Intro to Micro Flashcards
The microbial world includes
Virus
* Bacteria
* Fungus
* Parasites
Extremophiles
sychrophiles, thermophiles, radio resistant,
alkaliphiles, acidophiles, halophiles, xerophiles, barophiles, endoliths
Clinical Microbiology
Investigation of human infection by pathogens such as bacteria, viruses, fungi and protozoa
infection control, epidimiology
PROKARYOTE
Very small Bacteria & Archaea
no nucleaus
dna shape Circular (free floating)– 1 chromosome
no membrane enclosed organelles
Ribosomes- 70S (50S & a 30S subunit)
and divide through fission or budding
EUKARYOTE
Fairly large- Fungi, Plants & Animals
have a nucleas
dna is linear inside the nucleus - many chromosomes
membrane enclose organelles - mitochondria
Ribosomes- 80S (60S & 40S subunit)
divide via mitosis or Meiosis
Virus
type of cell
reproduction
shape
genetic structure
size
Non cellular
reproduces by Invades a host cell and takes over the cell causing it to make copies of the viral DNA/RNA.
Destroys the host cell releasing new viruses.
Icosahedron helix
Either RNA or DNA surrounded by a protein
coat smallest at <1um
bacteria
type of cell
reproduction
shape
genetic structure
size
Prokaryotic- unicellular
reproduces by fission- a form of asexual reproduction
Cell replicates its own DNA and splits in two
Coccus
Rod
spirochetes
Supercoiled DNA and RNA floating freely in cytoplasm
1-5 um
Fungus
reproduction
shape
genetic structure
size
Eukaryotic, Unicellular, multicellular
reproduces - Sexual or asexual spores, budding, rhizoids and fragmentation
Yeasts Molds
DNA contained inside a nucleus
4-10um
Parasite
Eukaryotic, Unicellular/multicellular
Amoeba-fission or asexual reproduction
Worms- sexual -lay eggs inside host bod
Amoeba
Eggs
worms
DNA contained inside a nucleus
4-10um - the biggest
Pili or Fimbriae
Flagella
Both are tubes of protein
(shorter than flagella)
* Both are virulence factors allow bacteria to attach to human cells or adhere & colonize surfaces
* Only conjugation pili are used to transfer genetic material between bacteria
* Neither have anything to do with bacterial motility
* Both are antigenic
Protein appendages for motility
* Are antigenic
Capsule
not all bacteria have one
* Made of polysaccharides
* Help bacteria adhere to tissue and surfaces
* Protects bacteria from immune system antibodies & phagocytosis
* Barrier against some antibiotics
* Antigenic can be negatively charged and could repel WBCs that are also neg charged
Plasma, Cytoplasmic or Cell Membrane
Phospholipid bi-layer
* Allows selective permeability & active transport of material in and out of the cell
* By passive diffusion of small molecules though bilayer
* Or through protein transport channels
* Proteins also provide enzymes needed for metabolism, phospholipid synthesis & DNA replication
* Proteins may be an anchor for pili or
flagella the phospolipid molecule has phosphate head - hydrophilic attracted to water
lipid tail - hydrophobic tail
Cell Wall
Provides shape, structural support & osmotic protection
* Composition depends on microorganism type
* Peptidoglycan (murein) & lipid (most bacteria)
* Chitin (fungus)
* Mycolic Acid (Mycobacteria or acid-fast bacteria)
* Mycoplasmas have no cell wall at all
* Confers gram staining characteristics of bacteria - antigenic
outer structures of the bacterial cell
Plasma, Cytoplasmic or Cell Membrane
Cell Wall
Capsule
Pili or Fimbriae
Flagella
INNER BACTERIAL CELL STRUCTURES
Cytoplasm
Ribosome
Chromosome
Endospore
Cytoplasm
Amorphous fluid with carbohydrates, proteins, enzymes, other metabolites, ribosomes & the bacterial chromosome
* If a bacteria has any plasmids -will be
found in the cytoplasm
Ribosome
Synthesize amino acids & proteins
* 70S made up of 30S & 50S subunits
* This difference from eukaryotic cells (80S made up of 60S & 40S) allow for the use of antibiotics that target only bacterial ribosome
Chromosome
A single negatively charged supercoiled double strand of DNA
* May also have small amounts of RNA, RNA polymerase
kept in the nucleoid
Endospore
Are formed by some bacteria as a survival response to adverse conditions in the environment
* Spores are resistant to dehydration, chemicals & temperature change
* Germinate under favorable nutritional conditions
* Bacterial spores are not reproductive structures
killed by autoclaving or glutaraldehyde
clostridium -
BACTERIAL TAXONOMY
Classification and grouping of organisms
* Based on genotype and phenotype
Phenotype is based on readily observable characteristics - shape color
* Macroscopic/microscopic morphology
* Staining characteristics
* Nutritional requirements
* Biochemical reactions
* Antigenic properties
* Resistance profiles
Genotype is the genetic makeup of an organism
