basic techniques in microbiology final
calibration constant
[(stage divisions)/(ocular division)] x 10 micrometers (***calibration constant) so... (5/10) x 10 micrometers = 5 micrometer calibration constant
differential plates
allow you to distinguish between different types of coliforms
anaerobes
can grow in the absence of O2
antibiotic resistance, how does it work?
it inactivates drug -pumps it out -change binding sites
phase contrast microscopy
live, unstained cells
what inhibits the the growth of fungus during fermentation?
salt and anoxia/anaerobiosis (life that is sustained without air)
you pipetted 500 micrometers of water into a micro centrifuge tube. you know that the tube weighed 1.5 g before adding the water. if the tube weighs 2.1 g after adding the water what is the percent error of your pipette?
% error = [(measured mass - expected mass)/expected mass]*100 2.1g - 1.5g = 0.6 g of measured water [(0.6 - 0.5)/0.5] *100 = 5% error
you prepare a wet mount from an unknown bacterial culture and measure a single bacillus cell length at 2 ocular divisions using the 40x objective. if your calibration constant for this objective is 2.5 micrometers, what is the actual length of the cell?
(stage divisions/ocular divisions) x 10 micrometers calibration constant. 20x = 5 micrometers 40x = 2.5 micrometers 100x = 1 micrometer so 2.5 micrometer length at 2 ocular division = 2.5 * 2 = 5 micrometers actual length of cell
calculating cell density
(total cells counted /5)x dilution times x 10,000 = _________ cells/mL -divide by 5 because you are averaging the cells per 1 mm squared across the five areas counted -multiply by the number of times diluted -10,000 is the volume factor of the chamber *contsant to convert to cells/mL
standard notations for mixed acid fermentation
-NG = no growth - (-) = growth, but no acid or gas - (+) = acid produced, but no gas - "+" with a circle around it = both acid and gas produced
sterile technique
-autoclave -bunsen burners -contact consciousness
when looking at live cells on a wet mount, how can you tell the difference between the movement of the cell and Brownian motion?
-brownian motion: random, short distance movement due to temperature. -streaming: all microbes move rapidly in the same direction -flagellum: recognized when a given cell moves in a single direction that is different from other neighbor cells
microbial metabolism- what does this mean?
-global biogeochemical cycling -bioremediation -understanding evolutionary past
coliforms are gram____________ bacteria. they __________ capable of lactose fermentation and they _________ form spores
-gram negative -are capable of lactose ferm -do not form spores enteric bacteria are gram negative, non-sporulating rods, facultative anaerobes some are known as coliforms coliforms are gram negative, aerobic or falcultative anaerobic rods, non-spore forming, growing at 35 C, fermenting lactose with the formation of acid and gas. most are enterobacteriaceae.
coliforms
-gram negative bacteria -aerobic or facultative anaerobic rods -non-spore forming -growing at 35C -fermenting lactose with the formation of acid and gas production -most are enterobacteriaceae
identification schemes from gram staining
-gram-positive cocci: * start with catalase test -gram-positive rods: *start with spore staining -gram-negative cocci: *done!!! -gram-negative rods: *start with lactose broth
gram staining method
-label slides -make smears from broth (let dry -wax pencil to outline -methanol fix -crystal violet for 30 sec -water rinse -iodine (mordant) for 20 sec -alcohol rinse for ~10 sec -immediately gently rinse with water -safranin for 1 min -rinse gently with water -observe in microscope with bright field
steps of salmonella lab
-pre-enrichment -selective enrichment -selective plating -confirmation media -identification
what are the 3 types of typical placement for endospores?
-terminal (end of cell) -central (middle of cell) -sunterminal (between middle and end of cell)
endospore formation
-vegetative cell's chromosomal DNA is replicated -vegetative cell's plasma pinches off between the replicated chromosomes, forming a "forespore" -a second membrane encloses the forespore with calcium and dipicolinic acid, forming a cortex between the inner and outer membrane -an external protein spore coat encloses the endospore -the endospore is released once the vegetative cell that generated it dies and disintegrates
micropipette
1 mL = 1000 microliters *P20 = 1-20 microliters *P200 = 20-200 microliters *P1000 = 200-1000 microliters more precise towards their upper range
if the digits on a p1000 micropipettor read: 074, what volume is expressed and in what units?
