Contents Page
Magic Bullet Factories: Isolating Antibiotic Producing Bacteria
Abstract, Introduction, Special Materials, Time required 3
Preparation, Making the agar medium 4
Part I: Sampling the Soil or Compost 5
Part II: Selecting a Bacterium with a Zone of Clearing 5
Part III: Adding the Cross-Streaks 6
Part IV: Finding Antibiotic Producing Bacteria 6
Healing from the Kitchen
Abstract, Introduction, Special Materials, Time required 7
Preparation, Making the Agar Medium 8
Part I: Making the Extracts 9
Part II: A Drop of Healing 9
Part III: Checking for Growth Inhibitors 10
Do Soaps Really Do It?
Abstract, Introduction, Special Materials, Time required 11
Prep: Making up the Plate Count Agar Medium 12
Part I: Asking a Question 12
Part II: Checking Results and Redesigning the Experiment 13
Genetic Exchange in Bacteria
Abstract, Introduction, Special Materials, Time required 14
Preparation: Making solutions and growing cultures 15
Part I: Plasmid Preparation 16
Part II: Making competent E.coli cells 17
Part III: the Transformation (genetic exchange) 17
Appendix, Culture Media, Supply Houses 19
"Magic Bullet" Factories: Isolating Antibiotic Producing Bacteria
Authors
Susan M. Merkel, Scott Kachlany, Kimberly Baldwin, Malik Williams
Section of Microbiology, Cornell University
Abstract:
This activity demonstrates where anti-microbial compounds come from
by allowing the students to isolate antibiotic producing bacteria from
soil or compost. Students spread soil or compost onto agar plates,
and incubate 1-2 days. Students then test colonies that show
a clearing zone against known Gram-positive and Gram-negative bacteria.
This lab activity was designed for and tested in middle or high school
biology classrooms.
Introduction
Since the early 20th century, antibiotic compounds have been used across
the world to fight against all kinds of bacterial diseases. These
low- molecular weight compounds are produced by microorganisms, such as
fungi or bacteria, to kill off other bacteria that are competing for similar
resources. In general, antibiotics work by inhibiting specific bacterial
enzymes. In this experiment, students will obtain soil or compost
from various sites to find naturally occurring antibiotic producing bacteria.
Special Materials:
Soil or compost samples
Petri plates with Plate Count or Starch -Casein Agar
Some of the following bacteria (available from Ward's Biology:
1-(800) 962-2660 or Carolina Biological Supply:
1-(800) 334-5551)
Gram-negative Gram-positive
Escherichia coli Staphylococcus epidermidis
Pseudomonas sp.
Bacillus subtilis
Time Required:
Prep-Day Obtain soil or compost from various sites. Prepare agar petri
plates.
(Time: 1- 2 hours).
Day 1 Mix soil or compost with tap water in a clean bottle and shake.
Dip a sterile cotton swab into soil suspension.
Spread on agar and incubate overnight (Time: 10 minutes).
Day 2 Look at plate for zones of clearing and select those colonies.
Pick a colony and streak a single line down the
center of the plate and incubate overnight
(Time: 10- 15 minutes).
Day 3 With a toothpick, pick known test bacteria (Staphylococcus, E.
coli, Pseudomonas, or Bacillus). Streak bacteria
from the inner streak to the outer edges and incubate overnight (Time:
10- 15 minutes).
Day 4 Observe plates for any zone of inhibition near the center streak
(Time: 10-15 minutes).
______________________________________________________________________________
Preparation: Making the Agar Medium
Instructor or students need to make Plate Count Agar or Starch-Casien
plates before isolating bacteria. Alternatively, these items can
be purchased from Ward's Biology: 1-(800) 962-2660 or Carolina Biological
Supply: 1-(800) 334-5551)
NOTE: This lab is written using Plate Count Agar. Starch-Casein
Agar produces more antibiotic producing isolates,
but they take 3-5 days to grow. The recipe for Starch-Casein
Agar is given in Appendix A.
Materials
The following recipe makes about 20 plates:
Equipment: 1 liter Erlenmeyer flask
balance, weighing boats and spoons
aluminum foil
hot plate or autoclave
protective gloves
20 sterile petri plates
Supplies: 11.75 g of Plate Count Agar (or Starch-Casein agar)
tap water
Procedure
1) Weigh out 11.75 g of Plate Count Agar (PCA) and place in Erlenmeyer
flask.
