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clinical micro lab midterm

Terms in this set (115)

Staph aureus
Catalase (+)

oxidase (-)

Gram + cocci in pairs/clusters

facultative anaerobe

Coagulase (+)

Beta-hemolytic

Growth and acid production on MSA

Nitrate reduction (+)

novobiocin -
MRSA
Same as Staph aureus, plus oxacillin/methicillin resistance
Corynebacterium diptheriae
Catalase (+)

Oxidase (-)

Gram + pleiomorphic, club shaped coccobacilli in pallisade arrangement

Nonmotile

Nonhemolytic

Needs to be sent to reference lab for further secondary testing to ID

sometimes form V shape in cells after division
Bacillus anthracis
Catalase (+)

Oxidase variable

Large, Gram + rods

Spore +

Nonmotile

Nonhemolytic

Lecithinase +

Naturally occurring strains are penicillin susceptible, but bioterror strains may be resistant
Haemophilus influenzae
Catalase +
Oxidase +
Facultative anaerobe
Gram -
pleomorphic
often with filamentous forms
may be faintly staining
Facultative anaerobe
Nonmotile
Growth on CHOC agar (charcoal)

Requires both X (heme) and V (NAD) factor for growth, will not grow on SBA

H2S (-)
Indole variable
Nitrate (+)
Arginine (-)
Glucose (+)
Lactose (-)
Streptococcus pneumoniae
Catalase (-)
Oxidase (-)
Gram +ve lancet shaped cocci in pairs
Alpha hemolytic
Bile soluble

if bile resistant but with typical colonies, confirm with optichin (Taxo p) susceptibility
Klebsiella pneumoniae
Catalase (+)
Oxidase variable
Gram -ve straight thick rods; short to medium with rounded ended
facultative anaerobe
Indole (-)
H2S (-)
Urease +
Non-motile
Lactose positive(pink on MAC or purple on EMB)
Ornithine -
Legionella pneumophila
Catalase (+)
Oxidase variable
Graim -, stain poorly, pleiomorphic slender rods of various length (usually coccobaccilli)
obligate aerobes
Growth on CHOC agar
No growth on blood agar

Ground glass appearance
Urease (-)
Nitrate reduction
Definitive ID requires direct fluorescent antibody microscopy or PCR
Acinetobacter baumanii
Catalase (+)
Oxidase (-)
Gram -, coccobacilli (singles, pairs or chains)
obligate aerobe
nonhemolytic

twitching motility
blue-gray colony center on EMB
citrate +
glucose+
Ornithine+
Mycobacterium spp
Gram + beaded or gram neutral rod
Mycolic acid, acid fast staining
Rods may be short to long, banded or beaded, or slightly curved
Catalase +

Growth in lysozyme (-)
Arylsulfatase liquid medium - pink/red after addition of sodium carbonate
Nitrate reduction +
Urease +
Neisseria gonorrhoeae
Catalse positive
Oxidase positive
Medium to large Gram
negativediplococci and cocci in tetrads, coffee bean shaped
Capnophilic (candle jar)
Nonmotile

Growth on MTM (modified Thayer Martin)oSuperoxolpositivewith 30% H2O2
Glucose +
Lactose -
Sucrose -
Maltose -
Staphylococcus saprophyticus
Catalase positive
Oxidase negative
Gram positivecocci in clusters
Weak to nonhemolytic

Novobiocin resistant
Growth and variable acid production on MSA
Coagulase -
Staphylococcus epidermidis
Catalase positive
Oxidase negative
Gram positive cocci in clusters
Weak to nonhemolytic

Novobiocin susceptible
Growth but no acid production on MSA
Coagulase -
Streptococcus pyogenes(group A)
Catalase negative
Oxidase negative
Gram positive cocci in pairs
Colony of >0.5 mm in diameter with sharp edges

Beta hemolytic
PYR positive
Bile-esculin negative
Hippurate negative
Streptococcus agalactiae(group B)
Catalase negative
Oxidase negative
Gram positivecocci in pairs
Small zone of hemolysis around translucent colony

Hippurate positive
CAMP positive
Bile-esculin negative
PYR negative
Enterococcus faecalis
Catalase negative
Oxidase negative
Gram positivecocci in pairs
Gamma or alpha hemolytic

Bile-esculin positive
PYR positive
Hemolytic colonies could be group A streptococci
Hippurate + (80-90%)
Haemophilus ducreyi
Catalase negative
Oxidase positive
Gram negative small coccoid to rod, may be faintly staining
Facultative anaerobe+ 5% CO2(growth in candle jar)

