problems associated with culturing microorganisms
•Found everywhere (rarely alone)
•Are invisible
•Grown under artificial conditions
differences between a pure, mixed and contaminated culture.
pure- 1 microb
Mixed- 2 or more known micro
contaminated- unwanted/ unknown microbs
the “six I’s” and the purpose of each.
•Inoculation-in container, manage in environment
•Incubation-controlled env Temp, PH, CO2, promote growth
•Isolation-seperate, pure
•Inspection-observing macroscopically/microscopically, see cell type
•Information gathering-•Biochemical tests, Enzyme characteristics, Genetic testing, Drug sensitivity, Immunological testing, overall profile
•Identification- determine identity, further research
Compare the various methods of isolating organisms and the advantages and disadvantages of each method
streak plate- economical, but require skills
Loop dilution- consist result but require more time/material
spread plate- only useful for dilute samples
Recall the three primary ways in which media can be classified and understand how each is used in the study of microorganisms
1.Physical form
-liquid
-semi solid for motility
-solid (liquefiable/ nonliquefiable)- isolating
2.Chemical composition (synthetic and complex)
3.Function(general purpose and enriched
What ingredients are used to solidify media, and what are the characteristics of each
-agar from red alga
liquid- broths
-semi solid-0.3 agar
-solid (liquefiable/ nonliquefiable)
Compare synthetic and complex media
Synthetic (defined) media: Each ingredient is known precisely
Complex (undefined) media: Not chemically defined. Often contains extracts from plants or animals
differences between general purpose, enriched, selective and differential media and the proper use of each
•General purpose media: Grow a wide variety of microbes (nonsynthetic)
•trypticase soy broth
• nutrient agar
•Enriched media: Contains extra nutrients for fastidious organisms (complex organic)
blood agar
•Selective media inhibits growth of all but one type of microbe
•Contains a selective agent (drug, NaCl…)
•Differential media allows all organisms to grow, but each appears different
•MacConkey agar detects fermentation of lactose(turn red)
various ways in which samples can be prepared for microscopic analysis and the advantages and disadvantages of each method.
simple staining- size and shape, 1 color
-basic dye(+)
acidic dye(-) nigrosine, india ink
differential staining- 2 or more dye (Biology of cell)
-gram-+ purple, -neg-red
-acid fast- hold fast to dye
-endospore- between spores and vegetable
structural staining
capsule
flagella
differences between positive and negative staining as well as simple and differential staining
positive staining- dye stick to specimen
negative staining - dye stick to boundary
simple- (1 dye
differential- (primary dye, counterstain)
examples of bacteria for which each type of stain would be especially useful.
simple staining-
-basic dye(+) - staphylococcus aureus
acidic dye(-) - bacillus and staphylococcus
differential staining-
-gram •Staphylococcus, E. Coli
-acid fast- Mycobacterium leprae
-endospore- Bacillus anthracis
Clostridium tetani
structural staining
capsule
flagella
Basic dye
have positive charge
Acidic dye
negative charge
Positive staining
stain cell with
crystal violet, methylene blue, safranin, malachite green
Negative staining
stain background with nigrosin and india ink
Simple staining
single dye, uncomplicated
Differential staining
2 dye to distinguish between cell type \
gram- positive thicker walls
acid fast
endospore
Gram stain
•Gram positive (appear purple)
•Staphylococcus
•Streptococcus
•Bacillus
•
•Gram negative (appear pink)
•E. Coli
•Salmonella
•Yersinia-plague
Acid-fast stain
Mycobacterium leprae
Mycobacterium tuberculosis
outer wall hold to dye, appear pink
non acid fast appear blue
Endospore stain
bacillum and clostridium
Structural staining
emphasize capsule, enddospore, flagella
cryptococcus
Capsule stain
observing unstructured layer of cell of bacteria and fungi
often india ink
Flagellar stain
revealing flagella from bacteria
Culture
or cultivate
Medium
environment to multiply
Inoculation
culture microorganism in medium
Isolation
individual bacterial cell separated from other
Incubation
temperature controlled
Colony
grows from 1 cell
Loop dilution
or pour plate
loop into liquid of agar tube then pored into petri dish to solidify
Streak plate
sample spread with inoculating loop
Spread plate
small volume liquid from diluted sample is spread by hocky stick
Pure culture
only microb from sample is present
Mixed culture
2 or more microorganism
Contaminated culture
unknown microb
Liquid media
do not solidify at temp above freezing
broth, milk, infusison
Semisolid media
thicker consistency, motility
Solid media
liquefiable-liquid at 100C solidify at 42 C
nonliquefiable
Agar
polysaccharide isolated from red alga
Synthetic (defined) media
pure chemical
Nonsynthetic (complex) media
can't be defined by chemical
animal extact, blood,
General purpose media
grow broad microb
nutrient agar, broth, heart infusions, soy agar TSA
Enriched media
complex substance blood, serum,
special growth factors
Growth factors
organic compounds like vitamins or amino acid that microbs can't snythesize themselves
Fastidious
bacteria that require growth factor and complex nurtients
Selective media
1 or more agents that inhibit some mico except 1
Differential media
several types of microorganism but bring out differences
Structure and function of a generalized prokaryotic cell.
cell membrane, cytoplasm, ribosome, chromosome
Prokaryotic flagella
spin
run. tumbles,
axial filaments, pili and fimbriae.