* DNA/RNA base sequence analysis
Taxonomy of all living things
Domain→Kingdom→Phylum→Class→Order→Family→Genus→Species
Bacterial nomenclature
starts at Family
* But the commonly used name is binomial- it only includes the Genus & species
Example: Salmonella typhi
* The family is Enterobacteriaceae, but the organism is named based on
* The Genus: Salmonella
* The Species: typhi
when writing the first letter is capitalized they must be italized when typing but underlined when handwritten**
Main subspecies classifications there are two
Subspecies are an even smaller grouping of bacterial species that are too closely related to be a completely different species but that have enough unrelated characteristics to make them not exactly the same.
Biotype or biovar: based on slight & specific biochemical or physiological differences among species of the same genus
Serotype or serovar: based on more specific antigenic differences among species of the same genus ex e coli 0157:h7 vc just e coli
BACTERIAL GENETICS
CHROMOSOMAL
Bacterial genes are found on a chromosome free floating in the cytoplasm
* A single, closed, circular piece of double-stranded DNA
* Supercoiled
* Encoded with directions for essential functions like protein synthesis, cell growth, replication and survival
* Some genes are always expressed = constitutive
* Some genes are silent and expressed only under certain conditions = inducible
BACTERIAL REPLICATION
Duplication of chromosomal DNA for insertion into a daughter cell
* Begins at specific positions in chromosomes called “origins” where initiator proteins bind to DNA to start the process
* At origin point, two replication forks form and enzymes move in opposite directions to reform two identical double strands of DNA. One for the original and one for the daughter cell
daughter cells are genetically identical to
parent - bacteria divides by binary fission
Mutations:
changes in the original nucleotide sequence of an organism’s genotype
* Can occur spontaneously
* Can be due to an error during replication
* Can be due to chemical factors in the environment
* Or by introduction of foreign DNA into the cell by mutagens such as plasmids, transposons or bacteriophages
* May not cause any detectible changes
* May provide a survival advantage like toxin production or resistance to antibiotics
* May kill the cell
PLASMIDS
MUTAGEN
Smaller circular double stranded DNA outside the chromosome but still in the cytoplasm – not essential for cell survival
* Bacteria can have 1 – 100’s of plasmids
* Can replicate independently of chromosome thru conjugationhorizontal transfer from one bacterial species to another
* Or replicate with the chromosome and be passed on to a daughter cell
* Contain genes encoding for: Antibiotic resistance, toxin production virulence factors, and proteins for transfer of
plasmid to other bacteria
TRANSPOSONS
MUTAGEN
jumping genes
Moveable genetic elements of DNA segments
* Often causes spontaneous mutations, can encode resistance to antibiotics
* Can only replicate with the chromosome or piggy-backed on a plasmid
do not replicate independently like plasmids
BACTERIOPHAGE
A virus that infects and replicates within bacteria by injecting their genome into the bacteria’s cytoplasm
Two types of viral genome replication cycles:
Lytic cycle
Lysogenic cycle
Lytic cycle
- Virulent viral bacteriophage-host cell is lysed
- Lysis releases newly replicated packages of viral DNA that can infect other bacteria
Lysogenic cycle
Temperate (not continuing to replicate) viral bacteriophage - host cell is not lysed
* Phage DNA combines with the bacterial chromosome, creating the prophage
* Prophage is replicated along with the host chromosomes
* Lysogenic cycle can continue indefinitely - Or become lytic
MECHANISMS OF GENE TRANSFER
IN BACTERIA
Vertical Transfer
* Normal replication of chromosome -binary fission - mother to daughter
* Organism replicates it genome and provides exact copies to descendants
Horizontal Transfer
* Donor contributes part of genome to recipient that are not descendants
– usually through plasmids, bacteriophages, or incorporation of free naked DNA
* Recipient will have a different genes than other descendants
* Acquired DNA is incorporated into the chromosome or plasmid by Genetic Recombination
Conjugation - Both plasmid and chromosomal
genes can be transferred. Need a fertility gene F+ resulting in two cells with double stranded DNA
Transduction - Transfer of bacterial genes by a bacteriophage
BACTERIAL CLASSIFICATION based on nutritional needs
Autotrophs and heterotrophs
Autotrophs
Don’t need preformed organic compounds for energy
* Get energy photosynthetically or by oxidation of inorganic compounds
* Do not need organic carbon.
