ap® investigation #7 - ap biology...

AP® InvestIgAtIon #7 Cell ProCesses: Mitosis and Meiosis – teaCher’s Guide

©2012, Ward’s Natural Science

All Rights Reserved, Printed in the U.S.A.

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Kit # 3674-07

abstraCt 1

General overview 1

reCordinG data 2

Material requireMents/CheCklist 4

CurriCuluM aliGnMent 5

learninG objeCtives 6

tiMe requireMents 5

safety PreCautions 7

Pre-lab PreParations 8

student Guide Contents

baCkGround 11

Part 1: Cell size & diffusion 13

Part 2: ModelinG osMosis in livinG Cells 17

Part 3: osMosis in livinG Plant Cells 21

assessMent questions/additional questions (oPtional) 24

Material safety data sheets

table of Contents

**AP® and the Advanced Placement Program are registered trademarks of the College Entrance Examination Board. The activity and materials in this kit were developed and prepared by WARD’S Natural Science Establishment, which bears sole responsibility for their contents..

WILL FIX ALL PAGE #’S ONCE EVERYTHING ELSE IS FINALIZED.



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Cell ProCesses: Mitosis and Meiosis – teaCher’s Guide Kit # 3674-07

AbstrACtIn this lab, students will examine and compare the phases of mitosis and meiosis in plant and animal cells. Students will then determine the relative time cells spend in each phase and calculate the distance between a specific gene and the chromosome centromere. Students will accomplish this by preparing slides, making observations using a compound microscope, and manipulating data.



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generAl overvIewThe College Board has revised the AP Biology curriculum to begin implementation in the fall of 2012. Advanced Placement (AP) is a registered trademark of the College Entrance Examination Board. The revisions were designed to reduce the range of topics covered, to allow more depth of study and increased conceptual understanding for students. There is a shift in laboratory emphasis from instructor-designed demonstrations to student-designed investigations. While students may be introduced to concepts and methods as before, it is expected that they will develop more independent inquiry skills. Lab investigations now incorporate more student-questioning and experimental design. To accomplish this, the College Board has decreased the minimum number of required labs from 12 to 8 while keeping the same time requirement (25% of instruction time devoted to laboratory study). The College Board has defined seven science practices that students must learn to apply over the course of laboratory study. In brief, students must:

1. Use models

2. Use mathematics (quantitative skills)

3. Formulate questions

4. Plan and execute data collection strategies

5. Analyze and evaluate data

6. Explain results

7. Generalize data across domains

The College Board published 13 recommended laboratories in the spring of 2011. They can be found at: http://advancesinap.collegeboard.org/science/biology/lab

Many of these laboratories are extensions of those described in the 12 classic labs that the College Board has used in the past. The materials provided in this lab activity have been prepared by Ward’s to adapt to the specifications outlined in AP Biology Investigative Labs: An Inquiry-Based Approach (2012, The College Board). Ward’s has provided instructions and materials in the AP Biology Investigation series that complement the descriptions in this College Board publication. We recommend that all teachers review the College Board material as well as the instructions here to get the best understanding of what the learning goals are. Ward’s has structured each new AP investigation to have at least three parts: Structured, Guided, and Open Inquiry. Depending on a teacher’s syllabus, s/he may choose to do all or only parts of the investigations in scheduled lab periods.

The College Board requires that a syllabus describe how students communicate their experimental designs and results. It is up to the teacher to define how this requirement will be met. Having students keep a laboratory notebook is one straightforward way to do this.



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reCordIng dAtA In A lAborAtory notebook

All of the Ward’s Investigations assume that students will keep a laboratory notebook for student-directed investigations. A brief outline of recommended practices to set up a notebook, and one possible format, are provided below.

1. A composition book with bound pages is highly recommended. These can be found in most stationary stores. Ward’s offers several options with pre-numbered pages (for instance, item numbers 32-8040 and 15-8332). This prevents pages from being lost or mixed up over the course of an experiment.

2. The title page should contain, at the minimum, the student’s name. Pages should be numbered in succession.

3. After the title page, two to six pages should be reserved for a table of contents to be updated as experiments are done so they are easily found.

4. All entries should be made in permanent ink. Mistakes should be crossed out with a single line and should be initialed and dated. This clearly documents the actual sequence of events and methods of calculation. When in doubt, over-explain. In research labs, clear documentation may be required to audit and repeat results or obtain a patent.

5. It is good practice to permanently adhere a laboratory safety contract to the front cover of the notebook as a constant reminder to be safe.

6. It is professional lab practice to sign and date the bottom of every page. The instructor or lab partner can also sign and date as a witness to the veracity of the recording.

7. Any photos, data print-outs, or other type of documentation should be firmly adhered in the notebook. It is professional practice to draw a line from the notebook page over the inserted material to indicate that there has been no tampering with the records.

For student-directed investigations, it is expected that the student will provide an experimental plan for the teacher to approve before beginning any experiment. The general plan format follows that of writing a grant to fund a research project.

1. Define the question or testable hypothesis.

2. Describe the background information. Include previous experiments.

3. Describe the experimental design with controls, variables, and observations.

4. Describe the possible results and how they would be interpreted.

5. List the materials and methods to be used.

6. Note potential safety issues.

(continued on next page)



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reCordIng dAtA In A lAborAtory notebook (continued)

After the plan is approved: 7. The step-by-step procedure should be documented in the lab notebook. This includes recording

the calculations of concentrations, etc., as well as the weights and volumes used.

8. The results should be recorded (including drawings, photos, data print outs, etc.).

9. The analysis of results should be recorded.

10. Draw conclusions based on how the results compared to the predictions.

11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance, and uncontrolled variables.

12. Further study direction should be considered.

The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following questions, which also might arise in peer review:

• Explain the purpose of a procedural step.

• Identify the independent variables and the dependent variables in an experiment.

• What results would you expect to see in the control group? The experimental group?

• How does XXXX concept account for YYYY findings?

• Describe a method to determine XXXX.



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MAterIAls ProvIded In kIt MAterIAls needed bUt not ProvIded

Units per kit

Description Garlic clove

8 Scalpels, disposable Compound microscope

1 box 72 Precleaned microscope slides, Absorbent wipes

1 box 100 22 mm plastic coverslips, Razor blades

1Live materials coupon for

Sordaria cross demo plate*Glass marking pens

8 Large wooden clothespin, 100 mL beaker

4 Prepared slide, fish mitosis Ruler

4 Prepared slide, onion root mitosis Toothpicks

15 Pipets, 6’’ grad. Box (or dark area to grow roots)

1 Hydrochloric acid, 1 M, 30 mL, Dissection scissors

1 Carbol fuchsin solution, 30 mL Timer

8 Disposable inoculating loop Forceps

600 Red pop beads Bunsen burner

600 Pink pop beads Goggles, aprons, and gloves

1 Sand oPtIonAl MAterIAls (not ProvIded)

32 Magnetic yellow centromeresPrepared sordaria squash slides (912200) or squash

cards (323377)32 Centrioles, clear NaC

1 70% isopropyl alcohol, 30 mL, pH 3 buffer and/or pH 10 buffer

1 Instructions (this document)Lectin

(phytohemmaglutinin PHA-m, increases mitosis)

Uric acid (decreases mitosis)

NaCl

MAterIAls CheCklIst

Call “Us” at 1.800.955.2660 for

Technical Assistance Visit “Us” on-line at

www.wardsci.com

for U.S. Customers

www.wardsci.ca

for Canadian Customers

or* - It is recommended that you redeem your coupon for live/

perishable materials as soon as possible and specify your preferred delivery date. Generally, for timely delivery, at least a week’s advance

notice is preferred.