2) Add 500 ml of distilled water to the flask and mix thoroughly. Cover with aluminum foil.
3) Boil contents on hot plate for 30 minutes, or autoclave at 121oC
for 20 minutes. Let cool for 5 minutes or until
easy to handle.
4) With protective gloves, pour PCA mixture into 20 petri plates and
let cool until solid (about 10 minutes)
NOTE: Use caution when pouring plates. To prevent contamination,
only open the petri plate as long as
absolutely necessary.
5) Label plate "PCA". Allow the plates to dry out by placing them
at room temperature for 48 hours.
Part I: Sampling the Soil or Compost
Materials
Each group should be provided with the following:
Equipment: 2 clean bottles with lids
Supplies: 2 Plate Count Agar petri plate per soil sample
2 sterile cotton swabs
100 ml water
Procedure for Day 1
Experiment
1) Mix 1 tablespoon of soil or compost and 50 ml of tap water in a
clean bottle and shake vigorously for 1 minute.
NOTE: Be sure to dispose of all contaminated objects to
a proper container. To prevent contamination, only open
the petri plate as long as absolutely necessary.
2) Take a sterile cotton swab and dip into soil suspension.
3) With swab, spread out suspension onto PCA media. To spread, pick a spot near the edge of the petri dish and with continuous, overlapping gentle strokes, cover the entire plate.
Control
4) Add 50 ml water to a clean bottle (the same water used to make the
soils solution).
5) Take a sterile cotton swab and dip into water.
6) With swab, spread out the water onto PCA media (as described above).
7) Be sure to label each plate with your name (initials), date, and material that is being spread onto the dish.
8) Incubate plates overnight at room temperature.
______________________________________________________________________________
Part II: Selecting a Bacterium with a Zone of Clearing
Materials
Each group should be provided with the following:
Supplies: plates from the previous day
8 sterile cotton swabs
toothpicks
8 PCA (Plate Count Agar) plates
Procedure for Day 2
1) Inspect plates for bacterial colonies surrounded by zones of clearing
(see diagram). This implies inhibition of growth
of other neighboring bacteria.
2) Use a toothpick to pick a colony at the center of the zone of inhibition,
then gently streak the toothpick in a single line
down the center of a new PCA plate (Figure 3).
3) Incubate the plates overnight.
_______________________________________________________________________________
Part III: Adding the Cross Streaks
Materials
Each group should be provided with the following:
Supplies: toothpicks
different strains of known test bacteria growing on PCA agar
(we used Esherichia coli, Staph. epidermidis, Ps. aeruginosa
and Bacillus subtilis)
Procedure for Day 3
1) With a sterile toothpick, pick a colony from one of the known test
bacteria plates. Begin streaking the petri plate from the inner edges
of the streak toward the outer edge of the plate (see figure), barely touching
the initial streak made on Day 2.
NOTE: Be careful when handling these and any bacteria.
Although these strains are not pathogenic, care should be taken to not
spread them around.
2) On the same dish, repeat step 2 with the other known test bacteria. Streak each bacterium about 1 inch from other streaks. Label each known test bacterium.
3) Incubate the plates overnight at room temperature.
_______________________________________________________________________________
Part IV: Finding Antibiotic Producing Bacteria
Materials
Each group should be provided with the following:
Supplies: plates from Day 3
Procedure for Day 4
1) Observe plates for any inhibition of the known test bacteria.
A positive result is indicated by a lack of growth near the centerline.
(Why?)
Healing From the Kitchen
Authors:
Susan M. Merkel, Scott Kachlany, Kimberly Baldwin, Malik Williams
Section of Microbiology, Cornell University
Abstract:
This activity demonstrates how some researchers find new anti-microbial
compounds by allowing the students to screen different herbs, spices, and
plants for the ability to inhibit bacterial growth. Student
spread different bacteria onto agar plates, and test extracts from these
various materials for growth inhibition using a Disk Diffusion Assay.
This lab activity was designed for and tested in middle or high school
biology classrooms.
Introduction:
For more than 5,000 years, herbal remedies have been used in many different
cultures to cure and inhibit disease. People have claimed to successfully
treat many different diseases, ranging from minor infections to cancer,
with different herbal concoctions. With the rise in antibiotic resistant
bacteria, doctors need more antibiotic and growth inhibiting compounds.
Many researchers are now looking to natural products. This activity
shows students how natural products are screened for anti-bacterial activity.
