Midterm Exam Labs I-VII Flashcards
brightfield microscope
-allows light rays to pass directly to the eye without being deflected by intervening opaque plate
stage
-horizontal platform that supports the microscope slide
stage adjustment
-clamping device used for holding and moving slide around on stage
neutral density filter
-needed to reduce intensity of light
ocular
- eyepiece
- 2 or more internal lenses and has mag of 10X
total magnification for compound microscope
-determined by multiplying power of ocular by the power of objective lens used
condenser
- located under stage
- collects and directs light from the lamp to slide being studied
- doesn’t affect magnification power
diaphragm
- within the condenser
- regulates amount of light that reaches slide
limit of magnification for most light microscopes
- 1000X
- set by intrinsic property of lenses=resolving power
resolving power
- ability to completely separate two objects in a microscopic field
- d=.5(lambda)/NA–>numerical apeture
numerical apeture
- describes how condenser lens concentrates and focuses light rays from light source
- maximized when light rays focused into a cone of light that then passes through specimen and onto objective lens
maximizing resolving power
- blue filter: shorter wavelength of resulting light provides max resolution
- keep condenser at highest position that allows max amount of light to enter objective lens and limit amount of light lost due to refraction
- diaphragm shouldn’t be stopped down too much
- immersion oil should be used for 100X lens because it forms a continuous lense system that limits loss of light due to refraction
parafocal and paracentral
- good quality microscopes are this
- which means image will remain both centered and in focus when changing from low power to high power objective lens
the resolving power of a microscope is a function of
-the magnifying power of the lenses and the wavelength of light
the coarse and fine focus knobs adjust the distance between
-the stage and the objective lens
a microscope that maintains focus when the objective magnification is increased is called
-parfocal
the most useful adjustment for increasing image contrast in low power magnification is
-closing down the diaphragm
before the oil immersion lens is rotated into place you should
-center the object of interest in the preceding lens and place a drop of oil on the slide
how do bacterial cells grow on a solid medium?
-as visible, discrete colonies
colony
- visible mass of cells usually resulting from the division of single cell
- can arise from more than once cell
advantage of agar media
- easily manipulated and not degraded by pathogenic bacteria
- solidifying agent
- could be added to rich broths to form a solid medium on which isolated colonies could develop after inoculation
- we usually use TSA (trypticase soy agar)
in what ways do the macroscopic features of bacterial colonies differ from those of molds?
-bacteria is smaller and round while molds are uneven in shape
why is the level of contamination measured as number of colonies rather than size of colonies?
-numbers show how many organisms were on a plate instead of how much grew from organisms on a plate
bacteria vs. eukaryotic microorganisms
-bacteria are smaller, have DNA not enclosed in nucleus, 70S ribosomes, have a cell wall composed of peptidoglycan, they lack mitochondria and chloroplasts but can still carry out respiration and photosynthesis and may have flagella that are simpler than those of eukaryotic cells but may also be more numerous
staining
- increases contrast between cell and surrounding medium, allowing observer to see more cellular detail, including some inclusions and various organelles
- usually results in cell death
- can also lead to undesirable artifacts
working distance
- distance between objective lens and slide
- decreases with increasing magnification
field of view
- area viewed through ocular
- decreases with increasing magnification
- center specimen before increasing magnification
best magnification for bacteria
-100X
best magnification for fungi
-10X or 40X
phase-contrast microscope
- able to differentiate transparent protoplasmic structures and enhance the contrast between a cell and its surroundings without staining
- used to view living cells and activities like motility
phase shift
- what happens when light in a microscope passes through a transparent object and is slowed down by 1/4 wavelength
- for a cell the phase shift without a reduction in amplitude results in the cell having a different refractive index than its surroundings
two types of light rays
-light rays passing through a transparent object emerge as either direct or diffracted rays
direct rays
- rays that pass through unaffected by medium
- unaltered in amplitude and phase
diffracted rays
- bent because they are retarded by the medium
- retarded 1/4 wavelength
coincidence
- direct and diffracted light waves brought into exact phase with each other, result is coincidence with the resultant amplitude of the converged waves being the sum of the two waves and
- increase in amplitude will result in increased brightness of the object in the field
interference
-two light waves of equal amplitude are in reverse phase (1/2 wavelength off), amplitudes will cancel out to produce a dark object
how do typical modern phase-contrast microscopes differ from conventional brightfield microscope?
