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261 terms

Micro Lab final

STUDY
PLAY
electric cord
dont dangle from table so that a foot gets caught and is pulled off table.
stage
horizontal platform
ocular, objective, condenser
three lens of the brightfield
condenser
lens that collects and directs the light from the lamp
diaphram
regulates the amount of light that reaches the slide
adjusting knobs
fine adjustment knob and coarse adjustment knob
magnification
ocular 10 times the objective 10, 40, 100
resolving power
ability to completely separate two objects in a microscopic field
limit of resolution
0.2 um for light microscopes
parfocal
image remains in focus when changing from low power objective lens to high power objective lens
lamp life is extended
when opening diaphram fist instead of increasing voltage
oil immersion
oil has the same refractive index as glass and will increase resolving power by reducing light lost from refraction
brightfield microscope
allows light ot pass directly to the eye
bacteria
have simple cell structure that lacks defined nucleus and no nuclear membrane
peptidoglycan
unique molecule in bacterial cell wall
spirochete
motile, using axial filaments, which are a type of flagella that originate from both ends of the cell and wrap around the cell body
heterotrophic
"other" + "eating" - organisms which feed on other plants and animals, as oposed to autotrophs that use CO2
fungus
eukaryotic, nucleus, produce exoenzymes and absorb thier nutrients, lack tissue definition, have cell wall of chitin, propogate (multiply) by spores
morphological characteristics
structural, the form and structure of an organism considered as a whole
yeasts
unicellular, lack hyphae, multiply by budding or fussion or both
molds
multicellular
hyphae
microscopic intertwining filaments of molds
mycelium
mass of hyphae; macroscopic fungal colony
psuedohyphae
chains of buds of yeasts
asexual fungi
sporangiosopres, chlamydospores; does not involve the union of cell nuclei
sexual fungi
zygospores, ascopores, basidiospores; union of two parental nuclei followed by meotic division
eumycota
the fungal kingdom, "True fungi"
Saccharomyces cervisiae
yeast used in bread making and alcohol; blastospores
colony characteristics of molds
cottony, different colors, round or irregular
dimorphic fungi
characteristics of both mold and yeast
goals of aseptic technique
no unwanted organisms in culture, you are not contaminated, no contaminates are left afterwards
work area disinfected
destroys vegetative cells and viruses, but may not destroy endospores
vegetative
active bacteria, not endospores; cells involved in obtaining nutrients,not reproduction
endospores
resting, resistant, dormant structure formed inside some bacteria that protects form adverse environment; dehyrated with thick cells walls with extra layers
endospore staining
dyes do not penetrate the wall
Shaffer-fulton
malachite green is heat steamed, counterstain with safranin
survival of endospore
resisitant to heat, lack of water, and exposure to chemicals and radiation
loop or needle
is sterylized by inserting into bunsen flame until it is red hot
culture tube
mouth is flamed prior to and after inserting a loop or needle
petri plate
cover is held diagonally over the plate to protect the surface from any contaminates in the air
work area
should be cleaned with disinfectant before and after work
inoculated petri plates
incubated upside down to prevent moisture from condensing on the agar surface and spoiling or spreading inoculated organism
mixed populations
how bacteria exist in nature
pure culture
contains only a single kind of bacteria; able to study morphological, cultural and physiological characteristics
obtain pure cultures
streak plate and pour plate dilute bacteria to a point where a single cell gives rise to single pure colony
streak plate
economy and time make this method best
agar
becomes liquid at 85 and resolidifies at 42. Good gel stability at high temperatures; made from polysaccharide of algae
quadrant streak
technique that spreads oranisms around plate in a way that dilutes concetrations
two reasons plates are incubated upside down
water will spread organism, may inhibit growth
condesation on plate cover
can be avoided by cooling agar to 50 before pouring
goals of smear
adhere cells to slide and insure shrinkage does not occure; prepare thin
reasons for thin slide prep
to visualize individual cells, their arrangment and details regarding microstructures; stains better
simple staining
