LABORATORY 1: WE’VE GOT CULTURE!
Growing microorganisms on a solid surface; streaking to isolate single colonies
This lab will require 1 class period to pour plates and 1 class period to streak bacteria.
BACKGROUND
Microorganisms are found essentially everywhere; we cannot see them because of their small
size. One way that microbiologists observe bacteria, without the aid of a microscope, is to
culture them on solid nutrient media. When one invisible bacterial cell lands on the nutrient agar
in a Petri dish it utilizes the nutrients and divides into millions of cells, which pile up and form a
visible colony. Studies of microbes have shown that different microbes have characteristic
colony types which can be used to distinguish them. Microbiologists use colony morphology to
initially describe an organism before confirming its identity by other more extensive tests.
In this experiment you will learn how to grow the bacterium Eschericia coli (E.coli) on a solid
agar surface. Agar is a polysaccharide obtained from red algae. It has poor nutritional value but
makes an excellent gelling agent. Nutrient agar is a mixture of agar and beef extract and proteins
that allow many microorganisms to grow in culture. Special dishes called Petri plates (named for
Julius Petri) are used as containers for nutrient agar medium when we wish to grow bacteria in
the laboratory.
In this experiment you will also be introduced to antibiotics and plasmid-borne resistance to
antibiotics. The antibiotic ampicillin, used in this experiment, blocks synthesis of the
peptidoglycan layer that lies between the E. coli inner and outer cell membrane. Thus, ampicillin
does not affect existing cells with intact cell envelopes, but kills dividing cells as they synthesize
new peptidoglycan.
The ampicillin resistance gene carried by the plasmid pAMP produces a protein, B-lactamase,
that disables the ampicillin molecule. B-lactamase cleaves a specific bond in the B-lactam ring,
a four-membered ring in the ampicillin molecule that is essential to its antibiotic action. Blactamase not only disables ampicillin within the bacterial cell, but because it leaks through the
cell envelope, it also disables ampicillin in the surrounding medium.
Microbiology students must use aseptic (sterile) technique when working with microorganisms.
Aseptic technique includes sterilizing instruments, supplies and media before use and then taking
measures to prevent subsequent contamination of the microorganism during the procedure. In
the classroom, students will sometimes us plastic instruments that are sterile when purchased;
they may also sterilize instruments using a flame or alcohol. Aseptic technique also protects the
student microbiologist from contamination with the microbe, which should be always treated as a
potential pathogen.
Below are some general guidelines for aseptic technique.
Before beginning work, the area is cleaned with an antiseptic to reduce the possibility of
contamination.
1. When using a loop to pick up any culture material, use either a plastic, sterile loop or sterilize
a metal loop by flaming just before use.
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2. Always flame the lip of the culture tube before inserting your sterile loop into the culture. This
destroys any contaminating cells that may have been inadvertently deposited near the lip of the
tube during previous transfers or by other means.
3. Keep all cultures covered with their lids when not making transfers. Do not lay the tube caps
or Petri plate lids on the bench top as this exposes the cultures to potential contamination. When
transferring cultures to and from Petri plates do not remove the lid completely from the plate,
instead lift it up only as far as you need to insert your transfer loop and inoculate your culture.
This will considerably lower the risk of an airborne contaminant falling onto the surface of the
growth medium.
4. Do not allow tube closures or Petri plate lids to touch anything except their respective culture
containers. This will prevent contamination of closures and therefore of the cultures.
5. Avoid talking while inoculating cultures; aerosols containing microflora from your mouth
(e.g. Streptococcus mutans) are produced whenever you talk.
LABORATORY OBJECTIVES
In this lab, students will learn:
a. to prepare sterile nutrient agar plates.
b. to use aseptic technique.
c. to grow bacteria on a solid surface.
d. to streak bacteria to isolate single colonies.
e. about antibiotics and plasmid-borne resistance to antibiotics.
PRE-LAB ACTIVITES
Define the following:
a. morphology
b. agar
c. gene
d. aseptic technique
e. bacterial colony
MATERIALS AND SUPPLIES
• Petri Dishes (3 sleeves/1 L agar)
• LB Agar (40g/1 L water)
• Autoclave
• Incubator
• Sterile innoculating loops or metal loops and flame (at least 1/student)
• Permanent markers
• Biohazard bag
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•
Each group of 4 students will need:
• 2 LB plates
• 2 LB plates + ampicillin
•
•
E coli Bacterial cultures (Ampicillin resistant and sensitive)
Ampicillin (100mg/ml); Use 1 ml/1 L agar; cool agar to 50oC before adding ampicillin
PROCEDURE
Day 1
Preparing LB Broth And LB Agar
For 1 Liter LB Agar, weigh 40g LB Agar, add to 1L H2O. Mix and autoclave for 20-30 minutes.
Let agar cool to approximately 50oC before adding antibiotics.
Pour approximately 15 ml agar into plate. After agar has solidified, stack plate and store in
plastic bags in the refrigerator.
Plate-streaking Technique for streaking plates for single colonies
Plan out manipulations before beginning to streak plates. Organize lab bench to allow plenty of
room and work quickly.
1. Use permanent marker to label bottom of each agar plate with your name and the date. Each
plate will have been previously marked to indicate whether it is plain LB agar (LB) or LB
agar + ampicillin (LB/amp).
2. Select the two LB plates. Mark one – pAMP for cells without plasmid and the other + pAMP
for cells with plasmid.
3. Select the two LB/amp plates. Mark one – pAMP for cells without plasmid and the other
+ pAMP for cells with plasmid.
4. Hold inoculating loop like a pencil.
To avoid contamination, do not place inoculating loop on lab bench.
5. When working from culture plate:
• Remove lid from E. coli culture plate with free hand. Do not place lid on lab bench.
Hold lid face down just above culture plate to help prevent contaminants from falling on
plate or lid.
•
Use loop tip to scrape up a visible cell mass from a colony. Do not gouge agar. Replace
culture plate lid, and proceed to Step 7.
6. Select LB – pAMP plate and lift top only enough to perform streaking as explained below
and shown in the diagram. Do not place top on lab bench.
7. Select LB – pAMP plate and lift top only enough to perform streaking as explained below
and shown in the diagram. Do not place top on lab bench.
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• Streak 1: Glide inoculating loop tip back and forth across the agar surface to make a streak
across top of plate. Avoid gouging agar.
• Streak 2. Draw loop tip once through primary streak and, without lifting loop, make a zigzag
streak scross one quarter of the agar surface.
• Streak 3: Draw loop tip once through the last line of the secondary streak, and make another
zigzag streak in the adjacent quarter of the plate without touching the previous streak.
• Streak 4: Draw tip once through the tertiary streak, and make a final zigzag streak in
remaining quarter of plate.
Start streak 2 here
Start here
Streak 1
Start streak 3 here
Start streak 4 here
8. Repeat Streaking Steps to streak E. coli onto LB/amp + pAMP plate.
9. Repeat to streak E. coli /pAMP onto LB – pAMP plate.
10. Repeat to streak E. coli/pAMP onto LB/amp + pAMP plate.
11. Place plates upside down in 37oC incubator and incubate 12-24 hours. (Plates are inverted to
prevent condensation that might collect on the lids from falling back on agar and causing
colonies to run together.)
12. Optimal growth of well-conformed, individual colonies is achieved in 12-24 hours. At this
point, colonies should range in diameter from 0.5 mm to 3 mm.
13. Take time for responsible cleanup.
a. Segregate bacterial cultures for proper disposal.
b. Wipe down lab bench with soapy water, 10% bleach solution, or disinfectant at end of lab.
c. Wash hands before leaving lab.
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RESULTS
Antibiotic-resistant Growth
If you are not able to observe plates on the day after streaking, store plates at 4oC to arrest E. coli
growth and to slow growth of any contaminating microbes. Wrap in Parafilm or plastic wrap to
retard drying.
Observe plates and use the matrix below to record which plates have bacterial growth and
which have no growth. On plates with growth, distinct, individual colonies should be observed
within one of the streaks.
LB/amp
LB
Transformed cells
+ pAMP
experiment
positive control
Wild-type cells
- pAMP
negative control
positive control
On the LB/amp plate, growth must be observed in the secondary streak to count as antibioticresistant growth. In a heavy inoculum, nonresistant cells in the primary streak may be isolated
from the antibiotic on a bed of other nonresistant cells.
On the LB/amp + pAMP plate, tiny “satellite” colonies may be observed radiating from the
edges of large, well-established colonies. These satellite colonies are not ampicillin-resistant,
but grow in an “antibiotic shadow”, where ampicillin in the media has been broken down by the
large resistant colony. Satellite colonies are generally a sign of antibiotic weakened by not
cooling medium enough before adding antibiotic, long-term storage of more than 30 days, or
overincubation.
CONCLUSIONS AND QUESTIONS
1. Were results as expected? Explain possible causes for variations from expected results.
2. What is the reason for the zigzag streaking pattern?
3. Why should the inoculating loop be resterilized between each new streak?
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4. Why should a new streak intersect the previous one only at a single point?
5. Describe the appearance of a single E. coli colony. Why can it be considered genetically
homogeneous?
6. Upcoming laboratories use cultures of E. coli cells derived from a single colony or from
several discrete parental colonies isolated as described in this experiment. Why is it
important to use this type of culture in genetic experiments?
7. E. coli strains containing the plasmid pAMP are resistant to ampicillin. Describe how this
plasmid functions to bring about resistance.
LABORATORY 2: PINK OR PURPLE BUGS
Using gram staining to classify bacteria
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This lab will require 1 class period.
BACKGROUND
The Gram Stain was devised in 1884 by Christian Gram to try to observe bacterial cells in
infected tissues. Today the Gram stain is used to group bacteria into two groups: gram negative
and gram-positive organisms. This is useful clinically to help identify the cause of illness and
guide appropriate treatment. During the staining procedure both gram positive and gram
negative bacteria take-up the primary purpre stain (crystal violet; Solution A). The cells are then
treated with potassium iodide (Solution B), which binds to the dye to form insoluble "purple
colored iodine-dye complexes" in the cytoplasm of the bacteria. Because gram positive and gram
negative bacterial membranes have different permeabilities to the insoluble complexes they are
affected differently, in the next step, by the decolorizing agent, Solution C, a mixture of acetone
and alcohol. While Gram-positive bacteria retain purple iodine-dye complexes after application
of the decolorizing agent, Gram-negative bacteria do not retain the purple complexes. With the 3
step Gram staining procedure that you will use, a red counterstain, safranin, is used with the
decolorization treatment, allowing you to visualize Gram-negative bacteria. Therefore, the gram
positive bacteria remain purple while gram negative bacteria are stained red. The 3-step gram
staining process is outlined in the diagram below.
Gram +
Gram Fix cells
Blue
Crystal violet
Blue
Purple
Iodide
Purple
(Solution A)
(Solution B)
Purple
Decolorizer +
Purple
Safranin
No color
(Solution C)
Pink
OBJECTIVES
Each student will learn to:
1. identify the 3 main types of bacteria according to their morphology.
2. describe the basic cell arrangements common to bacteria.
3. identify stained bacteria based on their morphology and gram stain color.
4. use the gram stain to classify two unknown bacteria from the environment and two known
bacteria from bacterial plates.
