Elementary Microbiology Laboratory (MICRB 107)

Introductory Microbiology Laboratory (MICRB 202): Fall 2003 1 Introductory Microbiology Laboratory (MICRB 202) Fall 2003

Lab Room 124 MCB W, F 8:00 – 9:50 AM

Instructor: Matthew P. Hoch, Ph.D. E-mail: mph [email protected] Assistant Professor of Biology

Campus Phone: 4016 Website: www.yk.psu.edu/~mph13/

Office/Lab: Office Rm 131 MCB / Biology Lab Rm 124 MCB

Office Hours: F 11:00 a.m. - 1:00 p.m.; F 3:00 p.m. - 5:00 p.m., or by appointment.

Course Description:

An introduction to fundamental research principles, technical skills and operational protocols of a microbiology laboratory. Specific experiments address topics in the study of cell structure and metabolism, microbial genetics, disease, applied microbiology and microbial interactions in natural environments.

Prerequisite:

MICRB 201, which may be taken concurrently. CHEM 12 (may be taken concurrent), or permission from the instructor based on prior experience and willingness to participate in a non-credit chemistry primer experience.

Course Goals and Objectives:

 Learn basic specimen preparation and microscopy skills used in studying cell structures.  Students will properly maintain and use a compound microscope to view and interpret slides with live or stained microorganism specimens.  Using a variety of staining methods, students will prepare microorganism specimens on slides.

 Practice aseptic technique at all times to protect personal and public health, as well as, to prevent contamination of cultures or other experimental procedures.  Students will properly maintain an uncluttered and disinfected work space.  All equipment used by the student to manipulate microorganisms will be sterilized using appropriate protocols which the student will have mastered (e.g. lighting Bunsen burner).  Students will correctly transfer pure cultures of microorganisms growing in either solid or liquid media. Introductory Microbiology Laboratory (MICRB 202): Fall 2003 2

 Understand the formulation and proper use of microbiological media and diagnostic test procedures.  Students will correctly explain the formulation of solid and liquid media designed as being non-selective versus selective and/or differential for specific groups of bacteria.  Students will correctly record and interpret macroscopic observations of cultures results.  Students will correctly explain the essential ingredients of media used in diagnostic tests for determining various biochemical phenotypes.

 Know how to enumerate microbes in a sample using both indirect culturing procedures and direct microscopy methods.  Using the appropriate pipette, or pipetting device, students will quantitatively dilute a culture or unknown liquid sample to specific dilution factors.  Students will correctly calculate CFU or PFU from plate counts based on known dilution factors and sample volumes used to inoculate plates.  Students will quantitatively prepare samples on slides for direct microscopic quantification of sample cell density, using both light and epifluorescence microscopy.

 Develop thinking skills relevant to the application of the scientific method in microbiology.  Students will formulate clear, answerable questions and testable hypotheses.  Student will correctly follow experimental protocols and be able to predict their expected results.  Student will properly collect and organize data into a presentable format (e.g. table, text description, or graph).  After assessing the validity and significance of the date, students will draw appropriate conclusions.

 Develop communication and interpersonal skills required for successful scientific collaboration and reporting of experimental results.  Students will need to work effectively as part of a team, because the performing of tasks, collection of data, and analysis of results will often be a shared responsibility among the class.  Students will participate in presentation and discussion of date in both oral and written formats.

 Practice proper laboratory safety, etiquette, and equipment care at all times.  Students will correctly follow all procedures and protocols described in the “Fire, Safety, and Lab Policies” document for this laboratory course. Introductory Microbiology Laboratory (MICRB 202): Fall 2003 3

Student Evaluation:

Your grade in the course will be based on your participation inside and outside of class, presentation and interpretation of results, preparation before lab meetings, assimilation of lab knowledge and skills, and your lab etiquette. How you participate in the course, as well as your ability to properly present and interpret results, will be documented by your lab notebook. Quizzes will evaluate your understanding of exercise background materials, objectives, experimental design, and protocols. Two practical exams will evaluate your overall knowledge, skills and related theory.

