Clinical Microbiology Lec Exam 1 Study Guide

Contributions to the field of Microbiology

• Leeuwenhoek

• Cohn

• Pasteur

• Koch

• Gram

• Linnaeus

• Leeuwenhoek→ simple handcrafted microscope to observe live microbes from teeth scraping, rainwater (he called them “animalcules”)

• Cohn → discovered endospores

• Pasteur → (1) Theory of Biogenesis/Microbacterial growth via broth & air microorganisms (2) Vaccination against anthrax & rabies

• Koch → linked anthrax to a spore forming bacillus

• Gram → staining technique to classify microbes

• Linnaeus → Latin nomenclature of organisms

Contributions to the field of Microbiology Continued

• Semmelweis

• Lister

• Nightingale

• Snow

• Jenner

• Ehrlich

• Fleming

• Semmelweis → hand-washing w/ chlorinated lime water reduced Puerperal fever (childbed fever) which causes death of hundred of mothers

• Lister → Antiseptic surgery (phenol)

• Nightingale → Nightingale School of Nurses

• Snow → Cholera and the beginning of epidemiology

• Jenner → vaccination against smallpox

• Ehrlich → the “magic bullet” and development of a
  chemotherapeutic agent against syphilis

• Fleming → first mass-produced antibiotic: Penicillin

Koch Postules

Experimental steps that demonstrate that a specific microbe causes a specific disease

• suspect pathogen must grown outside of host→ isolated in pure culture

Microbes Carry Out 3 Fundamental Processes That Sustain Life

1. Nitrogen fixation & O2 Production

2. Decompose plant & animal matter

3. Symbiotic relationships w/ plant & animals

4 Uses of Microbes

1. Research → E.coli

2. Food Production → dairy, bread, bevs

3. Biodegradation/bioremediation → sewage & wastewater treatment

4. Synthesis of Commercially valuable products → ethanol/ liquor, antibiotics, insect repellant

Microbes make up the normal microbiota of the human body found where?

Function

• skin and mucous membranes of the upper respiratory
  tract, intestinal, and urogenital tracts

• Function: immune function, digestion, and synthesis of vitamins

Examples of Pathogenic Microbes that Led to Pandemics

• Influenza A virus strain H1N1

• Yersinia Pestis → European plague in 14th century

• SARS CoV2

Know the Microbial World of Organisms (living) & Infectious Agents (Non-Living) and the sub divisions…

Emerging Infectious Diseases vs Re-Emerging Diseases

Emerging → new, common infectious disease recognized w/in past 35 years

• ie: ebola, SARS, Hepatitis C, Lyme disease, or AIDS

Re-emerging→ once under-control via vaccines/immunity, but now have re-occured its spread due to drug resistance, lapse in vaccination…

• ie: MDRTB, Malaria, Measles

Bacteria are…

3 Characteristics

• …single-cell/unicellular prokaryotes → w/o membrane bound nucleus

• Have rigid cell wall → peptidoglycan

• Multiply via asexual reproduction→ binary fission

• move using flagella

Fungi are…

3 Characteristics

• …Unicellular/multicellular Eukaryotes | ie: yeast, mold, mushrooms

• Cell wall → Chitin

• Sexual/asexual reproduction

• many are saprophytes → decomposers of organic matter

Protozoa are…examples..

3 Characteristics & Location

• …Unicellular Eukaryotes ; ie. live freely or as parasites, pathogenic to humans (malaria)

• No rigid cell wall

• Sexual/asexual reproduction

• Motile via cilia, flagella, or pseudopodia

• Location: aquatic/terrestrial environemnts

Parasitic helminths/worms Characteristics

(roundworm= || tapeworm= )

• multicellular eukaryotes

• roundworm= ancylostoma | tapeworm= taenia

• eggs & immature stages are Microscopic & found in blood, fecal, and urine samples ; can be seen as they are adults & matured

Why are viruses considered non-living

They are not made of cells, rather they are a piece of genetic matierial (DNA/RNA) inside a protein coat; They cannot produce their own energy, nor multiply without a host.