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CUrrICUlUM AlIgnMentBig Idea

Big Idea 3: Living systems store, retrieve, transmit, and respond to information essential to life processes.

Enduring Understandings

3A1: DNA, and in some cases RNA, is the primary source of heritable information.

3A2: In eukaryotes, heritable information is passed to the next generation via processes that include the cell cycle and mitosis or meiosis plus fertilization.

3A3: The chromosomal basis of inheritance provides an understanding of the pattern of passage (transmission) of genes from parent to offspring.

3C2: Biological systems have multiple processes that increase genetic variation.

Science Practices:

1.2 The student can describe representations and models of natural or man-made phenomena and systems in the domain.

5.3 The student can evaluate the evidence provided by data sets in relation to a particular scientific question.

6.2 The student can construct explanations of phenomena based on evidence produced through scientific practices.

6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.

7.1 The student can connect phenomena and models across spatial and temporal scales.

7.2 The student can connect concepts in and across domain(s) to generalize or extrapolate in and/or across enduring understandings and/or big ideas.

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Kit # 3674-07

This lab activity is aligned with the 2012 AP Biology Curriculum (registered trademark of the College Board). Listed below are the aligned Content Areas (Big Ideas and Enduring Understandings), the Science Practices, and the Learning Objectives of the lab as described in AP Biology Investigative Labs: An Inquiry Approach (2012). This is a publication of the College Board that can be found at http://advancesinap.collegeboard.org/science/biology/lab.



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leArnIng objeCtIvesThe student can make predictions about natural phenomena occurring during the cell cycle (3A2 & SP 6.4).

The student can describe the events that occur in the cell cycle (3A2 & SP 1.2).

The student is able to construct an explanation, using visual representations or narratives, as to how DNA in chromosomes is transmitted to the next generation via mitosis, or meiosis followed by fertilization (3A2 & SP 6.2).

The student is able to represent the connection between meiosis and increased genetic diversity necessary for evolution (3A2 & SP 7.1).

The student is able to evaluate evidence provided by data sets to support the claim that heritable information is passed from one generation to another generation through mitosis, or meiosis followed by fertilization (3A2 & SP 5.3).

The student is able to construct a representation that connects the process of meiosis to the passage of traits from parent to offspring (3A3 & SP 1.1, SP 7.2).

The student is able to construct an explanation of the multiple processes that increase variation within a population (3C2 & SP 6.2).

tIMe reqUIreMents

Part 1: Mitosis (Structured Inquiry)

1a: Modeling Mitosis 20 minutes

1b: Onion and fish prepared slides 45 minutes

1c: Garlic Root Tip Squash 45 minutes

Part 2: Meiosis (Guided Inquiry)

2a: Modeling Meiosis20 minutes* * – Optional: This part may be done alongside Part 1a to save time.

2b: Meiosis & Sordaria Crossing Over 45 minutes

Part 3: Open Inquiry

NOTE: If you choose to have chemicals that modulate mitosis available for student directed inquiry, order these far enough in advance to assure they arrive prior to the start of the lab. Students will have to re-grow garlic roots as part of their experiments.

Varies, depending on students’ experimental designs.

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sAfety PreCAUtIons Lab-Specific Safety

70% isopropyl alcohol is poisonous if ingested, and will irritate the eyes. Wear safety goggles.Read the MSDS for this chemical.

Hydrochloric acid is a mild irritant. Avoid contact with the skin and eyes. Poisonous if ingested. Read the MSDS for this chemical.

General Safety

The teacher should be familiar with safety practices and regulations in their school (district and state). The teacher should know what needs to be treated as hazardous waste and how to properly dispose of non-hazardous chemicals or biological material.

Consider establishing a safety contract that students and their parents must read and sign off on. This is a good way to identify students with allergies to things like latex so that you (and they) will be reminded of what particular things may be risks to individuals. A good practice is to include a copy of this contract in the student lab book (glued to the inside cover).

Students should know where all emergency equipment (safety shower, eyewash station, fire extinguisher, fire blanket, first aid kit etc.) is located.

Make sure students remove all dangling jewelry and tie back long hair before they begin.

Remind students to read all instructions, Material Safety Data Sheets (MSDSs) and live care sheets before starting the lab activities and to ask questions about safety and safe laboratory procedures. Appropriate MSDSs and live care sheets can be found on the last pages of this booklet. Additionally, the most updated versions of these resources can be found at www.wardsci.com, under Living Materials http://wardsci.com/article.asp?ai=1346. (Note that in this particular lab, there are no live materials that require a live care sheet.

In student directed investigations, make sure that collecting safety information (like MSDSs) is part of the experimental proposal.

As general laboratory practice, it is recommended that students wear proper protective equipment, such as gloves, safety goggles, and a lab apron.

At end of lab:

All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness.

Remind students to wash their hands thoroughly with soap and water before leaving the laboratory.

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Pre-lAborAtory PrePArAtIon1 week before lab:

Redeem your coupon for your Sordaria demonstration cross plate at least one week prior to starting the lab to allow sufficient time for delivery. If the line of growth between the agar cubes appears dark and well-developed, store the plate in a refrigerator, agar side up, until ready to use. If the line of growth appears to be light, incubate at room temperature until it resembles the plate in Figure __________.

1 week - 2 days before lab:

Prepare the Garlic Root Tips for Part 1c: Garlic Root Tip Squash

If time is a concern, you may grow the garlic root tips in advance. You may also have your students grow the root tips; the following steps are also included in the students’ procedure.

1. Obtain a jar or Petri dish.

2. Using a ruler, measure 1.5 cm starting at the bottom of the container and mark the measurement with a permanent marker.

3. Fill the jar with the fine sand to the mark on the jar. You may need to sift or swirl the sand back and forth to ensure you have distributed the sand evenly.

4. Add water to the jar to wet the sand.

5. Separate a clove of garlic into individual sections, or “toes”. Remove the paper-like skin from each section and any dried roots on the primordia (blunt end) with a razor blade.

The section used must have root primordia present or it will not produce root tips. The root tips will grow within 2 days for very fresh garlic, or three days for older garlic. Because garlic has a mitotic cycle of approximately 12.5 hours, it’s important to “plant” the garlic and harvest the root tips at approximately the same time of day in order to get the greatest percentage of meristematic cells undergoing mitosis.

6. Place the cloves into the container with the root primordia (blunt end) in the sand.

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7. Place the jar in a box or dark place until the root tips have grown to a length of about 4 to 5 mm. Several viable root tips will grow on each section of garlic.

It is important that the garlic grows in the dark to ensure that it produces roots rather than shoots.