* Example of an inorganic carbon source = Carbon dioxide
Heterotrophs
Require organic carbon for growth
* Obtain carbon from preformed carbon and nitrogen containing compounds or glucose
* Acquire energy by oxidizing or fermenting organic substances
* Often the same substance (like glucose) can be used as both the carbon & energy source
All bacteria that inhabit the human body are Heterotrophs
bacterial nutritional requirements and their purpose
Water - transport solutes across the cell , needed to move things in and out of the cell
energy source - ATP
nitrogen source - make proteins amino acids =nucleic acids
carbon source - make cellular constituents
Phosphate - nucleic acids
sulfur - protein synthesis
metal/ions - enzymatic activities
WENCPMS
Temperature requirments from lowest to hottest
psychrophiles - 10-20
mesophiles - 20-50 (pathogens, opportunists because they are closest to body temperature)
thermophiles - 50-80
extreme thermophiles - over 80
oxygen requirements -
our air is 21% O2 and 1% CO2
bacteria can be obligate aerobes
obligate anaerobes
aerotolerant anaerobes
faculative anaerobes
microaerophilic
capnophilic
Superoxide
is a toxic by-product of bacterial metabolism that happens in oxygen - causes cell damage and death
* Organisms exposed to oxygen need enzyme superoxide dismutase
* Enzyme converts superoxide and hydrogen to hydrogen peroxide and oxygen →less toxic
* If organism also produces catalase enzyme→ hydrogen peroxide will be converted to water and oxygen
* Or if organism produces peroxidase enzyme→ hydrogen peroxide will be converted to water and a hydroxide
compound
Strict Aerobes:
Grow ONLY in presence of oxygen
* Produce superoxide dismutase ,catalase, peroxidase to protect them from toxic superoxide
* These bacteria oxidize sugars
* ATP made mostly from oxidative phosphorylation but some substrate level phosphorylation as well
Facultative Anaerobes:
Grow with or without oxygen – grow faster in O2 so colonies will often appear larger in an aerobic condition - has enzyme superoxide dismutase
* Can shift between fermenting or oxidizing sugars depending on the presence of O2
* ATP made by both substrate level and oxidative phosphorylation
Strict (Obligate) Anaerobes:
Can’t grow in oxygen- because they lack superoxide dismutase, catalase, & peroxidase
* Ferment sugars
* ATP made from substrate phosphorylation only
* Must be grown in anaerobic jar with Gas pak added
Microaerophilic:
Grow in reduced oxygen levels – specifically 5 % O2
* Killed by high oxygen levels
* Require a Campy pak
* Oxidize sugars
Capnophilic
Grow best in 5 – 10% CO2
* Candle Jar
* CO2 Incubator
* Oxidize sugars
Aero tolerant anaerobes
Grow with or without oxygen (not killed by 02)
* Grow better without oxygen
* Uses fermentative metabolism regardless of whether O2 is present or not
Lag phase:
Bacteria adapt to growth conditions
* Maturing and not yet able to divide
* Synthesizing RNA, enzymes and other molecules
best time to grow bacteria will take 18-24 hours
time on x axis——
log number of bacteris on y axis
Log or Exponential phase:
Active cell division – cell doubling
* Best stage for biochemical testing
* But can’t continue indefinitely due to depleted nutrients and buildup of wastes
Stationary phase
Growth rate and death rate are equal