740 microliters
% error
= [(measured - expected)/expected] * 100 so... Wf - Wi = D D-1g / 1g = X X *100 = % error ***Wf = weight final (g) ***Wi = weight initial (g) ***D = difference (g)
total magnification
= ocular x objective ex. (10x objective) x (40x lens) = 400x total magnification
when interpreting an antibiogram in this class A. what growth pattern generally represents sensitivity to antibiotics? B. how can you tell if you have antibiotic resistant bacterial cultures?
A. clear zones B. small colonies in the clear zones. the size of the clearing zone is proportional to the efficiency of the antibiotic against a specific microbe. measure the diameter of clear zones around the disks and note whether there are resistant mutants (small colonies within the clear zones)
Blood Agar plates
BA: alpha hemolysis: -partial hemolysis -bacteria produce H2O2 and oxidize hemoglobin -get a pea green color beta hemolysis: -complete hemolysis -bacteria produce exotoxin streptolysin -get yellow/colorless regions Gamma: -no hemolysis
brilliant green agar
BGA differential for salmonella -contains lactose and sucrose -brilliant green dye inhibits bacteria (LPS layer of salmonella protects it) -Phenol red pH indicator *pH below 6.8 = yellow *pH above 6.8 = red *salmonella does not ferment lactose or sucrose, so colonies are red
biosafety levels
BSL-1: low individual risk, low community risk BSL-2: moderate individual risk, low community risk BSL-3: high individual risk, low community risk BSL-4: high individual risk, high community risk
pH
Potential (Power) of hydrogen - describes the acidity or alkalinity of an aqueous solution -negative of the base 10 log of the concentration of hydrogen ions (protons) -lots of available H+ (acidic), lots of Hydrogen bound in hydroxide ions OH-(alkaline)
Streptococcus thermophilus Agar
ST agar -contains sucrose, easily fermentable by streptococcus thermophilus
how do bile salts in tetrathionate brilliant green broth (TBGB) inhibit gram positive bacteria?
TBGB is a selective media that contains ingredients that allow for the proliferation of salmonella and inhibit the growth of competing microorganisms. tetrathionate inhibits many gram-negative bacteria, but salmonella possess an enzyme tetrathionate reductase which reduces tetrathionate to thiosulfate. the bile salts in the media inhibit gram positive bacteria by acting as detergent and damaging the cell membranes. gram-negative bacteria have an LPS which is resistant to detergents like bile salts, finally the Brilliant green dye inhibits bacteria by interacting with their nucleic acids (LPS prevents this) BILE SALTS work on gram pos like detergent sine they don't have a thick LPS
broad spectrum antibiotics
TETRACYCLINE -inhibits binding of aminoacyl-tRNA to the ribosome -stops protein synthesis STREPTOMYCIN -interferes with binding of fMet-tRNA to the small ribosomal subunit -effectively stops protein translation
Tomato Juice Agar
TJ agar -acidic, selects for acidophilus such as leuconostoc
name two types of culture media we have prepared so far. what is each type best suited for?
TSB (TRYPTICASE SOY BROTH): -used for short term growth -increase cell count quickly / large volume (biomass) TSA (TRYPTICASE SOY AGAR) SLANTS: -long term storage -minimal contamination TSA (TRYPTICASE SOY AGAR) PLATES: -isolate colonies -check purity of culture/ contamination check *incubated either aerobically or anaerobically in jars
TSA plate
Trypticase soy agar -isolate colonies -check purity of culture/ contamination check *incubated either aerobically or anaerobically in jars
TSA slants
Trypticase soy agar -long term storage -minimal contamination
narrow spectrum antibiotics
VANCOMYCIN AND PENICILLIN -target gram+ -inhibit peptidoglycan cell wall synthesis POLYMYXIN B -gram negative -disrupts cell membranes
lawn streak
a lot of streaking and rotate and more streaking!!!