Special Materials:
Equipment: weighing balance
Supplies: rubbing alcohol (or pure ethanol)
small tubes with tight lids (eppendorf tubes work well)
tetracycline antibiotic disks (available from
Fisher Scientific Company 1-(800) 766-7000)
1/4" sterile disks
Petri plates with Plate Count Agar
Gram(-) and Gram (+) bacteria types
(We used Escherichia coli and Staphylococcus epidermidis, available
from Ward's Biology: 1-(800) 962-2660
or
Carolina Biological Supply: 1-(800) 334-5551)
Some of the following plant materials (or anything else students
want to test): teas; spices; herbs; plant leaves,
stems or roots; mushrooms; etc. (NOTE: we got positive results
with garlic, cloves and cumin)
Time Required
Prep Day Prepare the petri plates with Plate Count Agar.
Day 1 Weigh out material and let it sit in a known volume of alcohol
overnight.
Day 2 Add one drop of each extract onto a sterile disk and allow to
dry. With sterile cotton swab, pick and spread
a known test bacterium onto Plate Count Agar. Place dry disks
on bacterial lawn and incubate overnight.
Day 3 Observe plates for any zones of clearing around the disk.
Clearing (growth inhibition) around the disk is
a positive result.
Prep: Making the Agar Medium
Instructor or students need to make Plate Count Agar plates before
isolating bacteria. Alternatively, these items
can be purchased from Carolina Biological Supply or Wards Biological
Supply.
Materials
The following recipe makes about 20 plates:
Equipment: 1 liter Erlenmeyer flask
balance, weighing boats and spoons
aluminum foil
hot plate or autoclave
protective gloves
20 sterile petri plates
Supplies: 11.75 g of Plate Count Agar
tap water
Procedure
1) Weigh out 11.75 g of Plate Count Agar (PCA) and place in Erlenmeyer
flask.
2) Add 500 ml of distilled water to the flask and mix thoroughly. Cover with aluminum foil.
3) Boil contents on hot plate for 30 minutes, or autoclave at 121oC for 20 minutes. Let cool for 5 minutes or until easy to handle.
4) With protective gloves, pour PCA mixture into 20 petri plates and
let cool until solid (about 10 minutes)
NOTE: Use caution when pouring plates. To prevent contamination,
only open the petri plate as long as absolutely necessary.
5) Label plate "PCA". Allow the plates to dry out by placing them at room temperature for 48 hours.
Part I: Making the Extracts
Materials
Each group should be provided with the following:
Equipment: balance, weighing boats and spoons
Supplies: 1 ml alcohol in eppendorf tubes
plant material of students' choosing
Procedure for Day 1
1) Weigh out 0.5 grams of material (or use volume measurement).
For comparative purposes, the same amount of
material should be used for each extraction.
2) Place weighed material into 1 ml of alcohol and allow mixture to
sit overnight at room temperature. (This permits the
active compounds to move into the acetone)
_______________________________________________________________________________
Part II: A Drop of Healing
The instructor needs to have 1/4" sterile disks, and tetracycline antibiotic
disks for this part. These can be purchased from Fisher Scientific
Supply.
Materials
Each group should be provided with the following:
Supplies: forceps
alcohol in a small beaker
1/4" sterile disks
tetracycline antibiotic disk
plastic or glass pipettes
tooth picks
sterile cotton swabs
Plate Count Agar petri plates (1 for each bacteria type
tested)
test bacteria (we used Staphylococcus epidermidis,
Eschericha coli and bacteria isolated from Compost)
Bacterial strains can be obtained from Wards or Carolina Biological
Supply.
Procedure for Day 2
Experiment
1) Obtain tubes from Part I. Using the plastic pipettes, put
one drop of the alcohol extract on a 1/4" sterile disk. Allow disk
to dry in a clean, empty petri dish with the lid slightly open for
at least 10 minutes. If necessary, add another drop and allow it
to dry.
2) With a sterile cotton swab, spread each bacteria type onto a different
Plate Count Agar plate (i.e. E.coli on one plate,
Strep. on another).
NOTE: Be sure to dispose of all contaminated objects to
its proper container.
To prevent contamination, only open the petri plate as long as
absolutely necessary.
3) Soak forceps in alcohol for a few minutes and let air dry.
4) Using clean forceps, place your sample disks onto the Plate Count
Agar from step 2
NOTE: Soak the forceps between each use.
