Lab Practical Review (Bio 286 - Microbiology) Flashcards
types of pathogens used in the laboratory
BSL-1 (most common; minimal hazard; examples include Escherichia coli, Bacillus cereus, and Saccharomyces cerevisiae) or BSL-2 (less common; moderate hazard; example includes Staphylococcus aureus)
general procedures followed in laboratory
wear appropriate lab attire (including lab coat)… no eating/drinking in lab… report accidents or incidents to lab professor… behave appropriately… bench cleaning (beginning and end of lab session)… handwashing (after lab sessions)… clean up spills… proper waste disposal (non-contaminated trash into regular trash bins, glass waste into glass container, staining waste into stain receptacle, contaminated waste into red bag containers)
dangerous equipment used in laboratory
bunsen burner (most dangerous); bacterial stocks
ocular lenses
eye pieces of microscope; each has a magnifying power of 10X
cord wrap
brackets to store the power cord around when done using the scope
objective lenses
secondary lenses to magnify the sample; range from 4X to 100X
course focus knob
used to rapidly move the stage (a sample) to focus
fine focus knob
used to tweak the focus… used with 40X and 100X objectives
mechanical stage controls
move the sample in the X and Y planes, with the focus on the Z plane
condenser iris
lever controlling how much light passes through the sample
power switch
turns the lamp on and off
light source
halogen lamp
total magnification
ocular lens x objective lens
total magnification achieved by different objectives
objective 4X x ocular 10X = 40X…
objective 10X x ocular 10X = 100X…
objective 40X x ocular 10X = 400X…
objective 100X x ocular 10X = 1000X
immersion oil was used on the
100X objective
why is immersion oil used?
to keep light from bending… to reduce light loss between the slide and the lens… to improve the resolution
calibrate microscope using a stage micrometer
scale divisions x 10 / # ocular divisions
use the reticle to measure a cell size
after calibrating the microscope to determine how many micrometers each division of the ocular ruler is, count how many ocular lines it takes to span the bacterial cell
gram negative bacteria
pink/red
gram positive bacteria
purple
cocci
spherical bacteria
streptococci
spherical bacteria that form a chain
staphylococci
Spherical shaped bacteria that form grape-like clusters
bacilli
Rod shaped bacteria
coccobacilli
a bacterium that is an oval rod
streptobacilli
rod-shaped bacteria occurring in chains
spirochete
spiral shaped bacteria
firmicutes
bacteria with gram positive cell wall
proteobacteria
bacteria with gram negative cell wall
eukaryotes
bacteria with gram positive cell wall but irregular bacterial shape
gram stain primary stain
crystal violet
gram stain mordant
gram’s iodine
gram stain decolorizer
acetone alcohol
gram stain secondary stain
safranin
crystal violet
used as a primary basic dye which can be taken up by the majority of bacteria due to its ability to rapidly permeate all cell walls
gram’s iodine
used as mordant to “seal” the Crystal Violet stain into the cell walls of the bacteria
acetone alcohol
used in gram stain to wash away crystal violet from gram negative bacteria; used as a decolorizer
safranin
counterstain in gram stain; used as a secondary stain after the initial crystal violet is washed away
capsules
clear structures surrounding the cells
capsules
clear structures surrounding the cells… function is to evade phagocytosis… composed of polysaccharides
capsule stain
stain used to stain cells that form capsules
india ink
stain used to visualize the capsule by making the background of the slide dark
india ink
stain used to visualize the capsule by making the background of the slide dark
endospore
function as “survival” cells to withstand harsh conditions… appear as greenish blue structures
vegetative cell
a cell that has not formed spores or other resting stages… appear as pink structures
gram stain
crystal violet -> iodine -> acetone alcohol -> safranin
endospore stain
malachite green (primary stain) -> heat (mordant) -> water (decolorizer) -> safranin (secondary stain)
organisms forming endospores
microbes of the Firmicutes (primarily Bacillus and Clostridium)
more endospores were made when grown on TSA