PRE-LAB ACTIVITIES
1. Describe the shape and draw the following three types of bacteria.
a. Bacilli
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b. Cocci
c. Spirilla
2. Give the meaning of the following prefixes:
a. diplo
b. tetrad
c. strepto
d. staphylo
3. What are mycoplasmas?
4. What is a mordant?
5. What is the history of the Gram staining procedure?
MATERIALS AND SUPPLIES
• Live bacteria, no more than 1 day old; examples of gram (+) and (-) bacteria
• Sterile toothpicks, small beaker for each group
• Bunsen burner
• Solutions A, B, C (1 vial of each/group of students)
• Microscope slides (at least 2/student)
• Microscopes
• Sharpie markers (1/group)
• Clothes pins (1 for each slide)
• Squirt bottle, containing rinsing water
PROCEDURE
Preparing and Fixing Bacteria for Staining
Before you can stain your cells you must fix them to the glass slide. If they are not fixed they
will wash away during the staining procedure. You will use heat to kill the bacteria and fix them
to the slide.
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1.
Label slide with your name and sample name.
2.
Place a small drop of water on the center of the slide. Use a sterile toothpick to obtain
some cells from colonies on your agar plate and mix thoroughly with the water drop.
Make a new slide for each colony and be sure to label the slides so you know which
colony the sample came from.
3.
Spread the drop on the slide to form a thin film. Do not make the smear too heavy. Draw
a circle on the bottom of the slide, marking the location of the bacteria. You won’t be
able to see them later.
4.
Allow the slides to dry in the air.
5.
When the film is dry, slowly pass the slide, film side up, three times through the bunsen
burner flame. Caution: Too much heat distorts the shapes and structures of the bacteria.
The slide should feel warm but not hot against the back of your hand. Cool the slide to
room temperature before staining.
Staining Procedure
Do this over a pan or sink.
Solutions A and B will stain your hands and clothes so use caution.
1.
Flood the fixed smear with Solution A (primary stain, Gram Crystal Violet) and stain for
1 minute.
2.
Remove the primary stain by washing gently with cold tap water.
3.
Flood the slide with the Solution B (Stabilized Gram Iodine). Retain on the slide for 1
minute. Do not use water to wash off iodine.
4.
Wash off the Iodine with decolorizer/counterstain Solution C. Add more
decolorizer/counterstain solution to the slide and stain for 30 seconds.
5.
Remove the decolorizer/counterstain solution by gently washing the slide with cold tap
water.
6.
Blot gently with paper towel or allow to air dry.
RESULTS
Examine your smears under the microscope and record whether the organism is Gram positive or
Gram negative. Can you determine the shape of the bacterium? Does it appear as individual
cells, clusters, or filaments? In the space below, draw, color, and classify your bacterial smears
according to their morphology and arrangement.
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QUESTIONS AND CONCLUSIONS
1. What is the purpose of heat-fixing bacteria?
2. What could happen to the bacteria if you heat-fix them too long?
3. What color do Gram positive bacteria stain and why?
4. What color do Gram negative bacteria stain and why?
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5. How are Gram positive bacteria structurally different from Gram negative bacteria?
6. Why is the Gram staining procedure not useful for staining mycoplasmas?
7. Explain why the Gram stain is called a:
a. complex stain
b. differential stain
8. Explain why some bacteria are Gram variable.
9. Why is it important that your bacteria be no more than 24 hours old?
10. What was the mordant used in Gram staining?
11. What was the name of the counterstain used in the Gram staining procedure?
12. What is the clinical importance of the Gram staining procedure?
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LABORATORY 3: LOOK WHAT’S GROWING HERE!!
Isolating Microorganisms from the Environment,
Followed by Gram Staining
This lab will require 3-4 days to grow the bacteria and 1 day to perform gram staining.
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BACKGROUND
In this experiment you will isolate microorganisms from the environment and grow them on a
solid substrate. After selecting a defined object or surface to swab for culture, you will use a
swab to inoculate sterile Petri dishes containing agar. You will survey the area for
microorganisms before and after disinfecting and then perform daily follow up surveys to follow
the course of repopulation of the area. After incubating your plate you will observe colony
morphology of different organisms that have grown. You will perform the Gram stain on some
of the bacteria to help identify them.
LABORATORY OBJECTIVES
Students will:
a. gain more experience with Aseptic technique
b. learn to grow bacteria on a solid surface
c. obtain more experience with identifying bacteria using the gram stain and colony
morphology
d. appreciate the effects of disinfectants
e. understand the role of microbes in their environment.
f. understand the role of microbiology in society.
PRE-LAB ACTIVITES
Use your textbook to answer the following questions (Chapters 1 and 26)
1. What microbial activities support human life?
2. How has the field of microbiology improved the quality of human life?
3. What is the meaning of normal flora?
4. What are the environmental benefits of microorganisms?
5. How are microbes involved in biogeochemical cycles?
6. Describe the microbial role in the following cycles:
a. Carbon cycle
b. Nitrogen cycle
c. Sulfur cycle
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MATERIALS AND SUPPLIES
• LB Agar plates (4 plates/pair of students)
• Sterile water
• Sterile swabs
• Household Disinfectant solutions
• 37oC incubator
• Permanent markers
• Gram stain Solutions: A,B,C
• Microscope slides (2-3/student)
• Microscopes
PROCEDURE
Day 1
1.
Work in pairs. You will each be given a Petri dish containing nutrient agar.
2.
Use a marker to label the bottom of your plate (the part containing the agar) with your
name, today’s date and “Day 1”. Draw a line on the plate to divide the plate in half.
Mark one half “before” and the other “after”.
3.
Select a survey area that you do not expect to be cleaned in the next couple of days,
perhaps a telephone receiver, a doorknob, or lock on your locker. Select an area that is
different from that of your lab partner. Wet a sterile swab with sterile water. Swab your
area and use this swab to inoculate the “before” half of your agar plate.
4.
Disinfect your selected survey area with one of the solutions provided. Write down the
name of your disinfectant___________________________. Allow the area to dry. Swab
the area again and inoculate the “after” half of your plate.
5.
Place the plate from your survey area in the 37oC incubator.
6.
You will repeat a swab of your survey tomorrow and the following day.
Day 2 and Day 3
1. Examine both your plate and your lab partner’s plate. Observe any colony types that may be
growing on your plates. It is important to note the colony differences in color, size, shape,
and texture.
2. Record your observations in the RESULTS section.
3. Save your plates to make Gram Stains of your bacteria. Follow the protocol in Laboratory 2.
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4. You will share a new plate with your partner on Days 2 and 3. Divide it in half with a
marker. Label one half with your name, the date and Day 2 or Day 3; your partner will use
the other half of the plate.
5. Wet a sterile swab with sterile water. Swab your area and use this swab to inoculate your
half of the agar plate. Your partner will do the same with his/her half of the plate.
6. Place the plate from your survey areas in the 37oC incubator.
CONSLUSIONS AND QUESTIONS
Record data on next page
1. Was there a significant decrease in the number of microbes after you disinfected your area?
2. How many different kinds of microbes grew on your plate? Check for the different colors
and textures.
3. Should you be concerned about the healthiness of your environment if your plate is covered
with bacteria? Explain your answer.
4. How is disinfecting different from sanitizing?
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Survey Area Results
YOUR PLATES
Describe survey area________________________________
Disinfected with
________________________________
Number of Colonies
Description of Colonies
Day 1
Before disinfecting
Day 1
After disinfecting
Day 2
Day 3
LAB
PARTNERÕS
PLATES
Describe survey area________________________________
Disinfected with
________________________________
Number of Colonies
Day 1
Before disinfecting
Day 1
After disinfecting
Day 2
Day 3
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Description of Colonies
CLASSROOM ACTIVITY 1: MICROBE IDENTIFICATION
This activity will require 1 class period.
PURPOSE: To identify the unknown bacteria using the fewest number of tests.
MATERIALS:
Packet of twelve labeled envelopes containing test results
Description of selected species hand-out.
Flow sheet
PROCEDURE: On the basis of where your unknown was found, select tests (envelopes) that
would be most useful in identifying your bacteria. Open one envelope at a time, recording it on your
flow sheet, until you have sufficient information to identify the unknown. The goal is to use the
fewest number of envelopes (tests).
Tests:
1. Gram Stain
Gram stain is a differential stain. Organisms that stain blue (purple) are called
gram positive; organisms that stain pink are called gram negative.
2. Cell Shape
Choose from round (coccus), Rod (bacillus) or Corkscrew (spirilla).
3. Cell Arrangement
Bacteria are found singly, in pairs, in chains, in a tetrad (like a cube) and in clusters.
4. Endospore Formation
Does the bacterium form an endospore? The endospore is a highly resistant body,
capable of surviving long periods as well as high temperatures and toxic chemicals.
5. Pigment Production
Some bacteria produce pigment that gives the colony a distinctive color.
6. Catalase Production
Catalase is an enzyme found in some bacteria that breaks down hydrogen peroxide
with the release of oxygen gas.
7. Glucose Fermentation – Uses Glucose for ATP
8. Lactose Fermentation – Uses Lactose for ATP
9. Voges Proskauer – Produces Acetoin or it doesn’t
10. Citrate Utilization – Uses citrate as a source of carbon
11. Gelatin Liquefaction – If gelatin, a protein, is hydrolyzed, then the medium will turn
to liquid.
Questions: How did you decide which envelopes to open? If you were to repeat this activity
would you go about it the same way? What would you change?
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DESCRIPTION OF SELECTED SPECIES
Alcaligenes faecalis: Gram negative short rods, 0.5 by 0.2-2.0 µm, usually occurring singly,
motile. Agar colonies opaque, entire, without pigment. Metabolism respiratory. Aerobic.
Citrate may or may not be utilized as carbon source. Indole not produced. Catalase positive.
Casein or gelatin not liquefied. Litmus milk alkaline. Source: dairy products, rotting eggs and
other foods, and in aquatic and terrestrial environments where they are involved in
decomposition.
Bacillus megaterium: Gram positive endospore forming rods 1.5 to 3.0 µm in diameter, tending
to occur in short chains. Motile. On nutrient agar growth is heaped and non-spreading, glossy or
moderately dull, colonies generally cream colored. Aerobic. Acid produced from glucose and
usually from arabinose, xylose, and mannitol. Acetoin not produced. Active liquefaction of
gelatin; catalase positive. Starch hydrolyzed. Citrate utilized. Source: widespread in nature;
spores occur in soil
Bacillus subtilis: Gram positive endospore forming rods occurring singly. Endospores oval.
Cells stain evenly; surface of free spore stains faintly. Colonies on agar media round or
irregular; surface full; becoming thick and opaque; may be wrinkled and may become creamcolored or brown. Active spreading occurs on agar with a moist surface. Aerobe. Metabolism
predominantly respiratory; acid may be produced from glucose, arabinose, xylose and mannitol.
Acetoin produced. Catalase positive. Starch and gelatin liquefied. Citrate utilized. Litmus milk
reduced. Source: Common in environment; endospores widespread.