Notebook Checks 42 % (7% per) Lab Quizzes (six) 18 % (3% per) Practical Exams 34 % (17% per) Lab Etiquette 6 %

Lab Notebook: (42%; 7% each check) This is the fundamental document you will create throughout the course. Everything you plan to do and what get accomplished goes in your lab notebook. By faithfully following the guidelines for your notebook entries, you should be prepared for each class; hence, you’ll increase quiz performance. I will also ask you to include (attach) descriptive, tabular, and graphical presentations of your data, interpretations and conclusions; sometimes this will be requested in a more formalized scientific report format.

For each lab period, get started by reading the exercises for the planned period – this will be a handout passed out the prior week. Then state questions to be addressed and outline experimental procedures used. State any predictions for your results. Enter all data in an organized manner; this may mean creating hand written tables in your notebook. It also helps to use subheadings under each experiment to clearly identify entry components for easy review of your work. You should include a section on problems when they happen. When interpreting results, you should explain undesired results. You should address all requests for data presentation and discussion points, as stated in the lab handout for that week. You will need to save a couple pages at the start of your notebook to be used for creating an ongoing table of contents with page numbering. Other details to include are outlined on the Lab Notebook Guideline and Marking sheet (see attached).

Lab Quizzes: (18%; 3 % each quiz) Periodic quizzes will be given to assess understanding of lab exercises and your level of preparation prior to that days lab exercise. Quiz questions are usually multiple choice and matching in format, and they are based on lab notes and lab handouts for that days exercise and those since the previous quiz or course start for Quiz 1. Quizzes will begin promptly at the start of the indicated Wednesday lab period (see schedule below).

Practical Exams: (34%; 17% each exam) Students will demonstrate common laboratory skills at the mid-point and end of the semester. Students will be graded on good technique in handling organisms in an aseptic manner, colony isolation, preparing specimens for microscopic observation, understanding of equipment function and use, and analysis and interpretation of various test results. Introductory Microbiology Laboratory (MICRB 202): Fall 2003 4 Lab Etiquette: (6 %) The instructor will evaluate you on a number of criteria throughout the semester. You will be expected to arrive on time, demonstrate a state of preparedness (know what's going on in the lab), follow fire and safety policies, perform work properly, clean and sanitize your bench area at the start and completion of each lab, take care of the equipment correctly and participate in class discussions.

Microscopes are to be cared for properly. Each student will be assigned a microscope and will be expected to keep it clean and in good working order. Failure to do so will result in loss of instructor evaluation points. NEVER disassemble your microscope.

Grades:

Letter grades are based on the total percentage of course marks accumulated as indicated below. Plus (+) or minus (-) designations will be assigned only at final grade submission, and will be based on the statistical distribution of marks, i.e. "the curve", within each posted letter grade range. [E.g.: total course marks equal to or above 89, the A-range, will always be either an A-, A, or A+; which +/- designation received within the A-range will depend on the statistical distribution and number of students within the A-range.

A 89 - 100 % B 77 - 88 % C 65 - 76 % D 53 - 64 % F < 52 %

Lab Attendance Policy:

This laboratory course, like most others, is all about being minds-on and hand-on. Considering the later, it should come as no surprise that you need to be at each lab. How else are you going to perform the experiment or interpret results? Therefore, attendance and participation is required for all labs. Assignments will not be accepted if you were absent from lab unless you properly follow the “Make-Up” Policy (see below).

"Make-Up" Policy:

Missed examinations, quizzes, labs, or assignments can only be made up, or waived, by prior arrangement and/or with proof of an excusable absence (e.g., documented medical reason, sanctioned University Function). A different set of questions may be used for any make up assignments or exams.

Lab Unit Packets:

Lab activity details will be provided at various times throughout the semester in the form of unit packets. That is to say, detailed write-ups of “specific activities” will be compiled together based on common themes and distributed to you as a “lab unit”. Some units may contain activities that span several periods over a few weeks; the Water Quality Testing is a good example of this. Please realize that there may be specific activities from different units running during the same week. This may seem frustrating at first, until you become familiar with the process. Also, activities within each unit are clearly designated by the lab period number when work starts on an activity, and lab period number is followed by a number Introductory Microbiology Laboratory (MICRB 202): Fall 2003 5 indicating the order of the activity in that period. What follows is a list of lab units and specific activities. You may want to set aside extra blank pages in your lab notebooks for each lab unit, according to its number of activities and their duration. Cross reference unit activities with the schedule below.

