Microscope Types

Types of Microscopes

• Compound microscopes use visible light through multiple lenses with a magnification range up to 2000x
• Stereo microscopes use visible light with two separate optical paths and give a stereoscopic view and the magnification range goes up to 300x
• Digital microscopes feature digital imaging
• Fluorescence microscopes use fluorescent dyes with a magnification range of up to 1000x and help view fluorescent objects
• Inverted microscopes are suitable for cell culture, viewing specimens from the bottom
• Automated imaging systems can be used for automated cell analysis
• Electron microscopes use electron beams and can magnify up to 10,000,000x
• Transmission Electron Microscopes (TEM) use an electron beam transmitted through the specimen
• Scanning Electron Microscopes (SEM) use an electron beam that scans the surface of the specimen, with probes, and offer magnifications up to 500,000x
• Scanning Probe Microscopes have a magnification range of up to 100,000x
• Atomic Force Microscopes (AFM) use a mechanical probe to scan the specimen's surface, providing magnifications up to 10,000x
• Scanning Tunneling Microscopes (STM) employ an electrical probe to scan the surface, with magnifications up to 100,000x
• Optical Microscopes use visible light and have a magnification of up to 2000x

Concepts in Microscopy

• Magnification is the ability to make objects appear larger
• As magnification increases, the field of view decreases
• As the magnification of the microscope increases, the depth of field decreases
• Resolution is the smallest distance between two objects that can be distinguished as separate entities
• Resolution is limited by the wavelength of light used and the numerical aperture of the objective lens
• Contrast is the difference in brightness or color between different parts of a sample
• Illumination refers to the light source used to illuminate the sample
• Different types of illumination (brightfield, darkfield, fluorescence) can highlight different parts of the sample
• Imaging techniques used in microscopy include brightfield, dark field, fluorescence, confocal, and electron microscopy
• Each technique has its advantages and limitations and is used to answer different scientific questions

Overview of Bacteria

• Bacteria are prokaryotes that do not have a true nucleus
• Bacteria are unicellular organisms that reproduce asexually
• Bacterial cells lack membrane-bound organelles like mitochondria and Golgi apparatus
• Bacteriology is known as the study of bacteria
• Pili/Pilus are used for bacterial attachment, movement, and genetic exchange
• The capsule is a polysaccharides or proteins layer that protects against host immune responses
• Flagella are whip-like structures used for motility
• Gram-positive bacteria retain the Gram stain and appear purple/dark blue
• Gram-negative bacteria do not retain the Gram stain and appear pink

Bacterial Classification and Taxonomy

• Bacteria, Archae, and Eukaryotes are the three domains in the classification of organisms
• Phylum and Class are based on genetic and metabolic characteristics
• Order, Family, Genus, and Species are based on genetic, metabolic, and morphological characteristics
• In binomial nomenclature, organisms are named by their Genus and species.
• For example, Escherichia coli is classified as follows: Domain: Bacteria, Phylum: Proteobacteria, Class: γ-proteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae, Genus: Escherichia, Species: coli
• For example, Humans are classified as follows: Domain: Eukarya, Kingdom: Animalia, Phylum: Chordata, Class: Mammalia, Order: Primata, Family: Hominidae, Group: Homo, Species: sapiens

Oxygen Demand

• Aerobic bacteria use oxygen as a final electron acceptor like Pseudomonas aeruginosa (G-), Bacillus subtilis(G+), and Mycobacterium tuberculosis (G+)
• Anaerobic bacteria are inhibited or killed by oxygen like Clostridium perfringens (G+).
• Facultative anaerobic bacteria can grow with or without oxygen, e.g., Escherichia coli (G-), Salmonella enterica (G-), and Staphylococcus aureus(G+)

Gram Staining

• Microscopic Appearance and Chemical Reactions in Gram Staining
• Crystal violet (primary dye) stains all cell walls
• Gram's iodine (mordant) helps the dye crystals to get trapped in the cell
• Alcohol (decolorizer) weakens the outer wall of Gram-negative cells, causing them to lose the dye
• Safranin (red dye counterstain) stains the colorless Gram-negative cells red

Gram-Positive vs Gram-Negative Cell Walls

• Gram-positive cell walls have one major layer, while Gram-negative cell walls have two major layers
• The chemical composition of Gram-positive cell walls includes peptidoglycan, teichoic acid, and lipoteichoic acid
• The chemical composition of Gram-negative cell walls includes lipopolysaccharide (LPS), lipoprotein, peptidoglycan, and porin proteins
• Gram-positive cell walls are thicker (20-80 nm) compared to Gram-negative cell walls (8-11 nm)
• Gram-positive bacteria lack an outer membrane, while Gram-negative bacteria have an outer membrane
• Gram-positive bacteria have a narrow periplasmic space, while Gram-negative bacteria have an extensive periplasmic space
• Gram-positive cell walls are more permeable to molecules than Gram-negative cell walls

Systematic Identification of Gram-Positive Bacteria

• Gram-positive bacteria are first divided based on morphology: Bacilli (rods), Cocci (spheres), or Branching filaments
• Catalase test, Bile Esculin test, and Hemolysis test are used to test for Gram-positive bacteria
• Bacilli can be aerobic (Listeria, Bacillus, Corynebacterium) or anaerobic (Clostridium)
• Cocci can be Staphylococcus or Streptococcus

