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&ANATOMY PHYSIOLOGYLab Manual

Customized for Morton University

John Smith

General Chemistry

Experiments

Jane Smith


A Laboratory Manual


John Smith

MICROBIOLOGY


Jane Smith

BIOLOGY Lab Manual


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John Smith

General Chemistry

Experiments

Jane Smith


A Laboratory Manual


John Smith

MICROBIOLOGY


Jane Smith

BIOLOGY Lab Manual

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2.10 Comparison of Four Bacillus Species77(A) B. cereus grown on sheep blood agar (SBA) produces distinctively large (up to 7 mm), gray, granular, irregular colonies. They often produce a “mousy” smell. Also note the distinctive extensions of growth along the streak line. (B) B. anthracis colonies on SBA resemble B. cereus, but are usually smaller and adhere to the medium more tenaciously. It must be handled at least in a BSL-2 laboratory, and sometimes BSL-3 if the cell density is high enough, so it is unlikely you will be testing its tenacity! (C) B. mycoides produces distinctive, rapidly spreading, rhizoid (note the branching) colonies. It is a common isolate from soil and is shown here on SBA. (D) This unknown Bacillus isolated as a laboratory contaminant produced a wrinkled, irregular colony with an irregular (wavy) margin on tryptic soy agar.

2.11 Effect of Age on Colony Morphology77(A) Close-up of Bacillus subtilis on sheep blood agar after 24 hours of incubation. (B) Close-up of Bacillus subtilis on sheep blood agar after 48 hours of growth. Note the wormlike appearance.

2.12 Clostridium sporogenes Grown Anaerobically on Sheep Blood Agar77These irregularly circular colonies have a raised center and a flat, spreading edge of tangled filaments (reminiscent of the mythological creature Medusa, who had snakes for hair!). They vary in size from 2 mm to 6 mm. C. sporogenes is found in soils worldwide.

A

B

A

B

C

D

SECTION 2 Microbial GrowthMICROBIOLOGY: Laboratory Theory & Application66

2


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232 Anatomy and Physiology Lab Manual School of Morton LAB 9 General and Special Senses 233

Exercise: Anatomy of the EyeSensation is broadly defined as the detection of changes in the inter-nal and external environments. Sensation may be conscious or sub-conscious, depending on the destination of the sensory information. For example, certain blood vessels have receptors that detect blood pressure. This information is taken to the brainstem, which makes changes as necessary to ensure blood pressure remains relatively con-stant. This information never makes it to the cerebral cortex, so you are not consciously aware of it.

The following exercises ask you to examine the anatomy and physiology of the special senses: vision, hearing and equilibrium, taste, and smell. You also will examine the general senses in this unit, which include touch, pain, and temperature.

The eye is a complex organ consisting of three components:

1. External structures, such as the eyelids (Figure 9.3),

2. Accessory structures, such as the lacrimal (LAK-rim-ul) gland (Figure 9.3), and

3. The eyeball (Figure 9.4).

Many of the external and accessory structures of the eye protect the delicate eyeball. Anteriorly, the eye is covered by the accessory structures known as the palpebrae (pal-PEE-bray), or eye-

lids. The internal surface of the eyelids and much of the anterior eyeball are covered with a thin mucous membrane called the conjunctiva (kahn-junk-TEE-vuh). Another accessory structure of the eye is the lacrimal apparatus, which produces and drains tears. The lacrimal apparatus consists of the lacrimal gland, located in the superolateral orbit, and the ducts that drain the tears it produces. The other major accessory structures are the extraocular muscles, which move the eyeball.