Control
5) Prepare control disks by placing 1 drop of alcohol on a sterile
disk and allowing them to dry.
6) Place a solvent control disk and a tetracycline-antibiotic disk onto the surface of each PCA plate from step 2, above.
7) Incubate the plates overnight at room temperature to allow the lawn
of bacteria to grow.
____________________________________________________________________
Part III: Checking for Growth Inhibitors
Materials
Each group should be provided with the following:
plates from Day 2
ruler (with mm scale)
Procedure for Day 3
1) Measure the zone of clearing (if any) around the antibiotic disks.
A zone of clearing indicates anti-microbial activity. (Why?)
Do Soaps Really Do It?
Authors:
Kristen Wolfe, Kimberly Baldwin, Malik Williams, Sue Merkel
Abstract:
This activity allows students to explore how effective commercial soaps
and detergents are, in relation to manufacturer claims that their soaps
are "anti-bacterial". Students will screen different commercial soaps for
the ability to inhibit bacterial growth. Students develop their
own experiments and controls to determine the effectiveness of a variety
of soap products. Most experiments will involve sampling a surface
for bacteria, then using different soaps on their skin or an inanimate
surface, and then resampling to test for the presence of bacteria.
This lab activity was designed for and tested in middle or high school
biology classrooms.
Introduction:
There has been increased interest in bacteria that lurk everywhere
in our homes and kitchens, ready to attack and cause disease. Many
soap manufacturers have jumped on the band wagon by providing products
that claim "new and improved anti-bacterial activity." But do these
products really work? Here is a chance for students to test and compare
different products for themselves, by sampling a surface, washing it with
soap, then sampling again. Most students will find ambiguous results
after the first day because: they did not have the proper controls, they
did not sample equal areas, or the swab was wet one time, but not the other.
This lab gives students a chance to redesign their labs to address the
ambiguities, and illustrates why controls are so critical.
Special Materials:
Equipment:
Supplies: Petri plates with Plate Count Agar
sterile cotton swabs
a collection of commercial soap products
Time Required
Prep Day Prepare the petri plates with Plate Count Agar.
Day 1 talk about bacteria and introduce students to Plate Count
Agar petri plates.
Have students formulate a hypothesis about one r more soap product,
(does it really kill bacteria?) and design
an experiment to test that hypothesis using cotton swabs and PCA plates.
Carry the experiment out.
Day 2 Observe the PCA plates. Talk about student experiments and
controls. Have student redesign their experiments,
based on the discussion. Carry out new experiment.
Day 3 Observe the PCA plates.
Prep: Making up the Plate Count Agar Medium
Instructor or students need to make Plate Count Agar plates before
isolating bacteria. Alternatively, these items can be purchased from
Ward's Biology: 1-(800) 962-2660 or Carolina Biological Supply: 1-(800)
334-5551)
Materials
The following recipe makes about 20 plates:
Equipment: 1 liter Erlenmeyer flask
balance, weighing boats and spoons
aluminum foil
hot plate or autoclave
protective gloves
20 sterile petri plates
Supplies: 11.75 g of Plate Count Agar
tap water
Procedure
1) Weigh out 11.75 g of Plate Count Agar (PCA) and place in Erlenmeyer
flask.
2) Add 500 ml of distilled water to the flask and mix thoroughly. Cover with aluminum foil.
3) Boil contents on hot plate for 30 minutes, or autoclave at 121oC for 20 minutes. Let cool for 5 minutes or until easy to handle.
4) With protective gloves, pour PCA mixture into 20 petri plates and
let cool until solid (about 10 minutes)
NOTE: Use caution when pouring plates. To prevent contamination,
only open the petri plate as long as absolutely necessary.
5) Label plate "PCA". Allow the plates to dry out by placing
them at room temperature for 48 hours.
Part I: Asking a Question
Materials
Each group should be provided with the following:
soap products
2-4 PCA plates
sterile cotton swabs
Procedure for Day 1
1) Discuss what bacteria are and how we try to control growth with
soaps. Have students develop a hypothesis about
one of the soaps, (EXAMPLE: Clean-O soap kills 100% of all bacteria
in 30 seconds!); and develop an experiment to
test the hypothesis (EXAMPLE: sample skin, apply Clean-O for 30 seconds;
sample skin again). Skin and surfaces
can be sampled by rubbing them with a sterile cotton swab, then rubbing
the swab on the surface of the PCA plate.
