Week 1: i. Microbial Classification & Taxonomy; Bacterial structure + The Prokaryotic Cell Cycle; Bacterial Growth, Physiology and Metabolism Flashcards
What is cyanobacteria?
- Cyanobacteria is the first unicellular form of life identified ≈ 3.6 billion years ago.
What is the molecular phylogeny tool? (3)
- Molecular Phylogeny is the tool that enables us to understand the complexity of life and the relationships between living forms.
- This is done by comparing the ribosomal RNA (rRNA) genes.
- rRNA is well conserved so helps identify relationships between organisms. In prokaryotes,
get 16SrRNA & in eukaryotes, get 18SrRNA.
- Based on rRNA analysis, life has evolved into 3 main lineages:
o Prokaryotes (unicellular; bacteria)
o Archae (unicellular microbes, not of clinical significance) &
o Eukaryotes (multicellular, more complex organisms like fungi, protozoa)
What are the differences between prokaryotes vs eukaryotes? (8)
What are the types of microbes? (4)
- Viruses
- Bacteria
- Fungi
- Parasites
What are the characteristics of viruses? (5)
What are the characteristics of bacteria? (5)
What are the characteristics of fungi? (3)
What are the characteristics of parasites? (4)
Bacterial Classification:
- STAINING REACTIONS:
- Gram staining: (2)
= Gram-positive (Dark blue/purple – Due to thick cell wall, peptidoglycan layer)
= Gram-negative (Pink – Due to thin cell wall)
What is acid-fast?
Used with organisms that stain poorly with Gram stain e.g. Mycobacterium tuberculosis
Ziehl Neelsen stain (End TB); Kinyoun stain (Nocardia)
- SIZE:
- Measured in ______ (micrometres)
microns
What are the shapes of bacterial classification? (3)
- Cocci, Bacilli, Spiral
- Bacilli can be cocco-bacillary (bacilli shaped but rounder), pleomorphic (different bacillary
sizes and shapes) or club shaped/chinese-lettering - Spiral shape can be tight or loose coils
Bacterial Classification:
- ARRANGEMENT =
- Cocci in chains (streptococci) or in clusters (staphylococci)
Bacterial Classification:
- CULTURABLE OR NON-CULTURABLE (ABILITY TO GROW) =
(2)
- Culturable: Dilute bacterial suspension in a solution, plate out on solid agar medium and
observe colonies to determine organism type (perform further tests for definitive result) - Unculturable: Bacteria that cannot be grown on any artificial non-living conditions (identify
using PCR, Polymerase chain reaction)
Bacterial Classification:
- GROWTH REQUIREMENTS (FOR CULTURABLE) =
Aero-tolerance (tolerance for O2) v/s Anaerobe (intolerance for O2) AND Fast v/s Slow growers
What is flagella?
Locomotion; antigen; immune evasion
What is fimbriae?
Adherence
What are pili?
Join adjacent bacteria cell for partial DNA transfer (conjugation)
What is the capsule (most polysaccharide) ?
Protects the cell from dehydration and nutrient loss; antiphagocytic; antigenic/immunogenic; biofilm formation
What is the cell wall (cytoskeleton = peptidoglycan) ?
Rigidity & shape; protection against osmotic pressure - prevention of lysis; antigenic
What is the cytoplasmic membrane (phospholipid bilayer) ?
Cell respiration; cell precursor synthesis; selectively permeable
What is cytoplasm?
Solvent for materials used in all cell functions
What is intracytoplasmic inclusions?
Intracellular storage bodies
What is a nucleoid?
Bacterial cell genetic material (chromosome- single, circular double stranded DNA)
What are plasmids?
Free small circular, double stranded DNA
What are ribosomes?
Site of protein synthesis
What are spores?
Protect species of genera Bacillus and Clostridium from unfavourable conditions.
The Bacterial Cell and Ultra-structural characteristics:
Bacterial nomenclature/Taxonomy: Naming of microorganisms 9
(8)
Microbial Classification and Taxonomy: Bacterial structure
- Introduced the world of microbes. D escribing the “tree of life”, gave broad overview of different forms of microbial life having relevance for the discipline of infectious diseases; microbes include: (4)
Viruses
Bacteria
Fungi
Parasites
- Focused more on bacteria: Small, unicellular organisms that, if harmful and disease producing (pathogenic) wreak havoc in the ______ ______.