Tan-yellow colonies
Non-motile
Requires X factor(hemin)
H2S negative
Indole negative
Escherichia coli
Catalase positive
Oxidase negative
Gram negative straight thick bacilli; short to medium length with rounded ends

Indole positive
H2S negative
Motile
Lactose positive (pink on MAC or metallic green on EMB)
Urease negative
Ornithine variable
Salmonella enterica
Catalase positive
Oxidase negative
Gram negative straight thick bacilli; short to medium length with rounded ends
facultative anaerobe


->indole negative
->H2S positive
->Motile
->Lactose negative (clear on MAC or pink/colorless on EMB)
->Ornithine positive
->Urease negative
->glucose (+)
->Sobitol (+)
->Mannitol (+)
->Sucrose (-)
->ONPG (-)
->Ornithine (+)
-> Lysine (+)
-> Arginine variable
-> Citrate (+)
->Phenylalanine (-)
Proteus mirabilis
Catalase positive
Oxidase negative
Gram negative straight thick bacilli; short to medium length with rounded ends

Swarming motility
Indole negative
H2S positive
Lactose negative (clear on MAC or pink/colorless on EMB)
Urease positive
Ornithine positive
Pseudomonas aeruginosa
Gram -ve rods
Aerobes
Catalase +
Oxidase +
Motile
Some pigments produced
Ubiquitous in environment
-> 4-42 degrees celsius
->antibiotic resistance
->moist surfaces
Cornyebacterium jeikeium
Gram + rods
Aerobes
Catalase +
Oxidase +
Nonmotile
Aeromonas Hydrophila
Gram - rods
facultative anaerobes
Catalase +
Oxidase +
Motile
Similar in shape to Vibrio
-> O129 resistance
Pasteurella multocida
gram - coccoid to rod
facultative anerobes
catalase +
oxidase +
nonmotile
cat scratch fever causer
vibrio vulnificus
gram - curves rods
facultative anaerobes
Catalase +
Oxidase +
Motile
TCBS growth, no acid
gulf waters
Clostridium tetani
Gram + rods often in pairs or short chains with rounded or pointed ends.
May stain Gram negative as culture ages
Catalase negative
Oxidase negative
Spore former
oval, subterminal
Obligate anaerobe

Lecithinase negative
Lipase negative
Indole variable (+ or -)
Fine swarming growth (may be difficult to see)
Hemolytic (may appear beta hemolytic)
H2S positive
Motile
Clostridium perfringens
Gram + large "boxcars" with no spores, may stain Gram negative as culture ages
Catalase negative
Oxidase negative
Requires specialized media to form spores
Obligate anaerobe
Large, smooth, regular convex colonies or rough, flat colonies with irregular edges

Lecithinase positive
Lipase negative
Indole negative
Motile
H2S positive
Reverse CAMP reaction
Double zone beta hemolysis
Glucose positive
Lactose positive
Sucrose positive
Maltose negative
Propionibacterium acnes
Gram positive, pleomorphic rods, often club shaped with one end rounded and other end tapered,in V's or Y's
Facultative anaerobes
Catalase positive
Oxidase negative

Beta hemolysis (21-79% of strains)
Nonmotile
Glucose positive
Maltose negative
Sucrose negative
Bile-esculin negative
Leptospira interrogans
Gram negative, helically shaped rods
Obligate aerobes
Oxidase positive
Catalase positive

Motile
Nonsaprophytic (doesnot ferment carbohydrates)
Mycobacterium ulcerans
Gram positivebeaded or gram neutral rod Based solely on chemical make-up of cell wall. Mycolic acid in cell wall resists Gram staining.
Rods may be short to long, banded or beaded, and/or slightly curved
Rods may appear pleomorphic or coccoid
Acid-fast positive
Catalase positive

Nonmotile
Slow grower
Growth in lysozyme negative
Arylsufatase liquid medium pink/red after addition of sodium carbonate
Edwardsiella tarda
Gram negative short, straight rods
Catalase positive
Oxidase negative
Facultative anaerobe

Motile
H2S positive
Indole positive
Ornithine positive
Citrate negative
VP negative
Phenylalanine negative
EMB -growth positive, acid positive (weak)
Serratia marcescens
Gram negative short rods
Catalase positive
Oxidase negative
Facultative anaerobe

Motile
H2S negative
Indole negative
Ornithine positive
Citrate positive
VP positive
Phenylalanine negative
Urease negative
Moraxella catarrhalis
Gram negative cocci, may stain dark, in singles, pairs or tetrads
Catalase positive
Oxidase positive
Capnophilic

