BIO2

CELLS, MOLECULES, & GENES

Laboratory Manual

Fall 2024

Department of Biological Sciences CSU, Sacramento

N. Ewing, E. Gonzalez-Orta, T. Landerholm,

K. McDonald, K. Mulligan, and H. Nguyen; 2015

Updated in 2022 by
S. Kumar, R. Cummings, B. Von der Mehden, and J. Mukerji;
And in 2023 by C. Fox

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 TABLE OF CONTENTS

SECTION 1: THE BIO2 LABORATORY DOCUMENTS .............................................. 3

LEARNING OBJECTIVES FOR THE BIO 2 LABORATORY............................................................................................................................ 4
LABORATORY SAFETY RULES (MANDATORY) AND BIO2 GUIDANCE ....................................................................................................... 5
HOW TO KEEP A LABORATORY NOTEBOOK ........................................................................................................................................... 8

SECTION 2: BUILDING ESSENTIAL LAB SKILLS ...................................................... 9

EXERCISE 1 – INTRODUCTION TO PIPETTING....................................................................................................................................... 10
EXERCISE 2 – PIPETTING FOR PRECISION AND ACCURACY .................................................................................................................. 14
EXERCISE 3 –STREAK PLATING FOR BACTERIAL ISOLATION AND SERIAL DILUTIONS ........................................................................... 19
EXERCISE 4 – ANALYSIS OF COLONY MORPHOLOGY & CELLULAR MORPHOLOGY............................................................................... 22

SECTION 3: THE RIVER CITY SCIENCE PROJECT ...................................................26

EXERCISE 6 – DETERMINATION OF COLONY FORMING UNITS (CFU) OF BACTERIA AND STREAK PLATING SELECTED COLONIES FOR
ISOLATION........................................................................................................................................................................................... 29
EXERCISE 7 – STAINING, COLONY & CELLULAR MORPHOLOGY (OF BACTERIA FROM THE AMERICAN RIVER) ..................................... 31
EXERCISE 8 – ISOLATION OF GENOMIC DNA FROM SOIL ..................................................................................................................... 35
EXERCISE 9 – PCR AMPLIFICATION FROM GENOMIC DNA (GDNA) ...................................................................................................... 39
EXERCISE 10 – ELECTROPHORESIS OF GENOMIC DNA & PCR PRODUCTS ............................................................................................ 41
EXERCISE 11 – TROUBLESHOOTING (REFLECTING ON HOW TO IMPROVE FUTURE EXPERIMENTS & RESULTS) .................................. 45

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 SECTION 1:
THE BIO2 LABORATORY DOCUMENTS

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 LEARNING OBJECTIVES FOR THE BIO 2 LABORATORY

CONCEPTS

• Learn about the geography of the American River drainage, riparian ecology, historical and current land
use, and the potential hazards and solutions that exist due to human influences along the drainage.

• Design experiments that will identify the different niches that bacteria occupy in the American River and
surrounding environments near Sac State.

• Become familiar with the laboratory facilities and general procedures that you may use during the
semester.

• Learn to keep a laboratory notebook.

COMPETENCIES

• Learn safety considerations that must be observed to work with bacteria in the lab.

• Perform the experiments and collect valid data.

• Organize and interpret data.

• Evaluate the results in terms of research done by others in the lab.

• Communicate both the preliminary planning stages and the final results of your research both orally
and in writing.

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LABORATORY SAFETY RULES (MANDATORY) and BIO2 GUIDANCE
Important note: We will be working with microorganisms of unknown identity. We will be using media and techniques to
minimize exposure to potentially hazardous organisms. These microbes should not cause problems in a person with a normal,
uncompromised immune system.

Please let the instructor know if you have any concerns regarding work with unknown specimens.

SAFETY ATTITUDES
1. USE COMMON SENSE: Most laboratory accidents occur while performing a simple task.
2. BE AWARE: Know the hazards before beginning. At a minimum, read the label and the Material Safety Data Sheet
(MSDS) for the chemicals you are using.
3. BE PREPARED: Answer the following question: “What’s the worst thing that can go wrong?”
4. BE PROTECTED: Know what practices and equipment can minimize exposure to hazards in your workplace. “What
should I do to be prepared for it?”

GENERAL SAFETY RULES

1. NEVER work alone in a laboratory.
2. NEVER drink, eat, chew gum, smoke or apply cosmetics in the laboratory.
3. KNOW the location of the emergency eye wash station and safety shower before starting work in the laboratory.
4. KNOW the location of all nearby sources of ignition when working with flammable chemicals.
5. WASH PROMPTLY whenever a chemical has contacted the skin and BEFORE LEAVING the laboratory with soap and
water.
6. DO NOT “SNIFF TEST”; Avoid inhalation of chemicals.
7. DO NOT MOUTH PIPETTE anything; use the proper tools/equipment (micropipette, roller pipet filler, etc.).
8. ALWAYS use proper aseptic technique when transferring and handling microbes. This includes not leaving culture dishes
open for an extended period of time and showing open culture plates to students without asking first.

CHEMICAL SPILL PROCEDURES

1. LEVEL 1: In the event of a chemical spill of known low toxicity inform the instructor and s/he will help you clean it up.
2. LEVEL 2: In the event of a chemical spill of known moderate toxicity move your classmates away and inform the
instructor - s/he will help you clean it up.
3. LEVEL 3: If the chemical spilled is unknown or extremely hazardous, inform the instructor immediately, evacuate, close
doors to the laboratory and wait for emergency personnel.
4. LEVEL 4: In case of a medical emergency, summon medical help immediately by contacting the Department of Public
Safety; phone from any campus phone.

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PROPER USE OF CHEMICALS
1. KNOW the chemical hazards of what you are using before you use it, as determined from the SDS and other appropriate
references.
2. KNOW the appropriate safeguards for every chemical, including required or suggested personal protective equipment
before using it.
3. KNOW the location and proper use of clean-up, safety and emergency equipment.
4. KNOW how and where to properly store the chemical when it is not in use.
5. KNOW the proper method of transporting chemicals within the facility.
6. KNOW appropriate procedures for emergencies, including evacuation routes, spill cleanup procedures, and proper waste
disposal.

GUIDELINES FOR HOUSEKEEPING

1. Access to emergency equipment, showers, eyewashes, and exits shall never be blocked by anything, even temporarily.
2. Whenever possible, chemicals will be stored in their original container and label integrity maintained. If chemicals are re-
containerized, the label on the new container will include all hazards and safety information as on the original container.
3. Keep all the work areas, especially benches and counter tops clear of clutter.
4. No chemicals are to be stored in aisles or stairwells, on desks or laboratory benches, on floors or in hallways, or to be left
overnight on shelves over the workbenches. Store all chemicals in a seismically safe manner. Closed cabinets and lipped or
retaining wired shelves are two methods used for seismic bracing.
5. Do not store extraneous materials in fume hoods. These materials will interfere with the airflow and jeopardize the safe
operation of the hood.
6. Properly label all waste.
7. Wastes shall be properly labeled and kept in their proper containers.
8. Promptly clean up all spills; properly dispose of the spilled chemical and cleanup materials. Do not dispose of toxic chemicals
in the sinks; place in properly labeled waste containers.
9. All working surfaces should be cleaned regularly.
10. Dispose of cracked or broken glassware immediately. Protect your hands when inserting or attaching glassware.
11. If you spill a culture or chemical, please inform the instructor immediately. The instructor will then give you instructions on
how to clean the particular spill. Please, do not attempt to clean a spill on your own without prior instruction.
12. It is the individual student’s responsibility to label and keep track of all samples in the lab. Pertinent information to be included
in on the label are the following: Last name, First Initial and Date.
13. Waste disposal:
• Petri Dishes - Red biohazard bin at front of class.
• Pipette tips/serological pipettes – Red biohazard bin
• Slides – small plastic medical sharps container on counter.
• Paper towel and lens paper-regular wastebasket.
• Gloves:
• If contaminated - Red Biohazard Box
• If non-contaminated - regular wastebasket.
• Chemicals: The Lab Manager will place designated waste containers for specific labs as necessary.