Lab Units: (Orientation , policy, and safety review is on Sept 2nd, prior to the start of labs.)

I. The Microbiology Laboratory & Cultivation of Bacteria 1.1 Introduction to Brightfield Microscopy 1.2 Introduction to Culture Media 1.3 Bacterial Colony Characteristics 1.4 Hunt Microbes from the Environment 2.1 Aseptic Technique in Transferring Bacteria 2.2 Plate Streak Method for Colony Isolation

II. Bacterial Morphology and Cellular Structures 2.3 Wet Mounts & Observing Live Bacteria 3.1 Motility Test 3.2 Smear Preparation & Simple Staining 3.3 Gram Stain 4.1 Endospore Stain 4.2 Capsule Stain

III. Physical Controls on Growth 5.1 Temperature Effects 5.2 UV Light: Lethal Effects

IV. Estimating Cell Density of Laboratory Cultures 6.1 Estimating Cell Density by Turbidity 6.2 Viable Plate Counts

V. Microbial Water Quality Analysis 8.1 Water Testing Sampling Design Discussion 9.1 Fecal Coliforms by Membrane Filtration 9.2 Most Probable Numbers of Total Coliforms 9.3 Heterotrophic Plate Count of Water Samples 9.4 Total Direct Count of Water Samples 10.1 Coliform Confirmations 11.1 Water Testing Data Compilation & Discussion

VI. Food and Beverage Microbiology 12.1 Yogurt 12.2 Wine or Vinegar

VII. Phage & Microbial Genetics: 15.1 Phage Isolation 15.2 Transduction 17.1 Bacterial Conjugation Introductory Microbiology Laboratory (MICRB 202): Fall 2003 6

VIII. Natural Microbiota and Non-Specific Host Defense 17.2 Effectiveness of Hand Scrubbing 19.1 Lytic Effect of Tears and Saliva 19.2 Antimicrobial sensitivity testing (Kirby Bauer)

IX. Identification of a Gram-Negative Intestinal Pathogen 19.3 Isolation & Phenotypic ID of Enteric Pathogens 21.1 Enterotube II Multiple Test System 23.1 Molecular ID of Enteric Pathogens

Lab Schedule:

Lab Date Deadlines* Specific Activities** Period 0 T, Sept 2nd Orientation , Policy, & Safety 1 W, Sept 3rd 1.1 Introduction to Brightfield Microscopy 1.2 Introduction to Culture Media 1.3 Bacterial Colony Characteristics 1.4 Hunt Microbes from the Environment 2 F, Sept 5th 1.4 Hunt Microbes from the Environment (day 2) 2.1 Aseptic Technique in Transferring Bacteria 2.2 Plate Streak Method for Colony Isolation 2.3 Wet Mounts & Observing Live Bacteria 3 W, Sept 10th Quiz 1 & 2.3 Plate Streak Method for Colony Isolation (day 2) Notebook 3.1 Motility Test (read result within 24 h) check 1 3.2 Smear Preparation & Simple Staining 3.3 Gram Stain 4 F, Sept 12th 4.1 Endospore Stain 4.2 Capsule Stain 5 W, Sept 17th 5.1 Temperature Effects 5.2 UV Light: Lethal Effects 6 F, Sept 19rd 5.1 Temperature Effects (day2) 5.2 UV Light: Lethal Effects (day 2)

7 W, Sept 24th Quiz 2 & 7.1 Estimating Cell Density by Turbidity Notebook 7.2 Viable Plate Counts check 2 8 F, Sept 26th 6.2 Viable Plate Counts(day 2) 8.1 Water Testing Sampling Design - Discussion 9 W, Oct,1st 9.1 Fecal Coliforms by Membrane Filtration 9.2 Most Probable Numbers of Total Coliforms 9.3 Heterotrophic Plate Count of Water Samples 9.4 Total Direct Count of Water Samples 10 F, Oct 3rd 9.1 Fecal Coliforms by Membrane Filtration (day 2) 9.2 Most Probable Numbers of Total Coliforms (day 2) Introductory Microbiology Laboratory (MICRB 202): Fall 2003 7 9.3 Heterotrophic Plate Count of Water Samples (day 2) 9.4 Total Direct Count of Water Samples (day 2) 10.1 Coliform Confirmations 11 W, Oct 8th 9.4 Total Direct Count of Water Samples (day 3) 10.1 Coliform Confirmations (day 2) 11.1 Water Testing Data Compilation & Discussion No F, Oct 10th Study Day Class 12 W, Oct 15th Quiz 3 & 12.1 Yogurt Notebook 12.2 Wine or Vinegar check 3 13 F, Oct 17th 12.1 Yogurt (day 2) 12.2 Wine or Vinegar (day 2) 14 W, Oct 22nd 12.2 Wine or Vinegar (day 3) Practical Exam Review