use of single stain to color bacterial cell
simple stains
methylene blue, basic fuchsin and crystal voilet are basic dyes
positive stains
bacteria are negatively charged
staining time
30 seconds to 2 minutes
pleomorphic, pleomorphism
irregularity of form, demonstrating several different shapes
metachromatic granules
distinct reddish-purple granules with cells that show up when the organisms are stained with methylene blue
palisade arangement
parallel arrangment of rod shaped cells "picket fence" (characteristic of corynebacteria)
capsule
slime layer, gycocalyx, protective role for certain bacteria like streptococcus pneumoniae, prevents phagocytosis by WBC, to attach to solid surfaces
shrinks capsule
heat fixing prior to staining
india ink
negative stain
Streptococcus pneumoniae
capsule producing to evade WBC phagocytosis in lungs
primary stain
for gram stain is crystal violet
differential stain
take advantage of the fact that cells or structures within cells display dissimilar staining reactions that can be distinguished by the use of different dyes
gram positive bacteria
retain crystal violet-iodine complex through decolorization with alcohol; appear purple
gram negative bacteria
alcohol removes the crystal violet iodine complex; counterstain of safranin makes bacteria appear pink
mordent
for gram stain is iodine; forms complex with crystal violet that is insoluble in gram positive cells
decolorization
alcohol leaches the dye-mordent complex from gram negative cells
gram positive cell wall
has thick peptidoglycan layer
gram negative cell wall
has thin petidoglycan layer
counterstain
for gram stain is safranin, to stain any gram negative cells in the smear
Mycobacterium smegmatis
acid fast bacteria that stains gram positive; representative of acid fast bacteria
gram positve
S. aureus
gram negative
E. coli
endospores
allow bacteria to survive environmental conditions that are not favorabe for growth; resistant to heat, radiation, acids, and many chemicals such as disinfectants
exosporium
protien coat that forms a protective barrier around the spore
low water content of spores
provides heat resistance for endospores
dipicolinic acid
forms gel that controls the amount of water that can enter the endospore, thus maintaining its dehydrated state
autoclave
121 degrees for 15-20 minutes to destroy endospores
heat
mordent in spore staining to facilitate the uptake of malachite green
Shaffer-Fulton method
utilizes malachite green to stain the endospore and safranin to stain the vegetative portion
Bacillus subtilis
endospore stain bacteria
acid fast
bacteria such as mycobacteria and some nocardia
mycolic acid
waxy material; complex lipid that is composed of fatty acids and fatty alcohols that have hydrocarbon chains up to 80 carbons in length; prevents routine stains
acid fast stain
diagnositc tool for Mycobacterium tuberculosis and Mycobacterium leprae (tuberculosis and leprecy)
Zeil-Neelsen method
carbolfushion mixed with phenol is heated, creates noxious fumes toxic to the eyes and mucous membranes
Kinyoun method
acid fast stain that is not heated, concentrations are increased for same results
acid-fast
carbolfuchsin mixed with phenol (for permeability), then decolorized with alcohol, counterstained with methylene blue
non acid fast
S. aureus
thioglycolate medium
test to determine the oxygen requirements by observing the nature of growth in the tube
parasitology
study of protozoans, parasitic worms (helminths), and parasitic arthropods such as lice, mites and ticks
sacodina
protozoa with locomotion by means of psuedopods
mastigophora
protozoa with locomotion by means of flagella
ciliata
protozoa with locomotion by means of cilia
sporozoa
nonmotile protozoa
according to motility
how protozoa are classified
trophozoite
motile form of protozoan, pathological form
cyst
after leaving the warm host, cools and curls, forms thick walls, becomes inactive
Entamoeba histolytica
representative sarcodina protozoan; moves by psuedopod
repesentative flagellate; mastigophora
Trichomonas vaginalis, Giardia lamblia, Trypanosoma gambiense
plasmodium
causes malaria
true
protozoans are classified according to their modes of locomotion
flagellate
mean of mobility for Giardia lamblia
False, cyst
laboratory identification of the parasitic protozoans is usually made from microscopic examination of the trophozoite stage.
true
specimens containing the parasitic trophozoite stage are more readily available for classroom study than those containing the cyst stage.