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 14. Computer usage: Laptop computers will be used in this class for the last two to three weeks of class.
a. You may use your own computer, but only for Bio2 lab-related work. Be sure to disinfect your workspace
accordingly prior to the use of the computer.
15. Microscopes: You will be using light microscopes in this class. Please be careful with the equipment, as we do not have funds
to replace any that we lose. You will be responsible for cleaning the microscope and putting them away at the end of each
class period. Students that do not take care of microscopes properly will face a 5-point deduction from their lab grade each
time improper care occurs.
16. Once you are done with the day’s experiments, please put your items away (microscopes, etc.), wipe down your bench with
70% ethanol , and wash your hands with soap and water.

PROTECTIVE CLOTHING AND EQUIPMENT

1. Eye protection worn while working with chemicals should meet the requirements of ANSI Z87.1.
2. If your vision requires the use of corrective lenses in glasses and you are required to wear eye protection, either: (1) wear
corrective glasses that meet the requirements of ANSI or (2) wear approved goggles over regular corrective glasses.
3. Avoid wearing contact lenses when working with chemicals. In the event of eye exposure, contact lenses may absorb and/or
trap chemicals against the eyes. Wear safety glasses instead of contact lenses.
4. When working with corrosives, wear gloves made of material known to be resistant to permeation by the corrosive chemical
and tested for the absence of pinhole leaks. Gloves will be provided.
5. Wear a laboratory coat any time you are performing an experiment in the lab. You will need to purchase a lab coatt by the
first lab exercise and store it in your assigned drawer in the laboratory classroom. Lab coats can be purchased from the campus
bookstore or online. Please inform your instructor if you are unable to acquire a lab coat.
6. Legs must be covered. Do not wear shorts or short skirts.
7. Closed-toed shoes are required. Do not wear shoes with open toes or constructed of woven material. High-heels are
discouraged.
8. Carefully inspect all protective equipment before using. Do not use defective protective equipment.

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 HOW TO KEEP A LABORATORY NOTEBOOK
The Laboratory Notebook is a permanent record of your experiments.
It is the property of the laboratory – not of the student/scientist.
The purpose of the notebook is to record the plan for your experiments, your thoughts on the execution and appropriateness of the
plan, and the results. The peer-reviewed scientific process requires that you report, not just the results and interpretation of your
work, but enough information for any skilled scientist to repeat the experiment independently. This includes you! A well-kept
notebook will allow you to monitor your daily efforts, troubleshoot things that don’t work out, and improve your experimental
protocols when things go particularly well.

How to keep a Laboratory Notebook:

1. Write out your plan for each day’s experiment in advance. Read the protocol and make an experimental plan prior to going
to the lab. Each new technique is written out in detail. Things that are repeated and unchanged can be referenced to the
latest page in the Notebook on which the protocol is written.
2. On the day of the experiment, record your observations on laboratory conditions, group member tasks, variations from
intended times and concentrations, etc. Record your data.
3. After completing the experiment, record your thoughts on the success or failure of the experiment. Was the data what you
expected? If not, why do you think it was different? If the data were what you expected, was the protocol followed without
deviation? If you deviated from the plan why do you think it might have given good results?

General Notebook Guidelines:

1. The notebook must have stitched or glued binding.
2. The notebook should be written in either blue or black ballpoint pen. Do not use pencil or gel-ink.
3. Your writing should be legible. No type written pages will be allowed in the lab notebook.
4. Whiteout/correction tape should not be used to correct mistakes. Please strike through
the word(s) you want to delete (e.g., mistake). You must also initial and date these changes.
5. Each page of the lab notebook should include the date and page number. Empty pages should be crossed out in pen. No
pages should be torn or removed.

Notebook Format:
Table of Contents: Leave the first 3 or so pages of the lab notebook blank. The table of contents should include the Lab exercise
number, Date, Title of lab exercise and Page number.

Before Each Lab:

The Title, Purpose, and Materials and Methods sections should be completed prior to the lab you are performing that day. Your
notebook will be checked at the start of lab. If this is not completed, you will not be permitted to participate in lab and this will
count as an unexcused absence.

Lab Notebook Entry:

1. Title: Title of Experiment, Lab number, and Date
2. Purpose: State the reason(s) why you are doing the experiment.
3. Materials and Methods: State what materials you used for your experiments. Please also write a detailed description of your
methods in this section as well. A person should be able to follow the instructions for the lab without reading the lab
manual.
4. Observations/Results. Write down the results and observations for your experiments. If it makes sense to do so, draw
pictures or include digital photographs of your results.
5. Conclusions: Write down the interpretation of the results for your experiment. Make sure to answer the following questions:
What do these results mean? What is the significance of your finding?

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 SECTION 2:
BUILDING ESSENTIAL LAB SKILLS

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 EXERCISE 1 – INTRODUCTION TO PIPETTING

Exercise 1 learning objectives:

1. Understand the need for accurate measurement and quantitative transfer.

Skills practiced:
1. Using the Gilson Pipetman micropipette and the thumb-roller pipet filler

BRIEF INTRODUCTION
allow accurate measurement of microliter (µl) volumes.

Conversions: 1 µl = 1/1000 milliliter (ml); 1000 µ1 = 1 ml; 1000 ml = 1 liter

Use the right pipettor for the job (low - max): P20: 2-20 µl
P200: 20-200 µl
P1000: 200-1000 µl

Volume Adjustment: The volume adjustment wheel in the handle changes the reading in the
front window. Be careful not to turn the wheel beyond the max volume.

Tips: A tip is placed on the end of the shaft. There are two sizes of tips: one for the P1000
(blue) and one for both the P200 and P20 (yellow).

Using the Micropipette:

1. Gently press down on the plunger to the first stop position.
2. Submerge the tip into the liquid & GENTLY let the plunger back up.
3. Remove the tip from the liquid and put it in the next container.
4. To expel the liquid, push the plunger down to the second stop position.

SOME DO'S AND DON'TS

These are expensive instruments and should be handled with extreme care.
• Always hold the instrument upright; DO NOT hold them horizontally or upside down.
• Move the plunger slowly so that liquid in the pipette tip does not splash into the shaft of the pipettor.
• If fluid enters the shaft, please call this to the attention of your instructor or a TA so that the instrument may be cleaned
without damage. Please do not attempt to remove the contaminating fluid yourself.
• When placing a new tip on the end of the shaft, do so with care and attention – don’t force it.

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PROCEDURE
A. Using a Micropipette

1. Check the top of the micropipette plunger buttons to make sure that you have the micropipette that you need. We have
three sizes: P-20, P-200, P-1000

2. Rotate the volume adjustment knob until the digital indicator reaches the desired volume.
3. Firmly, place a disposable tip on the shaft of the micropipette.

4. Press down the plunger to the First Stop. This part of the stroke is the calibrated volume that you see on the digital
micrometer.

5. Hold the micropipette vertically and immerse the disposable tip into the sample. It is only necessary to place the tip
several millimeters into the sample. If you place any part of the micropipette into a liquid other than the disposable tip,
you can damage the micropipette.

6. Allow the plunger button to return slowly to its original position. Do not allow it to snap up.

7. Wait a couple of seconds to ensure that the full volume of the sample is drawn into the tip.

8. Withdraw the tip from the sample.

9. To dispense the sample: place the tip against the side wall of the receiving tube and push the plunger down to the first
stop, then depress the plunger to the second stop to expel any residual sample in the tip.

10. While the plunger is still pushed down, remove the tip from the tube and allow the plunger to slowly return to its
original position.

11. Discard the disposable tip by pushing the ejector button. Never remove the tip with your hands. (Be careful where you
point. You should have a designated waste beaker for tips at your desk for each experiment.)

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 Important DOs AND DON’Ts:
a. Never rotate the volume adjustment knob past the upper or lower range of the micropipette.
b. Never lay the down on its side when it contains liquid. This liquid could run into the micropipette .
c. Never let the plunger snap back after withdrawing or ejecting fluid as this could damage the piston.

12. Complete the Micropipette Practice Handout provided by your instructor before moving on the next step.

13. Follow the procedure below to familiarize yourself with the use of the three micropipettes. You and your partner should
work together make sure you’re using the correct micropipette and setting the volume correctly. Take turns pipetting by
alternating between each tube.

a. Label 6 microcentrifuge tubes: A, B, C, D, E, and F

b. P1000: Using the table below, pipette the correct volume of each color into tubes A and B.

Tube A Tube B

Yellow water 735 µl 502 µl

Red water 233 µl 466 µl

c. P200: Using the table below, pipette the correct volume of each color into tubes C and D.

Tube C Tube D

Red water 53 µl 80 µl

Blue water 121 µl 94 µl

d. P20: Using the table below, pipette the correct volume of each color into tubes E and F.