F, Oct 24th ************** Practical Exam I *************** 15 W, Oct 29th Quiz 4 & 15.1 Phage Isolation Notebook 15.2 Transduction check 4 16 F, Oct 31st 15.1 Phage Isolation 15.2 Transduction 17 W, Nov 5th 17.1 Bacterial Conjugation 17.2 Effectiveness of Hand Scrubbing

18 F, Nov 7th 17.1 Bacteria Conjugation (day 2) 17.2 Effectiveness of Hand Scrubbing (day 2) 19 W, Nov 12th Quiz 5 & 19.1 Lytic Effect of Tears and Saliva Notebook 19.2 Antimicrobial Sensitivity Testing: Kirby Bauer check 5 19.3 Isolation & Phenotypic ID of Enteric Pathogens 20 F, Nov 14th 19.1 Lytic Effect of Tears and Saliva (day 2) 19.2 Antimicrobial Sensitivity Testing: Kirby Bauer(day 2) 19.3 Isolation & Phenotypic ID of Enteric Pathogens (day 2) 21 W, Nov 19th 19.3 Isolation & Phenotypic ID of Enteric Pathogens (day 3) 21.1 Molecular ID of Enteric Pathogens

22 F, Nov 21st 19.3 Isolation & Phenotypic ID of Enteric Pathogens (day 4) 21.1 Molecular ID of Enteric Pathogens (day 2)

23 W, Nov 26th Quiz 6 & 21.1 Molecular ID of Enteric Pathogens (day 3) Notebook 23.1 Enterotube II Multiple Test (read result within 24 h) check 6 No F, Nov 28th Thanksgiving Holiday Class 25 M, Dec 3rd 23.1 Enterotube II Multiple Test (day 2) 21.1 Molecular ID of Enteric Pathogens (day 4) 26 W, Dec 5th 21.1 Molecular ID of Enteric Pathogens (day 5) Introductory Microbiology Laboratory (MICRB 202): Fall 2003 8 27 W, Dec 10th Practical Exam Review F, Dec 12th *************** Practical Exam II ***************

* Quiz content includes materials for all exercises begun since the last quiz (since the start of class for Quiz 1) and materials for new exercises begun on the quiz day.

** For each lab period, new activities are given in boldface, and on-going activities from prior lab period(s) are given in italics. Refer back to the appropriate lab period handout, or your notebook, to see what work remains in an on-going lab.

NOTE: Topics for particular lecture dates are tentative; although, we will try to keep to this schedule. Any change to the syllabus will be presented in writing via a paper hardcopy or electronic format (e.g. website or Email).

Course Requirements:

1. You are responsible for your own laboratory safety. Thereby, you will need a lab coat and safety glass to be worn during labs.

2. You are responsible for having read and understood the day’s lab before the lab period!

3. You will need a hardback laboratory notebook for keeping a record of laboratory activities, results and discussions.

4. A PSU Access Account. Go to IST Rm 106 and make the request for your user-ID and password. It will take about 24 hours for activation, at which time I recommend you change your password.

5. A single Email address. I will send all course announcements and notes to your PSU user-ID address. This is also the easiest address for other PSU students to use in reaching you, as it is on the PSU Directory. I suggest you use the PSU Webmail.

6. You will keep in touch with me and other students in the class by Email. Check your Email frequently (i.e. daily) so you don’t miss important announcements.

7. The ability to save computer work to permanent space on the network, a diskette, CD, or zip- disk. You will have to supply the disc or CD.

