BIOD171_Microbiology_Lab_Notebook_Ramirez.docx

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BIO 171- Microbiology Lab Notebook Updated 2022/2023 Lab Notebook Bookmarks (click to navigate): Lab 1 Notebook Lab 2 Notebook Lab 3 Notebook Lab 4 Notebook Lab 5 Notebook Lab 6 Notebook Lab 7 Notebook Lab 8 Notebook Lab 9 Notebook
Lab 1 Notebook Back to Home Page Title: Introduction to Medical Microbiology Objective: The objective of this lab will be to familiarize myself with how to conduct experiments and practice proper safety procedures while in a Lab. Cultivation, Identification, and Evaluation are the 3 main principles discussed when conducting any experiment. Procedure: Our instructor introduces us to the foundation of microbiology lab with an introduction to basic equipment and safety rules. We are also show how to keep our Lab Notebook formatted. Notes: Basic Equipment ❖ Cleaning – need to work in a sterile environment to keep everyone healthy o Main tool: Autoclave 125 degrees C (looks like a giant dryer machine with a thick door) ▪ Come in variety of sizes ▪ Uses heat, pressure, and steam to sterilize environment ▪ Put tools in before you use for a lab OR put contaminated tools to kill bacteria ▪ Not just dry air, steam is more efficient ❖ Growing - how to grow the pathogens and bacteria? ❖ Two Main Ways:
➢ Fixed incubator 37 degrees C (Chamber- looks like a mini fridge) ▪ Stored Petri dishes, which contain the samples ▪ Airtight seal, and metal door to keep dark conditions ➢ Shaker incubator 37 degrees C ▪ Used to take a growth media, which is liquid nutrient broth ▪ After inoculating bacteria, put it in here, and shaker shakes and rotates culture in a swirling fashion ▪ Helps arete it and help bacteria grow under optimal conditions ❖ Visualizing- How do we see these bacteria and things? ➢ Microscopy ▪ Beneficial to seeing bacteria ❖ Storing ➢ Refrigerator 4 degree C ▪ Temperature slows bacterial growth, which helps preserve samples and keep them for long term. • Test tubes which may have liquid broth, petri dishes which are wrapped airtight, or test tube racks General Lab Safety ❖ Never eat or drink in lab ➢ Contamination risks ❖ Use PPE ➢ Latex (or nitrile (no latex) or thermal gloves (handle anything hot) or cold gloves (liquid nitrogen) ➢ Eyewear protection ➢ Lab coat (worn when working with chemicals that can be spilled)
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❖ Never leave lab while wearing PPE ➢ Spreads and containments other areas ▪ Public hallway, restrooms, cafeteria, etc. Results: In this Lab, the instructor was able to educate us on proper Lab room procedures and safety. We were given an introduction in learning the basic equipment we will use to cultivate, identify, and examine bacteria.
Lab 2 Notebook Back to Home Page Title: Introduction to Microscopy Objective: To become familiar with the basic components of a light microscope & how to load a sample for viewing. Procedure: Using standard light microscope. 1) Review parts & components 2) Load sample of glass slide onto microscope 3) Learn how to select different magnifications 4) Make necessary adjustments to optimize sample visualization Notes: ❖ Basic Idea Behind Microscopy ❖ Similar to a magnifying glass (need similar components) ➢ Lens (magnifies object) ➢ Object to observe ➢ Light source ❖ Basic Parts of Microscope: ➢ Eyepiece ➢ Arm/neck ➢ Objectives
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➢ Revolving nosepiece ➢ Stage ➢ Stage controls ➢ Stage clips ➢ Coarse/fine focus ➢ Iris diaphragm ➢ Light source ➢ Base ❖ Standard Light Microscope ➢ Uses standard halogen light bulb for lamination source ➢ Top Down Parts: ▪ Oculars (eye pieces): • 10x magnification • able to grab and separate them or push together to help fit the width of your eyes • Look with both eyes • See single circle within viewfinder ◆ If you see 2, may need to compress or expand eyepiece ▪ Arm/Neck • Grab to move (along w base) ▪ Objectives (different microscopes have different set-ups) • Lenses that provide part of magnification • Has revolving nose
