Study Guide for BIOL 2421 Lab Practicum (Subject to Change)

Skills Performance Tests – For each of the skills performance tests, you will be observed by instructors
and graded based on your ability to perform the skills properly and your results.
• Gram Stain (must be completed in less than 20 minutes)
o Perform a Gram Stain of an unknown bacterium properly.
 Students will be provided with a numbered heat-fixed smear on a slide. There are
three possibilities:
• Staphylococcus epidermidis only
• Escherichia coli only
• Mixed smear of S. epidermidis and E. coli
 Highly recommended for students to practice staining and finding a smear only
containing E. coli.
o Use a microscope to properly to view the Gram Stain.
 Microscopes will be cleaned before the lab exam.
 Students who get oil on the high power objective will have points deducted on
microscope usage.
 At the end of 20 minutes, instructors will find the smear for the students but will
deduct points for assistance.
• Students may not just give up and ask an instructor to find their smear
unless there is less than 5 minutes left on the timer for the Gram stian.
o Record your observations (shape, arrangement, Gram stain result)
 Students should observe multiple fields of view under oil immersion to ensure they
are seeing all the types of bacteria present in their smear.
 If the slide has only one type of bacterium present, students will only fill out one line
of the table.
 If the slide has both bacteria present, students will fill out both lines of the table.
 Students will not have to remember the name of the bacteria, but proper
terminology is required for shape and arrangement.
• Staphylococcus epidermidis
o Shape = coccus/cocci
o Arrangement = staphylococcus/staphylococci
o Gram stain result = Gram positive
• Escherichia coli
o Shape = bacillus/bacilli
o Arrangement = individual or scattered
o Gram stain result = Gram negative
o Instructors will observe and grade the quality of your Gram Stain after you have it focused
properly under the microscope.
 Students must write down their answer before asking an instructor to observe their
result.

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 Instructors are allowed (but not required) to tell students to “think about their
answer” one time if they don’t have the correct answer.
• Streak Plate (not timed)
o Students will be allowed 1 nutrient agar plate to perform the streak plate during the lab
practical.
o Students will print their full name and lab section on the bottom of the streak plate.
o Students may write/draw anything on the bottom of their plate to assist with performing the
streak plate technique.
o Students will use proper aseptic technique to prepare a streak plate to obtain isolated
colonies of a mixed culture of bacteria containing Serratia marcescens and Escherichia coli.
o After completing the streak plate, student will tape the lid to the base of the streak plate and
leave it where indicated on the lab bench.
o Streak plates will be graded after 18 to 24 hours of incubation.
o How streak plates are graded:
 There should be a visual decrease in the concentration of growth after the first
streak.
 Whichever streak is the last streak with growth, isolated colonies should be visible.
 There should be no visible signs of contamination on the streak plate other than the
bacteria provided in the mixed broth culture.
 The following will cause a minor deduction in the streak plate score:
• Gouging of the agar
• Drawing from an earlier streak when performing the last streak in the
middle
• Not printing your full name and lab section on the bottom of the petri plate
Identification and Analysis Questions – For each of the identification and analysis questions,
you will be asked to look at something through the microscope or in culture and identify or
analyze what you see to answer the question(s) at each station
.
• Identification of a mixed versus pure culture on a petri plate.
o Be able to determine if a culture is mixed or pure based on observing an agar plate.
• Identification of differential stains under the microscope and properties of the bacteria that give
them the colors seen under the microscope.
o Acid-fast stain
 Hot pink/fuchsia = acid-fast bacteria
 Blut to purple (depending on decolorization) = non-acid fast bacteria
 How are acid-fast bacteria different from non-acid bacteria?
o Spore stain
 Green = endospores
 Red = vegetative cells

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o Capsule stain
 Blue = background stain
 Colorless = capsule
 Red/Fuchsia = bacterial cell
 What advantages does producing a capsule provide bacteria?
• Identify an unknown bacterium based on the results the Kligler’s Iron Agar and Citrate Utilization
tests.
o Kligler’s Iron Agar (KIA) test
 Remember – RED = negative, YELLOW = positive
• Lactose utilization – look at the slant of the KIA tube
• Glucose utilization – look at the butt of the KIA tube
 Gas production
• Is their cracking, separation, or lifting of the agar in the butt
 H2 S production
• Positive = black coloration observed in the butt of the tube
• If the black obscures the ability to determine if the butt of the tube is red or
yellow, assume it is yellow
 If a bacterium is positive for H H2 S production, it is positive for gas production
whether or not observable cracks, separation, or lifting of the agar is visible.
o Based on observing of the KIA and Citrate utilization slants, students will have to determine
the identity for the unknown bacterium by comparing their observations with a table
provided.
• Evaluation of Motility Test Agar results
o Students should be able to observe a TTC motility test agar tube and determine if the
bacterium inoculated into the tube is positive or negative for motility.
• Evaluation of Kirby-Bauer test results to choose which antibiotic would be most effective against a
bacterium based on zone of inhibition diameter.
o Students should be able to observe the results of a Kirby-Bauer plate (or an image) and
determine which antibiotic would be most effective against the test culture.
• Evaluation of Water Bacteriology MPN results
o Students should be able to look at a set of MPN analysis results and determine the number
of tubes that are positive for acid and gas production at each dilution.
o Based on recorded observations of the number of tubes that are positive for acid and gas
production, students should be able to determine the Most Probable Number (MPN) result
for the water sample tested and the 95% confidence interval.