Brevibacterium linens: Gram positive rods. 0.6 to 2.5 µm in size. Non-motile on nutrient agar
growth of colonies is convex, glistening, entire and cream-colored. Metabolism predominantly
respiratory. Aerobic. No acid or gas produced from arabinose, destrin, glucose, ducitol,
glactose, lactose, fructose, maltose, manitol, sorbitol, sucrose or xylose. Catalase positive.
Active liquefaction in gelatin. Litmus milk reduced slowly. Source: widely distributed in and
especially on the surface of dairy products.
Citrobacter freundii: Gram negative rods, occurring primarily singly. Motile by peritrichous
flagella; non-encapsulated. Colonies on agar usually smooth, low convex, shiny, lacking color.
Facultative anaerobes. Glucose and other carbohydrates fermented with the production of acid
and gas. Lactose fermentation may be delayed or absent. Citrate used as sole carbon source.
Hydrogen sulfide produced; indole not produced. Catalase positive; oxidase negative. Gelatin
not liquefied. Source: Found in water, food, feces and urine.
Corynebacterium pseudodiphtheriticum: Gram positive, short regular rods. In stained smears
cells often occur singly or in rows with the long axes parallel. Non-motile. Colonies regular and
smooth, pigmented white. Metabolism respiratory. Aerobic. Acid not produced from any
carbohydrate tested. Catalase positive. Indole not produced. Urea hydrolyzed. Source: found
in nasopharyngeal mucosa of man. Not pathogenic.
Corynebacterium xerosis: Gram positive irregular rods, staining with occasional granules and
club forms, occurring singly. Non-motile. Small circular colonies on plain agar may be rough or
smooth, lacking pigment. Carbohydrate metabolism fermentative. Acid produced from glucose,
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maltose, galactose and sucrose. Anaerobic. Catalase positive. Nitrates produced from nitrates.
Urea not hydrolyzed. Indoled not produced. Source: presumably inhabits skin and mucous
membranes of man. Not pathogenic.
Enterobacter aerogenes: Gram negative rods 0.5 to 0.8 by 1.0 to 2.0 µm occurring singly.
Motile. Agar colonies thick, white, moist, round, smooth, entire. Metabolism fermentative.
Facultative anaerobes. Acid and gas produced from most carbohydrates, including glucose and
lactose, gelatin is slowly liquefied by most strains. Indole is usually not produced. Citrate and
acetate can be used as sole carbon source. The Voges Proskaur test is usually positive; Catalase
positive; oxidase negative. Source: found in the feces of man and other animals, sewage, soil,
water and dairy.
Escherichia coli: Gram negative straight rods 1.1 to 1.5 by 2.0 to 6.0 µm, occurring singly or in
pairs. Motile. Colonies on agar usually smooth, low, convex, shiny, entire, gray. Many strains
have capsules. Metabolism respiratory and fermentative. Faculative anaerobes. Acid and gas
produced from fermentation of glucose, lactose and other carbohydrates. Catalase positive.
Citrate not utilized. Source: found in the lower intestines of warm blooded animals.
Micrococcus luteus: Gram positive cocci, 1.0 – 2.0 µm in diameter, occurring singly, in pairs
and dividing in more than one plane to form tetrads, irregular clusters or regular packets of cells.
Non-motile. Colonies are round, smooth, entire, with yellow-orange pigment. Metabolism
strictly respiratory. Strict aerobe. Acid not detected from carbohydrates. Proteins and fats may
be hydrolyzed. Catalase produced. Source: common in soil, dust, water, skin of man and other
animals.
Micrococcus roseus: Gram positive spheres 1.0 – 2.5 µm in diameter, occurring singly, in pairs,
and dividing in more than on plane to form irregular clusters, tetrads or cubical packets. Some
strains are motile. Colonies pink or red, smooth, slightly convex with regular margins.
Metabolism strictly respiratory. Strict aerobe. No acid from carbohydrates detected. Nitrates
usually reduced to nitrate. Indole not produced. Catalase produced. Gelatin liquefied slowly.
No growth on citrate agar. Source: dust, water and salt-containing food.
Proteus vulgaris: Gram-negative usually straight rods 0.4 – 0.6 by 1.0 – 3.0 µm; usually
occurring singly, pairs or chains. Motile. Agar colonies gray, opaque, smooth; swarming
growth. Facultative anaerobes. Acid is formed regularly and rapidly from glucose and more
slowly from fructose, galactose, maltose and sucrose. Acid not formed from arabinose or
lactose. Acetoin is rarely formed. Gelatin is liquefied. Urea is hydrolyzed. Catalase protein;
oxidase negative. Citrate utilization negative. Source: found in fecal matter of many animals,
sewage and soil, especially where animal protein is decomposing.
Pseudomonas fluorescens: Gram-negative rods, usually 0.7 – 0.8 by 2.3 – 2.8 µm occurring
singly and in pairs. Motile. Agar colonies circular, smooth, translucent. Cultures produce
yellow-green pigment. Metabolism respiratory. Aerobic. Acid not produced from
carbohydrates. Citrate utilization positive. Alkaline reaction in litmus milk. Gelatin liquefied.
Catalase positive. Oxidase positive. Source: found in soil and water. Commonly associated
with spoilage of food.
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Sarcina lutea: Gram positive spheres, 1.0 – 1.5 µm in diameter, occurring in packets. Agar
colonies coarse, granular, circular, raised, moist, glistening and yellow in color. Metabolism
respiratory. Aerobic. No acid produced from glucose, lactose or sucrose. Alkaline reaction in
litmus milk. Gelatin liquefied. Source: air, soil and water; also found on skin surfaces.
Serratia marcescens: Gram negative, short rods, sometimes almost spherical. 0.5 by 0.5 to 1.0
µm, occurring singly and occasionally in chains. Motile. Agar colonies circular, thin, granular,
white becoming red. Fermentation metabolism. Facultative anaerobes. Acid produced from
glucose, sucrose, mannitol, maltose. Voges Proskaur Test positive. Acid not produced from
lactose. Citrate utilized as carbon source. Gelatin liquefied. Catalase positive. Litmus milk
reaction acid. Source: water, soil, milk, and foods, also found in silk worms and other insects.
Staphyococcus aureus: Gram positive spheres, 0.8 – 1.0 µm in diameter, occurring singly, in
pairs, in short chains and in irregular clumps. Non-motile. Agar colonies are circular, smooth,
glistening and range from white to orange in color. Facultative anaerobes. Acid produced from
glucose, lactose, sucrose, mannitol and glycerol. Acetoin is produced from glucose. Catalase
positive. Gelatin is liquefied. Litmus milk reaction is acid. Source: usually found on the skin
of humans and warm blooded animals.
Staphylococcus epidermis: Gram positive spheres, 0.5 – 1.5 µm in diameter, occurring singly,
in pairs and dividing in more than one plan to form irregular clusters; occasionally tetrads
observed. Colonies are circular, convex with a smooth or slightly granular surface, usually white
in color. Facultative anaerobes. Acid from glucose and usually from lactose and maltose.
Acetoin is produced from glucose. Citrate is not utilized. Source: mainly found on the skin and
mucous membranes of warm-blooded animals.
Streptococcus faecalis: Gram positive spheres, occurring singly or in chains. Cells 0.5 – 1.0 µm
in diameter. Non-motile. Colonies are smooth and entire; rarely pigmented. Facultative
anaerobes. Acid produced from glucose, sucrose, manitol, lactose, maltose, and mannitol.
Citrate is utilized. Acid produced in litmus milk. Catalase negative. Source: feces of humans
and warm blooded animals. Common in many food products, often unrelated to direct fecal
contamination.
Helpful Vocabulary
Aerobic – Strict air user
Anaerobic – Strict non-air user
Facultative Aerobic – may or may not use air
Hydrogen Sulfide – Gas that smells like rotten eggs
20
Indole – Formed from tryptophan
Litmus – Acid/base Indicator
Motile – It moves
Opaque – Can’t see through it
MICROBE IDENTIFICATION FLOWSHEET
YOUR
NAME______________________
Number of Microbe to ID_________
Where microbe was
found___________________________________________________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
21
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
22
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
_____________________
Next Characteristic:_______________________________
This eliminates:
_____________________
_____________________
_____________________
_____________________
_____________________
MICROBE NUMBER __________
_____________________
IS________________________________
23
LABORATORY 4: DON’T BE DECEIVED!
Effectiveness of disinfectants, antiseptics, and antibiotics in the control of microbial growth.
The Kirby-Bauer disc diffusion assay
24
BACKGROUND
Control of microorganisms is essential in order to prevent the spread of diseases and infection,
stop decomposition and spoilage, and prevent unwanted microbial contamination. Low
temperature, such as refrigeration or freezing, inhibits microbial growth by slowing down
microbial metabolism. Heating to high temperatures kills many but not all microbes. Chemical
agents such as disinfectants, antiseptics, and antibiotics are used to kill microorganisms. Natural
substances, such as, herbs and spices are also known to have antimicrobial effects. Many factors
influence the effectiveness of these chemicals and natural substances: the concentration of the
substance, the temperature at which it is used, the kinds of microorganisms present, and the
number of microorganisms present. There are two common antimicrobial modes of action for
disinfectants and antiseptics: 1) They may damage the lipids and proteins of the cytoplasmic
membrane of the microbe resulting in leakage of cellular materials 2) They may denature
microbial enzymes and other proteins by disrupting the hydrogen and disulfide bonds that give
the protein its 3-dimensional shape. Antimicrobial agents can be classified as either bactericidal
or bacteriostatic. A substance that is bactericidal kills bacteria; one that is bacteriostatic inhibits
bacterial growth.
In this experiment, you will compare and evaluate the effectiveness of various disinfectants,
antiseptics, herbs, and spices on two organisms, a Gram positive bacterium, Bacillus cereus and
a Gram negative bacterium, E. coli.
LABORATORY OBJECTIVES
Students will:
a.
b.
c.
d.
distinguish between disinfectants, antiseptics, antibiotics, and herbs.
distinguish between bactericidal and bacteriostatic action of chemical agents.
evaluate the effects of antiseptics, disinfectants, and herbs on selected bacterial cultures.
discuss the limitations of the disk dilution method.
PRE-LAB ACTIVITIES
1. Define the following terms:
a. disinfectant
b. antiseptic
c. antibiotic
d. herb
25
e. bacteriostatic
f. bactericidal
g. zone of inhibition
h. sanitization
i. sterile
j. autoclave
2. On the next page, complete the graphic organizer of antimicrobial agents.
26
27
TEACHER PREPARATION
1. Overnight broth cultures of:
A. Bacillus cereus
B. Escherichia coli
(Obtain from CORD)
2. Prepare a variety of herbal solutions (usually 0.5 g/10ml water). Aliquot into microfuge tube
one day prior to lab.
2.
Aliquot a variety of disinfectants and antiseptics into microfuge tubes. This should be done
one day in advance.
