1. Contact Info
Professor Carrie Kahr (Ms. Kahr)
ckahr@irsc.edu
Contact me through Angel
http://ckahr-IRSC@weebly.com
2. Lab Safety
 Lab coats are required. Get disposable coats. Lab coats will not be returned at the
end semester.
 Close-toe shoes are required.
 Gloves and safety glasses are recommended, but not required.
 No food or drink in lab.
 Tie long hair back.
 Clean lab table with ethanol and paper towel before & after each lab.
 Wash hands before leaving the lab.
3. Grades
 Grades are made up of 3 practicals. Test 3 is NOT cumulative.
 Make-ups of test 1 or 2 will be on the last day of class, directly after test 3.
 You must notify me within 24 hours of the missed test that you need to schedule
the make-up.
 You must sign the attendance sheet.
4. Studying
 Read the introductory material in lab exercises in book before class.
 You do not need to spend a lot of time on the Lab Manual’s methods/ procedures,
as they are frequently different than how we will do them. I will provide you with
a protocol for you to follow and study from.
 Re-write your class notes to study.
 Be able to describe what we did, why we did it, how we did it, and what the
results were and why.
 Take pictures.
 At my faculty website, you can hear how to pronounce microbe names.
 Use files in Angel. ** Google Chrome works best with Angel.
5. Class tour
 Drawers are numbered. This is your seat for the semester.
 You will use the microscope with the same number as your drawer.
 Find the plastic mat in cabinet. We place that on the lab bench.
 Incinerator.
 Where to put things: incubator box, autoclave cart, normal trash, biohazards,
broken glass, plate disposal container, and pipette container.
6. Lab manual is Special Edition for IRSC. The 11th & 12th edition are OK.
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Exercise 5: Ubiquity of Bacteria
OUR GOALS- 1.) To learn that bacteria are everywhere,
2.) Learn how to sample.
1. TSA plates (Tryptic soy agar) - general media.
2. Labeling agar plates
We label plates with ______________, _____________, _________________________,
and __________________________________.
We label plates ______________________________ so if lids are mixed up be still know
what we have.
We label plates _____________________________ so the view is not blocked.
3. We use aseptic technique when we sample and work with bacteria. Examples are:
1. _____________________________________________
2. _____________________________________________
4. We store plates __________________________________, so that water from
condensation during the incubation process will collect in the lid, not on the culture. It
can be very messy, and can contaminate other samples.
5. Positive controls & negative controls ______________________________________.
6. Follow protocol for sampling.
I will assign you a place to sample. Some of you will require swabs & some will not.
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Exercise 1: Bright-field Microscopy
OUR GOALS: 1.) Name the parts of the microscope,
2.) Learn how to focus on a slide,
3.) Develop skill to focus on slide with 40X within 90 seconds.
4.) Learn bacterial cell shapes and arrangement of cells.
1. Play game.
2. Demonstration by PK.
3. There are four objectives, _________________________, ___________________,
__________________, and ____________________.
4. Also, the ocular piece has a lens with ________________________.
5. Oil is only used with ____________________________.
6. ________________________________ only is used to clean the 100X lens. To clean
slides, use _______________________________________________________.
7. Bacteria cells have 4 main shapes: ____________________, ____________________,
______________________, and ________________________.
8. Cell arrangement of cocci can be in _______________, ______________, or
________________.
9. Cell arrangement of bacilli may be __________________, ________________, or
___________________.
10. Some cells have ______________________, which can be seen in the stained
specimen.
11. Follow protocol of Bright-field microscopy. View slides 1-11.
Slide 1-2, do not use oil. Use scanning and 10X only.
Slide 3-11, use oil and focus up to 100X.
Note shapes and arrangement of cells.
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Review Exercise 5
1. Review Positive / Negative controls.
2. What is a colony?
A CFU?
