Bio 205 L Final Review PDF

Bio 205 L Final Review Cheat codes: CIA’S, CAM, MDS Gram-Stain Procedure 1. Crystal violet 2. Iodine (mordant) 3. Alcohol (decolorizing step) 4. Saffranon CIA’s MOST IMPORTANT- ALCOHOL DECOLORIZING Acid Fast stain 1. Carbol fuschin (colors acid-fast red) 2. Alcohol (decolo...

Bio 205 L Final Review Cheat codes: CIA’S, CAM, MDS Gram-Stain Procedure 1. Crystal violet 2. Iodine (mordant) 3. Alcohol (decolorizing step) 4. Saffranon CIA’s MOST IMPORTANT- ALCOHOL DECOLORIZING Acid Fast stain 1. Carbol fuschin (colors acid-fast red) 2. Alcohol (decolorizing) 3. Methylene blue (colors non-acid fast blue) CAM (College of Arts and Media- acid used in art) Endospore stain 1. Malachite green (steam on, heat driven into cell, green) 2. Distilled water ( 3. Safranin (stains pink) My Doctor Sucks (MDS) Mordant: chemical bridge between cell wall and stain (iodine) E-coli- gram negative Staphylococcus epidermidis- gram positive Mycobacterium leprae- acid fast Bacillus subtilis- endospore Mycolic acid stains- red (baby bell cheese (waxy)) Vocab Parfocal- switch from one objective to the next, minimize focusing Immersion oil/glass- refraction minimization (they have the same refractive index) Scrubbing the desktop takes out- ALL of it (viruses, bacteria, etc) % bleach for bacterial spill- 10% Fomite- anything bacteria can be on Ubiquitous- everywhere Turbidity- cloudy BFF in lab- inoculating loop 6 factors: temp, moisture, pH, oxygen, nutrients, competition You turn a petri dish upside down because- prevents moisture from condensing, makes it easier to see the bacteria Pure culture- cell derived from 1 mother cell Streak plate- isolate individual bacteria from mixed culture  Bacteriacide drug- drug that kills te bacteria Bacteriostatic drug- stops growth but doesn’t kill Broad spectrum antibiotic- kills many things What affects the size of inhibition? - type, concentration of antibiotic, microbe, age of antibiotic 2 advantages to heat fixing- kills bacteria and glues peptidoglycan to the slide Endospore wall- red Endospore nucleus- green Endospore- formant bacterial cell Methylene- positive + stain Nigrosin- negative - stain Differential staining- tells two things apart Grams stain iodine- mordant, fixes crystal violet Chemical basis behind + stain- ions attach (- on cell wall attaches to + on stain) Chemical basis behind - stain- ions repel (- on cell wall repels the - stain) Prokaryotic- no organelles, circular chromosomes free floating, smaller, bacteria and archaea Eukaryotic- larger, plants, animals, fungi, protozoa, seaweed What info is obtained from a simple stain- phenotype, morphology, color Capsules- protect bacteria from phagocytosis 2 reasons why negative stain is used- morphology and cellular arrangement Selective media- allow growth of certain organisms while preventing the growth of different organisms Differential media- allow the growth of multiple organisms but have components that allow the researcher to differentiate between them Protozoa- unicellular eukaryotic microorganisms lacking a cell wall, trophozoite or cyst Immunoassay- quick tests that can detect the presence of specific molecules in a sample Innate immunity- nonspecific defenses, birth, nonspecific like physical and chemical barriers First line- skin, mucus membrane, normal microbiota Second line- phagocytic WBC, inflammation, fever (granulocytes, monocytes) Specific (3rd line)- specialized lymphocytes- T and B cells, antibodies (lymphocytes) Serum- removes everything from the blood, leaving a clot Plasma- everything besides RBCs Neutrophil- 50-70% WBCs, phagocytic on bacteria and fungi Eosinophils- attack parasites and intracellular bacteria Basophils- injury and allergic reaction, histamine