Applied Microbiology Lab Midterm

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What Biosafety Level lab are we in?

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Biology

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1

What Biosafety Level lab are we in?

Biosafety Level II lab

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Lab Apparel/PPE

  • lab coat with top two buttons buttoned at minimum

  • long pants cover the whole skin area

  • long hair tied back. ponytail tucked in back of lab coat

  • safety goggles

  • closed toe and closed heel shoes: no sandals, clogs, mules, or crocs

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Safety procedures

  • no food, drink, gum, or smoking

  • no putting on makeup

  • all belongings should be in the cubby (backpack, computer, etc.)

  • only thing at your station should be manual, pen, pencil, sharpie.

  • lab coat and writing utensils should be in Ziplock bag when not in use.

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How-to put-on PPE

  1. Wash hands

  2. Put on gloves right after washing hands

  3. Disinfect goggles with disinfectant and put them on

  4. Put on lab coat (button at least 2 buttons)

  5. Wipe down tabletop with disinfectant

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How-to- take- off PPE

  1. wipe down tabletop with disinfectant

  2. disinfect gloves with disinfectant

  3. unbutton lab coat

  4. remove gloves and dispose of

  5. remove lab coat

  6. wash hands thoroughly

  7. remove goggles and disinfect

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DISPOSAL- Biohazard box

  • gloves only in red biohazard box (NOT in black trash can or sharp containers)

  • Contaminated items (paper towels used for cleaning the bench or bacteria spills need to be disposed in red biohazard box in the front

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DISPOSAL- Regular trashcan

  • all contaminated paper towels (paper towels used to dry hands), petri dishes sleeves, cotton swab package

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DISPOSAL- Biohazard bags at back

  • contaminated plates (cannot exceed half full, tell TA)

  • contaminated enteropluri test tubes

  • contaminated GN-ID A & B Test strips

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DISPOSAL- Small bench top sharps container

  • contaminated razor blades, syringe needles, microscopic slides, covering slips, Kimwipes and cotton swabs

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DISPOSAL- Benchtop at the back

  • contaminated tubes (separate liquid(left) and solid(right) tubes

  • transfer your tubes from the white rack into the colored racks separately

  • all labels need to be removed from the tubes, and disposed into biohazard box

  • spray the white rack with disinfectant, and put them back in order next to the wall

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Micropipetting and the Basic Steps to Pipetting

  • measures in volume units of microliters (uL)

  • uses volumes between 0.5uL to 1000uL

  • uses disposable tips that should be discarded in sharps containers STEPS TO PIPETTING:

  1. Select a pipette based on the volume needed

  2. Adjust the volume on the pipette to the desired amount

  3. Attach tip snugly

  4. Depress plunger to first stop

  5. Place tip in fluid and slowly release plunger, leaving the tip in the fluid a few seconds after the plunger is fully released.

  6. Place the tip in the receiving tube, then eject the sample slowly by depressing the plunger to the second stop.

  7. Discard the tip using the ejector into a sharp's container.

<ul><li><p>measures in volume units of microliters (uL)</p></li><li><p>uses volumes between 0.5uL to 1000uL</p></li><li><p>uses disposable tips that should be discarded in sharps containers STEPS TO PIPETTING:</p></li></ul><ol><li><p>Select a pipette based on the volume needed</p></li><li><p>Adjust the volume on the pipette to the desired amount</p></li><li><p>Attach tip snugly</p></li><li><p>Depress plunger to first stop</p></li><li><p>Place tip in fluid and slowly release plunger, leaving the tip in the fluid a few seconds after the plunger is fully released.</p></li><li><p>Place the tip in the receiving tube, then eject the sample slowly by depressing the plunger to the second stop.</p></li><li><p>Discard the tip using the ejector into a sharp&apos;s container.</p></li></ol>
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Serial Dilutions

  • A dilution is performed by taking a portion (called an aliquot) of one solution and adding it to an appropriate amount of liquid (diluent) in order to lower the concentration of whatever is in the solution.

  • A series of subsequent dilutions is called a serial dilution.

  • the only thing that is changing in all of these solutions is concentration.

  • The only difference between the stock tube and the final dilution (the tube on the far right) is how much "stuff" is in the solution.

  • Looking at it , this should make sense. Thesolution on the far left (called the stock solution) is a very dark pink. With each dilution, the color of the solution is considerably lighter, representing the lower concentration of each diluted solution. This is because each time a dilution is performed, a small amount of that "stuff" is transferred to the next tube.

