Micro Lab Final

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medium

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mixture of nutrients
different orgs. require diff nutrients

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synthetic media

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composed of entirely of known chemically pure org/inorg compounds

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defined media

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composed of entirely known chemically pure org/inorg compounds
can be duplicated exactly

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complex media

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has rich assortment of soluble org/inorg compounds that meet requirements of growth of many orgs.
exact chem composition not known

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agar

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complex polysaccharide, solidifying agent
ADVANTAGES
1. mp ~ 88 C, solidify ~ 40 C
2. not used as a nutrient so it does not liquefy
3. high degree of transparency

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gelatin

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solidifying agent
DISADVANTAGES
1. melting point is 30 C
2. used as a nutrient by many microorg
> liquefaction

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selective media

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dyes, antibiotics and other chemicals added to a medium to inhibit the growth of certain types of microbes and permit the growth of org of interest

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differential media

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media has dyes or indicators that give some microbes easily recognizable characteristics

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enrichment media

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media has special substances that enhance the growth of a partic type of microbes, usually fastidious

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good smear

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1. has appropiate thickness to view indiv cells, monolayer
2. withstand repeated washings
3. cells will retain their original shapes after fixation and staining

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Heat fixing

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1. kills cells
2. the coagulation of cell protoplasm causes cell to adhere to slide during staining process

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simple stain

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bacteria have net neg charge at pH 7 so use positive basic dye
safranin o, crystal violet, methylene blue, basic fuschin

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bacteria genus produce endospores

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Bacillus
Clostridium
Sporolactobacillus
Desulfotomaculum
Sporosarcina
B,C,D,S, S

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endospores

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dense multilayered structures, a means of survival under adverse enviro conditions(heat, freezing, chem agents, desiccation, radiation), one endospore per cell
formed by sporogenesis.
germinate and give rise to vegetative bacterial cell
water content is lower, has dipicolinic acid & calcium
heating increases permeability of spore

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endospores features

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size:
shape: spherical, oval, cylindrical
position: terminal, subterminal, central

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Spore stain 1st stain

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malachite green

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spore stain decolorizer

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water

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spore stain counterstain

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safranin

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spore stain results

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spores: stained green
vegetative cells: red

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Gram stain factors

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1. smear preparation : if smear too thick will not decolorize properly, if overheated or heat fixed before aire dried cause cell wall rupture
2. concentration and freshness of reagents may affect the quality of the stain
3. excess water on slide will dilute reagents
4. reliable on cells from cultures in the exponential growth phase of growth
5. pH of culture medium affects gram rxn, ^ acidity gram positive > negative, ^ alkaline gram neg > gram pos

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acid fast stain

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Mycobacterium
for acid fast bacteria with mycolic acids in the cell wall
the lipids give waxy properties and makes difficult to stain
heat softens lipids of walls to make more penetrative
resist decolorization by alcohol

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Ziehl-Neelsen method

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acid-fast
apply heat to soften waxy cell wall to allow basic fuscin to penetrate
acid alcohol
counterstain w. methylene blue

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acid fast 1st dye

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basic fuschin

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decolorizering in acid fast

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acid alcohol

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counterstain acid fast

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methylene blue

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acid fast results

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fuscia - acid fast
blue - non acid fast

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fluorochromes

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staining methof detects acid-fsat cells
dye molecules emit visible light when stimulated w. ultraviolet light of a specific wavelength

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optimum temperature

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narrow temp range where most rapid growth occurs
~ temp of normal habitat

from 24 hr obs

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cardinal temperature

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min/max and optimum temp for growth

min/max from 72hr obs

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temperature ranges

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psychrophiles opt <15
mesophiles opt 25 - 40
thermophiles opt > 45 (50 - 60)
hyperthermophiles opt > 80

psychroduric & thermpduric - endure a temp
psychrotrophic - NL prokaryotes (btn psychrophiles and mesophiles) opt ~ 20 C

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temperature influence

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effect on cellular enzymes
function slowly at temps below optimum, decrease chem rxn rates, ^ viscosity of fluid and hardening of lipids
high temps cause denaturation of cellular proteins/enzymes and separation of DNA strands

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sterilization

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complete destruction of all microorganisms associated with a material or article
accomplished by physical and chem means
methods: heat, filtration, gas, radiation

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chemical sterilization disadvantage

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leaves residue of the germicide and are less effective against endospores

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sterilization methods

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Heat : [BADF] flaming, dry heat, boiling/ free flowing steam (Arnold sterilizer), Steam under pressure
Filtration: Diatomaceous earth (Candle), Sintered glass (Morton), Asbestos (Seitz), Membrane (Millipore& Nucleopore)
Gas: Ethylene oxide, B-propiolactone, formaldehyde

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flaming

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sterilize instantaneously
heated to red hot