Monocytes- enter peripheral tissues as macrophages Lymphocytes: T cels, B cells, targeted, specific immunity RBC- biconcave shape Macrophages- monocytes that came from the blood into infected tissue Epitopes- antigenic determinants, antibodies recognize and react Protozoa trophozoite- vegetative  Protozoa cyst- resting, dormant Parts of a Microscope ‘ Microscope: Total magnification= objective lens (4, 10, 40, 100) x ocular (10) “High and dry” lens= 40x Condenser- concentrates light onto a specimen and reduces light lost Iris diaphragm- mechanical device on the top of the condenser that can be opened or closed with a lever to regulate light column illuminating a specimen Resolution- the smallest distance between two points on a specimen that can still be distinguished as two separate entities 2 factors that enhance resolution- fine focus and diaphragm Simple stain: Most of bacteria is water- without stain, you can’t see much of the bacteria Simple positive: (dyes bind to negatively charged molecules) - Methylene blue  - Crystal violet - Safranin - Malachite green Take sample from broth or agar, pur onto slide with water, dry, heat fix, put on methylene blue, let sit one minute, wash with distilled water, blot with bibulous Simple negative: (acidic or capsule dyes) - Measurement of bacterial size - Presence and size of capsules - Congo red Get slide, put a drop of nigrosin on one end, transfer bacteria to the drop of nigrosin, gently mix, smear the dye over the whole slide, air dry the smear, do NOT heat fix negative (breaks capsules), set aside Gram’s stain: 1. Prepare smear 2. Cover with crystal violet for 1-2 mins 3. Gently rinse with water 4. Cover with Gram’s iodine for 1-2 mins 5. Gently rinse with water 6. Decolorize briefly with alcohol for 2-3 seconds 7. Immediately rinse off decolorizing alcohol 8. Cover with safranin counterstain for 1-2 mins 9. Rinse with water 10. Gently blot dry with bibulous paper, allow to air dry 11. Examine under oil immersion lens Acid-fast stain: Mycobacterium are identified, for diseases like TB and Leprosy acid fast- pink, not- green 1. Glass slide, prepare mixed smear 2. Allow to air dry, the heat fix 3. Place in staining rack 4. Cover with bibulous paper 5. Apply carbol fusion. Let sit for 10 mins, do not let it evaporate 6. Wash gently with water 7. Decolorize with acid-alcohol 8. Wash with water 9. Counterstain methylene blue for 2 mins 10. Wash with water 11. Blot dry Endospore stain: Spore- green (inside), vegetative- red (outside) 1. Prepare smear oh glass slide 2. Air dry, then heat fix  3. Place slide over boiling water 4. Flood smear with Malachite green, steam 10 mins, do not let evaporate 5. Wash with distilled water 6. Cover with safranin for 2 mins 7. Rinse with water 8. Blot dry pGLO: Pt. 1 Ampicillin (amp)- antibiotic that kills bacteria, the pGLO bacteria contain an ampicillin resistant gene Arabinose (ara)- the plasmid for it has been spliced on the GFP gene, so that the GFP gene turns on in the presence of arabinose pGLO plasmid- engineered plasmid that contains the green fluorescent protein (GFP) LB- nutrient rich medium for culturing bacteria LB/amp/+pGLO= grow and glow LB/amp/ara= all grow since there is no antibiotic on the plate LB/amp/-pGLO= nothing can grow w/o pGLO plasmid LB/-pGLO= all cells grow since there is no antibiotic on the plate Pt. 2 Isolating plasmid DNA from pGLO transformed cells in order to have a pure sample to amplify with PCR Pt. 3 DNA polymerase is the principal enzyme involved in the replication of DNA PCR- assembling millions of copies of a particular DNA segment, PCR: 1. Heat separates the DNA into a solution called dNTP. 2. TAQ polymerase builds two new strands of DNA using the original strands