MCB 101 Introductory Microbiology www life uiuc edumcb101

MCB 101 Introductory Microbiology www. life. uiuc. edu/mcb/101 - Syllabus Announcements Practice Exams Lecture Slides

MCB 101 Introductory Microbiology Exam Information for Spring 2019 2 hourly exams - worth 100 points each - given in lecture period on Feb. 22 and April 5 - machine graded multiple choice questions - practice exams will be posted on web site user name: your net. ID password: your UIUC password Exam 1 covers experiments 1 – 8 Exam 2 covers experiments 9 – 14 The Final Exam follows the same format, about 2/3 of the exam covers experiments 15 – 19 while the rest covers earlier material.

Course Instructor and Laboratory Coordinator: Dr. Kenneth Chapman kenchap@life. uiuc. edu 217 -244 -4941 Office: 241 Burrill Hall Office Hours: 3: 15 – 5 PM Monday, Wednesday 4: 00 – 5 PM Tuesday, 9: 00 – 11: 00 Thursday or by appointment Teaching Assistants Sec. A - MW 8 – 9: 50 am Brendan Sullivan bgs 4@illinois. edu Sec. B – MW 10– 11: 50 am Gregory Schwarz schwar 46@illinois. edu Sec. C - MW 3 – 4: 50 pm Kavjit Durairaj kavjitd 2@illinois. edu Sec. D – MW 5 – 6: 50 pm Dhaya Ponsingh dp 5@illinois. edu Sec. E - TR 8 – 9: 50 am Alex Phillips apphill 2@illinois. edu Sec. F - TR 10 – 11: 50 am Alex Phillips apphill 2@illinois. edu Sec. G - TR 1 – 2: 50 pm Dhaya Ponsingh dp 5@illinois. edu

Basis of Course Grade: Student grades in MCB 101 are based on total of 1000 points. While the instructor reserves the right to make minor corrections, the distribution of points will closely resemble the following: 12 Lectures (5 pts. each) LON-CAPA homework & quizzes LON-CAPA based labs Lab worksheets 2 hourly exams (100 pts. Each) 1 final exam (100 pts. ) Lab practical exam (4 parts) The Grading Scale A+ > 966 points B+ 899 – 867 points C+ 799 – 767 points D+ 699 – 667 points F < 599 points A B C D 50 points nominal 220 points nominal 40 points possible 290 points possible 200 points possible 100 points possible 966 – 933 866 – 833 766 – 733 666 – 633 ABCD- 932 – 900 832 – 800 732 – 700 632 – 600 Note: It is theoretically possible for a student to get more than 1000 points but the 1000 point scale given above will be used to determine final grades.

Pre-Lab Questions on LON-CAPA • • • Read Background and Procedures BEFORE lab. Answer questions in LON-CAPA folder on-line Pre-lab question folders open a week prior to due date Pre-lab folders are worth 10 points each Most pre-lab question folders close at 1 PM on Mondays There are NO MAKEUPS for missed Pre-lab Quizzes on LON-CAPA * About 5 questions, worth 10 points * 15 -20 minute time limit, (make sure your internet connection is good) * There are no make up quizzes

LON-CAPA Quizzes – Spring 2019 Quiz # Opens Due 1 10 AM 1/18 8: 30 AM 1/25 2 10 AM 1/25 8: 30 AM 2/1 3 10 AM 2/1 8: 30 AM 2/8 4 10 AM 2/8 8: 30 AM 2/15 5 10 AM 2/22 8: 30 AM 3/1 6 10 AM 3/1 8: 30 AM 3/8 7 10 AM 3/8 8: 30 AM 3/15 8 10 AM 3/15 8: 30 AM 3/29 9 10 AM 4/5 8: 30 AM 4/12 10 10 AM 4/12 8: 30 AM 4/19 11 10 AM 4/19 8: 30 AM 4/26 LON-CAPA Pre-Labs - Spring 2019 Pre-lab # Opens Due 1 8 AM 1/14 8 AM 1/23 2 8 AM 1/18 8 AM 1/30 3 8 AM 1/25 8 AM 2/4 4 8 AM 2/1 8 AM 2/11 5 8 AM 2/8 8 AM 2/18 6 8 AM 2/15 8 AM 2/25 7 8 AM 2/22 8 AM 3/4 8 8 AM 3/1 8 AM 3/11 9 8 AM 3/8 8 AM 3/25 10 8 AM 3/15 8 AM 4/1 11 8 AM 3/29 8 AM 4/8 12 8 AM 4/5 8 AM 4/15 13 8 AM 4/12 8 AM 4/22 Covers Lab Safety, Course Policies and Microscopes Microscopy, Simple and Differential Stains Aseptic Technique, Pure Culture, Media, Streak Plate Bacterial Nutrition, Enzymes and Adaptations Biochemical Tests for Bacterial ID Dilutions, Viable Plate Count, Enterobacteriaceae Bacterial Recombination Bacterial Growth Antibiotics, Disinfectants and Preservatives Pasteurization and Controlling Bacteria in Milk Fermented Milk Products, The Nitrogen Cycle Covers Microscopy More Microscopy Aseptic Technique, Pure Culture, Media, Streak Plate Bacterial Nutrition, Enzymes and Adaptations Biochemical Tests for Bacterial ID, Enterobacteriaeceae Dilutions, Viable Plate Count, Enterobacteriaceae Bacterial Recombination Bacterial Growth Control of Bacterial Growth - Antibiotics, Disinfectants, Preservatives Pasteurization Fermented Milk Products, The Nitrogen Cycle

