The ones that need review Flashcards
Concerns in testing:
Bias (experimenter/subject), record of procedure, reproducibility, statistical significance, qualitative/quantitative, correlation/causation
Define: specificity
True pos result from true pos sample
Random error of pH meter:
Contamination, improper mixing, improper reading
Variables for effectiveness of heat killing:
Temp, time, conductance
Autoclave - how does it work? what are the temp/pressure standards?
As pressure is increased, boiling point increases.
As boiling point increases, temperature increases.
15 PSI = 121ºC
T and A equation:
A = log (1/T)
DNA from absorbance equation:
[DNA] = A * dilution factor * 50
Absorbance for bacteria:
595-600 nm
A260/A280 of protein? DNA?
0.55
2, but 1.8 is good
Biuret assay:
Indirect protein assay. Measurement of colour change when CuSO4 reacts with peptide bonds under alkaline solutions. Turns purple (540 nm). Useful to 10 mg/mL.
Thumbs down: takes 15 minutes.
Bradford assay:
Indirect protein assay. Measurement of colour change when Coomassie Blue binds protein. Unbound dye has a max abs of 465 nm; bound dye has a max abs of 595 nm.
Thumbs down: Reaction varies with AA sequence (likes basic ones) and protein function. Upper limit for detection is 1 mg/mL. Dye may precipitate with detergent.
Resolution equation:
R = 0.61 λ / NA NA = n sin θ θ = half the angle that light enters the lens n = refractive index of the medium between subject and lens
Morphologies:
Cocci, bacilli, vibrio, spirochetes, spirillas
Arrangements and groupings:
Pairs (diploboth)
Clusters (staphylococci)
Chains (streptoboth)
Gram pos:
Lots of pep layers.
Teichoic acid makes it negative.
Ex: pneumonia, s. aureus, botulism, anthrax
Gram neg:
One pep layer, one phospholipid layer.
Ex: e. coli, gonorrhea, flu, cholera
Stain: Gram
CV, iodine, acetone, safranin.
Stain: acid fast
For fatty cell walls that repel Gram stains. Primary dye is carbol-fuschin, which has a high affinity for waxy mycolic acids. Decolour with acid-alcohol. Counterstain with methylene blue.
Positive stains red, negative stains blue.
Ex: tuberculosis, leprosy
Glycocalyx:
Sugars and proteins. Increases pathogenicity. May produce biofilms, which can help bacteria stick to a surface.
Ex: S. mutans and plaque (calcified capsules)
Negative stain:
Allows visualization of the glycocalyx. Nigrosin dye is bulky and negative charged so it doesn’t stick to bacteria. Thin film prep without fixing cells. Additional staining (CV) needed to visualize bacteria.
Leifson stain:
For thickening the flagellum.
CV and a mordant.
WHICH BACTERIA MAKE ENDOSPORES?
Gram pos. Bacillus (anthrax, insecticides) and clostridium (tetanus, botulinum, gangrene)
Endospore stain:
Visualization of endospores. Malachite green is steamed into the endospores. Counterstain with safranin.
What do aerobes do with free radicals?
Detoxify them with catalase
Characteristics of agar - temperatures
Solidify at 40, melt at 85.
MacConkey agar:
Selects against Gram pos using salts and CV.
Differential for lactose fermentors (pos = purple).
Mannitol salt agar (MSA):
Selective with high salt concentration.
Differential for alcohol fermentation (pos = yellow).
Useful for S. aureus.
Blood agar:
Enrichment and differential.
Hemolysis - alpha (discolouration), beta (clearing), gamma (none).
Saboraud dextrose agar:
High glucose. Selective for yeasts and molds bc high sugar and acidic pH.
SIM deep:
Differential.
Sulfur reduction: pos = black.
Indole production: add Kovak, pos = pink/red
Motility
Oxidase assay:
Tests for cytochrome 3 oxidase (making ATP using O2). Pos = purple.
Catalase assay:
Tests for catalase (H2O2 into water and oxygen). Pos = bubbles.
Biochemical identification using DNA:
PCR. Use a primer that binds a specific DNA species.
Antibiotic target: cell wall inhibitor
Penicillin and cephalosporins. Natural penicillin only works on gram neg. All contain beta-lactam ring. Bind and inhibit transpeptidases in the periplasmic space.
LYSIS.
Antibiotic target: protein synthesis inhibitor
Interfere with ribosome/mRNA/tRNA complex. Ex: chloramphenicol (broad), streptomycin (gram neg), neomycin (topical streptomycin), tetracycline (broad).
STASIS.
Side effects of protein synthesis inhibitors
Chloramphenicol can poison mitochondria and cause anemia. Bad for babies.
Streptomycin can cause deafness.
Tetracycline can yellow developing teeth, because it’s negatively charged and binds to calcium.
Antibiotic target: plasma membrane inhibitor
Polymyxins damage gram neg membranes. (Toxic to neurons and kidneys.)
Daptomycin depolarizes gram pos membranes.
Polysporin: Polymyxin B and bacitracin inhibit gram pos. Good as topical, not so much otherwise.
Antibiotic target: nucleic acid synthesis inhibitor
Rifamycin/rifampin: best against mycobacteria (TB, leprosy). Inhibits mRNA synthesis. Highly permeable because of -OHs and hydrophobicity.
Quinolines: inhibit DNA gyrase (replication). Broad spectrum. Ex: ciprofloxin.
Antibiotic target: metabolic inhibitor
Sulfonamides. Completely synthetic. Used in WWII to prevent gangrene. Blocked folate, which is used to produce thymine and uracil.
CFU and dilution:
1/DF = CFU/mL / desired count * volume plated
M-M equation: Km is often..
At phys pH.
Km of hexokinase? glycokinase?
30 uM, 10 mM.
kcat equation:
kcat = Vmax/[E]t
Km of ALDHs:
2: 80
1: 130
U:
Catalyzing 1 umol in 1 min
Enzyme activity equation:
ΔC/min = reaction volume/enzyme volume
U/V
Specific enzyme activity equation:
Activity / [protein].
U/g