as templates Thermocycler- the machine that alters the temperature to allow for the denaturing and synthesis of the DNA 1. Denaturing (heat separates DNA), 2. Annealing (RNA primers attach), 3. synthesis (DNApolymerase makes new strand) 1. DNA sample, 2. Thermocycler machine, 3. Epindorph (micro) tube, 4. RNA primers, 5. TAQ polymerase, 6. Free nucleotides (A,T,C,G) in solution, 7. Buffer solution Gel Electrophoresis Used to separate DNA based on their size. AN electric current is applied, and the the DNA molecules (- charge), move from the sample wells at the - pole, to the + wall on the other side In electrophoresis, the cathode is negative and the anode is positive Phylogenetic tree- shows how organisms evolved Unknown bacteria lab MacConkey Agar Both selective and differential Crystal violet and bile inhibit Gram + while selecting Gram - Incorporation of lactose and pH indicator (red)  Gram - divides into 2 groups: Lactose (pink) and non-lactose (clear) Mannitol Salt Agar Gram Positive Selective (for staphylococcus) and differential (some bacteria ferment mannitol- turn the pink agar yellow) Blood Agar Gram Positive Differential for bacterial enzymes on RBCs Alpha (partial hemolysis, green), beta (complete clearing, lysis, open), or gamma reaction (no lysis, red) Catalase Gram Positive Differentiate aerobic staph from strep Tests for the production of catalase, bubbles when hydrogen peroxide is placed on Esculin ID membrane Gram Positive Hydrolyze esculin into glucose and esculetin, produce dark precipitate Eosin-Methylene Blue Agar Gram Negative Selective and differential Inhibits Gram +, selects Gram -, Also contains lactose, lactose + is blue, metallic green is E. coli (also +) Oxidase Gram Negative The oxidase test uses an alternate molecule as an electron donor. When that molecule is present, the paper turns a dark blue color. If its blue, then the bacteria is positive for the enzyme cytochrome oxidase UTI ID Membrane Gram negative Can identify UTIs Put membrane on colonies grown on NA plate, then incubate -end of unknown lab experiments Plaque assay The amount of viruses produced in affected cells can be measured by a plaque assay Researcher puts single virus with a bunch of host cells The virus spreads and lysis cells, these are plaques ELISA Simulate testing of body fluids as if diagnosing the presence of HIV A positive (blue) result comes from the presence of the antigen in the fluid sample Controls help determine inconclusive results with human error False positives can result from recent vaccination False negatives can result from immune suppression, or to soon after having HIV  Pathogenic Eukaryotes Entamoeba histolytica- amoebiasis Giardia lamblia- giardiasis Trypanosoma brucei- sleeping sickness Penicillium spp. (spp means species pluralis, or multiple species) Aspergillus spp. Rhizopus spp. Saccharomyces spp. Taenia spp.- bladder worm or cysticercus Ascaris lumbricoides Infectious Diseases Bacillus anthracis- anthrax Clostridium botulinum- botulism Clostridium tetani- cause of tetanus Corynebacterium diphtheriae- diphtheria Haemophilus influenzae- bacterial meningitis Mycobacterium tuberculosis- TB Nisseria gonorrhoeae- gonorrhea Salmonella typhi- typhoid fever Shigella dysenteriae- dysentery Staphylococcus aureus- cause of boils and pimples Streptococcus pneumoniae- pneumonia Streptococcus pyogenes- scarlet fever, glomerulonephritis Plasmodium falciparum- malaria Streptococcal diseases A-hemolysis Strep. Pneumoniae- pneumonia, conjunctivitis Strep. Viridans- dental caries and endocarditis B-hemolysis Strep. Pyogenes- pharyngitis, rheumatic fever, glomerulonephritis Strep. Agalactiae- meningitis Y-hemolysis Strep. Enterococcus- endocarditis, UTI, abd infection