Tube E Tube F

Yellow water 4 µl 7 µl

Blue water 12 µl 9 µl

e. Visually compare Tubes A and B, Tubes C and D, and Tubes E and F. The sets of tubes should contain
the same total volume. Note any discrepancies in your notebook.

f. Set your P1000 to968 µl. draw up the liquid in Tube A. Was there any liquid remaining in the tube? Was
there air in your micropipette tip? Place the liquid back into Tube A.

g. Repeat “step f” with Tube B.

h. Set your P200 to 174 µl. Draw up the liquid in Tube C. Was there any liquid remaining in the tube? Was
there air in your micropipette tip? Place the liquid back into Tube C.
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 i. Repeat “step h” with Tube D.

j. Set your P20 to 16 µl. draw up the liquid in Tube E. Was there any liquid remaining in the tube? Was
there air in your micropipette tip? Place the liquid back into Tube E.

k. Repeat “step j” with Tube F.

B. Using a Thumb Roller Filler Pipet

1. Firmly place a disposable serological pipette into the plastic shaft of the pipet filler. Do not use excessive force.

2. If requiring a sterile pipette, peel back the paper from the cotton-plugged end, load onto the pipet filler and finish
removing paper packaging.

3. Place the tip of the serological pipet in the solution and roll your thumb on the wheel.

4. Draw until the fluid meniscus reaches the desired quantity.

5. Withdraw the tip from the sample.

6. To dispense the sample: place the tip against the side wall of the receiving tube and depress the extended button on the
top of the pipet filler.

7. Discard the disposable serological pipette in the appropriate waste receptacle.

8. Follow your instructor’s directions for practicing the transfer of known volumes.

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 EXERCISE 2 – PIPETTING FOR PRECISION AND
ACCURACY
Exercise 2 learning objectives:
1. The importance of proper pipetting technique to…
• protect your equipment,
• prevent contamination, and
• obtain reliable, reproducible results from your experiments.
2. How to modify your pipetting techniques to improve precision and accuracy.
3. Pipetting skillfully saves time, money, and frustration; this can accelerate your progress towards exciting scientific findings.

Skills practiced:
1. Transferring specific volumes of liquid efficiently and accurately.
2. Selecting the appropriate pipette for each volume-transfer.
3. Analyze the percent-error of the volume-transfers that you can achieve by practicing your pipetting skills.

BRIEF INTRODUCTION (Modified from: https://meschedl.github.io/MESPutnam_Open_Lab_Notebook/Pipetting/ )

Proficient pipetting is probably the most crucial skill in molecular biology lab-work, for three reasons:
• Accurate pipetting is crucial to the success of molecular projects. Mistakes during pipetting may cause your
experiments (e.g. PCR) to fail or to be irreproducible, and thus cause long delays and considerable expense
• Pipettes are delicate pieces of equipment with high accuracy, which can easily be knocked off their calibration.
Furthermore, they are expensive – each set of pipettes represent about $ 1,500.
• Pipettes are the main source of contamination in PCRs, and thus can cause a lot of problems.

Never (a.k.a., “Don’ts”)

• Never adjust the volume beyond the maximum setting / Never rotate the volume adjustor beyond the upper limit.
o This changes calibration (which makes the pipette ineffective) and can break the pipette completely.
• Never use without a tip.
o Liquid will get into piston, causing damage and contamination.
• Never turn a pipette horizontal nor upside down while a liquid-containing tip is attached.
o Liquid could run back into the piston, and damage/contaminate it.
• Never let plunger snap back after withdrawing or ejecting fluid
o Damages piston

Proper Use (a.k.a., “Do’s”)

• Use the correct pipette.
o Never adjust the volume beyond the maximum setting, but also
o do not use pipettes that are too large. Accuracy and precision drop rapidly towards the lower limit of a
pipette’s volume range.
• Adjust volume by turning the volume adjustment knob (or, for some models of pipette), the plunger.
• Be sure to locate the decimal point correctly when reading the volume setting (see Exercise 1 images, above).
• Always dial DOWN to the desired volume to avoid mechanical backlash affecting accuracy.
• Firmly seat the proper-sized tip on end of the pipette.
o (If the fit is loose, your pipette will draw up less volume than intended and liquid may drip from the tip.).
• The pipettes have a two-stop position plunger:
o Depressing to the first stop measures the desired volume.

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 o Depressing to the second stop introduces an additional volume of air to blow out any remaining fluid
from the tip.
• When withdrawing or expelling fluid, always hold the tube firmly between thumb and forefinger.
o Hold the tube close to eye level to observe the fluid-level changing in the tip (or in the tube, if your goal
is to pipette without disturbing a pellet, for example, as in our upcoming DNA isolation protocol.)
o Do NOT pipet with the tube in rack, nor when someone else is holding the tube.
• To withdraw fluid from a tube.
o Hold the pipette almost vertically (< 20o from vertical)
o Use your thumb to depress the plunger to first stop and hold.
o Dip tip 2-4 mm into the fluid.
• Do NOT push down to the bottom of the vial, otherwise the tip is blocked and your pipette will
draw less liquid than intended.
o Slowly and gently release your thumb.
• Do NOT release quickly: that creates aerosols (small droplets), which contaminate the pipette.
o Wait for a second or so, to confirm that all the liquid has been taken up.
• (This is especially important for highly viscous (“thick”) liquids).
o Slide pipette tip out along the inner wall of tube, to dislodge droplets adhering to the outside of the tip.
o Check that there is no air space at the very end of the tip.
o Learn the approximate levels that particular volumes fill the tip – this will allow you to check your
pipetting visually.
• To expel sample into tube
o Touch the tip to the inner wall of tube
• [or to the liquid at the bottom of the tube if there is already some in there; if you do this, be extra-
careful not to push the plunger past the first stop: that would create bubbles in your sample.]
o Slowly depress plunger to first stop, and
o Then, hover your tip slightly above the surface of your sample as you press the plunger to the second
stop, to expel any remaining fluid.
o While keeping the plunger at the second stop, slide the tip out of the tube, making sure you don’t suck up
any liquid and that there is no liquid still left in the tip.
• Eject the tip into your tip-waste container, by pressing the tip ejector button.
• Use pipette holders (storage racks), if provided.
o If pipette racks are not available, gently set your pipette on the bench-top, and do so ONLY when the
pipette does NOT have a liquid-filled tip attached to it.
• To Prevent Cross-Contamination
• Use a fresh tip each time
• Do NOT touch the tube with the pipette, only with the tip.
• Only touch the tip to internal surfaces of the tube (NOT the outside).
• If you suspect pipette-body contamination, wipe the outside of the pipette with ethanol.
• Draw up liquid slowly to prevent the formation of aerosols and/or bubbles.
• (Bubbles also distort the volume you intend to transfer.)

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PROCEDURE (PRELAB Write-up begins HERE)
Materials:
You will receive (per person):
• 6 empty microcentrifuge tubes
To share with your Lab partner:
• A 50-mL tube of colored water
• A set of micropipettes, and appropriate disposable tips for each micropipette
• A container for liquid waste
• A waste container for used pipette tips

General advice:
• Be careful to use the correct pipette for the volume that you are going to transfer.
• You don’t have to change tips for these exercises.
o [Although changing tips would be important if using multiple reagents, and if we were concerned about
preventing contamination of these samples.]
• As you work, look at the pipette tip when it is holding a volume so you can learn to check your pipetting visually.
o [You may even want to include some sketches in your lab notebook. (e.g., of how full different tip-sizes
look when they contain frequently-used volumes, such as 20 µl, 100 µl, 200 µl, etc.).]

A. SETUP (each person)

1. Label your empty tubes:

a. “1” and your initials. Leave this empty until part B.

b. “2” and your initials. Leave this empty until part B.

c. Label the remaining 4 tubes “A”, “B”, “C”, and “D”.

2. Transfer 1000 µl of colored water from your 50 mL tube into Tube “A”.

3. Repeat Step #2 for Tubes “B”, “C”, and “D”.

4. Visually check that Tubes “A”, “B”, “C”, and “D” each contain the same volume.

5. Transfer 200 µl of colored water from your 50 mL tube into Tubes “A”, “B”, “C”, and “D”.

6. Visually check that Tubes “A”, “B”, “C”, and “D” each contain the same volume.

7. Transfer 200 µl of colored water from your 50 mL tube into Tubes “A”, “B”, “C”, and “D”.

8. Visually check that Tubes “A”, “B”, “C”, and “D” each contain the same volume.

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B. TRIAL #1:
9. Add 500 µl from Tube “A” into your Tube labeled “1”.