2) Carry out the experiments, and incubate PCA plates overnight (or
more).
_______________________________________________________________________________
Part II: Checking Results and Redesigning the Experiment
Materials
Each group should be provided with the following:
soap products
2-4 PCA plates
sterile cotton swabs
Procedure for Day 2
1) Observe the plates. If your results are ambiguous, redesign
the experiment to better answer your original question.
Think about what controls you had, and which ones you may need.
2) Carry out the experiments, and incubate PCA plates overnight (or more).
3) Observe the plates and summarize your results.
Genetic Exchange in Bacteria
Authors
Susan M. Merkel and Adam M. Chazan
Section of Microbiology, Cornell University, Ithaca, NY
Abstract
This activity demonstrates the exchange of genetic information by bacterial
cells, using competent E. coli cells and a plasmid carrying a gene for
Green Fluorescent Protein (GFP). We describe a simplified method
for making competent E. coli and for carrying out the transformation.
Although this process is very common in nature and in research labs,
it is not always easy to observe. This obstacle is overcome by using
the GFP as a marker to visualize gene movement. Not only is GFP very
easy to detect, it is quite striking
to observe. This lab is meant for very advanced high school classes,
or introductory college level classes.
Introduction
The exchange of genetic information (DNA) in bacterial cells has helped
to drive evolution since life began. In more
recent times, scientists have developed tools that allow us to purposefully
move genes from cell to cell, paving the way for
recombinant DNA technology. There has been a renewed interest
in and concern about these processes, as the exchange
of DNA among bacteria contributes to the rise in antibiotic resistance
observed lately.
Special Materials
Equipment: tabletop centrifuge
20 - 200 ul pipettors
Supplies: a special strain of E. coli, carrying the GFP plasmid
a non-GFP strain of E. coli
ampicillan
100% ethanol
RNAase
plastic eppendorf tubes
Time Required
Prep Day For the plasmid preparation: Make TE buffer and Solution and
III (see Appendix for recipes).
Grow E. coli (GFP) in 4 ml LB+AMP broth at 37oC overnight.
For making competent cells:
Day 1 For the plasmid preparation: Prepare a fresh solution of NaOH/SDS
solution. Centrifuge the cells,
and extract the plasmid DNA. Rinse with EtOH. Store in
TE buffer.
For making competent cells:
Day 2: Carry out the genetic exchange through transformation. Spread cells on LB Agar plates. Incubate at 37oC overnight.
Day 3: Look for transformed colonies.
Preparation: Making solutions and growing cultures
Materials
Equipment: incubator
Supplies: 5 M potassium acetate
Glacial acetic acid
For the plasmid preparation:
Make TE buffer, GTE Solution and Acidic Solution. Grow
E. coli (GFP) in 4 ml LB+AMP broth at 37oC overnight.
(See appendix for medium recipe)
TE Buffer
1) Prepare 1 l of a 25 mM Tris-Cl (pH 8.0), and 10 mM EDTA (pH 8.0)
solution.
GTE (Glucose/Tris/EDTA) Solution
1) Add 50 mM glucose to 100 ml TE buffer. Autoclave for
15 minutes; store at 4oC.
Acidic Solution
1) CAREFULLY mix together 60 ml of 5 M potassium acetate, 11.5 ml of
glacial acetic acid, and 28.5 ml of H2O
Overnight Culture of E.coli (GFP)
NOTE: cultures of E.coli (GFP) should be grown in the presence of 50
ug/ml ampicillan, to help maintain the plasmid
in the cells.
1) Transfer a single colony of E.coli (GFP) into 4 ml (LB + 50 ug/ml ampicillan) broth. Shake (if possible) overnight at 37oC.
For making competent cells:
TSS buffer
Overnight Culture of E.coli ( no GFP)
1) Transfer a single colony of E. coli (no GFP) into 4 ml LB broth.
Shake overnight at 37oC.
Part I: Plasmid Preparation
Materials
Equipment: vortex (if possible)
table top centrifuge
Supplies: NaOH solution
Acidic solution
TE buffer
GTE solution
glacial acetic acid
100% ethanol
bucket of ice
Procedure for Day 1
Make NaOH/SDS Solution
1) Dilute 10N NaOH to a concentration of 0.2 N Na OH in 1% SDS (for
example, add 0.5 ml 10N NaOH to 25 ml 1% SDS). Prepare fresh immediately
before use by mixing ingredients in a test tube, and inverting 5 times
(do not vortex).