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dry heat

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requires high temp (160 -180 C) for 2 - 3 hours
dry heat dehydrates cell and destroys by oxidation of intracellular constituents
used for : glass, mineral oil, metal ojts
DISADVANTAGE:
long time
penetration of pourous material is very slow
high temp can damage

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Boiling (moist heat)

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for glassware and some culture medium (carbs, gelatin)
greater penetration than dry heat
kills cells by coagulation of proteins in protoplasm
DISADVANTAGE:
temp never rise above 100 C .: cant kill endospore

Arnold sterlizer (free flowing steam)

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Free flowing steam

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aka Arnold sterilizer accomplishes fractional sterilization , Tyndallization
steam for 20 mins 3 or 4 times separated by 24 hrs
intervals allow endospores to germinate and become vunerable to steaming at 100 C

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Steam Under Pressure

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aka Autoclave
15 pounds of pressure / sq inch
rises temp to 121 C
All air is evacuated and replaced by compressed steam
Sterilization in 15-20 mins (30-35 mins to heat up/cool)
used for culture media, glass, dilution blanks , discarded cultures

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kilit ampule

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tests if autoclave functions properly
sealed vial w. endospores of Bacillus stearothermophilis in nutrient broth + pH indicator BROMCRESOL PURPLE
put in autoclave and than incubated at 55 C, if autoclave works no change, purple
if autoclave does not work, medium becomes turbid, yellow (low pH from acids produced)

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Kilt results

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purple - autoclave works and endospores were killed
yellow - autoclave not functioning properly or not exposed long enough

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filtration

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used for sterilization of solns or media which cant be heated w/o altering their physical/chem means
[Cant See Me]
candle, Seitz, Morton
positive charge surface of filter psg adsorbs neg charged bacteria

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Candle filter

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diatomaceous earth compressed in cylinder

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Seitz filter

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pad of asbestos fibers in silver unit

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Morton Filter

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sintered glass filter

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millipore

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membrane filter (cellulose acetate)
diameter of pore 1 um to lesss than .005 um

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nucleopore

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polycarbonate
membrane filter

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gas sterilization

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gas agents sterilizate materials
used for plastics, petri dishes
ethylenes oxide, B-priopiolactone, formaldehyde
kills by alkalization of proteins and nucleic acids
can kill spores

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ethylene oxide

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depends on temp, humidity, and concentration of ethylene oxide
ADVANTAGES
good penetration
broad spectrum of activity
effective at low temps
does not damage materials exposed to it
DISADVANTAGE
takes long time 8 - 12 hours, plus wait for it to dissipate
in presence of O2, very flammable and explosive
toxic and residues are mutagenic

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control

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shows org are viable to grow under inoculation condition

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UV

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EMR that fall within wavelength 380 nm - 15 nm, practical range is 200nm- 380nm, 260 nm- 265nm is most potent
extreme range (15nm -200nm) are absorbed by air and unction only in vacum, "vacum ultraviolet"
direct, intense, for long
has low penetrating power
affects DNA; absorbed by N.A & proteins, damage pymaridine bases forming thymine dimers. suppresses DNA and cause mutations (mutagenic effect)
(DNA has repair enzymes and mechanisms to enable growth)
photosynthetic bacteria not ffected, use as energy source

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antibiotics

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antimicrobial agents that inhibit the growth of, or kill other microbes
exhibit selective toxicity
ADVANTAGE
reduces competition for space, nutrients...

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Antibiotics requirements

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1. sensitivity of infecting microorg to the antibiotic
2. stability of antibiotic in vivo
3. lack of toxic effects in patient
4. lack of allergic rxns in patient

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semisynthetic

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antibiotics chemicially modified forms produced originally by microorgs
synthesized the biochem structures of most naturally occuring antibiotics

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disc-plate method

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method to determine sensitivity of org to antibiotics
filter-paper disc w. defined concentrations of antibiotics is place on surfce of agar inoculated w. test org
antibiotic diffuses from disc into the agar medium
zone- of inhibition surrounded disc shows suseptability of org

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growth-inhibition zone size

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1. concentration of antibiotic disc
2. rate of diffusion of antibiotic into surrounding
3. sensitivity of org to antibiotic
4. growth rate of org
5. viscosity/density of medium
6. suitability of enviro conditions for growth of org

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Fungi needs

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need more sugar, less pH
SAB plates

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penicillin G

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affects gram + bacteria
inhibits cell wall synthesis by preventing synthesis of peptidoglycan
cell wall weakened > cell lysis

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Tetracycline

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affects gram + and -
inhibits protein chain elongation
interfers with attachment of tRNA to mRNA- ribosome complex
binds to 30S ribosome subunit of mRNA translation complex

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Streptomycin

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changes shape of 30S portion so mRNA is read incorrectly
inhibits protein chain synthesis