because TSA is less nutrient rich than BHIA, causing the cells to need to form more endospores in response to starvation conditions
acid-fast stain
a differential stain used to visualize acid-fast bacteria
acid-fast primary stain
basic fuchsin (+ phenol mordant, performed simultaneously)
acid-fast secondary stain
methylene blue
two pathogens that would appear violet/red with acid fast stain
Mycobacterium tuberculosis and Mycobacterium leprae
mycolic acid
compound within the cell walls of acid-fast bacteria (Mycobacterium tuberculosis and Mycobacterium leprae) that makes them waxy
flagella
external bacterial structures that function by providing motility to the cell
monotrichous flagella
single flagellum at one end
amphitrichous flagella
flagella at both ends
peritrichous flagella
flagella that cover the surface of a cell
lophotrichous flagella
cluster of flagella at one end of the cell
flagella provide motility through
spinning (like an electric motor)…rapidly rotating and providing thrust
flagella in eukaryotes
provide motility through wave-like motion
entamoeba histolytica
Causative agent of amoebic dysentery– a severe diarrheal disease associated with ingestion of contaminated food or water – part of SARCODINA
balantidium coli
The only ciliate human pathogen… these cells are transmitted by fecal contamination – part of CILIOPHORA
trichomonas vaginalis
Causes vaginitis and is an example of a sexually transmitted protozoan; only has a trophozoite form – part of MASTIGOPHORA
giardia lamblia
has a much reduced (nearly absent) mitochondria… Causes diarrheal diseases – part of EXCAVATA
plasmodium falciparum
causes malaria… ring stage trophozoite can be seen in some red blood cells – part of APICOMPLEXANS
fusarium verticilliodes
filamentous fungus widely distributed on plants and in the soil… a member of the class ASCOMYCETES and has SEPTATE hyphae… “football-shaped” macroconidia…may cause various infections in humans–one of the emerging causes of opportunistic mycoses
penicillium chrysogenum
filamentous fungus that is widespread… it can be found in soil, decaying vegetation, and the air…. It is a member of the class ASCOMYCETES and has SEPTATE hyphae… The conidia form on finger-like projections… may cause infections, particularly in immunocompromised hosts, but also produces the ANTIBIOTIC PENICILLIN
rhizopus stolonifer
cosmopolitan filamentous fungus found in soil, decaying fruit and vegetables, animal feces, and old bread (BLACK BREAD MOLD)… It is a member of the class ZYGOMYCETES and has COENCYTIC hyphae… common contaminants, but can also occasional causes of serious (and often fatal) infections in humans
syncephalastrum racemosum
filamentous fungus that is commonly isolated from soil and animal feces particularly in tropical and subtropical areas… It is a member of the class ZYGOMYCETES and has COENCYTIC hyphae. This heterothallic fungus requires a mating strain to produce zygospores… commonly considered as a contaminant and is very rarely associated with human disease
tube dilution factor (TDF)
sample + diluent / sample
serial dilution factor (SDF)
TDFa x TDFb x TDFc … (product of all TDFs)
CFU/mL
CFU x SDF / volume (mL) plated
plaques
holes in the bacterial lawn… caused by viral propagation overwhelming the host cells
microbial interaction represented by plaques
parasitism
virus (bacteriophage, T4)
caused the formation of plaques
lag phase
“flat” period of adjustment, enlargement; little growth
log phase
The period of exponential growth of bacterial population
stationary phase
period of equilibrium; microbial deaths balance production of new cells
death phase
population is decreasing at a logarithmic rate
growth rate (g)
log(2)/m [m = slope]
generation time (k)
1/g (x 60 if in hours)
m
log(y1) - log(y2) / x1 - x2
non-halotolerant
cannot withstand high salt concentrations
halotolerant
a microorganism that does not require NaCl for growth but can grow in the presence of NaCl
halophile
an organism that can grow in, or favors environments that have very high salt concentrations
acidophile
an organism that grows best at low pH; typically below pH 6
neutrophile
an organism that grows best at neutral pH, between pH 5.5 and 8
alkalinophile
an organism that grows best at basic/alkaline pH, between pH 8 - 11
psychrophile
bacteria that prefer cold, thriving at temperatures between 0 C and 25 C.