10. Add 10 µl from Tube “B” to Tube “1”.

11. Add 100 µl from Tube “C” to Tube “1”.

12. Add 600 µl from Tube “D” to Tube “1”.

13. Add 50 µl from Tube “B” to Tube “1”.

14. Add 20 µl from Tube “C” to Tube “1”.

15. Again, add 20 µl from Tube “C” to Tube “1”.

16. Remove 500 µl from Tube “1” and expel the liquid into your liquid-waste container.

17. Remove 200 µl from Tube “1” and expel the liquid into your liquid-waste container.

18. Remove 20 µl from Tube “1” and expel the liquid into your liquid-waste container.

19. Remove 200 µl from Tube “1” and expel the liquid into your liquid-waste container.

20. Remove 350 µl from Tube “1” and expel the liquid into your liquid-waste container.

21. Remove 15 µl from Tube “1” and expel the liquid into your liquid-waste container.

22. Repeat Step #21, being extra-careful to release the plunger slowly, as you pipette up the liquid from Tube “1”.

1. IMPORTANT: if you get an air-bubble, keep holding your pipette vertically, and refer to Part C:
complete Steps “bi” and “ci” BEFORE you discard the fluid from your tip!

C. ANALYSIS of TRIAL 1: How accurately did you pipette?

a. Remember to hold the pipette vertically during all of the following steps!

b. Depending on your results, include a sketch in your lab notebook of either: …

i. … your tip (if your tip took in air with your last volume-removal), OR

ii. … your tube (if there was some fluid left in the tube after your last volume-removal).

c. Did you draw air up with the last removal of liquid?

i. If yes, measure the volume of air by decreasing the volume-setting of your pipette, until all air
below the liquid is expelled from the tip.

d. Is some liquid left in the tube?

i. If yes, estimate by eye how much volume you think remains in the tube. Then, set your pipette to
that estimated value.

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 ii. Measure the volume of fluid left in the tube by drawing it up (slowly release the plunger), and
then adjust the pipette volume, either…

1. … increase the volume-setting of your pipette, until there is no fluid left in the tube, OR

2. … decrease the volume-setting of your pipette, until all air below the liquid is expelled
from the tip.

e. Note your results in your lab notebook.

f. Discuss your results with your Lab partner.

i. Sharing your experiences may reveal suggested modifications of techniques that could help
improve your results!

ii. Your instructor will be roving around to help provide guidance and feedback, also.)

g. Try again, to see whether you can improve your own previous “personal-best” accuracy.

D. TRIAL 2:
a. REPEAT Part “B” (TRIAL 1) above, using the empty microcentrifuge tube that you labeled “2”

b. REPEAT Part “C” analysis (above) for Trial. #2

i. Record your results as “ANALYSIS of TRIAL # 2”.

ii. Write any “notes to self” that you think may help you with future lab-work and pipetting.

iii. Clean up your work-station, as directed by your instructor, wash your hands, and check for
additional advice before leaving Lab.

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 EXERCISE 3 –STREAK PLATING FOR BACTERIAL
ISOLATION AND SERIAL DILUTIONS
Exercise 3 learning objectives:
1. The importance of aseptic technique when working with live bacterial cultures.
2. The importance of following established protocols when working with biohazardous material.
3. The goal of streak plating and when to use it.
4. How to calculate the dilution factor of a solution.

Skills practiced:
1. Aseptic transfer of a bacterial culture to another media.
2. Proper disposal of biohazardous waste.
3. Isolation of bacteria from a culture.
4. Making serial dilutions.

PROCEDURE
A. Transferring bacteria from one petri dish to another using a sterilized loop
a. Obtain:
• Petri dish with bacterial colonies
• five sterile Petri dishes with agar medium but without bacterial colonies
• wire loop
• Bunsen burner

b. Label your petri dish on the agar side (bottom), around the edge of the plate using a sharpie (see Figure 1):
• Write your name
• Date
• What you are culturing

c. Read through the instructions below and refer to Figure 2 before continuing with the
protocol.

Figure 1. Labelled Petri dish

19
d. CAUTION: BEFORE lighting the flame of your Bunsen burner:
• Place your ethanol spray bottle (EtOH) a safe distance AWAY from the Bunsen burner. ETOH is highly flammable.
• Make sure all papers and materials are far away from the flame and long hair is pulled back with a hair-tie.
• NEVER leave a flame unattended! If you and your lab partner must both leave the bench / take your eyes off the
flame, CLOSE all sterile supplies, and then turn off the gas. Re-light the flame when you are ready to resume work.
e. Light your Bunsen burner by turning on gas and using the striker lighter.
f. Sterilize your inoculating loop by placing the loop in the flame of your Bunsen burner until red hot.
g. Let cool for 15-30 seconds. If you do not let it cool, you will kill the bacteria.
h. Lift the lid of the Petri dish with colonies and cool the loop by touching it to an area of agar devoid of bacteria [meaning
BLANK agar, without any cells]..
i. Use the loop to collect cells from a single colony on the plate and quickly place the lid back onto the Petri dish.
• You only need a very small amount of bacteria! (Just lightly touching your loop to the colony will allow bacteria to
stick to your loop; you do not need to scrape up the whole colony.)

j. Open the other sterile Petri dish (with only agar medium) and rub the loop back and forth on section 1 of the agar surface
(see example below).
k. Re-sterilize and cool your inoculating loop.
l. Drag the loop across a small section of your first streak and then rub it back and forth through quadrant 2 in a zig-zag
pattern.
m. Re-sterilize and cool your inoculating loop.
n. Now drag the loop through a small section of the end of your streak from quadrant 2, then zig zag your loop through
quadrant 3.
o. Repeat steps f through n with a SECOND colony on your second plate.
p. Invert plates (agar side on top) and incubate Petri dishes of bacterial culture overnight at 37°C.

Figure 2: Streak Plating Procedure

In between each step the loop is flame sterilized and allowed to cool before proceeding to the next
step.
**REMEMBER: Streak plating is about diluting your original sample to get isolated cells, which
divide and grow into genetically identical colonies (right panel).

20
 B. Contamination Plate

a. For your third plate (five plates total with your partner), you and your partner will practice improper sterile
technique. Feel free to use different methods to contaminate your plates. This plate will (hopefully) give you a
positive control for what contamination can look like.

b. Once finished, place in incubator with other streaked plates.

I think we should move this section on serial dilutions back to exercise 1. Serial dilutions are not used otherwise in this exercise, and
so don’t fit conceptual
C. Serial Dilution of a Solution
Each individual should perform a serial dilution of Trypan Blue stock solution in DI water.

a. You will be performing 5 dilutions in series, each with a dilution factor of 1:4 for a final dilution factor of 1:1024.

b. Set up seven (7) 1.5 mL plastic microcentrifuge tubes in a test tube rack on the bench top. Label the top of each tube so
that you remember which position it is in relative to the others in your dilution series.

c. Using a P-200 handheld pipette, remove 200 µL of Trypan Blue 0.4% stock solution and place it in the first tube.

d. Now, place 150 µL of DI water in each of the remaining 6 tubes in your row of 7.

e. Transfer 50 µL of Trypan Blue solution from the first tube into the second. Use your pipette tip to gently mix the
solution by slowly partially-pressing and then partially-releasing the plunger button, with the tip submerged in the
solution. This is your initial dilution. Be sure to gently mix, each time after you transfer liquid into the next tube. Use
a new/clean tip for each transfer.

f. Repeat Step 5, but now transferring 50 µL from the second tube to the third, then from third to fourth and so on until
you have transferred into the sixth tube. This tube now contains a 10-5 dilution of the Trypan Blue solution that is in
your first tube. Be sure you are mixing each tube well before you move on to the next dilution. You should also be
discarding your pipet tips and getting a clean one after each transfer of liquid.

g. Leave the seventh tube without Trypan Blue. The water will serve as a control for the solution diluent.

h. Visually inspect the color change across the tubes. Compare your dilution series to others from your lab table.

21
EXERCISE 4 – ANALYSIS OF COLONY MORPHOLOGY &
CELLULAR MORPHOLOGY

Exercise 4 learning objectives:

1. The importance of being able to identify microorganisms properly using simple techniques.
2. The importance of following established protocols when working with biohazardous material.