8. Remove the garlic from the box approximately one half-hour before performing the experiment to expose the root tips to light.

2 to 3 mm of the root tip can be cut off and stored in 70% isopropyl alcohol for up to 1 week.

Immediately before lab:

1. Make copies of Student Guide.

Copy pages __ to __ of the student copymaster prior to starting class.

2. Count out beads.

In preparation for modeling mitosis (Part 1a) or meiosis (Part 2a), you may count out the beads for each group. Provide each lab group with the following:

• 40 red pop beads

• 40 pink pop beads

• 4 yellow magnetic centromeres

• 4 clear plastic centrioles

extensIon ACtIvIty: loss of Cell CyCle Control Obtain photos of normal and Philadelphia type leukemia chromosomes. If time permits, students may obtain these via computer searches. This can also be done as part of a classroom or homework assignment.

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Pre-lAborAtory PrePArAtIon (ContInUed)



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objeCtIves

Make predictions about natural phenomena occurring during the cell cycle,

Describe the events that occur in the cell cycle.

Construct an explanation, using visual representations or narratives, as to how DNA in chromosomes is transmitted to the next generation via mitosis, or meiosis followed by fertilization.

Represent the connection between meiosis and increased genetic diversity necessary for evolution.

Evaluate evidence provided by data sets to support the claim that heritable information is passed from one generation to another generation through mitosis, or meiosis followed by fertilization.

Construct a representation that connects the process of meiosis to the passage of traits from parent to offspring.

Construct an explanation of the multiple processes that increase variation within a population..

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bACkgroUndReproduction in living things can be achieved through either asexual or sexual processes. In asexual reproduction, offspring organisms are genetically similar to the parent organism with limited, if any, introduction of DNA from another individual. In sexual reproduction, offspring organisms are generated by combining one half of the DNA from each of two parent individuals to produce a diploid (2N) offspring that has two complete sets of chromosomes and genes. The process of sexual reproduction enables greater genetic variation in offspring than asexual reproduction.

Multicellular, diploid, eukaryotic organisms use both types of cellular reproduction. Growth and development of the organism occurs through asexual reproduction of cells through the process of mitosis where every normal daughter cell is diploid, carrying two copies of each chromosome and set of genes. In mitotic reproduction, these daughter cells are genetic replicates of both the parent cell and the sister cell. Most single cell eukaryotes reproduce by simple mitosis, producing more replicates of the parent organism.

Mitosis

The mitotic cell cycle is a highly regulated process that insures accuracy in DNA replication and equal division into daughter cells. Defects in accurate reproduction will cause abnormalities that will result in decreased viability of both the daughter cells and the multicellular organism through a variety of mechanisms (including apoptosis and cancer).

Mitosis is a continuous process that can be described by a sequence of stages. The stages are defined by molecular and cytosolic events. Visual inspection of dividing cells through the microscope has defined three main stages (Figure 1) that can be further subdivided (Figures 2, 3 on the next pages):

Mitosis: The cells are separating DNA equally for two future daughter cells.

Cytokinesis: The daughter cells are separated into two individual cells,

Interphase: The cells look quiescent,

Figure 1

please check figure #’s - these were renumbered, for better clarity

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bACkgroUnd (ContInUed)

notes In interphase, the cell may be arrested in this part of the cell cycle (referred to as the G

0 part of interphase) as a diploid cell (2N). If the

cell continues through the next mitotic cycle, interphase only appears to be quiescent. This is when the cell grows in preparation of another cycle (G

1 ), DNA is replicated (S phase, cell becomes 4N), the DNA

checked for errors and repaired prior to mitosis (G2).

Figure 2: Interphase cell in onion root tip.

Interphase Prophase Metaphase Anaphase Telophase Cytokinesis

Figure 3: Visually distinct stages of the mitotic cycle in the onion root tip.

Mitosi s is subdivided into four stages that can be identified through the microscope:

Figure 4: Prophase –early, in onion root tip,

• The replicated DNA condenses around histone proteins and supercoils to form visible chromosomes

• The nuclear membrane dissolves

• Centioles form and begin to migrate to opposite sides of the cell

• Microtubules organize around the centrioles to form mitotic spindles

• Spindle fibers attach to the kinetochore proteins at the centromere (holding sister chromotids together).




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notes


Figure 5: Metaphase

• Centrioles complete migration to opposite poles of the cell

• Sister chromatids line up along the midline of the cell

Figure 6: Anaphase

• Spindles shorten, pulling sister chromotids apart to opposite poles of the cell

Figure 7: Telophase

• Chromosomes de-condense

• 2 nuclear envelopes form around the separated chromosomes (each nucleus is 2N again)

Cytokensis generally follows and divides the cytosol and cellular membrane to yield two cells that are identical. In plant cells, small vesicles move along microtubules to the mid line. These vesicles fuse to form a cell plate, which grows to form the cell wall that separates the cells.

Control of the cell cycle

To maintain normal growth and development it is essential that cells divide only when and where needed. Therefore, the transition between the stages of the cell cycle is a tightly controlled process. Levels of of proteins called cyclins build up in the cell and bind to cyclin-dependent kinase, forming CDK complexes. These CDK molecules either add or remove phosphate groups from substrates to move the cell to the next stage of the cycle. In order to progress, however, the cell must pass through several “checkpoints.” These checkpoints assure that cells only divide when needed and when DNA duplication is completed and without errors.



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notes


The three main checkpoints are:

Restriction Point – this occurs in G1. After division the daughter cells will continue to grow in size for a short time but are halted at the restriction point. Here cells will either terminally dif ferentiate and enter what is termed G0, or the cells will be stimulated a by growth factor and continue through the cycle again.

G2/M Checkpoint - occurs between the end of G2 and the beginning of mitosis. Specialized molecules read the newly formed DNA and will delay the cell from entering mitosis if there are strand breaks or if inappropriate nucleotides are incorporated. The cell cycle will only continue if repairs can be made, otherwise, the cell will die without completing the cycle.

Metaphase/Anaphase Checkpoint - early in mitosis. Specialized proteins on each centromere, called the kinetochores, activate if microtubules are attached and appropriate force is being applied. Activation allows the chromosomes to separate. If activation does not occur, mitosis will halt and the cell will die without completing the cell cycle.

Mutations in cells that lead to the loss of cell cycle control result in a loss of control over growth, cell differentiation, and death. Mutations may accumulate over time causing the cell clones to become a tumor; the tumor may later become metastatic if the cell acquires the ability to establish separate masses in distant tissues. These cancerous mutations usually affect genes which encode proteins involved in the control of other genes (transcriptional regulation), cellular metabolism (speeding up or slowing down cell growth), and/or the cell cycle of division and growth. Also affected are hormones and their receptors, regulatory molecules such as cytokines and their receptors, and DNA repair mechanisms.

Meiosis

Sexual reproduction occurs through the production of gametes which contain only one set of genes (haploid, 1N). When two gametes fuse, a new diploid organism with a different complement of gene copies than either parent is produced. Haploid gametes are produced through the process of meiosis.

Figure 8: Cell cycle subdivisions and

check points

referecne to figure 8 somewhere in here?