mesophile
bacteria that prefers moderate temperature and develops best at temperatures between 25 C and 40 C
thermophile
bacteria that thrive best at high temperatures, between 40 C and 70 C
GasPak chamber
sodium citrate and sodium bicarbonate react to release carbon dioxide, and the activated charcoal in the packet binds free oxygen gas – removing oxygen and creating a carbon dioxide atmosphere, ie anaerobic conditions
organisms that can grow in GasPak chamber
anaerobes, facultative anaerobes, aerotolerant anaerobes
organisms that cannot grow in GasPak chamber
aerobes
organisms that could grow in GasPak chamber or in air
facultative anaerobes, aerotolerant anaerobes
methylene blue
oxygen indicator used in BAA plates
resazurin (or methylene blue)
oxygen indicator used in fluid thioglycolate tubes
aerobe
an organism that requires oxygen for respiration and can live only in the presence of oxygen
aerotolerant
an organism that does not utilize oxygen but can survive and grow in its presence
anaerobe
an organism that grows without air, or requires oxygen-free conditions to live
microaerophilic
an organism that can require a lower oxygen concentration than what is in air
facultative anaerobe
an organism that prefers an oxygen environment but is capable of living and growing in its absence
zone of inhibition
region around a chemical saturated disc, where bacteria are unable to grow due to adverse effects of the compound in the disc
most effective drug
forms the largest zone of inhibition
least effective drug
forms the smallest/no zone of inhibition
antiseptics
used on living surfaces (like skin); tend to be less toxic… include isopropanol (alcohol), iodine (oxidizer), dish soap (surfactant), and germicidal soap (phenolic)
antibiotics
produced by other microorganisms and tend to be used chemotherapeutically… include peptidoglycan attackers (ampicillin, cephalothin, penicillin) and 70s ribosome attackers (erythromycin, streptomycin, tetracycline)
disinfectants
used on inanimate objects and tend to be more toxic… include lysol (phenolic), roccal (quaternary ammonium compound), bleach (oxidizer), and aldehyde (alkylator)
resistant bacteria
small/no zone of inhibition
intermediate bacteria
intermediate sized zone of inhibition
susceptible bacteria
large zone of inhibition
thermal death time
the shortest amount of time needed to kill all microbes at a given temperature
thermal death point
the lowest temperature needed to kill all microbes in a given time
most heat-resistant bacteria
bacillus or clostridium because they form endospores
fecal coliforms
lactose-fermenting gamma proteobacteria; typically found in mammalian feces… appears blue on mFC plate
fecal streptococci
typically found in feces of animals (like ducks, not humans)… appears red on mEA plate
human contamination source
blue (coliforms) / red (streptococci) > 4:1
mixed contamination source
2:1 > blue (coliforms) / red (streptococci) < 4:1
unknown contamination source
0.7:1 > blue (coliforms) / red (streptococci) < 2:1
animal contamination source
blue (coliforms) / red (streptococci) < 0.7
mFC plate
used to detect fecal coliforms… contains lactose and resorcinol dye that makes coliforms appear blue… contains bile salts that inhibit growth of non-coliforms
mEA plate
used to detect fecal streptococci… contains metabolic poison sodium azide and tetrazolium red that makes streptococci appear red… sodium azide prevents growth of non-streptococci
transformation
process in which exogenous DNA is incorporated into another cell’s genome
in the transformation experiment, what microbe was used?
two strains of Acinetobacter calcoaceticus, one strain resistant to streptomycin and the other strain susceptible to streptomycin
in the transformation experiment, what antibiotic was used?
streptomycin
streptomycin targets
70S ribosome
does streptomycin work against yeast?
no because yeast are eukaryotes that have 80S ribosomes
why does UV kill?
it forms pyrimidine dimers, preventing proper base pairing in DNA… causing mutations in DNA sequence where enough mutations will kill the cell
UV wavelength used in germicidal lamps
260 nm (UVC)
where is UV light used at the wastewater treatment plant?
wastewater disinfection
what organisms were the most resistant to UV killing?
Bacillus cereus (most resistant because it forms endospores) > pseudomonas aeruginosa (because the green pigment protects it) > serratia marcescens (because the red pigment protects it) > staphylococcus aureus (most susceptible)
lichens exhibit
mutualism behaviour
termites and their hindgut microbiota exhibit
mutualism behaviour
cow and their ruminants exhibit
mutualism behaviour
staphylococcus aureus and clostridium sporogenes exhibit
commensalism behaviour
lactose fermentation (MacConkey plate) positive result
RED
lactose fermentation (MacConkey plate) negative result
tan/brown
citrate positive result
BLUE
citrate negative result
green
vogues proskauer positive result
RED
vogues proskauer negative result
brown
TSI H2S positive result
BLACK
TSI H2S negative result
no change
indole positive result
RED
indole negative result
yellow
urea positive result
PINK
urea negative result
yellow
MacConkey Agar
if the microbes ferment lactose then the acids they produce turn the indicator dyes red… a positive result is red and a negative result is no change
indole
if microbes breakdown amino acid tryptophan to indole, the indole reacts with Kovac’s reagent to turn bright red… a positive result is red and a negative result is yellow
