2
exercise two
Bacteria and Protista
Learning Outcomes
•
•
•
•
•
Learn how to culture and identify different bacterial strains.
Compare the effectiveness between hand sanitizer and soap and water.
Compare and contrast different phyla of algae with a focus on Phylum Chlorophyta.
Describe the significance of the Volvocine line.
Examine the similarities and differences between members of Kingdoms Protista and Fungi.
INTRODUCTION
Bacteria
Organisms classified into Domains Bacteria and Archaea, the prokaryotes, are the oldest and most abundant organisms
on Earth. Present for over 1 billion years before the evolution of eukaryotes, cyanobacteria produced enough oxygen in the
atmosphere to promote the development of diverse eukaryotic species including protists, fungi, animals and plants.
Bacteria are ubiquitous in nature, inhabiting environments that range from the frozen ice in Antarctica to the digestive
tracts of ruminant animals. To date, greater than 7,000 bacterial species have been identified, some of which are pathogenic
to humans (e.g. Yersinia pestis, the causative agent of plague or Helicobacter pylori, which promotes ulcer formation) while others are important for food production, manufacturing pharmaceuticals and the decomposition of dying/decaying material
(saprophytes). In addition, some bacterial species form symbiotic (mutualistic) associations with other organisms in which
both partners benefit from the relationship. For instance, lichens are a mutualistic pairing of a fungus and a green alga or
cyanobacteria. In this partnership, the fungus provides the green alga/cyanobacteria with protection while the green alga/
cyanobacteria provides the fungus with food. Overall, bacteria are a diverse group of organisms that play vital roles in the
ecosystem.
Prokaryotes in general, are smaller and have a much simpler internal organization, lacking the membrane-bound
­organelles (figure 2.1) which is a characteristic of eukaryotes. The composition of the genetic material in bacteria is also very
different; in contrast to the multiple linear chromosomes found in the nucleus of eukaryotes, bacteria possess a single, circular
chromosome in the nucleoid region of the cell. The main differences between prokaryotic and eukaryotic organisms are
­summarized in Table 2.1.
Bacteria are generally single-celled (unicellular) organisms; however, some species (primarily the cyanobacteria) are
multicellular, forming associations of various sizes, filaments or colonies. Their classification is usually based on morphology
and biochemistry. The morphological characteristics include (1) shape and (2) the differential thickness of their cell wall.
There are three main types of bacterial shapes coccus (spherical), bacillus (rod-shaped), or spirillum (helical-shaped) which
are shown in figure 2.2.
The role of the bacterial cell wall is to maintain the shape of the cell. Depending upon the thickness of the peptidoglycan (protective) layer in the cell wall, bacteria are classified as either gram-positive or gram-negative. Gram-positive bacteria
(e.g. Streptococcus and Micrococcus) have much simpler cell walls with a very thick peptidoglycan layer that is capable of
retaining the Crystal violet dye (purple) used during the gram-staining procedure. Gram-negative bacteria (e.g. Escherichia coli
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Pilus
Cytoplasm
Ribosomes
Nucleoid (DNA)
Plasma membrane
Cell wall
Capsule
Flagellum
Pili
Figure 2.1
General structure of a prokaryotic cell.
Table 2.1
Main Differences Between Prokaryotic and Eukaryotic Organisms
Characteristic
Prokaryotic
Eukaryotic
Cellularity
Unicellular
Most are multicellular but not all
Cell size
1 µm or less
10 µm or more
Chromosomes
Single, circular
Multiple, linear
Cell division/genetic recombination
Asexual: Binary Fission Conjugation
Mitosis Sexual: Meiosis Asexual (some plants)
Compartmentalization
Absent
Present
Mitochondria
Absent
Present
Nucleus
Absent; nucleoid region instead
Present
Ribosomes
Present
Present
Flagella
Present
Present
Photosynthesis
Yes
Yes
Cell wall
Peptidoglycan
Absent in animals, present in plants, fungi and some protists
b. Bacill
a. Cocci
c. Spirilla
Figure 2.2
Types of Bacteria: a) cocci, b) bacillus and c) sprillium.