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Meiosis consists of one round of DNA replication followed by two nuclear divisions, meiosis I and meiosis II. This results in the formation of four daughter cells, each with only half the number of chromosomes of the parent. When two gametes combine during fertilization to form a zygote, the diploid chromosome number is restored in the resulting organism. Typically, we think of gametes as cells that come from either a male (sperm) or a female (ova) that are specialized to fuse with each other in species specific way (mouse sperm cannot fertilize a whale ovum).

Under a microscope, mitosis and meiosis do not look very different. The phases of division are visually distinct and named as their visual look-alikes in mitosis. However, the way replicated DNA is divided is very different in meiosis than mitosis.

Prophase I:

The duplicated sister chromosomes condense and are attached at a centromere as in mitosis. Unlike mitosis, these sister pairs form a tetrad with duplicated chromosome homologs from both the maternally and paternally contributed chromosomes. At this point DNA may cross over between non-sister, chromatid homologs forming unique chromatids that contain DNA from both maternal and paternal chromosomes.

Metaphase I:

The tetrads align at the center of the cell and spindle microtubules attach to the kinetochores.

Anaphase I:

The tetrads separate into two, duplicate chromosomes that remain attached at the centromere which move to separate poles of the cell.

Anaphase I is followed by telophase 1 and cytokinesis, resulting in two daughter cells that each have 2N DNA.

Mitosis II follows immediately after meiosis I is completed without further duplication of the DNA. The single set of chromosomes (held together at the centromere) are separated on the second spindle thus forming daughter cells that are now 1N. A starting diploid germ cell has now formed 4 haploid gametes, each containing a mixture of DNA


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from both the mother and the father. This method of division produces gametes that vary greatly between each other with respect to the allele combinations available to pass to the next generation.

Figure 9 (reprinted from College Board) Crossover events can be observed in fungal asci resulting

from meiotic divisions

The example of meiosis that will be used in this investigation is Sordaria fimicola. S. fimicola is an ascomycete fungus that is haploid for the bulk of its life cycle; the haploids comprise the individual fungal filaments, called hyphae, which normally exist in a mass, called a mycelium, representing the “body” of the fungus, and the ascospores, from which mycelia develop. The only diploid portion of the life cycle of S. fimicola occurs when the nuclei of specialized hyphae come together.


notes


Figure 10: Life cycle of the fungus, Sordaria.



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notes


These hyphae fuse to form a diploid zygote. This zygote then undergoes meiosis to produce the haploid ascospores, yielding four haploid nuclei contained in a sac called an ascus. After meiosis I and II, the four haploid nuclei undergo mitosis, resulting in an ascus containing eight haploid ascospores from one diploid zygote. Many asci form inside a fruiting body called a perithecium (Figure_5_).

Wild types sordaria are a dark brown, but we will grow this culture in the presence of a tan mutant so that when mycelium from a wild type fuses with the tan to form a zygote, we will be able to observe the results of crossing over events that occurred as the zygote underwent meiotic division. Figure 6 shows a culture plate that was seeded with two squares of wild type sordaria containing agar and two squares of tan type sordaria containing agar. The mycelium grow out from these blocks so fusion will occur where the two types meet. This region of fusion is where you will find perithecium containing ascospores from zygotes that came from a wild type and a tan parent (arrows). After meiosis I and II, the haploid ascospores will be either tan or wild type. When the parathecium are gently ruptured, the ascospores will be in strings of 8 that can easily be scored as tan or wild.

Figure 11 Figure 12???

Insert SORDIA Image on Page 7 250-7052???

Figure 12: Meisosis with no crossing over



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How these ascospores are arranged within the ascus is a direct representation of whether or not crossing over has occurred between the centromere and the site for the gene for ascospore color. If no crossing over has occurred, the ascospores will be arranged in a 4:4 manner. If crossing over has occurred, they will occur in a 2:4:2 or 2:2:2:2 manner(s).

Figure 13: Meisosis with crossing over

By observing the ascospore arrangement, the percentage of asci exhibiting crossover can be determined. This frequency appears to be affected largely by the distance from the gene to the centromere. From the crossover frequency, the distance in map units from the gene for ascospore color, and the chromosome centromere, can be calculated.


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notessAfety PreCAUtIons

When working with the chromatography solvent, use a chemical hood or proper ventilation.

As general safe laboratory practice, it is recommended that you wear proper protective equipment, such as gloves, safety goggles, and a lab apron.

As general lab practice, read the lab through completely before starting, including any Material Safety Data Sheets (MSDSs) and live materials care sheets at the end of this booklet as well as any appropriate MSDSs for any additional substances you would like to test. One of the best sources is the vendor for the material. For example, when purchased at Wards, searching for the chemical on the Ward’s website will direct you to a link for the MSDS. (Note: There are no live materials care sheets included in this particular lab.)

At the end of the lab:

All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness.

Wash your hands thoroughly with soap and water before leaving the laboratory.

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ProCedUre tIPs

When performing this lab activity, all data should be recorded in a lab notebook. You will need to construct your own data tables, where appropriate, in order to accurately capture the data from the investigation.

If directed to do so by your teacher, this part of the lab may be done at the same time as Part 2 of the lab.

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PArt 1 – strUCtUred InqUIry

MAterIAls needed Per lAb groUP??? Red pop beads??? Pink pop beads?? Clear plastic cetrioles?? Magnetic yellow centromeres

PArt 1A – ProCedUre: ModelIng MItosIsUsing the pop beads (red and pink), the magnetic yellow centomeres, and the clear plastic centrioles, simulate each stage of mitosis.

To simulate Interphase – DNA replication:

1. Construct two strands of seven red pop beads and attach each strand to a yellow centromere. Repeat with two strands of seven pink pop beads and a yellow centromere. These will represent a homologous pair of chromosomes (red from the father and pink from the mother).

2. Picture an imaginary boundary in the center of your desk. This boundary will represent the nuclear membrane. Place the chromosomes in the center of the imaginary nucleus.

3. DNA replication occurs, producing a duplicate of each chromosome. Construct two chromosomes identical to the ones you made previously. Each of the duplicated chromosomes is called a chromatid. Join both red chromatids at the centromere to form a pair of sister chromatids. Repeat this process for the pink chromosome.

4. Place a pair of plastic centrioles, at a ninety degree angle, just outside of your nuclear membrane. The centrioles also replicated during interphase, so place another pair next to them in your cell.

TIP: It may be helpful to tape your centrioles together during the exercise.

5. Illustrate your simulation of interphase and in your own words provide a brief description of what happens during interphase.

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please fix/edi list for this all parts of this activity or remove completely

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To simulate Prophase:

1. Move your two pairs of centrioles to opposite poles (sides) of the cell (your desk).

2. Illustrate your simulation of prophase and in your own words provide a brief description of what happens during prophase.

To simulate Metaphase:

1. Center your chromosomes along an imaginary metaphase plate with the centrioles still at the opposite poles of the cells.

2. Illustrate your simulation of metaphase and in your own words provide a brief description of what happens during metaphase.