<ul><li><p>A dilution is performed by taking a portion (called an aliquot) of one solution and adding it to an appropriate amount of liquid (diluent) in order to lower the concentration of whatever is in the solution.</p></li><li><p>A series of subsequent dilutions is called a serial dilution.</p></li><li><p>the only thing that is changing in all of these solutions is concentration.</p></li><li><p>The only difference between the stock tube and the final dilution (the tube on the far right) is how much &quot;stuff&quot; is in the solution.</p></li><li><p>Looking at it , this should make sense. Thesolution on the far left (called the stock solution) is a very dark pink. With each dilution, the color of the solution is considerably lighter, representing the lower concentration of each diluted solution. This is because each time a dilution is performed, a small amount of that &quot;stuff&quot; is transferred to the next tube.</p></li></ul>
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Labeling Method

  • Petri dish is labelled on the bottom rather than on the lid. Write close to the edge of the bottom of the plate to preserve area to observe the bacteria culture after it has incubated.

  • Culture tube is labelled on the glass rather than on the cap. Write close to the bottom edge of the cap to preserve area to observe the bacteria culture. **Labels should include lab section, group initials, type of media, bacteria name, and the date.

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Growth Media

  • refers to a liquid, or gelatin, which supports the growth of microorganisms

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Liquid Media- NUTRIENT BROTH

  • mostly water with dissolved peptones and some beef extract. (Peptones are short chains of amino acids)

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Solid Media

  • are prepared by adding agar to liquid medium

  • adding agar to nutrient broth makes nutrient agar (NA)

  • is necessary for the isolation of individual colonies

  • can be set in petri dishes (plates)

  • can be set in tubes (solidifies at an angle) which is referred to as a slant

  • agar solidifies at 40 degrees C. (can't incubate at high temps w/out agar liquifying)

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Sterilization

  • treatment that eliminates all forms of life, including heat resistant spores and viruses

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Bacterial Colonies

  • a collection of many bacteria (several million) on a surface such as agar.

  • bacterial growth on slants can also vary greatly

<ul><li><p>a collection of many bacteria (several million) on a surface such as agar.</p></li><li><p>bacterial growth on slants can also vary greatly</p></li></ul>
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Different Streak patterns

a. tri- streak Isolation streak) b. lawn streak

<p>a. tri- streak Isolation streak) b. lawn streak</p>
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Bacteria Morphology

Bacilli- rods Cocci- spheres Streptococci- chains of spherical cells Diplococci- pair of spherical cells Staphylococci- clusters of spherical cells

<p>Bacilli- rods Cocci- spheres Streptococci- chains of spherical cells Diplococci- pair of spherical cells Staphylococci- clusters of spherical cells</p>
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Gram Stain

Gram Positive - purple color (thick peptidoglycan Gram Negative- pink (thin peptidoglycan)

  • gram stain process= crystal violet-> iodine solution-> rinsed w/ethyl alcohol

  • RESULTS: Bacillus licheniformis: large gram positive (purple rod) Escherichia coli: small gram negative (pink rod)

<p>Gram Positive - purple color (thick peptidoglycan Gram Negative- pink (thin peptidoglycan)</p><ul><li><p>gram stain process= crystal violet-&gt; iodine solution-&gt; rinsed w/ethyl alcohol</p></li><li><p>RESULTS: Bacillus licheniformis: large gram positive (purple rod) Escherichia coli: small gram negative (pink rod)</p></li></ul>
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Capsule Stain

Capsule: an envelope having a gelatinous consistency Procedure for visualizing the bacterial capsule- India ink (negative stain) THE BACKGROUND IS STAINED BUT NOT THE CELL

<p>Capsule: an envelope having a gelatinous consistency Procedure for visualizing the bacterial capsule- India ink (negative stain) THE BACKGROUND IS STAINED BUT NOT THE CELL</p>
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Selective vs. Differential Media

Selective- allows certain bacteria to grow but will inhibit others from growing Differential- contains substances that cause some bacteria to take on an appearance that distinguish them from other bacteria

<p>Selective- allows certain bacteria to grow but will inhibit others from growing Differential- contains substances that cause some bacteria to take on an appearance that distinguish them from other bacteria</p>
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Staphylococcus aureus

  • grows in mannitol-salt agar, appears yellow

<ul><li><p>grows in mannitol-salt agar, appears yellow</p></li></ul>
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Nutrient Agar

basic media used to grow our stains in our lab. (Low sensitivity and non-differential)

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MacConkey's Agar (MAC)

  • a differential and selective medium recommended for the use in the detection and isolation of all types of enteric bacteria. (Escherichia coli, Salmonella enterica)