- has a different type of diaphragm
- has a phase plate
diaphragm of phase-contrast microscope
-has annular stop that allows only a hollow cone of light rays to pass through the condenser to the specimen on the slide
how do you make a microscope function efficiently in both phase-contrast and brightfield situations?
- line up annular ring and phase rings so they are perfectly concentric
- adjust light source so maximum illumination is achieved for both phase-contrast and brightfield usage
- be able to shift back and forth easily between the two
light source adjustment for phase-contrast and brightfield microscopes
- blue light provides better images for both so make certain a blue filter is placed in filter holder or over the light source on the base
- more light required for phase-contrast than brightfield since so much light is blocked by annular stop
- evenness can be adjusted by removing lamp housing from microscope and focusing light spot on a piece of white paper
guidelines to be adhered to in all phase-contrast studies
- use only optically perfect slides and cover glasses
- be sure slides and cover glasses are clean
- use wet mount slides instead of hanging drop
- limit observations to living cells
bright phase microscopy
-making a bright image on a dark background that results from light rays in exact phase
dark phase microscopy
-making a dark image on a bright background that results from light rays in reverse phase
which two items can be used to check the alignment of the annulus and phase ring?
-centering telescope or optovar
why do we use aseptic technique?
- so we don’t introduce contaminating organisms
- insures organisms being handled don’t contaminate handler
- not contamination remains after working with the cultures
general aseptic procedure
- work area disinfected
- sterilization of loops or needles needed for inoculation
- inoculate media
- flame loop or needle again
- disinfect work area again
transfer from broth to broth
- heat loop
- shake tube to disperse organisms
- remove loopful of organisms
- cap removed from sterile broth and inoculated loop is inserted into sterile broth
- loop flamed
transfer from slant to slant
- heat loop
- pick up some culture
- smear culture in zig zag up sterile slant
- heat loop
transfer from agar to slant
- heat loop
- pick up organism of interest from plate
- transfer to slant in zig zag pattern
- heat loop
loops and needles
- loops used when streaking agar plates and slants
- needles used in stab cultures
pure cultures
- bacteria exist in natural environments in mixed populations so it is necessary to devise a way to isolate individual populations to obtain a pure culture
- consists of only one single kind of organism
- can study cultural, morphological, and physiological characteristics of an individual organism
streak plate
- involves diluting bacterial cells in a sample to an end point where a single cell divides giving rise to a pure colony
- used most often to obtain pure cultures
- take loopful and make dot
- make 6-7 lines radiating from that dot
- flame loop
- cool in center
- make 6-7 new lines in a new quadrant from end of last lines
- flame loop
- do this till you have four quadrants
- if colony arises not on streak lines there has been a contamination
pour plate
- involves diluting bacterial cells in a sample to an end point where a single cell divides giving rise to a pure colony
- inoculate bottom of plate with culture of interest
- pour agar over top
- swirl in small figure 8’s to mix
- multiple plates have been diluted to better obtain isolated colonies
subculturing technique
- want to take isolated colony and transfer it to a slant or broth to make a stock culture
- flame loop
- take small bit of isolated colony and only isolated colony
- inoculate slant or broth
- flame loop
what colony characteristics can be used for differentiation of bacterial species?
- color and shape
- i.e. serratia is gram negative rod while micrococcus luteus is gram positive coccus
why is dilution necessary for pure culture preparation?
-gives you isolated colonies
what advantage does streak plate method have over pour-plate method?
-streak plate is more economical in materials and time
what advantage does the pour-plate method have over streak plate method?