Nonmotile
H2S negative
Indole negative
Phenylalanine negative
Urease negative
Glucose negative
Lactose negative
Maltose negative
Sucrose negative
Nitrate positive
Erysipelothrix rhusiopathiae
Gram positivestraight to slightly curved rods-Catalase negative
Oxidasenegative
Facultative anaerobe and capnophilic

Nonmotile
H2S positive
Indole negative
Glucose positive
Lactose positive
Maltose positive
Sucrose positive
Mannitol negative
Acinetobacter baumanii special characteristic
Colony with blue-gray center on EMB
Pseudomonas aeruginosa
Flat, rough, metallic sheen, blue-green pigment (not always seen) on blood agar
Phenol Red Carbohydrates
tube test
(glucose, sucrose, lactose)
Yellow-positive
red-negative
MSA mannitol salt agar
yellow - positive for mannitol fermentation

red-negative for mannitol fermentation
Bile Esculine (e.g. E. facaelis)
black = positive

no color change = negative
Urease
orange tint = uninoculated

bright pink tint = positive

yellow tint = negative
MAC (MacConkey agar)
selects for gram negative

Positive - pink

negative - colorless
EMB
Positive (E. coli)= metallic green sheen

Positive(coliforms) = purple

Negative = pink/colorless
Ornithine decarboxylase
Violet/light purple reddish tint = uninoculated tube

dark opaque purple = positive

bright/translucent yellow = negative
TCBS agar
green agar

growth with vibrant yellow colonies = positive for acid

growth with regular appearance = negative for acid
PYR
Red = positive (E. facaelis and S. pyogenes)

no color change = negative
Hippurate
blue = positive (s. agalactiae)

no color change ( clear)= negative
CAMP test
S. aureus struck in a line on SBA plate, resulting in a line of B-hemolysis

S. agalactiae struck perpendicular to line of hemolysis, improving agalactiae's abilitiy to perform hemolysis, and gives a distinct arrowhead appearance of growth/hemolysis pointing towards the S. aureus line
BHI with 6.5% NaCl
What test can differentiate microorganisms based on their ability to grow in 6.5% NaCl?
methyl red
which test differentiates microorganisms based on their ability to produce and maintain stable acid end products from glucose fermentation?
nitrite
if a red color is produced after adding 2-3 drops of nitrate reagent A and 2-3 drops of reagent B, what is nitrate reduced to?
nitrogen gas or ammonia
if a no color is produced after adding 2-3 drops of nitrate reagent A and 2-3 drops of reagent B, what is nitrate reduced to?
if there was no color change after adding the nitrate reagents, a small amount of zinc dust is added and there was no color change, is this positive or negative for the reduction of nitrate to N2 gas or NH3?
positive
if there was no color change after adding the nitrate reagents, a small amount of zinc dust is added and there was a red color, is this positive or negative for the reduction of nitrate to N2 gas or NH3?
negative
optichin
what test differentiates microorganisms based on susceptibility to ethyl hydrocupreine hydrochloride?
phenylalanine
positive = green

negative = yellow
Listeria Monocytogenes
Tiny Gram + rods, sometimes in pairs but without chaining

Catalase (+)

Narrow zone of B-hemolysis

Tumbling motility in wet mount @ 25 C

Bile- Esculin (+)

CAMP (+)

Hippurate (+)

check motility at 25 C in SIM for umbrella shaped growth if wet mount unclear
Bacillus cereus
Large, Gram +ve rods in chains

Catalase (+)

Oxidase variable

facultative anaerobe

Spore former (not always present, if present will be seen in gram stain)

Nonmotile

Nonhemolytic

Lecithinase (+) weak

Naturally occuring strain penicillin susceptible

Mannitol (-)
Mycobacterium avium
Gram +ve beaded or gram neutral rod
->mycolic acid resists stain
Rods may be short or long, banded or beaded, slightly curved, may be pleomorphic or coccoid

Acid fast (+)

Catalase (+)

Nonmotile

Slow grower (10-14 days)

Growth in lysozyme (-)

Arylsulfatase liquid medium: Pink/red after addition of sodium carbonate

Urease (-)
Enterobacter aerogenes
Gram -ve, straight rods

Catalase (+)

Oxidase (-)

Motile

Indole (-)

(H2S) -

Urease (-)

Lactose positive (pink on MAC, purple on EMB)