TSA plate for coliform contamination
allows all bacteria to grow so we can obtain the total number cultural/colony forming units (CFU) * count all organotrophs
selective media
allows the growth of some microorganism while inhibiting the growth of others ex. -MRS, ST, TJ
skin flora
also complex and variable -different communities on local micro-environments FOREHEAD: acidic, rich with sebaceous glands -propionbacterium acnes -staphylococcus epidermidis ARMS & LEGS: (dry) -staphylococcus (variety) -micrococcus luteus
lactic acid fermentation
anaerobic metabolism that results in sugar (glucose) and the byproduct lactate from pyruvate typical species: -lactobacili -leuconostoc -streptococcus -bifidobacterium
lactic acid is produced by ______________ bacteria via fermentation and serves to preserve food _______________ the growth of other microbes
anaerobic, inhibiting lactic acid is produced by the fermentation of simple sugars by a variety of anaerobic microorganisms (lactobacillus, leuconostoc, streptococcus, and bifidobacterium) foods may be preserved by fermentation performed by nonpathogenic bacteria which produce organic acids under controlled conditions
does fermentation happen under oxic or anoxic conditions?
anoxic conditions
why would you streak a bacterial lawn?
antibiogram test
clearing zone
antibiotic sensitivity colonies found within zone of clearing = from resistant individuals no clearing = resistant population
selection pressure
antibiotics weed out susceptible strains, leaving resistant strains -resistance genes are easily transferred -quick bacterial generation time = quick spread -rise of super bugs "MRSA"
Hugh and leifson O/F test
asks whether facultative anaerobe or something else. stays green if not fermentation (no glucose eaten). mineral oil determines aerobic vs anaerobic conditions
circle what features you stain when performing a capsule stain. more than one option may be circled. a. the capsule b. the cell c. the slide itself d. flagella e. endospores
b. the cell c. the slide itself a capsule stain is a negative stain (meaning the cell is stained, the glass is stained, but the capsule is not) used to detect cells capable of producing an extracellular capsule.
what are exoenzymes and what is the reason they are used by bacterial organisms?
bacteria cannot bring nutrients inside their cells as large pieces. these cells can only import dissolved chemicals. one means for these cells to obtain dissolved solutes is to excrete extracellular enzymes (exoenzymes) into the surrounding medium. many are specific hydrolases that will degrade external insoluble polymers such as carbohydrates
why use specialized motility media to differentiate motile from non motile cultures?
can clearly see that some bacteria are mobile vs not mobile
facultative anaerobe
can grow in the absence of O2 but better when O2 is present
what is fermentation?
can happen with or without O2, but greater amounts of acidic products than oxidation
what is oxidation?
can only happen in the presence of O2
obligate (strict) aerobes
cannot grow in the absence of O2; O2 is required for respiration
Petrol-Hausser counting chamber
cell counting chamber -2 sides: add 10 microliters to each chamber
anaerobic
cellular respiration undergone without oxygen
MPN chart
choose the highest dilution (most dilute) giving positive results in all 5 replica tubes -select the series that shows changes between solutions
Superoxide dismutase & catalase
convert oxygen radicals to less toxic compounds -H2O2 -> further degraded to H2O and 1/2 O2
ryu stain
crystal violet in alcohol solution -leaves precipitate around flagella, making them visible using light microscopy
hemolysis
destruction of red blood cells -Alpha -Beta -Gamma -partial hemolysis: partial decomposition of hemoglobin of RBCs within area of bacterial growth -Complete hemolysis: complete breakdown of hemoglobin of RBCs in vicinity of bacterial growth. ***the presence or absence of hemolytic activity can help identify certain bacteria
capsule stain
detects cells capable of producing extracellular capsule NEGATIVE STAIN: stains everything except the capsule. -india ink stains glass -crystal violet stains cells
what is the difference between selective media and differential media?
differential media are media designed to make particular bacteria look different from others, whereas selective media inhibits the growth of some bacteria allowing others to grow.
when observing results from the microbial respiration lab, the agar deeps you had inoculated with E. coli had "broken up" and displayed areas of air between the pieces of agar. why did this happen?