MATERIALS AND SUPPLIES
The following supplies will be needed for each group of 4 students:
•
•
•
•
•
•
•
•
•
•
Four LB agar plates
A petri dish of ethyl alcohol
Petri dish containing sterile filter paper disks (obtain from CORD)
Two sterile swabs
Two forceps
2 antibiotics
2 disinfectants
2 antiseptics
2 herbs
Transparent metric ruler
PROCEDURE
Day 1
1. Work in groups of 4 students. One pair will work with the bacterium Esherichia coli and the
other pair with Bacillus cereus. Invert your Petri dishes. Using your marker divide the plates
into quadrants and label the four sectors as shown in the drawing. Be sure you write the
name of the antimicrobial agent that you have chosen in each quadrant. The numbered
sectors should correspond to the numbers you used to label the antiseptic, disinfectant,
antibiotic, and herbal tubes. Label two plates “E. coli” and two plates “B. cereus”.
28
2. Using the aseptic technique demonstrated by your instructor, remove a sterile swab and insert
it into either the E. coli or the B. cereus broth culture.
3. Use the inoculum on the swab to evenly spread bacteria over the entire surface of the nutrient
agar on your appropriate plates.
4. Choose 2 household disinfectants, 2 antiseptics, 2 antibiotics, and 2 herbs you would like to
test. You will test these same antimicrobial agents on both of your B. cereus plates and both
of your E. coli plates. You will test one antimicrobial agent per quadrant. Make sure that
you have labeled each quadrant with the antimicrobial agent chosen.
5. Remove the forceps from the alcohol and air dry.
6. Use the forceps to pick up a sterile blank disk and dip it into the antimicrobial agent. Be sure
to allow the excess solution to drain off.
7. Place the saturated disk in the appropriate sector of your plate. Gently press the disk down
with the tip of the forceps to ensure contact with the agar.
8. Repeat steps 5 through 7 for the remaining antimicrobial agents.
9. Invert the plates and incubate at 37oC overnight.
29
RESULTS
Day 2
Examine your plates for a zone of no growth
(zone of inhibition) around the disk for each
disinfectant, antiseptic, antibiotic, and herb for
both organisms. Following incubation, the zones
of inhibition should be measured around each disk.
Measure the diameter (in millimeters) of each
zone of inhibition and record this data in the table
below.
diameter
Diameter of zone of inhibition (mm)
Name of solution
E. coli
1
2
3
4
5
6
7
8
30
B. cereus
CONCLUSIONS AND QUESTIONS
1. List, in a separate column for each bacterium, the disinfectants, antiseptics, antibiotics, and
herbs in order of their effectiveness in inhibiting growth of bacteria? List in order from the
most effective to the least effective
E.coli
B. cereus
1. What are the active ingredient(s) in each antimicrobial solution tested?
2. Was each solution equally effective with both bacteria? If not, suggest why.
3. This is only one assay that can be used to assess the ability of a chemical to prevent bacteria
from growing. It is possible that a chemical does not inhibit growth in this assay but is still
able to inhibit growth under other conditions. Give two variables that might affect the ability
of a substance to inhibit growth in this assay.
4. This disk diffusion technique does not allow us to distinguish between bacteriostatic and
bactericidal effects of the antimicrobial chemicals. How might we determine whether the
bacteria were inhibited or killed?
31
5. Other than these antimicrobial agents used in this experiment what are some other methods
used to disinfect and sterilize?
6. Learn more about Bacillus cereus. Suppose you were given 2 identical broth cultures of
Bacillus cereus. You boiled one culture for 2 minutes and you autoclaved the other culture.
You then streaked a swab of each on a plate of nutrient agar. What are your predicted results
and why?
8. An HIV positive mother gives birth in a hospital. The delivery room staff usually cleans the
delivery area and then will disinfect with alcohol. After the delivery the staff washed all the
hard surfaces and this time disinfected with bleach. Why?
9. Why are plates inverted in the incubator?
10. Were any bacterial colonies observed within your zones of inhibition? Which microbial
agent or agents showed this characteristic and why?
CLASSROOM ACTIVITY 2: DISEASE DETECTIVES
32
This activity will require 1 class period.
PURPOSE: To learn about disease transmission and symptomotology.
MATERIALS:
Packet of 4 labeled envelopes containing information about each disease.
PROCEDURE: In this activity the class should divide itself into 4 groups. Each group of
students will be given an envelope labeled with a disease: Legionella, HIV, Cholera, or Typhoid
fever. The envelope contains a description of the disease symptoms and questionnaires
containing information obtained from patients with the disease. Students should discuss the
information provided and determine the answers to the following questions:
1.
Is this disease acute or chronic?
2.
Is the outbreak an epidemic or a pandemic?
3.
How is the disease spread?
4.
Can you identify a common source? If so, what is it?
5.
What is the vehicle of the infection?
6.
What measures would you recommend to curb the spread of disease?
33
34
LABORATORY 5: WE’VE GOT MORE CULTURE
Growing liquid cultures of bacteria; growth curves, effect of antibiotics
This lab will require 1 class period of preparation; 1 class to perform the lab and 2 class periods
for analyzing data.
BACKGROUND
Under laboratory conditions, bacterial growth follows a predictable course. After inoculating a
tube of sterile media with bacteria the cells first increase in size but the number of cells lags.
This initial lag phase is followed by a log phase during which bacteria grow exponentially. After
this period of exponential growth, nutrients run out, wastes build up and bacterial growth slows
until finally the number of cells begins to decline. One way to follow the growth of bacterial
cells in culture is to examine the turbidity of the bacterial suspension; as the cells grow, the
bacterial suspension becomes more turbid. In the lab we generally express the turbidity of the
bacterial suspension as absorbance, measured by the how much light is absorbed by the sample,
which is directly proportional to the density of the bacteria in the suspension: Absorbance
increases as the bacteria grow. Absorbance is measured with a spectrophotometer, an instrument
that is used to measure the amount of light absorbed by the bacterial suspension. In this
experiment you will construct a bacterial growth curve, using a spectrophotometer to follow the
growth of bacteria in a liquid culture.
LABORATORY OBJECTIVES
Each student should:
a. learn to use a spectrophotometer to collect data for a bacterial growth curve.
b. construct a growth curve from the data that are collected from an entire day and describe the
stages of bacterial growth.
c. understand the indirect relationship between percent transmission and absorbance.
d. understand that there is a direct relationship between the light absorbed and the amount of
bacteria in a suspension.
e. give examples of direct and indirect cell count methods.
PRE-LAB ACTIVITIES
1. Define the following terms:
a. spectrophotometer
b. turbidity
c. direct cell count method
35
d. indirect cell count method
e. binary fission
f. absorbance
g. transmittance
h. batch culture
i. continuous culture
2. Describe what occurs during each of the phases associated with a growth curve.
1. lag phase
2. log phase
3. stationary phase
4. death phase
3. Write a hypothesis for your experiment.
36
MATERIALS AND SUPPLIES
Day 1 – Pre-lab setup (1 day prior to the experiment)
•
•
•
•
•
•
•
Six sterile flasks containing 50 mL of LB broth. One flask will be used to zero the blank in
the spectrophotometer and the other 5 flasks will be assigned to different laboratory groups.
These same flasks will be used all day.
An overnight batch culture of E. coli will be made. This will be prepared by adding one
colony of E. coli to a sterile flask containing 25 mL of sterile broth.
One spectrophotometer
One box of cuvettes.
One box of kimwipes
100 µl of ampicillin (100mg/mL)
One shaker incubator. Set to 37oC before going home.
Day 2 – Growth curve lab
Set up the following in the front of the room.
•
•
•
•
•
•
•
•
•
•
Turn on spectrophotometer 30 minutes before class and set to the absorbance mode; set
wavelength to 550nm. Next to spectrophotometer, place the following:
One box of kimwipes
One box of cuvettes
One data-collecting chart for entire class
Set shaker incubator to 37oC
One ice water bath
One flask of batch E. coli
(1) 100 microliter pipette and box of matching sterile tips.
(2) 1000 microliter pipettes and a box of matching tips.
One waste beaker (500mL)
Divide the class into 5 groups of 5 to 6 students. Each group will need the following supplies:
•
•
•
•
•
One flask of sterile broth.
One cuvette
One permanent marker
One sterile plastic pipette
Each group will label their flask accordingly
• Group 1: 37oC with shaker
• Group 2: 37oC without shaker
• Group 3: 37oC with ampicillin and shaker
• Group 4: ice water bath
• Group 5: room temperature
37
PROCEDURE
When using a spectrophotometer, you should adjust the instrument to show an absorbance of 0
when a tube containing only sterile broth is placed in the sample holder.
3. Pipette 1 microliter of sterile broth into a cuvette and read absorbance. Press 0/ABS to zero
this reference blank in the spectrophotometer. Each group will use this same cuvette to set
the spectrophotometer throughout the day.
4. Groups 1 – 5 will inoculate their flask with 1ml of batch E.coli.
5. Group 3 will also add 50 microliters of ampicillin to their flask.
6. Each group will swirl their flask and pipette 1 ml of inoculated broth into a cuvette.
7. Place the cuvette into the spectrophotometer, read and record the absorbance.
8. Empty cuvette contents into the waste beaker.
9. Place labeled flask in the correct environment.
10. Repeat spectrophotometer readings every 30 minutes.
11. Record Absorbance readings on a chart.
12. Each succeeding class will continue this lab all day.
11. The teacher will also need to keep a tally of each group’s absorbance. Place this information
on the chart by the spectrophotometer.
Day 3 –Graph results
Materials needed:
Graph paper and 5 different colors of ink or pencil.
PROCEDURE:
1.
2.
3.
4.
Each group will share their data with the other groups.
Each student will graph the data for all 5 groups.
The time is on the X-axis and the absorbance is on the Y-axis.
When possible, label the lag, log, stationary, and death phases on each graph.
38
QUESTIONS
1. Why did you use sterile broth instead of distilled water as the reference blank?
2. Why was it important to have a blank as a reference?
3. Which inoculated flask showed the greatest change in absorbance? Why?
4. Which inoculated flask showed the least change in absorbance? Why?
5. Which flask contains the fastest growing bacteria? Do you think this same growth rate would
continue over the next 2 days? Why or why not?
6. What happens to the amount of light absorbed as the bacteria grows?
7. What happens to the amount of light transmitted as the amount of light absorbed increases?
8. Why will bacteria not immediately grow exponentially when added to a new environment?
39
40
LABORATORY 6: LEAF PRINTS
Using leaf Prints to investigate PPFMs (Pink-Pigmented Facultative Methylotrophs)
This lab will require 1 class period to make leaf prints and approximately 2 weeks to observe the
growth of PPFMs. An additional class period will be required if students prepare their own
plates.
BACKGROUND
The bacteria examined in this lab activity are members of the genus Methylobacterium. These
bacteria are distributed ubiquitously on the surfaces of plants. Colonies of the bacterium are pink
in color and have the somewhat unusual ability to utilize methanol as their sole source of carbon.
These traits are the basis of a popular nickname for the bacteria, PPFM. This acronym stands for
Pink-Pigmented Facultative Methylotroph. The bacteria are not pathogenic to their plant hosts,
and although they obtain all of their nutrient resources from their hosts, the relationship is not
one-sided. The bacteria take carbon (growing plants produce substantial amounts of methanol
(CH3OH) as they reshape their cell walls) and mineral nutrients, but they also contribute to plant
metabolism in a number of important ways. Bacterial enzymes like urease can contribute to plant
nitrogen metabolism. They stimulate seed germination. If the bacteria are removed from seeds,
the rate of germination falls. Adding bacteria back to the seeds restores their germinability. Even
the germination rate of old seeds can be enhanced by the addition of PPFM bacteria. PPFM
bacteria also stimulate the growth of plants, presumably because they produce the plant growth
regulator, cytokinin, as well as Vitamin B12. The ability of the bacteria to grow on methanol is
the basis for devising a selective medium for the PPFMs.