Axial filament- •AKA periplasmic flagella. Found in spirochetes (spiral shaped bacteria
•pilus- Small rigid tube used to transfer small amounts of bacterial DNA (conjugation)
-Typically found only in Gram negative bacteria
•Fimbriae are numerous small appendages used for attachment to surfaces/host cells
bacterial conjugation
Bacteria with pili transfer DNA
•S layer
Tight covering of proteins surrounding the cell, produced in harsh conditions
•Glycocalyx
A thick film composed of oils, sugars, and proteins that protect against dehydration and allows attachment to surfaces
Biofilm-
communities of bacterial cell work together
Gram positive-
thick Peptidoglycan layer
Teichoic acid
Lipoteichoic acid
Gram neg-
thin Peptidoglycan layer
LPS(lipopolysaccharide)-
atypical cell walls.
Mycobacterium , for tuberculosis. Need acid fast stain
cell membrane, cytoplasmic matrix, nucleoid, ribosomes, inclusions, granules, and cytoskeleton.
Cell membrane- phospholipid and protein( selective permeability)
•Cytoplasmic matrix provides water for biochemical reactions
•Nucleoid contains genetic material
•Ribosome- protein synthesis(Prokaryotic ribosomes 30S + 50S = 70S)
Inclusions- stores nutrients
Granules- crystals of inorganic compound
•Cytoskeleton provides support
how bacterial endospores form and germinate
Endospores are formed in response to poor growth conditions
Germinate happens with water
Vegetative cycle(active) and sporulation cycle
common bacterial shapes and sizes.
•Coccus 1-2 μM
•Bacillus 2-20 μM
•Spirillum10-200 μM
Bacterial species-
collection of bacterial cell that share similar patterns of traits
Subspecies, strain and type
subcategory
endosymbiotic theory
Prokaryotic cell live inside cell evolved into eukaryotic cell
eukaryotic cell structure
flagella, cilia, glycocalx, cell wall sometimes, nucleus, organelles, ribosomes, cytoskeleton
-ribosomes•Prokaryotic & Eukaryotic
Prokaryotic 30S & 50 = 70S
Eukaryotic 40S & 60S = 80S
Kingdom Protista.
Eukayote that is not fungus animal or plant
Simple eukaryotic cell that lack multicellular structure
Algea, protozoa,
trophozoite and a cyst.
•trophozoites (active, motile, feeding form) or cysts (inactive, resistant, survival structures)
Cyst form is generally more infectious as it can pass through the stomach
protozoan parasites life cycle.
Active trophozoite, encystment environment is poor , become cyst. Excystment becomes trophozoite
Heterotrophic
food in complex organic form
obligate intracellular parasites.
virus, no cell structure, depend on host cell, cause disease in animal, plant, fungi protozoa
properties of viruses
Not nearly as complex as cells
No cell membrane, no nucleus, no organelles
structure of a typical virus.
Protein coat (capsid) surrounding a small amount of genetic material
•Some viruses also have an envelope and protein spikes
•Capsids, •Helical capsids
composed of protein units called capsomers
•Helical capsids- capsomers wrapped around nucleic acid
•Coronavirus, •Rabies virus, •Influenza virus
•Icosahedral capsid vs helical capsid
•Icosahedral capsids have twenty sides, each made up of many capsomers- poliovirus
•Helical capsids consist of capsomers wrapped around nucleic acid
•Coronavirus
viral replication for both bacteriophage and animal viruses
Bacteriophage- injection of nucleic acid through cell wall
Animal virus- whole vuris engulfed/ fused
Absorption, penetration, uncoating, synthesis, assembly, release
lytic and lysogenic cycles
Lytic Cycle Ends With the Lysis of the Bacterial Cell
•Lysogenic phase--viral DNA splices into the host cell chromosome. Does not result in death of the host cell
cell culture, eggs, and animals in the culturing process.
