Biology 241 – Lab
LAB #1
(1st/21 Lab Sessions for Fall Quarter, 2008)
TOPICS TO BE COVERED:
»Orientation to the Lab and Lab Safety
»Cell Structure & Somatic Cell Life Cycle Review
»Histology Drawings Assignment Guidelines
»Microscopy Review
»Buccal Smear preparation and investigation
»Whitefish Blastula Mitotic Figures
DESIRED OUTCOMES:
After completing the activities described for this lab session, students should:
»Be able to locate all safety equipment in the Lab Room
»Have read LAB RULES and REGULATIONS handout; this is to be carried at all times.
»Have read and signed the INFORMED CONSENT AGREEMENT (witness same for Lab Partner)
»Recall the 4 main parts of a cell (plasmalemma, nucleus, cytoplasm, and extracellular compartment)
»Review the 4 main events of a Somatic Cell’s Life Cycle (G1, S, G2 (Interphase), and Cell Division)
»Be familiar with the Histology Drawings Assignment Guidelines and begin individual drawings
»Review how to carry, use, clean and store a compound light microscope
»Be able to identify the parts of the microscope and describe their function(s)
»Be proficient at viewing objects using all magnifications and calculating the total magnification (TM)
»Be able to perform a wet mount of cheek cells, simple staining, and visualization of cellular detail
»Have completed the first drawing (of 25 drawings) and labeling as per Assignment Guidelines.
»Identify the two events of somatic cell division (Mitosis and Cytokinesis); draw and label the 4
stages of Mitosis to review the significant nuclear activity of each stage and appreciate the
importance of Cytokinesis as the cytoplasmic activity involved in cell division
MATERIALS NEEDED:
»Fire Extinguisher(s), Fire Blanket, Eye Wash Station, First Aid Kit, Phone, 9-911 Procedures
»LAB RULES and REGULATIONS handouts; INFORMED CONSENT AGREEMENT sheets
»Models / Diagrams of Animal Cells; Wall Charts of Somatic Cell Division (“Mitosis”); 3-dimensional
models of the continuum of somatic cell division
»Histology Drawings Assignment – (see attached)
»Compound microscopes; lens paper; lens-cleaning solution; Wall Chart of Compound Microscope
(See also Figure 3.1 in your Photographic Atlas)
»Prepared slides of the letter “e”; clean microscope slides, coverslips, sterile tongue depressors; dropper
bottles of methylene blue
»Prepared slides of Whitefish blastula (serial-sections)
»Photographic Atlas, Ch. 2
Biology 241 – LAB #1 – continued
Page Two
Activity #1: Orientation to the Lab and Lab Safety
The location of all Lab safety equipment will be pointed out by your Instructor. Each person is responsible for periodically refreshing his/her knowledge of the whereabouts (and manners of use) of such equipment.
Each student will read the ten entries on the LAB RULES and REGULATIONS handout and will carry
this handout with him/her at all times. It constitutes the basis for responsible laboratory habits and behavior by
all Lab users (students, staff, and instructors, alike!) If you have any questions about the entries on this sheet,
please ask for a more full explanation/discussion.
Activity #2: Cell Structure & Somatic Cell Life Cycle Review
Since Biology 101 (or equivalent) is one of the pre-requisite academic courses for any student of the
200-level Human Anatomy & Physiology series of lecture/lab courses, it is expected that each student enrolled
in Biology 240/241 already has a working knowledge of the names, descriptions, locations, and functions of the
various components of a generic animal cell. These include:
Plasmalemma (aka Cell membrane)
Nucleus (including nuclear envelope)
Nucleoli
Cytosol
Cytoplasm how are these different?
