BISC209/S13: Assignment 209 Lab5

Materials and Methods

Due prior to Lab 6. Please submit an electronic copy to your dropbox and bring a hard copy to class.

Write the materials and methods section of your final paper with the following general sections:

• Acquiring a soil sample for bacterial community assessment.
  
• Enumerating bacterial soil community microorganisms.
  
• Culture Dependent Microbial Community Assessment of Functional Metabolic Diversity & Co-operative Community Behavior
  
• Isolation of select soil community bacteria to pure culture and testing Cultured Bacterial Isolates as examples of co-operation and competition among community members.
  
• Identification of culturable bacteria by 16S rRNA gene sequencing and Maldi-TOF Biotyper;
  

How to Write a Materials and Methods Section

It can be a daunting task to write a single paper on several months of experiments, particularly when those experiments result in a huge amount of disparate data. Turning an entire semester of lab work into a Materials and Methods section for your final paper can be particularly overwhelming, but it need not be. It is less so if you have kept a good lab notebook that contains all the information necessary to explain how to make or purchase reagents and clearly explains how you used these materials to accomplish your small and large goals.

It is also much easier if you think of your semester's work as a whole project that involved many experiments to gather specific evidence for aspects of the investigation. You must organize everything by goal (NOT tool!) and explain what you did to answer each of our experimental questions: How many microbes are in our soil community? Who are some of them and how diverse, metabolically and phylogenetically, are the community members we cultured? How do they compete and co-operate to function effectively as a community?

Organization of your M&M section by goal rather than by tool is essential. Do not write a chronological description of everything you did each day: A time-course description of the semester's lab work is NOT the way to organize and report the materials and methods applied to this project. The wiki protocols are instructions to “do this, then that” in an exact, detailed time-course set of instructions for YOU to follow. they tell you exactly what to do in lab and in what order, recognizing that you are new to most of these tools and materials. The detail in these protocols is far beyond anything needed in M&M and the focus is wrong. It would make your M&M section unreadable and unintelligible if you were to include all the wiki's trivial directives about what to do to tubes and plates at each step of every protocol. The wiki also lacks, in these detailed chronological lab day descriptions, lots of crucial information that you need in M&M. The wiki, largely, ignores all the behind the scenes work required to make or acquire the materials necessary to accomplish the work described. Materials in Methods should be none of that; rather, the M&M is divided into discrete sections (sometimes with subsections) that each begin with a title that states the smaller goal that will be accomplished and then explains how one accomplishes that goal from the starting material to the end point that is described in the title. Stress the goal and not the tool! For example, do not use the title “polymerase chain reaction” for a section in your M&M since that only describes the tool and says nothing about the context or goal. A better title would be, “Amplification of 16s rDNA by polymerase chain reaction (pcr)”.

Although it is important for your audience (who knows nothing, necessarily, about soil or bacteria) to be able to understand how the goal of each part of the experiment was reached, don’t spend time here explaining a lot of theory ( eg., how pcr works). M&M is not a “why does it work this way” section.

The focus of a good M&M section is on how each of the stated goals was accomplished. The ingredients and concentration information needed to make media, etc. or the information required to order reagents and use them properly must be included in a M&M section. Fortunately, if all that information has been previously published or can be found on a kit or reagent suppliers web-site, you need not repeat it-- as long as you include sufficient information in your M&M for a reader to find the supplier or manufacture's product information or you have the full citation to the paper or methods manual that describes how to make all the materials and to perform the testing. You MAY NOT cite the wiki for any information.