-requires less skill
before inoculating and pouring molten nutrient agar into a plate, why must the agar first be cooled to 50 degrees C?
-in order to avoid condensation and moisture
in which shapes of bacteria does motility occur?
-rods and spirochetes and almost never in cocci
flagella
- major organelles of motility in bacteria
- allow cells to move towards nutrients in environment or move away from harmful substances
- too small to be seen under light microscope
chemotaxis
-the complicated process that allows the flagellum to move
structure of flagella
- rigid filament that occurs in form of helix that constitutes main body
- filament connected to a hook that is attached to a shaft that’s inserted into series of rings whose number differs from gram positive to gram negative cells
- gram positive cells contain S and M rings situated in area of cell membrane
- gram negative cells have additional L and P rings associated with outer membrane and peptidoglycan of cell
- shaft, rings, and accessory proteins=basal body
proton motive force (pmf)
- rotates flagellum
- established when proteins associated with basal body transport protons across cell membrane creating a charge differential
- induces S and M rings to rotate which rotates shaft, hook, and filament
monotrichous (polar)
-on flagellum
lophotrichous
-multiple flagella from one side of cell
peritrichous
-flagella surrounding cell
amphitrichous
-multiple flagella on opposite sides
wet mounts
- can be used to determine motility
- drop of viable cells placed on slide and covered
- observed with phase-contrast microscope
- rapid swimming movement of cells in microscopic field conforms motility
- easily dry out due to evaporation
hanging drop technique
- can be used to determine motility
- drop of cells placed on cover glass placed over special slide with concave depression in center
- prevents evaporation
brownian motion
- caused by currents under cover glass
- motion due to molecular bombardment of cells causing cells to shake or jiggle but not move in any vectorial way
inoculating semi solid media
- can be used to determine motility
- agar concentration of .4% (1.5% normally) that doesn’t inhibit swimming
- stab semisolid media with inoculating needle
- motile organisms will swim away from line of inoculation into uninoculated surrounding media causing the media to be turbid
- nonmotile found only along line of inoculation
- has dye that turns red where there is growth
which bacterial species exhibited true motility on slides?
-proteus
which arrangement of flagella would you expect to be associated with highly motile species?
-monotrichous flagellar arrangement has less likelihood of tangling so would move greatest distance per unit time
directional motility
-directional movement that is several times the long dimension of the bacterium
water current movement
-all objects move in a straight line-not true motility
why are semisolid media sometimes preferred over slide techniques for evaluating bacterial motility?
-greater risk for contamination/infecting one’s self with a pathogenic organism when checking for motility
if SIM medium was used for motility determination for proteus vulgaris what noticeable change to the medium was observed?
-tube will turn black due to hydrogen sulfide
bacterial morphology
-look at figure 11.1 on page 93
true motility
-random movement in all directions
good smears are critical for discerning
- morphology of cells (rods, cocci, and commas)
- arrangement of cells (single cells, chains, bunches)
- internal structures (endospores, and cell inclusions
goals in preparing smears
- cause cells to adhere to slide so not washed off during staining
- make sure shrinkage doesn’t occur during staining
- prepare thin smears-thick smears trap stain and can obscure details about arrangement and presence of internal structures
steps in preparing smears
- if liquid source two loopfuls onto slide, if solid loopfuls of water on slide and then put source in to disperse organisms
- dry slide on heat thingy
- pass through flame 2-5 times to heat fix: kills cells and adheres it to slide
color changes ocurring at each step
-look at chart 14.1 on page 105
gram staining
- differential stain
- identifies gram positive (purple) vs. gram negative (red) by colors at end of process
- gram positive bacteria retain a purple dye complex whereas gram negative are decolorized and must be counterstained with red to be visualized
differential stain
