R.O Lecture 1 & 2 Flashcards
What are microbes?
Bacteria, fungi and viruses
Taxonomy
A method of grouping bacteria. The science of classification
Binomial Nomenclature
“two-name”
True or False the Three-domain classification system supersedes kingdom classification
True
Approaches to identifying bacteria
-Microscopy: Light v Electron
-Morphology-Staining: Gram/Spore etc.
-Biochemical Tests
-Growth conditions: Molecular Biology Techniques
Dichotomous Key
A tool used to identify bacteria.
-Uses Paired Statements “either-or” choice
-Can be useful for practical reasons but have limitations
-Molecular methods are most accurate for classification
Light Microscopy
Note Limit of resolution for bacterial cells using light microscopy.
-Bright-field
- Dark-field
- Confocal
- Phase-contrast
- Normarsky
- Fluorescence
Electron Microscopy
TEM: Transmission electron micrograph
Resolution to 1nm
Magnification up to 500,000 X Cross-sections, usually 1D
SEM: Scanning electron micrograph
Resolution to 20 nm
Magnification up to 50,000 X Surface/Exterior 3D views
Describe Specimen Preparation for Light Microscopy
- Wet Mount: Allows visualisation of microbial motility
- Smear: Loopful of media containing microbe of interest
- Air dry
- Heat fixation:
-Kills organism
-Adheres to slide
-Take up dyes more effectively
Name the Dyes used for staining techniques
-Cationic/basic (Methylene Blue, Crystal Violet, Safranin, Malachite Green)
-Anionic/acidic (Eosin, Picric Acid)
Name the types of stain used for staining techniques
- Simple (1 dye, basic cell shapes/ arrangements)
- Differential (2+ dyes, distinguishes between cell types/ parts of an organism)
- Special Stain
Explain Simple Stain
- Uses a single dye, do not distinguish organisms or structures by different staining reactions.
- Example - Methylene Blue, Safranin (red) and Crystal Violet (purple)
- Shows sizes, shapes and arrangements of cells.
Differential Stains
- Use two or more dyes that react differently with various kinds or parts of bacteria, allowing them to be distinguished.
- Example Gram Stain
- Distinguishes Gram negative and gram positive bacteria
Special Stains
- Identify various specialized structures
- Example Schaeffer Fulton Spore Stain
- Endospores retain malachite green stain, vegetative cells accept safranin counterstain and appear red.
- Allows visualisation of hard to stain bacterial endospores such as members of genera Cloistridium and Bacillus
- Spore stain is slightly modified as spores do not stain easily due to their resistant structures.
Describe the nature of the Catalase Biochemical Test
Hydrogen peroxide (H2O2) is poured over heavy growth of an organism on an agar slant; release of o2 bubbles indicates the presence of catalase, which oxidises H202 to H20 and O2
Name the methods of obtaining pure cultures.
Physical techniques: Streak Plate Method and Spread Plate Method
Explain the Spread Plate method
Isolating different bacterial species.
Can be used in combination with the streak plate method to isolate pure colonies from a mixed culture.
Name the types of growth media used in clinical setting.
Selective Media : Encourages growth of some microbes while surppressing growth of others. (may contain antibiotics ect.)
Diffferential Media: Includes a component that causes an observable change in the media in response to a particular biochemical reaction ( e.g. pH/colour change)
Enrichment Media: Contains ingredients that enhance the growth of particular microbes that may be present in relatively low numbers; doesn’t suppress others.
Name the molecular methods for bacterial identification/classification.
▪Genetic Homology:
-Base Composition
-DNA/RNA sequencing
-DNA Hybridisation
-Protein Profiles/Amino Acid Sequencing
-Ribosomal Properties (rRNA)
▪MALDI-ToF mass spectrometry
▪Immunological Reactions
▪Phage Typing
What does the Three-domain classification system consist of?
Bacteria, Archaea, Eukarya
Describe Prokaryotes
*Genetic Material is not membrane enclosed; circular chromosome
*Lack other membrane-enclosed organelles
*DNA is not associated with histone proteins
*Most cell walls contain the complex polysaccharide peptidoglycan
*Divide by binary fission; Asexual reproduction only
Describe Eukaryotes
*Membrane-enclosed nucleus; typically paired chromosomes
*Endoplasmic reticulum, Glogi apparatus, Lysomsomes ect.
*DNA packaged, tightly folded around histone proteins
*Cellulose/chitin found in plant/fungal cell walls
*Mitosis and/or meiosis; Sexual/Asexual reproduction
What is the diameter and length of bacteria
Diameter: 0.2 –2 µm; Length: 2 –8 µm
What are the 3 basic shapes of bacteria.
-Spherical (Coccus)
-Rod-like (Bacillus)
-Spiral
Other Shapes: Star, Spindle, Square, Triangular, Irregular
What way do bacteria divide/reproduce?
Bacilli divide only along their short axis.
Asexual reproduction only
What are the functions of the cell wall?
Functions:
- Maintains shape
- Prevents rupturing due to osmosis
- Anchorage for flagella
Often Porous
Can contribute to virulence (ability to cause damage to host)
Site of action for some antibiotics
Composition varies
Peptidoglycan
Most important cell wall component. Consists of a carbohydrate backbone which is made up of repeating units.