Viruses Characteristics

• Obligate intracellular parasite (infection microogranism that cannot survive outside a host) - neither prokaryotic or eukaryotic

• RNA or DNA w/in protein envelope

• Infect bacteria, plants, and animals (humans)

• Visible via electron microscopes (TEM)

Viroids vs Prions Characteristics

• both neither prokaryotic or eukaryotic

• Viroids: infectious agents made of single-stranded RNA

• Prions: infectious agents composed of proteins

Binomial Nomenclature

Two names: First name is the Capitalized Genus ; Second name is non capitalized species

• ie: Escherichia coli (Genus= Escherichia; species=coli)

Definition of species for prokaryotes

Species (for prokaryotes): A group of closely related isolates or strains (not identical!)

General Taxonomic ranks (Ascending Order) & Ranks for Escherichia coli (E. coli)

General (Ascending):

Species→ Genus→Family→Order→Class→Phylum→Domain

Escherichia Col (Ascending)i:

coli→Escherichia→Enterobacteriaceae→Enterobacteriales→Gammaproteobacteria→Proteobacteria→Bacteria

Three Domains & Where would you find Staphylococcus aureus, Penicillium chrysogenum, Giardia intestinalis, or Scistosoma mansoni?

1. Bacteria → staphylococcus aureus

2. Archaea →

3. Eucarya → Penicillium chrysogenum, Giardia intestinalis, scistosoma mansoni

Morphology Refers to _____ & Most common Morphology among bacteria include:

Morphology= Cell Shape

Common Morphology=

• Coccus (circle/spjere) &

• Bacillus (oval/rod shaped)

Multicellular arrangement in coccus _______,

Difference between arrangements…..

• Amount of connected cells:

• Diplo - two

• Strepto - chained

• Tetrads- 4 in one linear plane (square)

• Sarcinae - 4 in a 3D plane (cube)

• Staphylo- large 3D cluster

Multicellular arrangement in bacillus _______,

Difference between arrangements…..

• Single Bacillus

• Diplo

• Strepto

• Palisade

Bacteria Staining:

Direct Stain vs Negative Stain

• Direct stain → stains the cell

• Negative stain→ stains the background (capsule)

Function of the gram stain

• one of the first steps a CLT performs to identify bacteria

General Structural Components of a Typical Bacterial Cell

• Cell envelope: capsule→cell wall→plasma membrane

• Cytoplasm → plasma membrane= thick subst. filled w/ enzymes, ribosomes & nutrients (fluid portion of cytoplasm=cytosol)

• Nucleoid (contain DNA)

• Plasmid

• Inclusion

• Flagella

• Fimbria

• Pilus

Plasma Membrane Characteristics & Function

• Selectively permeable lipid bilayer barrier → polar head, non-polar fatty acid tail

• Surrounds cytoplasm & interact w/ external environment

• Proteins act as chemoreceptors & transport systems

Structural/functional characteristics of a typical bacterial cell:

Plasma Membrane Transport Proteins - Define the following & their source of Energy

• Simple Diffusion=

• Facilitated Diffusion/Passive Transport=

• Active Transport=

• Simple Diffusion= Gases (O2, CO2, N2), Small hydrophobic Molecules, and water pass freely → down concentration gradient (high to low concentration) until equilibrium is reached

• Facilitated Diffusion/Passive= protein transporter allow substance to move down its concentration gradient

• Active Transport: protein transport uses energy (proton motive force or ATP) to move a substance across membrane → against concentration gradient (low to high concentration)

Structural/functional characteristics of a typical bacterial cell:

• LPS Stand for ____ and is found where?

• Lipid A is an _____

• O _____ is …

• Lipopolysaccharide → Found in Cell wall of gram negative bacteria

• Lipid A is an endotoxin

• O polysaccharide - a diagnostic marker to identify species or strains

Structural/functional characteristics of a typical bacterial cell:

• The cell wall is composed of

• Functions of cell wall

Composed of:

• peptidoglycan

• Alternating NAD→NAM subunits to form glycan chain

• Tetrapeptide chains link glycan chains

• Peptide interbridge (Gram positive)

Function:

• protect cell from osmotic pressure→prevent cell lysis

• provides structure, shape

Atypical cell wall Characteristics

• acid-fast cell walls: → Waxy Lipids (mycolic acid) bound to the peptidoglycan

Structural/functional characteristics of a typical bacterial cell:

• Why do we apply the acid-fast stain to detect the causative agent of TB?

