Microscopy and Genetics Lab Notes
Mitochondrial DNA and Barr Bodies
The transcript begins with a discussion of X-chromosome inactivation concepts: "There's four for men. Women, only one out of those four is actually bimet. The others are known as bar bodies."
Barr bodies are inactivated X chromosomes; in females, one of the X chromosomes is inactivated in each cell, forming a Barr body.
The speaker then introduces mitochondrial DNA (mtDNA):
mtDNA is separate from nuclear DNA; it is circular and has its own genome.
mtDNA is inherited maternally: "Only women give mitochondrial DNA. Men do not give it."
The transcript asserts that 8% of you comes from mitochondrial DNA, describing it as an 8% contribution from mtDNA that comes through the maternal line.
Implication discussed: lineage tracing is easier through maternal lines due to mtDNA inheritance, whereas paternal DNA contributions become “muddled” after a few generations.
The speaker makes a numerical claim about genome contributions: "8% additional for women. That means we're all actually 46% dad, 54% mom." This is presented as a takeaway from the lecture, though it does not reflect standard human genetics (nuclear DNA is roughly 50/50; mtDNA is separate and maternally inherited).
The session transitions to the cell cycle and microscopy modules.
Microscopy logistics and safety
The instructor introduces the microscopes and emphasizes handling them carefully.
Two microscopes per table are required.
The heavy equipment is described as around 30 pounds; the instructor frames lifting as a workout to reinforce caution.
Proper grip and movement:
Hold with one hand on the neck and the other under the base.
Do not hold or slide by the ocular, stage, or dials.
When moving, lift and place rather than sliding to avoid knocking alignment.
Storage and organization:
When returning microscopes, place them alternating (sideways) with oculars oriented left then right for neighboring units (e.g., ocular left implies eyepiece to the right on the next unit).
Setup at each table:
Two per table; a labeling station may exist.
The instructor explains that this is an additional chapter added to the manual for the exercise in the syllabus.
Anatomy of the microscope and magnification
Key components (top to bottom):
Ocular lenses at the top.
Objective lenses below the nosepiece; each objective has a different magnification.
Magnification rule:
Total magnification is the product of the ocular magnification and the objective magnification:
Common objective codes and total magnifications (as described):
Scanning objective (red): 4x
Low power objective (yellow): 10x
High power objective (blue): 40x
Oil immersion objective: 100x (requires immersion oil)
Therefore, total magnifications are:
Scanning:
Low power:
High power:
Oil immersion:
Slide placement and stage:
The stage has clips to hold the slide; clips open to place the slide in the grooves.
Use the stage adjustment knobs (on the side) to move the stage left/right and up/down.
Do not move the stage by hand; use the adjustment knobs.
Ensure the sample is over the light before observing.
Setup sequence for viewing:
Start with the smallest objective (scanning/red) and bring the sample into view.
Adjust light intensity to a comfortable level; begin at roughly halfway power.
As magnification increases, light needed typically decreases; increase light as you switch to higher magnifications.
Focusing strategy and parfocal concept
Start with the stage as low as possible (all the way down) before focusing:
Move the stage down to its lowest position using the coarse adjustment knob.
Then slowly raise the stage toward you using the coarse adjustment knob to bring the image into focus.
Eyepiece and binocular setup:
Adjust the eyepieces so both eyes can view comfortably to reduce eye strain/migraine.
Focusing with different objectives:
On scanning (red) and low power (yellow): use only the coarse adjustment knob to focus.
On high power (blue) and oil immersion (100x): the fine adjustment knob is used, but very cautiously.
Parfocal principle: once you have a sample in focus on one objective, you should be close to in focus when you switch to the next objective. You may need a small coarse adjustment to recenter, and then fine-tune as needed.
Practical workflow when changing objectives:
Start on red (scanning) at lowest power, focus with coarse knob, then switch to yellow and recenter (use coarse knob), then to blue and finally to oil immersion if needed.
If the image goes blurry when switching objectives, revert to the previous objective, re-focus with the coarse knob, and then proceed to the next objective.
Safety and eye safety tips:
Do not look directly at the light source; keep light intensity at a safe level to avoid eye strain.
Treat the equipment with care to avoid misalignment or overheating the lamp.
E slide demonstration and practice observations
The instructor demonstrates viewing an E slide (letter E) to understand inversion and flipping of the image in a light microscope:
Place the slide so the E is above the light; adjust focus until the E appears clearly in the eyepiece.
Draw what you see through the microscope, then write how the E actually looks in real life (upright). Expect the image to be inverted and flipped; rotate and flip mentally or with the controls to compare with the original.
Students take turns getting the E into focus; others observe and compare the inverted view to the real orientation.
The activity emphasizes:
Practicing focusing across objectives (scanning, low, high).
Observing the inversion/flip characteristic of a compound light microscope.
Developing skill in drawing microscopic images and comparing them to real-world orientation.
Parfocal property and field of view concepts
Parfocal means you can switch objectives and the image remains approximately in focus; you should only use coarse adjustment at lower magnifications and expect minor refocusing when moving to higher magnifications.
Field of view (FOV) and light are related to magnification:
As magnification increases, the field of view decreases:
Light intensity entering the eye generally decreases with higher magnification; you must increase illumination at higher magnifications.