  • SELECTIVE: Gram negative bacteria

  • DIFFERENTIAL: Lactose fermentation

  • Lactose fermentation- red/pink colonies

  • Non-Lactose fermentation- white/clear colonies

<ul><li><p>a differential and selective medium recommended for the use in the detection and isolation of all types of enteric bacteria. (Escherichia coli, Salmonella enterica)</p></li><li><p>SELECTIVE: Gram negative bacteria</p></li><li><p>DIFFERENTIAL: Lactose fermentation</p></li></ul><ul><li><p>Lactose fermentation- red/pink colonies</p></li><li><p>Non-Lactose fermentation- white/clear colonies</p></li></ul>
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Eosin Methylene Blue Agar (EMB)

  • a weakly selective and differential plating medium. (Inhibit Gram positive growth)

  • SELECTIVE: enteric bacteria; gram positive cannot grow -DIFFERENTIAL: lactose and or sucrose fermentation

  • Sugar fermentation: dark blue/black colonies; E. coli: metallic green

  • Non- Fermenters: white/clear/slightly pink colors

<ul><li><p>a weakly selective and differential plating medium. (Inhibit Gram positive growth)</p></li><li><p>SELECTIVE: enteric bacteria; gram positive cannot grow -DIFFERENTIAL: lactose and or sucrose fermentation</p></li></ul><ul><li><p>Sugar fermentation: dark blue/black colonies; E. coli: metallic green</p></li><li><p>Non- Fermenters: white/clear/slightly pink colors</p></li></ul>
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Endo Agar

  • is deferential and slightly selective. (Inhibit of gram positive is achieved by the incorporation of sodium sulfite and basic fuchsin)

  • SELECTIVE: gram negative

  • DIFFERENTIAL: lactose fermenters/non lactose fermenters *Lactose fermenters: clear colonies

  • Non lactose fermenters: red colonies

<ul><li><p>is deferential and slightly selective. (Inhibit of gram positive is achieved by the incorporation of sodium sulfite and basic fuchsin)</p></li><li><p>SELECTIVE: gram negative</p></li><li><p>DIFFERENTIAL: lactose fermenters/non lactose fermenters *Lactose fermenters: clear colonies</p></li></ul><ul><li><p>Non lactose fermenters: red colonies</p></li></ul>
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Xylose lysine deoxycholate Agar (XLD)

  • a highly selective medium recommended for the isolation of Salmonella enterica and Shigella

  • SELECTIVE: gram negative -DIFFERENTIAL: hydrogen production *Sugar fermentation: media changes to a yellow color *No Xylose fermentation: colonies appear res (shigella) *Hydrogen sulfide production: black precipitate in colonies (Salmonella enterica)

<ul><li><p>a highly selective medium recommended for the isolation of Salmonella enterica and Shigella</p></li><li><p>SELECTIVE: gram negative -DIFFERENTIAL: hydrogen production *Sugar fermentation: media changes to a yellow color *No Xylose fermentation: colonies appear res (shigella) *Hydrogen sulfide production: black precipitate in colonies (Salmonella enterica)</p></li></ul>
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Mannitol Salt Agar (MSA)

  • selective for staphylococcus and is differential for staphylococcus aureus from other staphylococci.

  • SELECTIVE: salt tolerant organisms -DIFFERENTIAL: mannitol fermentation; changing the pH indicator

  • Growth on Media- organism is tolerant

  • Mannitol fermentation- media changes from yellow to red

  • Non-Mannitol fermentation- media remains red

<ul><li><p>selective for staphylococcus and is differential for staphylococcus aureus from other staphylococci.</p></li><li><p>SELECTIVE: salt tolerant organisms -DIFFERENTIAL: mannitol fermentation; changing the pH indicator</p></li></ul><ul><li><p>Growth on Media- organism is tolerant</p></li><li><p>Mannitol fermentation- media changes from yellow to red</p></li><li><p>Non-Mannitol fermentation- media remains red</p></li></ul>
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osmotic pressure

involves 6.5% NaCl Temp: 37 degrees C

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Oxidase Test

  • tests for the presence of cytochrome oxidase

  • Positive result- formation of a purple color within minutes

  • Negative result- no color change

<ul><li><p>tests for the presence of cytochrome oxidase</p></li></ul><ul><li><p>Positive result- formation of a purple color within minutes</p></li><li><p>Negative result- no color change</p></li></ul>
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Fermentation of sugars

Reversion: SUGAR-> PROTEINS-> pH CHANGE-> YELLOW-> pH CHANGE-> RED Temp: 37 degrees C

<p>Reversion: SUGAR-&gt; PROTEINS-&gt; pH CHANGE-&gt; YELLOW-&gt; pH CHANGE-&gt; RED Temp: 37 degrees C</p>
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Carbohydrate Fermentation Test

  • objectives

  • principles

  • procedure

  • result and interpretation

  • precautions and limitations

<ul><li><p>objectives</p></li><li><p>principles</p></li><li><p>procedure</p></li><li><p>result and interpretation</p></li><li><p>precautions and limitations</p></li></ul>
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Extracellular Enzymes

  • test for the presence of certain extracellular enzymes: alpha-amylase, and enzyme that catalyzes the breakdown of starch.