* The number of new bacteria created is limited by the declining nutrients and increasing toxic byproducts
* Result is a “smooth,” horizontal linear part of the curve during the stationary phase
Death phase
Bacteria run out of nutrients and only toxic
byproducts are present, so all bacteria die
* The # of organisms dying > number produced
The Generation Time -
The time it takes for a single bacteria to divide or double in number - also called the doubling time
Factors affecting growth rate:
* Species and strain, whether nutritional requirements are in media, presence of inhibitors, radiation, detergents or toxins
* Environmental factors: temperature, presence or absence of oxygen, osmotic pressure, pH
Function of bacterial Metabolism:
- To make energy in the form of ATP
Anabolism:
Uses energy (ATP) to make cell components like proteins and nucleic acids
Catabolism:
Makes energy (ATP) from breakdown of organic matter
Bacterial mechanisms of carbohydrate catabolism:
Two mechanisms used to breakdown carbohydrates: Oxidation
(also called Respiration) & Fermentation
SUMMARY OF OXIDATION
(AEROBIC RESPIRATION)
- Aerobic metabolic process used by obligate aerobes,
facultative anaerobes, microaerophiles & capnophiles
Achieved through: - Glycolysis (also called the Embden–Meyerhof pathway)
- Krebs Cycle (also called Citric Acid Cycle)
- The Electron Transport Chain (ETC)
Phosphorylation of ADP: - By substrate level in Glycolysis and Krebs
- And oxidative phosphorylation in the Electron transport chain
- More ATP (up to 38) & very little acid is produced by oxidative breakdown of glucose
BACTERIAL ANAEROBIC
METABOLISM OF GLUCOSE
Used in the absence of O2 or if organism can’t grow inO2
* In this case energy is made from glucose in one of two ways
fermentation or anaerobic respiration
FERMENTATION
Non-oxygen requiring way to get energy from the breakdown of glucose or other sugars
* Performed by strict anaerobe, facultative,
aerotolerant bacteria & yeast
* Glycolysis forms pyruvate then in the
absence of O2 & depending on the enzymes available, is converted into acids, gases, or alcohols -no Krebs cycle or ETC
* Purpose of fermentation reactions is to
regenerate NAD+ so glucose continues to be broken down by glycolysis
* Lots of acid but only 2 ATP produced in glycolysis – fermentation does not produce any extra ATP
Lactate dehydrogenase converts
pyruvate to lactate NADH reduced to NAD+ which can be reused in glycolysis
fermentation in identification
Considered an anaerobic process but facultative bacteria & yeast can choose to use it even when oxygen is present
Not all sugars can be fermented by all bacteria – E. coli is a lactose fermenter, but other similar organisms can’t do this
The end products are used as phenotypic markers & in bacterial
ID schemes
* Some biochemical test media used to ID bacteria have a carbohydrate & a pH indicator - Fermentation produces lots of acid and a color change in the pH indicator in the media
ANAEROBIC RESPIRATION
Anaerobic or aerotolerant organisms use anaerobic respiration facultative can switch from aerobic to anaerobic respiration or fermentation depending on O2 or if it has enzymes to ferment.
* Even though fermentation also happens without oxygen, it isn’t the same as anaerobic respiration.
* Anaerobic respiration starts with glycolysis it continues with acetyl CoA entering a modified cycle of Krebs.