Store tubes on ice.
Plasmid Preparation
1) Place GTE and Acidic solutions on ice.
2) Pour 1.5 ml of the overnight cultures into eppendorf tubes. Centrifuge at 12,000g for 1 minute (top speed on table top centrifuge). Remove broth and repeat.
3) Resuspend pellet in 100 ul ice cold GTE Solution. Vortex or shake by flicking tube with index finger.
4) Add 200 ul NaOH/SDS solution (freshly prepared). Invert tubes several times to mix. (This lyses the cells.)
5) Add 150 ul ice cold Acidic solution. Close tube and invert for 10 seconds. Place on ice for 5 minutes. (This helps to separate the double stranded DNA. The large chromosome can't get back together; the smaller plasmids can.)
6) Centrifuge at 12,000g for 15 minutes. Transfer supernatant
to fresh tube.
(The smaller plasmids should be dissolved in the supernatant.)
7) Precipitate DNA with 2 1/2 times volume cold ETOH. Vortex, and place on ice for 20 minutes.
8) Remove supernatant and allow pellet to dry by inverting over a paper
towel. (The plasmid DNA should appear
as a small white dot on the side of the tube.)
7) Rinse with 1 ml cold 70% EtOH, being careful not to disturb the pellet. Air dry again.
8) Dissolve the pellet in 50 ul TE buffer. Store at -20oC.
Part II: Making competent E.coli cells
Materials
Equipment: 1 liter Erlenmeyer flask
floor centrifuge/ centrifuge tubes
Supplies: TSS buffer
bucket of ice
Procedure for Day 1
1) Beginning early in the day, place 1 ml of the overnight culture
into 100 ml LB broth (use a 1 liter Erlenmeyer flask).
Shake at 37oC until solution is cloudy (absorbance = about 0.3;
this may take 3-4 hours)
2) Spin 20 ml culture at 10,000g for 10 minutes.
3) Pour off the supernatant.
4) Add 2 ml cold TSS buffer. Mix gently to resuspend the cells.
Place on ice.
Cells are now ready to take up DNA or can be stored at -70oC.
Part III: the Transformation (genetic exchange)
Materials
Equipment: 37oC incubator
timer
Supplies: eppendorf tubes
bucket of ice
10 mls LB+ 20 mM glucose broth
Procedure for Day 2
Experiment
1) Place 100 ul competent cells in sterile eppendorf tubes. Place
on ice.
2) Add 20 ul plasmid DNA solution to tube
3) Incubate on ice for 30 minutes.
4) Incubate at 37oC for 20 seconds.
5) Incubate on ice for 2 minutes.
6) Add 1.9 ml prewarmed LB+ 20 mM glucose broth to each tube. Incubate 60 minutes at 30oC. Shake.
7) Plate 0.1 ml cells on each of 5 - 10 LB (+ampicillan) plates. Incubate overnight at 30oC.
Controls
8) Plate 0.1 ml untransformed, competent E.coli (no GFP) cells on one
LB (+ampicillan) plate. Incubate overnight at 30oC.
9) Plate 10 ul of the plasmid preparation on one LB (+ampicillan) plates. Incubate overnight at 30oC.
Procedure for Day 3
1) View all the plates under long UV radiation to see if any cells
"picked up" the plasmids DNA with GFP gene. These colonies should
glow bright green under the UV lamp.
Appendix
Culture Media
Plate Count Agar (per liter) Starch-Casein Agar (per
liter)
5g Bacto Tryptone 10g
Soluble Starch
2.5g Bacto Yeast Extract 0.3g Vitamin
free casein
1g Bacto Dextrose (glucose) 2.0g
Sodium Nitrate
15g Bacto Agar 2.0g
Sodium chloride
1000 ml of water 2.0g Dipotassium
phosphate
0.05g Magnesium sulfate
7H20
0.02g Calcium carbonate
0.01g Ferrous sulfate/ 7H20
15.0g Bacto agar
1,000 ml Tap water
LB Broth (per liter)
10g Bacto Tryptone
5g Bacto Yeast Extract
10g Sodium Chloride
1000 ml water
Supply Houses
Ward's Biology: 1-(800) 962-2660
Carolina Biological Supply: 1-(800) 334-5551
Fisher Scientific Company 1-(800) 766-7000
MicroBiology Ambassadors-Fall 1997 page