Skills practiced:
1. Examine and record details of colony morphology
2. Simple staining to identify cellular morphology of bacteria.
3. Properly disposal of biohazardous waste.

PROCEDURE
A. Random Selection of Colonies from Plates.
1. Obtain a numbered grid and a mixed colony agar plate
2. Place the grid beneath the agar side of your plate
3. Randomly select 3 numbers
4. Pick a colony within the 3 randomly chosen numbers on your grid to assess morphological
characteristics. Record your data in your notebook, using the chart on page 22.
5. Assess morphology for an additional colony, using a colony from the plate used in
Exercise 2
B. Analysis of Colony Morphology

1. Important Safety Considerations

• DO NOT place any personal items (laptops, phones,
backpacks, purses, bags, etc.) on the bench top
during lab

• Wipe down your bench at the beginning and at the

end of lab with ethanol

• Which trash do I use for this stuff?

Biohazard trash: If it touched cultured, live bacteria,

it goes here (e.g., used swabs, toothpicks, petri
dishes with bacteria)
Broken-glass bin: Used microscope slides (the
bacteria will be heat-fixed, which kills them)
Regular trash: If it has not touched cultured, live
bacteria (and is not glass); for example, gloves,
paper towels, etc.

2. Examine colonies on the agar plate containing the unknown

bacteria from last week.

3. Complete the chart in Section E, which includes all

observable features described in the chart at the right
(“Characteristics of Bacterial Colony Morphology”). Use the dissecting microscope, if necessary.
22
C. Analysis of Cellular Morphology: Simple Staining Protocol

1. Same safety considerations as described above.

2. You will be performing the simple stain procedure to determine the cellular morphology of the bacteria you plated last
week (and, for fun, of the bacteria that live under your fingernails!) For comparison, we will be using prepared cultures of
S. epidermidis (sphere-shaped/cocci) and E. coli (rod-shaped/bacilli)

3. Perform the simple stain procedure:

a. Obtain a glass slide. Using the black grease/wax pen, draw a line and four evenly spaced circles on the slide, as
shown in the illustration below. The order of samples from the line over will be: unknown bacteria from your plate
from last week, S. epidermidis, E. coli, and “under-fingernail” swab.

b. Transfer a bacterial colony from your plate (from last week) onto the first circle in the slide to create a smear.
1. Use a P20 micropipetter to place 5ul of d-H2O onto the first circle on the slide.
2. Use a flame sterilized loop to pick a small colony from the Petri dish
Note: you should transfer a small amount of bacteria from the colony; too much can make it difficult
to visualize the bacteria under the microscope.
3. Mix the bacteria into the droplet of water on the slide.

c. Gently swirl the tube with S. epidermidis to mix. Dip flame sterilized loop into the liquid broth of S. epidermidis
culture, then touch the loop with bacteria in the second circle on your slide. You do not need to mix with water.
Flame-sterilize your loop again before putting it back on the table.

d. Using your loop, do the same for the E. coli culture as with S. epidermidis.
Note: Do not allow the bacteria between different circles to mix, as this will produce confusing results.

e. Using a toothpick, scrape under your fingernail. Apply the collected material to the fourth circle. Discard the used
toothpick in your biohazard bin.

f. Allow the slide to completely air dry for at least 10 minutes (you will have a brief lecture during this waiting
period). It is critical that the bacterial smear is completely dry before moving on. After it dries, use a clothespin to
quickly pass the slide through the flame of a Bunsen burner three times. This procedure will “heat fix” the cells to
the slide so they do not wash off when dyes and water are applied during the staining. It also kills the cells.

g. For the following step one person should be timing while others apply the reagent.
1. Add a drop or two of crystal violet to the entire surface of each circle. Let sit for 1 minute, and then
thoroughly rinse the slide into the waste beaker with water from your squeeze bottle (make sure the water flow
is gentle).
2. Lightly pat slide dry with lens paper.

23
 D. Microscope and Image Capture Protocol

1. Turn on the power of your microscope.

2. IMPORTANT: Plan how/where you will collect & save all of your cell-phone camera images from microscopy.

• Make sure both lab partners have access to the data. (e.g., email, text-massage, or file-share via OneDrive)

3. Place your slide on the conventional, upright microscope stage.

• Begin by using the objective lens with the lowest magnification, the 4x objective. Carefully focus the microscope
on the sample, first using the coarse focus, then the fine focus. Be sure that your sample is completely in focus
before moving to the 10x objective lens.
• Briefly view your slide to orient yourself to each of the four cell preparations. At this magnification, you will only
be able to see the color of your dye, but will not be able to distinguish actual cells.
• Move to the 40x objective lens (400X magnification) and adjust the focus. At this stage, you may be able to
identify individual cells, but will not be able to identify the shape of these cells, with the possible exception of your
fingernail scraping
• You will repeat the above steps for each circle on your side.

4. Use a cell phone camera (yours, a classmate’s, or your instructor’s) to capture a 400X image of your samples.
a. Repeat to capture representative images from all of your cell preparations.
b. Send your images to yourself using email or Dropbox, or transfer your files to a USB drive.

5. Be sure to clean up your microscope appropriately.

• See microscope care checklist at your lab station and your instructor for details.
• [As indicated on page 7 of the safety guidelines: "Students that do not take care of microscopes properly will
face a 5-point deduction from their lab grade each time improper care occurs."]

Microscope Checklist

o Remove slide from the microscope stage.

o If needed, clean objectives with lens paper.
o Set to the lowest objective (4X).
o Lower the stage using the coarse focus knob.
o Turn the power off.
o Unplug your microscope and properly stow the cord.
o Cover.
o Using two hands, put microscope back in/on designated shelf or cabinet.

24
 E. Results:

Colony Morphology (use proper terminology; see pg. 21)

Species Pigmentation Physical Traits

Pigmentation: Optical Property: Texture: Shape: Elevation: Margin:

Bacteria from plate

Random colony from grid plate #1 Pigmentation: Optical Property: Texture: Shape: Elevation: Margin:

Random colony from grid plate #2 Pigmentation: Optical Property: Texture: Shape: Elevation: Margin:

Random colony from grid plate #3 Pigmentation: Optical Property: Texture: Shape: Elevation: Margin:

Cellular Morphology (use proper terminology; see below)

Species Cell Arrangement Cell Shape

Bacteria from plate

S. epidermidis

E. coli

Under-fingernail Scraping

25
SECTION 3: THE RIVER CITY SCIENCE PROJECT

Overview of the River City Science Project methods

26
EXERCISE 5 – SEDIMENT SAMPLES, SERIAL DILUTION & PLATING OF SOIL
(Acquired from the American River by your Bio2 Teaching Team)

SERIAL DILUTION AND CULTURE OF BACTERIA ON AGAR PLATES

Exercise 5 learning objectives:

1. Understand the need to perform proper and relevant analysis of collected samples.
2. The importance of knowing what to expect in a sample and adapting your protocols to accommodate them.
3. Understand the need to use proper technique when processing samples in the laboratory.

Skills practiced:
1. Analysis of soil samples in the laboratory.
2. Preparation of samples collected in the field for further microbiological and molecular analysis.
3. Dilution of bacteria-rich soil samples.

27
PROCEDURE

A. Dilution and Plating of Bacteria from 1 Gram of River Soil

NOTE: Remember to WEAR GLOVES!
1. Label one conical tube :10-1 and three
microcentrifuge tubes with lids: 10-2,
10-3, and 10-4.

2. Add 9 ml of sterile H2O to the conical

tube using a 10ml serological
pipet and thumb roller.

3. Add 900µL of sterile H2O to each

microcentrifuge tube using a P1000
pipette and corresponding tips.

4. Weigh 1 gram of your soil sample.

Transfer the soil using a scoopula
that you have first sterilized with
ethanol. Add it to the conical
tube labeled 10-1.

5. Tighten the lid and mix well by

gently turning the tube upside-
down, then right-side-up a few times
until you get a
homogenized sample (non- scientifically, sludge).

6. Pipette 100µl of this [10-1] sludge using a clean pipette tip and transfer the sludge into the tube labeled 10-2. Continue
the serial dilutions until 10-4. Shake the tube gently and use a new tip before moving each sample.