Bacteriological Media
2 media will be used:
a. M9 + methanol: selective medium, consisting simply of inorganic salts and minerals from
which the bacteria must make everything they need for growth. As a carbon source, the
selective medium contains only methanol. Since most organisms cannot use one-carbon
compounds as a carbon source, PPFM bacteria are selected for by this medium.
a. TSA: Tryptic Soy Agar: A nonselective nutrient medium which contains a variety of
nutrients, minerals and carbon compounds. Many types of organisms can grow on this
medium.
LABORATORY OBJECTIVES
Students will learn that:
1. Although bacteria inhabit every conceivable niche in the environment, particular species of
bacteria have specific habitats.
2. Selective media can be used to isolate specific bacteria for study.
41
PRE-LAB ACTIVITIES
• Find out how urease contributes to plant nitrogen metabolism.
•
Find out what pigment is responsible for the pink appearance of PPFM.
•
Define the following:
1. Facultative
2. PPFM
•
What is the composition of M9+ethanol plates?
•
What is the composition of TSA plates?
TEACHER PREPARATION
Prepare TSA and M9 + ethanol plates or pick them up from CORD.
MATERIALS AND SUPPLIES
• M9 + Ethanol plates (1 per pair of students)
• TSA plates (1 per pair of students)
• Sharpie markers
• Parafilm to seal plates (2 strips per group)
• Various leaves collected by students
PROCEDURE FOR PREPARING PLATES
M9 + methanol
11.28 g M9 salts
15 g agar (plain, not nutrient)
1 L water
Stir
Autoclave
Cool to 50oC.
Add 5ml Methanol (0.5%)
Pour plates
42
TSA
40g Tryptic Soy Agar
1 L water
Stir
Autoclave
Pour plates
PROCEDURE FOR MAKING LEAF PRINTS OF PPFM
1. Working in pairs, take two plates, one containing the nonselective nutrient-rich medium
(TSA) and the other the selective medium (M9+methanol). Label your plates with your
names and the date.
2. To inoculate the plates with bacteria, lay your plant leaf on the surface of the medium and
impress it onto the agar using the eraser end of a pencil. After making an impression, the
leaves should be lifted carefully away from the medium and discarded. Note that a clear
imprint of the leaf should be visible on the surface of the medium. Both types of medium
should be inoculated in this way.
3. After inoculating the agar plates, close them and seal around the edge with Parafilm. Invert
your plates so condensation from the lid doesn't drip onto the leaf print and smear it.
4.
Incubate plates at room temperature while colonies develop. Once the plates are sealed, they
should not be opened again! Bacterial growth can be observed clearly through the lid of the
plate. Many types of bacteria are expected to grow--especially on the nonselective medium-and not knowing what all of them are, it is best to avoid direct contact with them.
5. Tape the leaf to the outside of the plate.
6. Describe below how your experiment was set up (include type of plant used and whether
upper or lower leaf surface was printed), what kind of observations you will make (what kind
of data you will collect), and what results you expect. The plates should be observed for 5 to
14 days.
TSA Plates
Type of leaf _________________________________________ upper or lower
Type of leaf _________________________________________ upper or lower
Expected results
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________
Actual results
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________
43
M9 + ethanol Plates
Type of leaf _________________________________________ upper or lower
Type of leaf _________________________________________ upper or lower
Expected results
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________
Actual results
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________
RESULTS AND QUESTIONS
1. What question(s) are you hoping to answer with this experiment?
2. Count the colonies present on each dish. In order to do this, create a grid on the underside of
the dish. The grid will make it easier to keep track of all the colonies counted.
3. Compare your dishes to others in the classroom. Some of you will find that fungi will grow
on the rich media and create a fuzzy overlay on the print. In some instances fungi will also
grow on the selective media plates; be sure you can distinguish bacteria from fungi.
4. Describe some qualitative attributes of the cultures growing on your plates, such as, colony
morphology (both size and shape), and colony color.
5. Is PPFM growth the same on all plates? What data lead you to this conclusion? What may be
the reasons for the variations of the growth of the PPFM bacteria on various plates?
6. Why do bacteria grow faster on the nonselective medium?
7. Why did only the PPFM bacteria grow on the selective media?
8. On the nonselective medium, what types of organisms are present?
44
9. Do all microorganisms grow at the same rate? What evidence does your experiment give?
Design Your Own Experiment
Hypotheses are presented below as examples of possible experiments.
Variation #1. Do only certain parts of plants have PPFMs?
Print different plant parts (leaves, stems, roots, flowers, etc.) side by side on one plate. Expected
results?
Variation #2. Can you wash off PPFMs?
Wash leaves with soap and water before printing them. Be sure to dry the leaves so that the extra
water will not make a mess of the agar. As a control, print unwashed leaves next to washed
leaves on the same plate. Expected results?
Variation #3. Are PPFMs the same on all plants?
Print various plants on the plate side by side. Expected results?
Variation #4. Will PPFMs grow in hot or cold temperatures?
Print the same plants on three different plates. One is incubated at room temperature, one in a
refrigerator (about 4o C on average) and one in an incubator (at about 37o C). Expected results?
Variation #5. Do PPFMs grow everywhere?
Make prints on selective medium of everyday objects other than plants. Pencils, fingers, stones
or sidewalks might be used. Plates are incubated as for leaf prints. Expected results?
45
46
LABORATORY 7: Part 1: Mello JELL-OTM, etc.
Protein Digestion by Enzymes
This lab was borrowed from the Science in the Real World: Microbes in Action Program. It has
been modified only slightly to fit with the other laboratories in this manual.
There are actually 3 separate labs described here; each will require one class period to do the lab
and one class to analyze the results.
INTRODUCTION
Foods are not only important nutrients for cells, they are also the cause of some stains in clothes.
Since some of the large complex molecules in food do not dissolve well in water, they are often
left in clothes after washing. Enzymes are proteins that break down complex molecules in food
to produce smaller molecules that are more soluble in water. For example, enzymes can break
down the protein gelatin, a major part of JELL-OTM. Manufacturers take advantage of the ability
of enzymes to break down food by adding them to detergents to enhance stain removal.
LABORATORY OBJECTIVES
You will observe the effect of enzymes present in detergents and cleaning solutions on JELLOTM.
PRE-LAB ACTIVITIES
1. Define:
a. enzyme
b. substrate
c. enzyme-substrate complex
d. active site
2. Explain how enzymes lower the activation energy of a chemical reaction.
3. Describe 5 general properties of enzymes.
47
4. What are coenzymes?
5. Describe the international system of naming enzymes (enzyme nomenclature).
6. Give 2 examples of enzymes that do not fit the international system of naming enzymes.
7. How does temperature generally affect enzyme activity?
8. Describe and give a cause of enzyme denaturation.
9. How does pH affect enzyme activity?
10. Gas gangrene can be potentially fatal. What are the cause, symptoms, and treatment for gas
gangrene? Describe the enzyme–substrate complex associated with this condition.
Cause:
Symptoms:
Treatment:
Enzyme-substrate complex:
11. How do enzyme inhibitors function?
12. Why should we not eat the fish caught from Logan Martin Lake (near Pell City, AL)?
48
Part 1: Mello JELL-OTM
Pre-Lab Set-up: Prepare plates
Dissolve 36 g JELL-O in 100 ml boiling water.
Check pH with pH paper or pH meter. Add crystalline sodium carbonate (1-1.5 grams) to raise
the pH to 8. One 3 oz. box makes approximately 10 plates.
Refrigerate. Wells are easier to cut when the JELL-O is firm.
Note: These plates are NOT edible as sodium carbonate is poisonous.
MATERIALS AND SUPPLIES for Part 1
For each group of 3-5 Students
1 plate containing JELL-OTM
1 plastic straw section
1 toothpick
1 marking pen
1 metric ruler
detergents with cleaning solutions each with their own pipette
distilled water with a pipette
PROCEDURE
1. Obtain a JELL-OTM filled plate. Label the plate on the bottom by writing (near the edge and
in small letters) your name, class period, and today’s date.
Caution: This JELL-OTM contains sodium carbonate and should NOT be eaten.
2. Place the plate right-side up on the template provided and remove the lid. Using the piece of
plastid soda straw, cut wells in the JELL-OTM using the template for a pattern.
3. Remove the plugs of JELL-OTM with a toothpick. Take care not to tear the layer of JELLOTM. Number the wells on the bottom of your plate, so that when face up they are as shown
on the template. (This means you write the numbers backward and counter-clockwise on the
bottom of your plate.)
49
4. Measure the diameter of the wells (in millimeters). Record these numbers in Table 1 as
“initial diameter”.
5. Decide within your group which detergents you would like to test. Record the detergent
number or letter in Table 1 next to the appropriate well number.
6. Use only the dropper that is in each solution. Do not exchange droppers between tubes!
7. Load distilled water into well #7. This will be your negative control. To load the wells,
place the pipette into the well and gently dispense just enough liquid to fill the well. Try not
to drop any liquid onto the surface of the JELL-OTM. If you do, note the location of the drop
by drawing a picture in your notebook.
8. Carefully load each of the wells with one of the detergent solutions.
9. Replace the lid on the plate. Do not turn the plate upside down. You will spill the solutions
that you just loaded into the wells.
10. Let the plate sit undisturbed for several hours or overnight at room temperature.
RESULTS
1. Using a pipette, remove the liquid from the wells and discard. Observe the wells in the plate.
Record any physical change in the JELL-OTM that you see around any of the wells.
2. Was there any change in the JELL-OTM around well #7? Explain.
50
3. Measure the largest diameter of each well in millimeters. The diameter of a well is the
distance from solid JELL-OTM on one side to the solid JELL-OTM on the other side. Record
this number in the table below as the “final diameter”. Calculate the change in diameter for
each detergent.
Well #
1
Initial
Diameter
mm
Final
Diameter
mm
Change
in Diameter
mm
2
mm
mm
mm
3
mm
mm
mm
4
mm
mm
mm
5
mm
mm
mm
6
mm
mm
mm
mm
mm
mm
7
Detergent Name
and Number of Letter
distilled water
4. Which products increased the diameter of the well?
5. Based on your observation and the information supplied in this lab, what ingredient in JELLOTM do you think was changed?
6. What ingredient in the detergent is probably responsible for the breakdown of the protein,
gelatin?
7. Enzyme names often end in “ase”. For example, lactase is the enzyme that breaks down the
milk sugar, lactose. Suggest a name for the enzyme that breaks down the protein, gelatin.
Part 2: In The Clear
BACKGROUND
Most foods contain a variety of nutrients, but foods usually have large amounts of protein,
carbohydrates and fat. Different foods have more or less of these three nutrients. Enzymes are
special chemicals that can recognize a specific type of molecule and can break the chemical bond
in that molecule. In the stomach and intestines a variety of enzymes act together to break down
the complex molecules in food. The simple molecules produced by the enzyme action can pass
through the intestines to the blood where they serve as food for the cells in the body.
