DIAGNOSTIC MICROBIOLOGY a summary of reviewers

BACTERIOLOGY
Important Characteristics of Bacteria
▪ Prokaryotic → No true nucleus
▪ Has both RNA and DNA
▪ Multiplies by binary fission (asexual reproduction)
▪ Measured in um (micra)
▪ Average size 0.4 to 2 micra
▪ Produce either exotoxins and endotoxins → Biologically produced poisons
▪ Mycoplasma – smallest living organism
Exotoxin Endotoxin
▪ Composition: Protein in nature ▪ Composition: Lipopolysaccharide
▪ Effect: Systemic ▪ Effect: Local
▪ Toxicity: High toxicity ▪ Toxicity: Low toxicity
▪ Stability to heating: Unstable (heat-labile) ▪ Stability to heating: Stable (heat-stable)
▪ Stimulates antitoxin production: YES ▪ Stimulates anti-toxin production: NO
▪ Excreted by living bacterial cell ▪ Limulus lysate test
▪ Examples: TSST-1, botulinum toxin, diphtheria toxin o Detects endotoxins in body fluids
o Cytotoxin – kills host cell o Reagent: Aqueous-extract from blood cells
o Enterotoxin – damages cells of the GIT of horseshoe crabs
o Neurotoxin – interferes with nerve impulses o Positive: Clumping or turbidity
▪ May be in enzyme form: coagulase
▪ Released by all major Gram positive except: Listeria
monocytogenes (produces endotoxin)
▪ Can be produced by Gram negative organisms such
as Vibrio and E. coli
Important Parts of the Bacterial Cell
1) Capsule
▪ Not common to all bacteria
▪ Anti-phagocytic – considered a virulence factor
▪ Slimy layer surrounding the cell wall
▪ Responsible for mucoid colonies
▪ Gives K or Vi antigen in serotyping
▪ Neufeld-Quellung capsular antigen (+)
o Capsular swelling due to Ag-Ab reaction
o Way of determination of capsulation
▪ Notable capsule positive organisms: B. anthracis, K. pneumoniae, H. influenzae, S. pneumoniae, N. meningitidis
o Can be used for vaccination efforts against these bacteria
2) Cell wall
▪ AKA. Peptidoglycan or murein layer
▪ Defines the shape of the bacteria
▪ Main component is peptidoglycan
▪ Point of anchorage for flagella
▪ Site of antibiotic action of beta-lactams
▪ Basis of Gram staining reactions
o Gram positive with rigid peptidoglycan layer (with teichoic acid and N-acetyl-glucosamine & N-acetyl-
muramic acid) – Impermeable to alcohol decolorization
o Gram negative with thinner peptidoglycan layer (with amino acids, phospholipids, &
lipopolysaccharides) Critically, NO teichoic acid – Permeable to alcohol decolorization
▪ Mycoplasma and ureaplasma → No cell wall; rely on their plasma membrane to maintain general shape
▪ Notable organism cell walls:
o S. pyogenes – Contains M protein, prevents phagocytosis
o Mycobacterium spp. – Contains mycolic acid, prevents digestion after phagocytosis
▪ Responsible for acid-fast property of the genus
3) Plasma membrane
▪ Surrounds the cytoplasm
▪ Site of energy synthesis
▪ Transport of nutrients in and out of the cell
▪ Function as Golgi apparatus & lysosome
4) Pili
▪ AKA. Fimbriae
▪ Not present in all bacteria
▪ Usually found in Gram negative bacteria like E. coli, N. gonorrhoeae, Pseudomonas spp.
▪ Common pili – attachment to host cell
o First step in establishing infection
o Also, for virulence (N. gonorrhoeae)
▪ Sex pili – gene conjugation (transfer of genetic material)
5) Endospores
▪ AKA. Spores
▪ Resistant structure enabling bacteria to withstand adverse conditions
▪ Best way to destroy spores is with autoclaving
▪ Cause of resistance: Calcium dipicolinate or dipicolinic acid
▪ Bacteria with spores: Bacillus spp. & Clostridium spp.