To simulate Anaphase:

1. Separate and move the centromeres of each chromosome toward opposite poles of the cell. Notice how the arm of each chromosome trails the centromeres to the poles.

2. Illustrate your simulation of anaphase and in your own words provide a brief description of what happens during anaphase.

To simulate Telophase and Cytokinesis:

1. Move one red strand and one pink strand to the centrioles they were heading toward during anaphase. Imagine a cleavage furrow developing between each nuclei and separating the cell into two daughter cells.

2. Note how each cell now contains one red and one pink chromosome, as well as one pair of centrioles, exactly like the cell with which you began.

3. Illustrate your simulation of telophase/cytokinesis and in your own words provide a brief description of what happens during telophase/cytokinesis.

ProCedUre – PArt 1A: ModelIng MItosIs (ContInUed)

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notes


ProCedUre– PArt 1b: observIng MItosIs In AnIMAl And PlAnt Cells

1. Obtain a compound microscope, the fish blastula slide and the onion mitosis slide.

2. Observe the prepared microscope slide of onion root tip first at 100X (10X objective and 10X ocular) then at 400X (40X objective, 10X ocular). Look for cells in mitosis.

Depending on the quality of your microscope you may be able to distinguish the various phases of mitosis.

Use oil immersion if available.

Using the mitosis illustration provided on page ___ as a guide, try to identify each phase of animal cell mitosis.

3. Observe the prepared microscope slide of the fish blastodisc mitosis first at 100X, then 400X. Look for cells in mitosis and classify the stages. Compare and record similarities and contrast differences between the animal and plant cell mitosis.

4. Examine at least three fields of view of the apical meristem of the onion root tip at 400X. In each view, count and record the number of cells in interphase and the various stages of mitosis.

5. Calculate the percentage of total cells counted in interphase and in each stage of mitosis. If the individual phases cannot be discerned, calculate the percentage in interphase and in mitosis.

Table 1

Stage# cells

field 1

# cells

field 2

# cells

field 3

Total #

cells

% total

number

of cells

Time

in each

stage

Interphase

Prophase

Metaphase

Anaphase

Telophase

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notes


ProCedUre– PArt 1b: observIng MItosIs In AnIMAl And PlAnt Cells (ContInUed)

6. Assuming that it takes an average of 24 hours (1,440 minutes) for onion root tip cells to complete the cell cycle, calculate the amount of time cells spent in each phase of the cycle. Use the formula provided below. Enter your results in Table 1.

% of cells in phase x 1,440 minutes = _______ minutes cell spent in phase

7. Make a pie chart representing the amount of time spent in each stage of mitosis.

Prophase is normally the longest phase of mitosis, due to the complexity of the events occurring during prophase. These complex events take a relatively long time for the cell to perform:chromatin condensing and thickening, nuclear membrane dissolving, and the early stages of spindle development.

ProCedUre– PArt 1C: gArlIC root tIP sqUAshDue to time constraints, your instructor may have grown garlic root tips in advance. If this is the case, begin with Step 8.

1. Obtain a jar or Petri dish.

2. Using a ruler, measure 1.5 cm starting at the bottom base of the jar and mark the measurement with a permanent marker.

3. Fill the jar with the fine sand to the mark on the jar. You may need to sift or swirl the sand back and forth to ensure you have distributed the sand evenly.

4. Add water to the jar to wet the sand.

5. Separate a clove of garlic into individual sections, or “toes.” Remove the paper-like skin from each section and any dried roots on the primordia (blunt end) with a razor blade.

The section used must have root primordia present or it will not produce root tips. The root tips will grow within two days for very fresh garlic, or three days for older garlic. Because garlic has a mitotic cycle of approximately 12.5 hours, it is important to “plant” the garlic and harvest the root tips at approximately the same time of day in order to get the greatest percentage of meristematic cells undergoing mitosis.

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notes


ProCedUre– PArt 1C: gArlIC root tIP sqUAsh

(ContInUed)

6. Place the cloves into the jar with the root primordia (blunt end) in the sand.

7. Place the jar in a box or dark place until the root tips have grown to a length of about 4 to 5 mm. Several viable root tips will grow on each section of garlic.

It is important that the garlic grows in the dark to ensure that it produces roots rather than shoots.

Remove the garlic from the box approximately one half-hour before performing the experiment to expose the root tips to light.

8. Blot as much excess water from the root tips as possible. Any excess water on the slide will affect your results. Do not allow the root tips to dry out, however.

9. Using a scalpel, cut off the end of one of the emergent root tips; the section should be approximately 1 to 2 mm long. Place the root tip on a clean microscope slide and apply two or three drops of hydrochloric acid (HCl) to the root tip.

10. Holding the slide with a clothespin, pass it through the flame of a Bunsen burner for five seconds.

Pass the slide through the flame of the Bunsen burner. Do not hold the slide directly in or over the flame.

11. Without harming the root tip, blot the specimen with a paper towel to remove the excess HCl.

You may wish to touch a corner of the paper towel to the drop on the slide and allow the paper towel to soak it up. This may not remove the liquid from the slide as effectively as blotting, but it will not disturb the root tip.

12. Add a few drops of carbol fuchsin stain, covering the root tip.

13. Pass the slide through the flame of the Bunsen burner for two minutes. Let the slide stand for one minute.

Pass the slide through the flame of the Bunsen burner. Do not hold the slide directly in or over the flame.

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notes

ProCedUre– PArt 1C: gArlIC root tIP sqUAsh

(ContInUed)

14. Without disturbing the specimen, use a paper towel to remove the excess stain.

15. Cover with a coverslip. Using a pencil eraser or other blunt instrument, gently press down on the coverslip to squash and spread out the root tip. Blot off the excess stain, if any, that may have come out from under the coverslip.

16. Observe the slide under a microscope at 100X. Locate the apical meristem. Examine the slide at 400X. Locate cells in the various stages of mitosis, and make sketches of what you find.



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PArt 2 – gUIded InqUIry: MeIosIs

MAterIAls needed Per lAb groUPPLEASE LIST FOR THIS PROCEDURE OR REMOVE COMPLETELY

PArt 2A – ProCedUre: ModelIng MeIosIs1. Using the colored pop beads follow a similar procedure as you

did for mitosis, but model meiosis I and meiosis II.

PArt 2b – ProCedUre: MeIosIs And Sordaria CrossIng over

1. Place a drop of water on each of the clean microscope slides with an inoculating loop. Within this procedure, you will prepare three slides to get an adequate sampling of hybrids.

2. With an inoculating loop, scrape several perithecia from the demo cross plate. Scrape the perithecia from the interface of the two crossing strains (Figure _6_) close to the edge of the plate and place in the drop of water on the slide. Avoid picking up agar along with perithecia; it will interfere with the results.

3. Cover the slide with a coverslip. Using a pencil eraser or other blunt instrument, gently press down on the coverslip to squash and spread out the perithecia. The pressure should be sufficient to squeeze asci from the perithecia, but not enough to crush the asci themselves.

