Midterm -1913L – Flashcards
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| Classifications of media |
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| 1) Non-selective - most organisms can grow (general purpose media) 2) Selective - Inhibits growth of some groups, but allows for growth of others. 3) Differential - Allows for identification of certain species or groups of organisms. 4) Enrichment - Used to isolate organisms from complex samples. |
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| Solidifying agent used in our lab |
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| Agar |
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| Difference between broth and solid media |
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| Broth does not contain agar; solid media does |
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| Fomite |
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| Inanimate object that can harbor pathogens |
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| Ubiquity of microorganisms |
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| Microorganisms are EVERYWHERE |
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| Aseptic technique |
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| Methodology used to reduce contamination of cultures, people, and workspace |
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| Innoculation |
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| To introduce a microorganism into |
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| Growth patterns in broth |
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| Pellicle, Turbidity, Sediment, and Flocculent |
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| Benefits of a digital pipette |
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| Allows the experimenter to transfer precise volumes of liquid from one container to another. |
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| Number of cells a colony arises from |
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| One (i.e. 1 colony = 1 cell) |
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| Understand the microscope and total magnification |
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| See PowerPoint from Lab 2 |
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| Streak plate |
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| Serves to: 1) Isolate pure colonies of bacteria from a mixed culture of bacterial species 2) Observe morphology of the isolated colonies for identification and classification |
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| Spread plate |
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| Used to evenly distribute bacterial cells over entire plate surface (2 possible results): 1) Spread concentrated samples to create an even lawn of bacterial growth 2) Spread diluted sample of bacteria so individual colonies can be seen *100?L is the standard amount used for spread plates* |
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| What is used with 100X on our microscopes |
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| Immersion oil (or Ooyle, if you're from AL) ;) |
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| Smear prep procedure |
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| Draw a circle on the back of a clean slide with marker Flame loop If making smear from an agar plate: -Apply a couple of drops of water to slide and smear a small amount of bacteria in the water -Flame loop If making smear from a broth culture: -Take a loopful of bacteria and smear on the slide (No Water) -Flame loop Make sure to spread bacteria evenly over most of the slide surface to create a thin smear Air dry smear completely Apply enough methanol to cover and fix the smear, let sit for 5-10 seconds Rinse with water |
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| Purpose of fixing |
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| Fixing serves 2 purposes: 1)Adheres the cells to the slide 2) Kills the organism |
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| Difference between simple and Gram staining |
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| Simple uses only ONE stain, while Gram uses a primary and a secondary stain. |
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| Gram stain steps and components |
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| Step 1: Flood slide with Crystal Violet for 30 seconds - 1 minute Wash with H2O -Acts as Primary Stain Step 2: Flood slide with Gram’s Iodine for 30 seconds - 1 minute Wash with H2O -Acts as Mordant Step 3: Decolorize with Acetone alcohol for a few seconds Wash with H2O -Acts as Decolorizer Step 4: Flood slide with Safranin for 30 seconds - 1 min Wash with H2O -Acts as Secondary Stain |
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| Purpose of Gram stain |
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| Differentiate between Gram-positive and Gram-negative bacteria. Also allows determination of (1) cell morphology, (2) size, and (3) arrangement. |
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| Gram stain results |
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| + = Blue (purple) - = Pink |
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| Purpose of endospore |
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| Allows bacterium to survive in poor environmental conditions |
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| Purpose of flagella |
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| Motility |
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| What characteristics can be determined from simple stain |
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| Cellular morphology Cellular arrangement |
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| Cell wall component that dictates Gram reaction |
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| Peptidoglycan |
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| Most critical Gram step |
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| Step 3 - decolorization |
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| Endospore staining procedure (Schaeffer-Fulton method) |
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| Step 1: Flood slide with Malachite Green -Acts as Primary Stain Step 2: Heat over steam bath for 5 min -Acts as Mordant Step 3: Rinse with Water for a few seconds -Acts as Decolorizer Step 4: Flood slide with Safranin for 2 min -Acts as Secondary Stain |
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| Acid Fast stain |
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| Used primarily to stain & identify members of the genera Mycobacterium and Nocardia -A differential stain used to detect the presence of mycolic acids in the cell wall -Cells typically don’t stain well with Gram Stain method -Acid-fast organisms stain pink and non-acid fast organisms stain light blue with these methods |
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| Capsule stain |
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| Used to visualize gelatinous coating that surrounds some bacteria SMEAR IS NOT HEATED or the capsule melts! Background is stained by one of the following stains: (a) Nigrosin (b) India Ink (c) Congo Red Counterstain with a basic stain to stain cells *Important to use older cultures so capsules have time to form* |
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| Flagella stain |
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| Many bacteria are motile due to production of flagella Flagella are very fine, hair-like structures that are difficult to stain due to their small size and delicate nature Requires the addition of mordant and stain to increase the diameter of flagella for microscopic observation |
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| Flagellar arrangements |
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| Monotrichous (single polar) Lophotrichous (clump of tuft at one or both poles) Amphitrichous (single flagella at each pole) Peritrichous (covered) |
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| Acid fast families |
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| Myobacterium Nocardia |
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| Spore-producing famililes |
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| Bacillus Clostridium |
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| Mannitol Salt Agar (MSA) |
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| Selective Agent: NaCl (Salt) -Selects for Staphyloccus spp. Differential Agent: Mannitol -Differentiates pathogenic from non pathogenic Staphylococcus spp. pH Indicator: Phenol Red -Indicates acid production due to mannitol fermentation |
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| Phenylethyl Alcohol Agar (PEA) |
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| Selects for Gram positives -Growth of Gram negative organisms should be reduced or inhibited Selective Agent: Phenylethyl Alcohol Not Differential |
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| Eosin Methylene Blue Agar (EMB) |
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| Selects for Gram negatives -Growth of Gram positive organisms should be reduced or inhibited Selective Agents: Eosin Y & Methylene Blue Differential Agent: Lactose -Differentiates lactose fermenters from lactose non fermenters Indicator: Eosin Y -Indicates acid production due to lactose fermentation E. coli is easily identified by the production of green, metallic sheen |
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| MacConkey Agar (MAC) |
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| Selective for Gram negatives -Growth of Gram positive organisms should be reduced or inhibited Selective Agents: Crystal Violet & Bile Salts Differential Agent: Lactose -Differentiates lactose fermenters from lactose non fermenters pH Indicator: Neutral Red -Indicates acid production due to lactose fermentation |
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| Sheep's Blood Agar (SBA) |
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| Selective agent: None Differential agent: Blood |
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| Hemolytic patterns |
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(beta) hemolysis - complete lysis of RBCs (alpha) hemolysis - partial lysis of RBCs (gamma) hemolysis - no lysis of RBCs |
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| Catalase test |
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| Identifies organisms that produce the enzyme catalase Differentiates families of Gram positive cocci -Catalase (+) Micrococcaceae from -Catalase (-) Streptococcaceae Substrate: hydrogen peroxide (+) test: bubbles form immediately (-) test: no bubbles |
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| Oxidase test |
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| Identifies bacteria that produce the respiratory enzyme cytochrome c oxidase Differentiates families of Gram negative rods -Oxidase (-) Enterobacteriaceae from -Oxidase (+) Pseudomonadaceae Reagent: Oxidase reagent (+) test: blue color appears within seconds (-) test: no color change |
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| Coagulase test |
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| Differentiates S. aureus from other Gram-positive cocci Substrate: rabbit plasma (+) test: formation of white clumps (-) test: milky white liquid (no clumps) |
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| Amylase test |
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| Media: Starch Agar Plate (SA) Reagent: (Lugol's) Iodine (I2KI - Iodine Potassium Iodide)After incubation, plate is flooded with iodine:(-) test: Iodine binds with remaining starch in the plate to produce a dark brown/black color(+) test: As a result of starch breakdown, a clear area or halo will be visible around the colony that produces amylase |
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| Urease test |
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| Differentiates organisms based on their ability to hydrolyze urea with urease Helpful in detecting rapid urease positive organisms such as Proteus sp. from other Enterobacteriaceae Media: Urea agar slant Positive reaction overcomes the buffer in the medium and changes the color of the slant (+) test: Hot pink slant (-) test: Peach or yellow slant pH Indicator: Phenol Red |
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| Aerotolerance |
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| The ability or inability of an organism to grow in the presence of molecular oxygen (O2) |
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| Classificaitons of aerotolerance |
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Obligate aerobe - requires O2 for growth Falcultative anaerobe - can grow in the presence or absence of O2 Obligate anaerobe - cannot grow in the presence of O2 Microaerophile - requires low levels of O2 Capnophile - requires elevated levels of CO2 |
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| Thioglycollate medium |
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| A liquid medium formulated to promote growth of a fastidious anaerobes without an anaerobe jar -Creates anaerobic conditions by reducing oxygen to water Oxygen Indicator: Resazurin If oxygen present = Pink/Red If no oxygen present = Colorless |
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| Anaerobic jar |