• 4 different objectives- just rotate and wait to hear click to know it is locked in place • Offers different powers of magnification ◆ Red (shortest)- 4x magnification. ◆ Yellow- 10 x ◆ Blue- 40x ◆ Grey- 100x ▪ Stage (Different microscopes have different ways to hold sample) • Flat surface where you place sample • 2 Basic ways of holding: ◆ Clamp holder- pinch medal bars to open arms on stage. Push slide on & slowly release. Bars hold slide ◆ Stage clips- medal clips that rotate on stage to hold slide in place ▪ Focus knobs • To adjust focus ◆ Outer ring- course adjustment ➢ Use to make large steps in changes of your focus ◆ Inside ring- fine focus ➢ See object but finetune it ▪ Iris/Diaphragm • Controls how much light is let into microscope & eye piece ▪ Base • Where microscope is set & steady ❖ How to See
➢ Power on Light Microscope ➢ Adjustment knob on left (help dim or lighten) ➢ Use diaphragm ❖ Visualization ➢ Types of Objectives ▪ Dry – 4, 10, 40x • No other medium is needed to view object ◆ Load sample and view immediately • If use oil, everything will be blurry & out of focus ▪ Oil- 100x • Oil required for imaging ◆ Gives better resolution on higher mags. • Add drop of immersion oil • Helps with light refraction, gives you better image • Use w/o oil sample looks bad ➢ Stage guide ▪ 2 knobs • 1) controls vertical axis (forward/backwards) • 2) left/right ➢ Intensity of light source: ▪ Start about midway on light power w diaphragm open ▪ Too bright= saturation • Can’t see lightly stained/opaque cell wall
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▪ Too dark= low visibility • May miss sample ➢ Magnification ▪ Working w small tissue or bacteria • Start at higher mag. ▪ Uncertain? • Start on lowest objective as possible ➢ [Power of object]] x [power of eyepiece] = Total magnification ▪ If a cell is 15 mm in diameter, using a 40x objective & 5x eyepiece the cell will now appear to the eye 200 times larger (200x) or ~3000mm in diameter ❖ How to Find Sample ➢ Know where stage is (control course knob-lower/raise stage) ▪ Start with lower & don’t hit glass w lens ➢ Slowly raise until comes into field ➢ Slowly adjust until clearer image ➢ Rotate ocular to use pointer to point to sample Results: The magnification= power of your objective x eyepiece
Lab 3 Notebook Back to Home Page Title: Mounting Techniques Objective: We will examine bacterial samples through various staining techniques using color and shape. Procedure: Dry Mount ❖ Clean slide (70% ethanol) ❖ Circle area on slide for easy location of specimen (optional) ❖ Apply the organism to slide: ❖ Air dry at room temperature until all moisture has evaporated Wet Mount ❖ Clean slide (70% ethanol) ❖ Circle area on slide with a wax/hydrophobic pen ❖ Apply the organism to slide: ❖ View under microscope ➢ Wet mount is ideal for viewing the motility of an organism. Do not dry out. Gram Staining ❖ Clean slide (70% ethanol) ❖ Apply organism to slide:
➢ Use sterile loop to spread ~1-3 drops onto slide ➢ Spread into a thin film ❖ Allow to air dry ❖ Fix organism to slide by passing 3x through a flame. Do NOT overheat slide! ❖ Flood the slide with crystal violet for 30-60 seconds ❖ Rinse slide with water ❖ Cover with Gram’s iodine for 30-60 secs. ❖ Rinse with water ❖ Counter stain with Safranin for 30 seconds ❖ Rinse with water ❖ Blot dry and examine under microscope Carefully dispose. Notes: ❖ NEGATIVE STAINING: • Commonly used to identify organisms with opaque structures ◆ Because they are so clean, don’t retain dye well/ if they do, so light and hard to see on microscope • Inverse ◆ Dark background via nigrosine dye ➢ Negatively charged • Negatively charged, repelled from membrane ❖ GRAM STAIN
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Gram Positive bacteria = PURPLE ▪ Thick peptidoglycan layer , retains crystal violet stain • Outside layer of bacteria is thick • Purple color dominates Gram Negative bacteria = PINK ▪ Thin (single) peptidoglycan layer, damaged by alcohol rinse step and crystal violet stain washed away ▪ Pink color derived from Safranin (secondary counterstain) ❖ EXAMPLES ➢ Stain color ▪ Positive (purple) ▪ Negative (pink) ➢ Shape ▪ A) Gram (-) Rods • Capsule shape ▪ B) Gram (+) Chains (cocci) • Spherical shape ▪ C) Gram (+) Clusters (cocci) • Grape-like clusters ❖ ACID FAST STAIN: ➢ Strong resistance to decolorization ◆ Commonly used to identify mycobacterium (TB) Carbolfuchsin dye retained (red dye)