51
Just as there are different classes of nutrients, there are different classes of enzymes. In general,
an enzyme recognizes the particular type of molecule that it will break down, but does not
recognize other molecules. For example, an enzyme that breaks down protein is called a
protease. It will break down most proteins, but will not break down fats or carbohydrates.
Enzymes that break down fats (lipids) are called lipases. The ability of an enzyme to act on only
one class of molecules means that it is specific for that type of molecule; therefore, we say that
enzymes show “specificity” for those particular molecules.
In this activity you will observe the action of enzymes on all three types of nutrients. Milk agar
plates contain large amounts of the protein, casein. Egg yolk agar plates are high in lipids, while
the starch plates contain the carbohydrate molecule, starch. These nutrients are all large
molecules that typically do not dissolve well in water. Therefore the plates appear cloudy.
Enzymes break down the large complex molecules to smaller molecules that are soluble. The
result is that the cloudiness of the plates disappears whenever an enzyme has broken down the
large molecule.
This lab demonstrates that the enzymes in detergents degrade biochemical compounds such as
lipids, proteins and starches found in food.
LABORATORY OBJECTIVES
Students will determine the types of enzymes that are present in various detergents and compare
these products in terms of enzyme activity.
PRE-LAB SET-UP: Prepare plates
Milk Agar (protein)
In one flask, 9.2 g nutrient agar
320 ml water
In another flask, 8 g non-fat dry milk
80 ml water
Stir
Autoclave
Cool agar to 50oC and add milk solution.
Pour plates.
Egg Agar (Fat)
16 g nutrient agar
400 ml water
Autoclave
Cool to 50oC.
Sterilize an egg in ethanol for 5 minutes; add egg yolk to the agar and mix.
Pour plates
52
Starch Agar (carbohydrate)
16 g nutrient agar
4 g starch
400 ml water
Stir
Autoclave
Pour plates
MATERIALS AND SUPPLIES For Part 2
Per group of 3-5 students:
1 egg yolk agar plate
1 milk agar plate
1 starch agar plate
1 plastic straw section
1 small container of alcohol
1 toothpick
1 metric ruler
Lugol’s iodine stain
1 marking pen
1 piece of aluminum foil
detergents and cleaning solutions each with their own pipette
distilled water with a pipette
PROCEDURE
1. Label each of the three agar plates on the bottom by writing (near the edge and in small
letters) your name, your class period and today’s date.
2. Using the template for a pattern, cut wells in each of the agar plates with the plastic soda
straw. In this lab, we are using nutrient agar plates so it is important to sterilize the straw in
alcohol in order to minimize the risk of transferring bacteria onto the plate. To do this, dip
the straw in the beaker of alcohol. Let the alcohol drain out of the straw back into the beaker.
When most of the alcohol has evaporated use the straw to cut wells in the agar. Remove the
agar plugs with a sterile toothpick.
53
3. For each of the three plates, number the wells as indicated on the template. Remember to
write on the bottom of the plate. (Write the numbers backwards and counterclockwise on the
bottom of your plate.)
4. Measure the diameter of the well (in millimeters). Record this value in the Data Table as the
initial diameter.
5. Decide within your group which of the available detergents you would like to test. Choose
six and record the names in your data table next to the appropriate well number. Use only
the dropper that is in each solution. Do not exchange dropper between tubes.
6. Fill well #7 with distilled water. This will be your control. To fill the wells, place the pipette
into the wells and gently dispense just enough liquid to fill the well. Try not to drop any
liquid onto the surface of the agar. If you do, note the location of the drop by drawing a
picture in your notebook.
7. Carefully load each of the wells 1-6 with one of the detergents.
8. Cover your plates and carefully place the three agar plates in a stack in the area designated by
your teacher. Do not turn the plates upside-down.
Observations
The yolk in egg yolk agar contains lipids and proteins the yellow color of the agar. The
breakdown of these lipids by enzymes called lipases leaves a clear ring around the wells of the
solution that contain lipid digesting enzymes.
Similarly, milk agar contains milk proteins (casein). The white color provided by casein
disappears in the presence of proteases.
Starch agar contains starch, a long chain carbohydrate, that can be digested by enzymes called
amylases. This agar is transparent and must be stained with Lugol’s iodine to show where the
starch is present (the purple stained area) and where it has been digested (clear).
9. In order to observe the digestion of starch in the agar by the detergent solutions, the starch
plate must be stained with the starch indicator, Lugol’s iodine. Before staining, remove the
liquid in each well using a dropping pipette. To stain, slowly flood the surface of the starch
agar with iodine, replace the lid, cover the plate with aluminum foil, and let it sit while you
observe the milk and egg agar plates.
10. Look for zones of clearing around the wells in the egg yolk, milk and starch agar plates.
Clearings are areas where the agar is still present and intact but it looks much clearer or
transparent than the surrounding agar. Record in the Data Table (next page) the presence and
the diameter of this clearing.
11. Compare data with your classmates.
54
Data Table
Well #
Detergent
Solution
Egg Yolk Agar
Initial/Final
Diameter (mm)
1
2
3
4
5
6
7
Change
in Diameter
Milk Agar
Initial/Final
Diameter (mm)
/
/
/
/
/
/
/
Change
in Diameter
/
/
/
/
/
/
/
Starch Agar
Initial/Final
Diameter
(mm)
Change
in Diameter
/
/
/
/
/
/
/
RESULTS
1. Using the data above, name the 3 detergents with the most enzyme activity among the six
that you chose.
2. Which if any, of the detergents digested the following nutrients?
Protein (milk agar)
Lipids (egg yolk agar)
Carbohydrates (starch agar)
3. Find the containers for these detergents and read the labels (always a good practice for the
informed consumer!). Do the labels help you determine what might account for the
differences observed among the different detergents? Explain your answer.
4. Think about food stains. Why would you want a detergent to be able to digest starches,
proteins and lipids?
55
5. Using the class data, list below the most active detergents for each category.
Starch Digestion
Protein Digestion
Lipid Digestion
6. Look at the class data and determine any trends that may be apparent regarding the types of
detergents, the names of the detergents, the ingredients listed on the labels, and name brands
versus generic brands. Discuss your conclusions as a class. Summarize that discussion here.
Part 3: Go to the Source
INTRODUCTION
Enzymes are biological catalysts and are produced by all living organisms. Most enzymes
increase the rate of a reaction by at least a million fold. Another way to say this is that it the
reaction would take one minute with an enzyme, it would require two years without an enzyme!
Enzymes are also specific. Each enzyme recognizes a specific types of chemical by its shape
and structure. It helps that particular chemical to react.
Enzymes are proteins that have a particular function in living cells. They break down complex
molecules in food to produce smaller molecules that can be taken up by cells. All cells are
surrounded by a membrane, the cytoplasmic membrane, which does not allow large molecules to
pass into the cell, but does allow small molecules to enter.
Bacteria are similar to human cells in some ways. For example, bacteria cannot use complex
food sources in their environment because these complex molecules cannot pass through the
bacterial cytoplasmic membrane into the cell. Some bacteria have solved this problem by
making enzymes that can break down complex food molecules and then secreting those
enzymes out into the environment. When these enzymes leave the bacterial cell they move into
56
the environment around that cell and break down complex foods to simple molecules. These
simple molecules then enter the bacterial cell providing it with the nutrients it needs to grow. In
order to take advantage of all the complex food molecules in the environment, the bacterial cell
would have to produce enzymes for all three major nutrients: proteins, carbohydrates and lipids.
Because enzymes act specifically on only one class of molecules, different enzymes are required
for different food molecules.
In today’s activity you will determine whether different strains of bacteria can secrete enzymes
into the environment.
LABORATORY OBJECTIVE
Students will see whether bacteria produce enzymes that can break down the protein in milk
agar, the lipid in egg yolk agar, or the carbohydrates in starch agar.
MATERIALS AND SUPPLIES For Part 3
Per student or team of students:
• 1 Bunsen burner (can be shared between 2 teams)
• 1 inoculating loop
• 1 metric ruler
• 1 starch agar plate
• 1 egg yolk agar plate
• 1 milk agar plate
• Lugol’s iodine
• 10% bleach solution (disinfectant)
• aluminum foil
• broth cultures of 3 different bacteria
PROCEDURE
Although we are using species of bacteria that are harmless to humans, all bacterial should be
handled with care. Wash your hands and disinfect your countertop before you begin and when
you finish the procedure!
1. Label each of the three plates by writing (near the edge and in small letters) your name,
class period, and today’s date on the bottom. Using the marker, divide the bottom of the
plate into three sections numbered 1, 2, 3 as shown below.
2
1
3
57
2. Place the plates right-side up on the counter. Using a micropipettor, transfer 100µL from
the broth culture to the well of the appropriate section of the plate. List the strains used
in the table on the right.
1.
2.
3.
3. Leave plates right side up at room temperature for 48 hours.
RESULTS
1. In order to observe the digestion of starch in the agar by bacteria, the plates must be
stained with the starch indicator, Lugol’s iodine. To stain, slowly flood the surface of the
starch agar with iodine, replace the lid, cover the plates with aluminum foil, and let it sit
while you observe the milk and egg yolk agar plates.
2. Observe the milk and egg yolk agar plates. Digest of nutrients in the agar can be
observed as a clear area around the growth of bacteria. Record your assessment of the
digestive abilities of each species of bacteria on each type of nutrient agar plate. For
positive results, use (+), (++), or (+++) to indicate the degree of digestion. For negative
results, use (-) to indicate no digestion of nutrient.
Bacteria
Egg Yolk Agar
Milk Agar
58
Starch Agar
QUESTIONS AND CONCLUSIONS
1. What type of chemical compound, secreted by bacteria, causes the digestion of nutrients?
2. Do some bacteria secrete digestive enzymes outside the cell? How do you know this?
3. Milk has a high concentration of a particular protein. Which kind(s) of bacteria digested
milk protein? How do you know?
4. Egg yolk has a high concentration of lipids. Which kind(s) of bacteria digested the lipids
found in egg yolk? How do you know?
5. Which kind(s) of bacteria digested starch? How do you know?
6. Did any of the bacteria digest all three nutrients? If so, which?
7. You are the head chemist at a detergent company. You are to select one bacterial strain to
use as the organism to produce secreted enzymes to use as an additive for a new detergent.
Which of the bacterial strains that you or your classmates tested would you choose? Why?
Spot Be Gone!
How does all of this relate to the real world and the practical application of laundry detergent?
Design a real world application using this information that would test the effectiveness of
detergents on food stains. Follow the steps below to begin your laboratory development.
1. Pose a question that you can answer by performing an experiment.
2. Develop a hypothesis using the if, then format.
3. Identify the variables. What is the independent variable? What is the dependent
variable?
4. What would be an appropriate control?
5. Design your experiment. Be creative!
59
60
LABORATORY 8: OIL EATING BUGS
Bioremediation of oil using penicillium and pseudomonas
and commercial drain cleaners
This lab consists of 2 parts; each part will require 1 class period to set up and several days to
observe results.