-reactions take advantage of fact that cells or structures within cells display dissimilar staining reactions that can be distinguished by use of different dyes
primary stain
-crystal violet
mordant
- gram’s iodine
- fixes the stain into the cell wall
- combines with crystal violet and forms insoluble complex in gram-positive cells
decolorization
- alcohol
- gram positive cells retain crystal violet-iodine complex
counterstain
- safranin
- applied so that the decolorized gram-negative cells will be able to be visualized
peptidoglycan
- gram positive cells have thick layer that comprises the cell wall
- these thick tightly linked molecules in gram positive cells trap the crystal violet iodine complexes preventing their removal when correctly decolorized
- gram negative cells have much thinner layer of this and have lipopolysaccharide in their cell walls
gram-variable bacteria
-some bacteria are this because some cells retain the crystal violet stain while others won’t and will appear red
acid-fast bacteria
- unique cell wall made of waxy lipids
- cells may appear either nonreactive or gram positive
- but have a special acid-fast staining technique that can also be done
several factors that can affect outcome of gram staining procedure
- cultures must be 16-18 hours old: conversion from one to the other can happen if any older
- thin smears are critical
- decolorization is most critical step: if over applied dye-mordant complex can be removed from gram positive cells causing them to be incorrectly identified as gram negative
gram staining procedure steps
- apply crystal violet for 30 seconds
- wash for 2 seconds
- apply grams iodine for 1 minute
- wash for 2 seconds
- decolorize for 10-15 seconds or until solvent flows colorlessly
- wash for 2 seconds
- apply safranin for 1 minute
- wash for 2 seconds
- blot dry between two sheets of paper towel
working stock culture
- allows you to determine cell morphology, gram reaction of unknown, and whether culture forms pigment
- also presence of glycocalyx, endospores, or cytoplasmic granules
- use stocks as gram stain controls on unknowns
pleomorphism
- pertains to irregularity of form
- demonstrating several different shapes
metachromatic granules
-distinct reddish-purple granules within cells that show up with the organisms are stained with methylene blue
palisade arrangement
-pertains to a parallel arrangement of rod-shaped cells
endospores
- when species of bacteria belonging to genera Bacillus and Clostridium exhaust essential nutrients they undergo complex developmental cycle that produces these
- allow bacteria to survive environmental conditions that aren’t favorable for growth
- if nutrients become available the endospore goes through process of germination to form new vegetative cell and growth resumes
- not actively metabolizing very dehydrated structures that are resistant to heat, radiation, acids and many chemicals that would normally harm them
obligate (strict) aerobes
- metabolism requires oxygen to grow
- carry out respiration in which oxygen is used as terminal electron acceptor in electron transport chain
- ex: pseudomonas and micrococcus and many bacillus
- grow at top of tube
microaerophiles
- prefer oxygen of 2-10% necessary for respiratory metabolism
- ex: helicobacter pylori
- grow just below surface of tube
facultative anaerobes
- grow very well aerobically but can also grow anaerobically if oxygen isn’t present
- carry out respiration under aerobic conditions and ferment under anaerobic conditions
- ex: e. coli
- grow all along tube at any postion
aerotolerant anerobes/obligate fermentors
- can tolerate oxygen and grow in it’s presence but do not require oxygen for energy production
- use fermentation strictly
- ex: streptococci that produce food by fermenting, enterococcus faecalis, and streptococcus pyogenes
- grow all along the tube at any position
- have superoxide dismutase but lack catalase8
obligate (strict) anerobes
- cannot tolerate oxygen
- carry out fermentation/anaerobic respiration in which inorganic compounds replace oxygen in electron transport as terminal electron acceptor
- ex: clostridium, methanococcus, and bacteroides
- grow well below the surface of the tube near the bottom
most aerobes
- have systems that will convert toxic forms of oxygen to less harmful compounds
- catalase: degrade hydrogen peroxide
- superoxide dismutase will act on superoxide anion and convert it to oxygen and hydrogen peroxide
- these lack in obligate anaerobes
cultivation of obligate anerobes