Observe the slide under a microscope at 100X. Locate the asci. Then view the slide at 400X to determine the color of the ascospores. The slide preparation should show collapsed perithecia and asci clusters (rosettes), with mature ascospores in various arrangements. Immature ascospores will all be light colored. Since Sordaria fimicola is homothallic, the preparation will show both hybrid and self-fertilized perithecia, however, fertilization will not occur very far from the line of contact between the two varieties.

4. Count approximately 50 hybrid asci from at least three fields of view, preferably from different slides. Record the data in your laboratory sheet or notebook.

q

ProCedUre tIPs

If directed to do so by your teacher, this part of the lab may be done at the same time as Part 1a of the lab.

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Figure __: Sordaria squash



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notes

5. Calculate the frequency (%) of asci crossing-over.

% Cross over = # showing cross overtotal counted

x 100

TIP: The percentage of crossing-over must be divided by two, since only half the ascospores in each hybrid ascus are the result of crossing over.

6. Determine the number of map units between the centromere and the gene for ascospore color.

7. Compare your data with the class and make an account for the classrooms data versus published data about sordaria crossing over. The published distance between the tan gene and the centromere is 26 map units.

8. Disposeoflabmaterials.

NOTE: Properly dispose of sharps.

When finished with the sordaria plate, please dispose of it in one of the following ways:

• Use a 20% bleach solution for 10 minutes.

• Place the organism in 70% isopropanol alcohol for 24 hours.

• Autoclave the organism at 121 °C for 15 minutes in an autoclave bag. The Petri dish will melt in the autoclave so be sure to bag your organism and close securely before autoclaving.

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PArt 2b – ProCedUre: MeIosIs And Sordaria CrossIng over (ContInUed)



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PArt 2 AssessMent qUestIons 1. How does meiosis lead to genetic variability within a population? Use S. fimicola as an example.

Meiosis leads to genetic variability through the segregation of gene alleles, the independent assortment of genes, and crossing-over, as well as the variability that results from the combination of the genetic material from the gametes of two genetically different individuals.

2. How does genetic variability represent an adaptive advantage for organisms that reproduce sexually?

With increased variability among individuals in a population comes an increased probability that the population would be able to survive under changing environmental and evolutionary pressures. This ability, in turn, gives the species a better chance of surviving and thriving in the future.

3. Why is S. fimicola an ideal organism for the demonstration of crossing-over?

The fact that it displays both haploid and diploid stages of reproduction allows scientists to easily manipulate different strains of the organism. The colored ascospores are easily identified and the ascospore patterns readily indicate when crossing-over has occurred.

4. Research cell division in prokaryotic and eukaryotic cells, compare and contrast the characteristics of mitosis in each.

Unlike eukaryotic cells, prokaryotic cells utilize neither mitosis nor meiosis. Prokaryotic cells replicate through a process known as binary fission. The prokaryotic DNA, which is found free in the cell and not confined in a membrane-bound nucleus, replicates and each copy attaches to a different part of the cell’s plasma membrane. The cell grows and, when it is approximately double its original size, the membrane grows inward, dividing the cell into two genetically identical daughter cells.

5. Using what you know about mitosis, genetic transcription, cell cycle, and the two karyotype images answer the following questions:

How do cells ensure DNA replication is accurate?

The DNA polymerase has a 3’---5’ exonucluease domain. This domain is responsible for proofreading the DNA and reads in the opposite direction of DNA replication.

According to the images, on which chromosomes did the mutation occur? By which mechanism of cell replication do you think the Philadelphia chromosome occurs? Explain.

????

Assuming that it takes an average of 12 hours for CML cells to complete the cell cycle, how many cells will be produced within a week (7days)?

4,096 cells will develop in one week (168 hours/week)

Would you agree that cancer is the disease of mitosis? Explain.

Answers will vary, but the students should be strongly considering cancer as a disease of mitosis.



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PArt 3: Cell ProCesses: MItosIs And MeIosIs

oPen InqUIry: desIgn An exPerIMentWhat questions occurred to you as you investigated mitosis and meiosis? Now that you are familiar with a mitotic system (onion root tip squash) and a meiotic system (Sordaria ascus formation using a tan mutant) design an experiment to investigate one of your questions.

Questions may involve differences in mitotic rates in different cell types, what types of environmental signals would inhibit or accelerate mitosis, what types of environmental conditions would result in the de-regulation of mitosis or meiosis, are there conditions that would inhibit or accelerate crossing over during meiosis, how might mitosis proceed differently in a cancerous cell line, how are germ cells that produce haploid daughter cells different from somatic cells that produce diploid cells?

See optional materials not provided for some agents that affect mitotic rate in some systems.

Before starting your experiment, plan your investigation in your lab notebook. Have your teacher check over and initial your experiment design. Once your design is approved, investigate your hypothesis. Be sure to record all observations and data in your laboratory sheet or notebook.

Use the following steps when designing your experiment.

1. Define the question or testable hypothesis.

2. Describe the background information. Include previous experiments.

3. Describe the experimental design with controls, variables, and observations.

4. Describe the possible results and how they would be interpreted.

5. List the materials and methods to be used.

6. Note potential safety issues.

After the plan is approved by your teacher:

7. The step by step procedure should be documented in the lab notebook. This includes recording the calculations of

exPerIMent desIgn tIPs

The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following ques-tions, which also might arise in peer review:

Explain the purpose of a procedural step.

Identify the independent variables and the dependent variables in an experiment.

What results would you expect to see in the control group? The experimental group?

How does XXXX concept account for YYYY findings?

Describe a method to determine XXXX.

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concentrations, etc. as well as the weights and volumes used.

8. The results should be recorded (including drawings, photos, data print outs).

9. The analysis of results should be recorded.

10. Draw conclusions based on how the results compared to the predictions.

11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance and uncontrolled variables.

12. Further study direction should be considered.

notes

PArt 3: oPen InqUIry (ContInUed)



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extensIon ACtIvIty: loss of Cell CyCle ControlNOTE: Cancer was chosen as the example for this exercise because it demonstrates what occurs when a cell loses control of the cell cycle. There is a possibility that your student will have a family member with cancer, or who died of cancer. It may be appropriate to suggest that they discuss the issue with their family or seek the advice of a medical practitioner.

ProCedUreWhen performing this lab activity, all data should be recorded in a legal scientific notebook or on the laboratory sheet provided. Students will need to construct their own data tables, where appropriate, in order to neatly and accurately capture the data from the lab activity and their investigations.

Leukemia is a type of cancer that is characterized by the uncontrolled growth of a specific type of leukocyte (white blood cell). The Philadelphia chromosome causes several kinds of leukemia; for example, Chronic Myelogenous Leukemia (CML). During this part of the lab, compare karyotypes between normal and CML.

Search the Internet for images of normal and CML karyotypes. Be sure to print the image as it will be needed for the next step.

Compare and contrast between a normal chromosomes vs. CML chromosomes.

Note. Be sure both karyotypes are from the same sex, either both male or both female.

notes



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MAterIAl sAfety dAtA sheets


Material Safety Data Sheet Page 1 of 2

© 2008, Scholar Chemistry. All Rights Reserved. 1/12/2012

Carbol Fuchsin Stain MSDS # 164.00 Section 1: Product and Company Identification

Carbol Fuchsin Stain Synonyms/General Names: Carbol Fuchsin, Biological Stain Product Use: For educational use only Manufacturer: Columbus Chemical Industries, Inc., Columbus, WI 53925.

