Prokaryotic & Eukaryotic Cells: Anatomy and Microscopy

Functional Anatomy of Prokaryotic and Eukaryotic Cells, Microscopy and Staining

• Lecture 5 covers functional anatomy of prokaryotic and eukaryotic cells, microscopy, and staining techniques.

The Nucleoid

• Bacterial chromosome: typically a circular, double-stranded DNA thread with the cell's genetic information.
• Bacterial chromosome is not enclosed within a nuclear envelope (membrane).
• Bacterial chromosome has no associated histones.
• Plasmids are small extrachromosomal circles of DNA.
• Plasmids often carry non-crucial genes, such as those encoding antibiotic resistance or toxin production.
• Plasmids replicate independently of the chromosome.
• Plasmids may be transferred to other bacteria.
• Plasmids are used in biotechnology for gene manipulation.

Ribosomes

• Ribosomes are present in both eukaryotic and prokaryotic cells, and are where protein synthesis takes place.
• Ribosomes are made of protein and ribosomal RNA (rRNA).
• Prokaryotic ribosomes (70S) differ from eukaryotic ribosomes (80S) in the number of proteins and rRNA molecules.
• A prokaryotic ribosome 70S consists of a 50S (large) subunit and a 30S (small) subunit.
• Eukaryotic ribosomes are 80S
• 80S consists of a large 60S subunit and a small 40S subunit
• Some 80S ribosomes are membrane-bound on the endoplasmic reticulum, others free in the cytoplasm
• 70S ribosomes found in chloroplasts and mitochondria
• Ribosomes' antibiotic targets: antibiotics that interfere with prokaryotic ribosomal function and inhibit protein synthesis.
• Streptomycin and Gentamicin attach to the 30S subunit, interfering with protein synthesis.
• Erythromycin and Chloramphenicol attach to the 50S subunit, interfering with protein synthesis.

Inclusions

• Inclusions contain reserve deposits of certain nutrients.
• Polysaccharide granules are for energy reserves.
• Sulfur granules are energy reserves.
• Carboxysomes contain enzymes for CO2 fixation during photosynthesis.
• Gas vacuoles maintain buoyancy.
• Magnetosomes align bacteria along the Earth's magnetic field, helping them move downward toward a suitable environment and destroy H2O2.

Endospores

• Vegetative growth occurs through binary fission, increasing the cell number.
• Endospore formation is a survival mechanism in gram-positive bacteria under unfavorable conditions, and is not for reproduction.
• A nutritional depletion triggers endospore formation.
• Endospores are highly durable, dehydrated cells that are extremely resistant to harsh conditions such as desiccation, heat, chemicals, and radiation.
• Endospores can survive in a dormant state for thousands of years; some 7500-year-old spores have been germinated.
• Endospores are produced by members of the Bacillus and Clostridium genera.
• Sporulation is the process of endospore formation.
• Germination is when an endospore returns to a vegetative state.
• Endospores are important in the food industry.

Formation of Endospores by Sporulation

• Sporulation is when endospore formation takes place in a vegetative cell.
• Spore septum formation is when the chromosome and cytoplasm are isolated by the plasma membrane.
• Spore septum develops into a double-layered structure called a forespore.
• Proteins surround the outside membrane, forming the spore coat, which provides resistance to harsh chemicals.
• The original cell is degraded, and the endospore is released.
• Endospores' enzymes break down the extra layers surrounding the endospore under favorable conditions, then water enters, and metabolism resumes (1:1 ratio not reproduction).

Flagella and Cilia

• Flagella and cilia are projections used for movement or to move substances along the cell surface.
• Flagella are long projections and few in number.
• Cilia are short projections and numerous.
• Both flagella and cilia consist of microtubules made of the protein tubulin.
• Microtubules are organized as 9 pairs in a ring, plus 2 microtubules in the center (9 + 2 array).
• Flagella move in a wavelike manner.

The Cell Wall and Glycocalyx

• The cell wall is found in plants, algae, and fungi.
• The cell wall is made of carbohydrates like cellulose (plants and algae) and chitin (fungi).
• The glycocalyx is carbohydrates bonded to proteins and lipids in the plasma membrane.
• The glycocalyx found in animal cells.
• The glycocalyx strengthens the cell surface and helps cells attach to each other.