Cytoskeleton
∙Microfilaments
∙Microtubules
∙Intermediate filaments
Ribosomes
SER (Smooth Endoplasmic Reticulum)
RER (Rough Endoplasmic Reticulum)
Golgi Apparatus (aka Golgi Body)
Lysosomes
Peroxisomes
Centrosome
∙Centrioles (2)
∙Pericentriolar region
Mitochondria
Microvilli
Cilia
Flagellum
Vesicle
Inclusions
Extracellular
compartment
Each student should be able to identify these sub-cellular structures on a model, on a diagram, or on a chart and
be conversant about their functions and interrelationships within the cell.
In addition, each student should be able to describe events occurring in the cell during the 4 phases into
which the Life Cycle of a Cell is divided: G1, S, G2, (collectively known as Interphase) and Cell Division (in which
nuclear events are collectively called Mitosis and cytoplasmic events are called Cytokinesis. Also, we will be looking more
closely into the 4 major subdivisions of Mitosis (Prophase, Metaphase, Anaphase, + Telophase) in Activity #6 of this Lab.)
Please review these basic concepts if they are fuzzy in your mind; and please LEARN them now if you have not
ever done so during your past preparation for this course!)
Activity #3: Histology Drawings Assignment
The Histology Drawings Assignment follows on the next several pages. These pages include Instructions, a Template Drawing Sheet (from which additional copies should be made), a List of Labels to include on the
drawings, a Legend of Tissues/Organs from which the prepared slides have been made, and other useful information. You will start today generating drawings; but will spend the next five lab sessions completing this classic significant assignment. Remember: another way of getting at the nature of Histology (aka “the study of
tissues”) is to refer to it as “microscopic anatomy”. It is, after all, the study of cellular ultrastructure and function.
In a nutshell, here are the REQUIREMENTS for this assignment:
1. You are responsible for 25 drawings from laboratory observations.
2. Each drawing you generate should be clearly labeled (see the List of Labels)
3. Include in your drawings, details that will aid you in future identification of that particular tissue.
4. Make copies of the Template Drawing Sheet for your future drawings; make the copies single-side.
5. On your cover-sheet, include Your Name, Lab Section/Time, Instructor’s Name, Quarter/Year
Biology 241 – Lab #1 – continued
Page Three
6. Draw what you see, but also “project in” what you know is there! Ah-ha! Histology drawing is both a
“science” and an “art”. (For example, in a mitotic figure, you will not see the actual centrioles within
the organelle called a centrosome (from which the mitotic spindle microtubules emanate); however, you
will definintely be able to discern the pericentriolar region, thus you will label it!
7. Your drawing should be a representation of the field of view in your microscope. The circular outlines
on your drawing sheets represent the field of view in your microscope. All tissue drawings should
fill the circle. Labels and leader-lines should be neatly added to clearly indicate salient structures.
8. When completed, your drawings and cover-page should be simply stapled together in the upper lefthand corner as a flat packet; or in a THIN (not to exceed 1 cm) 3-ring binder or 3-prong notebook or
pocketed folder. (Please: NO PLASTIC PAGE PROTECTORS. These make it awkward for making
comments and grading!)
9. You will be graded on completeness, accuracy, and neatness. Each drawing will start with +4 points
and each omission or incorrect item will carry a deduction of ½ point. Thus, if the TM is forgotten (- ½),
the tissue sub-type/Type naming is incomplete (-½), and if the Basement Membrane is misidentified (-½). The
drawing will now be given 2½ out of 4 points, or 2½ ⁄ 4. If there are NO errors nor omissions, a 4/4 will result.
10. Special Note: Be sure that the tissue you are drawing is really the tissue of interest!! Since slides are
from real organisms, the tissue you need to observe will be connected to other tissues; or the particular tissue type may only be found in a specific area of the specimen. So, be sure you have the correct
view in your microscope. When in doubt, ASK the Instructor to check before you spend time drawing!
Activity #4: Microscopy Review
Working with a microscope is a true privilege and carries quite a few responsibilities with it. These include: (1) being aware that these important investigative tools cost between $1500 - $2000 apiece; therefore
great care MUST be taken to ensure they stay in top working-condition; (2) applying the general guidelines for
use/handling of the scopes; and (3) knowing the names/functions of all the components.