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and Serratia), on the other hand, possess a much thinner layer of peptidoglycan in their cell wall and thus, a reduced affinity
for Crystal violet. Instead, these bacteria stain dark pink from the Safranin dye also added during the gram staining process
(figure 2.3 and 2.4). Although gram-negative bacteria have less peptidoglycan, their cell walls are more complex due to the
presence of lipopolysaccharides which secrete potent toxins.
On the other hand, the biochemical characteristics refer to (1) whether or not they use oxygen for cellular respiration
(aerobic vs. anaerobic) and (2) whether they can use light to generate their own carbon sources (autotrophy) or if they
require organic molecules to obtain carbon (heterotrophy).
Peptide side
chains
Cell wall
(peptidoglycan)
Plasma
membrane
Protein
Gram-positive
bacteria
Lipopolysaccharides
Outer
membrane
Cell wall
Peptidoglycan
Gram-negative
bacteria
Plasma
membrane
Figure 2.3
Gram-negative vs. Gram-positive bacteria.
10 µm
Figure 2.4
Gram-positive (purple) and Gram-negative (pink) gram stain cells.
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Task 1—Bacterial Identification
A. Identifying Bacterial Types
1. View the prepared slides of the three bacterial shapes (bacillus, coccus, and spirillum) as well as the gram stained
bacteria located in the slide box at your table. Draw what you see in the spaces provided below.
Magnification:
Magnification:
bacillus
Magnification:
Magnification:
coccus
spirillum
Magnification:
Gram-negative bacteria
Gram-positive bacteria
Color: ___________
Color: ___________
B. Identification of Bacteria Cultured from Hands
Different bacterial species require different environments for growth. In fact, there are over 100 trillion bacteria
that live on or in humans (Costello et al., 2009), some of which aid in nutrition while others resist pathogens and
­maintain a normal, healthy flora. Listed in figure 2.5 are a few of the most common types of bacteria found in or on the
human body.
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Common Bacteria in or on Humans
Skin
Eye
Ear
Mouth Nose
Intestinal tract
Genital tract
Streptococcus
Corynebacterium sp., Staphylococcus sp., Streptococcus sp., Escherichia coli, Mycobacterium sp.
Corynebacterium sp., Neisseria sp., Bacillus sp., Staphylococcus sp., Streptococcus sp.
Staphylococcus sp., Streptococcus sp., Corynebacterium sp., Bacillus sp.
Streptococcus sp., Staphylococcus sp., Lactobacillus sp., Corynebacterium sp., Fusobacterium sp., Vibrio sp., Haemophilus sp.
Corynebacterium sp., Staphylococcus sp., Streptococcus sp.
Lactobacillus sp., Escherichia coli, Bacillus sp., Clostridum sp., Pseudomonds sp., Bacteroides sp., Streptococcus sp.
Lactobacillus sp., Staphylococcus sp., Streptococcus sp., Clostridum sp., Peptostreptococcus sp., Escherichia coli
Escherichia coli
Lactobacillus
Corynebacterium
Figure 2.5
Bacteria commonly found on/in humans.
In addition to environment, bacterial species also differ in their nutrient requirements. This factor makes it is possible
to isolate bacterial species by growing them on agar that has or is missing a particular nutrient necessary for growth of specific
bacterial species.
During last week’s lab, you cultured bacteria present on your hands “before” and “after” washing them with soap or
disinfecting them with hand sanitizer. Both agents are advertised as effective means of removing dirt, grease and certain bacterial strains, the main difference being that soap requires water for use while hand sanitizer, which is alcohol based, does not.
The purpose of the experiment you setup last week is twofold: (1) to compare the effectiveness of both disinfecting agents in
killing bacteria and (2) to identify the different strains of bacteria that are present on your hands when they have not been
washed/sanitized.
Figure 2.6
A. Blood agar and B. MacConkey agar plates after a 24 hour incubation period.
B: Identification of Bacteria Cultured from Hands
Procedure
1.
2.
3.
Obtain your group’s plates from the refrigerator.
Observe the growth and appearance of the colonies on all plates.