Gram Positive Cocci-Catalase Test

• The Catalase ability of bacteria produces the catalase enzyme to break down hydrogen peroxide into water and oxygen
• The enzyme forms gas bubbles when reacting with 3% H2O2
• Catalase protects intra-phagocytic microbes by destroying hydrogen peroxide produced by the phagocyte

Gram Positive Cocci-Catalase Positive-Staphylococcus

• Coagulase test helps differentiate Staphylococcus species and detects production of coagulase enzyme
• The bacterium works in conjugation with normal plasma components to convert fibrinogen to fibrin and causes clotting of plasma
• Staphylococcus aureus is Coagulase positive and sensitive to Novobiocin
• Staphylococcus epidermidis is Coagulase negative and resistant to Novobiocin
• Staphylococcus saprophyticus is resistant to Novobiocin

Staphylococcus aureus Details

• Staphylococcus aureus can grow under both aerobic and anaerobic conditions
• Structural properties include a capsule for protection against phagocytosis, presence of Protein A, and non-motility
• Staphylococcus aureus can colonize the skin and nose
• Exhibits a wide range of temperature tolerance and high concentration of salt
• Enzymes such as Catalase protect from peroxides, and Coagulase converts fibrinogen to insoluble fibrin that forms clot and protect
• Produces heat and acid resistant Enterotoxins that are responsible for food poisoning
• Its Exfoliative toxins A and B can cause superficial skin to peel off causing scalded skin syndrome
• Toxin shock syndrome toxin is heat and protease resistant and can affect multiorgan pathology
• Staphylococcus aureus is a cause of both community-acquired and nosocomial infections
• Antibiotic-resistant strains (MRSA) are common

Clinical Diseases of S. aureus

• Pyogenic Diseases are localized skin infections with pus-filled vesicles
• Pyogenic Diseases can cause Wound infections characterized by erythema and pus
• Pyogenic Diseases can cause Endocarditis that infects the linings of the heart
• Pyogenic Diseases can cause Septic arthritis characterized by swollen, red joints with an accumulation of pus
• Scalded skin syndrome is a toxin-mediated disease that causes the peel off of skin in young children
• Toxic shock syndrome is a toxin-mediated one that affects multiple organs
• Aureus infections include pyogenic and toxin-mediated infections
• epidermidis are opportunistic infections, such as catheter-associated infections and surgical site infections
• saprophyticus causes urinary tract infections, particularly in sexually active young women

Gram Positive Cocci-Catalase Negative-Streptococcus

• Hemolysis test differentiates Streptococcus species based on their hemolytic properties
• Hemolysis is the lysis of red blood cells
• Extracellular enzymes called hemolysins radially diffuse outwards from the colonies causing complete or partial lysis of RBCs
• Bacitracin is a cyclic polypeptide antibiotic known as Bacitracin is used to prevent wound infections, treat pneumonia and empyema in infants, and to treat skin and eye infections.

Streptococcus pyogenes (Group A) Details

• Streptococcus pyogenes grows under aerobic conditions
• has a Hyaluronic acid Capsule that provides protection against phagocytosis and clearance as a structural property
• It can colonize the skin and throat
• It has M proteins, in the cell wall which blocks complement by mediating phagocytosis
• There are four distinct heat labile streptococcal pyrogenic exotoxins that enhance release of proinflammatory cytokines responsible for clinical manifestations

Streptococcus agalactiae – Group B

• Streptococcus agalactiae requires aerobic conditions
• Properties include: Outer Polysaccharide capsule that protects from phagocytic clearance and Sialic Acid blocks complement mediated phagocytosis
• Reservoir as UTI
• Can cause Neonatal diseases like neonatal meningitis and neonatal pneumonia
• Neonates are at high risk of infection via mother during delivery

Streptococcus pneumoniae details

• Streptococcus pneumoniae grows under aerobic conditions
• Properties are its outer polysaccharide capsule protects it from phagocytic clearance and surface protein adhesins bind the bacteria to epithelial cells
• Have IgA is a protease
• It is a Diplococcus
• Reservoir is the Respiratory tract
• Can cause Pneumonia, Meningitis, and Ostitis media
• It is a virulent member of the viridans streptococci

Viridans Streptococci

• They grow under aerobic conditions
• They have relatively virulence but are opportunistic pathogens that form Biofilms
• Its Reservoir is Normal Oral flora
• They cause Dental Caries and Subacute endocarditis
• There is Ubiquitous colonization of mucosal surface restricted to areas near the mouth
• They are NOT found on skin

Bile Esculin Test

• Bile salts are the selective ingredient, while esculin is the differential component
• The Bile Esculin Test tests for the Streptococcus species
• Enterococcus hydrolyze esculin to products react with ferric citrate in the medium to produce insoluble iron salts, resulting in medium blackening

Enterococcus – Group D

• Requires aerobic conditions
• Is a relatively virulent but opportunistic pathogen
• Antibiotic resistance inhibits effective antibiotic therapy
• Colonizes the GIT of humans and animals