* Electrons formed in Krebs enter the ETC, but O2 is not the terminal electron acceptor - instead, other molecules like nitrate (NO3), sulfate or carbon dioxide are used as final electron acceptors
* ATP in ETC by oxidative phosphorylation but in < #s max of 36
growing bacteria
Media can include
* Solid form -agar in plastic petri dishes
* Fluid form - broths in tubes
can be grown using a patient swab or by transferring colony from another sample - subculture - purity plate
WAYS TO ASSESS GROWING
BACTERIA
Increased size or increased numbers -qualitative
Direct plate estimation – semi qualitative
* Estimate the number of viable bacteria present
* Bacteria growth is described as light, moderate or heavy depending on which quadrant it grows up to on solid agar media - 4 quadrant plate estimate
Direct plate count – quantitative *measure count
* Grow known dilutions of broth culture
* Count colony forming unit per milliliter (CFU/mL) - calibrated loop
* Provides bacterial cell count of viable organisms
Turbidometric-quantitative cfu/ml
* Using photometer to measure turbidity it makes a broth
photometer is used to compare the amount of light that goes through
* Does not distinguish between live or dead organisms
Radiometric, Colorimetric or Fluorescence - Measure CO2 produced by bacteria as they grow
blood culture
types of incubators
35ºC – 37ºC aerobic (O2)
* 35ºC – 37ºC with 5% CO2
* 42 ºC or 30 ºC aerobic (O2)
Anaerobic Containers:
To create anaerobic conditions jar must include
* GasPak™ EZ Container System Sachets - Envelope gas generator (Anaerobe Container System Sachets white)
* Inorganic carbonate, activated carbon, ascorbic acid and water
* produce an anaerobic atmosphere within 2.5 hours
To monitor anaerobic conditions jar must include
* Methylene blue oxidation- reduction indicator strip - Verifies that anaerobic conditions have been achieved - Changes from blue to white in anaerobic conditions
Campylobacter Jars -
(Microaerophilic)
Campy Container System Sachets (are blue)
* Produce a microaerophilic atmosphere within 2 hours
* Does not have a chemical indicator
* Must use a biological indicator that proper growth conditions were achieved
* Include a known microaerophilic organism in the jar
Candle Jar - (Capnophile)
5-7% CO2
* As flame burns, oxygen decreases and flame goes out
* CO2 increases due to the flame
* Use of white unscented candle(scented inhibitory to certain bacteria)
* Humidity provided by the moisture evaporating from the media within the jar
Origin of Microbial Biota:
A human fetus is in a sterile environment in the womb
* At delivery it is exposed to microorganisms on their mother’s body & in the environment
* Each organism finds the area of the baby’s body where it can colonize and become the predominant organism
Colonization:
is the growth of microbiota in or on a body site without the production of damage or notable symptoms
Symbiosis:
Any association of two or more organisms living
together
* If both organisms benefit from the relationship = mutualism
* If the organisms benefit but there is no benefit or harm to the host = commensalism
* If the organisms benefits at the expense of the host = parasitism
Benefits of Normal flora
Bacteria produce vitamins that the human body can’t like B12 & K.
* Help digest food
* Helps prevent infection by out-competing invaders for nutrients & production of antibiotics that kill other organisms
* Helps develop our immune system – induce production of natural antibodies & stimulate low levels of circulating antibodies to enhance immunity
* Induce growth of lymphatic tissue and the wall of the Caecum in the GI tract
Resident Microbiota (Microscopic organisms of a region(e.g., respiratory, urinary, gastrointestinal tract) harbored by normal, healthy individuals.
Microorganisms that colonize an area for months or years
Transient Microbiota
Microorganisms that are present at a site temporarily.
* Come to “visit” but don’t stay because they are eliminated by the host immune system or competing resident biota
Opportunistic Microbiota
Resident microorganisms can cause disease when their habitat is altered or the host’s immune system is compromised
like yeast that can lead to yeast infection if left unchecked
* Compromised due to immunosuppressive drugs, chemotherapy and radiation
* Or due to diseases that cause immune disorders
True pathogen vs Opportunistic pathogen
True pathogens are organisms that cause disease in a healthy immunocompetent individual a high percentage of the time (in nearly all situations)
* Opportunistic pathogens are organisms that are usually normal flora and under usual conditions do not cause disease but can cause infections if changes occur in the host immune system or habitat alteration
Pathogenicity
the ability of a microbe to produce disease in an individual
Infection
the invasion and multiplication of
microorganisms in/on body tissues
Damage to the infected cells or organs is called disease
Types of Infection: EELNOZ
Endogenous: source is from inside host (reactivation of a dormant virus or bacteria) - herpes causing cold sores or TB
Exogenous: source is from outside the host
Latrogenic: source acquired during a medical procedure
Nosocomial: source acquired while in hospital
Opportunistic
Zoonotic: source acquired from an insect or animal
Virulence =
ability of a microbe to cause disease or the
degree of pathogenicity
* Measured by # of organisms needed to cause an infection
* Organisms that cause infection with a low infective dose are more virulent than organisms that need a high dose
Virulence factors =
varied mechanisms that allow microbes
to persist in a host and cause disease – can be coded in the genome of chromosome or acquired from plasmids, transposons or phage
Ability to Resist Phagocytosis
Ability to Evade Antibodies
Adhesion to Host Cells & Tissues:
Ability to Survive Intracellularly & Proliferate
Bacterial Ability to Produce Toxins
Exotoxins
endotoxins
Ability to Resist Phagocytosis:
Host phagocytic cells ingest invading microbes
* Organism’s with a capsule can hide cell surface structures
(antigens) recognized by receptors on the surface of the phagocytic cell
* Capsule & WBC are negatively charged therefore repel
* Capsule may also inhibit the activation of complement by masking structures which bind to complement
* Bacteria can release material in tissue that kill phagocytes
* Examples: hemolysin (lyse red blood cells), leucocidins (kill leucocytes)
* Organism with pili attach to target cell and resist phagocytes
Ability to Evade Antibodies
- Some bacteria produce immunoglobulin protease –degrades Ab
- Or can change their antigen structure so antibodies can’t bind
- Protein A on bacteria, binds to FC portion of Ab & neutralizes it.