trophozoite
stage that cause pathological conditions in the host
flagella
Giardia is identified by this
tsetse fly
transmits african sleeping sickness
Giardia
found in humans and animals, especially beavers
plasmodium species
malaria
Entamoeba histolytica
dysentery with fluid feces
Typanosoma gambiense
drowsiness, coma and death
giardia lamblia
diarrhea, abdominal cramps; beaver fever
Ascaris lumbricoides
ova must live in soil for several weeks before able to infect humans
nematodes
parasitic round worms
Trichinosis
diagnosed by clinical symptoms, serology tests and muscle biopsy
Nectator americanus
burrow through the soft skin on the sides of the feet
Trichinella spiralis
females bear fully developed, viable larvae instead of depositing ova
Taenia
tapeworms
tapeworm infestation
result of ingestion of encysted larvae in the improperly cooked flesh of an intermediate host animal
measly beef
beef visibly infested with Taenia cysts
Taenia ova
tapeworm stage not infectious for humans
distal progottids
more mature than the proximal on the tapeworm
free living
nonparasitic protozoans
observable traits, phenotype
Color of colonies, number of colonies, distribution of colonies on the plate.
good choices for genetic transformation experiments
non toxic, reproduces quickly, single cell organism
LB amp and LB amp/ara
plates that the transformed organisms will be found
Genetically transformed cells
cells that have taken up the pGLO plasmid express the ampicillin resistance gene—these cells can survive on the plates which contain ampicillin.
determine success of transformation
The LB/amp (-) pGLO and the LB/amp (+) pGLO plates should be directly compared.
ampicillin
antibiotic that only the organisms that took the plasmid will be able to survive
arabinose
sugar that regulates the expression of GFP, green florescent protien
pGLO plamid
GFP, ampicillin resistance, arabinose gene regulation system
control plate
guide your interpretation of results in an experiment; usually a plate of your original without manipulation
bacterial lawn
so many bacterial on a plate that individual colonies are not present, looks like a complete covering of growth
protien that the plasmid encodes
source of the florescences
how to determine ampicillin resistance
take some of the bacteria growing on the LB plate and
streak them on an LB/amp plate.
arabinose sugar
turn on the expression of the GFP gene.
two factors that cause bacteria to glow green
The sugar arabinose turns on expression of the GFP gene by binding to a regulatory protein. When arabinose
is present, it facilitates transcription of the gene by RNA polymerase. Exposure to UV light causes GFP to
resonate, thereby giving off energy in the form of green light.
advantage of turning off a gene
Gene regulation allows for adaptation to differing conditions and prevents wasteful overproduction of unneeded proteins.
E. coli
gram negative rod, nonspore forming, ferment lactose and live in the colon of humans
S. aureus
gram positive cocci, cause of staph infections, golden color, commensal on skin, produce enterotoxin
coliform
commonly-used bacterial indicator of sanitary quality of foods and water; almost exclusively of fecal origin and their presence is thus an effective confirmation of fecal contamination; E. coli and Enterbacter aerogenes
positive result for E. coli
metallic green colonies
b. subtilis
gram positive rod, endospore forming, nonpathogenic, found in soil, flagellated
saprophytes
nonpathogenic bacteria that may be found in drinking water
Vibrio cholerae, Salmonella typhi, Shingella dysenteriae
pathogens in contaminiated water
E. coli
bacteria that indicates likelihood fecal contamination in water and the potential for serious disease
E. coli is a good indicator
occurs primarily in the intestines of of humans and warm-blooded animals and is not found routinely in soil or water; easily identified by microbiological tests; not as fastidous as the intestinal pathogens and survives a little longer in water samples
presumptive, confirmed and completed
three tests performed to determine coliform cound
presumptive test
see if the water contains any lactose fermenting bacteria that produce gas
Most probable number
15 tube method, pattern of positive tubes and chart is used to ascertain the MPN of coliforms in 100 ml of water
confirmed test
plates of Levine EMB agar or Endo agar are inoculated from positive tubes to see if the organism is gram negative; media inhibits growth of gram positive
EMB agar
coliform produces small colonies with dark centers (nucleated colonies)
Endo agar
contains a fuchsin sulfite indicator that makes coliforms produce reddish colonies and confirms presence of lactose fermenting, gram negative bacteria; nonfermenters do not affect the color of the medium (lt pink)
