Lab 3

Objective of Lab 3: Identifying and Understanding Research Methods

Most readers find it difficult to understand the methods (also called techniques or procedures) used by researchers and the application of those methods to solve microbiological problems. This process becomes easier as each reader develops a mental "database" of technical information. This week you'll learn to find explanations for the methods used in microbiology and build your own mental database.

It's important to recognize the difference between research methods and research results. Research methods are like recipes, research results like foods prepared according to the recipes.

For example, the basic method for making bread includes a list of materials and procedures - activate the yeast, mix it with the other ingredients, incubate the mixture to let the bread rise one or more times, shape the loaves and bake them in an oven. This method can be stored in someone's head, or written down. The experiment (the preparation of a particular bread, with particular ingredients and a particular size, shape, etc.) is one specific result of applying that method.

It's essential to understand the methods employed by a group of researchers before trying to understand how they were used in any particular situation, to produce any particular outcome (set of data, or research results).

Extracting information from your research article and supplementing that information

Now go back to your list of the Methods used by the authors of your article. A few methods can be considered "standard" for a scientific discipline. For microbiology these include making solutions and basic microbiological media, streaking plates (including viable counts), and growing bacteria in simple batch cultures. You have learned these techniques in undergraduate labs so they do not require further explanation in your Report.

Next, make sure that you can understand the other methods used by your researchers. The information provided in the Methods (Techniques, Procedures) section of your article is probably not adequate for this purpose. It may only state that a method was used, citing a reference for the use of that method, or it may give a brief account of changes to a published method. Research articles (like yours or the ones it cites) often state what was done (e.g. x mL of solution A was added to y mL of solution B) but do not explain why it was done or how the method works.

Fortunately, other resources are available. They include method books written by experienced researchers (many now available online), web sites provided by the vendors of scientific equipment and supplies, and "protocols" or even method explanations posted online by individual labs. Use these resources carefully - remember that they are usually not peer reviewed!

The publications listed below contain explanations of research methods pertinent to this course. In Report 1, you will be required to cite such sources for three (3) of the non-standard methods used by the authors of your article (including the method you will explain in Report 1). Your Instructor or TA will assign those three methods in Lab 5.

How will I know if I've found the right resources and understood a method?

Review the methods that you identified during Lab 2. Make sure that you can answer the questions listed below. SDS-PAGE of proteins is used as an example to illustrate how these questions could be answered.

• How is each method performed? (e.g. proteins are dissolved in SDS-containing solution (so that they become uniform in charge/mass ratio and shape) and loaded into wells of a gel, a voltage is applied across the gel, the proteins separate according to their mass, and the gel is stained to reveal their positions).
• What data are obtained? (e.g. the relative distances migrated by the proteins (the electrophoretic mobilities) are measured and recorded)
• How are the data interpreted? (e.g. a graph of electrophoretic mobility versus the log of molecular weight is plotted for proteins of known molecular weight, and the molecular weights of "unknown" proteins are deduced using this standard curve) , and
• What information is gained by using this method? (SDS-PAGE is used to separate mixtures of proteins (revealing the mixture components) and to determine their molecular weights).

During the next week, use this information to begin preparing Report 1. Use Mendeley or another reference management program to record the citation information as you work on your project! Note that course lab manuals are NOT acceptable citations because they are not "published" documents.

Method Books

The series BioMethods, published by Birkhäuser of Basel, Switzerland.

BioMethods is a series of monographs, each with a distinct editor or editors, containing chapters written by experts on particular research techniques. They are designed for new users of research techniques and are usually very readable. The subjects covered by the BioMethods series can be obtained by performing either (1) a Basic title search in TRELLIS or (2) by using the Advanced Search feature by typing in the name of the series, selecting phrase from the 1st drop down menu, and then Series from the 2nd drop down menu. This approach displays the results by volume title which can be handy. Unfortunately the titles of the volumes in this series do not include information on their specific topics, so you will have to look further at each individual entry to determine what it covers.

The series Methods and Tools in Biosciences and Medicine is a continuation of the BioMethods series and is also published by Birkhäuser as well as Springer.

Colowick, S.P., Series Editor

Methods in Enzymology QP601 C733 Academic Press, NY Methods in Enzymology contains descriptions and evaluations of many research techniques, including sub-cellular fractionation, enzyme purification, enzyme assay techniques and many others. All volumes in this series are shelved together on the library's second floor and the last five volumes are on limited (3 day) circulation. Volumes 1-244 (1955-94) are also available on CD-ROM.

The series Methods in Molecular Biology, published by Humana Press, Totowa, N.J., USA

Methods in Molecular Biology is a series of monographs, each with a distinct editor or editors, containing chapters written by experts on particular research techniques. They are designed for new users of research techniques and are usually very readable. The subjects covered by the Methods in Molecular Biology series can be obtained by clicking on the Advanced Search in TRELLIS. Type "methods in molecular biology" in the 1st box, select "as a phrase" in the 1st drop down menu, and then in the last pull down menu select "series". Each volume's title indicates its contents. As well the more recently published volumes include a link to either the Table of Contents or even better, if available electronically, the full text of the volume itself.

The Practical Approach Series of books, published by IRL Press, Oxford, England

The Practical Approach books are monographs, each with a distinct editor or editors, containing chapters written by experts on particular research techniques. They are designed for new users of research techniques and are usually very readable. A list of the subjects covered by the Practical Approach series can be obtained by performing a "Search By Title" search of the Main Catalogue using Trellis and asking for "practical approach". Editions in this series particularly pertinent to the course have been placed on three day reserve.

The following monographs are also useful sources of technical information.

• D. Freifelder Physical Biochemistry: Applications to Biochemistry and Molecular Biology, Second Edition QH 345.F72 1982
• P. Gerhardt, Editor in Chief Methods for general and molecular bacteriology QR 65.M26 1994
• E. Harlow and D. Lane Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press QR186.7 .H37 1999
• J. Sambrook, E.F. Fritsch and T. Maniatis, Eds. Molecular Cloning: A Laboratory Manual, Second Edition QH442.2 S26 1989, Vol. 1, 2 and 3
• R.K. Scopes Protein Purification: Principles and Practice, Second Edition QP551 S4257 1987
• R.K. Scopes Protein Purification: Principles and Practice, Third Edition QP551 S4257 1994

Please inform Janet Wood if you find other references that should be added to this list!