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Primary way of distinguishing normal flora from pathogenic bacteria isolated from the gut
lactose fermentation
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media used to determine ability to ferment lactose
phenol red broth
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EMB plate
- eosin methylene blue
- encourages Gram (-) growth
- dyed colonies if lactose fermented
- contains peptone, lactose, sucrose, and eosin Y and methylene blue
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phenol red broth
- differential test
- distinguishes Enterobacteriacceae from other Gram(-) rods
- shows fermentation by pH change and gas production
glucose and sucrose were used in lab
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citrate test
- determines what can live on electron transport chain and Krebs cycle alone
- citrate as sole C source
- ammonium phosphate as sole nitrogen source
- green -> blue : POSITIVE
- growth -> citrate being utilized
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SIM media
- Sulfur reduction (black ppt from sodium thiosulfate)
- Indole production from tryptophan (red layer after Kovacs' reagent -> presence of tryptophanase)
- Motility (movement from stab location)
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2 enzymes involved in sulfur reduction
- cysteine desulfurase
- thiosulfate reductase
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enzyme involved in indole production
tryptophanase (tryptophan -> pyruvate, ammonia, indole)
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6 general categories of API20E test
- 1) fermentation
- 2) decarboxylase
- 3) deamination
- 4) utilization
- 5) hydrolytic
- 6) reduction
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How do carb fermentation tests work in the API20E system?
- pH indicator for acid production from fermentation
- includes VP test
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API20E decarboxylase test
- pH indicator
- breakdown of R, K, ornithine
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API20E tryptophan deaminase test
tryptophan -- (tryptophan deaminase)--> product+FeCl3 -> color change
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API20E hydrolysis reaction (+4 tests)
- breakdown with enzyme+water
- 1)
-gal - 2) urease
- 3) indole
- 4) gelatinase
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positive ONPG test
bacteria can hydrolyze lactose
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indication of positive indole reaction
bacteria can hydrolyze tryptophan
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indication of positive gelatinase reaction
bacteria can hydrolyze gelatin
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nitrate reduction test
- red : POSITIVE
- reduces nitrate to nitrite
- anaerobic
yellow after Zn : reduces nitrate to N 2 gas
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sulfate reduction test
- black : POSITIVE
- organism reduces sulfur
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4 defining characteristics of an enteric
- 1) Gram (-) bacilli
- 2) ferment glucose
- 3) reduce nitrate
- 4) oxidase (-)
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Enterobacteriaceae represent what % of the normal intestinal flora?
10%
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severity/prevalence of diarrheal diseases
- 1.5M deaths of children/year
- 2B cases per year
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coliforms
- lactose fermenters
- "like E. coli"
- normal intestinal flora
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5 genera of coliforms
- 1) Escherichia
- 2) Enterobacter
- 3) Klebsiella
- 4) Serratia
- 5) Citrobacter
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prototypical coliform
E. coli
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O in E. coli O157:H7
outer antigen
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H in E. coli O157:H7
flagellar protein
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E. coli O157:H7
- dangerous pathogen with virulence plasmids
- causes hemorrhagic colitis, hemolytic uremic syndrome
- damage due to toxin produced
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3 genera within Enterobacteriaceae that are obligate pathogens
- 1) Salmonella
- 2) Shigella
- 3) Yersinia
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2 major diseases caused by Salmonella
- 1) typhoid fever
- 2) gastroenteritis (diarrhea)
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2 major diseases caused by Shigella
- 1) dysentery
- 2) gastroenteritis?