The Nucleus

• The nucleus is a double membrane structure (nuclear envelope) enclosing the cell's DNA.
• The nucleus contains DNA complexed with histone for DNA packaging and gene regulation.

Endoplasmic Reticulum

• The endoplasmic reticulum is a folded transport network within the cell.
• Rough ER is studded with ribosomes and is the site of protein synthesis.
• Smooth ER lacks ribosomes and synthesizes cell membranes, fats, and hormones.

Golgi Complex

• The Golgi complex is a transport organelle.
• The Golgi complex modifies proteins from the ER.
• The Golgi complex transports modified proteins to the plasma membrane via secretory vesicles.

Mitochondria

• Mitochondria contain inner folds of the inner mitochondrial membrane and fluid called the matrix.
• Mitochondria are involved in cellular respiration (ATP production).
• Mitochondria contain 70S ribosomes and circular DNA.
• Mitochondria can reproduce on their own.

Chloroplasts

• Chloroplasts are the location of photosynthesis.
• Chloroplasts contain flattened membranes (thylakoids) that contain chlorophyll.
• Chloroplasts contain 70S ribosomes and circular DNA.
• Chloroplasts can reproduce on their own.

Microscopy and Staining

• A smear is a thin film of material containing microorganisms, spread over a slide.
• Fixing a smear must precede staining to attach the microorganisms to the slide, kill the microorganisms, and preserve the microbes with minimal distortion.
• Microscopes are used to magnify small objects.
• Resolution (resolving power) refers to the ability of lenses to distinguish fine detail and structure.
• The size of a specimen determines which microscopes can be used effectively.
• The metric system is used when measuring microorganisms units relate to each other with a factor of 10.
• One meter (m) equals 10 decimeters (dm), 100 centimeters (cm), or 1000 millimeters (mm).

Types of Microscopy

• Light microscopy uses visible light to observe specimens.
• Electron microscopy uses a focused beam of electrons to observe specimens.

Compound Light Microscopy

• In a compound light microscope, the image from the objective lens is magnified again by the ocular lens.
• Total magnification = objective lens x ocular lens.
• Specimens must contrast sharply with their medium, and is achieved by staining.
• Limited to approximately 1500x.

Bright-Field Light Microscopy

• Visible light illuminates the specimen.
• Often bright-field light microscopy requires staining.
• Best for observing the outward appearance and few internal structures.
• Oil becomes part of the optics of the glass of the microscope.
• Adding oil has the same effect as increasing the objective lens diameter, improving the resolving power of the lenses.

Dark-Field Microscopy

• A microscope scatters light from the illuminator.
• Specimen appears light against a black background.
• Best used for examining live microorganisms in liquid that are invisible in the ordinary light microscope, cannot be stained by standard methods, or distorted by staining.

Phase-Contrast Microscopy

• Phase-contrast microscopy allows examination of structures inside live cells via a special condenser.
• Best for detailed examination of living organisms and internal structures.
• Fixation (dead cells) or staining is not required.

Fluorescence Microscopy

• Flourescence microscopy uses blue or ultraviolet light to illuminate specimens that will fluoresce.
• Fluorescence is the ability of a substance to give off one color of light when exposed to another.
• Very useful for the rapid detection of pathogenic microorganisms in clinical samples.
• Organisms fluoresce naturally under UV light or can be stained with fluorochromes.
• Fluorochrome auramine O glows yellow when exposed to UV light; absorbed by Mycobacterium tuberculosis.
• Fluorescent antibodies are fluorochromes chemically combined with an antibody and is used to detect unknown bacterium.

Confocal Microscopy

• Each plane in a specimen is illuminated for confocal miscroscopy, and a three-dimensional image is constructed with a computer.
• Cells are stained with fluorochrome dyes.
• Light is used to excite a single plane of a specimen.
• Clear two-dimensional images can be obtained.

Electron Microscopy

• Electron microscopy, a microscope, uses a focused beam of electrons to observe small specimens.
• Used for images too small to be seen with light microscopes, for example viruses and internal cellular structures.
• Images are black and white, and are often enhanced by adding color digitally.
• Use electromagnetic lenses to focus electron beams.