- By understanding bacterial structure and form, a _______ classification of bacteria can be derived. Naming of bacteria using taxonomy is complex, for the sake of simplicity, we will focus on ______ ________ of bacteria using genus and species designations only
Morphological
Binomial classification
Bacterial physiology: (4)
- Nutrition
- Environmental factors affecting survival
- Growth and multiplication
- Metabolism
What are the Minimum requirements for growth and multiplication? (4)
- Water (80%)
- Carbon
- Nitrogen
- Inorganic salts
Requirements for growth of pathogenic (disease causing) species:
- Organic compounds and growth factors (bacterial vitamins)
o Essential:
o Accessory:
o Essential: Growth does not occur in their absence.
o Accessory: Enhances growth without being really necessary for some bacteria.
Requirements for growth of pathogenic (disease causing) species:
- Growth factors:
Produced by body fluids and tissues ‘in vivo’ and from yeast extract, blood and blood products ‘in vitro’ include: B complex vitamins
Thiamine (B1), Riboflavin (B2), Nicotinic acid (B3), Folic acid (B9, Folate), B12
- Based on nutritional requirements microbes are classified as:
Autotrophs:
Synthesize organic compounds using CO2 (source of carbon) and N2 (no medical importance).
- Based on nutritional requirements microbes are classified as:
Heterotrophs:
Unable to synthesize own metabolites and depend on preformed organic compounds as source of carbon (All pathogens).
Bacterial Growth Refers to:
- Increase in the number of microbes, rather than an increase in size of a microbe = (4)
- Involves increase in cell mass and number of ribosomes, duplication of bacterial chromosome, synthesis of new cell and plasma membrane, partitioning of the two chromosomes, septum formation and cell division.
- When growth reaches critical mass, cell divides.
- Bacteria grow and divide by binary fission (asexual reproduction) and nuclear division
precedes cell division. - “In vivo” growth of bacteria depends on our nutritional status, immunity, humoral factors, pH, redox potential…
What is generation time (Population doubling time) ?
- Interval of time between two cell divisions OR Time
required for a bacterium to give rise to two daughter cells
under optimum conditions
“The hypothetical culture begins with one cell having a __ min generation time”
20
- Knowledge of microbial growth is useful for understanding population dynamics and control in infectious diseases and food preservation.
Why is this important? (3)
o Treating life threatening infectious diseases
o Disinfection of living (animate) or non-living (inanimate) surfaces
o Informing processes related to cell division in higher organisms
How can microbial growth be expressed?
Microbial growth can be expressed as the number of cells in a bacterial generation using the power of 2𝑛, where n is the number of doublings that have occurred as shown in the illustration.
Factors Affecting Bacterial Growth: (7)
- Temperature
- Atmospheric O2 and CO2
- H ion concentration
- Moisture and drying
- Osmotic Pressure
- Mechanical and sonic stress
- Radiation
Classification by temperature:
- Mesophilic:
- Psychrotrophilic:
- Thermophilic:
- Mesophilic: 25 - 40 ‘C (Pathogens)
- Psychrotrophilic: Can grow at 0 - 7 ‘C , Optimum 20 - 30 ‘C (Cold loving) - Listeria
monocytogenes - Thermophilic: 55 - 88 ‘C - Bacillus sterothermophilus (used to test efficacy of sterilization)
Classification by oxygen requirement:
- Strict obligate aerobe:
- Strict obligate anaerobe:
- Microaerophilic:
- Strict obligate aerobe: Love O2 (P. aeruginosa, Staphylococcus spp)
- Strict obligate anaerobe: Don’t love O2, may die (Bacteroides fragilis , Clostridium spp)
- Microaerophilic: Best in low O2 (Helicobacter, Campylobacter spp)
Classification by CO2 requirement:
- Facultative aerobe:
- Aerotolerant anaerobe:
- Capnophilic:
- Facultative aerobe: Can grow in O2 also (most aerobes)
- Aerotolerant anaerobe: May tolerate O2 exposure (Streptococcus pyogenes, Clostridium
perfringens) - Capnophilic: Needs CO2 (5 10%) (Neisseria spp, Streptococcus pneumoniae, Brucella
abortus)
Classification by H ion (pH) requirement: (4)
- Human body - Neutral (pH 7.3-7.5)
- Human pathogens - Neutrophiles (pH 5.4-8.5)
- Lactobacillus spp - Acidophile (pH 0.1-5.4)
Acidic products of bacterial metabolism interfere with growth.