3. How can you tell different kinds of bacteria apart?
4. How can we identify a fungal colony?
5. If there do not seem to be any molds (fungi) on a plate, why is it wrong to assume that?
6. What are the 4 shapes of bacteria?
7. Bacteria contain a cell wall made of _______________________________________.
Review Exercise 1 Bright-field Microscopy
1. Point to microscope parts.
2. Time slide (#13) for focusing in 90 seconds.
3. Terms:
Focus:
Parfocal:
Contrast:
Resolving power/ Resolution:
4. Ways to increase resolution:
1.
2.
3.
4. ____________ for 100X lens. Prevents light from refracting.
5. Calculating magnification: Total Magnification = Ocular (10X) X Objective
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Exercise 2 & 3 Darkfield & Phase-Contrast Microscopy
OUR GOALS: 1.) To learn to set up the microscope for dark-field & phase contrast
microscopy.
2.) To observe true motility of organisms in pond water with dark-field.
3.) To observe current, Brownian motion and true motility of bacteria
with phase contrast.
4.) To understand how light is manipulated in dark-field & phase contrast
microscopy.
5.) Prepare wet mount slides.
1. Dark-field and phase-contrast (next exercise) are used to observe protist and bacteria
that are ______________________. Staining cells ___________________ cells.
2. There are three types of movement observed with a microscope:
1. ______________________ caused by water evaporation.
2. ______________________ caused by molecules hitting non-motile bacteria.
3. ______________________ caused by flagella.
3. Demonstration by PK: Dark-field, phase-contrast, wet mount.
4. Follow the protocol for dark-field microscopy & observe pond water.
5. Follow the protocol for phase-contrast & observe bacteria movement by current,
Brownian motion, and true motility.
Exercise 14: Motility Determination by Tube Method
OUR GOALS 1.) To observe motility through the tube method.
2.) To compare this method with wet mount.
3.) Compare agar with SIM.
1. Review of Lab safety.
Don’t leave tools sit in the __________________________________ because
they will burn you when you reach for them.
Don’t wave ______________________ to cool them.
Remember to use ___________________________________ and open lid of
culture partially.
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2. Demonstration of tube inoculation.
3. Compare agar and SIM ____________________________________________.
SIM is less concentrated, so more liquid. __________________ is a liquid.
_____________________ is a solid.
Review Exercise 2 & 3 Dark-field & Phase-contrast
1. Time slide (#13) for focusing in 90 seconds.
2. Set microscope up for dark-field.
3. Set microscope up for phase-contrast.
4. Movement results
Movement
Pond protists
Staphylococcus sp.
Pseudomonas
aeruginosa
Water current
Brownian
True motility
5. Comparison of Microscopy
Microscopy
Organism Light
Blue filter
Oil
Condenser
Brightfield
Darkfield
PhaseContrast
6. Darkfield shows the _____________________________________ of specimen, and
the field of view is ____________________ where there no objects are.
Phase-Contrast shows the _________________________________ structures, and the
field of view is many shades of __________________.
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Review Exercise 14: Motility Determination by Tube Method
1. Compare methods of motility determination
Method
Advantage
Disadvantage
1.
Wet Mount
(darkfield & phase contrast)
2.
Tube Method
2. Why do we want to determine motility for bacteria?
3. Results
Bacteria:
Moltility yes or no?
Bacteria:
Moltility yes or no?
4. The tube that has bacteria with motility looks _________________________ and the
tunnel left by the stab is _________________________. In the tube with non-motile
bacteria the tunnel left by the stab is _________________.
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Exercise 9: Pure Culture Technique
OUR GOALS: 1.) To perform streak plate with the quadrant method.
2.) Review colony and CFU.
3.) Introduce selective media.
1. Bacteria exist in normal environments in mixed population.
2. Reasons to isolate bacteria:
1.
2.
3.
4.
3. TSA (tryptic soy agar) is a _____________________________________ media which
allows many different kinds of bacterial and fungal growth. EMB _________________
_____________________ is selective for gram negative bacteria. That means that it does
allow gram _________ to grow, but not gram ___________.