Lacrimal gland

Lacrimal canals

Lacrimal caruncle

Lacrimal sac

Nasolacrimal duct

Lateral canthus

Medial canthus

Superior palpebra

Inferior palpebra

FIGURE 9.3 External and accessory structures of the eye

◗◗ Eye models

◗◗ Preserved eyeballs

◗◗ Dissection equipment

◗◗ Dissection trays

◗◗ Snellen vision chart

◗◗ Dark green or blue paper

◗◗ Ruler

MaterialsThe eyeball itself is a hollow organ with three distinct tunics, or tissue layers (Figure 9.4):

1. Fibrous tunic. The outermost layer of the eyeball consists mostly of dense irregular connective tissue. It is avascular (lacks a blood supply) and consists of two parts:a. Cornea. The clear cornea makes up the anterior one-sixth of the fibrous tunic and is one of the

refractory media of the eyeball (it bends light coming into the eye). b. Sclera. The sclera (SKLAIR-uh) is the white part of the eyeball, which makes up the posterior five-

sixths of the fibrous tunic. It is white because of numerous collagen fibers that contribute to its thickness and toughness (in the same way a joint capsule or a ligament is tough and white).

2. Vascular tunic. Also called the uvea (YOO-vee-uh), the vascular tunic carries most of the blood supply to the tissues of the eye. It is composed of three main parts:a. Iris. The pigmented iris is the most anterior portion of the uvea. It consists of muscle fibers arranged

around an opening called the pupil. As the fibers contract, the pupil either constricts or dilates.b. Ciliary body. The ciliary body is located at the anterior aspect of the eye. It is made chiefly of the

ciliary muscle, which controls the shape of the lens. The muscle attaches to the lens via small sus-pensory ligaments.

c. Choroid. The highly vascular choroid makes up the posterior part of the vascular tunic. The choroid is brown in color to prevent light scattering in the eye.

3. Sensory tunic. This layer consists of the retina and the optic nerve. The retina is a thin, delicate structure that contains photoreceptors called rods and cones. a. Rods. Rods are scattered throughout the retina and are responsible for vision in dim light and for

peripheral vision. b. Cones. Cones are concentrated at the posterior portion of the retina and are found in highest

numbers in an area called the macula lutea (MAK-yoo-luh LOO-tee-uh). At the center of the macula lutea is the fovea centralis (FOH-vee-uh sen-TRAL-iz), which contains only cones. Cones are responsible for color and high-acuity (sharp) vision in bright light.

Anterior cavity(containing aqueous humor)

Optic nerve

Conjunctiva

Optic disc

Rods and cones

Choroid

Posterior cavity(containing vitreous humor)

Retina

Choroid

Sclera

Pupil

Ciliary body

Suspensory ligaments

Fovea centralisIris

LensCornea

Iris

Macula lutea

FIGURE 9.4 Eye sagittal section

Chapter 3 | The Building Blocks of Life: Understanding Microscopy and Cells) 67Biology in the Laboratory Morton University66

Elodea is a common plant that lives in freshwater habitats such as ponds and lakes. The leaves of Elodea are only a few cells thick and allow light to pass through the leaf without special preparation techniques. Refer to Figure 3.17 and Table 3.3 for references to plant cell anatomy.

1 Procure a microscope, a blank slide, coverslips, and a pipette. Carefully remove a single healthy leaf from the Elodea. Place the leaf in a drop of water on the blank slide with the top surface facing upward. (The cells on the upper surface are much larger and easier to observe.) Place a coverslip over the Elodea. Periodically check the leaf, making sure it does not dry out. If the leaf begins to dry, add a drop of water with a pipette.

2 Examine the leaf surface with the scanning and low-power objectives. Focus through the cell layers of the Elodea. Describe and sketch Elodea in the space pro-vided below.

3 Using the high-power objective, examine a single cell of Elodea. Attempt to locate the structures indicated in Figure 3.18. The gray-colored nucleus may be difficult to locate. The nucleus may become more evident if a drop of iodine is placed upon the leaf. In a good prepara-tion, the nucleolus may be evident. Carefully notice if

K ingdom Plantae includes some of the most conspicuous organisms on Earth. The plant kingdom con-tains approximately 280,000 species of multicellular, photosynthetic autotrophs. Plants vary in size

and complexity from the minute duckweed to the giant redwood tree. Two of the primary cell types found in plants are the collenchyma cells, which provide support in actively growing plants, and the epidermal cells, which cover and protect the underlying cells and tissues in leaves and stems.