During this first Lab, we will review the general guidelines for use/handling of the scopes; as well as a
brief overview of the components of each microscope and hints for proper use. Remember, we use light microscopes, which means that they employ light shining through a specimen to illuminate it; then the light is refracted through a series of lenses (both objective and ocular lenses) to magnify the specimen. Here is a list of parts
about which you should already be conversant: Base; Body; Arm; Ocular lenses (10X); Objective lenses (4X:
scanning, 10X: low, 40X: high – dry, 100X: oil immersion) mounted on the Turret (aka Nosepiece); Stage with
mechanical operating knobs; Illuminating source; Condenser with Iris diaphragm; Coarse adjustment knob;
Fine adjustment knob. (Please refer to Figure 3.1 in your Photographic Atlas.)
Remember: When using the microscope:
1) carry it using TWO HANDS to support it - one firmly grasps the arm, and the other hand supports the base;
2) ALWAYS start viewing the specimen ON SCANNING POWER, then switch to higher power(s). DO NOT
use the Coarse adjustment knob with any power other than scanning.
3) clean the lenses with lens paper only. Use of paper towels or cloth will result in SCRATCHING the lenses.
When storing the microscope (and upon removing it from storage to use it):
1) the turret is positioned so that the lowest power objective (4X) is aligned for use;
2) the mechanical stage clasps are “centered” and NOT shifted to/hanging over on either the Right nor Left;
3) the illuminating source (light) switch is in the OFF position;
4) the electrical cord is loosely coiled, secured, and hung gentle over the oculars;
5) the entire scope is CLEAN - there is NO oil residue, no slide left mounted on the stage, and dust free.
You will be responsible for the identification and brief functional description of each of the parts of
your microscope; as well as for the proper “closing down” of the scope before storing it in the cupboard.
Your Instructor will “quiz” you on parts, hints for use, focusing techniques, and close-down procedures.
Biology 241 – LAB #1 – continued
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Activity #5: Buccal Smear Preparation and Investigation
To identify the 4 major components of a cell, you will be looking at cells from the superficial epithelial
layer of the buccal mucosa – that is, the “cheek region” of oral mucosal membrane lining the inside of your
mouth! Specifically, you will remove some of the cells from the superficial layer of squamous epithelial cells;
they will be “disrupted” from the “sheet of cells”; and thus, once stained, visible as separate and single cells
(however, they originated from a stratified squamous epithelial tissue that is part of the oral mucosa).
To prepare your slide: Your Instructor will first demonstrate this technique to the entire class; but then each
student should follow these steps:
(1) Put a 10”-12” length of paper towel on your lab bench to use as your “placemat”.
(2) From the middle drawer, remove a clean microscope slide and cover-slip and place both on the placemat.
(3) Using a sterile tongue depressor, gently scrape the inside of one cheek once or twice, then “drag” the blunt
end down along the buccal aspect of your mandibular molar teeth (this allows you to retrieve some bacteria that
lurks there!)
(4) Now, tap the used end of the tongue depressor onto the middle of the clean slide (you may not see much, but
there are plenty of cells (your own AND bacteria!!) and saliva there!
(5) Place a small drop of methylene blue (the dropper bottle will be at your Lab Table) onto the “wet specimen”.
(6) Place the coverslip over the drop of dye, and soak up any excess blue fluid with a torn-off corner of your
placemat. Remember: by capillary action, you can draw off quite a bit of the “excess blue” without harming
the preparation.
(7) Start looking at your slide on the scope – being sure to get the image in focus on scanning power (TM =
40X) initially; then, switch objectives going to low power next (TM = 100X); and finally, switch objectives
again going to high-dry (TM = 400X). (You will NOT be using the oil-immersion objective, nor any oil.
There is enough resolution at 400X.)