In the space provided below, you should draw what your Blood Agar and MacConkey plates look like. Make notes of
what color the colonies are. If you used hand soap, you should draw in the space below what your group member’s plate
looked like for those that used hand sanitizer or vice versa.
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Table 2.2
Comparison of Different Bacteria Strains
Occurrence on the skin
Appearance on
Blood Agar
Appearance on
MacConkey Agar
+
nearly 100%
small white colonies
no growth
spherical
+
~ 25%
“gold,” or yellowishwhite colonies
no growth
Streptococcus pyogenes
spherical
+
rare, > 5%
colonies exhibit large
zones of β-hemolysis
no growth
Corynebacteria
rod-shaped
+
nearly 100%
colonies exhibit a small
zone of β-hemolysis
no growth
Escherichia coli
rod-shaped
−
rare, > 5%
Bacterial Strain
Shape
Gram Stain (+/−)
Staphylococcus
epidermidis
spherical
Staphylococcus aureus
pink colonies
Blood agar plates
Before
After Soap
Before
After Sanitizer
Before
After Sanitizer
MacConkey agar plates
Before
4.
After Soap
Now it’s time to mount your bacteria onto a slide. If within your group, everyone had growth of each one of the
bacterial strains from Table 2.2 on the blood agar plate then you can do a total of 3 slides in the group. This way each
group member can mount one of the three bacterial strains. If someone had growth on the MacConkey Agar plate
then you can have a 4th slide.
a. Obtain a new slide.
b. Using a toothpick, transfer distilled water onto the slide. Do this about 2–3 times. Do not put a drop of water or it
will take too long to dry.
c. Using a toothpick, transfer a small amount of one colony of the bacterial strain you’re responsible for and add it to
the water that is on the slide. Mix the water and bacteria well. You will notice that the water will become cloudy.
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d. Place the slide on the staining tray and allow it to air-dry for about 5–10 minutes.
e. Using forceps pick up the slide and pass it 3 times over an ethanol lamp to heat fix the bacteria to the slide. Be
careful not to leave it in the flame too long or you will kill your bacteria.
f. Place the slide on the staining tray and add 2 drops of methylene blue to cover the sample of bacteria.
g. Leave the slide undisturbed for 1 minute.
h.Pick up the slide with forceps and hold it at a 45 degree angle above the staining tray while you rinse off the excess
dye with distilled water.
i. Place the slide on a paper towel and fold it over to lightly pat dry any excess water.
j. Examine the slide under the microscope. Keep in mind that your slide does not have a cover slip so you must be
very careful NOT to let the microscope objectives touch the actual slide.
k. Fill in the table below for question 1.
Questions
1. List the color of the colonies based on how they looked on the plate originally. You should also write the bacteria
shape you saw for each slide after you looked at it under the microscope. Based on the colony color and shape, write
what bacterial strain it most likely is. You can use Table 2.2 to help you.
Colony color
Bacteria Shape
Bacterial Strain
Slide 1
Slide 2
Slide 3
Slide 4 (optional)
2.
What ingredient(s) present in MacConkey agar inhibits the growth of gram positive bacteria?
3.
Streptococcus pyogenes and Corynebacteria form zones of β-hemolysis. What is β-hemolysis? What exactly does the
bacteria release that causes that type of growth on the blood agar plate?
4.
Did each group member’s “before” plates contain all the same bacterial strains? If not, which strain(s) was common to
all group members?
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5.
Which treatment, hand washing or hand sanitizer, was more effective at eliminating the bacteria on the hands?
i. Would eliminating all the bacteria on the hands be harmful? Explain.
ii.Which treatment would be best to remove dirt from a person’s hands? Why?
Kingdom Protista
Members of the Kingdom Protista are the earliest known eukaryotes, with fossils estimated to be 1.5 billion years old.
