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lab attire
lab coats, gloves, goggles, long pants, long hair should be tied back, closed-toe shoes
aseptic technique
limits the presence and spread of potentially harmful organisms
prevents exposure of yourself and others to potentially harmful bacteria
reduces the posibility of contaminating your sample
two-tube transfer
1. Sterilize loop
2. Uncap tubes
3. Sterilize tubes
4. Transfer from culture to fresh medium
5. Sterilize tubes
6. Recap tubes
7. Sterilize loop
staining
useful for identifying morphology, size, and arrangement of the bacterial sample
simple, differential, structural
catioinic
basic dyes, + charged chromophores
+ ion exhibits color
methylene blue or crystal violet
anionic
acidic dyes, - charged chromophores
- ion exhibits color
acid fuchsin, congo red, nigrosin
negative stain
stain doesn't penetrate the cell
- negatively charged stain repelled by - charge at cell surface
- transparent cells against a colored background
good for examining morphology and size of bacterial cells
- cell are NOT heat fixed, so distortion of the bacterial cells is minimized
useful for organisms that don't stain easily
nigrosin: black anionic dye
simple stain
one dye used to stain all cells present
- we will use crystal violet (cationic dye)
useful in distinguishing different morphologies, arrangement, and relative sizes
colors the cells and provides contrast when using bright field microscopy
+ charge of the dye interacts with the - charge on bacterial cell wall
smear preparation [from a broth]
1. aseptically transfer 1 loopful of culture to a clean slide and spread to maximum thinness
2. allow to air dry
3. heat fix by holding over microincinerator for 10 seconds - USE CLOTHESPIN!!
smear preparation [from slant or plate]
1. add 1 loopful of water to the slide FIRST
2. aseptically collect bacterial sample from plate or slant
- just touch the loop to the bacterial growth on the plate, slant, mix well to suspend
3. allow to air dry
4. heat fix by holding over microincinerator for 1o seconds - USE CLOTHESPIN!!
microscope
know the parts
exercise 2 slide 14
illumination [bright-field microscopy]
light source
condensor: directs light towards the objective lens in bright field microscopy
iris diaphragm: adjusts the diameter of the cone of light so that it just fills the objective lens
magnification [bright-field microscopy]
ocular: 10x
objectives: 4x, 10x, 40x & 100x
resolution [bright-field microscopy]
resolution is the smallest distance between two objects which can be seen as separate
resolving power: 0.2 mm
average microbe: 1.0 mm
oil immersion
100x = oil immersion
MUST use oil
oil has the same refractive index as glass
-oil stops refraction of light between specimen and lens
resolution
d(resolution) = lamda/(2NA)
cocci
spherical bacteria
bacilli
Rod shaped bacteria
differential stain
detects differences between organisms through the use of many dyes and reagents
- gram stain
structural stain
confirm structural characteristics of cells
Hans Christian Gram (mid 1880s)
developed the gram stain
danish physician and scientist
gram stain
used crystal violet to stain bacterial cells
when cleaning up sample with ethanol - found some bacterial cells would not retain purple
- considered the stain a "failure"
now the most widely used & important stain in bacteriology
peptidoglycan layer
differentiates bacteria into 2 groups based on the thickness of the peptidoglycan cell wall
primary dye [gram stain]
crystal violet
mordant [gram stain]
Gram's iodine
decolorizer [gram stain]
95% ethanol
secondary dye/counterstain [gram stain]
safranin
gram negative
stain pink
gram positive
stain purple
acid-fast staining: Ziehl-Neelsen Method
differential stain
acid-fast microorganisms
have high mycolic acid content in their cell walls - waxy and resists staining
steam is used to force the primary dye to penetrate the waxy cell wall
fastness = to retain the dye when challenged by a decolorizer
primary dye [acid-fast stain]
carbol fuchsin
[need steam]
decolorizer [acid-fast stain]
acid alcohol
methylene blue [acid-fast stain]
secondary dye/ counterstain
acid-fast
cerise or pink/violet because of the high wax content they retain the dye after decolorization
acid-fast negative
blue because the cells will readily decolorize and be counterstained with methylene blue
acid fast genera
Mycobacterium and Nocardia
-M. tuberculosis: causative agent of tb
-M. leprae: causative agent of Hansen's disease (leprosy)
spore staining: Schaeffer-Fulton Method
structural stain used to detect dormant forms of bacteria [endospores]
- these structures can be released called - free spores
- to force the primary dye into the resistant endospore steaming is used
- stained spores are then resistant to decolorization
primary dye [spore staining]
malachite green
- steam
decolorizer [spore staining]
water
secondary dye / counterstain [spore staining]
safranin