Results: BACTERIA MENTIONED i) Staphylococcus aureus (gram (+) cluster structure) ii) Escherichia coli (e coli) (gram (-) rod shape) iii) Bacillus subtilis (gram (+) rod shape) iv) Pseudomonas aeruginosa (gram (-) rod shape) v) Streptococcus (gram (+) chain structure) Lab 4 Notebook
Back to Home Page Title: Growth Media Objective: To understand the types and uses of growth media for the isolation and identification of unknown bacterial samples. Procedure: This procedure will be a 4-phase Dilution Streaking: Clonal Isolation - Begin with LB plate because it is a non-selective, so we can get something growing. 1) Use sterile loop spread culture in are #1 2) Using a NEW sterile loop, drag through the end of area #1 (ONCE ) 3) Using a NEW sterile loop, drag though the end of area #2 (ONCE ) 4) Using a NEW sterile loop drag through the end of area #3 (ONCE ) - Use a back and forth pattern to dilute the culture in each zone - Invert plate and incubate overnight at 37 degree C Quadrant Growth: Rapid test for multiple isolates 1. Using sterile loop, spread unknown culture A in area #1 2. Using a NEW sterile loop, spread unknown culture B in area #2 3. Using NEW sterile loop, spread unknown culture C in area #3 4. Using a NEW sterile loop spread unknown culture D in area #4 - Use a back and forth pattern to dilute the culture in each zone - Invert plate and incubate overnight at 37 degree C
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Notes: Growth Media ❖ Non-Selective Media: Important for the expansion of unknown bacteria ➢ Anything will grow on these media plates ➢ Provides carbon source and nutrient ❖ Selective Media: Used to eliminate irrelevant bacteria from mixed cultures ➢ Can inhibit one (Gram (-) or Gram (+)) while promoting the growth of the other ▪ May only be able to grow Gram (+) or on a different type of plate, only grow Gram (-) ❖ Differential Media: Used to distinguish between species of the same group ➢ Differentiate between Gram (-) bacteria A vs. Gram (-) bacteria B Plates ❖ LB: most common nutrient agar ➢ Light/pale yellow ➢ Invert plate (helps stick to agar and keep it contamination-free) ❖ Blood Agar Plate: contains RBCs ➢ RBCs are a nutrient source ➢ Derivative of BAP: ▪ TSAYE: Tryptic Soy Agar Yeast Extract (when you remove RBCs) • Complex, general purpose media • Contains tryptic soy & yeast as nutrient source for plate ❖ MaConkey Media ➢ Selective ➢ Slight cloudiness, red
▪ Lactose in it ➢ Only Gram (-) will grow, inhibits Gram (+) ❖ SMAC: Sorbitol MaConkey Agar ➢ Pink color and more translucent ▪ Sorbitol in it ➢ Selective ▪ Selects for Gram (-) bacteria ➢ Differential ▪ Selects between E. Coli (standard K12) vs. E. Coli (0157), which is known as potent pathogen ❖ EMB: Eosin Methylene Blue ➢ Selective ▪ Grows Gram (-) bacteria ➢ Looks dark red, but when tilted a bluish/greenish sheen ➢ Contain eosin & methylene blue ➢ Can be used as differential ▪ Differentiate different subgroups of gram (-) bacteria based on their ability to ferment lactose ➢ If you have strong lactose fermentation ability- colonies turn green ➢ If you have partial lactose fermentation ability- colonies turn pink Four Phase Dilution Streaking ❖ Start in zone 1 ➢ Encourages bacterial growth in its highest concentration ❖ Move to zone 2
➢ Moving a little bit of bacteria with a clean loop ➢ Done a dilution factor ❖ Repeat for zone 3 ➢ Diluting bacteria even further ➢ Spreading bacteria farther and farther apart ❖ Zone 4 ➢ Thinned out bacteria enough where you may start to see clonal isolates growing on LB agar plate ▪ Look like dots growing on plate (single colony growth) Results: 4-Phase Dilution Streaking Results ❖ Zone 1 growth ❖ See line going through to Zone 2 ❖ In-between Zone 2 & 3, have nice single colony isolates ❖ Can re-inoculate a single colony in a standard growth media, and grow it up in larger scale ❖ Quadrant Growth Results ❖ Zone 1: E. Coli Strain ➢ Grew very rapidly ➢ Took up entire quadrant ❖ Zone 2: Staph aureus sample ➢ Uniformed, long growth pattern ❖ Zone 3: Strep sample