Procedure 1

Representative Plant Tissues

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1231

23

●●●

❏❏ Compound microscope❏❏ Blank slide and coverslip❏❏ Pipette❏❏ Prepared slide of plant epidermal tissue

❏❏ Specimen of onion skin

Materials

Epidermal tissue in plants is usually made up of a single layer of living cells. They serve to cover and protect roots, stems, leaves, flowers, and fruits. Plant epidermis is composed of a closely packed single layer of living cells. Epidermal cells do not perform photosynthesis and do not contain chloroplasts.

1 Procure a microscope, prepared slide, and specimen.

Observing Eukaryotic Cells in Plant TissueEXERCISE

3.7Procedure 2

Elodea

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●●●

❏❏ Compound microscope❏❏ Living specimen of Elodea❏❏ Blank slides and coverslips❏❏ Pipette

Materials

Vacuole

Golgicomplex

Chloroplast

Cell wall

Cell (plasma) membrane

Rough endoplasmicreticulum

Nucleus

Nucleolus

Nuclear membrane(envelope)

Peroxisome

Smoothendoplasmicreticulum

Mitochondrion

FIGURE 3.17 Typical eukaryotic plant cell.

Elodea Magnification ________________

______________________________________

______________________________________

______________________________________

Wet mount of onion skin

Magnification __________________________

______________________________________

______________________________________

Plant epidermal tissue

Magnification __________________________

______________________________________

______________________________________

FIGURE 3.16 Epidermal cells from onion skin.

2002



John Smith

231

Senses and Sheep Eyeball Dissection

LAB

99Learning Outcomes

◗◗ Students will be able to measure bone length and height using the metric system.

◗◗ Students will use anatomical position terminology correctly.

◗◗ Students will correctly identify the major organ systems in the human body.

IntroductionIn this lab you will examine the senses—sight, hearing, taste, touch, and smell. You will be able to determine if your sensory pathways are in working order. You will dissect a sheep eye and learn the major anatomical features of the eye.

Pre-lab Exercise: SightEye AnatomyExtrinsic Muscles of the Eye

1. Label Figure 9-1 with the following:

extrinsic muscles inferior oblique inferior rectus lateral rectus medial rectus superior oblique superior rectus

trochlea (a piece of cartilage that acts as a pulley for the superior oblique)

FIGURE 9-1 Extrinsic muscles of the eye.


Jane Smith

BIOLOGY Lab Manual

65

The microscope is one of the most important and frequently used tools in the biological sciences. It allows the user to peer into the world of the cell as well as discover the fascinating world of microscopic

organisms. A typical compound microscope is capable of extending the vision of the observer more than a thousand times (Fig. 3.1). Other microscopes, such as the transmission electron microscope, can magnify objects up to 1 million times. Since its invention more than 300 years ago, the microscope has greatly improved our understanding of the cell, tissues, disease, and ecology.

The most commonly used microscope in the biology laboratory today is the light microscope. A simple light microscope can have a single lens, similar to the early microscopes. Compound microscopes use two sets of lenses to magnify an object. They are capable of a magnification range of 10–2,0002 and a resolution of 300 nanometers.

The Invisible WorldUnderstanding Microscopy 3

At the completion of this chapter, the student will be able to:

77 Discuss the importance of the microscope in biology.

77 Identify and describe the function of the parts of a compound micro scope.

77 Properly handle and care for a microscope and stereomicroscope.

77 Exhibit the proper technique when using and focusing a microscope.

77 Determine the total magnification of a compound microscope using different objectives.

77 Properly prepare a wet mount.

OBJECTIVES

FIGURE 3.1 The compound microscope is an essential instrument in the biology laboratory.

65

We focus on four core course areas:Anatomy & Physiology, General Biology, Microbiology, and General Chemistry.


