Functional Anatomy of Cells

Functional Anatomy of Cells

• Lecture 5 studies the anatomy of prokaryotic and eukaryotic cells
• Lecture 5 also covers the use of microscopy and staining techniques to observe cells

The Nucleoid

• Bacterial chromosomes are circular and contain the cell's genetic information
• Bacterial chromosomes are not enclosed within a nuclear envelope and have no associated histones
• Plasmids are small, extrachromosomal DNA circles that carry non-crucial genes
• Plasmids may encode antibiotic resistance or produce toxins
• Plasmids replicate independently and can be transferred to other bacteria
• Plasmids are used in biotechnology for gene manipulation

Ribosomes

• Ribosomes found in both eukaryotic and prokaryotic cells are responsible for protein synthesis
• 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 they contain
• Prokaryotic ribosomes are 70S and made of 50S (large) and 30S (small) subunits

Ribosomes: Eukaryotes

• Eukaryotes have 80S ribosomes
• 80S ribosomes consist of a large 60S subunit with a small 40S subunit
• Some 80S ribosomes are membrane-bound, attaching to the endoplasmic reticulum
• Other 80S ribosomes are free in the cytoplasm
• 70S ribosomes are present in chloroplasts and mitochondria

Ribosomes: Antibiotic Targets

• Some antibiotics interfere with prokaryotic ribosomal function by inhibiting protein synthesis
• Streptomycin and Gentamicin attach to the 30S ribosome subunit, interfering with protein synthesis
• Erythromycin and Chloramphenicol attach to the 50S ribosome subunit, interfering with protein synthesis

Inclusions

• Inclusions store reserve deposits of certain nutrients
• Polysaccharide granules store energy reserves
• Sulfur granules also store energy reserves
• Carboxysomes contain enzymes for carbon dioxide fixation during photosynthesis
• Gas vacuoles maintain buoyancy
• Magnetosomes align bacteria along magnetic fields, helping them find a suitable environment and destroy hydrogen peroxide

Endospores

• During vegetative growth, binary fission increases cell numbers
• Endospore formation occurs in gram-positive bacteria under unfavorable survival conditions but is not reproduction
• Nutrient depletion triggers endospore formation
• Endospores are durable and dehydrated, making them resistant to desiccation, heat, chemicals, and radiation
• They can survive in a dormant state for thousands of years; 7500-year-old spores have been germinated
• Members of Bacillus and Clostridium genera produce endospores
• Sporulation is endospore formation
• Germination is when the endospore returns to its vegetative state
• Endospores are important to the food industry

Formation of Endospores by Sporulation

• Endospore formation takes place in a vegetative cell
• Spore septum forms when the chromosome and cytoplasm get isolated by the plasma membrane
• The spore septum becomes a double-layered forespore
• Proteins surround the outside membrane, forming the spore coat that provides resistance to harsh chemicals
• The original cell degrades, and the endospore gets released
• Endospores, under favorable conditions, break down extra layers surrounding the endospore
• Water enters, and metabolism resumes in a 1:1 ratio, which is not reproduction

Eukaryotic Cell Structures

• Vacuoles, cell walls, and chloroplasts only exist in plant cells
• Peroxisomes, the Nucleus, endoplasmic reticulum, smooth and rough, microtubules, and microfilaments are in both plant and animal cells
• Centrosome, lysosomes, basal bodies, and flagellum are unique to animal cells

Flagella and Cilia

• Flagella and cilia are projections used for movement and moving substances
• Flagella are long projections
• Cilia are short and numerous projections
• Flagella and cilia consist of microtubules, which are made of tubulin protein
• Microtubules are organized into 9 pairs around a central pair (9 + 2 array)
• Flagella move in a wavelike manner

The Cell Wall & Glycocalyx

• Cell walls are found only in plants, algae, and fungi
• Cell walls are comprised of cellulose and chitin
• Glycocalyx is made of carbohydrates bonded to proteins and lipids
• Glycocalyx strengthens the cell surface, and helps cells attach to each other

The Nucleus

• The nucleus has a double membrane structure (nuclear envelope)
• DNA is complexed with histone, which allows for DNA packaging and gene regulation

Endoplasmic Reticulum

• The endoplasmic reticulum is a folded transport network
• Rough ER has ribosomes that are responsible for protein synthesis
• Smooth ER has no ribosomes and synthesizes cell membranes, fats, and hormones

Golgi Complex

• The golgi complex transports organelles
• The golgi complex modifies proteins from the ER
• The golgi complex transports modified proteins through secretory vesicles to the plasma membrane

Mitochondria

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

Chloroplasts

• Chloroplasts are the site of photosynthesis
• They have flattened membranes (thylakoids) that contain chlorophyll
• Chloroplasts contain 70S ribosomes and circular DNA
• Chloroplasts can reproduce independently

Microscopy: Smear and Fixing

• A smear is a thin film of material containing microorganisms spread over a slide
• Fixing must happen before staining
• Fixing attaches microorganisms to the slide, kills them, and preserves their parts with minimal distortion

Microscopy

• Microscopes magnify small objects
• Resolution (resolving power) is the lenses' ability to distinguish fine detail and structure
• Resolving power of 0.5 µm means objects 0.3 µm apart blur together
• The size of a specimen determines which microscope can be used, and when it is effective

