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Biology 240L Final Exam

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University of Waterloo
BIOL 240
Cheryl Duxbury

Biology 240L Final Exam Lab Safety:  inoculating loop/needle should be heated to a red-hot stage  Disposal of lab material: o Glass Graduated Pipettes- discard with tip pointing down into tall plastic container; don’t include Pasteur pipettes or plastic pipette tips o Glass Microscope Slides- small labeled trays above workbench (don’t clean slides) o Small Disposable Items- includes swabs, plastic/glass Pasteur pipettes, dispose in small bucket on above workbench o Large Disposable Items- includes plastic petri dishes, dispose in large trays on side bench o Other Glassware- dispose in large trays on side bench (remove all labels/markings, and separate agar from broth tubes)  Working with microorganisms classified as Biosafety Level 2- class 2 microorganisms in lab manual found with bold and asterisk; A class 2 Safety Cabinet used in lab (has HEPA filters)  Use of Bacti-cinerator: inoculating loop and needle sterilizer- used when working with level 2 pathogen. They’re designed to eliminate aerosols (potential source of contamination). Wait 10 mins after incinerator is on for optimal sterilizing temperature. Keep loop in heater for minimum of 10 seconds; microorganisms should be destroyed in 10-15 seconds; don’t need a glowing loop to ensure sterility Emergency Procedures:  Fire and Explosion: Fire extinguishers: o Types of Fires: A-ordinary combustibles, B- flammable liquids, C- electrical fires, D- combustible materials o Operation of fire extinguishers (PASS)- Pull out pin, Aim pivoted horn at base of fire, Squeeze trigger at top of extinguisher, Sweep back and forth covering fire with entire contents of extinguisher o Water-type and Soda-acid extinguishers- good for wood, paper, textile, rubbish fires (Type A). Never use water on electrical fires (Type C) o Carbon Dioxide extinguishers- used on most fires (Types A,B,C) except on sodium, potassium, magnesium and aluminum (Type D) o Dry chemical extinguishers- good for flammable liquids and electrical fires (Types A,B,C)  If clothes catch on fire; stop, drop and roll (don’t run), or have another person smother flames with fire blanket  Acid or Chemical Spills- consult MSDS, and wash with a lot of water  Safety Shower/ Eye Wash- hand-held safety shower/eye wash units; used for large spills; wash eyes for at least15 minutes Experiments 1. Simple Staining  Increase contrast by using colour compound stain to see microorganism under conventional light microscope  Simple staining- single stain used so all cells stain same colour, but can distinguish based on morphology  Common dyes: methylene blue (blue), basic fuchsin (red), and crystal violet (purple)  Two types of staining procedures: positive and negative o Positive: for light microscopy, stain is basic has positively charged (cationic) chromophore which attracts to the slightly negatively charged outer surface of bacteria  Used Loeffler’s Methylene Blue stain for this experiment  Flame loop in coolest part (yellow), then in hot (blue cone) until it glows red at 45 degree angle  Use Aseptic technique to inoculate tube and transfer bacterial broth on microscope slide, heat fix, cover with 1-3 drops, and gently wash off with water 2. Operation of the Microscope  Magnification achieved by 2 lenses: objective and ocular (eyepiece) lens; objective magnifies specimen to produce real image which is projected to ocular lens  Light directed upwards through condenser which has a lens that concentrates light; the amount of light is adjusted to match the numerical aperture of the objective by turning the notched ring on the condenser  Lowering the condenser decreases the amount of light that reaches the object  When using 100X oil immersion objective, make sure condenser lens is in the uppermost position and set iris diaphragm to 100  Ocular/eyepiece lens has a magnification of 10X  The oil on oil immersion lens improves resolution of the magnified image; resolving power/resolution is the ability to distinguish two images as separate (max. on light microscope is 0.2 µm)  Most modern microscopes are parafocal- when a specimen is in focus using one lens it remains in focus when switching to another objective lens  When returning, make sure ocular lens are facing the back/handle side, and leave stage raised  Results: Staphylococcus epidermidis, group/clusters of coccus; Bacillus subtilis, chains of rods 3. Basic Bacterial Shapes  Bacteria- 3 general shapes: rod (bacillus), round (coccus), and corkscrew or spiral (spirochete or spirillum). Other type: comma shaped (vibrio)- variations due to age/composition of the culture media Characteristic