Transmission Electron Microscopy

• Transmission electron microscope provides high magnifications of thin sections of a specimen.
• Images are produced on a viewing screen and saved digitally.
• Transmission electron microscopy only shows thin sections and no 3-D aspect.
• Specimen is fixed, dehydrated, and viewed under high vacuum.
• Treatment can cause distortion, leading to the appearance of additional structures called artifacts

Scanning Electron Microscopy

• Scanning electron microscopy provides a three-dimensional view.
• Scanning electron gun produces a beam of electrons that scans of an entire specimen's surface.
• Images are produced on a viewing screen and saved digitally.
• Specimen is fixed, dehydrated, and viewed under high vacuum.

Preparing Smears for Staining

• Staining is coloring microorganisms with a dye that emphasizes certain structures.
• Smear is a preparing a thin film of a material containing microorganisms spread over a slide.
• Fixing (attaching) a smear must precede staining to attach microorganisms to the slide, killing them and preserving microbes with minimal distortion.
• Bacterial cells a negative charge, so basic dyes adhere to them.
• When The background is stained, not the cell, it is called negative staining and uses acidic dyes.
• Simple stains and Different Stains.

Simple Stains

• Highlights the entire microorganism to visualize cell shapes and structures.
• A mordant may be used to hold the stain or coat the specimen to intensify staining.

Differential Stains

• Used to distinguish between bacteria, and it reacts differently with different bacteria.
• Examples include Gram stain, and Acid-fast stain.

Gram Stain

• The gram stain classifies bacteria into gram-positive or gram-negative. Developed by Hans Christian Gram.
• Gram-positive bacteria have a thick peptidoglycan structure, which stain purple.
• Gram-negative bacteria have thin peptidoglycan cell walls, an outer membrane of lipopolysaccharides, and phospholipids; they stain pink/red.
• Gram staining is an important staining technique in medical microbiology; can be used to detect to bacteria in specimens, can identify an unknown bacteria and provide information for treatment
• Not universally applicable, doesn't stain for all bacteria.

Gram Stain Steps

• First application is crystal violet (primary stain).
• Second application is iodine (mordant).
• Third step is alcohol wash for decolorization.
• Fourth step is application of safranin for counterstaining.
• Alcohol wash and safranin causes gram-positive to get/stay purple and gram-negative stain pink.

Atypical Cell Walls

• Acid-fast cell walls.
• Similar to gram-positive cell walls (thick peptidoglycan).
• Mycolic acid bound to peptidoglycan prevents uptake of dyes.
• Arabinogalactan polysaccharide that holds mycolic acid and peptidoglycan.
• Mycobacterium and Nocardia are acid-fast genera.

Acid-Fast Stain

• The acid-fast stain only binds to bacteria with a waxy material in their cell walls that is not decolorized by acid-alcohol.
• Used for the identification of Mycobacterium such as Mycobacterium tuberculosis and Mycobacterium leprae.

Acid-Fast Stain Procedure

• Heat gently, cool, slide wash, de-colorizing agent.
• Then apply carbolfuchsin stain to fixed smear and counterstain-methylene blue
• Acid-fast stain binds strongly to bacteria with a waxy material in their cell wall.

Acid-Fast Stain Colors

• Primary Stain of Heat Carbolfuchsin: Red, both non and acid fast.
• Decolorizing Agent of Acid-Alcohol, only acid fast is red after process.
• Counterstain of Methylene Blue, non-acid-fast is blue after process.

Special Stains: Endospore

• Endospores are resistant, dormant structures that cannot be stained by ordinary methods.
• Schaeffer-Fulton endospore stain is common.
• Involves primary stain of malachite green used with heat, decolorization of cells with water, then counterstain with safranin.
• Spores appear green, and cells either red or pink.

Question 1

• The coronavirus (0.1 µm) would not be clearly observable using a microscope with a resolutioon of 0.5 µm since they are smaller.

Question 2

• Gram-negative cell walls contain an LPS-rich outer membrane; gram-positive cell walls do not.

Question 3

• The likely outcome is that the culture will die and/or there is a loss distortion by the process.

Question 4

• A student neglects to apply it and both gram-positive and gram-negative bacterial cells will be stained purple.

Question 5

• The bacterial cells will likely wash away during the staining process.