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Microbiology Morton University Microbial Growth/Lab 2 Colony Morphology 6968

Procedure 1Sampling of Bacterial Colony Features

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❏❏ Lab coat❏❏ Disposable gloves❏❏ Chemical eye protection❏❏ Per Student Group❏❏ (Optional) colony counter, stereo (dissecting) microscope, or hand lens

❏❏ Metric ruler❏❏ Plates from Exercise 2-1❏❏ (Optional) tryptic soy agar or brain-heart infusion agar streak plate cultures of any of the following:

❏❏ Bacillus subtilis❏❏ Corynebacterium xerosis

Materials

Today you will be viewing colony characteristics on the plates saved from Exercise 2-1 and (if available) prepared streak plates provided by your instruc-tor. Figures 2.4 through 2.30 show a variety of bacterial colony forms and characteristics. Where applicable, contrasting environmental factors are indicated.

1 Working with your group, use the terms in Figure 2.4 and in the text to describe some representative colonies on your plates from Exercise 2-1 (if not already described) and the pure cultures supplied. Figures 2.5 through 2.30 may also be useful. Measure colony diameters (in mm) with a ruler and include them with your descriptions in the table on the data sheet, page 77. If you see a distinctive feature that has not been given a name, make up one! Just make it descriptive and easily understood by others. That’s what the early microbiologists did to compile the list you have been given. (Note: Remember that many microorganisms are opportunistic pathogens, so be sure to handle the plates carefully. Do not open plates with BSL-2 organisms on them or those containing fuzzy growth, because a fuzzy appearance suggests fungal growth containing spores that can spread easily and contaminate the laboratory and other cultures. If you are in doubt, check with your instructor.)

2 Unless you have been instructed to save today’s cultures for future exercises (such as Exercise 2-3), discard all plates in an appropriate autoclave container.

TheoryWhen a single bacterial cell is deposited on an appropriate solid nutrient medium, it begins to divide. One cell makes two, two make four, four make eight . . . one million make two million, and so on. Eventually a visible mass of cells—a colony—appears where the original cell was deposited. Color, size, shape, and texture of microbial growth are determined by the genetic makeup of the organism (in many cases by yet unknown mechanisms), but are also greatly influenced by environmental factors, including nutrient availability, temperature, and incubation time.

Colony morphological characteristics may be viewed with the naked eye, a hand lens, a stereo (dissecting) microscope, or a colony counter (Fig. 2.3). The seven basic categories include colony size, shape, margin (edge), surface, elevation, texture, and optical properties (Fig. 2.4).

1. Size is simply a measurement of the colony’s dimensions—the diame-ter if circular or length and width if shaped otherwise.

2. Shape may be described as round (circular), irregular, or punctiform (tiny, pinpoint).

3. The margin may be entire (smooth, with no irregularities), undulate (wavy), lobate (lobed), filamentous (unbranched strands), or rhizoid (branched like roots).

4. The surface may be smooth, rough, wrinkled (rugose), shiny, or dull.

5. The texture may be moist, mucoid (sticky), butyrous (buttery), or dry.

6. Elevations include flat, raised, convex, pulvinate (very convex), and umbonate (raised in the center).

7. Other useful features include color and optical properties such as opaque (you can’t see through it) and translucent (light passes through).

Features such as colony shape, margin, surface, texture (shiny or dull), and color are best viewed by observing from above while holding the plate level with the lid off (if it is safe to do so), but rocking it back and forth slightly so reflected light hits it at different angles. If allowed to do so, you may also check texture by touching the growth with an inoculating loop or wooden stick. Be sure to flame the loop afterward or dispose of the wooden stick properly.

Elevations are best viewed with the plate tilted slightly at eye level. Opacity and translucence are best viewed by placing the plate on a colony counter or holding it (lid on) so it is illuminated from behind (transmitted light). Colony dimensions are best measured from the plate’s base rather than through the lid.

When reporting colony morphology, it is important to include the medium and the incubation time and temperature, all of which can affect a colony’s appearance.