7. Acquire two culture plates containing R2a Agar:

a. For one, label: 10-3 soil, the date, and your group’s identification.
b. For the other, label: 10-4 soil, the date, and your group’s identification.

8. Using a P1000 P200 micropipette, transfer 100ul of the 10-3 solution/sludge and add to the 10-3 agar plate. (Remember
to shake the tube gently before moving the sample. )Use a cell spreader to evenly spread the soil solution on the agar.
Cover this plate with the lid. Make sure the plate is lying flat.

9. Repeat steps 6 and 7 with the 10-4 agar plate.

10. To ensure the solution has plenty of time to soak into the agar, turn your plate in to your instructor with the agar on the
bottom and lid on top. Once the plates are dry, your instructor will flip them (so that the agar is on top) and put them in
the incubator.

11. Plates will be incubated at room temperature (~23°C) for 1-3 days. Your Bio2 Teaching Team will remove the plates at
the appropriate time and store them in the refrigerator.

B. Clean up procedures
1. Dispose of all river sediment dilutions into the regular trashcan.
2. Dispose of all remaining tubes into the regular trashcan.
3. Spray and wipe down your work area.

28
 EXERCISE 6 – DETERMINATION OF COLONY
FORMING UNITS (CFU) OF BACTERIA AND STREAK
PLATING SELECTED COLONIES FOR ISOLATION

Exercise 6 learning objectives:

1. The importance of identifying and assessing the bacterial diversity in the soil of the American River.
2. The importance of pre-reviewing complex laboratory protocols before performing them.

Skills practiced:
1. Quantitative sampling of bacteria from a population to assess population diversity.
2. Bacterial streak plating on growth medium to purify selected isolates.

PROCEDURE
A. Determine the Number of Culturable Bacterial Colonies in Your River Samples

1. Using the dilution values of the sediment that you placed onto your R2a agar plates and the number of total colonies that
were formed from them, calculate the total number of colony-forming units (CFUs) in each dilution.

2. From the two sediment samples, estimate the average (between the two plates) CFUs for your river location.

3. Compare the values determined for the other locations along the river and theorize what ecological conditions might be
responsible for the variation.

B. Streak Randomly-Selected Bacterial Colonies for Isolation on Fresh Media Plates

1. For your pair, choose a total of 4 colonies randomly, using a random number generator and grid, to perform further
analyses. You may circle these colonies on your perti dish, as you will be transferring them to a new agar plate. Each
person will streak plate two unique colonies: One per plate.

2. Collect two R2a agar-filled Petri dishes each.

3. Take a picture of your dilution plate (from last week) with your randomly chosen colonies circled in permanent marker.
(You will refer back to this picture to determine whether or not you got a pure sample during next week’s lab!)

29
4. Using careful sterile technique, transfer each isolate from your soil plate to your streak isolation plate.
a. Transferring bacteria using your flame sterilized loop:
Once complete, you and your partner will have a total of four agar plates, with one unique colony streaked
on each.

Please NOTE that BOTH partners will perform these steps: each partner will inoculate 2 colonies on to their
2 separate plates. (TOTAL per team = 4 colonies on 4 plates.)

1. Use the sterile loop to choose one of your randomly selected colonies from your dilution plates.

2. Go through the streaking procedure as in Exercise 3 Part B .

• REMEMBER to sterilize the loop, between each time you streak.

o This is important, because we are diluting the bacteria with each subsequent streak.
By your third streak for that bacterial isolate, you will be spreading so few cells that
single cells will “land” far enough apart to produce separate, distinct colonies after
incubating the plate (shown in figure below).
• Also, always touch sterile agar with your sterile loop, BEFORE touching your colony. (This
cools the loop enough that the hot metal will not kill the cells.)
Complete the procedure as in the image on the right, for both of your isolates.

b. Invert and place your plate on the tray at the front of class. Your instructor will incubate them at room
temperature for 2-3 days, then transfer to 4°C until your next lab session.

Before incubation After 1-3 days of

(streaking technique) incubation

30
 EXERCISE 7 – STAINING, COLONY & CELLULAR
MORPHOLOGY (OF BACTERIA FROM THE
AMERICAN RIVER)

Exercise 7 learning objectives:

1. Understand the principles of colony morphology.
2. Understand the principles of cell morphology and simple staining of bacteria.
3. Understand the principles of high and low magnification microscopes, digital cameras and image software.
4. Learn the process of troubleshooting your own data and procedures.

Skills practiced:
1. Crystal Violet simple stain procedure.
2. Microscopic analysis of collected samples.
3. Troubleshooting your data and procedures and applying it to genomic DNA isolation.

PROCEDURE
A. Examine Colony Morphology

1. Important Safety Considerations

• Do not place any personal items (laptops,
phones, backpacks, purses, bags, etc.) on
the bench top during lab

• Wipe down your bench at the beginning

and at the end of lab with ethanol

Which trash do I use for this stuff?

Biohazard trash: If it touched cultured, live bacteria, it

goes here (e.g., used swabs, toothpicks, petri
dishes with bacteria)
Broken-glass bin: Used microscope slides (the
bacteria will be heat-fixed, which kills them)
Regular trash: If it has not touched cultured, live
bacteria (and is not glass); for example, gloves,
paper towels, etc.

2. By eye, examine a representative, isolated colony on each of the four plates you and your partner streaked last
week.

This figure shows the predicted growth

pattern after streak plating and incubation
at room temperature for 1-3 days.

31
 3. Complete the “Colony Morphology” chart in your lab notebook for each of the 4 colonies.
• [See section “E” for the chart.]
• Use the dissecting microscope if necessary.

B. Simple Staining Protocol

1. Collect a clean microscope slide. Use the wax pencil to draw a line at one end of the slide, followed by four
circles (similar to the diagram on page 23).

2. Place each of your 4 isolates onto a separate spot on the microscope slide (one in each circle).
a. Use a P20 micropipette to place 5 µl of d-H2O onto the slide.
b. Use a sterile flame sterilized loop to pick a small colony from the Petri dish
Note: you should transfer a small amount of bacteria from the colony; too much of the colony can make it
difficult to visualize the bacteria under the microscope.
c. Mix the bacteria into the droplet of water on the slide.
d. Take note of which isolate is in which circle.
e. Flame sterilizing your loop before moving on to the next colony/setting the loop on your desk.

3. For the following steps, one person should be timing while the other applies the reagents.
a. Follow the simple stain protocol in Exercise 4 to stain each of your slide preparations.

C. Microscope and Image Capture Protocol

1. Turn on the power of your microscope.

2. Before you begin, your instructor will share digital images of various cellular morphologies at 1000X.

• NOTE: in the steps that follow, you will only go up to 400X, so you will be observing COLONY
morphology of the bacteria (~ hundreds of cells), rather than single-cellular morphology of the bacteria.

3. IMPORTANT: Plan how/where you will collect and save all of your cell-phone camera images from
microscopy. Make sure both lab partners have access to the data. (e.g., email, text-message, or file-share
via OneDrive)

4. Place your slide on the conventional, upright microscope stage.

• Rotate the turret so your 4x objective lens is selected. (total magnification = 40X)
• View your slide, bring your samples into the field of view, and orient yourself to each of the four cell
preparations. At this magnification, you will only be able to see the color of your dye, but will not be able
to distinguish actual cells.
• Repeat this process with the 10x objective.
• Move to the 40X objective lens (400X magnification) and focus the lens well. At this stage, you may be
able to identify individual cells, but will not be able to identify the shape of these cells.
• You will repeat the above steps for each circle on your side.

5. Use a cell phone camera (yours, a classmate’s, or your instructor’s) to capture a 400X image of your samples.
• Repeat to capture representative images from all of your cell preparations.
• Send your images to yourself using email or Dropbox, or transfer your files to a USB drive.

6. Be sure to clean up your microscope appropriately.

• See microscope care checklist at your lab station and your instructor for details.
32
• [As indicated in the safety guidelines: "Students that do not take care of microscopes properly will face
a 5-point deduction from their lab grade each time improper care occurs."]

33
 D. DATA

Be sure to include the following charts in your lab notebook. You will need data about colony and cellular
morphology for your final report.