Although it is not possible to know exactly how eukaryotic cells arose, the endosymbiotic theory (figure 2.7) proposes that a
primitive eukaryotic cell engulfed an aerobic bacterium that had the necessary enzymes to derive energy from oxygen. In the
increasingly oxygenated Earth, aerobic respiration conferred a selective advantage on the eukaryotic host. Similarly, other
cells may have also engulfed photosynthetic bacteria, enabling them to become autotrophic. These aerobic and photosynthetic bacteria gave rise to modern-day mitochondria and chloroplasts respectively. Evidence in favor of the endosymbiotic
Chloroplast
Eukaryotic cell with chloroplast
and mitochondrion
Endosymbiosis
Photosynthetic bacterium
Mitochondrion
Eukaryotic cell with mitochondrion
Aerobic
bacterium
Endosymbiosis
Internal membrane system
Ancestral
eukaryotic cell
Figure 2.7
Endosymbiotic theory.
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theory is compelling since both mitochondria and chloroplasts possess characteristics similar to that of bacteria. Mitochondria
and chloroplasts have their own circular DNA, are surrounded by double membranes and divide by binary fission. It is believed
that a series of endosymbiotic events gave rise to the different organelles that characterize eukaryotic cells today. Of all the eukaryotic kingdoms, Kingdom Protista is the most diverse, consisting of organisms that lack distinguishing
characteristics of fungi, animals or plants. Members of this group are both unicellular and multicellular organisms that vary in
size, means of reproduction, locomotion and nutritional strategies. Protists are also broadly separated into three main groups,
(1) algae (plant-like), (2) slime molds (fungus-like) and (3) protozoans (animal-like), as illustrated in figure 2.8. In the
upcoming exercises you will examine representative species from these three groups.
Animals
Choanoflagellates
Fungi
Plants
Green algae
Brown algae
Diatoms
Water molds
Ancestral
eukaryote
Amoebas
Radiolarians
Foraminiferans
Ciliates
Dinoflagellates
Apicomplexa
Cellular slime molds
Acellular slime molds
Euglenids
Primitive parasitic organisms
Figure 2.8
Kingdom Protista cladogram.
Task 3—Examining members of the Kingdom Protista
A. Algae
Algae are an aquatic group of autotrophic organisms that commonly occupy marine and freshwater environments. Algae
are classified into 5 phyla, Chlorophyta, Phaeophyta, Rhodophyta, Chrysophyta and Euglenophyta, and can be differentiated
based on the types of pigments that each possesses. In addition to pigmentation, the different algal species also have ­disparate
modes of cellular organization (ranging from unicellular, filamentous to colonial), reproductive mechanisms (some reproduce
sexually, while others can reproduce both sexually and asexually) as well as the composition of their cell walls (see Table 2.3).
The differences between algal groups are enormous, but in this task you will focus on traits present in members of
­Phylum Chlorophyta, also known as the Volvocine line. The Volvocine line includes five genera (Chlamydomonas, Gonium,
Pandorina, Eudorina, and Volvox) of related organisms that show progressive changes in cell aggregation and specialization.
Chlamydomonas, for example, is a single celled, motile alga with a stigma (eyespot) that functions in the absorption of light.
Reproduction in Chlamydomonas is usually asexual except during times of environmental stress, when the organism produces
identically sized and shaped gametes (isogamy) for sexual reproduction. At the other end of the spectrum is Volvox, which in
contrast to Chlamydomonas, is colonial, has specialized cells for reproduction and is oogamous, (gametes produced are not
identical; one gamete is small and motile while the other is large and non-motile). While all members of the Volvocine line
can reproduce both sexually and asexually, oogamy is unique to Volvox. Similarly, while each genus possesses eyespots to sense
light and flagella for movement, cell polarity only becomes evident later in the Volvocine line, beginning with Pandorina.