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Hydrolysis of Starch

Temp: 37 degrees C Test for amylase using Gram's iodine solution Positive result: clear zones surrounding bacterial growth indicated the absence of starch. Negative result: a starch-iodine complex produces a deep purple to black color

<p>Temp: 37 degrees C Test for amylase using Gram&apos;s iodine solution Positive result: clear zones surrounding bacterial growth indicated the absence of starch. Negative result: a starch-iodine complex produces a deep purple to black color</p>
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Hydrolysis of Triglycerides

MA: spirit blue agar plate Temp: 25 degrees C Positive result: cleaning of the emulsion Negative result: precipitation of the blue dye

<p>MA: spirit blue agar plate Temp: 25 degrees C Positive result: cleaning of the emulsion Negative result: precipitation of the blue dye</p>
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Catalase Production

positive: production of gas negative: no bubbling

<p>positive: production of gas negative: no bubbling</p>
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Sulfur Metabolism

  • bacteria that are able to use sulfur will produce hydrogen sulfide or H2S.

  • uses media allied Klinger's Iron Agar (KIA)

  • Temp: 37 degrees C

<ul><li><p>bacteria that are able to use sulfur will produce hydrogen sulfide or H2S.</p></li><li><p>uses media allied Klinger&apos;s Iron Agar (KIA)</p></li><li><p>Temp: 37 degrees C</p></li></ul>
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Protease production

Temp: 37*C -Positive: If culture remains liquid or liquefied, gelatin hydrolysis occurred by proteases produced by the inoculums -Negative: Gelatin solidifies, then no protease was present

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Ryo or KOH String Test

  • The alkaline solution of KOH dissolves the gram-neg. outer envelope, causing the bacteria to become sticky and tacky, thus generating a slimy emulsion. ● Positive: Stringy bacteria, predicting a gram-neg. Stain result. ● Negative: Non-stringy bacteria predicting a gram-positive stain result.

<ul><li><p>The alkaline solution of KOH dissolves the gram-neg. outer envelope, causing the bacteria to become sticky and tacky, thus generating a slimy emulsion. ● Positive: Stringy bacteria, predicting a gram-neg. Stain result. ● Negative: Non-stringy bacteria predicting a gram-positive stain result.</p></li></ul>
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Alpha, Beta, and Gamma Hemolysis

  • Alpha hemolysis: green coloring under bacteria colonies; partial lysis of cells

  • Beta hemolysis: Clearing around the colonies; complete lysis of cells

  • Gamma Hemolysis: Growth on plate without any lytic action (non-hemolytic) Temp 37*C

<ul><li><p>Alpha hemolysis: green coloring under bacteria colonies; partial lysis of cells</p></li><li><p>Beta hemolysis: Clearing around the colonies; complete lysis of cells</p></li><li><p>Gamma Hemolysis: Growth on plate without any lytic action (non-hemolytic) Temp 37*C</p></li></ul>
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Microbial Biofilms

Test the ability of bacteria to form biofilm using the dye crystal violet Temp 37*C

<p>Test the ability of bacteria to form biofilm using the dye crystal violet Temp 37*C</p>
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Motility Test Medium (MTM)

Semisolid agar media to detect motility Temp 37*C ● No growth is an inconclusive result ● Non-motile growth is only visible along the line of inoculation ● Motile growth disperse away from the stab line of inoculation (positive result)

<p>Semisolid agar media to detect motility Temp 37*C ● No growth is an inconclusive result ● Non-motile growth is only visible along the line of inoculation ● Motile growth disperse away from the stab line of inoculation (positive result)</p>
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Antimicrobial Susceptibility Testing

  • An important technique to determine the effectiveness of antibacterial agents for a partial target organism is sensitivity disc assay.

  • This assay is also called the Kirby-Bauer method after the inventors.

  • The MIC is the lowest concentration of a compound that prevents growth of a particular organism. -Bactericidal: death to an organism -Bacteriostatic: block and slow the growth or metabolisms of the organism

  • Temp 37 degrees C

<ul><li><p>An important technique to determine the effectiveness of antibacterial agents for a partial target organism is sensitivity disc assay.</p></li><li><p>This assay is also called the Kirby-Bauer method after the inventors.</p></li><li><p>The MIC is the lowest concentration of a compound that prevents growth of a particular organism. -Bactericidal: death to an organism -Bacteriostatic: block and slow the growth or metabolisms of the organism</p></li><li><p>Temp 37 degrees C</p></li></ul>
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