discoloration, cracks or bubbles means oxygen was able to slowly diffuse into the agar as it cooled but did not reach the bottom of the tube. E. coli produced gas that wasn't able to escape the agar so it caused the agar fracturing.
aerotolerant anaerobes
do not use O2 but can tolerate it
citrate test
does only ammonia and citrate support growth?
true or false: according to your lab manual, you're expected to observe streptococcus thermophiles in your sauerkraut samples while observing leuconostoc sp in your yogurt samples?
false: commercial yogurt are made from milk fermented by pure cultures of lactobacillus delbrueckii bulgaricus and streptococcus thermophilus where as leuconostoc and lactobacillus sp are found in sauerkraut.
sauerkraut
fermented cabbage -anoxic/anaerobic conditions; salt, anoxia inhibit growth of fungus -acid tolerant bacteria: -leuconostic mesenteroids -lactobacillus sp lactic acid accumulation eventually inhibits all microbial activity, preserving your delicious sauerkraut
yogurt
fermented milk species: -lactobacillus delbrueckii bulgaricus -lactobacillus acidophilus -bifidobacterium sp *probiotic
colony morphology
form: basic shape of the entire colony Elevation: shape of the cross section of the colony Margin: shape of the edge of the colony also look at color, texture, translucence, and physical consistency
gram negative stain
have a basic lipid bilayer (hydrophilic heads and hydrophobic side chains/tails) as the outer membrane layer and a thin peptidoglycan layer as the middle layer in the cell membrane. because the peptidoglycan layer is thinner crystal violet doesn't bind to it. the second dye, safranin binds to most things including gram + bacteria but you can't see the safranin in gram + because the crystal violet binds to it and it is darker.
gram positive stain
have a thick outer membrane layer of peptidoglycan which absorbs more of the stain and to set/fix resulting a darker purple color. purple= pos
why do we use heat instead of methanol to fix endospore stain?
heat drives the stain into the endospore
capsule
help defend bacteria from predators (e.g.protists) or can, in some cases, increase virulence the microbe. can also promote adhesion to favorable environments.
what is broken down in Beta hemolysis and what protein is responsible?
hemoglobin in blood is broken down by exoprotein (hemolysin)
what is the difference between homolactic and heterolactic bacteria?
homolactic: produce lactic acid as their main fermentation byproduct. (lactobacillus) heterolactic: produce approximately equal amounts of lactic acid, ethanol, and CO2 (leuconostoc)
bright field microscopy
inanimate objects -stained or pigmented cells
violet red bile petrifilm plate
indicator for E. coli - red colonies formed are not e. coli, bubbles near blue colonies indicate gas production and E. coli *count coliforms & E. coli
the IMViC test that determine whether an organism can metabolize tryptophan is called the ___________test.
indole indole test determines whether an organism metabolizes tryptophan, an aromatic amino acid, the enzyme trytophanase splits tryptophan in an aromatic part (indole), pyruvate, and ammonia. the indole does not further metabolize and accumulates in the medium, uses a reagent called kovac's reagent, when it reacts a red color product is formed.
mordant
inorganic oxide that combines with dye or stain and fixes it.
TSA deeps
insulated while molten, mixed and allowed to solidify -bottom of tube provides an anaerobic environment when incubated aerobically
sugar broths + phenol red test
interpretation A. gas in durham tube = surge fermentation = (+) B. sugar fermentation = lower pH = yellow color = (+) C. sugar oxidation = no pH change = red color (-) D. metabolize protein only = raise in pH = fuchsia color = (-) E. inoculated control = no change = (-)
antibiogram
is a report used to determine the optimal antibiotic house to kill bacteria -used in hospitals to determine treatment -sample is taken from patient to determine efficiency of antibiotics
VP test
is anaerobic fermentation producing a neutral product (acetoin)? -same media as methyl-red.
methyl red test
is anaerobic fermentation producing acidic products (acetate, lactate, etc.)? -same media as VP
indole test
is tryptophan metabolized?