Coccus Bacillus Sprillium Shape of Cell Round Rod Spiral Motility Most nonmotile Most motile Most motile Arrangement Singly (micrococcus) Singly Singly spirochetes Pairs (diplococcus) Pairs (diplobacillus) Chains Chains (streptococcus) (streptobacillus) Groups of four (Gaffkya) Groups (cubes) of eight (sarcina) Clusters or packets (staphylococcus) Spores None Produced by several None different species  Calculate magnification of specimen drawing: size of specimen in drawing (in µm) – 1mm= 1000 µm/ estimated or actual size of image (in µm)  Results: E.coli- short individual rods, B.subtilis- chains of rods, S.epidermidis- groups of coccus, Rhodospirillium rubrum- singular, spiral 4. The Gram Stain  Devised by Christian Gram, most widely used in bacteriology, used to differentiate types of bacteria depending on their abilities to retain a certain dye (differential staining technique)  Bacteria divided into Gram-positive and Gram-Negative  Steps: stain fixed smear with primary stain of crystal violet, then add Gram’s iodine stain (mordant which fixes primary stain in bacterial cells), wash stained smear with decolourizing agent 95% ethyl alcohol, then counterstain with safranin  Gram-positive not easily decolourized and therefore retains purple colour from crystal violet, Gram-negative organisms are decolourized and stained red/pink from safranin  Age of cultures and acidity or alkalinity (pH) of medium which bacteria are grown will affect reaction to Gram’s stain o Older cultures of Gram-positive organisms and those in acid medium appear either Gram-negative or Gram-variable (both Gram-positive and Gram-negative cells of same organism in same culture) o Gram-negative organisms may appear Gram-variable due to age/medium used  Results: S.epidermidis- cluster of coccus, Gram positive (purple); E.coli- singular rods, Gram negative (pink) 5. The Acid-Fast Stain (Kinyoun Method)  Similar to Gram stain, also differential stain used to identify tuberculosis bacillus, Mycobacterium tuberculosis and Mycobacterium leprae  Particularly bacteria in genus Mycobacterium, do not stain readily by simple stain or Gram stain due to waxy cell walls (large lipoidal material- mycolic acids) that are impermeable to most stains  Ziehl-Neelsen procedure smear is flooded with carbolfuchsin (red dye with 5% phenol) which has high affinity for lipid-rich material of bacterial cell wall, then heated to facilitate penetration; once stained they retain the dye even when treated with decolourizing agent- acid-fast due to this property . Methylene blue used to counterstain to observe non-acid fast organisms  Acid-fast organisms appear red and non-acid fast organisms appear blue  In Kinyoun modification called a cold-stain, concentrations of phenol and carbolfuchsin are increased and detergent is added so heating isn’t necessary  All acid-fast organisms are Gram-positive, but not all Gram positive organisms are acid- fast  Procedure: Mycobacteria would be on agar slant, and quite waxy due to mycolic acids in cell wall, prepare heat fix smear, flame loop to prevent sputtering, in fumehood add 4-5 drops of carbolfuchsin and stain for 5-10 mins and keep slide moist by adding more stain, rinse with tap water then decolourize with acid-alcohol, rinse with tap water and counterstain with methylene blue, rinse again with water, blot dry and examine on oil- immersion objective  Results: S.epidermidis , acid-alcohol fastness negative, M.tuberculosis, acid-alcohol fastness positive 6. The Spore Stain (Schaeffer-Fulton Method)  Environmental conditions too harsh to permit further vegetative growth and reproduction, certain species such as Bacillus and Clostridium are capable of condensing vital cellular components into an endospore  Vegetative cells slow down, lose moisture, and withdraws substance into one area which it surrounds with a thick impermeable wall during sporulation; remnants of vegetative cell fall away and endospore (spore) remains highly resistant to environmental influences  Free spores resist ordinary dyes, but once stained it will resist decolourization by various solvents  Primary stain malachite green driven into cell (for 30 mins) with heat, then counterstained with safranin to give vegetative cells a contrasting colour (light red); must over heat fix by running slide through flame approx. 