1. Colony Morphology
Please refer to this chart when completing your data table.
Optical Texture Margin Shape Elevation
Isolate Number Pigmentation
Property

1
2
3
4

2. Cellular Morphology – Refer to 1000X digital images that your instructor will project on screen.

REMEMBER …

Microscope Checklist
o Remove slide from the microscope stage.
o If needed, clean objectives with lens paper.
o Set to the lowest objective (4X).
o Lower the stage using the coarse focus knob.
o Turn the power off.
o Unplug your microscope and properly stow the cord.
o Cover.
o Using two hands, put microscope back in/on designated shelf or cabinet

34
 EXERCISE 8 – ISOLATION OF GENOMIC DNA FROM
SOIL
[from Cordova Creek and American River soil]

Exercise 8 learning objectives:

1. The importance of identifying and assessing the bacterial diversity in the soil of the American River.
2. The importance of pre-reviewing complex laboratory protocols before performing them.
3. Learn the proper procedure to isolate genomic DNA from collected samples.

Skills practiced:
1. Isolation of genomic DNA from soil samples
2. Preparation of collected field samples for further molecular analysis.

PROCEDURE
A. Genomic DNA Isolation from River Soil
NOTE: WEAR GLOVES AT ALL TIMES! YOU DO NOT WANT TO CONTAMINATE THE SOIL COMMUNITY
WITH YOUR OWN FLORA!

Refer to overview figure on next page as you read the detailed protocol that follows.

35
Sub-headers of the protocol are listed on the left-hand side in the figure. (NOT all steps are shown.)

I. Prepare Sample & Lyse Cells:

1. Add 250mg of soil sample to the “PowerBead” Tube (the tube that contains small beads). Gently centrifuge the tube
using the low power centrifuge at the back of the room. Be sure to label your tube and balance the centrifuge.
The “PowerBead” tube contains a buffer to help disperse the soil particles, dissolve humic acids (from
decomposing material in soil), and protects nucleic acids (i.e. our DNA!)
What is another reason we use buffers?

2. Add 800μl of Solution CD1.

C1 contains SDS (an anionic detergent) & agents for cell lysis. SDS will break down fatty acids and lipids in the
cell membranes.
3. Secure in a vortex fitted with a 2 ml tube holder assembly and vortex at maximum speed for 15 minutes.
This step causes all of the bacterial cells to lyse.

36
 4. After vortexing, centrifuge the PowerBead tubes at 15,000 x g for 1 minute.
a. CAUTION:
1. Do not exceed 15,000 x g or the tubes may break!
2. (Be sure to balance the centrifuge: you will need to wait for at least one other group to spin at the same time.)
5. Transfer the supernatant to a clean 2mL microcentrifuge tube
The supernatant = the liquid containing your lysed bacterial cells. Expect 500-600ul.
{The supernatant may still contain some soil particles.]

II. Remove Cellular Debris:

6. Add 200 µL of solution CD2 to the tube from step 5 and vortex for 5 seconds.

7. Incubate the solution on ice (4°C) for 5 minutes.

During this incubation, CD2 will precipitate non-DNA organic & inorganic matter (i.e. humic acids, proteins, cell
debris). These compounds will interfere with future experiments with our DNA and will cause our final DNA to be
“impure.” (Precipitate may not be seen, in some cases.)

8. Centrifuge the tube at 15,000 x g for 1 minute.

The dense, non-DNA matter will collect at the bottom of the tube.

9. Carefully transfer ~500-600 µl of supernatant into a clean 2 mL microcentrifuge tube. …

a. … DO NOT touch or disturb the pellet!
* It is better to transfer a bit less volume, instead of risking pipetting up the pellet. DO NOT transfer more than 700 µl!
The pellet contains the non-DNA matter that must be removed. The supernatant contains the DNA.
If the pellet is disturbed, it will cause the final DNA product to be impure.

III. Bind DNA to Spin-Column Filter:

10. Add 600 µl of solution CD3 to the supernatant from Step 9. Vortex for 5 seconds.
a. … NOTE: Be careful not to overfill the tube!
CD3 contains high concentrations of salts. DNA binds tightly to silica when at high salt concentrations. The filter
in the following step is composed of silica.
Note:
• Now we will filter out and discard liquid (a.k.a., “flow-through”) via 3 repeated spins: (Steps 11, 13, & 15).
• We must do this in 3 repeated spins because the tube cannot contain the whole volume at once.
o (We have more liquid than will fit in the tube (Step 10 = 600 uL of supernatant + 600 uL of CD3).
• In Steps 11-16, discard liquid that flows through the column. [BUT in a later step, we will SAVE flow-through.]

11. Load 650 µl of the solution from Step 10 into a spin filter (i.e., an “MB spin column”) and
centrifuge at 15,000 x g for 1 minute.

12. Discard the flow-through, but keep the collection tube. (i.e. pour out the liquid, but keep the tube).

13. Load an additional 650 µL of solution into the same spin filter and
centrifuge at 15,000 x g for 1 minute.

14. Discard the flow-through.

15. Load any remaining solution you have from Step 10 into the same spin filter and
centrifuge at 15,000 x g for 1 minute.

16. Discard the flow-through. KEEP the filter!! Avoid splashing any flow-through onto spin-column.
The DNA is now bound to the silica on the spin-column filter. Contaminants passed through the filter to be discarded.

17. Centrifuge your spin-filter one more time at 15,000 x g for 1 minute. NOTE: we did NOT add any liquid for this spin.
This ensures that all of the lysate has passed through the filter.

37
IV. Wash DNA-bound Filter:

18. Place the spin-column into a CLEAN 2mL collection tube.

19. Add 500 μl of solution EA into the spin-column.

Solution EA (a wash buffer) removes protein & other non-aqueous contaminants from the spin filter membrane.

20. Centrifuge at 15,000 x g for 1 minute.

21. Discard flow-through and place the spin column back into the same 2ml collection tube.

22. Add 500 µL of solution C5 to the spin filter and centrifuge at 15,000 x g for 1 minute.
C5 is an ethanol-based solution used to further purify the DNA that is bound to the silica filter.

23. Discard the flowthrough.

24. Centrifuge the spin filter again at 15,000 x g for 2 minutes.

This step ensures the C5 ethanol solution is completely removed from the filter. Be sure not to splash any of the
flowthrough onto the filter – ethanol can interfere with PCR reactions.

25. Place the spin filter into a clean 1.5 mL microcentrifruge tube.
• Take care: Avoid splashing any solution from the previous centrifugation onto the spin filter!

V. Elute DNA from Filter:

26. Add 100 µl of solution C6 to the center of the white filter membrane. Make sure the solution is applied directly to the
entire filter surface and not to the sides of the tube
C6 is a sterile elution buffer. This will release the DNA on the filter and the flowthrough will contain our DNA.
[Enrichment info: Most buffers contain a chelating agent (a molecule that “sops up” ions that DNA-digesting enzymes require
as co-factors.) Chelating agents help prevent a DNA sample from getting digested. NOTE: Buffer C6 does NOT contain a
chelating agent. Instead, we will protect your eluted DNA from being degraded by storing it in a freezer, until you use it PCR
and gel electrophoresis, on future days of Lab.].

27. Centrifuge the tube at 15,000 x g for 1 minute.

28. Discard the spin filter. KEEP the tube that contains your eluted DNA!
**Remember: the DNA has moved through the filter into your tube, so you do not want to accidentally throw
this away after all of your hard work! **

The DNA in your tube is now pure and ready for PCR and downstream applications!

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 EXERCISE 9 – PCR AMPLIFICATION FROM GENOMIC
DNA (gDNA)

Exercise 9 learning objectives:

1. Understand how PCR targets specific areas of the genome to produce billions of copies of the desired product.
2. Understand the purpose of each reagent in PCR.
3. Understand the importance of utilizing proper positive and negative controls.

Skills practiced:
1. Set up a PCR reaction and understand the different components of a reaction.

PROCEDURE
A. Amplification of the 16S Small Ribosomal Subunit Gene
PRECAUTIONS: Please wear gloves during the entire procedure to protect PCR from foreign DNA and nucleases
that could be on your hands. Also, ALWAYS USE FILTER TIPS when doing PCR.

1. Make sure that you have the following reagents in your ice bucket before starting: PCR Master Mix without primers
(which already contains buffer, 10mM dNTPs, 2.5mM MgCl2, 10% DMSO and Taq DNA polymerase), 10 µM PCR
primers (27F and 1492R), and your DNA template (your prep from last lab). You should also have nuclease free water
in the rack on your lab bench. A positive control (E.coli DNA) will be at the front desk and shared among groups.