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Nystatin

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affects fungi
bind to ergosterol in cell membrane and makes pore which cause K+ leakage

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flagella

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organelles of locomotion
diameter of bacterial flagella (20nm) is below limits of resolution on light microscope, need special staining where stain is deposited on flagella to increase their diameter

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Brownian motion

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vibratory movement in a limited area due to molecular bombardment

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true motility

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definite and continuous movement in a given direction

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flagella class

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peritrichous- uniformly distributed
monotrichous- 1 at 1 pole
lophotrichous- many at 1 pole
amphitrichous- many at each pole

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monotrichous

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one flagella at one pole

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peritrichous

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flagella uniformly distributed
i.e Pseudomonas vulgaris

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amphitrichous

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several flagella at each pole

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lophotrichous

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many flagella at one pole of cell. Pseudomonas fluorescens

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flagella movement

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vibration
rapid, darting zigzag : polar
tumbling motion, straight line, tumble : peritrichous

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methods for determining motility

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hanging drop method
phase contrast
motility test media

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isolate pure culture

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progeny of single cell
done by spread, pour, streak plate

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streak plate

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dilution process used to obtain pure cultures
each section of streaked plate becomes successively less populated with cells
depostit indiv cells far enough part so that each cells grows into isolated colony
ASSUME: one colony arises from one cell

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fastidious

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orgs are highly restrictive, and only grow if certain complex org materials are supplied

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nutrition

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can use a given nutrient only if microb has specific enzyme system
extensive enzyme systems allow use of variety of org/inorg compounds and enables them to synthesize vitamins and other essential growth factors
essential macronutrients : C, H, N, O, P, S, K, H20 and energy

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heterotrophic

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unable to use CO2 as C source or atm N as N source
C and N must be provided in medium

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yeast extract

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source of org nutrients and rich in B vitamins

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peptone

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org C and N and good source of AAs
small amounts of vits and carbs

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sterile saline

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growth cultures are centrifuged and resuspended in sterile saline
removes many nutrients from original growth medium to minimize growth carry over of nutrients
.: growth is due to nutrients supplied by media

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oxygen requirements

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obligate aerobes- grow only in presence of free O2, enzyme sys requires O2 is terminal e acceptor

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obligate anaerobes

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grow only in absence of O2, O2 is lethal

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falcultative aerobes

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grow in presence or absence of O2
more efficent if O2 is present

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microaerophiles

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require low concentration of O2, O2 is required as terminal e acceptor

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aerotolerant anaerobes

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grow in presence or absence of O2
does not use O2 as terminal e acceptor

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special techniques for obligate anaerobes

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O2 must be excluded from enviro
mechanical exclusion of O2
use of reducing agents substrates that combine w. free O2
1. anaerobic jar
2. GasPak Pouch
3. Bray dish

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anaerobic jar

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sachet contains dry chemicals and ascorbic acid which is activated w. exposure to air, placed in jar w. indicator strip
jar is tightly sealed to prevent entry of atm O2,
O2 > CO2
white indicator = no O2 present

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anaerobic indicator strip

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pad saturated w. methylene blue soln
colorless in absence of O2
green/blue color in presence of O2

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GasPak Pouch

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create anaerobic enviro
transparetn plastic GasPak Pouch and reagent sachet w.inorg carbonate, activated C, ascorbic acid and H2O methylene blue indicator tablet
produces CO2

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Bray dish

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chems pyrogallol and sodium carbonate
inoculated agar plate is placed on Bray dish and sealed
absorption of O2 and release of CO2

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Thioglycollate broth

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determine O2 requirements of bacteria
nutrient broth w. sodium thioglycollate, a strong reducing agent that inds free O2, and agar to slow diffusion of O2 through medium
methylene blue indicator, O2 green color, yellow no O2

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deep tubes of agar media

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inoculated while agar is still molten and bacteria can be distributed throughout media
position of growth indicates O2 requirements

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rxns depend on

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temp, pH, enzyme concentrat, substrate concentration, ezyme affinity for substrate

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extracellular enzyme

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exoenzyme
secreted from cell wall and function in enviro, break down complex nutrients in the enviro by hydrolysis

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intracellular enzyme

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endoenzyme
function within the cell
responsible for production of energy for cell and synthesis of protopasmic requirements of the cell

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casein hydrolysis

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casein is protein in milk
ests if bacteria produce proteolytic exoenzyme CASINASE, which hydrolyses casein to produce soluble and transparent derivatives (peptides and AAs)
positive test: clear zone around growth

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indole production

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tests if bacteria produce tryptophanase which breaks tryptophan into indole, pyruvate (> glycolytic or Kreb's cycleto produce CO2, H2O and energy) and ammonia ( > make AAs)
indole detected by KOVAC's reagent and HCl.to form rosindole red dye layer