ApplicationRecognizing different bacterial growth morphologies on agar plates is a useful step in the identification process. It is often the first indication that one organism is different from another. Once purity of a colony has been confirmed by an appropriate staining procedure (this is not always done), cells can be transferred to a sterile medium, grown, and maintained as a pure culture, which then acts as a source of that microbe for identification or other purposes.

Colony MorphologyEXERCISE

2.1

2.3 Colony Counter n Subtle differences in colony shape and size can best be viewed with magnification, such as is provided by a colony counter. The transmitted light and magnifying glass allow observation of greater detail; however, colony color and many other features are best determined with reflected light. The grid in the background is a counting aid; each big square is 1 square centimeter.

Convex Umbonate Plateau Flat

Round Irregular Spindle

Elevation

Margin

Whole colony

Filamentous Rhizoid

Smooth,entire

RhizoidIrregular(erose)

Lobate Filamentous(Filiform)

Raised Raised,spreading

edge

Flat, raisedmargin Growth into

medium

2.4 A Sampling of Bacterial Colony Features n These terms are used to describe colony morphology. Descriptions also should include color, size, surface characteristics, texture, and optical properties (opaque or translucent). See the text for details.

48 Chemistry Lab Manual Morton University Lab 4 Elements and the Periodic Table 49

Part A: Flame Tests for Metal Ions

1 Label six test tubes 1 through 6.

2 To each test tube, add 10 drops of the appropriate metal ion solution as indicated below:

77 Test Tube 1: barium ion solution77 Test Tube 2: calcium ion solution77 Test Tube 3: lithium ion solution77 Test Tube 4: sodium ion solution77 Test Tube 5: potassium ion solution77 Test Tube 6: strontium ion solution

3 Clean a flame test wire by dipping it into a small amount of 6 M HCl and holding it in the flame of a Bunsen burner. Repeat this step two times.

4 Dip the test wire into test tube 1. Hold the wire loop into the tip of the burner flame. On the data sheet, page 55, record the color of the flame you observe.

5 Repeat steps 3 and 4 on the samples in test tubes 2 through 6. On the data sheet, record the color of the flame produced as each sample is heated.

NOTE: DO NOT discard the solutions in the test tubes. You will use them in Part B.

Part B: Reactions of Metal Ions

1 Prepare a boiling-water bath on a hot plate using a half-filled 250 mL beaker. Retain the water bath for Parts B and D.

2 To each of the test tubes used for the flame tests in Part A, add 5 drops of aqueous ammonium carbonate [(NH4)2CO3]. If a solid forms, record ppt (precipitate) in the table on the data sheet, page 55. If no solid forms, record NR (no reaction) in the table.

3 Dispose of the test tube contents as directed by your instructor.

4 Refill each test tube with 10 drops of the appropriate metal ion solution listed in Part A, step 2.

5 To each test tube, add 5 drops of aqueous ammonium hydrogen phosphate [(NH4)2HPO4]. Record your observations as ppt or NR on the data sheet table.


7 Again, refill each test tube with 10 drops of the appropriate metal ion solution.

8 To each test tube, add 5 drops of ammonium sulfate [(NH4)2SO4]. For any test tube in which you observe no precipitate formation, warm the solution by placing the test tube in the boiling-water bath for up to 5 minutes. Record your observations as ppt or NR on the data sheet table, including whether additional heat was necessary (` heat).


For example, you will use a “sodium ion solution” rather than elemental sodium. The periodic law applies to these ions in the same way that it applies to the parent atoms because elements within a group form ions that bear identical charges.

In Part A, you will perform a flame test on six solutions, each containing a single alkali or alkaline earth metal ion. A flame test involves placing a small amount of solution on a metal loop and holding the loop in the tip of a flame. As the substance is heated, the color of the flame is observed (Fig. 4.1). Each metal ion produces a different color, providing information that should help you distinguish between the six metal ions you examine.

