Microbiology: Staining Techniques

• Tutoring is available:
  • Mondays: 10:00 AM - 2:00 PM
  • Wednesdays: 10:00 AM - 1:00 PM & 1:30 PM - 5:00 PM
  • Thursdays: 10:00 AM - 1:00 PM & 1:30 PM - 5:00 PM
• Written Test 1 : 02/27 (2 weeks)
• Practical test 1: 03/04-03/11 (2 sessions) covering Gram stain and streaking on 5 media types (BA, MS, MSA, PEA, NA)
• Lecture exam 1: 03/04 (lectures 1-4)

Smear Prep

• Prepare smear and let it dry
• Turn on hot plates for steaming
• Prepare smear with 1/3 tap water

Different Staining Types

• Endospore staining
• Capsule/negative staining

Gram Staining

• Used to determine if cells are Gram-positive or Gram-negative, also gives shape and arrangement
• Gram-positive cells (e.g., Staphylococcus) appear as cocci in clusters
  • They exhibit clusters, chains, diplococci, and tetrads
• Gram-negative cells appear as bacilli in single arrangements
  • They exhibit single, diplobacilli, chains, and palisades arrangements

Acid-Fast Stain (ZN Method)

• Carbol Fushsin: Primary stain
• Heat
• Acid-alcohol: Decolorizer
• Methylene Blue: Counter stain
• Acid-fast positive results in red
• Acid-fast negative results in blue

Bacterial Characterization Sessions

• Session 1: Microscopy
• Session 2: Inoculation and transfer techniques aseptically
• Session 3: Preparation and staining slides for characteristics
• Further characterization includes differential media, biochemical tests, and other future tests

Bacterial Cell Structures

• All bacteria contain a cell membrane, bacterial chromosome/nucleoid, ribosomes, and cytoplasm
• Some bacteria have S layers, fimbriae, cell walls, outer membranes, cytoskeleton, pili, glycocalyx, inclusions/granules, microcompartments, nanotubes/nanowires, plasmids, endospores, intracellular membranes, flagellum

External Structures of Bacteria

• Flagella enables movement and tumbling/running
• Fimbriae facilitates attachment
• Pili enables conjugation and exchange of plasmid DNAs
• Nanotubes/nanowires facilitate the transport of amino acids or smaller molecules
• S-layer is a shield of repeated proteins produced in hostile environments
• Glycocalyx is a polysaccharide layer that could be organized as a slime layer or capsule

Cell Envelope

• Cell membrane: All bacteria have it
• Cell wall presents in some bacteria; made of peptidoglycan
• Outer membrane found in Gram-negative bacteria only

Internal Structures

• Cytoplasm
• Bacterial chromosomes and plasmids
• Ribosomes (70S; unique to bacteria)
• Cytoskeleton
• Inclusion bodies, for food storage
• Microcompartments, for storing enzymes
• Endospores which are dormant bodies resistant to extreme conditions

Endospores

• Internal structures are present inside the cell
• Resistant structures formed by bacterial species
• Resistant to heat, radiation, desiccation, and chemical disinfection
• Formed by vegetative cells through sporulation
• Not for reproduction, but for survival
• Sporulation occurs under harsh conditions
• Increase the virulence of cells

Sporulation Process in Bacillus Species

• Hostile environment sensing: Vegetative cell begins to be depleted of nutrients
• DNA replication: Chromosome is duplicated and separated
• Forespore and cell separation: Cell is septated into a sporangium and forespore
• Engulfment of forespore: Sporangium engulfs forespore for further development
• Endospore layer synthesis: Sporangium begins to actively synthesize endospore layers around forespore
• Cortex, Endospore coat, Chromosome begin to be involved
• Layer deposition: Cortex and outer coat layers are deposited
• Endospore coats involved: Exosporium, Endospore coat, Cortex, Core
• Endospore ready: Mature endospore
• Free endospore: Free endospore is released with the loss of the sporangium
• Germination: Endospore swells and releases vegetative cell

Endospores

• Spores produced by Bacillus, Clostridium, and Sporosarcina
• Vegetative cell are metabolically active
• Sporulation induced by certain environmental conditions
• Endospores resist extremes of heat, drying, freezing, radiation, and chemicals that would kill vegetative cells

Medical Significance of Bacterial Endospores

• Bacillus anthracis can cause anthrax
• Clostridium tetani can cause tetanus
• Clostridium perfringens can cause gas gangrene
• Clostridium botulinum can cause botulism
• Clostridium difficile: "C. diff," causes a gastrointestinal disease (nosocomial)

Endospore Staining

• These endospores are difficult to stain and can often be seen as clear bodies within bacterial cells
• Major spore-forming Bacillus and Clostridium
• Location of spore within the vegetative cell helps identify species within those genera
• Staining technique heats the primary stain (malachite green) into the endospores
• Once stained, endospores are difficult to decolorize
• Vegetative cells are then stained with a counterstain, like safranin
• Types of endospores: terminal, sub-terminal and central

Endospore Staining Steps

• Apply malachite green (primary stain), apply heat (mordant), apply water (decolorizer), apply safranin (counterstain)
• Malachite Green for 5 minutes and Safranin for one minute

Today’s Activities: Endospore Staining

• Bacillus subtilis( 48 hrs slant)
• Bacillus cereus (48 hrs slant)
• Requires staining tray, slide holder, forceps, water bottle, hot plate, newspaper, can containing water, Bunsen burner, loop, Malachite Green (primary), Safranin

Endospores

• Endospores are resistant to antibiotics, most disinfectants, and physical agents such as radiation, boiling, and drying
• Bacterial endospores can be destroyed at high temperatures in their spore state
• Otherwise by triggering endospore germination back to vegetative cells to be easily killed through traditional sterilization

External Structures

• Flagella for motility
• Pili for transfer of genetic material
• Fimbriae for attachment
• Glycocalyx as a slim layer or capsule, aiding virulence

Glycocalyx

• Glycocalyx is a capsule type that protects the cell against immune cells and enables attachment
• There is also the slime layer which enable attachment and aggregation of bacterial cells

Capsules

• Protect against white blood cells (phagocytes) by way of its glycocalyx
• Biofilms prevent bacteria from becoming dislodged (IE plaque on teeth)
• Responsible for persistent colonization on devices like catheters, IUDs, and pacemakers

Capsule Stain

• Capsule is polysaccharide-containing structure on the outside of cell
• Composed of fibrous polysaccharides or globular glycoproteins Capsule is in a tight matrix, firmly attached to wall, slime layer less organized/easily deformed
• Enables protection and can lead to biofilms (ecosystem with millions bacteria)
• Example organisms include Strep. pneumoniae, C. perfringens, Klebsiella pneumoniae, E. coli
• Bacterial capsules are non-ionic, so no acidic or basic stain will adhere
• Best way to visualize is with acidic stain
• It is recommended to have bacteria that are old
• No heat-fix is required, as it can distort capsules
• Structures can help increase bacteria's virulence

Capsule/Negative Stain

• Negative staining is a technique where the dark background makes the cell itself visible
• Heat-fixing not used
• Bacteria used for this stain are Staphylococcus aureus and Micrococcus luteus
• https://www.youtube.com/watch?v=avveXgPWVJ8
• Capsules formed by Klebsiella pneumoniae, Bacillus spp., Staphylococcus and Streptococcus spp., E. coli, Pseudomonas aeruginosa and Neisseria spp

Learn about smear preparation, Gram staining, and Acid-Fast staining techniques in microbiology. Understand how to differentiate between Gram-positive and Gram-negative cells and their arrangements. Also includes a practice exam schedule.