Buffers are used to stabilize pH - Vibrio cholerae - Alkalophile ( pH 8-12)
Moisture and drying:
- Water(constitutes 80% of total bacterial cell weight) is essential for bacterial _____; Drying is lethal to bacterial cells.
protoplasm
- Effect of drying varies: (3)
Osmotic Effect:
- Osmotic pressure(OP): Cells are 80 - 90% water
- Isotonic solution:
- Hypertonic solution:
- Hypotonic solution:
Mechanical and Sonic Stress and Radiation:
- Cell wall may be ruptured by grinding and ______ shaking
- May be disintegrated by exposure to ______ vibration
- X rays and gamma rays exposure _____
vigorous
ultrasonic
lethal
Bacteria are grown on laboratory media that support their growth
There are various types of culture media within the lab (solid agar medium, broth medium with bacterial suspension, liquid-nutrient broth, semi-solid agar) that help enhance bacterial growth.
Culture medium: (3)
- Must be sterile
- Contain appropriate nutrients
- Must be incubated at appropriate temperature
Bacterial Growth Curve:
- When a bacterium is added to a suitable culture medium, it goes through definite _____ ______.
- If bacteria is quantified at intervals after _______ and plotted in relation to time and temperature, a _____ curve is obtained.
growth phases
inoculation
growth
What is the LAG phase? (6)
What is the clinical significance of the LAG phase?
Clinical significance: Incubation period of diseases
What is the LOG phase? (5)
LOG phase
o Cells most susceptible to adverse environmental factors: (3)
▪ Irradiation
▪ Antibiotics that affect protein, DNA and cell wall synthesis. Active as cells rapidly divide in culture where proteins are rapidly synthesized, B-lactams effective when cells are making peptidoglycan
▪ Disinfectants
What is the clinical significance of the stationary phase? (7)
What is the clinical significance of the stationary phase?
Clinical significance: Toxemia (multiple virulence factors which play a role)
What is the PHASE OF DECLINE/DEATH? (6)
o Nutritional exhaustion and toxin accumulation ↑
o Number of cells ↓ due to death of cells caused by autolytic enzymes
o Death rate> growth rate
o Involution forms (with ageing)
o Irreversible loss of the ability to reproduce
o Often, death rate slows due to accumulation of resistant cells (resistant bacteria
show extended survival)
What is the clinical significance of the decline/death phase?
Clinical significance: Phase of convalescence
Autolytic enzymes can break down human tissue and cellular debris to spread to nutrient rich tissues. However, during the death phase this does not happen and cells die.
What is bacterial metabolism? (2)
- Series of changes of a substance/food (carbohydrate, fat, protein), taking place within the
cell from absorption to elimination = SUM TOTAL OF CHEMICAL REACTIONS IN A CELL. - Absorbed nutrients are utilized and eliminated by certain metabolic pathways
Bacterial metabolism =
Catabolism:
Breakdown of macromolecules into simpler micromolecules to release energy. Production of
precursor metabolites used in biosynthesis
Bacterial metabolism =
Anabolism:
Basic building process of blocks utilized in biosynthesis of various cellular structures (monomers and polymers). Assemble subunits of macromolecules that make up cell structures, using ATP to drive reactions
Microbes use variety of compounds:
- Synthesize subunits using the central ______ pathways.