Triple Sugar Iron Agar (slant)
if ammonia is produced then the pH of the media becomes neutral – red and if lactose/sucrose is fermented then the tube will remain yellow… a positive result is yellow (A) and a negative result is red (K)
triple sugar iron agar (butt)
the butt of the TSI is always yellow – a positive result is yellow (A)
triple sugar iron agar (gas)
gases produced during fermentation will appear as bubbles or cracks… a positive result is cracking and a negative result is no change/no cracking
triple sugar iron agar (H2S)
hydrogen sulfide produced from breakdown of amino acid cysteine reacts with the iron to form ferric sulfide which is black… a positive result is black and a negative result is no change
MRVP (methyl red)
turns red in the presence of organic acids… a positive result is red and a negative result is yellow
MRVP (voges proskauer)
detects acetoin to turn red in its presence… a positive result is red and a negative result is coppery brown/no change
citrate
at acidic pH (citrate present) the media is green and if the citrate is metabolized the pH will turn blue… a positive result is blue and a negative result is green
NaCl broth positive result
GROWTH – CLOUDY
NaCl broth positive result
GROWTH – CLOUDY
NaCl broth negative result
no growth – clear
bile esculin (BE) slant positive result
BLACK
bile esculin (BE) slant negative result
tan
optochin positive result
SUSCEPTIBLE
optochin negative result
resistant
DNase (HCl) production positive result
CLEARING
DNase (HCl) production negative result
no clearing
mannitol salt agar
selective and differential media; contains 6.5% NaCl, phenol red dye, and mannitol…. microbes that are able to grow may ferment mannitol or not (if they do, the acids they produce turn the indicator dyes in the media yellow)
catalase
enzyme that decomposes hydrogen peroxide to water and oxygen gas– when H2O2 is placed on the microbes, the production of bubbles indicates a positive reaction
EF broth
media containing a fermentable sugar (glucose) and an acid indicator (bromcresol purple)– glucose fermentation results in acid end products, which cause the media to turn yellow… the media also contains a metabolic poison sodium azide, allowing only azide-resistant microbes to grow
NaCl broth
contains 6.5% NaCl, allowing only halotolerant microbes to grow and turn the media cloudy
bile esculin slant
media used to detect the metabolism of a compound called esculin– if it is converted to esculetin, it will react with iron in the media and turn completely black… bile acids in this media inhibit the growth of most gram positive organisms
DNase
breaks down DNA to nucleotides by adding HCl to the DNA plate after microbes have been cultured– the low pH causes DNA to precipitate within the media, turning the late cloudy. any areas where DNA has hydrolyzed will not precipitate, leaving clear areas around growth of positive cultures
sheep blood agar
some bacteria break down blood cells using toxins called hemolysins… resulting in three patterns of hemolysis clearings- alpha, beta, and gamma… additionally, bacitracin and optochin are antibiotics used to determine susceptibility
alpha hemolysis
partial breakdown of red blood cells– resulting in smoky green color (no longer red but no clearing)
beta hemolysis
complete breakdown of red blood cells, resulting in complete clearing– can read text through the plates
gamma hemolysis
no clearing, the plates have growth on them but are just as red
bacitracin
an inhibitor of peptidoglycan biosynthesis that group A streptococci are particularly susceptible to
optochin
a copper containing compound that pneumococci are particularly susceptible to
amylase exoenzyme
exoenzyme used to break down polymer amylose (starch) [polysaccharide]… visualized by a clear zone around bacteria after addition of iodine… performed by bacillus cereus
DNase exoenzyme
exoenzyme used to break down polymer DNA [polynucleotide]… visualized by clear zone around colonies after addition of HCl… performed by staphylococcus aureus
gelatinase exoenzyme
exoenzyme used to break down polymer gelatin [protein]… visualized by gelatin not solidifying when on ice… performed by staphylococcus aureus, bacillus cereus, and pseudomonas aeruginosa
lipase exoenzyme
exoenzyme used to break down polymer lipid (triglyceride) [lipid]… visualized by less opaque media and a halo around the bacterial growth… performed by pseudomonas aeruginosa and staphylococcus aureus
lysozyme
targets peptidoglycan of bacteria… an important component of immune defense… found in eggs, tears, saliva, and can be commercially made
E.coli is resistant to lysozyme
because it is gram negative and has less peptidoglycan in its cell wall
M.luteus
gram positive bacterium that is a normal inhabitant of human microbiota
neutrophil
most common white blood cell… main phagocytic cells in circulation
lymphocyte
second most common white blood cell… includes B-cells and T-cells of adaptive immune response
monocyte
immune cells that differentiate into tissue macrophage and dendritic cells… important antigen presenting cells
eosinophil
cells filled with pink stained granules… deal with worm infections and are also associated with type I hypersensitivity (allergies)
basophile
least common/rarest white blood cell… filled with purple stained granules… associated with anemia and some microbial infections
