Quiz 1 Study Guide
Know the concepts listed below. You should be able to describe each one without looking
back into the lab manual. You should also be able to visualize the outcome of each
staining procedure (ie. Bacillus subtilis + gram stain Æ what shape, gram reaction will you
see?). You should be starting to memorize the gram staining characteristics of each
bacteria, their size and morphology so that you are able to make a educated guess upon
seeing an unknown stained bacteria with brightfield microscopy.
Regarding the quiz, think about which questions could possibly be asked! There are only
so many things one could actually quiz on for each section.
Exercise 1: Principles of aseptic technique
Pure vs. mixed cultures
pg. 3
Exercise 2: Aseptic methods for entering culture tubes and transferring cells
Simple rules (5) of aseptic technique
pg. 5
Exercise 3: Principles and care of the light microscope
Identify all parts of the microscope
Objective lenses, the ocular and their magnifications
Calculation of the limit of resolution and numerical aperture
Function of immersion oil
Rules for handling and storing microscopes
pg. 14
pg. 15
pg. 15
pg. 15-16
pg. 16-17
Exercise 4: Introduction to staining microorganisms
Simple stain: basic and acidic dyes
Differential stain: gram and acid-fast stains
Structural stain: endospore, flagella, capsule, and inclusion stains
pg. 21
pg. 21
pg. 21
Exercise 5: Smear preparation, fixation, and simple staining with basic dyes
Cultures: Staphylococcus epidermidis, Esherichia coli, Saccharomyces cerevisiae
Type of rod shaped bacteria
pg. 28
Arrangements of spherical shaped bacteria
pg. 28
Simplified procedure: label bottom of slide, make smear, fix cells, stain pg. 29-31
Exercise 6: Observing live bacteria using wet-mount method + phase contrast
Advantages and disadvantages of brightfield vs. phase-contrast
pg. 35
Phase advantage: observe live, motile cells, and unstained cells, simple preps
Phase disadvantage: low contrast, no fine details
Brightfield advantage: observe finer details (endospores, peptidoglycans, etc)
Brightfield disadvantage: longer prep time (staining!), dead cells, non-motile
Principle of image formation in phase contrast microscopy
pg. 35-37
Size of prokaryotic vs. eukaryotic cells
pg. 40
Exercise 7: Gram stain: A differential stain
Cultures: Pseudomonas fluorescens, Bacillus cereus, unknown (Ex 7B)
Components of the Gram stain and their function
Interpretation of the gram stain
Gram staining procedure
False gram negative staining (ie. when a culture should be + but looks -)
pg. 52-54
pg. 51+lecture
Exercise 9: Bacterial endospores
Cultures: Bacillus subtilis, Lactobacillus platarum
Terms: central, terminal or free endospore, vegetative cell, sporangium
Genera of bacteria that form endospores
Physical properties of endospores (resistance to sterilization)
Gram and malachite green prodecure for staining of endospores
pg. 67
pg. 67-68
pg. 67-68
pg. 69-71
Exercise 10: Bacterial flagella and motility (not 10A)
Cultures: Proteus mirabilis, Pseudomonas auruginosa
Chemotaxis and chemoreceptors
Motility exerpiments using soft-agar deeps and agar plates
Aerobic, faculatative anaerobic, and obligate bacteria (an-- or aerobes)
pg. 79
pg. 82-83
pg. 82-83
Exercise 12: Sterilization principles and methods
Understand 6 different types of sterilization (Table 12-1)
Pasteurization; tyndallization
Thermal death time vs. thermal death point
pg. 100
lecture
pg. 100
Exercise 13: Preparing culture media
Complex vs. defined media
Peptones, tryptones and yeast extract
Difference and examples of liquid, solid and semi-solid media
Preparing liquid broth (TSB) and agar deeps, slants and plates
pg. 107
pg. 107-108
pg. 108-114
Exercise 14: Separating microbes on streak plates
Colony, colony forming unit (CFU), confluent growth,
Procedure for T-streaking microbes
Morphology of colony (Table 14-1)
pg. 119
pg. 119-125
pg. 120
pg. 51
Exercise 15: Determining culture purity
Cultures: mixture of Esherichia coli + Staphylococcus aureus
Pure vs. mixed cultures
pg. 131
Know the function of the primary and secondary streak plates
pg. 131
How purity was determined:
pg. 131-132
Primary T streak, secondary T streak, morphology of colonies, gram staining
Exercise 16: Agar-slant and agar-deep cultures
Cultures: Esherichia coli, Bacillus subtilis, and Saccharomyces cerevesiae
Know difference between TSA and SAB
pg. 529 & 527
Know the purpose agar slant and agar deep cultures are made
pg. 143
Procedure for inoculating agar slants and agar deep cultures
pg. 144-145
Consider what could happen if obligate aerobes are inoculated in the agar deep culture
E. coli = facultative anaerobe; B. subtilis = obligate aerobe, S. cerevesiae = aerobe
Exercise 17: Broth cultures
Cultures: Bacills subtilis, Escherichia coli, Saccharomyces cerevisiae, Penicillium notatum
Turbidity vs. pellicle vs. pellet(button) vs. mycelia
pg. 151 & 154
Procedure for inoculating broth cultures
pg. 152-153
Know the characteristic microbial growth pattern for each microbe
pg. 154
Exercise 20: Counting viable cells: serial dilution and spread plates
Viable #, CFU, “30-300 rule” and sampling error
pg. 175
Understand the use of serial dilutions and plating
pg. 175
Know the difference between “dilution” and “dilution factor”
pg. 176
Procedure for preparing serial dilution plates
pg. 180-182
Know how to calculate CFU/mL from # colonies/plate and the dilution
pg. 184
Be prepared to do a dilution problem(s) like those you saw in the homework assignment
This is a guide I put together. It is not complete, so you should fill in the spaces with
observations you have seen under the brightfield (cell shape, size) or dissection
microscopes (colony morphology). I wouldn’t worry about all the spaces being filled in, as
I’m not going to ask tricky questions…ie. I will not ask if Penicillium notatum is gram
positive or negative. Each microbe was chosen for a certain characteristic that is possesses
for the lab exercises. Thus, not all microbes were seen under the brightfield or their
colonies were not observed under the dissection microscope. The information to complete
this table is not readily available in the manual but I would expect you to be able to fill in
most of the table since you have been taking observations of what you have been seeing.
Microbe
Shape of
microbe
E. coli
short rod
B. subtilis
long rod
B. cereus
long rod
L. planatarum rods
P. notatum
N/A
P. mirabilis
rod
P. fluorescens very small rod
S. cerevisiae
oval w/
budding
S. aureus
cocci
S. epidermidis cocci
Growth
pattern
chains
chains
single/pairs
N/A
N/A
single cells
clusters
clusters
Gram +/- Forms spores? Colony
morphology
no
+
yes
+
yes
+
no
N/A N/A
green,
filamentous
N/A
waves
N/A no
rd, creamy,
glossy
+
sm. yellow, rd
+
sm. white, rd