Phenylalanine negative

nitrate reduction +

Citrate (+)
Neisseria meningitidis
Gram -ve, medium to large diplococci and cocci in tetrads, coffee bean shaped

Oxidase (+)

Catalase (+)

Non-hemolytic

Growth on MTM (superoxol negatibe with 30% H2O2)

Glucose (+)

Maltose (+)

Sucrose (-)

LActose (-)
E. coli O157:H7
Gram negative

Straight thick bacilli; short to medium length with rounded ends

Catalase positive

Oxidase negative

Facultative anaerobe

->Indole positive
->H2S negative
->Motile
->Lactose positive (metallic green on EMB)
->Sucrose positive
->Mannitol positive
->Glucose positive
->Sorbitol negative (clear on SMAC)
->ONPG positive
->Ornithine variable
->Arginine negative
->Lysine positive
->Urease negative
->Citrate negative
->Phenylalanine negative
Shigella dysenteriae
Gram -ve, straight thick bacilli; short to medium length with rounded ends

catalase (+)

Oxidase (-)

Facultative anaerobe

->Indole variable
->H2S negative
->Motility:
-Nonmotile at 37°C
-Motile at room temp (25°C)
->Mannitol positive
->Lactose negative (pink/colorless on EMB)
->Sorbitol variable
->Glucose positive
->Sucrose negative
->ONPG negative
->Ornithine negative
->Lysine negative
->Arginine negative
->Urease negative
->Citrate negative
->Phenylalanine negativ
Yersinia enterocolitica
Gm -ve, Straight thick bacilli, short to medium length with rounded ends

Catalase (+)

Oxidase (-)

Facultative anaerobe

-Indole variable
-H2S negative
-Non
-motile
-Lactose negative (pink/colorless on EMB)
-Sucrose positive
-Mannitol positive
-Sorbitol positive
-Glucose positive
-ONPG positive
-Ornithine positive
-Lysine negative
-Arginine negative
-Urease positive
-Citrate negative
-Phenylalanine negative
Campylobacter jejuni
Gm -ve, comma shaped rod

Microaerophilic

Grows @ 37-42C

Catalase (+)

Oxidase (+)

-Indole negative
-H2S negative
-Motile
-Negative for all carbohydrates(sugars)
-ONPG negative
-Ornithine negative
-Lysine negative
-Urease negative
-Hippurate positive
Campylobacter coli
Gm -ve, comma shaped rod

microaerophilic

Grows @ 37-42C

Catalase (+)

Oxidase (+)

-Indole negative
-H2S negative
-Motile
-Negative for all carbohydrates
-ONPG negative
-Ornithine negative
-Lysine negative
-Urease negative
-Hippurate negative
Vibrio Cholerae
Gram -ve, mix of curved and straight rods

facultative anaerobe

Grows @ 20-37C

Catalase (+)

Oxidase (+)

-Indole positive
-H2S negative
-Motile
-Citrate positive
-Lactose negative
-Mannitol positive
-Sorbitol negative
-Glucose positive
-Sucrose positive (TCBS)
-ONPG positive
-Ornithine positive
-Lysine positive
-Arginine negative
-Urease negative
Vibrio parahemolyticus
Gram -ve, straight rods

Facultative anaerobe

Grows @ 20-37C

Catalase (+)

Oxidase (+)

-Indole positive
-H2S negative
-Motile
-Citrate negative
-Lactose negative
-Mannitol positive
-Sorbitol negative-Glucose positive
-Sucrose positive (TCBS)
-ONPG positive
-Ornithine positive
-Lysine positive
-Arginine negative
-Urease negative
Helicobacter pylori
Gram -ve, spiral rod

Microaerophilic

Grows @ 30-37C

Catalase (+)

Oxidase (+)

-Indole negative
-H2S variable
-Motile
-Negative for all carbohydrates
-ONPG negative
-Ornithine positive
-Urease positive
-Hippurate variable
Aeromonas caviae
Gram -ve, straight rods with rounded ends in singles, pairs, or short chains

Facultative anaerobe

Grows @ 20-37C

Catalase (+)

Oxidase (+)