8. A fundamental working knowledge of Excel, or other spreadsheet software package, for data manipulation and graphing.

9. You will need a fundamental working knowledge of Word, or other word processing software, for the writing of a scientific report.

10. Anticipate out-of-class lab time. Yes, this means coming into the lab at unscheduled times to make observations, record results, and possibly inoculate cultures. It’s the nature of those little Introductory Microbiology Laboratory (MICRB 202): Fall 2003 9 beasts to grow really fast – they just can’t wait for our schedule. An alternative, is to extend scheduled hours and cold-store your results; however the latter can lead to ambiguities.

11. Participate class and bring your most positive attitude.

Class Guidelines:

I would like everyone to participate in making our class times a nice comfortable place to learn throughout the next fifteen weeks. To this end, please, let us all adopt the following guidelines on appropriate and considerate behavior.  Unless you are waiting for an organ transplant, or a birth is imminent, please turn off all electronic devices (e.g. cell phones, pagers, etc) that could generate distracting noises prior to entering class.

 There will be classes when computers are used, but at other class times please refrain from typing Emails or chat applications on your personal or lab computers. The excess “click-clack” of a keyboard is distracting to others and the instructor.

 I encourage you to fill the room with questions and discussion on the topic at hand. Please ask questions freely, and if not in class then out of class, either verbally or via Email. When others ask, or answer, questions in class, please give them your full attention and respect.

 If you have a comment or complaint regarding any aspect of the course other than the course content or activity being discussed, please wait to speak with me until after class, outside of class, or by Email. This saves valuable class time and is much more discrete, particular on issues of grade disputes or any other potential misunderstanding.

 Please don’t study or read other course material in class. If you find yourself in a time management crises come see me before class instead.

 Also, refer to “Promoting a Vibrant Learning Culture” at www.psu.edu/oue/vibrant.html.

Academic Integrity:

Academic dishonesty, aka cheating, is not something you want on your record, nor do you want to lower your grade or fail the course. This first year of university is a time to focus on developing study skills and learning your strengths and weaknesses. Students that cheat are not challenging themselves, and such cowardly behavior now, will leave them even more ill prepared for their future. If you ever feel the urge to cheat in any form, that is your signal to seek help from your peers, instructor, and/or the learning center. Faculty will have great respect for the hard working honest student that feels they have learned something, regardless of any grade outcome. Preservation of a high standard of academic integrity is, for many individuals, a source of self-confidence and pride.

To possess academic integrity means you don’t cheat. So what is cheating?  Copying someone else’s assignment or and submitting it as yours. Introductory Microbiology Laboratory (MICRB 202): Fall 2003 10  Referring to another student or sources during a quiz or exam. It is NOT cheating to work with other students while studying for quizzes and exams. It is NOT cheating to seek assistance from another student, a tutor, or your instructor, in order to understand an in-class activity or out-of-class assignment.

If the instructor suspects you of cheating, they will immediately notify you to meet informally after class or at some other time outside of class. At that time, the instructor will inform the student of the accusation and its circumstances, and the student will have the opportunity to explain their view point or situation. The instructor will then decide whether to proceed with a formal academic sanction. Notification of an academic sanction will involve a formal meeting between accused student and instructor. At this formal meeting, the student will be presented with an academic sanction form that explains the accusation and the sanctions, such as a failing grade on an assignment, quiz, or exam. The formal meeting will be arranged by e-mail or phone call. The student may agree and sign the form or contest the allegations through the university authorities. If a student is absent from the formal meeting time, they will be given three additional days to contact and meet with the instructor. Failure to formally meet with the instructor will be deemed as the student's acceptance of academic sanction, and waives the student's right to contest the allegations of academic dishonesty. Note, that any student(s) assisting or facilitating others in cheating will be subject to equal penalty and treated according to these same guidelines The official guideline on this policy can be found in the Academic Administrative Policies Manual, which is available on the PSU website: www.yk.psu.edu, or www.psu.edu/dept/oue/aappm/ , refer to section G9.

Disability Statement: Please see me immediately after the first class if you have a documented disability that may require special accommodations for this course. I will do all I can to accommodate your needs and/or arrange to get you any additional required assistance.