endospores
spores are green inside pink bacterial cells
free spores
small green oval bodies [free]
if no spores
only pink cells will be observed
genera of spore forming bacteria
Bacillus: gram + rod
- B. anthracis
Clostridium: gram + rod
- C. botulinum
- C. tetani
-C. difficile
pure culture
contains a single microbial species
environmental & patient samples
almost always have a mix of microbes
pure culture techniques
aim to dilute bacterial samples in order to separate individual cells that can grow into isolated colonies when plated
growth medium and agar
a variety of different types of media are available
- general purpose
- selective
- differential
general purpose medium
used when trying to isolate microbes from a mixed culture
solid media
are often preferred when trying to obtain pure cultures
-agar is solidifying agent derived from seaweed
microbes are unable to metabolized agar
pour plate
this method can be used in both a quantitative and non-quantitative way depending on the dilution method used
- by diluting individual cells are played which can grow into colonies
- culture is diluted in molten agar (> 40 degrees C)
- once diluted agar is mixed well it is poured into an empty sterile petri dish and allowed to solidify (40 C)
- colonies can be found on top of agar, embedded in agar, and underneath agar
spread plate isolation
brother culture diluted in a series of tubes - serial dilution
- quantitative or non-quantitative as the dilution is performed with an inoculating loop
- colonies will only form on the surface of the agar
streak plate technique
method to separate a single species from a mixed species population
- sample is plated across the surface of the agar in 3 zones
- isolation of 3 mixed species
- primary streak
general purpose media
support growth of variety of microorganisms
- good for maintaining cultures
- includes: nutrient agar (NA), tryptic soy agar (TSA), brain heart infusion (BHI)
selective media
use inhibitors to prevent growth of certain organisms
- includes: phenyl ethyl agar (PEA), eosin methylene blue (EMB), MacConkey agar (mac)
differential media
allow growth of many microbes, but differentiation is seen by indicators that detect changes that have occurred
- dye, reagents, blood cells, culture conditions
- includes: EMB, blood agar, macconkey agar
combination media
are both selective and differential
complex media
undefined, exact chemical composition not know [box cake mix]
- usually have nutrients supplied as extracts or digests from natural sources
- BHI, TSA
defined
known chemical composition for each component [homemade cake]
phenylethyl Alcohol Agar (PEA)
- selective only
- inhibitor: phenyethyl alcohol
- prevents growth of gram -
- often used to select for gram + species in patient samples
- staphylococci
- get rid of contaminants like E. coli
blood agar (BA)
- differential only
-indicator: red blood cells
- TSA base w/ 5% sheep's red blood cells added
- blood agar can be useful in differentiating hemolysis patterns of microorganisms
-lysis of red blood cells
- beta: best
- alpha: average
- gamma: trash
macconkey agar (Mac)
- selective and differential = combination
- indicator: neutral red
- differentiate lactose fermenters from non-fermenters
- inhibitor: crystal violet and bile salts
- prevent growth of gram +
- used for identification of gram - enterics
- enterobacteriaceae
eosin methylene blue (EMB)
- selective and differential = combination
-inhibitors: eosin & methylene blue
- prevents growth of gram +
- indicator: eosin & methylene blue
- differentiate lactose fermenters from non-fermenters
- used to screen for coliforms
-fecal colliforms
- can also be used when testing for UTI's
testing for the motility of bacteria
- organisms that have flagella are motile
- not all species produce flagella
- motility can be tested using motility media
- a motility is detectable due to the use of a soft agar (decreased agar concentration)
- a dye indicator may or may not be used to aid in the detection of motility
-motility media are inoculated by a single line stab
- motile: organisms can swim through the soft agar and spread away from inoculation stab line
- non-motile: organisms remain confirmed to the path of inoculation by the soft gel of the medium
viable plate count
- method for diluting a broth culture so that a small sample of it can be plated
- individual cells are isolated
- spread plate technique used
- when colonies grow they can be counted to determine the original concentration of the starting sample
- viable cells: are those that can replicate and form colonies
- this method does not detect dead cells
important formulas
- dilution factor: a/a+b
- a: volume being transferred
- b: volume being added to
- total tube dilution: the total dilution of the original sample in each of the tubes
- you must multiply the combined dilution of the previous tube by the dilution factor of your transfer