Levine EMB agar
contains methylene blue that inhibits the growth of gram negative and E. coli will produce small colonies with green metallic sheen; E. aerogenes colonies are larger and no sheen
completed test for coliform
inoculate a nutrient agar slant and lactose broth with Durham tube. gram stain is made from slant
catalase
enzyme that catalyze the decomposition of hydrogen peroxide to water and oxygen
enumeration of bacteria
standard plate count
number of bacteria is important
in milk, bladder infection, in food
microscopic count
diluted sample of cells is counted under a microscope, counts live and dead; doesnt require growth in alternative media
MPN
count of coliform determined by statistical relationship
standard plate count (SPC)
serial dilution, a portion is plated, dilution that produces CFUs between 30-300 are counted and multiplied by dilution factor
SPC
only counts live cells and CFUs may distort bacteria that grow in chains, media may bias bacteria that prefer other conditions
indirect method
determines growth and increase in number by method that is not a count
spectrophotometer
measures turbidity by absorance or optical density
elevated temperatures
cause protiens (enzymes) to denature and unfold
thermal death point
is the temperature at which an organism is killed in 10 minutes
thermal death time
time required to kill a suspension of cells or spores at a given temperature
Clostridium botulinum and Clostridium perfringens
two bacteria involved in food poisoning
anthrax, tetanus, C. diff, gangrene
four disease caused by spore forming bacteria
260 nm
most germacidal UV wavelength, at this wavelength DNA maximally absorbs UV light
pyrimidine dimers
form when cells and their DNA are exposed to UV light
dimers
cause DNA to become deformed and polymerase is unable to replicate DNA strands beyond the formation
plastic
can block UV light
reasons endospores are resistant to UV light
DNA is protected by a small, acid soluble protien that binds to DNA and unique spore photo product is generated by UV light and functions in enzymatic repair of damage DNA
reason for covering half of the plate
unexposed area of plate to compare with
SOS system
enzymatic removal of dimers and inserts in thier place new pyrimidine molecules
Kirby Bauer procedure
sensitivity testing method for testing antibiotics and antimicrobials
antimicrobials
inhibit or kill microorganisms
antibiotics
antimicrobials that are produced by microorganisms
penicillin and streptomycin
examples of antibiotics
semisynthetic
altered antibiotics to make them more effective intheir mode of action
synthetics
chemically synthesized in the laboratory and are not the result of microbial synthesis
sulfa drugs
example of sythetics
Kirby Bauer
streak plate uniformly, paper disks containing concentrations of antibiotics/antimicrobials are deposited on agar surface, antimicrobial diffusion causes a concentration grandient and inhibits growth
zone of inhibition
zone around the disk where no growth occurs
zone size variation
due to diffusibility of agent, size of inoculum, type of medium and effectiveness of antibiotic/anitmicrobial
Kirby bauer method
standardized system that takes zone size variations into consideration
narrow spectrum antibiotics
Drugs effective against only one or a specific family of microorganisms.
broad spectrum antibiotics
are effective against a wide range of bacteria
extra outer membrane
reason some gram negative bacteria are sometimes more resistant to antibiotics
outer membrane of gram negative
can restrict antibiotics with cytoplasmic targets
increase dosage or durations
if bacterial isolates show intermediate resistance to an antibiotic
sodium hypochlorite
common household bleach, used in hospitals to disinfect
disinfectant
used to kill or inhibit growth of microorganisms on inanimate objects, not effective on endospores
antiseptic
used to kill or inhibit growth of microorganisms in living tissue, not effective on endospores
sterilants or sporocides
destroy all microbial life, including endospores
sanitizers
reduce microbial numbers to a safe level but do not completely eliminate them
bacteriostatic
inhibits the growth of bacteria but does not kill them
alcohol and betadine
examples of antiseptics
bleach and lysol
examples of disinfectants
SIM media
semisolid medium used to determine motlity
gelatin deep
observe liquefaction to determine if organism produced protease, an enzyme that degrades protien
nutrient broth
observe the nature of growth to determine characteristics of growth like surface, subsurface, sediment and turbidity, faky or flocculent
nitrate broth
observe color change to determine if nitrate was reduced to a gas or to nitrite; gas= dark red; zinc will react if nitrate is still present by turning black; nitrate reductase
