BIOL 221 Lab Practical Study Guide copy

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Apr 3, 2024

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DISCLAIMER: This is a summary of all the exercises covered in the 1st half of the lab. It doesn’t include the level of detail displayed in the lab manual or Brightspace Presentations. It is supplemental study material only. BIOL 221 Lab Practical 1 Study Guide Exercise 00: Lab Safety, PPE, and Biosafety Levels

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Exercise 1: Basic Microscopy Techniques Familiarizing yourself with the Light Microscope Find these on the microscope: arm, base, ocular lenses (10x), rotating nosepiece with four objectives (4x, 10x, 40x, and 100x), fine and coarse adjustment knobs, stage and stage controls, iris diaphragm, condenser, light source, light control/rheostat Resolving power - distinguish two adjacent objects from each other Resolution - clarity of an image How to improve resolution: - Concentrate light using condenser (focus condenser lens) - Use light with shorter wavelength - Use immersion oil (same refractive index as glass) Contrast - distinguish from background Total Magnification = ocular magnification x objective magnification Working Distance - distance between slide and objective lens - smaller working distance = higher magnification

Correct Microscope Storage 1. Clean each objective lens (len paper and lens cleaner) 2. Start with the lower-power objective (4x) all the way through the high-power objective (100x) 2. Clean the ocular lenses 3. Once all lenses are clean, turn the rotating nosepiece to the 4x objective 4. Lower the stage all the way down 5. Wrap the microscope cord 6. Carefully store your microscope back in its location (hold it by the base and the arm), and place the microscope cover on top. Eukaryotes vs Prokaryotes General Characteristics Under the Microscope: examples Prokaryote - E. coli Letter slide Eukaryote - onion root

Wet Mount Procedure Examples: Some things to think about: - Wet mounts allow us to see ____ (movement), unlike stains that we heat fix. - Green color is from chlorophyll. How might some of these pond organisms get some of their energy? (hint: how do plants do it?) *** Slides and Coverslips go in the SHARPS CONTAINER

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Exercise 2: Aseptic Technique Turbidity - cloudiness of media that indicates growth Contamination - unintended introduction of microbes such as bacteria, mold, and fungi Aseptic technique example (LB broth): 1) Flame the loop until red hot 2) Take off cap of bacteria test tube with pinky 3) Flame mouth of tube, dip loop, flame mouth of tube again, put cap back on 4) Take off cap of LB broth tube you want to inoculate with pinky 5) Flame mouth of tube, dip loop into broth, flame mouth of tube, put cap back on 6) Flame the loop until red hot again **aseptic technique should always be applied, regardless of the media (tubes, plates, etc). It is just a good practice to insure there is no contamination Exercise 3: Gram Stain and Simple Stain Gram Negative vs Gram Positive

Gram Stain Dyes/Reagents: How They Work 1) Crystal violet dyes all cells. 2) Iodine traps crystal violet in the thick peptidoglycan layer of gram positive cells. 3) Alcohol wash breaks the outer membrane of gram negative cells, releasing all purple color from them. 4) Safranin dyes the colorless gram negative cells pink. Examples: Gram positive (S. epidermidis) Gram Negative (E. coli) Simple Stain - Same bacteria application, heat fixing, and drying procedure as gram stain (refer to lab manual for full procedure in detail) - Chose either crystal violet or safranin to stain bacteria - Will only give us shape and arrangement - Will NOT tell you what gram it is

Examples of Shape and Arrangement: Other Types of Stains:

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Exercise 4: Bacterial Isolation Colony: large mass of bacteria derived from a single cell Bacterial isolation: technique used to separate one species from another based on morphological differences. Streaking: technique that dilutes bacteria to the point where you can obtain single colonies Pure culture: contains one species of bacteria T Streak Technique Steps Steps 1-3 shown on blood agar plates 1) Using aseptic technique, dip the loop into the broth. Spread the loop back and forth in zone 1 of the plate. 2) Flame the loop. Let it cool. Starting in zone 1, continue streaking on to zone 2 by dragging your loop in a tight zig-zag. 3) Flame the loop. Starting in zone 2, continue streaking in zone 3 by dragging your loop in a more spaced out zig-zag. - If the loop is too hot, tap loop on zone 3 until the sizzle sound stops. If you try to streak when the loop is hot, you will kill bacteria. ***Plate labeling: last name, first name initials, date, division ID, name of media, and name of bacteria.

Exercise 5: Enumerating Bacteria Spread Plating (Titer) Plate 3 Plate 4 Plate 5 Units: CFU/ml T = titer = # of cells (CFU) per mL in the original sample N = # of colonies appearing on the plate after incubation DF = dilution factor V = volume plated ***Only calculate titer for plates with in between 20-300 colonies

Direct Microscopic Count (DMC How to Read a Pipette

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Exercise 6: Selective and Differential Uninoculated MacConkey , MSA , and Snyder Snyder Agar Selective: low pH of 4.5 used to select acid tolerating organisms - Inhibits non-acid tolerating organisms Differential: uses glucose and bromocresol green to distinguish between: - Glucose fermenters: Yellow (+) - Non-glucose fermenters: Green (-) acid sensitive vs acid tolerant (-) glucose fermentation both acid tolerant (-) glucose fermentation vs (+) glucose fermentation