(8) Identify individual squamous cells. These are very flat, polygon-shaped cells: think of a fried-egg and how
the white of the egg distributes fairly evenly around the large, centrally-placed yolk. Easily identifiable will
be a darkly-staining plasmalemma and centrally-placed, round to oval nucleus; fairly dense perinuclear region, and, often, bacteria visible on the cell’s surface. (Note the huge size difference between the one squamous
epithelial cell and the multiple bacteria!) Quite often, mucous threads are visible, as the protein mucin is secreted as a component of saliva and is stained by methylene blue.
(9) Look even MORE closely: do you see one or more nucleoli within the nucleus? Can you identify any other
organelles within the cell? Do you see that some cells look “folded over” – much like when you flip a fried
egg and a portion of one side of the white area gets folded over. On your specimen, this folded-over area
will be “doubly-dense” for the blue stain color. Also, scan for a field-of-view that shows three, four, and
more of these cells still joined together as a “sheet” (or cohesive layer) of cells; these cells still have their
shared “cell junctions” intact.
If bacteria are present, can you tell if their morphology is bacillus (rod), cocci (round), or spirillis (spiral)?
(10) Using the first circle on your Histology Drawings Assignment sheet, draw what you see and add labels,
making sure to use leader-lines to indicate the structure you are labeling. Draw at least one isolated squamous epithelial cell, and one “clump” of three, four, or multiple squamous epithelial cells. (Note: be sure
to show that these cells are NOT “round”; rather, they are polygonal (meaning “many sided”) and thus their
outermost boundary is quite “geometric”: therefore, use straight lines indicating 5, 6, 7, 8 sides.)
Activity #6: Whitefish Mitotic Figures
Reproductively speaking, there are two types of cells: somatic cells and sex cells. It is the fate of somatic cells to undergo the process of replacing themselves as they complete their life cycle and thus maintain the
health and integrity of tissues that face continual wear and tear. This process of replacing themselves is known
as somatic cell division and is the type of division in which a single starting cell duplicates itself to produce two
daughter cells, identical to each other and to the original cell (2n → 2 ∙2n). Somatic cell division consists of
mitosis (the nuclear events) and cytokinesis (the cytoplasmic events).
Biology 241 – LAB #1 – continued
Page Five
(Sex cell division gives rise to gametes; utilizes two successive divisions; and consists of Meiosis and Cytokinesis. It may
be represented 2n→ 4∙1n and the resulting cells are genetically not identical to each other nor to the original cell. )
Mitosis + Cytokinesis are parts of the larger Life Cycle of a Cell (G1 phase, S phase, G2 phase - which
are collectively known as interphase). Please review the progression of events of this cycle in Chapter 3 of
your textbook, and refer to Figure 2.24 in your Atlas. Also, review the four phases of the Mitotic (M) phase:
Prophase, Metaphase, Anaphase, Telophase as to the significant characteristics of each phase.
Now, select a slide from the Blue Box labeled WHITEFISH MITOSIS SLIDES. You will notice that
each of these slides has approximately 16 “small circles” of tissue mounted under the cover slip. These are
actually “serial sections” of one blastula (early stage in the development of a zygote – aka a very young, developing
“fish egg”) of a small fish called “Whitefish”. Each “circle of tissue” is a cross-section of one of these very small,
developing fish eggs containing approximately 32 – 64 cells in various stages of somatic cell division. Note that
every stage of the cell cycle may not be visible within EACH of the cross-sections of the blastula; hence, you
may have to change the field of view several times and utilize many of the 16 or so cross-sections. Also, these
blastulas were treated with a chemical called colchicine which arrests cell-division in “mid-action”. Sort of like
freeze-tag: in whatever position the nuclear envelope, nucleoli, chromatin, chromosomes, chromatids (aka nuclear events) or centrosomes, microtubules, spindle apparatus, etc. (aka cytoplasmic events) are when colchicine
reaches the cell, that’s how the position “freezes” or stays. Then, once stained, those “positions” are fixed and
can be analyzed. Mitosis is a continuum of steps NOT a “stop action” type of process. However, to “study” it,
it is useful to break it into four distinctive phases.