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Urea hydrolysis

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tests for enzyme UREASE which hydrolyses urea to form ammonia (makes alkaline, ^ pH) and carbon dioxide
phenol red pH indicator (yellow/orange) 6.8- 8.1 (purple)
positive test: urea slant color change from yellow orange > purple

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lipid hydrolysis

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tests for exoenzyme lipase which hydrolyzes lipids into glycerol and FAs
FAs lower pH causing dark color change around growth
positive test:Spirit blue agar darkens around growth

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starch hydrolysis

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test for extracellular enzyme alpha-amylase which hydrolyses starch into maltose, glucose and dextrins
plate is flooded w. iodine soln on starch agar plate (starch forms dark brown absorption complex w. iodine)
positive test: where starch was hydrolyzed, medium color will be yellowish brown like iodine

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citrate utilization

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used as only C source on Simmon's citrate agar
tests for enzyme CITRATE LYASE which require a divalent cation Mg/Mn for activity
breaks citrate > oxaloacetate (forms pyruvates and CO2) annd acetate
pH indicator bromthymol blue : geen a neutral pH > blue at alkaline pH
positive result: growth on medium, medium color change from green to blue (sodiumcarbonate)

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respiration metabolism of glucose overall rxn

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glucose + 6O2 + 38 ADP + 38 Pi >>> 6CO2 + 6H2O + 38 ATP
aerobic

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respiration pathway phases

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1. glycolysis
2. Kreb's cycle
3. oxidative phosphorylation

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fermentation

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does not invlve oxidative phosphorylation
anaerobic process
requires org compound as terminal e acceptor

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pyruvate

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intermediate in glucose degradation
made by glycolysis

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fermentation broth

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fermentation broth is nutrient broth w. single carb source and pH indicator phenol red
Durham tube traps gas bubbles = gas production

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carb fermentation test

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produce acidic end products from carb fermentation causes phenol red pH indicator to change color from red > yellow
grow in fermentation but do not use carb > turbidity w. no color change/gas
tests for diasccharide-splitting enzymes sucrase(sucrose), lactase(lactose), maltase(maltose) than fermentatino of monosaccharides
acid production: color change from red to yellow and turbid
gas production: gas buble in Durham tube

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methyl red test

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mixed acid fermentation of glucose yield acids; lactic, succinic, acetic, formic (pH decreases to below 4.5)
positive test: bright red color
use pH indicator methyl red

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Vogues-Proskauer test

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tests for fermentation of glucose by butylene glycol fermentation
forms intermediate ACETOIN which reacts w. alpha-naphthol and KOH. ACETOIN is oxydized to form diacetyl which reacts w. creatine in KOH to form red-colored complex at surface

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O-F test

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oxidation-fermentation test
determines if carb is used y respiration (less acids are produces and peptone breakdown neutralizes acids)
pH indicator bromthymol blue. change color from dark green to yellow at acidic pH
sterile oil covers one
oxidative - yellow medium in unoiled tube
flacultative (oxid&ferment) - yellow med in oiled & unoiled tubes

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catalase test

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enzyme catalase catalyzes decomposition of hydrogen peroxide to H2O and O gas
hydrogen peroxide is formed in oxidative-reduction rxns in respiration, toxic to bacteria
one molecule is substrate, other is donor
positive test: gas bubles is released

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cytochrome oxidase

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tests for terminal link cytochrome c and cytochrome oxidase, a lipoprotein complex
during respiration e are transferred throught oxi-reduct rxns to terminal e acceptor O2
cytochrome oxidase mediates transfer of e from reduced cytochrome c to molecular oxygen
tetramethyl-p-phenylenediamine dihydrochloride donates e to oxidized cytochrome c to form indophenol (purple color) in presence of O2 on filter paper

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nitrate reductase

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nitrate is used as terminal e acceptor instead of O2 in anaerobic respiration, catalyzed by nitrates reducatase
nitrate> nitrite or ammonia or N gas
nitrate > nitrite detected by adding sulfanic acid which forms a diazonum salt which reacts w. alpha-napththylamine to create red azo dye
no red, zinc added to reduce nitrate to nitrite to form red azo dye (neg. test)
no color change= all nitrate converted to ammonia/N gas, gas buble in Durham tube

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hydrogen sulfide production

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produced by reduction of inorg sulfur compounds
1) tests for breakdown of thiosufate to sulfite and hydrogen sulfide by enzyme thiosulfate reductase
sulfur is terminal e acceptor, anaerobic respiration
2) test for reakdown of cysteine to pyruvate, ammonia, hydrogen sulfide by cysteine desulfurase
anaerobic catabolism
indicator ferrous ammoniam sulfate > ferrous sulfide, black ppt
positive test: SIM agar blackens