In Part B, you will examine the behavior of the six metal ions in three different chemical reactions. For example, you will observe the separate reactions of a barium ion solution with solutions of ammonium carbonate, ammonium hydrogen phosphate, and am-monium sulfate. You may observe no reaction for the barium ions with these substances, or you may observe the formation of a white solid. A solid formed when two solutions undergo a chemical reaction is called a precipitate. The formation of a precipitate generally causes the solution to turn cloudy because the grains of solid are very tiny and remain suspended in the liquid (Fig. 4.2). Large crystals are rarely observed during a precipitation reaction.

You will be able to use your results from Part B to classify the six metal ions into two families based on similar patterns of reactivity. For example, if barium and potassium ions both form white solids when mixed with the same substances, then they belong in the same chemical family. If they display different reactivities, then they belong in different families.

In Part C, you will examine the distinct behavior of three halogen ions under identical reaction condi-tions. When mixed with bleach (sodium hypochlorite), the halogen ions undergo a similar reaction but each exhibits a different color. Thus, observing the color produced by this reaction should allow you to distin-guish between the three halogen ions.

In Part D, you will apply the testing procedures and data you gather from Parts A through C to a mys-tery solution in order to identify a metal ion and a halogen ion present.

Technique TipFlame tests can be difficult to read, so you may wish to repeat your flame tests several times in order to verify your observations. In addition, sodium often is present as an impurity in solutions of various alkali and alkaline earth metal ions. Sodium produces an orange-yellow flame test (Fig. 4.1). When present as an impurity, the sodium should produce only a weak orange-yellow color, and the color of the primary component of the solution should dominate. A true sample of sodium should produce an intense orange-yellow color during a flame test.

FIGURE 4.1 Flame tests involve holding a sample in a flame and observing the color produced.

FIGURE 4.2 When a precipitate forms, the solution changes appearance from clear to cloudy.

Hydrochloric acid (HCl) is a strong acid. Handle with caution! Certain metal ions are toxic. Use caution and follow all instructor safety precautions when handling these chemicals!

SAFETY NOTE

Materials❏❏ Test tubes

❏❏ Flame test wire

❏❏ Bunsen burner

❏❏ Hot plate

❏❏ 250 mL beaker

❏❏ Striker

❏❏ Test tube holder

A Laboratory Manual


John Smith

MICROBIOLOGY

Microorganisms are extraordinarily diverse, and every species demonstrates a unique combination of characteristics, some of which can be easily observed. In this section we illustrate some of those

characteristics and factors that affect them.You will begin this section with an exercise intended to sensitize you to the diversity of microbial

populations living all around us. Allowing for variables, such as the growth medium and incubation conditions, much can be determined about an organism by simply looking at the colonies it produces, or its appearance on slants or in broths. Distinguishing growth patterns on or in different media is an important skill—one that you can use as you progress through the semester. Note the growth characteristics of all the organisms provided for your laboratory exercises, and jot them down or even sketch them. When the time comes to identify your unknown species, you may find your records very useful.

Next, you will examine microbial nutritional diversity by growing bacteria on media with varying amounts of carbon and nitrogen resources. Following that, you will look at some environmental factors affecting microbial growth, such as oxygen, temperature, pH, and osmotic pressure. Finally, you will examine some physical and chemical microbial control agents and systems, that is, ways in which humans can control bacterial growth.

67

Microbial Growth 2

General Chemistry

Experiments

Jane Smith


The periodic law states that the physical and chemical properties of the elements recur in a

repeating pattern when they are ordered by increas-ing atomic number. On the periodic table (inside front cover), elements are arranged by increasing atomic number into horizontal rows and vertical columns. The rows are called periods, and the columns are called groups. This arrangement yields an important pattern: elements with similar properties are located within the same group. The pattern is a direct result of the fact that elements within a group possess the same number of valence electrons, those found in the outermost energy level of the atom. It is the valence electrons that are primarily responsible for an element’s reactivity. For that reason, a group on the periodic table can be thought of as a “family” of elements that share many chemical properties. Some of the groups on the periodic table provided with this text are labeled with the family name for ele-ments found within that group.