- specialized conditions that eliminate oxygen and therefore its toxic forms
- anerobic incubators or anaerobic jars that employ chemical catalysts to eliminate oxygen
- can also be cultivated in specialized media that contain chemicals such as thioglycolate, which reacts with oxygen to create anaerobic conditions
candle jars
- enhance cultivation of some bacteria like streptococci that need less oxygen that what’s found in atmosphere
- CO2 increases to 3.5% and O2 decreases
FTM
- fluid thioglycollate medium
- rich liquid medium that supports growth of aerobic and anaerobic bacteria
- glucose, cysteine, and sodium thioglycollate to reduce oxidation/reduction potential
- contains resazurin (dye) that becomes pink in presence of oxygen so top will be pink
- contains small amount of agar that helps localize the organisms and favors anaerobiosis in bottom of tube
TGYA shake
- used to determine oxygen requirements of different bacteria
- inoculated in liquefied state, shake to mix organisms, and allowed to solidify
- after incubation oxygen requirements determined on where growth occurs in tube
Brewer’s Anaerobic Agar
- contains thioglycollate, a reducing agent, and resazurin (dye),
- to ensure oxygen free environment to incubate must use gaspak anerobic jar
gaspak anerobic jar
- generates hydrogen which removes oxygen by forming water
- palladium pellets catalyze the reaction at room temperature
- produces CO2
- indicator strip of methylene blue becomes colorless in jar in total absence of oxygen
psychrophiles
- optimal growth between -5 and 20 C
- supercooled waters of Arctic and Antarctic
mesophiles
- optimal growth between 20 and 50 C
- most bacteria
- most pathogens grow between 35 and 40 C
thermophiles
- optimal growth between 50 and 80 C
- occur in soils where midday temps can reach greater than 50 or in compost piles where fermentation activity can cause temperatures to exceed 60-65 C
hyperthermophiles
- growth optimum above 80 C
- many Archaea occupy environments that are heated by volcanic activity where water is superheated above 100 C
psychotrophs
- bacteria will be in only one of these classes but can grow at temperatures either higher or lower than their optima
- i.e. proteus, pseudomonas, campylobacter, and leuconostoc can grow at 4 C (refrigerator temperatures) and can cause food spoilage
- max temperature for growth is greater than 20 C
temperature effects on metabolic factors
- enzymes: have minimum, optimal, and maximum for performance. above maximum enzymes denature and can also happen below minimum
- cell membranes and transport affected: temperatures decrease, transport of nutrients into cell decreases due to fluidity changes in membrane. temperature increases above maximum, membrane lipids can be destroyed resulting in damage to membrane and death of organism
- ribosomes: increase in temperature and they cease to function
serratia marescenes
-produces optimal pigment at 25 C
pH effects on growth
- affects proteins and other charged molecules in cell
- each org has optimal pH
- if pH exceeds optimum for org, solubility of charged molecules can be adversely affected and molecules can precipitate out of solution
neutrophiles
- most bacteria are this
- grow at neutral pH
acidophiles
- bacteria that grow at acidic pH values
- ex: thiobacillus thiooxidans
- most fungi and yeast (4-6)
alkaliphiles
- bacteria that grow at basic or alkaline conditions
- found groing in environments such as soda lakes and high carbonate soils
- many bacillus are this
- some bacteria are alkaline tolerant (Alcaligenes faecalis)
uses of pH
- because it can influence or inhibit growth has been used in food preservation
- fermentation of foods can yield acids like lactic and acetic acid which lower pH thus preventing growth of many microorganisms and spoilage
- i.e. pickles, yogurt, some cheeses
- fungi are often what spoil acidic foods
water activity (A_w)
- growth of bacteria profoundly affected by availability of water in environment
- determined by measuring ratio of water vapor pressure of a solution to the water vapor pressure of pure water
- values vary between 0 and 1 and the closer the value is to 1, the more water is available to cell
- water activity and availability decrease with increases in concentration of solutes like salts because water becomes involved in breaking ionic bonds and forming solvation shells
- most bacteria grow best when water activity is around .9 to 1.
how would pH of culture medium be influenced by sugar fermentation? by urea hydrolysis?
