Microbiology Laboratory Schedule MW, Fall 2012
Laboratory text: Microbiology: A Case Study Approach, 2nd Ed.
Barry Chess (626) 585-7166, bxchess@pasadena.edu
Date
M 8/27
W 8/29
M 9/3
W 9/5
M 9/10
W 9/12
M 9/17
W 9/19
M 9/24
W 9/26
M 10/1
W 10/3
M 10/8
W 10/10
M 10/15
W 10/17
M 10/22
W 10/24
M
W
M
W
M
W
10/29
10/31
11/5
11/7
11/12
11/14
M
W
M
W
M
W
11/19
11/21
11/26
11/28
12/3
12/5
Assignment
Orientation and lab safety (Ex 1).
Microscopy (Ex 2*). Identification of Fungi (Ex 5)
Labor Day: Campus Closed
Identification of Algae and Protozoa (Ex 3, 4). Negative stain (Ex 9*)
Lab Exam #1. Begin Staining, Simple stain, Gram stain
Continue Gram staining (Ex 45). Eukaryotic Assignment due.
Endospore and Acid-fast staining (Ex 11*). Complete staining.
Inoculate plates for environmental sampling (Ex 6*). Demonstration of Aseptic
Techniques (Ex 7*). Pure Culture Techniques (Ex 8*).
Evaluate Ex 6. Evaluate Ex 8 and subculture. Complete hanging drop slide and inoculate
motility media (Ex 48, 62).
Evaluate motility media results. Complete Ex 8. Outside of class: Read Appendix C.
Lab Ex #2. Gram Stain Practicum.
Inoculate for temperature (Ex 14*), pH (Ex 15*) and osmotic pressure (Ex 16*) effects on
growth.
Lecture Exam #1.
Evaluate Ex 14, 15, 16. Inoculate for the effects of UV light (Ex 17*).
Evaluate Ex 17 (UV light). Inoculate for hand scrubbing (Ex 19*) and Kirby Bauer (Ex
20*).
Evaluate Ex 20 (Kirby Bauer). Begin bacterial transformation (Ex 24*).
Complete Bacterial Transformation (Ex 24). Begin Epidemiology (Ex 22*).
Complete Epidemiology (Ex 22). Begin Identification of Enteric Bacteria (Ex 31*),
Staphylococci (Ex 32*), and Streptococci (Ex 33*).
Continue Ex 31, 32, 33.
Lecture Exam #2.
Continue Ex 31, 32, 33.
Lab Ex #3. Begin Major unknowns: Inoculate streak plates.
Veterans Day: Campus Closed
Complete Ex 31, 32, 33. Continue unknowns. Inoculate slants for growth. Gram stain of
unknowns
Evaluate unknowns. Inoculate carbohydrates, biochemical and cultural tests.
Lecture Exam #3.
Evaluate tests as time and readiness allows. Continue inoculations.
Continue evaluation of unknowns.
Lecture Exam #4. Special testing, other procedures by request.
Continue staining, complete identification. Discard unknowns, locker check-out
Content of lab exams:
Lab Exam #1: General lab instructions, Ex 1, 2,
Lab Ex #2:
Ex 6, 7, 8, 9, 11, 48, Appendix. C
Lab Exam #3: Ex 14, 15, 16, 17, 19, 20, 22, 24
(40 pts)
(55 pts)
(70 pts)
Evaluation: Lab points are added to lecture points to determine your overall point total. Available lab
points are as follows:
Lab exam #1
40 points
Eukaryotic assignment
25 points
Lab exam #2
55 points
Gram stain practicum
10 points
Lab exam #3
70 points
Minor unknown report
15 points
Major unknown report
40 points
Laboratory techniques
40 points
Online “surprise” quizzes
20 points
Total
315 points
As you can see this adds up to 315 points total for lab. In order to pass the course you must receive at
least 70% of the points available in lab.
Online content: The website for your lab book has animations and quizzes that may help you as you
study for each laboratory exercise. For each lab marked with an * in the lab schedule you are responsible
for completing the online PreQuiz (although you will not need to turn in the quiz). These quizzes are
designed to ensure that you have spent adequate time studying the lab exercise prior to coming to lab.
On four occasions throughout the semester you will have a ‘surprise’ quiz worth five points. This quiz
will be very similar to the quiz that you see online. If you make sure to study the lab, complete the quiz
(and remember the answers) this is essentially a free 20 points.
To get to the book’s website, go to www:mhhe.com/chess2e. You will be redirected to the proper site.
From there, click on the Online Learning Center (student edition). A dropdown menu will lead you to the
exercise and quiz you’re looking for. A final note: No excuses for broken computers, lost web
connections etc. Take care of the quiz early and it won’t be a problem.
Other Laboratory Information: More than any other introductory biological science, the microbiology
laboratory requires that even the beginning student learn and practice a series of unique techniques and
concepts from the very first day of class. We will be constantly concerned with how you handle yourself
in the laboratory, including your grasp of these fundamental techniques, your ability to follow written and
verbal directions, and your skills in observation and analysis.
Attendance: Attendance at lab sessions is critical to your success. The skills in the micro lab are unique
and the laboratory has been designed so that these techniques are constantly reinforced through repetition.
There is no way to compensate for missed laboratory time by simply reading the manual. Attendance is
monitored during lab, as is consistently arriving late or leaving early. The instructor may drop you from
class if you miss three labs. Tardiness is disruptive to class, causing you to miss out on demonstrations
and explanations, and may limit your access to important laboratory tests.
Laboratory materials: Each student is expected to wear laboratory protection (smock, jacket, shirt or
apron) at all times. This garment must be used exclusively in the lab. A bulky sweater, coat, or
sweatshirt is not acceptable. Lab partners sharing a locker are responsible for the locker contents. Keep it
locked to prevent the inadvertent borrowing and misplacing of supplies by students from other classes.
Safety: Laboratory safety is one of our most important concerns, both in terms of your safety over the
next several weeks as well as your ability to develop habits that will protect you throughout your career.
The basic aseptic and sterile practices and precautions are very similar to those you will encounter in the
clinical setting. They are designed to help you develop the confidence, dexterity and understanding to
apply proper sterile techniques to your work here as well as in your future profession.
Your work during lab will be observed and evaluated according to 1) the appropriate use and mastery of
proper laboratory techniques and 2) its independence. Although several exercises include group
assignments, you are still expected to do your own inoculations, observations, lab reports, unknowns,
slides, and to work with your assigned microscope and not a shared one. Do not constantly rely on others
in class to explain procedures or to do your work. It places an undue burden on them and interferes with
an independent learning environment. Students will be warned about displaying careless, hazardous or
faulty behavior or techniques and directed to improve. If there is no improvement, steps will be taken to
initiate a drop.
The PCC microbiology lab is rated as a class 1-2 biosafety facility, a level consistent with open bench
inoculations. Because certain experiments utilize primary pathogens (those that can cause disease in
healthy individuals), lab access is generally restricted to enrolled students and assistants. General safety
and organizational considerations in a class 1-2 laboratory include.