-Indole positive
-H2S negative
-Motile
-Sucrose positive
-Mannitol positive
-Sorbitol negative
-Glucose positive
-Ornithine negative
-ONPG positive
-Ornithine negative
-Lysine negative
-Arginine positive
-Urease negative
clostridium difficile
Gram +ve, rods often in pairs or short chains with rounded/pointed ends
->commonly pleiomorphic, may gram stain negative as culture ages

obligate anaerobe

Catalase negative

Oxidase negative

Spore former -, oval subterminal

-Glossy, grey, circular colonies with a rough edge
-Non-hemolytic
-Indole negative
-H2S positive
-Motile
-Lactose negative
-Sucrose negative
-Mannitol positive
-Sorbitol negative
-Glucose positive
-Urease negative
-Lecithinase negative
-Lipase negative
-Reverse CAMP reaction negative
Clostridium sporogenes (stand in for C. botulinum)
Gram =ve, rods in pairs or short chains, same as C. difficile

Spore former -, oval subterminal

obligate anaerobe

Catalase (-)

Oxidase (-)

-Large (3 mm), irregularly circular, smooth, greyish, translucent with a filamentousedgethat may spread
-Beta hemolytic
-Indole variable (may give violet color)
-H2S positive
-Motile
-Lactose negative
-Sucrose negative
-Mannitol negative
-Sorbitol negative
-Glucose positive
-Urease negative
-Lecithinase variable
-Lipase positive
-Reverse CAMP reaction negative
CHROMagar
nonselective differentiated medium for the isolation, differentiation, and enumeration of urinary tract pathogens

E. coli, enterococci, Klebsiella/Enterobacter/Serratia (KES) and the Proteus-Morganella-Providencia (PMP) groups frequently encountered in UTI's

Specially selected peptones supply the nutrients in BBL CHROMagar, called chromogens

chromogen mix consists of artificial substrates (chromogens), which release differently colored compound upon degradation by specific microbial enzymes

inhibits proteus swarming

dilution is required before plating the bacteria

do not incubate in capnophilic atmosphere
CHROMagar reagents
16.1g Chromopeptone

1.3g Chromogen Mix

15.0 g Agar
CHROMagar results
1.) E. coli
->appearance on agar: Dark rose to pink, transparent colonies, medium to large size, with or without halos in the medium

2.)KES group
->appearance: Medium-blue to dark blue colonies
->secondary test: BBL crystal E/NF for genera differentiation

3.) PMP group
->appearance: pale to beige colonies surrounded by brown halos
->secondary tests: Indole, H2S, ODC, BBL Crystal E/NF

4.)Enterococcus
->appearance: blue-green small colonies

5.) S. agalactiae
->appearance: Light blue-green to light blue, pinpoint to small colonies, with/without halos
->secondary tests: PYR

6.) S. saprophyticus
->appearance: Light pink to rose, small opaque colonies with/without halos
->secondary tests: 5 ug Novobiocin disc

7.) yeasts
->appearance: natural (cream) pigmentation
->secondary test: appropriate biochemical or serological ID methods
CHROMagar caveats
E. coli colonies that are dark rose to pink but are pinpoint to small in size require DMACA spot indole test

Vary rarely, Listeria monocytogenes or other Listeria may be present in urine, it will produce blue to blue-green colonies, PYR (-), and mimmicks S. agalactiae

Aeromonas hydrophila rarely may produce rose colonies, differentiated from E. coli with oxidase test

will not support fastidious organism growth

must minimize exposure of BBL CHROMagar medium to light before and during incubation, may destroy the chromogens
Dr. Kary Banks Mullis
improved greatly on PCR

called the father of modern PCR
DNA sequencing
2 methods were invented around 1976, only one is still used today widely: the chain-termination method invented by Fred Sanger
->other metod is Maxam-Gilbert chemical degradation method, still used specifically for DNA-protein interactions
Sanger sequencing
1.) Uses DNAP to synthesize a second DNA strand that is labeled.
2.) DNAP always adds new bases to the 3' end of a primer that is base paired to template DNA
->DNAP is modified to eliminate its editing functions

3.) Also used chain terminator nucleotides: dideoxy nucleotides (ddNTPS), which lack the -OH group on the 3' C of the deoxyribose
4.) When DNAP inserts one of these ddNTPS into the growing DNA chain, the chain terminates, as noth can be added to its 3' end
Sanger sequencin reaction
Template DNA is usually ssDNA

Sequencing is done by having 4 separate reactions, one for each DNA base

All 4 reactions contain the 4 normal dNTPS, but each reaction also contains one of the ddNTPS

In each reaction, DNAP creates the second strand beginning at the primer

When DNAP reaches a base for which some ddNTP is present, the chain will either:
-> terminate if a ddNTP is added, or
-> continue if the corresponding dNTP is added
->which one that is added is random, based on ratio of dNTP to ddNTP in the tube
Electrophoresis
Newly synthesized DNA from the 4 reactions of Sanger sequencing is then run (in separate lanes) on an elctrophoresis gel