24 Hour Emergency Information Telephone Numbers CHEMTREC (USA): 800-424-9300 CANUTEC (Canada): 613-424-6666

ScholAR Chemistry; 5100 W. Henrietta Rd, Rochester, NY 14586; (866) 260-0501; www.Scholarchemistry.com

Section 2: Hazards Identification Opaque red liquid, characteristic phenol odor HMIS (0 to 4) WARNING! Moderately toxic by ingestion and severely corrosive to body tissue. Target organs: Central nervous system, liver, kidneys, eyes. This material is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).

Section 3: Composition / Information on Ingredients Ethyl Alcohol, (64-17-5), 6-7%. Phenol, 90%, Liquified, (108-95-2), 4-5%. Fuchsin Basic, (632-99-5), <1%. Water, (7732-18-5), 87-88%. Section 4: First Aid Measures

Always seek professional medical attention after first aid measures are provided. Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally. Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing. Ingestion: Call Poison Control immediately. Rinse mouth with cold water. Give victim 1-2 cups of water or milk to drink. Induce vomiting immediately. Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures When heated to decomposition, emits acrid fumes. 0 Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 2 0

Firefighters should wear full fire fighting turn-out gear and respiratory protection (SCBA). Cool container with water spray. Material is not sensitive to mechanical impact or static discharge.

Section 6: Accidental Release Measures Use personal protection recommended in Section 8. Isolate the hazard area and deny entry to unnecessary and unprotected personnel. Remove all ignition sources and ventilate area. Contain spill with sand or absorbent material and place material in a sealed bag or container for disposal. Wash spill area after pickup is complete. See Section 13 for disposal information.

Section 7: Handling and Storage Green Handling: Use with adequate ventilation and do not breathe dust or vapor. Avoid contact with skin, eyes, or clothing. Wash

hands thoroughly after handling. Storage: Store in General Storage Area [Green Storage] with other items with no specific storage hazards. Store in a cool, dry,

well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal Protection Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Exposure guidelines: Ethyl Alcohol: OSHA PEL: 1900 mg/m3 and ACGIH TLV: 1000 ppm, STEL: N/A. Phenol: OSHA PEL: 19 mg/m3, ACGIH: TLV: 19 mg/m3, STEL: 60 mg/m3 ceiling/skin. Fuchsin Basic Stain: OSHA PEL: N/A, ACGIH: TLV: N/A, STEL: N/A

Health 2 Fire Hazard 0 Reactivity 0



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MAterIAl sAfety dAtA sheets

Material Safety Data Sheet Page 2 of 2


MSDS # 164.00 Carbol Fuchsin Stain Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula Mixture. Appearance Opaque red liquid.. Molecular weight N/A. Odor Characteristic phenol odor. Specific Gravity 0.9846 g/mL @ 20°C. Odor Threshold N/A. Vapor Density (air=1) 0.7 (water). Solubility Complete. Melting Point 0°C. Evaporation rate N/A (Butyl acetate = 1). Boiling Point/Range 100°C. Partition Coefficient N/A (log POW). Vapor Pressure (20°C) N/A. pH N/A. Flash Point: 118ºC (244 ºF) CC Ethanol. LEL 4.0% Ethanol. Autoignition Temp.: N/A. UEL 20.0% Ethanol.

N/A = Not available or applicable

Section 10: Stability and Reactivity Stability: Stable under normal conditions of use and storage. Incompatibility: Oxidizing agents, acids, halogens, calcium hypochlorite. Shelf life: Indefinite if stored properly.

Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching. Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of mucous membranes, coughing, wheezing, shortness of breath, Chronic Effects: No information found. Sensitization: none expected Ethyl Alcohol: LD50 [oral, rat]; 7060 mg/kg; LC50 [rat]; 20,000 mg/l (10 hours); LD50 Dermal [rabbit]; 20 mg/24H MOD Phenol: LD50 [oral, rat]; 317 mg/kg; LC50 [rat]; 316 mg/m3; LD50 Dermal [rabbit]; 500mg/24 hrs/severe. Fuchsin Basic Stain: LD50 [oral, rat]; N/A; LC50 [rat]; N/A; LD50 Dermal [rabbit]; N/A Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information Ecotoxicity (aquatic and terrestrial): Toxic to terrestrial and aquatic plants and animals. Do not release to the environment

Section 13: Disposal Considerations Check with all applicable local, regional, and national laws and regulations. Local regulations may be more stringent than regional or national regulations. Small amounts of this material may be suitable for sanitary sewer or trash disposal.

Section 14: Transport Information DOT Shipping Name: Not regulated by DOT. Canada TDG: Not regulated by TDG. DOT Hazard Class: Hazard Class: Identification Number: UN Number:

Section 15: Regulatory Information EINECS: Not listed . WHMIS Canada: Not WHMIS Controlled. TSCA: All components are listed or are exempt. California Proposition 65: Not listed. The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all the information required by the Controlled Products Regulations.

Section 16: Other Information Current Issue Date: January 12, 2012 Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any particular use.



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MAterIAl sAfety dAtA sheetsMaterial Safety Data Sheet Page 1 of 2


Hydrochloric Acid Solution, 1.0M MSDS # 338.00 Section 1: Product and Company Identification

Hydrochloric Acid Solution, 1.0M Synonyms/General Names: Muriatic Acid; Hydrochloric Acid Solution, 1N Product Use: For educational use only Manufacturer: Columbus Chemical Industries, Inc., Columbus, WI 53925.



Section 2: Hazards Identification Clear colorless liquid; pungent odor. HMIS (0 to 4) WARNING! Strongly corrosive to body tissue and moderately toxic by ingestion. Target organs: Respiratory system, eyes, skin, lungs. This material is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).

Section 3: Composition / Information on Ingredients Hydrochloric Acid, 37% (7647-01-0), 9-10%. Water (7732-18-5), 90-91%. Section 4: First Aid Measures

Always seek professional medical attention after first aid measures are provided. Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally. Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing. Ingestion: Call Poison Control immediately. Do not induce vomiting. Rinse mouth with cold water. Give victim 1-2 cups of

water or milk to drink. Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures When heated to decomposition, emits acrid fumes. 0 Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 2 0

Firefighters should wear full fire fighting turn-out gear and respiratory protection (SCBA). Cool container with water spray. Material is not sensitive to mechanical impact or static discharge.


Section 7: Handling and Storage White Handling: Use with adequate ventilation and do not breathe dust or vapor. Avoid contact with skin, eyes, or clothing. Wash

hands thoroughly after handling. Storage: Store in Corrosive Area [White Storage] with other corrosive items. Store in a dedicated corrosive cabinet. Store in a

cool, dry, well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal Protection Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Use NIOSH-approved respirator with an acid/organic cartridge. Exposure guidelines Hydrochloric Acid: OSHA PEL: 5 ppm ceiling and ACGIH TLV: 2 ppm ceiling, STEL: N/A.