If lacking enzymes, end products must be supplied. _______ bacteria require growth factors. - Excrete _______ enzymes, transport subunits into cell. Degrade appropriate precursor metabolites.
metabolic
Fastidious
hydrolytic
Implications of microbial metabolism: (5)
- Food production
- Biofuels
- Important in laboratory
- Invaluable models for research
- Unique pathways potential drug targeting
Glucose is centre of cell metabolism pathways:
- Three major pathways:
o Glycolysis
o Tricarboxylic Acid cycle (TCA)
o Pentose Phosphate
What is the process of glycolysis?
o Glycolysis (Embden Meyerhof Parnas (EMP),
is the most common)
- Splits glucose (6C) to two pyruvates (3C). Generates modest ATP, reducing power, precursors.
What is the TCA cycle? (2)
o Tricarboxylic Acid cycle (TCA)
- Oxidizes pyruvates from glycolysis
- Generates reducing power, precursor metabolites
What is pentose phosphate used for?
o Pentose Phosphate
Primary role is production of precursors metabolites, NADPH.
- Key outcomes of metabolic pathways: (3)
o ATP
o Reducing power: NADH, FADH, NADPH
o Precursor metabolites
What is the use of oxidation?
- Aerobes obtain energy using 02 as a hydrogen acceptor.
What is the process of fermentation?
- Anaerobes use electron donor & acceptor as nitrates and sulphites
- Products produced during fermentation: (4)
o Acids (lactic, formic, pyruvic)
o Alcohols
o Gases-H2,CO2
o Phosphates (rich), bonds are transferred to ADP to form ATP
Facultative bacteria can use both pathways, which are?
Oxidation and Fermentation
Oxidation Reduction Potential (redox potential - Eh):
- Oxidizing or reducing system accepts or loses ______ respectively
- Strict Anaerobes require a low __
electrons
Eh
What is Koch’s Postulate?
- Formulated by Robert Koch in 1887, in an attempt to determine the cause of infectious
disease as evidence required to establish an etiologic relationship between microorganisms and
disease. CAUSE AND EFFECT RELATIONSHIP BETWEEN BACTERIA AND DISEASE.
What are the components of Koch’s Postulate? (4)
o Microorganisms must be isolated from every diseased patient (Association)
o It must be isolated in vitro in pure culture (Isolation)
o When inoculated, the pure organism must cause disease in a healthy susceptible
animal (Causation)
o The organism must be recovered from the infected animal (Re-Isolation)
- Modifications to the postulates were made by _____ and _____ to address non-culturable microorganisms (causative agent of leprosy).
ESTABLISHMENT OF THE CRITERIA FOR A MICROORGANISM BEING THE CAUSE OF A DISEASE. ROLE OF MICROBES IN RELATION TO PATHOGENESIS
Fredericks and Relman
Role of Microbiology Laboratory in Diagnosis of Infectious Diseases
- Offers tests that:
Identify microorganisms from clinical specimens either, directly (visually, using a microscope, growth in culture) OR direct detection from specimens using genetic probes or serological indirect detection of antibodies/antigens to the microbe)
- General phenotypic, non-nucleic-acid-based types of tests include:
o Microscopy: (2)
▪ Stained smears (e.g. Gram, Ziehl Neelsen, Auramine);
▪ Wet preparations (for presence of cells, bacteria, yeasts, parasites or parasitic eggs)
- General phenotypic, non-nucleic-acid-based types of tests include:
o Culture and Susceptibility testing: (4)
- Characteristic of organism’s growth on culture media (colony size, colour and shape)
- Manual or automated systems or chromatographic method (HPLC)
- Mass spectrometry detects inorganic or organic compounds
Example: Proteins according to mass. Specific pathogens have unique proteins and relative mass (Identifies within 24 hours vs 24-72 hours in conventional way)
NEED A CULTURED ORGANISM.
- MALDI-TOF: Matrix assisted laser desorption ionization time to flight
(bacteria, mycobacteria, yeast, molds)
- General genotypic types of tests include: (2)
o Nucleic acid based:
Detect organism directly from specimen using probe specific DNA or RNA sequences PCR, RT PCR, LCR.
o Serological tests:
Immunologic tests such as: Latex agglutination, Enzyme immunoassay (ELISA), Immunofluorescent antibody (IFA)
Summary of Microbiology Lab Testing Techniques:
