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microbial growth in a liquid medium
turn turbid (cloudy).
(No colony shown, not sure if bacterial/fungal)
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differentiate bacterial from fungal growth on a solid medium.
bacterial is more # than fungal,
bacteria is smaller than fungal,
bacteria look wet & fungal looks dry
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how a cell is related to a colony.
One cell produce 2, 4, 8 that creates a mound of cell (colony)
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Tube with liquid growth media with microbes growing is
culture
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mound of bacteria on petri dish with many cells are
colony
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Negative pressure room vs positive pressure room
negative- keep pathogen in (may have mold)
positive- keep pathogen out
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loop or needle when transferring bacteria from one medium to another.
Loop- remove bacterial from liquid
Needle- remove from solid culture
Doesn’t matter the medium
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pure, mixed and contaminated culture
Pure- container growing single species of microbbes whose identity is known
Mixed culture- container growing 2 or more different known species of microbs
Contaminated- Medium that once held a pure (single or mixed) culture but now contains unwanted microorganism
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advantage and a disadvantage for streak plates versus loop dilutions
Streak plate
advantage- few minutes, single plate media
Disadvantage- skill required
Loop
Advantage- good result
Disadvantage- more time and material
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if the agar is cut during the course of streaking the plate.
Would not grow on top of agar
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loop dilution temperatures which agar melts, solidifies when bacterial cells may be added to warm agar.
Agar melts at 100C,
solidify at 42C
Bacterial cell can be added at 50 C agar
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what a subculture is
make a second generation culture from well established colony of organisms
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Color of Ecoli, Micrococcus luteus, Serratia marcescens
Ecoli -white
Micrococcus luteus- yellow
Serratia marcescens- red
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laboratory technique that exclude unwanted microbes are called
asceptic
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three different isolation techniques
streak plate- require skills
loop dilution-require material and time
spread plate- sample need dilution or medium is selective
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three most commonly encountered microbial morphologies
Cocci, bacilli/rod, spirilla
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morphology & arrangements
Morphology- study of organism structure
Arrangement- manner which bacterial cell associate with one another, by product of cell division
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Most common arrangements for both cocci and rods
Cocci arrangement -
Single plane- diplococcus(2) and streptococcus
2 perpendicular plane- tetrad(4) and sarcina (packet)
Several plane- staphylococcus and micrococcus
Rod arrangement- division along transverse plane
Diplobacilli (2)
Streptobacilli(line)
Palisade (next to each other)
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how the contrast between bacterial cells and their background is increased
Through stain, ionic interaction between chromophores and cells create coloring
Bacterial cells are transparent
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negatively stained would differ from positively stained.
Negative stain= dark background (stain is repelled by negative bacteria)
Simple stain= stain cell (positive stain stain negative bacteria)
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two negatively charged (acidic) stains and three positively charged (basic) stains.
Negative acidic stain= nigrosin and india ink
Positive charged basic stain= methylene blue, crystal violet, malachite green, safranin
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advantage negative staining has over simple staining
Negative stain--no heat so size of cell same
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information that can be obtained from either negative or simple staining.
Size, shape, arrangement
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how a simple stain differs from a differential stain.
Differential stain tells structural differences like gram positive and gram negative with outer membrane
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Gram stain, primary stain, mordant, decolorizer and counterstain.
Primary stain- crystal violet, staining
Mordant- grams iodine, fix a dye by creating insoluble complex in thick peptidoglycan of gram +
Decolorizer- ethyl alcohol, dissolve lipid in outer membrane of gram -
(clear)
Counterstain- safranin- increase contrast of gram -
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why the time spent decolorizing a smear is critical to the outcome of the stain.
Alcohol too long- remove crystal violet from gram + and turn pink
Alcohol not long enough- gram - will appear purple
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color of gram negative vs gram positive
Gram neg = pink
Gram positive purple
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three factors that may affect the outcome of Gram staining
Young culture should be used ( old may stain - due to peptidoglycan layer)
Smear must not be too thick (can trap violet leading to false positive)
Decolorize right time (too long will remove all gram + and become pink, too little gram neg will be purple)
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shape and Gram of Ecoli, S aureus, L innocuous, B megaterium
Ecoli- Rod, gram - (pink)
Staphylococcus aureus- cocci, gram +
Listeria innocuous- rod, gram +
Bacillus megaterium- rod, gram +
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what refers to arrangement of bacterial cell
palisades
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steps in gram stain
crystal violet 30 sec
wash 3 sec
grams iodine 1 min
alcohol 8 sec
wash 3 sec
safranin 1 min
wash 3 sec
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S aureus after mordant, decolorization color
S aureus is gram + so all purple
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Ecoli after crystal violet, grams iodine, ethyl alcohol, safranin
crystal- purple
gram iodine- purple
ethyl alcohol- clear
safranin- pink
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Endospore, bacterial genera, advantage
-small dormant, resistant derivative of a bacterial cell that germinates under favorable growth conditions into vegetative cell.
-Bacterial genera bacillus and clostridium are typical spore formers
-Heat, cold, chemical resistant to preserve genetic material
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why endospore forming bacteria are difficult to stain
Hard to stain because protective nature of endospore prevent dye penetration
-Heat used as mordant to drive primary stain into endospore, decolorized with water to remove green from vegetative cell but not endospore, safranin used to counter stain vegetative cell and sporagium
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two genera of endospore forming bacteria and disease they cause
Bacillus anthracis- anthrax
Clostridium tetani- tetanus
Clostraidium botulinum- botulism
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names of the primary stain, counterstain and decolorizing agent for endospore staining.
primary stain- malachite green and heat (30sec)
decolorizing agent- water (30 sec)
Counterstain- safranin (30 sec)
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Result for bacillus megaterium endospore staining
green endospore with pink bacilli
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what color is endospore? in endospore stain
green, vegetative cell pink
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Endospore stain is what kind of stain
structural
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color of endospore and nonendospore in each reagent
Endospore
Heat fix- clear
malachite green + heat- green
water green
safranin- pink + green
Nonendospore
Heat- clear
malachite green + heat- green
water- clear
safranin- pink
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why older culture used for endospore stain?
young may not form endospore cuz vegetative cell not subjected to stress to form sporulation
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endospore stain on bacillus megaterium but forget to add heat
no stain
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primary genus of bacteria identified using an acid-fast stain
Genus Mycobacterium
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diseases caused by the two primary pathogens in the genus. of acid fast
Mycobacterium. tuberculosis - tuberculosis (TB)
Mycobacterium. leprae -leprosy
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mycolic acid? and how this compound interferes with the staining process
-compound found in the cell wall of acid fast bacteria
-Prevents most stain from penetrating (light purple when gram stained)
Heat is used with carbol fuschin
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acid-fast
referring to the property of mycobacteria to retain carbol fuchsin even in the presence of acid alcohol. Used to diagnose tubercolosis
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acid-fast and non-acid fast cell look like after the staining process.
Acid fast- purple
Non acid fast- blue
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procedure for acid-fast staining of the primary stain, counterstain and decolorizing agent,
2 loop S. aureus and little M smegmatis, break with needle, air dry and heat fix
Flood with carbol fuschin
Microwave 30 sec
Decolorize with acid alcohol 8 sec (remove color from non acid fast
Rinse with water
Counterstain methylene blue 30 sec
Rinse
Blot
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acid-fast cell and a non-acid-fast cell would look like at each point in the staining process.
Acid fast
Heat fix (clear), carbolfuschin (purple), acid alcohol (purple), methylene blue (purple)
Non acid fast
Heat fix (clear), carbolfuschin (purple), acid alcohol (clear), methylene blue (blue)
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what kind of stain is acid fast
differential stain
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differences between a wet mount and a hanging drop slide and the advantages a hanging drop slide offers.
Wet mount- liquid on slide covered with slip
Hanging drop slide- cover glass with concave slide
Advantage- no drying, no water currents, reduce contamination
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how to prepare wet mount slide
2 loop of culture on slide with cover glass on top,
low light.
Can’t use oil,
dispose in biohazard
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how to prepare hanging drop slide
petroleum jelly on cover glass,
liquid bacteria on glass,
depression is facing downward and cover glass is on top, low light focus on edge of drop,
depression slide in disinfectant
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Brownian movement how it differs from true motility
passive nondirectional motion by microscopic particles. From bumped by water molecule, visible particles are suspended
True motility- runs(straight line) tumbles (change in direction)
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how motility media is used to determine motility.
Semisolid medium is inoculated with stab.
If restricted to stab, nonmotile,
if away from stab, motile
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evaluate a tube of motility media and determine motile or non-motile.
cloudy= motile, line = nonmotile
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results for Micrococus luteus & Proteus vulgaris motility
M luteus- non motile
P. vulgaris -motile
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advantages and disadvantage of motility medium and hangdrop
motility medium- less likely to dry, more observation time
hang drop- no incubation needed
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difference between complex and defined media.
Complex media- one or more ingredient are not precisely known (extract of animal, plant, yeast)
EX: trypticase soy agar and nutrient broth
Defined media- precisely known chemical composition
EX minimal agar
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importance of carbon, energy, nitrogen, minerals, vitamins, and growth factors in growth media.
Carbon- backbone of all organic molecule
Energy- required to assemble raw material found in media into biomolecule
Nitrogen- synthesis of amino acid, nucleotide (meat and peptones used)
Mineral- cofactor or enzymatic reaction. Part of structure of cytochromes, bacteriochlorophyll and vitamins (meat and yeast)
Vitamins- cofactors in enzyme catalyzed reaction (meat and yeast)
Growth factor- for growth of fastidious organism
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differentiate between autotrophs and heterotrophs,
Autotrophs-use C02 as carbon source
Heterotrophs- obtain carbon from organic compound, carb and protein
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Chemoorganotrophs
Chemoorganotrophs- obtain energy from organic molecule by fermentation/respiration
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Chemolithotrophs
Chemolithotrophs- obtain energy from inorganic ion, oxidizing inorganic substrates like sulfur or iron
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Photoautotroph
obtain energy through photosynthesis and carbon from C02
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Photoheterotroph
light as energy carbon is through organic molecule like glutamate
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selective media
Selective media- one or more ingredient that inhibit growth of most bacteria, only single group to grow
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example selective media
EMB(eosin methylene blue) agar inhibit gram + bacteria
7.5% sodium chloride in mannitol salt agar selects for staphylococcus but inhibit other bacteria
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differential media
Differential medium allow all species to grow but has special ingredient so some bacteria appear different.
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differential media example
Staphylococcus aureus on mannitol salt agar, ferments media to produce acid, turn yellow
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why is agar better than gelatin?
Media use agar because few bacteria can use it as nutrient
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how media is prepared from dehydrated powder, raise or lower the pH and how long media is generally autoclaved.
# of container X volume = milliters needed
Calculate powder needed for 1 L weigh powder,
If contain agar- boil and add extra water to balance evaporation
PH is low use NaOH 1N OR 0.1nN
PH is high use HCL
Autoclave immediately
Steralization at 121C 15lb per square inch psi
Small load 15 min
Full autoclave 30 min
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raise or lower the pH for prepared from dehydrated powder
PH is low use NaOH 1N OR 0.1nN
PH is high use HCL