Mannitol Salt Agar (MSA) Selective: 7.5% NaCl selects for salt tolerant organisms - Inhibits non-salt tolerating organisms Differential: uses mannitol and phenol red to distinguish between: - Mannitol fermenters: yellow, acidic (+) - Non mannitol fermenters: red, neutral (-) salt tolerant (-) mannitol fermentation vs salt sensitive salt sensitive vs salt tolerant (+) mannitol fermentation MacConkey Agar Selective: bile salts and crystal violet select for gram negative - Inhibits gram positive growth Differential: uses lactose and neutral red to distinguish between: - Lactose fermenters: pink, acidic (+) - Non-lactose fermenters: yellow, basic (-) gram negative (-) lactose fermentation vs gram positive Gram negative (+) lactose fermentation vs gram negative (-) lactose fermentation

***Tricks for Remembering Differential Color Changes - Snyder: Lemonade is sugary and yellow (yellow colonies are glucose fermenters). Kale is green and bitter because it is not sweet (green colonies are not glucose fermenters). - MSA: Lemonade is sugary and yellow (yellow colonies are mannitol fermenters). Dry red wine is gross because it is not sweet (red colonies are not mannitol fermenters). - MacConkey: Strawberry milk contains lactose (pink colonies are lactose fermenters). No lactose in lemonade (yellow colonies do not ferment lactose) Exercise 7: Oxygen Requirements Fluid Thioglycollate - agar and reducing agent creates oxygen gradient - Oxygen indicator: resazurin dye (oxygen = pink ring) Exercise 8: Carbon Requirements Citrate Utilization Test - uses bromothymol blue (basic) indicator to see if citrase is being utilized to convert ammonia salt into ammonia - Stab streak technique on Simmon’s Citrate Slant - Citrase produced and converts ammonia salt into ammonia: Blue (+) - No citrase utilization, no growth: Green (-)

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Methyl Red (MR) - tests for acid production from glucose fermentation - Sugar to lactic acid, acetic acid, or succinic acid - 3 drops of methyl red show results: - acid formation: red (+) - no acid: no color change (-) Voges Proskauer (VP) - tests for alcohol production from glucose fermentation - Pyruvic acid → acetoin → EtOH - 15 drops Barrit’s A and 15 drops Barrit’s B show results: - Alcohol formation : red ring at top of tube (+) - No alcohol formation: no color change (-) Phenol Red Lactose Broth - uses phenol red to indicate lactose fermentation and a durham tube to indicate CO2 gas formation. Results: - Ferments lactose, CO2: yellow (+), with gas bubble - Ferments lactose: yellow (+), no bubble - No lactose fermentation: red (-) , no bubble - Ammonia produced from proteins: fuschia, no bubble - If no carbon fermentation, there is peptone utilization

Exercise 9: Enzymatic Tests Catalase Test - uses H2O2 3% to see if an organism can detoxify and use oxygen. Majority of facultative anaerobes and aerobes use catalase to break down toxic H2O2 metabolite into harmless O2 and water. Results: - Catalase utilized: bubbles formed (+) - No catalase utilized: no bubbles (-) Oxidase - tests for cytochrome C oxidase using p=phenylenediamine (oxidase reagent and artificial e- donor). Results: - Oxidase positive: Blue (+) - Oxidase negative: no color (-) Tryptophanase Test Tests from tryptophanase by inoculating tryptone broth with organism and observing the color after adding Kovac’s reagent. - Tryptophan → indole, pyruvic acid, and ammonia - Indole reacts with Kovac’s reagent to produce red Results: - Tryptophanase (indole produced): Red (+) - No tryptophanase: no color change (-)

Phenylalanine Deaminase Test Tests for production of phenylalanine deaminase (phenylalanine → phenylpyruvic acid and ammonia). Organism is stab-streaked on phenylalanine slant. Ferric Chloride 10% added to see results. Results: - Deaminase positive: Green (+) - Deaminase negative: no color change (-) Exercise 10: Bloodstream Infections Urease Test - test for hydrolysis of urea into ammonia by urease. Makes it easier to live in acidic places. Results: - Urease present: pink (+), basic - No urease: no color change (-)

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Coagulase - inoculate rabbit plasma with an organism. Clots = fibrinogen → fibrin Results: - Coagulase: clots, pathogen (+) - No coagulase: no clots, non-pathogenic (-) CAMP Factor - streak blood agar with S. aureus and organism. The CAMP test is a test to identify β -hemolytic streptococci based on their formation of a substance that enlarges the area of hemolysis formed by the β -hemolysin elaborated from S. aureus. Results: - CAMP positive: triangle clearing (+) - CAMP negative: no clearing (-) In this picture, the vertical line is S. aureus and the 2 horizontal lines are 2 different organisms being tested. (In lab, out positive control was S. agalactiae)

Blood Agar Only differential: uses 5% sheep’s blood to distinguish between: - Beta hemolysis: Complete lysis of red blood cells - Alpha hemolysis: Not a true lysis. Green/khaki color halo surrounding growth due to reduction of hemoglobin into methemoglobin. If the plate is left in the incubator for too long, it may show signs of hemolysis. Always look for color to determine alpha hemolysis. - Gamma hemolysis: No lysis of red blood cells

BIOL 221 Lab Practical Study Guide copy

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