5 times  Spores stain green and vegetative cells stain red  Results: C.sporogenes- has spores (still inside vegetative cells which surround it), B.megaterium- has spores (spores outside of vegetative cells) 7. The Negative Stain  Uses acid-dye nigrosin (India ink) which has negative charges on its chromophore and therefore will be repelled by the negatively charged surface of bacteria- cell remains unstained (clear) and background is coloured (dark grey/black)  Does not involve heat fixing and therefore little distortion of cells; natural size/shape can be seen; also a way to see bacteria that are difficult to stain such as Mycobacterium  Place small drop of Nigrosin near one end of glass slide and mix bacteria in this drop, then use edge of another spreading slide to spread drop out into smear from black to grey, then let air dry  Cells are unstained because blackish colloidal particles of Nigrosin will not penetrate the interior of cells  Results: B.megaterium clear, rod shaped, short chains 8. The Capsule Stain  Cell wall of certain bacteria species often surrounded by an envelope of mucilaginous substances- capsule, slime layer, or glycocalyx  Capsule usually consists of polysaccharides, polypeptides, and/or carbohydrate which accumulate on cell surface  Alcian blue dye is water soluble and believed to form linkages with acid groups of acidic mucopolysaccharides staining them blue; the capsule will appear blue  Among disease producing bacteria, heavy capsule often indicates highly virulent form of the organism because capsule makes destruction of microbe by phagocytic cells difficult – used to diagnose pneumonia and other diseases  May also be detected using negative stain (used in this experiment); polysaccharides are water soluble and uncharged, simple stain’s will not adhere to it, therefore stain the bacteria and background but not the capsule- “negative” capsule stain  After negative stain flood with crystal violet, then add water and allow stain to float up (prevents stain from forming a precipitate and sticking to the slide), don’t blot dry(not heat fixed), then flood with Gram’s safranin and float this off too  Results: K. pneumonia -Cells appear pink/red and capsule is clear zone around cell 9. Culture Media Preparation and Sterilization  Medium whose chemical constituents are known is a chemically defined medium; media for fastidious organisms (human pathogens) may contain complex materials (digest of plants etc.) whose composition is not clearly defined known as complex media  Nutrient broth/agar commonly used since it supports growth of large number of organisms and is called all purpose or general purpose medium, in contrast to selective or differential media  Selective media contain chemical substances which prevent growth of one or more groups of bacteria without inhibiting the growth of the desired one  Differential media contain chemicals which permit differentiation between types of bacteria  Nutrient broth contains beef extract and peptone (partially digest protein)  Solid medium made by adding agar to broth medium; agar made of complex carbs, galactan, extracted from marine alga of genus Gelidium, it’s not used as food by most microorganisms and it melts at 100 degrees and remains liquid until 43 degrees; once solidified it’s reheated to 100 degrees to cause liquefaction  “Sterile” and “sterilized” mean free of all life, including viruses  The principal lethal agent in steam sterilization is heat; the enzymes and cellular proteins are irreversibly destroyed  Autoclave used to sterilize bacteriological media and surgical equipment- “pressure cooker.” Build up temperature by building up steam pressure and prevent boiling. Normal steam sterilization temperature kept at 121 degrees (pressure gauge at 15-16 pounds/square inch) for 15-20 minutes  Add 500 mL distilled water to 1L Erlenmeyer flask, add ½ L nutrient broth, use magnetic stirrer to prevent lumping, add agar-amount of agar needed is 15g/L (1.5g/100mL), set flask on heating-stir plate and bring it to a boil stirring constantly, remove from heat and dispense into bottles, place cap but do not tighten (contents expand during sterilization with steam- don’t want it to explode/improper sterilize). Also incubate broth tubes non- sterile at room temp. and other half nutrient broths for sterilization  Results: Non-sterile broth showed growth and broth was foggy/turbid, while sterile broth appears transparent  Use molten agar and pour into petri dishes aseptically, once solidified expose one to laboratory air and other to cultivate organism sample of choice with wet cotton swab, then incubate plates at room temp. for a week  Results: laboratory had many different types of contaminants from microorganisms of varying morphologies, and lab bench shelf had only 1 type of organism visible that was yellow and mucoid 10. Selective, Differential, and Enriched Media  To help isolate organisms found in minority, various enrichment and selective culturing methods available to enhance growth of some organisms or inhibit growth of others  Selective Media: contain chemicals that don’t affect growth of microorganism you wish to isolate but discourage growth of other microorganisms