Why would you streak for isolation?
isolating a colony
obligate (strict) anaerobes
killed by the presence of O2
Bacto Motility Agar
low % agar gel in tube contains TTC(s,3,5-triphenyltetrazolium chloride) -aerobic respirators reduce TTC, which forms a red precipitate -motile cells spread from the point of inoculation
please draw on the figure below the proper streaking technique for a TSA plate with a bacteriological loop in order to get isolated bacteria colonies.
make sure each streak has been sterilized to drag out the small sub same of bacteria
why do we test for coliforms?
monitor contamination (found in feces, want to confirm water is not contaminated) -are they present or absent? -is e.coli present specifically? -how many bacteria are present?
match the flagella arrangements shown below to the proper terminology.
monotrichous: only one flagella is present lophotrichous: flagella are an arraignment of clustered flagella at one of both ends amphitrichous: one flagella on both sides of the cell peritrichous: flagella are dispersed all over the cell.
why is it best to select from dilution plates showing between 30-300 colonies per plate?
most diluted plates will have lesser colonies and the ones not diluted enough will be too high many to count (TMC)
what is the precipitate in the motility agar
motility media generally contain chemicals that make it easy to see where growth has occurred. 2,3,5-triphenyltetrazolium chloride (TTC). bacteria capable of aerobic respiration will use TTC as an alternative electron acceptor instead of oxygen. upon chemical reduction, them TTC changes color to red and precipitates as formazan salt.
peptidoglycan
murine is a netlike macromolecule made of short peptides (amino acids) and glycan chains (sugars). forms a crystal lattice structure
antibiotic resistance, how does it develop?
natural occurrence -mutations -often transferred
does washing with soap remove all microbes?
no
flagella arrangement
observe the number of flagella, the location relative to the cell, and the location relative to each other Monotrichous = single, polar flagellum. Lophotrichous = tufts of flagella at one end. Amphitrichous = single flagella at both ends. Peritrichous = flagella over entire cell. Amphilophotrichous = tufts of flagella at both ends.
suppose you inoculate an Eosin Methylene Blue (EMB) agar plate with a water sample of unknown purity and observe bright green colonies with a metallic sheen following incubation at 37 C for 24 hours. what conclusion would you draw from this result?
obviously gram negative since EMB is selective for gram negative since gram positives are inhibited by these dyes. some colonies of microbes with vigorous lactose fermentation and acid production may appear metallic with a green sheen
selective plates
only allow growth of certain coliforms of interest
microaerophile
only grow when O2 is in low concentration
what are aerobic conditions?
oxic conditions: plenty of O2 for the cell to utilize (top of the tube)
over the duration of exercise 14's fermentations, the pH of both the yogurt and the sauerkraut liquid ____________, indicating a rise in ______________.
pH decreases rise in acidity lactic acid accumulates until all microbial activity is inhibited, thereby preserving the the sauerkraut for long periods
cytochrome-c oxidase
part of the electron transport chain -cellular respiration in aerobic bacteria *transfers electrons from cytochrome-c to O2 molecule to produce H2O
C. aperture diaphragm control H. fine focus adjustment knob J. objective lens L. ocular objectives *** F. phase ring adjustment B. condenser D. collector lens G. stage adjustment knobs
please identify the parts C, H, J, and L from the picture
during the process of making sauerkraut, explain one way in which the microbial community changed?
presence of salt and lactic acid limit growth of undesirable bacteria, resulting in a succession of microorganisms. at first only slightly tolerant of low pH and lactic acid, during their growth the O2 is consumed. as the pH drops , the growth of other bacteria that are more acid and lactate tolerant occur (leuconostoc and lactobacillus sp) eventually the low pH and the lactic acid will inhibit leuconostoc and the lactobacillus will dominate the sauerkraut.
in gram staining, a thick peptidoglycan layer on the cell wall stains __________ indicating a gram ______________ bacterium.
purple gram positive very thick peptidoglycan of gram positive cells prevent the loss of color
presence-absence broth
purple = negative yellow + gas bubbles = positive= contamination
resazurin
redox indicator turns blue when oxidized turns pink when partly reduced, and colorless when fully reduced
microbial respiration
refers to how oxygen is used. -obligate aerobes -obligate anaerobes -facultative anaerobes -aerotolerant anaerobes -microaerophiles *free oxygen is toxic to cells, bacteria need specific proteins to use O2 & protect themselves from it
what are two components of the rye stain?