The DNA bands fall into a ladder like sequence, spaced on base apart. The sequence can be read from the bottom of the gel up

automated sequencers use 4 different fluersecent dyes as tags attached to the dideoxy nucleotides and run all 4 reactions in the same lane of the gel
->sequencers todat use capillary electrophoresis instead of slab gells
->radioactive nucleotides (32P) are used for non-automated sequencing

Sequencing reaction usually produce about 500-1000 bp of good sequence

the separation of DNA based on size by migration through an agarose gel driven by electrical gradient
Next Generation sequencing
Applications:
-> sequencing of whole bacterial genomes in a single run
->sequencing genomes of individuals
->metagenomics: sequencing DNA extracted from environmental samples
->looking for rare variants ina single amplified region, in tumors, or in viral infections
->transcriptome sequencing: total cellular mRNA converted to cDNA
Pyrosequencing Biochemistry
In DNA synthesis, a dNTP is attached to the 3' end of the growing DNA strand. The two phosphatases on the end are release as pyrophosphate (PPi)

ATP sulfurylase uses PPi and adenosine 4'-phosphosulfate to make ATP

Luciferase is the enzyme that causes fireflies to glow. It uses luciferin to oxyluciferin and releasing visible light
->light released is proportional to the number of nucleotides added to the new DNA strand

After the reaction has completed, apyrase is added to destroy any leftover dNTPS
Pyrosequencing process
1.) 4 dNTPS are added one at a time, with apyrase degradation and washing in between

2.) The amount of light released is proportional to the number of bases added. Thus, if the sequence has 2 A's in a row, both get added and twice as much light is released as would have happened with only 1 A

3.) the pyrosequencing machine cycles between the 4 dNTPS many times, building yp the complete sequence.
->about 300bp of sequence is possible ( vs 800-1000 bp with sanger)

4.) light detected with a charge-coupled device (CCD) camera
454 Technology
1.) DNA is sheared into 300-800 bp fragments, ends are polished by removing unpaired bases at each end

2.) adapters are added to each end. The DNA is amde single-stranded at this point

3.) One adapter contains biotin, which binds to a streptavidin-coated bead.
->ratio of beads to DNA molecules is controlled so that most beads get only a single DNA attached to them

4.) Oil is added to the beads and emulsion is created. PCR is then performed, each aqueous droplet forms its own micro-reactor. Each beads ends up coated with about a million identical copies of original DNA

5.) After the emulsion PCR, oil is removed and the beads are put into a "picotiter" plate. Each well is jsut big enough to hold a single bead

6.) pyrosequencing enzymes are attached to much smaller beads, which are thn added to each well

7.) the plate is then repeatedly washed with each of the 4 dNTP's, plus other necessary reagents, in a repeating cycle

8.) The plate is coupled to a fiber optic chip. a CCD camera records the light flashes from eachwell
Illumina sequencing chemistry
This method uses the basic Sanger idea of "sequencing by synthesis" of the second strand of a DNA molecule. Starting with a primer, new bases are added one at a time, with fluorescent tags used to determine which base was added

The fluorescent tags block the 3'-OH of the new nucleotide, and so the next base can only be added when the tag is removed.
->unlike pyrosequencing, you never have to worry about how many adjacent bases of the same type are present

cycled for 50-100 times
Illumina massively parallel system
The idea is to put 2 different adapters on each end of the DNA, then bind it to a slide coated with the complementary sequences for each primer. This allows "bridge PCR", producing a small spot of amplified DNA on the slide.

The slide contains millions of individual DNA spots. The spots are visualized during the sequencing run, using the fluorescence of the nucleotide being added
Sequence assembly
DNA is sequenced in very small fragments: at most, 1000 bp. Compare this to the size of the human genome: 3,000,000,000 bp. How to get the complete sequence

n the early days (1980's), genome sequencing was done by chromosome walking(aka primer walking): sequence a region, then make primers from the ends to extend the sequence. Repeat until the target gene was reached.
-> identified cystic fibrosis gene this way
->still useful for short DNA molcules, 1-10 kbp
Shotgun sequencing
Shotgun sequencingis what is typically done today: DNA is fragmented randomly and enough fragments are sequenced so each base is read 10 times or more on average. The overlapping fragments ("reads") are then assembled into a complete sequence

For large genomes, hierarchical shotgun sequencingis a useful technique: first break up the genome into an ordered set of cloned fragments (scaffolds), usually BAC clones. Each BAC is shotgun sequenced separately.