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| Provides an anaerobic environment as a result of an added anaerobic gas pack Oxygen indicator: Methylene Blue If oxygen present = blue If no oxygen present = colorless (white) |
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| Most prevalent mutation caused by UV |
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| Thymine dimers |
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| UV Radiation |
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| Short wave length Mutagenic because it excites nucleic acid bases in DNA Penetrates glass and plastic poorly Prolonged exposure is lethal to cells |
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| Membrane filtration |
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| Used to detect and directly count fecal coliforms in water Normally use a 0.45 µm filter (0.22-0.45) It is very important to know the size of your target organism or compound to choose the correct filter E. coli ~ 1 ?m X 2 ?m Coliform counts are calculated per 100 mL of water Media: Endo broth Membrane filter is transferred to sterile Petri dish with sterile pad saturated with Endo broth |
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| Fecal bacteria |
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| Coliforms & Fecal Streptococci (including Enterococci) are used as possible indicator organisms for sewage contamination. -Coliforms are comprised of many organisms including the fecal coliform Escherichia coli. -E. coli is a normal intestinal colonizer of humans and other warm-bloodied animals . -The EPA recommends Total Coliform counts for drinking water, E. coli counts for recreational fresh water & Enterococci for recreational salt water. |
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| Endo Broth |
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| Selects for Gram(-) organisms Selective Agents: Sodium Sulfite & Basic Fuchsin Differential Agent: Lactose Indicators: Sodium Sulfite & Basic Fuchsin Lactose fermenters = metallic sheen OR dark pink Nonlactose fermenters = light pink |
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| Enumerating bacteria |
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| Bacterial populations can be enumerated in two ways: (1) Direct - actual number of cells (2) Indirect - relative amounts compared to beginning of experiment |
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| Purpose of performing viable plate count |
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| To obtain actual numbers of viable cells |
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| CFU stands for |
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| colony forming units |
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| OCD stands for |
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| original cell density |
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| Countable plate considerations |
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| 30-300 CFU. Below or above this number is considered statistically unreliable |
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| Serial dillutions |
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| -Systematically reduces cell density, producing at least one dilution that will yield a countable plate -Provides a reference to link the unknown (original cell density) with the known number of colonies on the plate -In a serial dilution, each transfer reduces the amount of original broth in the solution -Each dilution is given a dilution factor -The dilution factor of each new solution is compounded by the dilution factor of the previous tube |
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| Original Cell Density |
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| After incubation the colony forming units (CFU) are counted -The term CFU is used since more than one cell could form one colony -Only a plate with a colony count between 30-300 CFU can be used to determine OCD -Plates below or above this range are considered statistically unreliable -OCD = CFU/FDF (combination of volume plated and dilution factor = final dilution factor) -Ex: 47 CFU growing on 10-6 plate OCD = 47/10-6 OCD = 47 x 106 = 4.7 x 107 (correct scientific notation) |
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| Optimal temperature |
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| Temperature at which growth rate is maximum (optimal) |
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| Temperature classifictions |
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| Psychrophiles: -10 to 20°C Psychrotrophs: 0°C to 30°C -Their optimum temperature falls in the mesophilic range, however, they can grow at lower temperatures at slower rates -These are typically food spoilage organisms Mesophiles: 10°C to 50°C Thermophiles: 40°C to 75°C Hyperthermophiles: >75°C |
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| IMViC |
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| Series of tests used to differentiate Enterobacteriaceae -Includes: Indole Methyl Red Voges-Proskauer Citrate |
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| Indole test |
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| Media: Tryptone broth containing tryptophan Purpose: Determine bacteria’s ability to produce indole from tryptophan Reagent: Kovac’s Reagent (after incubation) 5 drops of reagent (+) reaction = red ring (-) reaction = yellow or colorless ring |
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| Methyl Red test |
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| Media: MRVP broth Bacteria are inoculated into MRVP broth, divided after incubation Purpose: Detects organisms capable of producing stable acid end products from mixed acid fermentation of glucose Reagent: Methyl Red Reagent Usually combined with VP test (+) reaction = media turns red (-) reaction = no color change |
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| Voges-Proskauer test |
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| Media: MRVP broth Bacteria are inoculated into MRVP broth, divided after incubation Purpose: Detects production of acetoin & 2,3-butanediol Reagents: ?-naphthol is added FIRST (mix well) 40% potassium hydroxide added SECOND (mix well) Usually combined with Methyl Red test (+) reaction = red color change (-) reaction = no color change |
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| Citrate test |
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| Media: Simmon’s Citrate agar Purpose: Determine bacteria’s ability to utilize citrate as sole carbon source pH indicator: Bromthymol blue Increase in pH = color change to blue (+) reaction = blue agar (-) reaction = green agar |