Culture spills and other accidents must be reported to the instructor immediately. Broken glass
and live bacterial cells are a potentially dangerous combination and must be handled with extreme
care. Generally this involves saturating the spill with disinfectant and covering it with paper
towels. After several minutes the paper towels can be disposed of in the biohazard bag while
broken glass can be disposed in an appropriate sharps container.

Cultures and test tubes containing media must be carried upright in a test tube rack. Always bring
your test tube rack with you when retrieving cultures from the front table. Most test tube closures
are not liquid-tight so it is important that you do not excessively tilt, shake or lay your tubes
horizontally. When grasping a tube, always be sure to hold it by the tube, and not the top, plug or
lid. Petri dishes should be carried in a metal sleeve if there are more than can be safely carried by
hand (2-3).

No bare feet are allowed in the laboratory. Long hair must be tied back as it is a source of
contamination and a fire hazard.

Routinely disinfect benchtops with amphyl before and after each laboratory session and degerm
hands before, during and after lab sessions

Only needed materials should be on your work table -lab book and locker contents- do not lay
book bags, purses, cellphones (!!!) and other uneeded items on the freshly disinfected lab bench.

Staining techniques must be done only on black lab benches. Clean up spills on the bench and hot
plates using special stain remover.

Learn how both refrigeration and incubation are used to control the growth of microbes. Do not
place or retrieve items from the refrigerators, incubators or cold room without approval of the
instructor of lab technician.
Handling of cultures:
Proper handling and final disposal of cultures and biohazardous laboratory materials are an
essential part of your training.
A culture is a container of microorganisms growing in media. All cultures, as well as items that
come into contact with cultural material, should be treated as though they contain pathogens. The
final decontamination of any culture requires the following routine considerations.
Viable cultures that are no longer needed must be properly prepared for decontamination. The
contents of culture tubes, plates etc. should never be touched by hand and students should make no
attempt to disinfect or clean these items themselves.
Recyclable supplies such as tubes should have all marking removed from their outsides,
be securely plugged, and be placed on the discards cart near the prep room door in a rack
with similar sized tubes. Pipettes and hanging drop slides must be placed in appropriate
containers for recycling.
Non-recyclable supplies such as Petri dishes should be placed in the biohazard bag for
sterilization and disposal. Swabs, tongue depressors and other hard objects should be
placed in an appropriate hard sided container. It is not necessary to remove markings from
these materials.
Safe materials such as lens paper or paper towels used in the cleaning of your hands or the
lab bench may be disposed of in the regular trash. Inoculating tools will be sterilized by
flaming and need not be discarded.
Microscopes: Each student is responsible for the care of his/her assigned microscope. This includes
cleaning of the lenses using lens paper and (if the lens is especially dirty) a small amount of ethyl alcohol,
which is the only acceptable solvent for the cleaning of microscope lenses in this lab. Get into the habit
of keeping your lenses clean! They are the very first thing that should be checked if you are having
difficulty focusing on a specimen. Keep the outside surfaces of the microscope clean as well and take
care to cover the scope when you are staining. When returning the microscope to the storage cabinets, be
sure to clean the lenses and store the scope with the low power objective in place, the course focus racked
all the way down, the slide holder centered and the power cord secured.
Slides: Slides should be purchased in the bookstore. You will use about ½ box over the course of the
semester so chipping in with your lab partner has advantages. Hanging drop (depression) slides should
not be treated like ordinary slides. They can be reused for many years. Never store them in lockers or
dispose of them along with ordinary slides.
Lab organization:




Because of the number of laboratory sections routinely scheduled, some of the
burden for keeping the lab neat and orderly will fall on you. This includes.
Return shared items (cultures, amphyl, staining bottles) to their proper places. Do not place
desktop and shared supplies (oil bottles, staining kits) in your locker.
The main lab sink is not for cleaning slides; it is for hand washing and general cleaning. Please
use the foot pedal, it is intended to facilitate aseptic procedures.
Adequately mark inoculated materials with name and lab time. Do not assume you are
recognizable by initials. Improperly marked materials may be lost or inadvertently disposed of.
Tubes may be labeled using labeling tape, grease pencils or sharpies (which require alcohol to
remove), and be sure to label the tube itself and not the lid, cap or plug. Do not use masking tape
as the adhesive leaves permanent marks. Regardless of which labeling method you use, be sure to
completely remove all marks from tubes prior to placing them in the disposal racks.