Procedures for 101 Lab 242 Burrill Hall • • • Read the Background and Procedures (do prelab) Arrive on time Put Coats and Backpacks by door Turn off phone, i. Pods, radios etc. Be ready to listen to TA presentation Wipe down work area with disinfectant Work efficiently Clean up and disinfect work area Wash hands before leaving

Lab Worksheets Your Results and Conclusions • You must turn in a lab worksheet reporting your observations and conclusions each lab period to get credit for doing the experiments. • Lab Worksheets are found at the back of the lab manual. For example, page 211 is removed from the lab manual and used on the first day of class. • There are no formal lab reports this semester, but the instructions on how to do a lab report are still found in the lab manual.

Microscopy January 14/15 – 16/17 Experiment 1 Basic Techniques in Microscopy January 23/24 Experiment 2 The Simple Stain January 28/29 Experiment 3 The Gram Stain January 30/31 Experiment 4 Additional Stains for Microscopy Homework Assignments Prelab-1 on LON-CAPA, due by 8: 00 AM Wed. January 23. (covers pages 1 – 18) Quiz 1 on LON-CAPA, due by 8: 30 AM Friday, January 25. (covers pages i – xvi and 1 – 15)

Experiment 1 2 3 4 - Basic Techniques in Microscopy Simple Stains The Gram Stain Special Stains

Microscope Parts - Specimen Stage (holds sample under lens) Slide holder (hold sample on stage) Stage X/Y-axis travel knobs (moves sample to place objects under the lens)

Microscope Parts - Light Condenser (this lens focuses light on the specimen) Field lens (collects light) Light Bulb control dial Iris Diaphragm (controls amount of light )

Microscope Parts - Lenses Ocular Lens (2 nd level of magnification) Objective Lens (1 st level of magnification) Focus knobs The large outer ring is coarse adjustment. The smaller inner knob is for fine adjustment.

Microscope Parts - Lenses Binocular Lenses are to be used with both eyes open. The distance between the lenses can be adjusted to match your interpupillary distance. Rotating Nosepiece When using a parfocal microscope you should not move the stage when changing lenses.

Uses of Different Objective Lenses • 10 X Scanning Find the object • 40 X High-Dry Focus on the object • 100 X Oil immersion Maximum Magnification

Viewing Cells Using Microscopy • Start with 10 X objective (coarse focus) • Move to 40 X objective (fine focus) (Don’t move stage when changing lenses!) • Move to 100 X objective Add immersion oil (fine focus) (Do not dip the 40 X objective in the oil!) • Observe cells • Record observations • Discard or put away slide, clean the microscope (wipe oil off 100 X objective)

Microscopic Observations Magnification and Resolution Magnification Total magnification = objective x ocular Example: if objective = 40 X and ocular = 10 X Total Magnification = 40 X x 10 X = 400 X The practical limit of magnification with a light microscope is 1000 X for brightfield microscopy One can magnify the image more than 1000 X, but this just gives an enlarged image with no better resolution.