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MSDS # 338.00 Hydrochloric Acid, 1.0M Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula HCl. Appearance Clear, colorless liquid. Molecular weight 36.46. Odor Pungent odor. Specific Gravity 1.01 g/mL @ 20°C. Odor Threshold N/A. Vapor Density (air=1) 0.7. Solubility Completely soluble in water. Melting Point 0°C. Evaporation rate < 1 (Butyl acetate = 1). Boiling Point/Range 100°C. Partition Coefficient N/A. (log POW). Vapor Pressure (20°C) 14. pH 1, acid (corrosive). Flash Point: N/A. LEL N/A. Autoignition Temp.: N/A. UEL N/A.

Section 10: Stability and Reactivity Avoid heat and ignition sources. Stability: Stable under normal conditions of use and storage. Incompatibility: Alkalis, strong bases, metals, amines, carbonates, metal oxides, cyanides, sulfides, sulfites and formaldehyde. Shelf life: Indefinite, store in a cool, dry environment.

Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, damage to cornea, conjunctivitis, loss of vision. Skin: Redness, blistering, burning, itching, tissue destruction with slow healing. Ingestion: Nausea, vomiting, burning, diarrhea, ulceration, convulsions, shock. Inhalation: Coughing, wheezing, shortness of breath, headache, spasm, inflammation and edema of bronchi, pneumonitis. Chronic Effects: Repeated/prolonged skin contact may cause thickening, blackening or cracking. Repeated eye exposure may cause corneal erosion or loss of vision. Sensitization: none expected Hydrochloric Acid: LD50 [oral, rabbit]; 900 mg/kg; LC50 [rat]; 3124 ppm (1 hour); LD50 Dermal [rabbit]; N/A Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information Ecotoxicity (aquatic and terrestrial): LC50 - 282 mg/l - 96 h - Gambusia affinis (Mosquito fish)

Section 13: Disposal Considerations Check with all applicable local, regional, and national laws and regulations. Local regulations may be more stringent than regional or national regulations. Small amounts of this material may be suitable for sanitary sewer disposal after being neutralized to pH 7.

Section 14: Transport Information DOT Shipping Name: Hydrochloric Acid. Canada TDG: Hydrochloric Acid. DOT Hazard Class: 8, pg II . Hazard Class: 8, pg II. Identification Number: UN1789. UN Number: UN1789.

Section 15: Regulatory Information EINECS: Listed (231-595-7). WHMIS Canada: CLASS E: Corrosive liquid. TSCA: All components are listed or are exempt. California Proposition 65: Not listed. The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all the information required by the Controlled Products Regulations.

Section 16: Other Information Current Issue Date: December 20, 2011 Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any particular use.




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Isopropyl Alcohol, 70% MSDS # 384.00 Section 1: Product and Company Identification

Isopropyl Alcohol, 70% Synonyms/General Names: N/A Product Use: For educational use only Manufacturer: Columbus Chemical Industries, Inc., Columbus, WI 53925.



Section 2: Hazards Identification Clear, colorless liquid, alcohol odor. HMIS (0 to 4) WARNING! Flammable liquid and slightly toxic by ingestion. Flammable liquid, keep away from all ignition sources. Target organs: Central nervous system, liver, kidneys. This material is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).

Section 3: Composition / Information on Ingredients Isopropyl Alcohol (67-63-0), 64%. Water (7732-18-5), 36%.

Section 4: First Aid Measures Always seek professional medical attention after first aid measures are provided.

Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally. Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing. Ingestion: Call Poison Control immediately. Aspiration hazard. Rinse mouth with cold water. Give victim 1-2 tbsp of

activated charcoal mixed with 8 oz water. Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures Class IB Flammable Liquid. When heated to decomposition, emits acrid fumes 3 Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 1 0 Firefighters should wear full fire fighting turn-out gear and respiratory protection (SCBA). Cool container with water spray. Material is not sensitive to mechanical impact. Material is sensitive to static discharge.


Section 7: Handling and Storage Red Handling: Use with adequate ventilation and do not breathe dust or vapor. Avoid contact with skin, eyes, or clothing. Wash

hands thoroughly after handling. Storage: Store in Flammable Area [Red Storage] with other flammable materials and away from any strong oxidizers. Store in a

dedicated flammables cabinet. Store in a cool, dry, well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal Protection Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Use NIOSH-approved respirator with an acid/organic cartridge. Exposure guidelines: Isopropyl Alcohol: OSHA PEL: 980 mg/m3, ACGIH TLV: 492 mg/m3, STEL: 984 mg/m3.




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MSDS # 384.00 Isopropyl Alcohol, 70% Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula CH3CHOHCH3. Appearance Clear, colorless liquid. Molecular weight 60.10. Odor Alcohol odor. Specific Gravity 0.877 g/mL @ 20°C. Odor Threshold N/A Vapor Density (air=1) 2.1. Solubility Completely soluble in water. Melting Point -88°C. Evaporation rate > 1 (Butyl acetate = 1). Boiling Point/Range 83°C. Partition Coefficient N/A. (log POW). Vapor Pressure (20°C) 33 mm Hg. pH N/A. Flash Point: 11.7°C (53°F) CC. LEL 2%. Autoignition Temp.: 399°C (750°F). UEL 12.7 %.

N/A = Not available or applicable

Section 10: Stability and Reactivity Stability: Stable under normal conditions of use. Avoid heat and ignition sources. Incompatibility: Oxidizing materials, caustics, aluminum, metal, oleum, chlorinated compounds. Shelf life: Fair shelf life, store in a cool, dry environment.

Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Stinging pain, watering of eyes, inflammation of eyelids and conjunctivitis. Skin: Insensitivity to pain, feel of coolness or cold, skin looks white and feels hard and cold. Ingestion: Breath has sweet, organic odor, mental confusion, drowsiness, nausea, vomiting and headache. Inhalation: Rapid irregular breathing, headache, fatigue, mental confusion, nausea and vomiting, giddiness and poor judgment, convulsions and death. Chronic Effects: Repeated/prolonged skin contact may cause dryness or rashes. Sensitization: none expected Isopropyl Alcohol: LD50 [oral, rat]; 5045 mg/kg; LC50 [rat]; 16,000 mg/l (4hours); LD50 Dermal [rabbit]; 500mg/24H Mild Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information Ecotoxicity (aquatic and terrestrial): Toxic to aquatic and terrestrial plants and animals. Do not release into environment.

Section 13: Disposal Considerations Check with all applicable local, regional, and national laws and regulations. Local regulations may be more stringent than regional or national regulations. Small amounts of this material may be suitable for sanitary sewer or trash disposal.

Section 14: Transport Information DOT Shipping Name: Isopropano.l Canada TDG: Isopropanol . DOT Hazard Class: 3, pg II. Hazard Class: 3, pg II . Identification Number: UN1219. UN Number: UN1219.

Section 15: Regulatory Information EINECS: Listed (200-661-7). WHMIS Canada:B2, D2B: Flammable liquid, Toxic material: eye irritant. TSCA: All components are listed or are exempt. California Proposition 65: Not listed. The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all the information required by the Controlled Products Regulations.

Section 16: Other Information Current Issue Date: December 21, 2011 Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any particular use.

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