BACKGROUND
Biodegradation is a natural process of the digestion of oil by microorganisms, which can be
expedited by feeding nutrients to existing oil-digesting microbes to stimulate their growth or by
adding more microbes. Bioremediation describes the use of microorganisms to recycle organic
materials. Under careful controlled conditions, bioremediation can be a practical and cost
effective method to remove hydrocarbons from contaminated surfaces. Two real life applications
of bioremediation are cleaning up oil spills on ocean shores and unclogging a drain in a kitchen
sink.
There are two related experiments in this lab. In this first experiment you will determine if the
microorganisms penicillium and pseudomonas will digest vegetable- and petroleum-based oils.
In the second experiment you will use drain cleaners, containing bacteria (unknown to you), to
discover if they will digest the same oils used in the first experiment. In both experiments you
will use a color indicator, triphenyltetrazolium (tetraZ), to give you quick results, regarding the
breakdown of the oil. TetraZ turns pink as oil is metabolized. By-products of oil degradation
reduce the compound; when it is reduced its color changes from a pale yellow to pink/red. After
observing the formation of color, you will continue to observe your samples for a longer period
of time to actually look for the disappearance of the oil.
Microorganisms used in bioremediation include bacteria, fungi and yeast. They all use oil as an
energy source just as other higher organisms, including people, do, however, microorganisms
can use mineral oil and petroleum based oils as food while animals cannot. Animals can only
digest oils of animal and vegetable origin. In these experiments you will discover which types of
oil the microbes that you are using will digest.
While there is much information you can obtain from these experiments, the experiments
themselves are quite simple. They contain three basic components:
• Oil: vegetable and petroleum-based.
• A source of oil-degrading microbes
• liquid cultures of a Penicillium species and a Pseudomonas species
• Commercial drain cleaners that contains bacteria.
• 2,3,5 triphenyltetrazolium chloride, which is colorless until reduced by the oil degrading
bacteria
You will mix these three components, incubate them and observe what happens over a couple of
weeks, making notes daily to document your observations.
Be sure to use a variety of oils in your class.
61
LABORATORY OBJECTIVES
Each student should be able to:
a. understand the process of bioremediation
b. understand how a color indicator such as tetraZ is used to give an indirect measurement
c. give real life applications of bioremediation
PRE-LABORATORY ACTIVITIES
1. Define the following terms:
•
Biodegradation
•
Bioremediation
•
Recalcitrance
•
Bioaugmentation
•
Genetically engineered organisms
2. Use the internet to learn about the Exxon Valdez oil spill of 1989. Where did this oil spill
take place and what were the different methods used to solve the problem?
3. Why are bacteria often ineffective in cleaning up large clumps of oil?
4. What are scientists doing to help increase the rate of bioremediation by bacteria?
5. Compare the advantages and disadvantages of cleaning up oil spills by using genetically
engineered organisms and by using bioaugmentation of a species common to the area.
6. Your car has a freon leak in the car air conditioner. By law, the leak has to be fixed before
more freon can be added. What is the reason for this?
7. How are microorganisms used in the treatment of sewage?
62
MATERIALS AND SUPPLIES
• 10 tubes + caps/group (5 tubes for each lab)
• permanent markers
• test tube racks (1/group)
• sterile water
• 0.2% tetraZ solution (30 ml/group)
• liquid cultures of penicillium and pseudomonas (2.5 ml/group)
• various oils in dropper bottles
• Solutions of Drain care and RidX, prepared according to package (25 ml/group)
PROCEDURE
Lab 1 Oil degradation using liquid cultures of penicillium and pseudomonas.
1. Work in groups of 4-6, students. Each group will use both penicillium and pseudomonas to
see what happens when incubated with 1 type of vegetable oil and 1 type of petroleum-based
oil. Each group should have a different type of vegetable oil. You will add TetraZ indicator
to your tubes to indirectly observe the oil degradation. What control sample(s) should you set
up?
2. Obtain 5 tubes for your group. Label your test tubes with a sharpie marker. On each tube,
give the following info: tube number (1-5), group number, type of oil, type of microorganism
and today’s date.
3. Have you decided on your control? Hint: what happens if you don’t add any bacteria? Set
up tubes that have tetraZ but don’t have any added pen or pseudo. This will remind you of
why you must use sterile water in your samples.
4. Use the table below to add the oil, bacteria and tetraZ to your tubes. Be sure that you use
sterile technique when setting up your samples. Pipette tetraZ first. Then add oil. Add
bacteria last.
Sample Set-up
tetraZ
_______
oil
1
4 ml
5 drops
2
4 ml
3
4 ml
4
4 ml
control
4 ml
#
_______
oil
pen
culture
pseudo water
culture
0.5 ml
0.5 ml
5 drops
5 drops
0.5 ml
5 drops
5 drops
0.5 ml
0.5 ml
63
5. Cap tubes. Use your finger to thump the tubes to mix the contents.
6. Incubate without shaking at 30oC.
7. Use the Data Table below to record your daily observations for about 10 days. In addition to
describing any color change, make notes of overall appearance.
8. Document the appearance of your samples immediately after setting them up.
Take some time to formulate a hypothesis or two for your experiment.
Hypothesis:____________________________________________________________________
______________________________________________________________________________
_____________________________________________________________________________
RESULTS: Data Table
date
date
date
date
64
date
date
Lab 1
CONCLUSIONS AND QUESTIONS
1. Compare the penicillium and psuedomonas samples.
2. Compare the oils that you used with those of the other groups in your class.
3. Compare the vegetable oils with the petroleum based oil.
4. What conclusions can you make from this lab?
5. What experiments could you do to answer new questions that have arisen from your results?
6. Did you results support your hypothesis?
Save your samples at room temperature so that you can compare them with Lab 2.
Lab 2 Oil degradation using commercial drain cleaners that contain bacteria.
PROCEDURE
In this experiment you will use commercial drain cleaners (Rid-X and DrainCare) that contain
bacteria to observe the biodegradation effect on vegetable and petroleum-based oils. Again, you
will add TetraZ indicator to your tubes.
1. Number five tubes 1-4 plus 1 control.
2. Label your test tubes with a sharpie marker, e.g.
Rid-X + olive oil
4/4/05
Group 1
65
3. Use the table below to add the oil, drain cleaner and tetraZ to your tubes. Pipette tetraZ or
water first. Then add Oil. Add drain cleaner last. Again, set up the appropriate control.
_______
oil
#
tetraZ
_______
oil
1
2 ml
5 drops
2
2 ml
5 drops
3
2 ml
5 drops
4
2 ml
5 drops
control
RidX
Drain
Care
water
2 ml
2 ml
2 ml
2 ml
2 ml
5 drops
4. Cap tubes. Mix the samples by tapping the tube with your finger.
5. Incubate without shaking at 30oC.
6. Use the Data Table on the next page for recording your daily observations for about 10 days.
In addition to describing any color change, make notes of overall appearance.
7. Document the appearance of your samples now.
8. Formulate a hypothesis for this experiment.
Hypothesis:____________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
66
RESULTS
Data Table
date
date
date
date
date
date
Lab 2
CONCLUSIONS AND QUESTIONS
1. Compare the effects of the different drain cleaners on the oils that you used.
2. Compare the oils that you used with those of the other groups in your class.
3. How do the vegetable oils compare with the petroleum based oil?
4. What conclusions can you make from this lab?
5. Did your results support your hypothesis?
6. Now compare your results with the drain cleaners with your results with penicillium and
pseudomonas cultures.
67
68
LABORATORY 9: HYPHAE, HYPHAE EVERYWHERE
This Lab Will Take Two Days (One Week Apart)
BACKGROUND
Fungi are multicellular, heterotrophic, and eukaryotic organisms. Unlike plant cells whose cell
walls are composed of cellulose, the cell walls of fungi are usually surrounded by a cell wall
composed of chitin. Many fungi are familiar; we see fungi growing on bread, cheese, and fruit.
People also use fungi to make many products: bread, wine, beer, and cheese.
Microscopic fungi can consist of molds, yeasts, or both. Structurally, molds form long,
branching filaments called hyphae as they grow. A mass of hyphae is known as a mycelium.
Their reproductive structures are spores and each spore is capable of germinating into a new
fungal colony when the conditions are favorable.
Lactophenol cotton blue (LPCB) is an important eucaryotic stain for fungi. It will stain the
fungal cell walls blue and enable the fungal hyphae to be seen more easily against a light
background. The phenol in the LPCB will kill the mold and the lactic acid will preserve the
fungal structures.
LABORATORY OBJECTIVES
Each student will:
b. describe the morphology of fungi
c. describe both sexual and asexual reproduction in fungi
d. grow and stain bread mold (Rhizopus nigricans)
PRE-LAB ACTIVITIES (FROM CHAPTER 11)
1. Define the following terms:
a. fungi
b. mycology
c. mold
d. yeast
e. spores
f. hyphae
g. mycelium
h. aerial hyphae
i. vegetative hyphae
j. opportunistic fungi
69
k. mycoses
l. sporangium
m. sporangiophore
n. stolons
o. rhizoids
p. zygospore
2. Explain the difference between asexual spores and sexual spores in fungi.
3. Describe the difference between septate and nonseptate hyphae. Sketch an example of each.
4. Compare the morphology between molds and yeasts.
5. Give 3 general characteristics of mold spores.
6. What are dimorphic fungi?
7. Give 3 characteristics that are used to classify fungi.
8. How are fungi beneficial to our society and environment?
9. Give 4 methods used to control fungal pathogens in plants?
Part 1 Growing hyphae
MATERIALS AND SUPPLIES
(for each student group of 4-6 students)
•
•
•
4 non-sterile petri plates
white bread
beaker of H2O with dropper
70
PROCEDURE
1. Label 4 petri plates 1-4 and with your group name and date.
2. Place a small sample of bread into each petri dish.
3. Plate 1 will not receive any water.
4. Plates 2 – 4 will receive varying amounts of water; each group will decide on the amount.
5. Put the lids on the dishes and place in a dark cabinet.
Seven Days Later
Part 2 Staining and visualizing hyphae
MATERIALS and supplies
(for each student group of 4-6 students)
•
•
•
•
5 glass slides
LPCB stain
clear transparent tape
microscope
PROCEDURE
1. Place a small drop of LPCB stain in the middle of a glass slide.
2. Obtain a 4cm strip of clear tape and hold firmly between your thumb and index finger on
both hands. Avoid contaminating with fingerprints.
3. Firmly press the sticky side to the surface of the bread mold.
4. Gently pull the tape from the surface and place it on the slide with LPCB stain. Press the tape
as smoothly as you can without pressing down too firmly. You do not want to disturb the
hyphae.
5. Observe under both low and high power. Look for hyphae, mycelium, sporangia,
sporangiophores, stolons, rhizoids, and zygospores.
6. Draw your results on the back of this page.
71
72
LABORATORY 10
McWane Center Laboratory
HIV:
The Life Cycle of a Virus
INTRODUCTION
In general, viruses are nonliving agents and each particle contains nucleic acid
surrounded by a capsid protein. The nucleic acid can be RNA or DNA. Viruses are much
smaller than cells; about 100 to 1000 fold smaller and can only grow inside living cells.
