soil 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 pairs (one 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)solution.
Gather or find the following materials:
5 sterile 13 x 100 size culture tubes with caps
1 sterile disposable plastic individually wrapped 1ml pipet,
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 appropriate solid medium 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, turning on the fan, placing the plates in the hood, and positioning the covers so they are ajar for 10 minutes or until the medium surface shows no visible moisture.
4. 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, transfer 100μL of the 1:100 dilution to the tube labeled 10-3, mix well by vortexing.
6. Using a new tip, transfer 100 μL ml 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 100μL aliquots (after mixing well) from each dilution to the next tube of water until you have carried the dilution to 10-7.
8. Use your P200 micropipet and a new tip to transfer 100μL of the 10-3 dilution and dispense it to the center of the dilute nutrient agar plate labeled 10-3. 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 with the 10-4 through 10-7 dilutions of bacteria.
10. Allow the moisture to be absorbed into the agar before inverting the plates and putting a labeled piece of your team color tape around the set. Incubating the set of standard plate count plates at RT until next week in a rack designated by your instructor.
Next week you will count the number of colonies on these plates, observe the variability in colonies on each plate and, possibly, select one or more of these colonies for isolation and further study.
Calculating the number of bacteria per gram of soil
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 that with between 30 and 300 colonies per plate give accurate calculations.
CFU = number counted on plate/(diluent plated*dilution of plate counted)
Aseptic Transfer of Soil or Soil Extract to Begin Enrichment for Specific Bacterial Groups from a Mixed Population
Isolation & Study of Culturable 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. 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 not only enriched nutrient and dilute nutrient agar but also a variety of defined media that will favor slower growing 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 a few of the culturable 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 part of this two part project, we will isolate genomic soil DNA and id both culturable and unculturable bacteria by 16S rRNA gene sequencing.
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 Spore Forming, Nitrogen Cycling, and Methylotrophic Bacteria from Soil
Our goal is to isolate culturable bacteria that belong to three important groups of soil bacteria: the spore formers, the nitrogen cyclers, and 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 Isolation and Enrichment Media for Soil Bacteria from Mixed Populations 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.
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 us 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 the growth you observe in your lab notebook. There is a data table in the form of an Excel spread sheet that you can download from the Data folder in your First Class Lab conference that will help you keep track of the characteristics of each of your isolates and allow you to share your data with your team.
This project is an investigative one that will require outside of lab time. Because bacteria have widely varying generation times, they will form colonies at different rates. 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 alt 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.
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 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.
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
There is good advice and general information about keeping a lab notebook in the BISC209/S11: Resources section of this wiki. In addition to the general information found there, we offer here more specific advice about how to organize the protocols and data you will gather this semester. It is your choice to follow, or not to follow, these directions. 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 and time spend now keeping a clear, well organized notebook will save you valuable time later.
Add notebook keeping advice here
Assignment
Revisit the greenhouse (hours and more information found at: | http://www.wellesley.edu/WCBG/Visit/info.html ) with your group or on your own, using the downloadable greenhouse map to guide you.
Link to the Wellesley College Margaret Ferguson Greenhouses: [[8]]
You can download a pdf file of a greenhouse map and tour route here: [[9]]
Improve the notes you took during the soil sampling visit paying special attention to what's different about the habitat you are investigating and how it is different from others in the Wellesley College Greenhouses.
Start doing a little research on the soil bacteria that we seek: the endospore formers, the nitrogen cyclers, and the methylotrophs. Look carefully at the recipes for the enrichment media on which we are culturing bacteria from this soil community. Try to ascertain how the bacteria that will grow on this medium will be selected or differentiated from other microorganisms likely to be found in this soil community. The use of specific types of selective, differential, enrichment or all purpose media has been carefully thought out by your instructors to maximize the chance that you will culture bacteria from specific desired groups with the physical or metabolic characteristics that we seek. There are many reference articles that your instructor will provide and a lot of information on media and BISC209/S11: Culture Media in the Protocols section of this wiki that will help you figure out how the bacteria of our desired groups are being enriched, selected, and differentiated.
Read the introduction to the soil communities and diversity project found at BISC209/S11:Project1 and all of the LAB 2 protocols and information found at BISC209/S11: Lab2. Outline or make a flow chart of your lab work for LAB 2 in your lab notebook. If you are unfamiliar with keeping a lab notebook, refer to the BISC209/S11:Resources section of the wiki where you will find some guidelines.
Homework Questions
You can download a .doc file of these questions at [[Media:]]
You will turn in the answers to these questions at the beginning of the next lab:
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