Shotgun sequencing of random DNA fragments necessarily misses some regions altogether

thus it is necessary to close gaps between contigs, and to re-sequence areas with low quality scores. This process is called finishing. It can take up to 1/2 of all the effort involved in a genome sequencing project.

Mostly hand work: identify the bad areas and sequence them by primer walking.

Once a sequence is completed, it is usually analyzed by finding the genes and other features on it: annotation. We will discuss this later.
PCR
purpose:
->to amplify large quantities of DNA (ug quantities)10^-6 from small quantities (fg quantities)10^-15
->to analyze a single DNA fragments out of large complex mixture
->Alter DNA sequence-directed mutagenesis

Overview:
1.) Temperature cycling
->denaturation (94-95C), Taq half life 40 min at 95C, 10 min at 97.5
->Annealing (44-65C)
->extension (72C)

2.) Every cycle DNA between primers is duplicated

gives exponential amplification, 30 cycles should yield 1 billion copies in theory
PCR components
1.) template DNA

2.) Flanking primers

3.) thermo-stable polymerase (Taq polymerase)

4.)dNTP
->(dATP, dTTP, dCTP, dGTP)

5.) PCR buffer (mg2+)

6.) thermal cyclers
Typical PCR Run
1.) 3 min at 95C

2.) 30 sec at 95C

3.) 1 min at 55C

4.) 2 min at 72C

5.) Repeat step 2-29 times

6.) 4 min 72C

7.) 0 min 4C

8.) End
Plateau Effect in PCR
The actual yield is much lower than the theoretical yield
PCR primers
paired flanking primers

17-28 bp in length

GC content is 50-60%

melting temp: between 55-80C

Avoid simple sequences (string of G's)

Avoid primer self complementary (hairpins, homodimers, heterodimers)
PCR buffer
Components:
-> 20mM Tris-HCL pH 8.4
-> 50 mM KCl
-> 1.5 mM MgCl2
Magnesium in PCR
Mg ions form complexes with dNTPs, primers, and DNA templates. So the optimal concentration of MgCl2 has to be selected for each experiment
->too few Mg2+ cations result in a low yield PCR product
->too many increase the yield of non-specific products and promote misincorporation
Taq polymerase
Half life
->40 min

3'->5' exo nuclease activity for proofreading

Fidelity (error rate)
->Taq 1/10,000 nt
API 20 E identification system
used for Enterobacteriaceae and other non-fastidious Gram -ve rods

20 microtubes that contain dehydrated substrates

microtubes are inoculated with a bacterial suspension that reconstitutes the media.

During incubation, metabolism results in color changes that are either spontaneous or revealed with addition of reagents
API procedure
1.) add ~5mL distilled H2O to the bottom tray of incubation box
2.) Remove a test strip frm its package and place in the incubation box
3.) Prepare bacterial suspension by adding one colony from an isolated plate that is 18-24 hrs old, to 5 mL of sterile 0.85% NaCl, mix to homogenize
4.)Using sterile pipete, fill both the tube and cupule of the CIT, VP, and GEL tests with bacterial suspension. Fill only the tube of the other tests
5.) Create anaerobic conditions by adding mineral oil to the wells marked ADH, LDC, ODC, H2S, and URE
6.) cover incubation tray with lid and incubate at 37C for 18-24 hrs
API tests requiring additional reagents
1.) TDA
->add 1 drop TDA reagent to the TDA well, the reaction will be immediate
->positive: reddish brown
->negative: yellow

2.) IND
-> add 1 drop of kovacs reagent to the IND well (immediate reaction)
->positive: red
->negative: colorless, pale green, or yellow

3.) VP
-> add 1 drop of VPA and VPB reagents, wait 10 minutes for color to develop
->positive: pink/red
->negative:colorless
API results
tests are separated into gorups of 3, with a value of 1,2 or 4 assigned to each test

Add together values corresponding to + reaction within each group of 3 tests to generate a 7 digit profile number
B-lactam disk
disk impregnated with benzylpenicillin which contains B-lactam ring.

When the B-lactamase enzyme is produced by an organism, the beta lactam ring of benzylpenicillin is hydrolyzed to form penicilloic acid, inactivating the antibiotic

the fall in pH is indicated by the brom cresol purple indicator changing from purple to yellow

Positive result: Color change from blue to yellow, indicates B-lactamase production

negative rsult: No color change

usually reacts in 1-10 minutes
BD directigen EZ group A strep test
can detect Strep from throat swabs

reagents:
->reagent A(red cap) 4 drops
->reagent B(yellow cap) 4 drops

Collect specimen and put the swab in the test tube with the reagents, leave swab in test tube for 1 minute.