For this particular assignment you should:

First describe any of the procedures we have completed to date that are common to the basic divisions: Each should be separately titled. If you did used a procedure (such as making a soil extract) more than once, there is no need to describe it multiple times. Separately title each protocol, stressing the goal rather than the tool in the title. Order them by deciding which goal must be accomplished first. (Note that you do NOT have to write up protocols that you have not performed yet for this assignment although you will need to include these in your final paper.) After describing general protocols common to different lines of our investigation, create categories that describe the lines of investigation that we are using to provide evidence for abundance, diversity (richness), community behavior (co-operation and competition). What you actually did to provide evidence for those investigative goals included, enumeration of microbes (abundance), testing the microbial population as a whole for functional metabolic capacity (diversity) and prevalence of certain useful capacities (community behavior). You also isolated and identified (using two different methods) a few of the bacterial members of the soil community of selected groups (diversity?) and tested those members for a few physical or metabolic capabilities (diversity, cooperation and/or competition).

The best way to learn how to write M&M properly is to look at published papers that use similar tools and see how they described the use of those tools. If you find a reference to a publication that may describe a protocol you performed, you must look up that reference and be sure they did the procedure the way you did and, if there are slight modifications, describe them.

How to Hierachially Organize the M&M section of this project?

To help you with this assignment we have provided an organization scheme for your M&M section for you. Notice that the scheme is based on the description of the project found at Media:schema_soil_community_project.pdf on the project home BISC209/S13:Project. The outline is one way to organize this paper, you may have a more creative ideas and should feel free to use them.

This list is to help you know what to include in your final M&M section.

Acquiring a Soil Sample for Bacterial Community Identification and Assessment

• Acquisition of soil sample
  
• Making a Soil Extract
  

Enumeration of Bacterial Soil Community microorganisms

• By Culture-Independent direct count of nucleic-acid aggregates (nuclei or bacterial chromosome)using Dapi
• By Culture-Dependent plate count

Culture Dependent Microbial Community Assessment of Functional Metabolic Diversity & Co-operative Community Behavior

• Community Physiological profiling: Carbon source utilization: Community metabolic diversity (CMD), carbon utilization patterns
  
• Prevalence of Starch, Cellulose, and Phosphate digesters or processors

Isolation of select soil community bacteria to pure culture and testing Cultured Bacterial Isolates as examples of co-operation and competition among community member

• Isolation of Nitrogen Cycling bacteria to Pure Culture
  
• Isolation of Spore forming Bacteria to pure culture
  
• Antibiotic Production/Sensitivity Testing
  
• Mutualistic and Antagonistic Interaction Assessment
  
• Functional testing for contribution to the nitrogen cycle
  
• Functional testing for other select metabolic pathways
  

Identification of culturable bacteria by 16S rRNA gene sequencing and Maldi-TOF Biotyper

• Amplification of 16s rDNA by polymerase chain reaction (pcr) and identification of isolates by Genewiz?
  
• Identification of Isolates by MALDI-TOF

EXAMPLE: The following is a description of isolation of one of the nitrogen cycling bacteria, Azotobacteria, to pure culture.
You are allowed to use the paragraph exactly as written in your M&M section use it as a model for any other medium you need to include in M&M for this paperl

Isolation of Nitrogen fixing Bacteria from Soil. Bacteria with the ability to fix nitrogen aerobically were initially enriched and selected for by making a 2% solution of sieved soil (obtained as described previously in M&M) in Azotobacter medium (Azoto medium) Azoto medium is a defined liquid medium of 0.08% K2HPO4; 0.02% KH2PO4, 0.02% MgSO4 0.01% CaSO4; 0.0015% FeSO4/7H2O; 0.00025% g NaMoO3; 0.5% sucrose. The cultures were incubated at 27 °C for 1-2 week(s) in the dark. Observation of a slimy growth on the air-liquid interface or a pellicle on the surface of the medium indicated potential nitrogen fixers were present. A sample of the slime was diluted in sterile water and vortexed to break up microbes embedded in the biofilm. Serial streaking for isolation on solid Azoto medium (1.5% agar) over several weeks produced isolated slimy colonies. These colonies were isolated to pure culture and maintained aerobically on either Azoto medium or nutrient agar (0.3% Beef extract, 0.5% Peptone, 1.5% Agar; pH 6.6- 7.0 at 25°C).

Note that:
Rather than explaining what you did to your “tubes” or “plates”, you change the focus to how the goal was accomplished.