o Example: Incorporation of sodium azide selectively isolates lactic acid bacteria such as streptococcus which lack a cytochrome system and are unaffected by sodium azide but prevent growth of organisms with this system o Selective agents can be dyes (crystal violet), or high concentration of NaCl, bile salts, antibiotics, specific sugars; KF Streptococcal Agar  Differential Media: Contains dyes/chemicals which allow observer to distinguish between types of bacterial colonies that have developed after incubation o Example: Eosin Methylene Blue (EMB- selective and differential) used to isolate and detect Enterobacteriace and related coliform rods such as E.coli which produces small colonies with dark centers and green metallic sheen o Enterobacter sp. Produce large pink mucoid colonies with dark centers and rarely show metallic sheen o Contains lactose as energy source so organisms that have enzyme β-galactosidase which break down lactose can grow, and organisms that don’t have the enzyme may be suppressed o Dyes eosin Y and methylene blue suppress the growth of various gram-positive organisms  Enriched Media: addition of components such as blood serum, extracts of plants/animals to nutrient broth or agar to provide additional nutrients to support the growth of fastidious heterotrophs (require specific nutrients such as vitamins and their stringent nutritional requirements termed fastidious)  Complex media: exact chemical composition is not known (eg. Extracts, partially digested proteins (peptone) etc.) – Ex. Tryptic Soy Agar  Results: Medium Bacterial Species Description of Growth Amount of Growth Appearance of Appearance of Growth Adjacent Medium Escherichia coli + + + + Opaque, cream/yellow Tryptic Soy Agar Enterobacter + + + + Opaque, aerogenes cream/yellow Staphylococcus + + + + Opaque, epidermidis white/cream Streptococcus + + + + Translucent, white faecalis Escherichia coli + + + + Green with metallic sheen EMB Agar Enterobacter + + + + Pink with dark aerogenes circles Staphylococcus - epidermidis Streptococcus - faecalis Escherichia coli - Enterobacter - KF Streptococcal aerogenes Agar Staphylococcus - epidermidis Streptococcus + + + + Pink faecalis 11. Streak-Plate Method – Isolation of Pure Cultures  Isolation in pure cultures- culture consists of only 1 type of microorganism to study a specific species  3 dilution methods: streak-plate, spread-plate, pour-plate  Streak plate is dilution technique that spreads culture over surface of agar plate  Used a four-way streak plate method, streaking plate but not overlapping for first quarter, then sterilize loop and cross over first section then continue streaking without overlapping, keep turning 90 degrees until whole plate has been streaked; incubated at 30 degrees for 24 hours in an inverted position  Results: prepared gram stain: E.coli- short rods, pink (gram-negative), larger than staphylococcus; S.aureus- groups of cocci, purple (gram-positive), smaller than E.coli; Mixed- E.coli (pink/red, rods, clustered) had larger colonies than S.aureus (purple, coccus, all connected) 12. The Pour-Plate Method  Another way to obtain isolated colonies from a mixed population; dilute specimen into a series of fluid-agar medium which is poured into petri dishes – make several dilutions to get distinct separate colonies  Colonies are visible in and on the agar on pour plate  Transfer 1 drop of mixed bacteria to tube 1 ofstolten nutrient agar, then 2 drops from tube 1 to tube 2, and tube 1 is poured into 1 petri dish; transfer 3 drops from tube 2 to tube 3 and tube 2 poured into 2 petri dish, and tube 3 poured into 3 petri dish; sterile nutrient agar poured into control petri dish- incubated for 48 hours at 30 degrees  Results: Plate 1- heavy growth, small colonies inside, and larger colonies on surface of agar, white/yellow colour; Plate 2- medium amount of growth, white/yellow, very large colonies on surface, some small colonies inside agar; Plate 3- Little to no growth, 3 very small colonies, yellow/white; control- no growth since it is poured from sterile broth 13. Aseptic Technique in Pipette Handling  Pipettes used to transfer bacterial suspensions or create serial dilutions  Open package at mouth portion and never at tip, attach bulb (not holding pipette at tip), put pipette in water after taking out air from the bulb, press S to apply suction, press E to st empty, keep pipette close to flame, transfer 10mL of water to 1 test tube, then 9mL to 2 test tube from 1 , 8mL to 3 from 2 , and 7mL to 4 from 3 , 6mL to 5 from 4 , th th th then 1 mL from 5 to nutrient broth; mix and incubate at room temperature for 1 week  Results: If bacterial growth, tubidity is detected; no turbidity therefore no visible
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