Cell culture- mono layer. plaque= cell lysed
Egg- any tissue available in developing egg
Animal- slow expensive
prion
abnormal, pathogenic agents that are transmissible and are able to induce abnormal folding of specific normal cellular (found in brain)
chemical composition of a bacterial cell
Sulfur, Phosphorous, Oxygen, Nitrogen, Carbon, Hydrogen (SPONCH)
•Macronutrient:
large quantities, primarily for cell structure (Proteins, carbohydrates) DNA, lipid, protein
•Micronutrient:
Small quantities for microbial metabolism. Often serve as co-factors for enzymes (Magnesium, Zinc)
•Organic nutrients:
Contain both carbon and hydrogen. Organic molecules are living things or the products of living things (Sugars, lipids, alcohols)
•Inorganic nutrients:
Do not contain both carbon and hydrogen (NaCl, H2O, CO2)
•Autotrophs:
use the carbon in CO2 and convert it into organic compounds (plants, algae, photosynthetic bacteria)
•Heterotrophs:
bodies of other organisms through decomposition or parasitism
•Saprobes
Free living (decomposers)
•Facultative parasite
May adapt to a host (Pseudomonas aeruginosa- cystic fibrosis)
Obligate parasite:
Must grow within a host (viruses, Mycobacterium leprae)
•Heterotrophs
Derive both carbon and hydrogen from other organic compounds. Several groups exist.
Prefix
•Auto, Hetero, Photo, Sapro, Halo, Thermo, Psychro, Meso, Aero, Anaero, Baro
Prefix
•Auto self
•Hetero other
•Photo light
•Sapro rotten
•Halo salt
•Thermo heat
•Psychro cold
•Meso intermediate
•Aero air
•Anaero no air
•Baro Pressure
Suffix
troph, phile, bios, tolerant/duric
troph feed
phile love
bios live
tolerant/duric survive (but not grow)
•Thermophile:
heat loving
•Psychroduric:
tolerant of cold temperatures
•Obligate/strict:
must have a specific set of conditions for survival
•Fastidious:
Requires specialized growth conditions
•Facultative:
adapt to a wide range of conditions
•Facultative anaerobe:
can live with or without oxygen
•Obligate halophile:
Can only survive in the presence of a high concentration of salt
•Diffusion:
Molecules naturally move from high concentration to low concentration with no expenditure of energy (rarely works as a way of gathering food)
Osmosis-
Cell membranes are semipermeable, most molecules are kept out, but water can diffuse in either direction
•Hypotonic solution Hypertonic solution :Isotonic solution: T
•Hypotonic solution: The solution outside the cell has a lower concentration of solutes than is found inside the cell. Net water movement is into the cell by osmosis. Cell walls
•Hypertonic solution: The solution outside the cell has a higher concentration of solutes than is found inside the cell. Net water movement is out of the cell by osmosis. Contractile vacuoles.
•Isotonic solution: The solution outside the cell has the same concentration of solutes as that found inside the cell. There is no net water movement.
•facilitated diffusion
•facilitated diffusion- Protein channels in the cell membrane allow specific molecules to enter and exit quickly with no energy used
-Better than diffusion but still not adequate
-Requires a separate protein channel for each type of molecule
-A gradient (high to low concentration must still exist
Active transport-
nergy is used to move molecules from a low concentration to a higher concentration
Phagocytosis-Pinocytosis-
ingest cell
ingest liquid
Cardinal temperature
•Minimum: No growth below
•Maximum: No growth above
•Optimum: Growth occurs most quickly
•Psychrophiles: •Mesophiles:•Thermophiles: •Hyperthermophiles:
•Psychrophiles: Optimum below 15°C
•Mesophiles: Optimum between 10°C and 50°C
•Thermophiles: Optimum 45°C to 80°C
•Hyperthermophiles: Optimum above 80°C
enzymes that bacteria use to detoxify the products of oxygen metabolism
Bacterial species that live in aerobic environments must possess enzymes to deal with the toxic byproducts of oxygen based metabolism (02-, H2O2, OH-). Two enzymes generally get the job done
•Superoxide dismutase converts O2--> H2O2
•Catalase converts H2O2 -> H2O and O2
aerobes
posses catalase to dismutes and use oxygen in metabolism
obligate anaerobes
posesses neither, live in environment without oxygen
faculatative anaerobe
will use oxygen is available, not necessary
bacteria pH & Salt concentration:
6 and pH 8
1% NaCl
Symbiotic associations
icrobes are closely associated (same place and time)
-termites and trichonymphs
Comensalism:
One member benefits, the other is not harmed (cow and bird)
-Staphylococcus aureus produces growth factors as it grows. Haemophilus colonies grow next to a streak of S. aureus
•Parasitism:
One member benefits at the expense of another
•Novel Coronavirus SARS-CoV-2 in the process of killing a cell
•Nonsymbiotic associations:
Members of the association are not always closely linked in time/space
-Syntrophy (crossfeeding):
Multiple species may work together to complete a task (decompose pig)
Amensalism:
Organisms work against one another