- plate dilution: amount plated (in mL)/ 1mL
standard plate count
Following incubation: count the plate that seems to have between 30 and 300 colonies
calculate the dilution factors (DF): between the counted plate and the original culture
- multiply the plate count by the reciprocal/inverse of the dilution factors
- results are reported as CFU/mL
biochemical tests
agar plate tests for exoenzymes:
- lipase
- milk agar
- starch agar
tube media tests for other metabolic processes:
- litmus milk
- sugar fermentation
- kligler's iron agar
litmus milk
contains:
- lactose
- casein and other peptones
- litmus pH indicator
tests for:
- fermentation of lactose
- metabolism of proteins
- degradation of casein
- litmus reduction
litmus milk: fermentation
fermentation of lactose produces acids that reduce the pH of the litmus milk solution
- litmus milk turns pink at acidic pH
litmus milk: peptone deamination
some bacteria may instead deaminate peptones, releasing ammonia into solution
- this rases the pH of the litmus milk, turning the solution blue
peptone deamination is an oxygen dependent process, so the color change may be limited to the top of the tube
litmus milk: reduction (redox)
litmus milk only functions as a pH indicator in an oxidized redox states - loses its color when reduced
- if the bacterial culture produces a reducing environment, then the litmus milk will go white as the litmus indicator is reduced
a band of color will persist at the top of the media, where oxygen can penetrate and re-oxidize the litmus indicator
litmus milk: proteolysis
some bacteria produce the caseinase enzyme
- if casein is degraded, the milk will turn clear
litmus milk: curding
if enough acid is produced from lactose fermentation, milk proteins may become denatured
- some curds completely solidify the milk, or may incompletely curdle the milk, leading to a lumpy consistency
curding does not always occur with acid production
phenol red (PR) sugar fermentation broth
contains one sugar: glucose, lactose, or mannitol
indicators: phenol red and a durham tube
- detect fermentation of included sugar
if an organism ferments:
- phenol red turns yellow
- gas formation: bubble collects in durham tube
if an organism can't ferment:
- peptone degradation releases ammonia
- phenol red indicator turning a shade of "hot pink" called cerise
Kligler's Iron Agar (KIA)
tests for: lactose fermentation, glucose fermentation, sulfur reduction
carbohydrates sources: 1.0% lactose, 0.1% glucose
other ingredients: 1.0% peptone (source of ammonia & sulfur via cysteine)
indicators:
- phenol red = pH indicator
- iron from ferric ammonium citrate - combines with H2S to form a black precipitate
KIA media: lactose fermenters
acid can be from fermentation of glucose (0.1%) and lactose (1.0%)
NH3 is released by the breakdown of peptone (1%) in the presence of oxygen
if both glucose and lactose are fermented, acid products will be produced in excess of NH3, and the entire tube will turn yellow
1% lactose + 0.1% glucose > 1% pepton (1.1% - 1%)
fermentation can result in the formation of gaseous end products which can lead to lifting or cracking of the media in the tube
KIA media: glucose only
glucose fermentation only: amount of NH3 produced by breakdown of peptone exceeds the amount of acid produced by glucose fermentation
breakdown of peptone will only take place in the presence of oxygen
deamination of peptones keep only the upper part of the slant at neutral/alkaline pH
butt of the tube will stay yellow due to the glucose fermentation, as oxygen can not penetrate far enough into the butt for peptone deamination to reverse acidification
0.1% glucose < 1.0% peptone
KIA media: reversion
some lactose fermenting bacteria produce weaker, less stable acids that can be oxidized into neutral end products
- leads to reversion of part of the slant back to neutral/alkaline pH
often observed with Enterobacter aerogenes, which uses the 2,3-butanediol pathway for lactose fermentation
- 2,3-butanediol products are not as stable as the products of the mixed acid fermentation pathway [lactate, acetate, succinate & formate], and can readily be neutralized in aerobic environments
KIA media: sulfur reduction
reduction of sulfur produces H2S which combines with iron in the medium to form a black precipitate
- precipitate will obscure color in the bottom of the tube
H2S will only form as a byproduct of sulfur reduction in an acidic environment
- black precipitate indicates that fermentation and acid production have occurred
lipase plate
differential media
tests for the enzyme lipase
- hydrolyzes fats > glycerol and fatty acids
indicator: spirit blue
hydrolysis of lipids will produce a dark blue zone around the growth with no oily surface
- + = blue color
- - = no color
milk agar plate
differential media
tests for enzyme caseinase
- hydrolyzes casein (milk production) > amino acid products
casein gives milk its white color loses its color when broken down - clearing