Because they share similar chemical properties, we often can identify which elements belong to the same family (i.e., group) by examining some of their chemical reactions. In this experiment, you will examine the reactivity of elements in three families: alkali metals, alkaline earth metals, and halogens. Like brothers and sisters, elements within a family still have individual characteristics that make them unique. In addition to identifying “family traits,” you will use your experiments to identify distinguishing characteristics of the individual elements within a group.

It is important to note that you will not use pure samples of elements during this lab. Pure alkali metals, for example, can explode when they come into contact with water in the air! During the lab, you will use aqueous (“water-based”) solutions that con-tain ions, which are charged forms of the elements.

ElementsLAB 4

47

Students should be able to:

• Describe the periodic law.

• Apply the periodic law to identify elements that share similar properties based on their locations on the periodic table.

• Properly perform flame tests.

• Use chemical changes to identify elements that share similar properties.

• Use chemical changes to distinguish between different elements.

• Apply data obtained in this experiment to identify metal and halogen ion components of a mystery sample.

Objectives

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1

Microorganisms Are Everywhere Background: Nutrient Agar Plates are generally made with some kind of nutrient mixture that includes vitamins, minerals, and sugar. These plates allow bacteria and fungi to grow when stored at appropriate temperatures. A new plate will have no microorganisms on it of any kind, and will be referred to as sterile. The jelly-‐like substance found in the plate is made from agar, which is a seaweed derived material that is a time-‐tested staple in microbiology. Agar melts into liquid at boiling, and once it solidifies at room temperature, it can be incubated (meaning stored for growth) at very high temperatures before re-‐liquefying. There are other kinds of agar plates which can be made from many kinds of recipes. Originally, bacteriologists cooked all manner of kitchen materials in a stew, which was boiled, strained, and then used as a growth medium. It is still possible to make some of these exotic agars today, including oatmeal molasses media and beef peptone broth!

Insert Figure 1.1 and Figure 1.2 w/caption: Agar plates with lids. Objective: Use agar plates to investigate bacterial and fungal presence on common objects. Materials: Nutrient Agar Plate Sterile Cotton Swabs Permanent Marker Experiment:

1. Obtain a plate from the instructor. On the bottom of the plate (meaning the part with the solid agar), label with your name, section, and create 4 quadrants. Number each quadrant.

2. Select four items/organisms/body parts to swab. Open a single sterile swab and firmly rub your chosen test object. If you are swabbing yourself, please remember to be specifically label the location: arm, elbow, ear, mouth, etc. and use good judgement about selecting such an area!

3. Gently swipe the swab back and forth in one of the quadrants on the agar surface. Do NOT tear the agar-‐ it is firm to the touch but too much friction will cause it to rip. You only need to transfer the microorganisms to the surface-‐ not embed them in the media.

4. Repeat the swabbing and swiping for each of the next three quadrants. 5. Invert the plate* (meaning turn it so the lid is on the bottom) and store in an incubator at 37C for at

least 24 hours. 6. Review the diversity of growth in your next lab.

Insert Figure 1.3 w/Caption: Incubator showing inner and outer doors.

*Tip: The plates are grown upside down to prevent the condensation that forms on the lid from falling on the plates and ruining the experiment. Drops of water falling on the agar surface will cause bacteria, yeasts and fungi to slide or spread and will prohibit meaningful conclusions of experiments. Insert Figure 1.4 and Figure 1.5 w/Caption: Bottom view and top view of plate growth.

Insert space to answer questions.

LAB 1: MICROORGANISMS ARE EVERYWHERE

Background Nutrient agar plates are generally made with some kind of nutrient mixture that includes vitamins, minerals, and sugar. These plates allow bacteria and fungi to grow when stored at appropriate temperatures. A new plate will have no microorganisms on it of any kind and will be referred to as sterile. The jelly-like substance found in the plate is made from agar, which is a seaweed derived material that is a time-tested staple in microbiology. Agar melts into liquid at boiling and once it solidifies at room temperature, it can be incubated (meaning stored for growth) at very high temperatures before re-liquefying. There are other kinds of agar plates which can be made from many kinds of recipes. Originally, bacteriologists cooked all manner of kitchen materials in a stew, which was boiled, strained, and then used as a growth medium. It is still possible to make some of these exotic agars today, including oatmeal molasses media and beef peptone broth!