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➢ Not as heavy of growth (compared to 1 & 2), but bacteria grew nicely ❖ Zone 4: Pseudomonas strain ➢ Considerably less growth ➢ See same colonies seen in dilution phase ▪ Culture wasn’t growing as strongly as others ▪ But able to isolate and have culture growing on plate to use later ❖ Blood Agar Plate ❖ 1 st area (bottom quadrant): E. Coli ➢ Darker No hemolysis ❖ 2 nd area (top quadrant): Staph aureus ➢ Exhibits beta hemolysis Red agar now clear ❖ EMB Plate (differential) ❖ Differentiate Gram (-) bacteria that can either ferment lactose vs. cannot) ❖ Top quadrant streaked with E. Coli ➢ Able to grow ➢ Has partial green, metallic color ❖ Bottom quadrant streaked with Gram (-) Pseudomonas ➢ Does not ferment lactose There is No color change
Lab 5 Notebook Back to Home Page Title: Testing Bacteria for Antibiotic Objective: Determine the threshold of antibiotic sensitivity across bacteria Kirby-Bauer method Procedure: • Streak bacteria A across LB agar plate for confluent growth using a sterile L spreader • Evenly place paper antibiotic discs on plate • Invert plate and incubate at 37 C overnight • Measure zone of inhibition • Compare results with sensitivity chart Notes: 1) There will be some light disc and have no aura around them, others will have a sone of clearing 2) White zone around disc a) Indicates bacteria is susceptible to antibiotic and bacteria was killed 3) See disc that you can see LB agar and bacteria growing all the way up the disc 4) Basic Test of Kirby-Bauer method a) Looking at antibiotic sensitivity i) Staph ii) E. Coli
iii) Gram (+) or (-) The Process • Coat surface area with bacteria to have a uniform growth • Place the disc to see if bacteria is resistant or possible antibiotic • If there are zones that overlap measure radius of zone that doesn’t overlap and multiply by 2 Results: 1) List diameter of zone of inhibition for each disc (mm) a) Erythromycin: 31mm i) Falls under susceptible (good antibiotic to use if having a staph infection) b) Gentamicin: 9 mm i) Falls under resistant (wouldn’t do any good) c) Oxacillin: no zone of inhibition at all (highly resistant staph strain to oxacillin) d) Penicillin: some zoon of inhibition but small (resistant) 2) Reference chart to determine sensitivity level: a) Resistant > Intermediate > Susceptible
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Lab 6 Notebook Back to Home Page Title: Enzymatic Assays Objective: To profile bacterial populations based on key enzymatic reactions and determine growth/metabolic characteristics Procedure: Coagulase Test Procedure ❖ Using a sterile look carefully pick colony from plate ❖ Add colony to tube of rabbit plasma, incubate overnight at 37 degrees C ❖ (Next day) examine samples for precipitants ➢ Coagulase negative= no precipitant ➢ Coagulase positive= presence of fibrin aggregates (clots) Oxidase Test Procedure: ❖ Using sterile loop pick and isolated colony from BAP ❖ Smear directly onto the reaction area of the slide ❖ Examine test area for color change w/in 20 secs. ➢ Oxidase negative= no color change ➢ Oxidase positive= purple color Lipase Test Procedure:
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❖ Using sterile loop unknown culture onto spirit blue plates ❖ Streak colony across plate, incubate overnight at 37 degrees C ❖ (Next day) examine samples for precipitants ➢ Lipase negative= no change ➢ Lipase positive= reduction in color (zone of clearance) ▪ Began to hydrolyze fatty acids ▪ Changes pH/acidity of media Catalase Test Procedure: ❖ Using sterile loop carefully pick colony from plate ❖ Smear colony directly onto glass slide ❖ Add 2 drops of hydrogen peroxide to each smear ❖ Examine tests area ➢ Catalase negative= no bubbles ➢ Catalase positive= bubbles Notes: ❖ Oxidase Test: qualitative test to determine the presence or absence of cytochrome c oxidase activity in bacteria ➢ Cytochrome c oxidase ▪ Component of electron transport chain in aerobic bacteria (ie. Pseudomonas) ➢ Commonly used in hospital setting ❖ Catalase Test: Qualitative test to determine the presence or absence of catalase, an enzyme used to break down hydrogen peroxide ➢ Catalase ▪ Vital to have in the survival of oxygen rich environment