Describe the outer membrane
-Primarily Gram negatives (Gram-positive bacteria lack an outer membrane)
-Phospholipid bilayer external to cell wall
-Some protective qualities (e.g. Penicillin)
-Contains Lipopolysaccharide (LPS)
Descirbe the Periplasmic Space
-Primarily Gram negatives
-Between Cell Membrane and Cell Wall
- Metabolism Site
Periplasm
- Is part of cell wall in Gram Positives
- Consits of peptidoglycan, proteins and metabolites
Teichoic Acid
Another component of cell wall.
-Gram positive only
-Glycerol, phosphates, ribitol
-Polymer, 30 units long, extends through cell wall
-Attachment sites for phages/Ion movement
What are the steps to Gram Stain
- Crystal Violet (1 minute) Drain, rinse. All purple
- Iodine is added. Iodine acts as mordant to set stain.
- Decolorise with alcohol and rinse with water.
- Safranin - Drain rinse and blot.
Gram + = purple
Gram - = red (pink)
Gram Positive Bacteria (reaction to stain)
Thick peptidoglycan layer: Retain crystal violet dye following alcohol destain.
- purple
Gram Negative Bacteria (reaction to stain)
Relatively thin peptidoglycan layer:
Lose Crystal violet dye following alcohol destain
(pink)
Acid-fast Bacteria
(Reaction to carbon fushsin stain)
Thick cell wall, 60% lipid: Retain carbon fushsin stain following acid-alohol destain
Examples include Mycobacterium species
Carbol fuchsin is used as the primary stain dye to detect acid-fast bacteria because it is more soluble in the cells’ wall lipids than in the acid alcohol.
Cell Membrane
*Fluid-mosaic model: Phospholipid bilayer and proteins
*Regulates entry/exit of materials
*Synthesizes cell wall components, assists with DNA replication
*Protein secretion, respiration, captures ATP
Name the internal bacterial structures
(Cell wall and membrane) Nucleoid and Plasmids Ribosomes
Inclusion Bodies
Spores
Nuclear Region (Nucleoid)
Nucleoid: Usually single large circular chromosome. Circuluar shape for protection
No nuclear envelope/histones(DNA-associated proteins)
Plasmids
Are seperate to the chromosome.
*Usually circular, dsDNA, extrachromosomal
*Replicate independently
*Can be gained/lost/transferred
*Additional genes
Ribosomes
- Involved in protein translation
-Consist of protein and rRNA (ribosomal RNA) components
-Sites of Protein Synthesis
-Cytoplasm
-Eukaryotic ribosomes(80S) v Prokaryotic ribosomes(70S)
-Antibiotic target (e.g. Streptomycin, erythromycin) Prokaryote specificity
Inclusion Bodies (reserve deposits/storehouses)
Granules and Vesicles/vacuoles
Granules
Tightly packed single substance that does not dissolve readily in the cytoplasm; not membrane enclosed; contents can be metabolised to produce energy/supply other metabolic processes E.g. Glycogen granules, polyphosphate granules
Vesicles/vacuoles
Membrane enclosed structures found in certain bacteria. May contain lipids orgas or iron etc.
What leads to the development of highly resistant endospores?
*Nutrient depletion can lead to the development of highly resistant endospores
Describe endospores
*Dehydrated cells with thick walls and additional layers
*Unique to Bacteria: Occurs in Bacillus, Clostridiumand some other Gram-positives (Not the same as fungal spores)
*Not a reproductive mechanism; survival
*Endospores are not metabolically active
*Resistant to heat, drought, many chemicals, radiation
What are the 3 stages of germination?
1) Activation:Damage to Spore Coat
2) Germination Proper: Spore’s enzymes break down peptidoglycan, Water enters
3) Outgrowth: Metabolism resumes if adequate nutrients are available
Name the pathogenic spore formers
Bacillus anthracis, Clostridium botulinum, Clostridium tetani, Clostridium difficile
Can be resistant to usual methods of sterilisation/food preservation: heating, freezing, chemicals, desiccation, radiation etc.
Autoclaving
Moist heat sterilisation (121*C, 15mins)
Name the external Bacterial structures
Flagella, Pili, Glycocalyx
What are the possible arrangements of the flagella?
Arrangements:
- Monotrichous: single, polar
- Lophotrichous: >2 flagella at one/both poles
- Amphitrichous: One at each pole
- Peritrichous: Distributed all over.
Note differences between gram-negative and gram-positive flagella
How do flagella motility
Rotation of flagella
Run: Anti-clockwise, then Tumble: Clockwise
What are the types of pili ?
Pili (fimbrae) and specialised pili.
* These are shorter structures than the flagella
* Conjugation Pili: Transfer of Genetic Material
* Attachment Pili
Glycocalyx
All polysaccharide containing substances external to cell wall
Capsule :
Protective Structure, can contribute to virulence Complex polysaccharides arranged in a loose gel
Slime Layer: Thinner than Capsule Less tightly bound to cell wall Protects against drying, traps nutrients, adherence, protection