Note in the example above your protocol tells you to add 0.5gm of soil to 25 ml of liquid Azotobacter medium. The M&M version I wrote tells you the approximate ratio of soil to medium (2%) since there is about 25ml in the tube when you are done 0.5g/25ml is x=100ml = 2%.

Don’t end a section of M&M until you make sure that the goal stated in your M&M section title has been clearly accomplished.

A second example, NOT for one of the bacterial groups we attempted to isolate for this project! But if you substitute the groups we isolated, the format is similar.

Isolation of Denitrifying Methylotrophic Bacteria from Soil
Denitrifying methyloptrophic bacteria were initially enriched and selected from an approximately 2% (wt/vol) soil sample (obtained as described previously in M&M) in liquid denitrifying methylotrophic medium with methanol (DMMM: 1% Freshwater Base [FWB: 10% NaCl, 4% MgCl2*6H20; 1% CaCl2*2H2O; 2% KH2PO4 (acidic); 5% KCl]; 0.02M 3-N-morpholino propanesulfonic acid (MOPS C7H15NO4S pH 7.2), 0.2mM Na2SO4; 0.15mM K3PO4 pH 7.2; 5.0 mM NH4Cl, 0.5% KNO3; pH 7); 1% vitamin mix (Sigma product #M7150 -Murashige and Skoog Vitamin Powder); 0.25% methanol) at 30C in anaerobic conditions achieved by displacing as much air as possible with medium in a tightly sealed culture tube. Observation of turbidity and bubbles of nitrogen gas were used to indicate when secondary enrichment/selection was appropriate. A ~2% subculture was made in liquid DMMM using the same culture conditions. Production of nitrogen gas was used to determine when to subculture the secondary enrichment/selection to solid DMM medium (DMMM without methanol +1.5% agar). Oxygen was reduced and methanol was available during culture by incubation in a closed jar with a methanol gas enriched atmosphere and incubated at 30C. Serial streaking for isolation on DMM solid medium on sequential subcultures over several weeks produced isolated clear, glassy, drop-of-water colonies that were selected by subculture and isolation streaking. A pure culture of bacteria resulted, presumed to be a denitrifying methylotroph, proposed to be Hyphomicrobia. A pure culture of this isolate was maintained aerobically on solid medium: peptone yeast calcium maintenance for Hyphomicrobium (PYCM: 0.25% peptone; 0.05% yeast extract; 1 mM CaCl2; 2 mM MgSO4; 1% agar) at room temperature.

Notice some things I did and didn’t do in the example above. A protocol would tell you to add 0.5gm of soil to a screw capped tube of liquid medium and then add medium until it is very full. The M&M version I wrote tells you the approximate ratio of soil to medium (2%). Because there is probably about 25ml in the tube, when you have filled it completely, I'm approximating that 0.5g/25ml is x=100ml = 2%. Although you should NOT use the word “tube”, I just broke that rule. Why? Note that I didn’t say that I added soil to a “tube” or that I incubated the “tube”, which are the uses I want you to avoid. I need, however, in this situation to describe how I achieved a somewhat anaerobic environment without making the reader think I used a GasPak or anaerobic incubator since both of those alternatives are expensive and not necessary here. Note that I didn’t give you all the ingredients for DMM. Can the reader make DMM from the information given? Yes, since I have given IN THE SAME SECTION the recipe for DMMM and told you that DMM is DMMM without methanol and with added agar of 1.5% (which would be 15g/L if you wanted to make that much).