- fermentation produces acids and would decrease pH
- urea hydrolysis produces ammonia which would increase pH
osmosis
- water diffuses from areas of low solute concentration where water is more plentiful to areas of high solute concentration where water is less available
- water naturally diffuses into cell
hypotonic
- medium where solute concentrations outside of cell are lower than cytoplasm
- generally don’t harm bacteria because rigid cell wall protects the membrane from being damaged by osmotic pressure exerted against it
isotonic
- environments where solute concentration is the same inside and outside the cell
- animal cells require this
hypertonic
- environment when solute concentration is greater on outside of cell relative to cytoplasm
- cell undergoes plasmolysis when this happens resulting in a loss of water, dehydration of the cytoplasm and shrinkage of the cell membrane away from cell wall
halophiles
- require high concentrations of sodium chloride to grow
- halophilic bacteria require 15-30% to grow and maintain integrity of cell walls
halotolerant
- capable of growth in moderate concentrations of salt
- ex: staphylococcus aureus (11%)
osmophiles
- able to grow in environments where sugar concentrations are excessive
- ex: xeromyces
complex media
- when we don’t know specific nutrient requirements needed to grow
- to cultivate need medium using rich extracts of meat or plants that would supply all amino acids, nucleotide bases, vitamins, or other growth factors
- exact composition and amounts of the components aren’t known
- ex: nutrient agar
defined medium
- specific chemical composition is known and individual components weighed out exactly
- used for organisms in which we know what specific nutritional requirements they need to grow
all medum need these
- carbon source
- energy source
- nitrogen
- minerals
- vitamins
- growth factors
- water
heterotrophs vs. autotrophs
- heterotrophs obtain carbon from organic compounds
- autotrophs derive carbon from fixing CO2
chemoorganotrophs
-derive energy needs from breakdown of organic molecules by fermentation or respiration
chemolithotrophs
-oxidize inorganic ions to obtain energy to fix CO2
photoautotrophs
-contain photosynthetic pigments such as chlorophyll or bacteriochlorophyll that convert solar energy into chemical energy then used to fix CO2
photoheterotrophs
-derive energy requirements from photosynthesis but carbon needs from growth of organic molecules such as succinate or glutamate
selective medium
-media made with components that will allow certain bacteria to grow but will inhibit others from growing
differential medium
-contains substances that cause some bacteria to take on appearance that distinguishes them from other bacteria
autoclaving
- heating media to 121 C for 15 minutes at 15 psi
- sterilizes media
techniques in counting bacteria
- microscopic counts
- most probable number (MPN)
- standard plate count (SPC)
microscopic counts
- sample diluted then counted with microscope
- determines both living and dead cells
most probable number
- determined by relationship of some growth parameter to statistical probability
- drinking water uses this technique by using tubes that show acid and gas that are then compared to statistical tables that give numbers of coliforms present
- specific conditions in media used which may exclude certain bacteria in counts
coliforms
- found in intestines of humans and warm-blooded animals
- ferment lactose and produce acid and gas
- presence in water suggests potential for disease
- gram negative, non endospore forming, facultative anaerobic, rods, ferment lactose to produce acid gas in 40 hours at 35 C
- lactose fermentation with the formation of acid gas provies the basis for determining total coliform count
standard plate count
- viable count
- diluted sample in series of dilution blanks plated onto media and numbers of colonies counted after incubation
- assumed that bacterial cells diluted to end point where single cell divides giving rise to visible colony on plate
- number of bacteria in original sample determined by multiplying the number of colonies by the dilution factor
- reported as colony forming units (CFUs)
- greater than 300, overcrowding inhibits growth, less than 30, sampling error
indirect methods
- growth can be related to some parameter that increases with cell division
- growing cells increase protein, nucleic acid content, and mass
- thus measurements of protein, DNA and dry weight can be used to monitor growth
- turbitidy can also be determined and related to growth