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• Hydrogen peroxide can easily be formed • Hydrogen peroxide not broken down, can form oxygen radicals (detrimental to bacterial cell- lead to cell killing quickly ➢ Used to differentiate staff from strep (both gram (+) bacteria, both have round/spherical shape, and yet only staff is catalase positive ) ➢ Differential test ❖ Coagulase Test: Qualitative test to determine the presence or absence of coagulase, an enzyme that plays a role in formation of blood clots or clotting in general ➢ Coagulase ▪ Binds CRF (coagulase reacting factor) ▪ Interacts with fibrinogen to form fibrin (aka blood clots) ▪ Positive of coagulase ➢ Staph aureus- most common coagulase positive bacteria ▪ Has resistance to antibiotics/antibodies ❖ Lipase Test: Qualitative test to determine the presence or absence of lipase, an enzyme that hydrolyzes triglycerides (fatty acids/lips) ➢ B subtills ▪ Lipase positive bacteria Results: ❖ Oxidase Test Results: ❖ Negative Control ❖ E. Coli (gram (-) ➢ Negative for oxidase activity
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❖ Pseudomonas ➢ Positive for oxidase activity ❖ Catalase Test Results: ❖ Staph aureus (gram (+) ➢ Catalase positive ❖ Streptococcus (gram (+) ➢ Catalase negative ❖ Coagulase Test Results: ❖ Staph epidermidis ➢ Coagulase negative (liquid) ❖ Staph aureus ➢ Coagulase positive (clotted)
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Lab 7 Notebook Back to Home Page Title: Secondary Assays for Bacterial Characterization Objective: To profile bacterial populations based on additional biochemical approaches to be better determine growth/metabolic characteristics Procedure: Indole Test Procedure #2: liquid culture 1) Using a sterile loop pick an isolated colony from BAP 2) Inoculate bacteria in media broth a. Tryptophan or peptone-based broth 3) Grow at 37 degrees C overnight a. Goes from clear, transparent media to cloudiness (indicates culture has grown) 4) Add ~5 drops of Kovac’s reagent to culture 5) Examine reaction a. Indole negative= yellow color i. Just a yellow broth (no color change_ b. Indole positive= red/pink orange color i. Strong += bright pink band at the top ii. Weak += slight pink color sample
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1. Tryptophan not being broken down as efficiently Indole Test Procedure #1: 1) Using a sterile loop pick an isolated colony from BAP 2) Smear directly onto the reaction area of the slide 3) Examine test area for color change within 20 seconds a. Indole negative= yellow color i. Tryptophan not broken down b. Indole positive = red/pink orange color i. Tryptophan being broken down ii. Don’t always get a perfect color shade 1) Fill all tubes with bacteria culture a. Fill CIT, VP, and GEL entirely b. Add mineral oil to ADH, LDC, ODC, H25, and URE = anaerobic 2) Add 5 mL distilled water into tray, add API strip and cover with lid 3) Incubate at 37 degrees C overnight TSI Test Procedure: 1) Use sterile loop to inoculate slant 2) Press loop into agar slant 3) While removing loop, streak slanted side of agar with loop 4) 37 degrees C overnight 5) (Next day) examine samples