73
Human Immunodeficiency virus (HIV) is the infectious RNA virus that causes Acquired
Immune Deficiency Syndrome (AIDS). AIDS is a public health concern, with an estimate of
36.1 million people living with HIV/AID and an estimate 21.8 million people who have died
from AIDS since the beginning of the epidemic. For HIV to enter a human cell, the viral
envelope protein, gp160, must interact with CD4, a human cell surface receptor. gp160 is made
up of two portions, which both function to allow HIV to enter a cell: gp120 and gp41, the
transmembrane tail. HIV destroys immune cells thus leading to death by infection. It is usually
the destruction of the immune system which leads to susceptibility to other infections like the flu,
Kaposi’s Sarcoma, and the cytomegalovirus.
A detection method, known as Western Blotting, can be used to detect the presence of
viral proteins from human samples. In this lab, we will look for the presence of gp41 as an
indicator of HIV infection. Students will also perform a mock fluid exchange to simulate the
spread of an infectious disease such as HIV and use computer exercises to learn about the
epidemiology of the disease.
Additional information can be found on certain web sites (seen below) and can be used to help
with the understanding of the biochemistry and public health issues dealing with this disease.
http://www.avert.org/young.htm
http://www.avert.org/hivquiz.htm
Information on AIDS and young people and a quiz
http://www.roche-hiv.com/infoactive_static/infoactive_static.htm
Information on the life cycle of HIV
http://www.cdc.gov
Information on the statistics dealing with HIV/AIDS
http://www.biology.arizona.edu/immunology/activities/western_blot/west1.html
Western Blot activity
http://www.nyhallsci.org/whataboutaids/main.html
http://www.pbs.org/wgbh/nova/aids/
General HIV/AIDS information
I. Western Blot and Coomassie Staining
You will be using SDS-PAGE (polyacrylamide gel electrophoresis) to determine if six patients
express the gp41 protein. It should run at about 41kDa. The CD4 receptor interacts with the
HIV protein gp120, while gp41 interacts with a fusion domain to allow HIV to enter the T cell.
You will perform a Western Blot to detect the presence of the
gp41. The patient stories can be found on the last page of the protocol.
WEAR GLOVES AT ALL TIMES!!!
Procedure A. Preparation of Samples for SDS-PAGE gel
1. Each group will have samples of 40 µl protein from three patients.
2. Tap the tube to mix the sample.
3. Spin briefly (few seconds) in microcentrifuge to collect sample at bottom of tube.
4. Boil samples for 5 minutes in the hot block at 95°C.
74
Assembly of the Precast Gel (See Figure on Page 6 of Protocol)
5. Each group will have a gel box and a pre-cast gel.
6. Rotate the cams outward to release the electrode assembly. Remove electrode assembly
from the clamping frame.
7. Cut along the black line on the pre-cast gel, and then remove the tab along the bottom of
the precast gel.
8. Place the pre-cast gel cassette into the slot at the bottom of the electrode assembly. The
short glass piece should face inward.
9. Place a “buffer dam” on the other side of the electrode assembly, make sure the buffer
dam is oriented correctly.
10. Transfer the electrode assembly and gels into the clamping frame.
11. Press down on the electrode assembly while closing the 2 cam levers of the clamping
frame.
12. Lower the electrode assembly and clamping frame into the mini tank. Fill the inner
chamber with 125 mls of 1x running buffer so that the buffer is between the short and
long glass plates. Now you can remove the comb.
13. Add 200 mls of 1x running buffer to the mini tank.
Loading the Gel
14. Load 5 µl of the protein marker into a well 1 and well 6 of the precast gel.
15. Load 10 µl of the protein/sample buffer mix into wells 2-4 and wells 7-10 of the pre-cast
gel. Your instructor will provide a demonstration. The samples in wells 1-4 will be used
for the Western Blot procedure. The samples in wells 6-10 will be used for the
Coomassie staining procedure.
75
Running the Gel
16. Align the electrode plugs and jacks and place the lid on the top of the mini tank.
17. Attach the black and red wires to the power supply, matching black wire to black outlet
and red wire to red outlet.
76
18. Electrophorese gel for 45 minutes at 200V.
Removing the Gel
19. Turn off power and disconnect wires.
20. Remove the lid from the mini tank and lift out the electrode assembly and clamping
frames. Open the cams of the clamping box, and pull the electrode assembly out of the
clamping frame and remove the gel cassettes.
21. Use a spatula to pry the glass plates apart very carefully!!
22. Slide a spatula under the gel to carefully lift it up. Cut gel between wells 4 and 6. Half of
the gel will be used in the Coomassie Stain procedure. The other half of the gel will be
used in a Western Blot. Western Blot is a used to transfer proteins from gel to
nitrocellulose membrane in order to detect proteins of interest with anti-bodies.
Procedure B. Transfer of Protein from Gel to Membrane-see pictures on next page
All groups will transfer gels at the same time with the same apparatus.
1. Remove lid of the transfer apparatus and cathode (by squeezing the white handles
towards each other).
2. Place presoaked filter paper onto the platform (anode). Roll over the paper with a glass
rod to get rid of any bubbles. This is very important!
3. Put one piece of presoaked nitrocellulose membrane on top of the filter paper. Roll over
the membrane with a glass rod to get rid of bubbles.
4. Place the gel directly onto the membrane; making sure it is in the center of the
membrane. Roll over the gel with a glass rod to get rid of bubbles.
5. Place another piece of filter paper on top of the gel and roll out any bubbles.
6. Place the cathode on top of the stack. Press to engage latches. Then place the safety
cover on top of the unit. Plug into the power supply (red wire to red outlet and black wire
to black outlet).
7. Transfer for 20 minutes at 10-20V.
8. After transfer proceed to Western Blot procedure.
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Procedure C. Western Blot
1. Pour 25 mls of Blocking Buffer (5% B.S.A.) into small plastic container. Add your
transferred nitrocellulose membrane.
2. Block membrane in Blocking Buffer for 15 minutes at room temperature on the orbital
shaker.
3. Pour Blocking Buffer back into tube.
4. Incubate membrane with 25 mls of diluted primary antibody to protein of interest for 30
minutes at room temperature on the orbital shaker. (This protein is analogous to the viral
gp41 surface protein.)
5. Pour the diluted antibody back into the tube.
6. Wash membrane with 25 mls of wash buffer 3 times (5 minutes each = total time is 15
min) at room temperature on the orbital shaker.
7. Incubate membrane with 25 mls of diluted secondary antibody for 30 minutes at room
temperature on the orbital shaker.
8. Wash membrane with 25 mls of wash buffer 2 times, then wash with dH2O once (5
minutes each = total time is 15 min) at room temperature on the orbital shaker.
9. Develop membrane with TMB substrate for 5-10 minutes (pour just enough TMB to
cover the gel), rinse with distilled water, allow to dry, and analyze.
This is a picture of what your protein marker will look like. Below is the list of proteins in the
sample, as well as their molecular weights.
Myosin
209,000 daltons
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Beta-galactosidase
Bovine serum albumin
Ovalbumin
Carbonic anhydrase
Soybean trypsin inhibitor
Lysozyme
Aprotinin
124,000
80,000
49,100
34,800
28,900
20,600
7,100
Procedure D. Coomassie Stain
1. Add 50 mls of distilled water to a small plastic container. Add your gel. Rinse for 15
minutes. Pour the water down the sink, carefully so as not to lose your gel!
2. Pour just enough of the Coomassie Blue stain to cover your gel.
3. Incubate for 1 hour on the orbital shaker.
4. Pour off the Coomassie Blue stain. Add 50 mls of Destain buffer and allow to incubate
on the shaker for 1 hour.
5. Analyze gel.
II. Fluid Exchange
In this mock fluid exchange, each student will receive a tube filled with a mock fluid.
The students will share with 3 different tubes (or people) making sure to take the fluid from the
other tube as well as pass some of their fluid to the tube they share with.
The students will keep track of which tube shares with which.
At the end, phenolphthalein will be added to the tubes, and the tubes containing NaOH will
change to pink and are “infected.” This shows the students that we started with only one infected
person (only one tube containing 0.1M NaOH) and many more have become infected.
Your tube number:
Tube # of 1st sharing:
Tube # of 2nd sharing:
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.
Tube # of 3rd sharing:
.
III.
Epidemiology
In this exercise, students will surf the web doing research in small groups. Each group
will be assigned to research statistics on HIV infection and deaths due to AIDS in
different countries, states, and counties. Any additional statistics you are interested in
looking up in any other part of the world is also fine. Write your results on the bottom of
the page. Use the back of the page if you need to.
Here are some places to start looking:
http://www.cdc.gov
http://www.unaids.org
Group 1: Jefferson County, Alabama
Sweden
China
Mexico
Group 2: The state of Alabama
India
France
Japan
Group 3: Montgomery County, Alabama
South Africa
The state of California
Canada
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Group 4: Lee County, Alabama
The state of New York
Nigeria
Brazil
Group 1 and 2:
1. Suzi went to a party one night and the guy she’d been trying to get to notice her
finally did. They went into the bedroom in the back together after they both had some
alcohol and they ended up having unprotected sex. Did this cost Suzi her life?
2. Bob is an average student, but lately he’s been feeling down in the dumps. Bob met
this guy on the way home from school. This guy says he knows how to make Bob feel better.
Bob ends up sharing a needle to take drugs with this guy. Was this such a
good idea?
3. Felicia’s normally not very wild, but her parents went out of town this weekend and
she’s been partying since they left. She starts experimenting with things she never has
before – drugs and sex. Was Felicia safe? Do you think she used clean needles and
condoms?
Group 3 and 4:
1. Rodney is the captain of the hockey team. One day at practice he and another team
member crashed into each other. There was blood everywhere. Rodney hears a rumor
that this person he ran into may be infected with HIV and he wants to know if he is
infected. Can you be sure of your answer at this time? What should Rodney do if he comes
back negative at this time?
2. Jeanette is in a monogamous relationship, or so she thinks. She catches her boyfriend
in bed with one of her so called friends! Now Jeanette is worried and wants to take an HIV
test. What are her results?
3. Brooke just found out that she is HIV positive, and she isn’t sure exactly when she got
infected. She just had a daughter a couple of months ago and is worried that she may
have passed the infection on to her daughter. Is it possible for Brooke’s daughter to be
infected? What are the results?
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HIV TRUE OR FALSE
♦ You can get HIV from a toilet seat.
♦ You cannot get HIV from a blood transfusion.
♦ You can get HIV from giving blood.
♦ Some athletes are more likely to get HIV than non-athletes.
♦ You can get HIV from a mosquito bite.
♦ You cannot get HIV from drinking or eating after someone who is
HIV positive.
♦ You cannot get HIV from oral sex.
♦ Birth control pills keep you safe from HIV.
♦ You can contract HIV from coming into contact with someone’s
sweat who is infected with HIV.
♦ You can get HIV from an HIV vaccine.
♦ You cannot get HIV from kissing someone.
♦ You can get HIV from getting a tattoo or body piercing.
♦ Condoms protect you from getting HIV during sex.
♦ A baby born from an HIV infected mother can also get HIV.
♦ Sharing a hairbrush can spread HIV.
♦ You can contract HIV through sharing drug needles.
♦ You cannot get HIV by sharing toothbrushes.
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