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major diseases caused by Yersinia enterocolitica and Yersinia pseudotuberculosis
- 1) gastroenteritis
- 2) tuberculosis-like sickness
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Yersinia pestis
- causes bubonic plague
- spread by flea bites
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3 groups classified by temperature requirements
- 1) thermophiles
- 2) mesophiles
- 3) psychrophiles
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sterilize vs disinfect
all dead vs all dangerous dead
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3 most difficult to kill organisms
- 1) endospores
- 2) mycobacteria
- 3) protozoan cysts
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What happens when a cell is subjected to heat?
proteins are denatured
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thermal death time
how long it takes to get the microbe # to 0 at a certain T
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dry vs moist heat
- dry heat takes longer, requires higher T
- moist heat takes lower T, & is more penetrating
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boiling
only kills vegetative cells
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autoclave
- sterilizes
- kills all but prions
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Pasteurization
kills some bacteria & viruses but not all
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dry heat oven
can sterilize, but takes longer, with higher T
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incineration
burns all organisms to ash
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dessication
drying so microbes don't grow
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lyophilization
freeze-drying
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5 types of peripheral blood WBCs
- 1) neutrophil
- 2) basophil
- 3) eosinophil
- 4) monocyte
- 5) lymphocyte
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3 types of granulocytes
- 1) neutrophils
- 2) basophils
- 3) eosinophils
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2 types of agranulocytes
- 1) monocytes
- 2) lymphocytes
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neutrophil (appearance, fcn, %)
- segmented lobes of nucleus, neutral-staining granules
- phagocytize bacteria
- 55-65%
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eosinophil (appearance, fcn, %)
- 2 lobes in nucleus, granules stain red
- allergic, parasitic reactions
- 1-3%
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basophil
- dark staining granules
- similar to mast cells, inflammatory response
- 0.5-1%
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monocyte (appearance, fcn, %)
- largest of leukocytes, horseshoe-shaped nucleus
- blood form of macrophages (phagocytosis)
- 3-7%
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lymphocyte (appearance, fcn, %)
- spherical nucleus that takes up most of the cell
- specific acquired immunity (T/B cells)
- 25-33%
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elevated neutrophil count
bacterial infection
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elevated eosinophil count
parasitic infection
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5 non-specific defenses of eyes
- 1) tears (mech)
- 2) blinking (mech)
- 3) salt (chem)
- 4) lysozyme (chem)
- 5) peptides (chem)
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trachoma
- most common eye infection
- leading cause of blindness
- repeated infections cause scarring that scratches cornea
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ophthalmia neonatorum
swelling (inflammation) or infection of the tissue lining the eyelids in a newborn
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antigen
substance that evokes the production of one or more antibodies
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epitope
the part of an antigen that is recognised by the immune system
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K antigen
bacterial capsular antigen, a surface antigen external to the cell wall
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O antigen
one occurring in the lipopolysaccharide layer of the wall of gram-negative bacteria
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H antigen
- 1. a bacterial flagellar antigen important in the serological classification of enteric bacilli.
- 2. the precursor of the A and B blood group antigens; normal type O individuals lack the enzyme to convert it to A or B antigens.
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antibody
Y-shaped protein produced by B-cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses
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Fab region
- fragment antigen-binding
- region on an antibody that binds to antigens
- composed of one constant and one variable domain of each of the heavy and the light chain
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Fc region
- fragment crystallizable region
- tail region of an antibody that interacts with cell surface receptors
- allows antibodies to activate the immune system
- composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains
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immunoassay
specific type of biochemical test that measures the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances
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serology
study of blood serum and other bodily fluids
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precipitation reaction
occur between soluble antigens and homologous antibodies precipitate forms when the two are at concentrations that allow maximum crosslinking in the antigen–antibody complex that forms
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immunodiffusion assay
In one well is the antiserum; in the other is the sample with unknown antigen composition. The two diffuse out of their respective wells toward each other. If the sample has an antigen that will react with the antibody, then a precipitation line will form at the region of optimal proportions as their diffusion paths pass.
If no precipitation line forms, the test is considered negative. That is, the sample has no antigen that will react with the antibodies.
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agglutination reaction
Particulate antigens (such as whole cells) may combine with homologous antibodies to form visible clumps (agglutinates)
used for blood typing
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ELISA
Enzyme Linked ImmunoSorbant Assay
- 1) specific Ag coated onto wells, unbound washed off, well blocked
- 2) sample w/ unknown Ab added to wells
- 3) secondary Ab added to react with primary Ab, unbound washed off
- 4) substrate added to react w/ secondary Ab (peroxidase) for color change
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