Exercise 10: Smear Preparation
OUR GOALS 1.) To learn what makes a good smear.
2.) To understand Heat Fixing a Slide.
1. Smears should be _____________________, which means there is a small amount of
bacteria rather than thick layers.
2. There are 3 reasons we “heat fix” a slide:
1.
2.
3.
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Exercise 11: Simple Staining
OUR GOALS: 1.) To understand why we would use a simple stain.
1. Simple staining
1.
2.
2. The paper we use to dry stained slides is ___________________________ paper.
3. Methylene blue contains a __________________________, a charged colored cation,
which attaches to negatively charged ________________________________________.
4. Demonstration of smear prep and simple stain.
Review Exercise 9: Pure Culture Technique
1. What is a colony, CFU?
2. What are the reasons for isolating colonies.
3. Notable colonies:
TSA plateEMB plate1.
2.
3.
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Review Exercise 10: Smear Preparation
1. Thick smears trap __________________, and the many layers of cells prevent
individual cells from being seen. Therefore, smears should be _____________________.
2. Heat fixing the smear is important because:
1.
2.
3.
3. We have learned about three types of paper/ tissue:
1. _____________________ paper is for cleaning microscope objectives.
2. _____________________ is for cleaning oil from slides and microscope stage.
3. _____________________ paper is for drying slides from stain.
Exercise 12: Gram Staining
1. Gram staining is very useful, because it can show you:
1.
2.


We will learn later that antibiotics can target parts of the bacteria cell wall. Some
antibiotics are effective on gram negative, and some are effective on gram
positive.
Also, Gram + / - can tell you a lot about virulence factors that you will learn about
in lecture.
2. The purple crystal violet stain is held in the thick layer of _______________________
in gram positive cells. PURPLE-POSITIVE-PEPTIDOGLYCAN
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3.
Reagent/ Step
Purpose
COLOR
Gram +
Pre-stain
Gram -
XXXXXXXX
Crystal Violet &
wash
Gram’s Iodine
Alcohol & wash
Safranin & wash
4. The step that requires the most skill and attention is the _________________________
with alcohol. If you leave the decolorizer on too long, the crystal violet will wash out of
the thick peptidoglycan layer of the gram positive, as well as the thin layer of
peptidoglycan in gram negative. Then all bacteria will appear gram ________________.
Also, if you do not decolorize long enough, the crystal violet will remain in the thin layer
of peptidoglycan of gram negative. Then, all bacteria will appear gram ______________.
5. Read the Stain: Gram reaction (+ or -), shape.
6. Demonstration of the gram stain by PK.
Exercise 13: Acid Fast Staining
1. Some bacteria can not be diagnostically stained by the gram stain. They have a high
amount of waxy material called ___________________________________________.
2. Two examples of bacteria that have mycolic acid are ___________________________
and ______________________________.
3. The __________________________________ stain is a diagnostic tool for the
identification of Mycobacterium ______________________________ and
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Mycobacterium ________________________ (causing the disease leprosy).
4.
Reagent/ Step
Pre-stain
Purpose
COLOR
Acid Fast
non-Acid Fast
(Acid Fast +)
(Acid Fast -)
XXXXXXXX
Carbolfuchsin &
wash
Acid-Alcohol &
wash
Methylene blue &
wash
5. Read the stain: Acid fast, shape. Non-Acid fast, shape.
You do not get any information about Gram stain quality from this stain.
Review Exercises 11, 12 & 13
1.
Stain
Color
Location
Stain type
Notes
Simple
Gram
Acid-Fast
3. Bacteria used
Staphylococcus aureus: gram +, coccus
Pseudomonas aeruginosa: gram -, rod
Mycobacterium phlei: acid-fast, rod
Pseudomonas aeruginosa: non-acid fast, rod
Staphylococcus aureus: non-acid fast, cocci
Bacillus subtilis: non-acid fast, rod
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