Adhesion to Host Cells & Tissues:
Most microbes must attach to host cells before infection occurs
* Surface structures that cause attachment are called adhesins – host cells must have the corresponding receptors for the adhesins
* Main adhesin in bacteria are capsules or pili (fimbriae)
* But host can make antibodies to the adhesins to prevent attachment
Ability to Survive Intracellularly & Proliferate:
- Some bacteria can live in phagocytic or tissue cell after being engulfed via a cell vacuole into the cell
- Can make a substance that neutralizes the lysozyme enzyme made in the vacuole to kill it - or escape from the vacuole into the cell’s cytoplasm
- Continue to replicate inside host cell & are protected from the host immune system
- Some bacteria multiply and continue to penetrate other cells in the host tissue = invasion
- Some spread to other distant organs or tissues = dissemination
- Examples of enzymes made by bacteria that allow them to disseminate: hyaluronidase , collagenase or kinase
Bacterial Ability to Produce Toxins:
Poisonous substances that cause damage to host cells or tissues
* Two main types Exotoxins & Endotoxins
Exotoxins:
Can be produced by gram negative and gram positive organisms
* Gene for making toxin found on plasmid
* Secreted by organism into the environment – or when the cell lyses
* Can cause formation of antibodies or antitoxin by immune system
* Damage to tissue and connective tissue helps organism spread
* Are heat labile
Types of Exotoxins: have a defined action to specific cells
Cytotoxin: damage to host cells
Neurotoxin: damage to nervous tissue
Enterotoxin: specifically damage the intestinal mucosa
Endotoxins:
Chromosomally mediated
* Heat stabile
* Part of the cell wall (LPS –lipopolysaccharide portion)of gram negative bacteria
* Released only when the bacteria is killed & cell wall breaks apart
* Not specific in their activity on host cells
* Not as potent
* Stimulate inflammatory response in the host innate immune response
* lowering blood pressure, bleeding, high fever, increase or decrease in different WBCs. Can sometimes become shock, organ failure & death
labelling
when labelling the plate label the bottom one around the edge - need to be able to see the growth
if the slant growth label the other side
Specimen number
* Mailbox number
* Date
* Atmosphere of incubation if not O2
35oC
streaking of liquid media
if you took too much sample it wont dry
streaking
turn left 30
go into the prior quadrant 2 times
the fourth quad takes up the the edge and the middle
INOCULATING A COLONY TO A SOLID AGAR TUBE
MEDIA
put the loop directly inside - shows motility
SYSTEM FOR ANAEROBIC CONDITIONS
Plates are placed into a container
* A special pouch is added to the container with the plates before it is closed.
* A chemical reaction occurs in the pouch that removes all O2 from the container environment.
* A special Methylene blue oxidation- reduction indicator strip is also added to ensure ANO2 conditions have been reached
* The strip starts blue and turns white when ANO2
conditions have been reached
WET PREPARATION FOR MOTILITY
saline on the slide with organism and cover