Take out the swab, insert the tube tip on the test tube, dspose of the swab in biohazard bin

Add 3 drops of liquid specimen from the test tube to the oval on the test trip

wait 5 mins, no more than 10

positive: 2 pink or red stripes at the C and T on the test strip.

negative: only one pink or red strip at the C

test invalid if only stripe at T or no stripe at all
ELISA
1.) Antigen is added to the wells of a microplatestrip and incubated to allow binding, after which unbound antigen is washed away. The detergent for washing also blocks the binding of all unused proteins binding sites in the wells, preventing nonspecific binding of antibody

2.) Primary antibody solution is added to the wells and incubated to allow the Ab to bind the Ag, unbound primary Ab is washed away

3.) Enzyme-labelled secondary antibody solution is added to the wells and incubated to allow 2ndary Ab to bind to the primary Ab, washing step repeated

4.) Chromogenic enzyme substrate is added to the wells and incubated to allow color to develop, results are then evaluated. Colorless wells are negative, Blue wells are positive
Indole Spot Test
reagent: DMACA (p-Dimethylaminocinnamaldehyde) Indole reagent are used for determining if bacteria can produce indole by tryptophan deamination

DMACA color change is rapid

reaction indicates the presence of tryptophanase that reacts with tryptophan to produce indole, the indole produced reacts with DMACA to form a blue-green color

Positive: blue to blue green color within 2 mins

negative: any other color than blue to blue green, usually pink to red

procedure:
1.) moisten bibulous paper with drop of indole reagent
2.)remove an isolated colony from 18-24 hr culture with sterile loop and smear on moistened paper
3.) using loop, pick up and smear 5 suspect colonies onto the Test-latex containing circle and mix this into the test latex reagent. spread to cover the circle
4.) observe for color change
PYR test
PYR is a rapid colorimetric method based on activity of the enzyme pyrolidonyl arylamidase

L-pyroglutamic acid beta naphthylamide is impregnated into the test disk as the substrate for detecting the enzyme

hydrolysis of substrate leads to beta-naphthylamide which combines with PYR reagent DMACA to form bright pink to cherry red color

Positive PYR is presumptive of Grop A strep (pyogenes) and group D enterococci (E. facaelis)

Disc on plate method, with PYR reagent added after bacteria allowed to react on disc
Staphyloslide
S. aureus agglutination test

1.) mix latex reagent by shaking, expel any latex from the dropper for complete mixing

2.) dispense 1 drop of test latex onto one circle, 1 drop of control latex onto another circle

3.) using loop, smear suspect colonies onto test latex and mix well to cover circle
->repeat for control latex

4.)pick up and hand rock the card for up to 20 sec and obserrve for agglutination under normal lighting

positive: agglutination of the blue latex particles occuring within 20 sec in test circle, none in the control. Noticeable clearing of the blue background is posiive

negative: no agglutination occurs in 20 sec and smooth suspension remains, with no clearing of the blue background in the test latex after 20 seconds
Subtyping
RFLP (restriciton Fragment Length polymorphisms)
->PCR then endonuclease restriction and electrophoresis
->same equipment as PCR, but DNA is restricted before electrophoresis
RFLP advantages
Same equipment as PCR

low cost

simple protocol

very little training needed
RFLP disadvantages
low discrimatory power

inconsistencies in how fragments move through gel
Pulsed-field Gel Electrophoresis
PFGE

Electrophoresis of large DNA fragemnts restricted with endonucleases

Gold standed for foodborne bacterial outbreaks (CDC)

USed worldwide

good for short term surveillance, such as outbreaks
PFGE advantages
High level discrimination

High reproductibility
PFGE disadvantages
Equipment cost

time requirement

high level training needed

some strains do not produce patterns
Multi-locus Sequence typing (MLST)
typing system based solely of sequence variation

Shows genetic variation from multiple chromosome locations (alleles or loci)
->usually housekeeping (conserved) genes)

acceptance as a subtyping method is growing quickly
MLST advantages
can characterize all strains

good for longitudinal studies

SImple PCR and databse requirement

no electrophoresis standardization required
MLST disadvantages
Lack PFGE's dscrimatory power

not useful for short term surveillance due to conservation of genes

sequencing large numbers of genes becomes expensive
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