Metrics

• Measuring microorganisms requires the metric system
• Units relate by factors of 10 in the metric system
• One meter (m) equals 10 decimeters (dm) one hundred centimeters (cm) or one thousand millimeters (mm)

Types of Microscopy

• Light microscopy observes specimens with visible light
  • Types of light microscopy include bright-field, darkfield, phase-contrast, fluorescence, and confocal
• Electron microscopy uses a focused beam of electrons
  • Types of electron microscopy include transmission and scanning

Light Microscopy

• Condenser lenses focus light through the specimen
• Objective and ocular lens magnify the specimen

Compound Light Microscopy

• In a compound light microscope, the objective lens magnifies an image that is further magnified by the ocular lens
• Total magnification equals objective lens power multiplied by ocular lens power.
• Staining is used to make specimens contrast sharply with their medium
• Compound light microscopy limitation is approximately 1500x

Bright-Field Light Microscopy

• Visible light illuminates the specimen
• Staining is often required
• Best for observing outward appearance and few internal structures
• Oil becomes part of the optics of the glass for better resolution
• Oil immersion is effective in improving the resolving power of the lenses to see things clearly

Dark-field Microscopy

• A microscope scatters light from the illuminator
• The specimen appears white against a black background
• It is best used for examining live microorganisms in liquid

Phase-contrast Microscopy

• Allows examination of structures inside live cells
• A special condenser is required
• Best for detailed examination of living organisms and internal structures
• No fixation or staining is required

Fluorescence Microscopy

• Uses blue or ultraviolet light as a source to illuminate specimens that will fluoresce
• Fluorescence is the ability of a substance to give off one color when exposed to another color
• Useful for the rapid detection of pathogenic microorganisms in clinical samples
  • Organisms fluoresce naturally under UV light.
  • Organisms are stained with a fluorochrome dye.
  • Fluorochrome auramine O (dye) glows yellow when exposed to UV and is absorbed by Mycobacterium Tuberculosis
• M. tuberculosis bacteria appear as yellow cells in a sputum sample stained with fluorochrome auramine O
• Fluorescent antibodies contain fluorochrome chemically combined with antibody which can be used in detecting unknown bacteria

Confocal Microscopy

• Each plane in a specimen is illuminated, and a 3D 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

• Microscopes use a focused beam of electrons to observe specimens

Transmission Electron Microscopy

• An electron microscope provides high magnifications of thin sections of a specimen
• Images are saved digitally on a screen
• Only provides a 2D image
• Vacuum fixation and dehydration can cause distortion of structures called artifacts

Scanning Electron Microscopy

• A SEM provides three-dimensional imaging
• An electron gun produces a beam of electrons that scans the speciman
• The images are then displayed and saved digitally
• The specimen is put in a high vacumn for viewing

Preparing Smears for Staining

• Staining colors microorganisms emphasize structures
• A smear is a thin film of material containing microorganisms across a spread slide
• Fixing (attaching) a smear must happen before staining to adhere to the slide
• Bacterial cells have a negative charge, so basic dyes adhere to them and the background is stained with acidic dyes
• Simple or differential stains may be used

Simple Stains

• A simple stain uses a single basic dye, such as methylene blue, carbolfuchsin, crystal violet, or safranin
• Simple staining highlights the entire microorganism to visualize cell shapes and structures
• A mordant may hold the stain or coat the specimen to intensify staining

Differential Stains

• Differential stains distinguish between bacteria and react differently with various bacteria
• Gram stain
• The acid-fast stain

Gram Stain

• It classifies bacteria into gram-positive and gram-negative
  • Gram-positive bacteria have thick peptidoglycan cell walls and stain purple
  • Gram-negative bacteria have thin peptidoglycan cell walls and an outer membrane of lipopolysaccharides/phospholipids and they stain pink/red -Primary stain is crystal violet, next lodine mordant, then Alcohol and finally safranin
• Gram stain is important in the identification of bacterias for the diagnosis of an illness

Atypical Cell Walls

• Acid-Fast Cell Walls
  • Similiar to gram positve cells but also have a thick layer of mycolic acid also attached is Arabinogalactan -Prevents the uptake of dye.

Acid-Fast Stain

• Binds only to bacteria with a waxy material in their cell walls, not decolorized by acid-alcohol to visualize cell shapes and structures with use of heat -Apply carbolfuchsin stain to fixed smear -Wash with slide wash with water -Apply a decolouring agent -Finally Counterstain methylene blue

Special Stains: Endospore

• Endospores are resistant to staining than ordinary methods, use Schaeffer-Fulton endospore stain
• Requires primary stain, malachite green, usually with heat to penetrate the endospore
• Decolourize will cells with water then Counterstain will with safranin -Spores appear green within red or pink cells

Question 1

• Coronavirus (0.1µm) is unable to be clearly observable with a microscope with resolution of 0.5 µm

Question 2

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

Question 3

• In the case a student failed to fix a bacterial specimen, the likely outcome will be cellular components of the sample wash away during staining

Question 4

• If alcohol is not applied during gram staining, the outcome will be both gram-positive and gram-negative bacterial cells will stain purple

Question 5

• If a student failed to fix a bacterial specimen the most likely outcome will be that the bacterial cells will likely wash away during the staining process