• Ziehl-Neelsen acid-fast stain detects mycobacterium species specifically

• TB= mycobacterium→ rod-shaped acid fast call → stained pink/red

Structural/functional characteristics of a typical bacterial cell:

• The capsule or slime layer can surround the bacterial cells, and it is composed of ___

• Capsule vs Slime Layer

• Function of Capsule or Slime Layer ____

• In the case of pathogenic species/strains, why is the capsule referred to as a virulence factor ___

• Termed Glycocalyx→ sticky substance made of polysaccharides, polypeptides or both.

• Capsule: Organized and firmly attached to the cell wall

• Slime layer: Unorganized and loosely attached to the cell wall

• Function: protect from desiccation (ext. dehydration) ; enable pathogens to escape or survive phagocytosis (virulence factor)

Structural/functional characteristics of a typical bacterial cell:

• Pili can enable attachment (fimbriae), ____, or _____

• Projections outside of bacteria

• enable attachment & biofilm formation, motility, & genetic exchange during DNA transfer (plasmids) →

• this process of DNA transfer gives bacteria (1) ability to produce toxin & (2) resistance to antibiotics

The pathogen that colonizes the mucous membrane of the reproductive tract by attaching with fimbriae (adherence) is termed

Neisseria gonorrhoeae

Proteus species are involved in nosocomial urinary tract infections

Proteus vulgaris

Structural/functional characteristics of a typical bacterial cell:

• What is the function of flagella? ____ What is their arrangement called? ____

• Flagella allow movement, which in turn can enable escape from
  host defense or systemic infections

• Peritrichous

• Monotrichous

• Lophotrichous

• Amphitrichous
  

  
  

• What is the axial filament found in the periplasmic space of spirochetes? ____

• The axial filament is made of endoflagella and found in
  spirochetes
  ▪ Causes the bacterial cell to rotate like a cork screw

Structural/functional characteristics of a typical bacterial cell:

• Where in a bacterial cell would you find the chromosome? _____

• What is the function of the ribosomes? ____

• Chromosome is found in the nucleoid (not membrane bound)

• Ribosome: composed of protein & RNA→ function: site of protein synthesis

Cell Motility =

Taxis

Structural/functional characteristics of a typical bacterial cell:

• Certain genera of bacteria (Bacillus and Clostridium) produce endospores, why? ___

• survival (not reproduction) in volatile environmental conditions

Structural/functional characteristics of a typical bacterial cell:

Endospore Characteristics

• Is an endospore metabolically active? ___

• An endospore can be resistant to ____

• Not metabolically active

• endospores are resistant to heat, radiation, chemicals, desiccation, boiling water

Structural/functional characteristics of a typical bacterial cell:

Endospore Characteristics:

Sporulation is ____.

Germination is ____.

• Sporulation = vegetative cell → endospore spore

• Germination = endospore → vegetative cell

What is the location of an endospore within the sporangium? ___

• Central

• Terminal

• Subterminal

Do bacterial cells store inorganic/organic material for later use? _____ What do they store? ___

Yes:

▪ Glycogen
▪ Carbon (poly-β-hydroxybutyrate - PHB)
▪ Phosphate (Volutin)
▪ Sulfur
▪ Nitrogen

Microbial growth =

an increase in number of cells rather than an increase in size

Groups of Bacteria according to the following factors:

• sources of energy and carbon, oxygen requirements, temperature, pH, and salts

Type→ Energy source → Carbon Source

Type→ Energy source → Carbon Source

Photoautotroph → sunlight → CO2

Photoheterotroph → sunlight → Organic compounds

Chemolithoautotroph → Inorganic chemicals (H2, NH2, Fe2+)→ CO2

Chemoorganoheterotroph → Organic compounds (Sugar, amino acids,..) → Organic compounds

Chemical Requirements for Growth

Capnophile

• Capnophiles are microbes that require higher concentration of
  carbon dioxide (up to ~10%) in addition to low oxygen levels

  
  

• A chemoorganoheterotroph is ___

• Chemo- → gets energy from chemical compounds

• Organo- → gets electrons from organic compounds

• Heterotroph → gets carbon from organic compounds

Mesophile

• human pathogens that survive/grow in human body temperature (37 degrees celsius)

• Range: 20-45 degree celsius

Acidophile

Grow best in acidic habitats (low pH)

less than 5.5 ph on a scale from 0-14 (7 is neutral)

Do halophiles require salts to grow? __

Yes, Halophiles require high concentrations of salt (NaCl) to grow.