Resolution is the sharpness or clarity of an image. Resolution describes the quality of the image a microscope can produce when it is focused on an object, the ability to see fine details. Resolving Power (RP) (AKA the Resolving limit ) is a measurement of the smallest distance between two objects that appear clear and distinct from each other. A small RP means good resolution. Resolving Power Equation RP = 2 NA As and NA , RP and resolution improves

Resolving Power Equation RP = 2 NA is the wavelength, or color, of the light that is used to illuminate the specimen. Blue light has a shorter wavelength than red light and therefore gives better resolution. Numerical aperture (NA) is a measure of the amount of light gathered by objective

Resolving Power Line

$Numerical Aperture Higher numerical aperture means more light reaches objective Immersion oil captures diffracted$

Numerical Aperture Higher numerical aperture means more light reaches objective Immersion oil captures diffracted light (refractive index of glass) NA

1) For light microscopy with the high power oil-immersion objective lens, if the numerical aperture is 1. 25 and the wavelength of the light used to illuminate the specimen is 450 nm, what is the limit of resolution? R. P. = __________ nm A. 450 nm B. 0. 18 mm C. 1. 25 nm D. 1. 125 mm E. 720 nm

2) 250 nm = _____ microns A. 250 D. 0. 25 (mm) B. 25 C. 2. 5 E. 0. 025

Experiment 2 Simple Stain • Basic stains are cationic (the chromaphore has + charge) • The dye binds to the negatively charged cell surface • Bacterial cells are colored against a brightly lighted background • Commonly Used Dyes: methylene blue crystal violet carbolfuchsin safranin

Experiment – 2 The Simple Stain - Put drop of water on clean microscope slide. Make a smear of bacterial cells Allow the smear to dry Attach the cells to the glass by heating gently Stain the sample. Wash off the excess dye. Blot dry without wiping off the cells. Examine cells with microscope, use low power. Switch to high power oil immersion lens.

Preparing a Smear & Simple Stain - Common Problems Problem: It seems to be taking forever for the drop of water to evaporate. Reason: Large drops of water take a long time to dry. Solution: Use a smaller drop of water. To make the smear (Use your inoculating loop to put a drop of water on the slide. ) Don’t pass the slide through the flame before the drop is dry. Boiling the water can disrupt cell structure.

Common Problems With the Simple Stain. Smear is too thick to see the shape of individual cells. - Make another smear but don’t use such a big glob of cells. - Spread your smear out on the surface of the slide. - Look at the edge of the smear where cells may be less crowded.

3) Which one of the following is NOT a commonly used basic dye? A. Carbolfuchsin C. Crystal Violet E. Methylene Blue B. Safranin D. Congo Red

3) Which one of the following is NOT a commonly used basic dye? A. Carbolfuchsin C. Crystal Violet E. Methylene Blue B. Safranin D. Congo Red Congo red is an acidic dye that is used for a negative stain, such as the capsule stain. We will look at this in Experiment 4.

4) Which one of the following types of cells is the largest? A. Red Blood Cell B. White Blood Cell C. Paramecium D. Corynebacterium diphtheria E. Trypanosoma brucei

4) Which one of the following types of cells is the largest? A. Red Blood Cell ( ~ 6 – 8 mm) B. White Blood Cell ( ~ 9 – 12 mm) C. Paramecium ( ~ 100 – 300 mm) D. Corynebacterium diphtheria (< 1 mm) E. Trypanosoma brucei ( ~ 10 x 2 mm)

Common Problems With the Simple Stain. Can’t quite focus because the focus knob won’t turn enough. DON’T TRY TO FORCE THE FOCUSING KNOB TO TURN!

Focusing the Microscope - The lens system does not move when you adjust the focus knobs. - Adjusting the focus moves the stage up or down. - The image is in focus when the specimen is positioned at the proper working distance. - As magnification increases, working distance decreases. - The coarse adjustment focus knob (big outer wheel) lets you move the stage up and down quickly. - The fine adjustment knob (smaller inner wheel) moves the stage slowly.

Focusing the Microscope Turning the focus knobs moves the stage up or down by means of a rack and pinion. There are two rack and pinion systems in your microscope, one for coarse focus adjustment and one for the fine focus. If you are turning the focus knob and it stops moving freely, STOP. Do not try to force the focus knob to move a little bit further. You could damage the gears.

Problem Focusing on High Power You are able to focus on your specimen on low power but you just can’t get a clear image on high power. One possible cause: slide is upside down As magnification increases , working distance decreases. For the oil immersion, high power lens, the working distance is less than the thickness of a slide.