ryu stain is crystal violet in an alcoholic solution, during staining a solution is added that upon evaporation leaves a precipitate around the flagella, increasing the thickness, also contains tannic acid and aluminum potassium phosphate (mordants) and phenol (anti fungal agent to help preserve the stain)
what is the counterstain for an endospore and what are we using it for?
safranin
why might you have observed a dense microbial population during period 2 of the cabbage juice observation but not during period 1?
sauer kraut is produced by salting cabbage and then fermenting it in an anoxic condition. the presence of the salt and the lactic acid produced help limit the growth of some undesirable bacteria, while anaerobiosis helps limit the growth of fungi. the fermentation of sauerkraut results in a succession of microorganisms
why would an endospore be created?
scarcity of food, change of water content, temperature changes, and to make copies of its chromosome
tetrathionate brilliant green bile broth (TBGB)
selective for samonella -bile salts inhibit growth of gram-positive *interferes with membrane; gram-negative lipopolysaccharide layer (LPS) protects -tetrathionate inhibited Gram-negative *salmonella possess the enzyme tetrathionate reductase -brilliant green dye inhibits bacteria *LPS layer of salmonella protects it *interacts with nucleic acids of other bacteria
Mitis Salivarius MSA
selective: -contains crystal violet and potassium tellurite -inhibits most gram-negative bacilli and most gram-positive bacteria except streptococcus differential: -trypan blue -different blue shades -varies by species *S. mitis: small blue colonies *S. salivarius "gumdrop" colonies *enterococcus: darkblue/black shiny colonies
electron transport chain
series of complexes that transfer electron from donors to acceptors. in respiration this reduces O2 to H2O
what are flagella?
slender, hair-like extensions used by bacteria for locomotion
fluid thyioglycollate medium
sodium thioglycollate consumes O2 and permits the growth of obligate anaerobes
what are some advantages of cell counting (direct counting)?
speed is the main advantage. it is a quick way to measure what's currently present in a sample, alive or dead. it is also advantageous for larger things - yeast, RBCs
what us a major advantage of directly counting cells?
speed is the main advantage. it is a quick way to measure what's currently present in a sample, alive or dead. it is also advantageous for larger things - yeast, RBCs
you want to prepare a serial dilution to a factor of 10^-4 from a stock culture of yeast. each dilution tube should contain 10 mL of liquid once prepared. outline, with a figure, how you would prepare these dilutions.
stock water sample pipette 1 ml to 10^-1 dilution tube, mix tube by vortexing each dilution, with sterile technique pipetting to the next dilution step until 10^-4 is reached.
explain how streaking a bacterial lawn is different from streaking for isolation.
streak lawns are useful for antibiograms and deciding if there is resistant strains or not, isolation streaking is more useful for colony identity streaking for isolation you use one streak and then drag sub colonies out of it (sterile) and keep dragging. lawn streaking is just literally covering the plate in bacteria.
what enzyme rapidly degrades hydrogen peroxide into water and oxygen a. cytochrome-c oxidase b. catalase c. peroxidase d. aceytl-C hydrase
superoxide dismutase converts the superoxide radical to hydrogen peroxide and oxygen, but catalase rapidly degrades the hydrogen peroxide to water and oxygen
name 2 of the 3 typical placements for endospores and where they are located.
terminal: end of cell central: middle of cell subterminal: between the middle and the end
what is the point of inoculation in Bacto motility agars?
test for mobility/ from the origin of the needle poke
what do I mean with the term cell motility?
the ability for the cell to move apart from streaming/ browning motion. some can glide but most use flagella to propel themselves
which of this pics shows motile cells? how can you tell?
the bottom one shows motile cells since it turned the agar red/pink
what function does cytochrome c oxidase perform? why is this function important?
the electron transport chain of most O2 respiring microbes contains an enzyme named cytochrome c oxidase which receives the electrons from cytochrome c and donates them to O2 to form water. (a membrane bound enzyme used in respiration to transfer electrons to O2)
A. forespore B. endospore
the following is an image of clostridium. what features are indicated by the arrows in the following image?