2. The Master Mix (MM) in your ice bucket will be pre-measured for you, so that you have enough for 4 reactions. Do
not leave the MM at room temperature. While we are only performing 3 reactions, we have supplied enough for 1 extra
PCR reaction in case any mistakes are made. You will be performing the following 3 PCR reactions: DNA template
extracted in previous lab, positive control (E.coli DNA), and negative control (nuclease-free water).

ATTENTION: THE FOLLOWING STEPS ARE ALL DONE ON ICE OR IN A FROZEN RACK.

3. Make the Master Mix according to calculations in a clean, sterile 1.5 ml microfuge tube. Add reagents in the order
listed on table. DO NOT ADD DNA TO MASTER MIX. Mix well by tapping tube or pipetting up and down.

TABLE 1: REACTION SETUP

Reagent Master mix (4 reactions* pre- Final Concentration NOTE
measured in 1.5ml tube “MM” - * Per reaction (=50 µl
primers must be added) total in each small
PCR tube)
Master Mix w/o primers 152 µl (pre-measured, in ice bucket) NA 38 µl
27F Primer (10 µM) 20 µl must be added to MM 1 µM 5 µl
1492R Primer (10 µM) 20 µl must be added to MM 1 µM 5 µl
DNA template DO NOT ADD TO MASTER ~5-10 ng 2 µl
MIX! (Instead, add unique template
DNA to each reaction. (2 µl)

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4. Label 3 sterile PCR tubes (small tubes) using a thin-Sharpie, making sure to also indicate your group #. If labeling with
numbers, make sure to have a key handy to decode. Record the key in your notes, and include in your lab notebook.

5. Aliquot 48µl of Master Mix into each of the sterile, thin-walled PCR tubes labeled in the previous step. Make sure to
aliquot MM before DNA, positive, or negative controls are added.

6. Add 2 µl of genomic DNA template into the appropriate PCR tube with MM already added. Close tube immediately to
prevent contamination.

IMPORTANT: KEEP your tube of genomic DNA! (We will use it during the Gel Electrophoresis lab).

7. To your positive control PCR tube, add 2 µl from the tube at the instructor’s desk labeled “+DNA.” This tube contains
E. coli DNA.

8. In your negative control PCR reaction tube, simply add 2 µl of nuclease-free water from the PCR tube labelled “H2O”
at your bench

9. Keep prepared PCR tubes in ice bucket. Your instructor will collect your PCR reaction tubes, place them in the
thermocycler, and run the program below.

Thermocycler Program: “Bio2 16S PCR”

1. Initial denaturation: 95°C for 2 minutes
2. Three-step cycling procedure repeated 30 times:
• Denaturation: 95°C for 1 minute
• Annealing: 53°C for 30 seconds
• Extension: 72°C for 90 seconds
3. Final Extension: 72°C for 5 minutes
4. Final Hold: 4°C for infinite time.

10. Clean up protocol

a. Dispose of MM and primer tubes in trash.
b. Empty ice buckets in sink once PCR tubes have been collected by your instructor
c. Dispose of all plates in the appropriate biohazard waste container.
d. Spray and wipe down work bench.

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 EXERCISE 10 – ELECTROPHORESIS OF GENOMIC DNA
& PCR PRODUCTS

Exercise 10 learning objectives:

1. Understand the principles of gel electrophoresis.
2. Understand how to use gel electrophoresis to analyze PCR products.
3. Understand the principles of troubleshooting your own data and procedures.

Skills practiced:
2. Prepare and pour an agarose gel.
3. Prepare samples for gel electrophoresis using an agarose gel.
4. Gel image capture.
5. Analysis of gel image.

PROCEDURE
A. Agarose Gel Electrophoresis

PRECAUTION: WEAR GLOVES AT ALL TIMES. BE CAREFUL WITH HOT AGAROSE!

1. Prepare and run gel

b. Obtain a gel tray, and using masking tape, seal the open sides of the tray. Make sure that the tape is on the tray
securely. Ask professor for assistance if unsure of the proper way to tape a gel tray.

c. Measure 45ml of tempered 1.0% Agarose with GelRed. GelRed is a dye that is used to stain and visualize DNA.

d. Pour the melted gel agarose into the sealed gel tray. Place comb vertically towards one end of the gel. This will
form wells in order to load samples.

e. Wait until agarose gel is solidified, about 15-20 minutes. You will notice a change in opacity in the agarose.
f. While you are waiting for your gel to solidify, prepare your samples to load into the gel. You have a total of 4
samples: your soil genomic DNA (used as your PCR template), gDNA PCR product, PCR positive control, and
PCR negative control;.

To prepare samples for loading on your gel:).

1. Label the tops of 4 microcentrifuge tubes, #1-4, using Sharpie permanent marker.
2. Place 2 µl of loading dye in each tube.
(Tubes are clean and empty, so you can use the same tip to add dye to all tubes).
3. Then, using a different tip for each sample, mix 5 µl of your samples with the dye in each 1.5 mL tube.

• If droplets collect on the sides of your tubes, you may need to centrifuge your tubes so all of the liquid
collects in the bottom.

g. By now your gel should be solidified. Gently remove the comb and tape.

h. Place your gel gently into gel rig. IMPORTANT: Make sure the wells are closest to the BLACK electrode:
DNA’s backbone contains negatively-charged phosphate groups, so DNA migrates towards the positive RED
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 electrode. If you place your gel into the rig backwards your samples will migrate off the gel, leaving you with no
results to interpret.

i. Fill the gel rig to the “fill” line with 1X TAE buffer. Your gel must be completely submerged and the wells filled
with buffer.

j. Draw a sketch (diagram) of your gel in your lab notebook, indicating which sample you will load into each well.
Lanes 1-4 will contain 7 µl of one of your samples, lane 5 will contain 5 µl of the pre-mixed DNA ladder 1kb”,
and lanes 6-8 will be empty).

k. Load 5 µl of the DNA ladder in the FIFTH lane.

• Remember: set your pipette to 5 µl, to load Marker into Lane #5!

• (DNA ladder is provided for you in the PCR tube labeled “1kb”.)

l. Load 7 µl of each of your samples, in sequential order, into the gel.

• Use a new tip for each sample.

• Remember: reset your pipette from 5 µl to 7 µl, to load 7µl of each of your samples!

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Diagram of agarose gel and sample order
1 kb DNA Ladder
(NEB 1 kb DNA Ladder)

43
 m. Place cover on gel rig and plug electrodes into power supply.

n. Set power supply to 100 V, and a timer for 60 min.

• After you start the power, bubbles should begin to form in the buffer near the electrodes. There will be
twice as many bubbles near the BLACK electrode.
• Run power through the gel until you see the loading dye migrating to the bottom of the gel (about 45-
60 minutes).

o. Turn off power supply and carefully remove gel. (Carry the gel carefully & hold it level … gels break if dropped!)

p. Photograph gel using Gel Documentation System (your instructor will show you how to do this).

q. Tape the photograph of the gel in your notes to include in your lab notebook and analyze the results. Answer any
supplemental questions provided by your instructor.

1. Clean up protocol
a. Return your gDNA and PCR samples to your instructor.
b. Dispose gels in the trash after photographing.
c. Rinse used TAE buffer down the sink.
d. Rinse your gel rig and tray with DI water. DO NOT wipe or touch the inside of the rig or you risk breaking
expensive wires. Set upside down on a paper towel at your bench to dry.
e. Dispose of all microcentrifuge tubes and micropipette tips used during the lab.
f. Clean all glassware and wipe down your work space.

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 EXERCISE 11 – TROUBLESHOOTING (REFLECTING ON
HOW TO IMPROVE FUTURE EXPERIMENTS &
RESULTS)

Exercise 11 learning objectives:

1. Learn the process of troubleshooting your own data and procedures.

Skills practiced:

2. Troubleshooting your data and procedures, and practice applying it to:

• Serial dilutions,
• Sterile technique,
• Determining CFU in a culture of unknown concentration
• (revising your serial dilution strategy to improve accuracy of your CFU counts),
• Plating and culturing bacteria
• Genomic DNA isolation,
• PCR amplification of a sequence of interest, and
• Gel electrophoresis for analysis of DNA.

PROCEDURE

• During Lab, you will complete several worksheets that guide your through the process of troubleshooting the protocols your
performed in Bio2 Lab.
• You may wish to read the worksheets ahead of time, and begin thinking about your responses (and questions you would like
to ask your classmates and your instructor.)
• There is no pre-lab writeup due for this week.

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