[Insert Figures 1.1A and B] Figure 1.1 Agar plates with lids.

Objective

Use agar plates to investigate bacterial and fungal presence on common objects.

Materials

Nutrient agar plate

Sterile cotton swabs

Permanent marker

Experiment

1. Obtain a plate from the instructor. On the bottom of the plate (meaning the part with

the solid agar), label with your name, section, and create 4 quadrants. Number each

quadrant.

2. Select four items/organisms/body parts to swab. Open a single sterile swab and firmly

rub your chosen test object. If you are swabbing yourself, please remember to be

specifically label the location: arm, elbow, ear, mouth, etc. and use good judgement

about selecting such an area!


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An Inquiry-based Guideto Experimentation in

Microbiology

Customized for Passaic County Community College

Erica Foote

LAB 1

Microorganisms Are Everywhere

1

BackgroundNutrient agar plates are generally made with some kind of nutrient mixture that includes vitamins, minerals, and sugar. These plates allow bacteria and fungi to grow when stored at appropriate tempera-tures. A new plate will have no microorganisms on it of any kind and will be referred to as sterile. The jelly-like substance found in the plate is made from agar, which is a seaweed derived material that is a time-tested staple in microbiology. Agar melts into liquid at boiling and once it solidifies at room temperature, it can be incubated (meaning stored for growth) at very high temperatures before re-liquefying. There are other kinds of agar plates which can be made from many kinds of recipes. Originally, bacteriologists cooked all manner of kitchen materials in a stew, which was boiled, strained, and then used as a growth medium. It is still possible to make some of these exotic agars today, including oatmeal molasses media and beef peptone broth!

FIGURE 1.1 Agar plates with lids.

Students should be able to:

•• Use agar plates to investigate bacterial and fungal presence on common objects.

Objective

2 An Inquiry-Based Guide to Experimentation in Microbiology Passaic County Community College

Materials❏❏ Nutrient agar plate

❏❏ Sterile cotton swabs

❏❏ Permanent marker

FIGURE 1.2 Incubator showing inner and outer doors.

FIGURE 1.3 Plate growth: (A) bottom view; (B) top view.

Experiment

1 Obtain a plate from the instructor. On the bottom of the plate (meaning the part with the solid agar), label with your name, section, and create 4 quadrants. Number each quadrant.

2 Select four items/organisms/body parts to swab. Open a single sterile swab and firmly rub your chosen test object. If you are swabbing yourself, please remember to be specifically label the location: arm, elbow, ear, mouth, etc. and use good judgement about selecting such an area!

3 Gently swipe the swab back and forth in one of the quadrants on the agar surface. Do NOT tear the agar—it is firm to the touch but too much friction will cause it to rip. You only need to transfer the microorganisms to the surface—not embed them in the media.

4 Repeat the swabbing and swiping for each of the next three quadrants.

5 Invert the plate,* meaning turn it so the lid is on the bottom, and store in an incubator at 37°C for at least 24 hours.

6 Review the diversity of growth in your next lab.

A B

NOTE: The plates are grown upside down to prevent the condensation that forms on the lid from falling on the plates and ruining the experiment. Drops of water falling on the agar surface will cause bacteria, yeasts and fungi to slide or spread and will prohibit meaningful conclusions of experiments.

Lab 1 Microorganisms Are Everywhere 3

Lab 1 Questions

Name Date Section

1 Why are there different colors, sizes, and shapes visible on the plate after growth?

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2 What object or body area had the most microorganisms? Which had the greatest diversity?

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3 If a plate is left open on the bench (meaning the lid is off) for an hour, and nothing is swabbed on the surface, but this plate is incubated alongside all the others in the lab, will anything grow? If so, where did these organisms come from?

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4 Is it possible to grow an infectious organism from the environment by performing this experiment? Why or why not?

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