▪ Location of spores:
o C. tetani – Terminal
o C. botulinum – Sub-terminal
o B. anthracis – Central
6) Flagella
▪ For locomotion
▪ Many bacilli and spiral organisms are motile while true motility is seldom observed in cocci
▪ Best observed at room temperature (25oC)
MOTILE E. coli, C. tetani, H. pylori
NON-MOTILE C. diphtheriae, C. perfringens, B. anthracis,
Staphylococci
TYPES OF MOTILITY
TRUE MOTILITY PSEUDO MOTILITY
▪ Due to flagella ▪ Brownian movement
CHARACTERISTIC MOTILITY
TUMBLING MOTILITY Listeria monocytogenes
GLIDING MOTILITY Capnocytophaga gingivalis
DARTING MOTILITY Campylobacter jejuni
CORKSCREW MOTILITY Spirochetes
TWITCHING MOTILITY Kingella kingae
SHOOTING STAR MOTILITY Vibrio cholerae
TYPES OF FLAGELLA
MONOTRICHOUS Single flagellum at one end
AMPHITRICHOUS Single flagellum at both ends
ATRICHOUS Absence of flagellum
LOPHOTRICHOUS Tuft of flagella at one or both ends
PERITRICHOUS Surrounded with flagella
AXIAL FILAMENTS / PERIPLASMIC FLAGELLA Spirochetes → Causes corkscrew motility
▪ Ways of demonstrating motility:
o Hanging-drop method
o Use of flagellar stains (Grey’s or Leifson’s)
o Use of semi-solid media (SIM)
▪ Parts external to the cell wall:
o Flagella
o Cell wall
o Pili or fimbriae
7) Inclusion bodies
▪ These are accumulated food reserves; serves as energy source
▪ Notable organisms with inclusion bodies:
o Corynebacterium diphtheriae – Babes-Ernst granules
o Mycobacterium tuberculosis – Much granules
▪ Notable organisms with inclusions:
o Yersinia pestis – Bipolar bodies (safety pin appearance due to Wayson stain)
o Nocardia & Actinomycetes – Sulfur granules
 8) Nucleoid
▪ The DNA in the bacterial cell is generally confined to the central region
▪ Bacteria do not have a nuclear membrane
▪ Chromosome – dsDNA
▪ Plasmid – Extrachromosomal DNA which carries antibiotic-resistant genes
o Large plasmid confers resistance to beta-lactam antibiotics (cell wall inhibitors)
▪ Responsible for ESBL organisms like K. pneumoniae & E. coli
o Small plasmid confers resistance to chloramphenicol & tetracycline
▪ Bacterial gene transfer summary:
o Conjugation – Plasmid-mediated via sex pili
▪ Transposon – Mobile, jumping genes for Gram negative bacteria (ESBL positive)
o Transduction – Bacteriophage-mediated
▪ Passes the TOX gene of C. diphtheriae
o Transformation – Naked-DNA
▪ Causes the virulence of S. pneumoniae
9) Ribosome
▪ Site of protein synthesis
▪ Gives the cytoplasm of bacteria a granular appearance in electron microscopy
▪ Bacterial ribosomes – 70S region
▪ Fungal ribosomes – 80S region
History of Bacteriology
▪ Anton van Leeuwenhoek
o First to describe bacteria
o Father of Microbiology & Microscopy
▪ Robert Koch
o Father of Germ Theory
Definition of Terms
▪ Normal flora
o AKA. colonizers, microbiota, microbiome
o Organisms which normally inhabit unsterile parts of an organism
▪ Biofilms
o Adhere to prosthetics
o Characteristically not removed by the immune system
o Organisms which cause biofilms: Staphylococcus epidermidis, Candida albicans, Pseudomonas spp.
▪ Vector
o Living organism which transfers the bacterium to a host
▪ Vehicle
o Non-living organism
Phenotype vs Genotype
Phenotype Genotype
Observable characteristics Requires gene analysis
Gram stain & acid-fast stain methods PCR, RT-PCR, LCR, Nucleic acid base amplification methods
Colony & biochemical characteristics Southern blot, FISH
Serotyping & drug resistance Restriction fragment length polymorphism
Bacterial Physiology
1) Nutritional requirement
▪ Phototrophic – uses light as an energy source
▪ Chemotrophic – uses chemical compounds as energy source
o Autotrophs / lithotrops – uses inorganic compounds as carbon source
o Heterotrophs / organotrophs – uses organic compounds as carbon sources (most bacteria)
▪ Note: Bacteria use carbon to synthesize extracellular components
2) Oxygen requirement
▪ Obligate aerobes – require O2 for growth
o Examples: Brucella spp., Francisella spp., Mycobacterium spp.