General tips:
Please look at the Methods sections of published scientific research papers (there are many in the References tool in Sakai) to see how the protocols in this wiki differ from Materials and Methods descriptions in a scientific paper. Notice that M&M paragraphs can be very short, particularly if you have used a kit or a previously published protocol for a part or all of the experiment. If some part of your work is published elsewhere, such as in a journal article's methods section or in a kit manufacturer's website, you may reference the kit manufacturer or use a journal article citation to the previously published directions. When you can reference a reagent manufacturer's web site (such as the New England BioLab's website directions for using Phusion® High-Fidelity PCR Master Mix with HF Buffer-- the reagent we used to amplify our target DNA by pcr (found at: | http://www.neb.com/nebecomm/ManualFiles/manualF-531.pdf), the manufacturer does not include the sequences of the primers we used (since those are unique to your target DNA) nor the exact thermal cycler program (since that is tweaked for the length of the DNA section you want to copy and for the relative CG content of the template DNA). Therefore, you must give more than a brief citation and web address. In the case of the pcr amplification using a commercial master mix, you must be specific about the template DNA, the primer information for amplifying 16s rDNA, and give the exact thermal cycler program you used.

Remember that all separately sectioned protocols are independent; therefore, you can not assume your reader knows what soil sample or extract you mean unless you say, "soil sample obtained as described in Materials and Methods: Acquiring a Soil Sample for Bacterial Community Identification and Assessment".

In order to write about how you accomplished your experimental goals succinctly and clearly, you must thoroughly understand what part of your goal is accomplished in each step. It does not require thorough understanding to follow the directions in the wiki and end up with a successful experiment; however, to write about your experiments in such a way that someone who is not part of this course can understand what you did and how it contributed to your overall goal... that's is not so easy. If you made sure that every time you left lab you understood how each part of each day's work fits into the project's goals, writing M&M, as well as the other parts of this paper, will be manageable.

Effective Concentration: You should use concentration rather than volume or weight terms whenever possible. Examples are % (vol/vol or wt/vol), or wt/vol terms such as mg/ml, or molarity. For example, instead of saying, “we combined 1 gm of soil with 100 mL of water”, say, “A 1% soil extract was prepared in sterile water”. You can assume your reader is a savvy enough laboratorian so that if he/she wants to follow this protocol that means that he/she must weigh out 1 gram of soil and dilute it in a total volume of 100 mL of sterile water. Note that you have changed the focus away from what YOU DID to how the goal was achieved (making a soil extract) and you haven’t given details that any laboratorian should know (like weighing, mixing, etc).

Sometimes it is not possible to give concentration; therefore, you must use volume or ratios of volumes. For example, when you resuspend cells in liquid medium, your reader needs to know approximately how concentrated the cells should be. If you make them too dilute or too concentrated it will negatively affect the goal, but sometimes (such as in our transformation) you don’t know the concentration. Volume is all you can do there.

Avoid using the words “tube” or “plate" Tubes are just pieces of glass, and plates are empty pieces of plastic; neither is an accurate way to describe what is happening in a culture or a step in an experiment. If you find yourself mentioning tubes or plates, stop and ask yourself, “what’s in the tube or plate?” and rephrase with a brief description of only the crucial info.

Using a kit: When you use a kit, you often don’t have all the information that you need to explain to your reader how to make all the reagents because they are proprietary, so you do the next best thing; you include where to buy the kit and a link to the manufacturers website for the directions on how to use the kit to accomplish the goal. Kit M&M descriptions are simple and short; For example: PCR products were removed of unincorporated dNPTs, primers, and other small fragments of DNA through use of Epoch BIoLabs GenCatch PCR Purification Kit (http://www.epochbiolabs.com/). This clean-up was accomplished by following the manufacturer’s directions.

Miscellaneous:
RPM’s are not universal but “g” force or rcf’s (relative centrifugal force) are terms that denote a centrifuge speed that can be achieved in any kind of centrifuge. Please use the universal terms, not the ones that only matter in a particular rotor in a particular centrifuge. If you set rpm on our microcentrifuges, you can find out the equivalent rcf's (g) by using the toggle in the control panel. Write this equivalent down in your lab notebook.

Use the full formal terms first time in each separately titled section. If there are acronyms that you want to use later, introduce them with the full term, ie. Polymerase chain reaction (pcr), nutrient agar (NA), Luria-Bertani (LB), etc.

There is an extensive handout on writing a Methods section in the Resources section of this wiki.