how do you sterilize your loop
the loop is sterilized via a Bunsen burner. the Bunsen burner is started via a striker and an open gas valve connected to the Bunsen burner. the flame should have no yellow in it and the ship of the flame (blue) should be the hottest part of the flame. proceed to hold the loop in the flame until red hot making sure the entirety of the metal coil on the loop has turned red hot. then as the loop cools for roughly 20 seconds to a moderate temp, insert loo into you sterile culture media. be sure that the loop is flame sterilized and maintain a conscious knowledge of contact
what is the purpose of the pre-enrichment step in the salmonella lab?
the pre-enrichment of salmonella, lactose broth is commonly used, most salmonella do not use lactose, but all salmonella are acid tolerant, in pre-enrichment media, lactose fermenting microbes drop the pH and eventually die off, while salmonella survives.
why did we use thyioglycollate medium last lab period?
the thyioglycollate medium contains resazurin, a redox indicator which turns blue when oxidized, pink when partially reduced, and colorless when fully reduced. used to assess the respiration methods used. *** reducing agent reduced oxygen to water creating an anaerobic environment
what are heterolactic bacteria?
they produce approximately equal amounts of lactic acid, ethanol, and CO2 as fermentation byproducts
what are homolactic bacteria?
they produce lactic acid as their primary fermentation byproduct
you inoculate 2 O/F tubes for a Hugh-Leifson test with an unknown bacterial culture and cover one tube with sterile mineral oil. when you check your results during the next lab period, you see that the liquid in the tube covered in mineral oil is green while the uncovered tube is yellow. what do these results tell you about your unknown bacteria?
this indicates oxidative metabolism with aerobic respiration.
why are endospores formed?
to deal with a variety of environmental stressors (food scarcity, change in water content, change pf temp) and reproductive factors (make copies of chromosomes)
TSB media
triplicase soy broth: large volume (biomass) short term -used for short term growth -increase cell count quickly *tryptone (digested casein) and soutane (digested soybean) *amino acids and other nitrogenous compounds *glucose (energy source) *sodium chloride (maintain osmotic equilibrium) *dipotassium phosphate (buffer to maintain pH)
commensal relationship
type of symbiosis in which one party benefits and the other party is neither harmed nor gains benefits. as opposed to parasitism or mutualism
you get results from a TSIA slant that is classic for salmonella, which shows a red/pink slant, a yellow butt and a black precipitate. what does the appearance of the TSIA slant specifically tell you about the organism
typical salmonella slants produces alkaline (red) slants and acid (yellow) butt, with or without production of H2S (blackened agar). meaning glucose fermentation is happening being a K/A or a K/A/H2S = acid, alkaline and hydrogen production
endospore
ultimate survival mechanism -allow cell to survive desiccation, nutrient deficiency, etc -formed by vegetative cell- a daughter cell within the mother cell -once conditions are favorable, an endospore can germinate into a new vegetative cell
how do we increase numbers of salmonella in contaminated food to detectable levels?
use a lactose broth -salmonella does not use lactose -fermenters do- lower pH -salmonella is acid tolerant -fermenters drop pH and eventually die, but salmonella lives
counting cells
use hemacytometer with newbauer grid. count all cells within a corner region of the grid - repeat for all 4 corners and for the middle
what does the citrate test verify?
verifies the ability of the cells to grow with citrate as the sole source of carbon and with ammonia as the sole source of nitrogen, a negative test means no growth. the medium contains an acid base indicator that turns blue in alkaline conditions. because fermentation of an organic acid salt such as sodium citrate causes the pH to rise, whenever cells grow and utilize citrate
mouth flora
very complex and variable population -create biofilms -differnet communities on teeth, tongue, and mucosal surfaces microbes between teeth: -streptococcus sp -enterrococcus s. -actinomyces -fusobacerium nucleatum
beta hemolysis: is a clearing of the blood surrounding a colony and occurs when the hemoglobin of the blood is destroyed by an exotoxin protein (hemolysin) produced by certain bacteria.
what type of hemolysis is evidenced on the plate below?