cell turbidity
- can be measured in spectrophotometer which measure absorbance or optical density
- living and dead cells contribute
microorganisms in food
- doesn’t mean it’s spoiled
- some used in production of certain foods (yogurt, sauerkraut, summer sausage) by microbial fermentation
- pasteurization or smoking significantly reduce amounts in end product
during processing and preparation
- foods can become contaminated with bacteria
- not necessarily harmful or pathogenic
- naturally associated with some foods when harvested (potatoes, beets, green beans)
- even after washing some may exist and be preserved when food is frozen
thermoduric bacteria
-bacteria that that survives high temperatures (as in pasteurization) and goes on to spoil the food
bacteria in hamburger
-many are harmless saprophytes that come from environment where processing occurs
transmission of disease via food
- 76 million become sick, 300,000 hospitalized and 5,000 die from foodborne illnesses
- foodborne illnesses usually result because pathogenic bacteria or their toxins are introduced into food products during processing, handling, or preparation
botulism food poisoning
-ingestion of toxin clostridium botulinum whose endospores grow in improperly home-canned food
during processing and preparation
- foods can become contaminated with bacteria
- not necessarily harmful or pathogenic
- naturally associated with some foods when harvested (potatoes, beets, green beans)
- even after washing some may exist and be preserved when food is frozen
coliform counts
- one method to ascertain if food is contaminated with fecal bacteria and has potential to spread disease
- coliforms are organisms like e. coli that occur in the intestines of humans and warm blooded animals
- presence in food or water indicates that fecal contamination has occurred and that there is a high potential for spread of serious disease
high coliform counts indicate potential for finding which intestinal pathogens
-e. coli, salmonella, shigella, vibrio cholerae, and intestinal viruses
why is e. coli a good indicator of fecal contamination and a good test organism?
- occurs primarily in intestines of humans and some warm blooded animals
- not found routinely in soil or water
- can be easily identified with microbiological tests
- not as fastidious as other intestinal pathogens and thus survives a little longer in water samples
e. coli
- found in intestines of cattle and can become associated with meat if fecal material from the animals’ intestines contaminates meat during butchering
- serious illness results from eating improperly cooked hamburger because cooking temperatures are insufficient to kill the organism
coliform counts
- one method to ascertain if food is contaminated with fecal bacteria and has potential to spread disease
- coliforms are organisms like e. coli that occur in the intestines of humans and warm blooded animals
coliforms on EMB agar
- gram negative lactose fermentors form small colonies with dark centers (nucleated)
- e. coli give green metallic sheen
coliforms on endo agar
- produce reddish colonies 43
- e. coli give green metallic sheen
tests to determine coliform count
- presumptive test
- confirmed test
- completed test
IMViC tests
-distinguishes if coliform present in completed test is e. coli or e. aerogenes
confirmed test
- plates of levine EMB agar or Endo agar inoculated from positive tubes to see if organisms that are producing the gas are gram-negative
- these media inhibit growth of gram positive bacteria
completed test
- concern is to determine isolate from igar plates truly matches definition of coliform
- if gas produced in lactose tube and slide from agar slant reveals gram negative, non-spore-forming rod can be certain it’s a coliform
IMViC tests
-distinguishes if coliform present in completed test is e. coli or e. aerogenes
what might cause a false positive presumptive test?
-two or more non-coliform bacteria working synergistically to produce sufficient gas in the tube
baking sterilization
-oven, dry heat, 180 C for 2-3 hours, glassware
filtration sterilization
-filter, heat sensitive solutions
chemical methods of sterilization
-phenols, alcohols, halogens, quaternary ammonium compounds, aldehydes, ethylene oxide gas
radiation sterilzation
-UV at 260 nm, gamma radiation from cobalt 60-plasticware, antibiotics, vitamins, and food
EMB agar
-contains methylene blue which inhibits gram-positive bacteria
endo agar
-contains fuchsin sulfite indicator that makes identification of lactose fermenters easy