• What is meant by optimum growth temperature? ___

• temperature where there is highest growth rate

Do obligate anaerobes grow and multiply in presence of oxygen? ___

• obligate anaerobes would not grow in presence of oxygen

• oxygen is toxic to them; lack enzyme to neutralize oxygen byproducts
  

What is the function of the enzyme catalase or SOD? ___

• Aerobic metabolism forms inflammation inducing reactive oxygen species (O2-/H2O2)

• Microbes use SOD and/or catalase as protective mechanisms against R.O.S.
  ▪ SOD converts O2- to O2 and H2O2
  ▪ Catalase converts H2O2 to O2 and H2O

What happens to a bacterial cell when placed in isotonic, hypotonic, or hypertonic conditions? ___

Isotonic- no change

Hypotonic: the bacterial cell gains water and swells to the limit of its
cell wall

Hypertonic: the cell loses water and shrinks (shriveling of the cytoplasm)

In the environment, microbes typically live in mixed communities form _____

biofilms → complex structures & regulated interactions

Define

Pure culture

Mixed culture,

Colony (CFU)

• Pure culture: A culture containing only one species or strain of microorganism.

  • Example: Only E. coli growing in a culture.

• Mixed culture: A culture containing two or more different species or strains of microorganisms.

  • Example: E. coli + S. aureus in the same culture.

• Colony (CFU): A visible mass of microorganisms growing on a solid surface, usually originating from one cell or a group of cells.

Inoculum=

a sample bacteria

Provide examples of clinical specimens/methods to collect microbes in the laboratory

• Sterile broths

• Sterile Agar media: Slants, Deep, Petriplate →

• What is the purpose of collecting clinical specimens? ___

• to identify type bacteria/pathogen in a patient and prescribe the right antibiotics

Proper collection of clinical specimens, transport or handling is important to prevent ___

Techniques to isolate the suspected pathogen in pure culture are used in combination with selective/differential media (i.e., MacConkey, MSA, or BAP) (review them!!)

• Quadrant streaking technique→ help separate species to detect the colonies of a pathogen

• Serial Dilutions → Pour plates → Spread Plate Methods

• MacConkey agar (MAC) → Selects for Gram-negative bacteria and differentiates lactose fermenters.

  • 🩷 Pink colonies = lactose fermenter

  • Example: E. coli

• Mannitol Salt Agar (MSA) → Selects for salt-tolerant bacteria, especially Staphylococcus, and differentiates mannitol fermentation.

  • 🟡 Yellow = mannitol fermenter

  • Example: S. aureus

• Blood Agar Plate (BAP) → Enriched and differential medium used to observe hemolysis.

MAC → Gram − + lactose
MSA → Staph + mannitol
BAP → Blood + hemolysis

Definition generation time ___

• Bacterial growth: An increase in the number of bacterial cells, usually through binary fission.

• Generation time: The time required for one bacterial cell to divide and produce two cells, or for a bacterial population to double in number.

o Bacterial cells undergo cell division by a mechanism know as ___

• Binary Fission → one cell divides into two genetically similar daughter cells

How would you use this simple equation

Nt = N0 x 2^n

To calculate the total number of cells in a population, we multiply the original number of cells by 2^n
▪ Nt = N0 x 2^n
▪ Nt = number of cells in a population
at a given time
▪ N0 = initial number of cells in the
population
▪ n = number of generations (division)

Phases of a bacterial growth curve ___

1. Lag Phase

2. Log (exponential) Phase

3. Stationary Phase

4. Death (decline) Phase

Several methods are used to measure microbial growth (What are they?)

(Direct vs Indirect Methods)

Direct Methods:

• Direct Microscopic Count → Hemocytometer

• Electronic counters (Coulter Counter & Flow Cytometry)

• Plate Counts

• Filtration

• MPN

Indirect Methods:

• Turbidity→ spectrophotometer

• Metabolic activity

• Dry Weight