B. strict anaerobe C. facultative anaerobe A: strict aerobe D: microaerophile
what type of microbial respiration is demonstrated in this figure?
what are anaerobic conditions?
without oxygen, bottom of tube or being covered and anoxic
several drops of mineral oil are added to the top of one glucose O/F test media following inoculation in order to: a. test for the fermentation of the mineral oil b. maintain an aerobic environment c. maintain an anaerobic environment d. protect the bacteria from incubation
C. maintain an anaerobic environment inoculation on glucose oxidation/fermentation media is a standardized test known as the Hugh and leifson test. results from this indicate candidate organism for the microbial production of ethanol, or other desirable fermentation products (lactate, pyruvate, ethanol, CO2) initially both tubes are green, if tube with overlay turns yellow this indicates fermentation, if the open tube is yellow that means aerotolerant fermentor, but if tube remains green the microbe is an obligate anaerobic fermentor. if open tube is yellow but closed tube is green = oxidation metabolism with aerobic respiration.
you find an average number of colonies on three replicate plates is 150. the dilution factor was 10-3 and you used 0.1 mL to inoculate each plate. calculate the CFU per mL.
CFU = n / (D*X) n = the number of colonies per plate D= the dilution factor X= the volume of liquid inoculated on the plate in mL 150/ (0.001*0.1)= 1,500,000= 1.5 * 10^6 cells per mL
when would you use CFUs instead of direct counting?
CFUs (colony forming units) are used for viable bacteria- living things. it eliminates dead cells or debris. can also help differentiate between what is in the sample.
what are two characteristics that you can use to describe colony morphology?
Colony form: circular, irregular, filamentous, rhizoid Colony elevation: raised, convex, pulvinate (strongly convex), concave, flat, umbonate Colony margin: Entire, undulate, filiform, curled, lobate, smooth circular, saw-blade edged, gently curved edges Transparence: transparent, cloudy Surface: smooth, rough, wrinkled Color: Other optical properties: shiny, dull, opaque Physical consistency: soft hard, sticky, mucoid
what is the name of the simple stain that we used to help visualize our microorganisms via bright-field microscopy? A. methylene blue B. gram safranin C. seafoam green D. crystal violet
D. crystal violet simple staining techniques use general purpose stains. they allow the staining of all microorganisms that have been smeared on glass slides and allow easy observation by bright field microscopy use crystal violet but many stains can be used.
On an EMB plate, you count 20 dark purple colonies and 40 colorless/pink colonies. a. what percent of the colonies on this plate are Gram-negative? b. what percent of the colonies on this plate are lactose-fermenting coliforms?
EMB agar test is selective because it inhibits gram positive but allows gram negative bacteria to grow. it is differential because it differentiates lactose fermenting microbes (E. coli) from non-lactose fermenting microbes (salmonella) lactose fermentors = blue/black vigorous lactose fermentors = green sheen non lactose fermentors = colorless or pink the colonies on the EMB plates will determine the total number of gram negative cells. coliforms can be distinguished from non-coliform gram negative bacteria by lactose fermentation capability. dark green and blue-black colonies are lactose fermentors whereas colorless or pink colonies are lactose non-fermentors. A. 100 percent are gram negative B. 1/3 of the colonies are dark purple (lactose fermentors) (20+40=60), 20/60= 1/3
describe how the Eosin methylene Blue (EMB) is selective and how it is differential.
EMB is selective in that it inhibits gram positive growth but allows gram negative bacteria to grow. it is differential because it differentiates between lactose fermentors (dark blue) and non-lactose fermentors (colorless or pink)
differential and selective media
EMB, VRB, and TSA plates
Eosin methylene blue
EMB: inhibits gram positive, allows gram negative to grow -lactose fermenter or not lactose fermenter * count gram-negative spp. & lactose-fermenting coliforms
IMViC test
Indole Methyl red VP Citrate
deMan-Rogosa-Sharpe agar
MRS -selects for lactobacillus sp
what is the stain used for endospores?
Malachite green MG