nitrate reductase
enzyme that reduces nitrate
fermentation tubes
observe color change and gas collection to determine if organism ferments a sugar
durham tube
inverted tube used to collect gas when organism produces gas during fermentation
yellow color fermentation tube
organism ferments the sugar
red color fermentation tube
organism does not ferment the sugar
methyl red test
3-4 drops of methyl red into MRVP tube to detect acid produced by other fermentation acidic byproducts, red band across top indicates mixed acid fermentation
VP test
color change to pink or red indicates organism fermented butanediol
Sim citrate slant
detects citrate degrading; color change from green to blue to determine if organism used citrate as sole carbon source and produced ammonia
oxidase test
observe color change to dark purple to determine presence of cytochome oxidase
E. coli
control for tryptophan degradation test
Tryptophan degradation test
observe presence of red band indicating the presence of indole, determines the organism can degrade amino acid to indole, pyruvic acid and ammonia
Kovacs reagent
forms a deep red color if indole is present
tryptophanase
enzyme that degrades the amino acid tryptophan
urea hydrolysis
observe a color change to determine if the organism can breakdown urea into CO2 and ammonia
urease
enzyme that breaks down urea, indirectly measured by the presence of ammonia
bright pink
positive color for urease, ammonia present
Proteus vulgaris
control organism for urea hydrolysis test
starch hydrolysis
iodine complexes with starch and blue indicates presence of starch and that the organism did not hydrolyse starch, clear area around organism indicates organism hydrolyses starch
B. subtilis
control for starch hydrolysis
casein hydrolysis
observe clear area around growth indicates organism hydrolyzes casein
proteases
enzymes that degrade protien molecules such as casein in the casein hydrolysis test
B. subtilis
control for casein hydrolysis
fat hydrolysis
observe color change to dark blue to determine if unknown produces lipase
lipase
cleaves fatty acid from glycerol and lowers the pH of the agar to produce dark blue precipitate
spirit blue agar
agar that is used in fat hydrolysis test
S. aureus
control for fat hydrolysis test
Kleigers iron agar slant
observe color change to black precipitate to determine if organism can breakdown the amino acid cysteine to hydrogen sulfide
cystein desulfurase
enzyme that breaks down amino acid cysteine
litmus milk test
organisms that ferment the lactose will turn litmus pink and organisms that degrade casein will release ammonia and turn the litmus blue
Streptococcus mutans
cause of tooth plaque
destroyed or shrunk
what happens to capsules if they are heat fixed prior to staining?
heat and phenol
help carbolfuchsin penetrate the cell wall of acid fast bacteria
malachite green and safranin
two stains used for endospore staining
heat
mordent in shaffer-fulton endospore stain
mycolic acid
substance in the cell wall of mycobacteria and some norcardia that significantly affects staining properties
tuberculosis and lepracy
acid fast is a diagnostic tool for these diseases
differential stain
stains to react differently with different types of bacteria, used to distinguish bacterial types. Gram stain, acid-fast stain
Grams iodine
blue/black areas indicate presence of starch
Proteus vulgaris
changes slant from yellow to bright pink/cerise
nitrate reduction test
zinc powder is used to confirm a negative result
E. coli
indole positive
positive for lactose fermenting organism
yellow with gas bubbles
durham tube sugar fermentations
glucose tube, lactose tube, mannitol tube
B. subtilis
control for starch hydrolysis
ferrous salts + hydrogen sulfide
insoluble black precipitate
TSI (Kliglers iron agar)
prepared as a slant and inoculated by streaking and stabbing
VP test (butanediol fermentation)
needs 25 minutes for chemical reaction to occur
mixed acid fermentation (MR test)
MRVP tube + 3-4 drops of methyl red
tryptophan degradation
kovacs reasgent interacts with indole
spirit blue agar
fat hydrolysis plate
narrow your search
important to complete morphological and characterizations before physiological testing
pH indicators
often added to test to detect acid/base byproducts of the specific degration, fermentation, metabolism
exoenzymes
an enzyme that is secreted by a cell and that works outside of that cell. It is usually used for breaking up large molecules that would not be able to enter the cell otherwise
fermentation
enzymatic degration of carbohydrates final electron acceptor is an organic molecule, oxygen is not required
respiration
enzymatic degration of carbohydrates final electron acceptor is an inorganic molecule like oxygen