- + = clear around colony
- - = no clearing around colony
starch agar
differential media
test for the enzyme amylase
- breaks down starch into simple sugars
add iodine after colony growth as the indicator
- iodine + starch = purple
tests that yield results within minutes
catalase
oxidase
tube media tests for other metabolic processes
nitrate broth
gelatin
urea broth
phenyalanine
SIM
IMViC
fermentation vs respiration
conversion of organic molecules to an energy source (ATP)
glucose > pyruvate via glycolysis
respiration
final electron acceptor is an inorganic molecule
- oxygen > aerobic respiration
- nitrate, sulfate > anaerobic respiration
general pathway for respiration
glycolysis > kreb's cycle > electron transport chain
transfer of electrons to molecules with a more positive potential
we are indirectly testing for the presence of an ETC
catalase test
during aerobic metabolism, reactive oxygen species (ROS) may be produced:
- superoxide radicals
- hydrogen peroxide (H2O2)
some bacteria have enzymes capable of breaking down these toxins (aerobic & facultative anaerobes)
- superoxide dismutase (SOD)
- catalase
detects catalase
- converts hydrogen peroxide > water & oxygen
- 2 H2O2 > 2H2O + O2
add 3% hydrogen peroxide to the edge of the colony
- oxygen product will be seen as bubbles coming from the sample
cytochromes C oxidase
main function is to remove electrons from cytochrome c (oxidize) and transfer them to oxygen (reduces)
- cytochrome c > cytochrome c oxidase > oxygen
however, it can also reduce cytochrome c by oxidizing a chromogenic reducing agent
- these reducing agents develop color when they become oxidized
- dimenthyl-p-phenylenediamine
oxidase test
detects cytochrome oxidase
- transfers electrons to final electron acceptor - oxygen
indicator: dimethyl-p-phenylenediamine hydrochloride (an aromatic amine)
in the presence of cytochrome c oxidase, reagent is oxidized and turns dark blue to black
nitrate respiration
utilizes nitrate (NO3) as final inorganic electron acceptor
requires enzyme nitrate reductase to convert nitrate to nitrite
some bacteria have other enzymes to reduce nitrate all the way down to N2 (denitrification)
others can reduce nitrate to ammonium for incorporation (assimilatory nitrate reduction)
nitrate broth
detects nitrate reductase which reduces nitrate to nitrite (NO3 > NO2)
nitrate I (sulfanilic acid) and nitrate II (dimethyl-alpha-napthylamine) react with NO2 > brick red color
if nitrate is not used and is residual, then zinc powder will catalyze the reaction
- a red result after Zn addition indicates that nitrate was not used = the organism did not have the enzyme
- if there is no reaction after zinc is added that means it was reduced all the way to nitrogen gas no zinc was used
urea broth
detects urease
- degrades urea > 2 ammonia (NH3) and carbon dioxide (CO2)
- increases pH - more alkaline
contains urea and the pH indicator phenol red
- as pH increases > 8.1 > cerise
- neutral pH > red
- as pH decreases > yellow
gelatin
detects with presence of gelatinase
- catalyzes the hydrolysis of gelatin > amino acids
- gelatin solidifies in low temperatures
- need ice bath
phenyalanine slant
medium detects production of phenylalanine deaminase
- phenylalanine > phenylpyruvic acid (PPA) + ammonia (NH3)
add 5-10 drops of 10% ferric chloride (FeCl3) reagent
- in the presence of PPA, FeCl3 appears a deep green color
SIM
s: sulfide
- H2S produced by bacterium reacts with Fe in medium to produce FeS
i: indole
- add 3-5 drops of Kovak's reagent
m: motility
fermentation
final electron acceptor is an organic molecule
- does not produce as much energy as respiration
pyruvate > general end products are organic acids and gases (CO2, H2)
- mixed acid fermentation
- 2,3-butanediol fermentation
can also be used as a general term for the breakdown of polysaccharides into monomers that can be fermented
ex: lactose > glucose & galactose > pyruvate
MR-VP broth
methyl red (MR) and Vogues-Proskauer (VP) test must be done separately
methyl red (MR): tests for mixed acid fermenters
add 3-4 drops of methyl red reagent
- detects high acid production, lowering pH to 4.4 or less
a low acid concentration does not allow retention of the red color
vogues-proskauer (VP): tests for 2,3-butanediol fermenters
- glucose > acetoin > 2,3-butanediol
2,3-butanediol fermenters produce the acetoin intermediate
- acetoin can interact with VP I and VP II (Barritt's reagents)
add 10 drops of VP I (alpha napthol) to intensify the red color
add 10 drops of VP II (KOH), produces an alkaline condition that favores acetoin oxidation > diacetal and O2 is mad, presence of O2 produces a red color
tyrptone broth
detects tryptophanase
- hydrolyzes tyrptophan > indole, pyruvate and ammonia (NH3)
add 3-5 drops of Kovak's reagents (DMABA and HCI dissolved in amyl or butyl alcohol)
- reagent is nonpolar & extracts indole to top
- indole + kovac's regeant = cerise