Contrast tends to increase with magnification, aiding in distinguishing features from the background.
The practical implication: to study cell dimensions, you estimate sizes by comparing to the field of view using a grid slide.
Field of view calculations with grid slides
Grid slides used to measure field of view (FOV) in terms of box units on the slide:
The grid has boxes with spacing that is either 1 mm or 2 mm between grid lines (the instruction notes a choice between 1 mm or 2 mm spacing per box).
An example from the transcript: one grid box width is about 2.5 units, and the total FOV can be estimated as roughly 6.25 units in the example discussed.
Observations summarized from the discussion:
For a 1 mm grid spacing, some groups reported seeing about four boxes across the FOV.
For a 2 mm grid spacing, some groups reported seeing about two boxes across the FOV.
Some notes mention seeing about three to six boxes across depending on alignment and the exact magnification, illustrating that FOV is not perfectly uniform across experiments and units.
Practical takeaway:
When you increase magnification, the width of the field visible through the objective shrinks; you can estimate cell sizes by counting how many grid boxes fit within the FOV.
This helps in approximating cell dimensions for different cell types (spherical cells, elongated muscle cells, amorphous white blood cells, etc.).
Worked example (based on the transcript):
If a grid box is 1 mm and FOV spans about four such boxes, then FOV ≈ 4 mm. If a box is 2 mm and you see about two boxes, then FOV ≈ 4 mm. The numbers in the lecture vary slightly by group and exact setup, illustrating the practical variability of field measurements.
Wet mount procedure and cheek cell observation
Wet mount setup:
Gather slides, cover slips, a cheek swab (sterile), gloves, and a bottle of water.
Place a small drop of water on the edge of a glass slide.
Use a sterile tongue depressor to collect cheek cells by rubbing the inside of the cheek, then mix cells into the drop of water on the slide.
Place a cover slip at a slight angle to the slide to minimize air bubbles.
Position the slide on the stage above the light and bring the cells into focus.
Observation goals:
Look for cheek cells under the microscope; cheek cells are often hard to visualize and may require high power to identify.
Document what you observe and then compare with typical cell structures.
Cleanup:
After observation, dispose of slides in the glass-only disposable bin.
Clean the instrument with alcohol swabs and return to its place.
When finished, wipe down with an alcohol swab and ensure the microscope is turned off and unplugged.
Wet mount observations: nucleus and membranes
The wet mount example yields visible cellular features such as:
Nucleus (observed as a dark-staining region) inside the cell.
Outer membranes outlining the cell periphery.
Inner components of the cell that appear protected by membranes.
Common student observations:
Some samples show a dense nucleus; others show amorphous cell bodies with membranes visible.
The appearance of the nucleus and membranes helps identify basic cell organization and supports later discussions of cell anatomy.
Practical tips for the lab session and references
Practice and repetition:
Start all focusing sequences with the lowest magnification and the stage as far down as possible to improve the chance of quickly achieving in-focus images.
Alternate between students in a group to share focus responsibility and ensure everyone practices using coarse and fine adjustments.
Safety and etiquette in class:
Wear a lab coat for wet mounts; gloves are provided for handling slides and materials.
Clean up devices after use and place slides in the correct disposal bins.
Classroom logistics and materials:
The course includes a heavy emphasis on microscopy and physiology, with upcoming practice of different cell types and tissues.
The instructor mentions the need to review the subcycle (cell cycle) in preparation for next week.
Textbook and manual references:
Principles of Anatomy and Physiology, 16th edition (for core text references).
Lab Manual: Human Anatomy and Physiology, 8th edition by Mary,
used for exercises and practical questions.
Miscellaneous notes from the session:
The instructor emphasizes that the E slide and the process of flipping and rotating the image helps students understand image orientation in a microscope.
The lab leader demonstrates proper handling, careful stage manipulation, and the importance of not forcing the knobs when focusing.
The class session closes with reminders about the upcoming lab coat and lab manual requirements and the plan to begin with attendance and pre-lab quiz in the next lab.
Quick reference: key terms and concepts
Barr bodies: inactivated X chromosomes in female cells (X-inactivation).
Mitochondrial DNA (mtDNA): circular DNA inherited maternally; used for tracing maternal lineage.
Parfocal: the ability to stay in reasonable focus when switching objectives.
Coarse vs fine adjustment knobs: coarse for major focusing at low magnification; fine for precise focusing at high magnification.
Magnification and light: higher magnification reduces field of view and incoming light; increase light intensity as magnification increases.
Field of view (FOV): the diameter of the visible area through the microscope; decreases with higher magnification.
Wet mount: a simple slide preparation using a drop of water to observe living cells (e.g., cheek cells).
E slide exercise: practice understanding image orientation (inverted and flipped) under a compound light microscope.
Grid slides: used to estimate cell size by comparing sample size to known grid box dimensions.
Glassware and disposal: use glass-only disposal for slides and clean equipment after use.
References and materials mentioned
Textbooks:
Principles of Anatomy and Physiology, 16th edition.
Human Anatomy and Physiology Lab Manual, 8th edition by Mary.
Class logistics:
Lab sections and attendance policies were discussed, along with the need to bring lab coats and lab manuals to the next session.