▪ Obligate anaerobes – require a lack of O2 for growth
o Examples: Fusobacterium spp., Bacteroides spp., Clostridium spp., Veillonella spp., Actinomyces spp.
▪ Facultative anaerobes – can live with or without O2; most pathogens are facultative anaerobes
o Examples: Staphylococcus spp.
▪ Microaerophiles – requires a small amount of O2 for growth
o Example: Campylobacter spp.
 ▪ Aerotolerant – does not grow well but survives in the presence of O2
o Example: Lactobacillus spp.
▪ Extremophiles – able to survive in unusual conditions (absence of oxygen & increased temperature)
o Example: Bacillus infernus
▪ Capnophilic – require increased amounts of CO2 for growth
o Example: Neisseria spp. & Haemophilus spp.
o Conditions: 5 – 10% CO2; Candle jar provides 3% CO2
ANAEROBIC VS MICROAEROPHILIC
Uses GasPak jar to achieve oxygen conditions Uses CampyGas to achieve oxygen conditions
Conditions: 5% CO2, 10% H2, 85% N2 Conditions: 5% O2, 10% CO2, 85% N2
EXAMPLES PER OXYGEN OBLIGATE AEROBES FACULTATIVE MICROAEROPHILES OBLIGATE
REQUIREMENT ANAEROBES ANAEROBES
Gram positive B. cereus Staphylococcus spp.; B. Streptococci spp. Clostridium spp.
anthracis; Corynebacterium
spp.; Listeria spp.; Actinomyces
spp.
Gram negative Neisseria spp., Pseudomonas Most Gram negative rods Spirochetes, Campylobacter Bacteroides spp.
spp., Bordetella spp., Legionella spp.
spp., Brucella spp.
Acid fast Mycobacterium & Nocardia

OXYGEN REQUIREMENT OXYGEN CARBON DIOXIDE

AEROBES 21% 0.03%
ANAEROBES 0% 5 – 10%
CAPNOPHILIC 15% 5 – 10%
MICROAEROPHILIC 5 – 10% 8 – 10%
3) Temperature
▪ Optimum temperature – temperature at which a bacterium grows best
▪ Psychrophilic – require 0 – 20oC for growth (cold-loving)
o Can tolerate cold enrichment: incubation at 4oC for several weeks
▪ Notable cold enrichment positive organisms: Listeria monocytogenes (coleslaw food poison),
Yersinia enterocolitica (blood bag contaminant)
▪ Mesophilic – require 20 – 45oC for growth (most pathogenic organisms)
▪ Thermophilic – 50 – 125oC (heat-loving)
o Notable thermophilic organisms: Bacillus stearothermophilus (autoclave indicator), Thermus aquaticus
(source of Taq polymerase for PCR)
▪ Thermoduric – bacteria which do not usually grow at high temperature but can withstand exposure to high
temperature
▪ Incubation temperature for molds – 25oC
▪ Incubation temperature for bacteria – 35oC
▪ Incubation temperature for viruses – 37oC
4) pH requirement
▪ Acidophilic – requires acid pH for growth
o Example: Lactobacillus spp. (can grow on tomato juice agar media pH 5.0) & fungi (can grow on
Sabouraud’s dextrose agar pH 5.6)
▪ Neutrophilic – requires a pH between 7.2 to 7.4 for growth
o Examples: Most pathogenic bacteria
▪ Basophilic – requires basic pH for growth
o Example: Vibrio spp. (can grow on alkaline peptone water pH 8.6)
5) Moisture
▪ Humidophilic – requires >70% moisture to prevent death
6) Salt concentration
▪ Halophilic – requires increased salt content
▪ Notable halophilic organisms:
o Enterococci spp. (tolerate 6.5% NaCl)
o Staphylococcus aureus (tolerate 7.5% NaCl)
o Vibrio spp. (requires 8 – 10% NaCl)
7) Bacterial metabolism
▪ Respiration (aerobic process)
o Kreb’s cycle (aerobic process)
o Electron transport chain (aerobic process)
o Glucose → CO2 and H2O (can not be detected by pH indicator)
 ▪ Oxidation (aerobic process)
o Used by non-fermentative organisms
o Glucose → Acid (can be detected by pH indicator)
▪ Fermentation (anaerobic process)
o Used by fermentative organisms
o Glycolysis (EMP)
o Glucose → Acid / alcohol
Stages of Bacteria Growth