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Table 2.3
Different Types of Algae
Phylum
Chlorophyta
Phaeophyta
Rhodophyta
Chrysophyta
Euglenophyta
Common name
Green algae
Brown algae
Red algae
Diatoms
Euglenoids
Pigments present
Chlorophyll a, b
Fucoxanthin
Phycobilins
Chlorophyll a,c
Xanthophyll
Chlorophyll a,b
Cell wall composition
Cellulose
Cellulose
Cellulose
Calcium carbonate
(some species)
Silicon dioxide (glass)
Protein
Distinctive structures
present
Stigma (some species)
holdfasts = rootlike
structures used for
attachment
Cellular organization
Unicellular
Filamentous
Colonial
Filamentous
Unicellular
Colonial
Filamentous (most
species)
Unicellular
Unicellular
Movement
Sessile & motile
Sessile
Sessile – attached
Motile
Motile – flagella
Example(s)
Chlamydomonas
Cladophora
Marcocystis (Kelp)
Fucus
Polysiphonia
Porphyra
diatomaceous earth
diatoms
Euglena
Stigma
Colony of cells
20-celled colony
(a)
(b)
Daughter colonies
Parent colony
Zygote
(c)
Figure 2.9
Phylum Chlorophyta: (a) Pandorina, a colonial green alga (400X). Pandorina forms small clumps of flagellated cells. (b) Eudorina (200X). This
colonial alga has many flagellated cells clustered in a gelatinous sphere. (c) Volvox (100X). A colony of Volvox is among the most complex of green
algae. Hundreds of flagellated cells are held together by thin cytoplasmic strands in a gelatinous sphere. Volvox reproduces asexually by producing
daughter colonies. It also produces motile sperm and large eggs that fuse to form a zygote for sexual reproduction.
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Centrate diatom
Diatoms
(b)
(a)
Pennate diatom
Striae
Raphe
Central nodule
(c)
Figure 2.10
Phylum Chrysophyta: (a) Strew of diatoms (40X). Diatoms are photosynthetic, mostly unicellular organisms with unique double shells made
of opaline silica, often ornately marked. The 11,500 species of diatoms have many shapes, including (b) centrate, round forms (400X) and
(c) pennate, elongated forms (400X).
Stipe
Receptacle
Holdfast
Pneumatocyst
Blade
Lamina
(a)
(b)
(b)
Figure 2.11
Phylum Phaeophyta: (a) Nerocystis. Brown algae, including kelps, are the most conspicuous seaweed and can form massive, complex organisms
with anchoring holdfasts and extensive photosynthetic blades. (b) Live Fucus (rockweed). The ends of the branches of this brown alga have
swollen receptacles dotted with conceptacles containing sex organs.
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Flagellum
Stigma
Second
flagellum
Reservoir
Basal body
Contractile
vacuole
Pellicle
Euglena
Tetraspores
Nucleus
Chloroplast
Paramylon
granule
(a)
(a)
(b)
(b)
Figure 2.12
Figure 2.13
Phylum Euglenophyta: (a) Generalized euglenoid. Some euglenoids are
autotrophic, some are heterotrophic, and some can alternate their feeding modes.
(b) Euglena (100X). These bright green Euglena are swimming among detritus.
Phylum Rhodophyta: Polysiphonia
Notes
•
•
o NOT contaminate the living samples by mixing the caps between the samples.
D
Do NOT completely close the caps on any of the living samples.
Task 3—EXAMINING MEMBERS OF THE KINGDOM PROTISTA
Procedure 1
1.
2.
3.
Prepare wet mounts of the Protists listed in table 2.4.
• Using a plastic pipette, place a drop from the tube that contains the live organism on a new slide.
• Position the edge of a coverslip against the water drop (at a 45° angle) and then slowly lower the coverslip onto the
slide. This is called a wet mount.
Observe the slides under a compound light microscope.
• Note: If you have a hard time finding live organisms, you can use the preserved slides that are located on your table.
Complete table 2.4 below.
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Table 2.4
The Volvocine Line
Genus ã
Characteristic å
Chlamydomonas
Gonium
Pandorina
Eudorina
Volvox
Number of cells
present in the field of
view
How many cells
make up the colony?
Cell Specialization
(Unicellular,
Filamentous,
Colonial)
Isogamy vs. oogamy
Drawing
(note magnification)
Questions
1. Explain the significance of the increased cell specialization of the Volvocine line.
2.
How does the stigma help algae survive?
3.
Which one of the organisms from the volvocine line is the simplest?
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4.
Which one of the organisms from the volvocine line is the most complex?
B. Protozoa
Protozoans (proto = first and zoa = animal) are unicellular, heterotrophic organisms that occupy marine, freshwater
and terrestrial environments. Members of this group are generally characterized by their mode of locomotion; (1) ameboid –
use psuedopods (Phylum Rhizopoda e.g. Amoeba), (2) ciliate – use cilia (Phylum Ciliophora e.g. Paramecium) and
(3) ­flagellate – use flagella (Phylum Sarcomastigophora e.g. Trypanosoma). In addition, some protozoans also possess a food
vacuole which is used to digest and absorb ingested materials and contractile vacuoles that function in expelling water.
­Reproduction in these organisms varies, but most genera reproduce asexually and sexually. Endoplasmic
reticulum
Food vacuole
Pseudopods
Mitochondria
Plasma
membrane
Amoeba
Nucleus
Nucleolus
(b)
(a)
(a)
Anterior contractile vacuole
Cilia
Food vacuole
Micronucleus
Gullet
Macronucleus
Vacuole
Pellicle
Posterior
contractile
vacuole
(c)
(c)
Cilium
Oral groove
Cytoproct
Contractile
vacuole
(d)
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Trypanosomes
(f)
(f)
(e)
(e)
Figure 2.14
Protozoa: (a) Generalized Amoeba (phylum Rhizopoda). Many amoebas are parasites but occur in all major environments, including soils. They
lack cell walls and have no sexual reproduction. Amoebas use pseudopodia to move and to capture prey. (b) Live Amoeba (40X), one of which is
surrounding a Paramecium. (c) Generalized Paramecium (phylum Ciliophora). (d) Paramecium (400X). All ciliates have cilia and two types of
nuclei—micronuclei and macronuclei. (e) (a) Sarcomastigophorans are commonly called flagellates because they have flagella. Trypanosomes
(phylum Sarcomastigophora) are common parasitic flagellates that cause African sleeping sickness and Chagas’ disease, believed to have led to
Charles Darwin’s death. They are spread by infection from biting insects such as mosquitoes and tsetse flies. (f) Trypanosoma cruzi (200X).
Procedure 2
1.
2.
3.
Prepare wet mounts of the Amoeba and Paramecium.
• Using a plastic pipette, place a drop from the tube that contains the live organism on a new slide.
• Position the edge of a coverslip against the water drop (at a 45° angle) and then slowly lower the coverslip onto
the slide.
View each specimen under the compound light microscope.
Complete Table 2.5 below.
4.
If prepared slides are available, compare these to your wet mounts.
Table 2.5
Protozoa
Phylum
Genus
Rhizopoda
Amoeba
Ciliophora
Paramecium
Description
Drawing (note magnification)
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Questions
1. Why is a contractile vacuole harder to see than a food vacuole?
2.
Compare and contrast the movement of Amoeba and Paramecium.
C. Myxomycota
Myxomycota, more commonly known as slime molds, are brightly-colored (yellow or orange), heterotrophic organisms
that exhibit amoeboid movement. Like fungi (mushrooms), slime molds are multinucleate, feed on dead/decaying material
(they are decomposers) and reproduce via spores produced in sporangia. However, in contrast to fungi, the cell walls of slime
molds are not made of chitin but instead are composed of cellulose.
Figure 2.15
Plasmodial slime mold, Physarum.
Figure 2.16
Basidiomycota, Amanita phalloides (death cap mushroom; usually fatal when eaten).
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Procedure 3
1.
Examine both Physarum (plasmodial slime mold) and Coprinus (button mushroom) with a dissecting microscope.
Phylum
Genus
Common Name
Myxomycota
Physarum
Slime Mold (Protist)
Basidiomycota
Coprinus
Button Mushroom
(Fungi)
Description
Drawing (note magnification)
1.
Why do you think that slime molds and fungus used to be classified in the same group?
2.
What are some differences between slime molds and fungus?
Task 4—FAST PLANTS
Check your fast plants and record any changes in your Fast Plant Chart in Appendix I. If they are dry, make sure to
water them.
Task 5—BASIL PLANTS
Check on your basil plants and record your data. If they are dry, make sure to water them.
REFERENCE
Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R. 2009. Bacterial community
variation in human body habitats across space and time. Science 326:1694–1697.
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