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a. Red to yellow: fermentation of sugars i. Empty space in bottom 1. gas has been produced and pushing agar up tube b. If tube hasn’t turn black i. gas is produced, but not hydrogen sulfide gas c. Color gradient (pink color at down and migrates down to yellow/orange color i. Only have glucose fermentation only ii. No black color/no changes in agar for gas pocket 1. No gas is being produced d. Dark color (black) i. Hydrogen sulfide gas is being produced by these bacteria e. Negative control i. Untreated/uninoculated sample ii. Absence of any bacteria Notes: - Indole Test: Biochemical test to determine a bacterial species ability to convert (breakdown) tryptophan into indole o Comes on a card that has 4 quadrants where bacterial sample is placed ▪ One sample per quadrant o Indole ▪ Responsible for regulating various aspects of bacterial physiology • Spore formation, plasmid stability, & drug resistance - TSI Test (Triple Sugar Iron): agar is used to assess the ability of bacteria to ferment sugars and/or produce hydrogen sulfide
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o Commonly used to test for enteric bacteria ▪ Ie: Salmonella or shigella o Slants contain a pH indicator ▪ Slants usually red (no bacteria growing in it) ▪ Bacteria may be able to break down sugars & form hydrogen sulfide gas (black color) - API Test (Analytical Profile Index): Broad, multi-panel test used for rapid characterization of bacterial species Results: TSI Test Results: - Negative control remains red (native color as pH indicator) - E. Coli (gram (-) bacteria) has a solid yellow color all the way up slant o Able to ferment all three types of sugar o Produces gas, but not Hydrogen Sulfide gas Salmonella (gram (-) bacteria) 2 distinct color changes (yellow at bottom & black on slant) o Ferments all three sugars o Produces Hydrogen sulfide gas Indole Test Results: - E. Coli (gram (-) bacteria) is positive for tryptophan breakdown into indole - Pseudomonas (gram (-) bacteria) is negative for indole - Salmonella (gram (-) bacteria) is negative for indole
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Lab 8 Notebook Back to Home Page Title: Biochemical Assays for Bacterial Antigen Detection Objective: To understand the assays available for detecting bacterial or vital antigens. Procedure: ELISA Procedure: Sandwich ELISA ❖ Each well is labeled with an antibody ➢ Already adhered to bottom on well ❖ Add a patient sample ➢ Serum or cultures that were grown ❖ If antigen is present & recognized by antibody, will be bound ❖ Follow with Incubation with a secondary antibody that also recognizes the antigen ➢ Why this is called a sandwich: antigen is sandwiched between 2 different antibodies ➢ Secondary antibody has a tag on it ▪ Either enzyme or substrate that will react once activated ❖ Wash away any unbound secondary antibody ❖ Everything is activated & get a colorimetric change ➢ Some may have dark color, light color of yellow, or won’t change at all ▪ Color intensity= abundance of antigen
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▪ Light yellow= low amounts of antigen present ▪ Dark yellow= high amounts of antigen present ❖ Western Blot Procedure: Western Blot 1: Loading the gel ➢ Mix samples with a loading dye ▪ SDS: negatively charged dye (has dark purple color) • Proteins are charged, so dye binds to it ▪ Load into a well of a polyacrylamide gel ▪ Marker (has color red) • Standards run from (-) terminal to (+) terminal ◆ Protein mixed with SDS is (-) charge ➢ Uses charge repulsion to push protein samples through the gel down towards bottom 2: Running the gel ➢ 35 mAmps/gel ➢ Samples go from purple boxes that were loaded in each well down to bottom, to a purple smear ▪ Dye front 3: Transferring the gel ➢ 25 V for ~75 minutes ➢ Use a nitrocellulose membrane that matches size of gel ➢ Set up sandwich, where paper on the outside to sandwich the gel and membrane together ➢ Soak this buffers
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4: Block the gel ➢ 2% BSN for ~45 minutes at RT ➢ Place on rocker ▪ Rocks sample back and forth while it’s in solution to ensure it covers entire membrane ➢ Prevent any non-specific binding of antibody to prevent from binding to membrane ▪ Influences signal that you get at the end 5: Primary Antibody ➢ TBST/BSA for ~45 minutes at RT ▪ If looking for specific protein or antigen, use primary antibody that is directed towards that antigen ➢ Place on rocker 6: Secondary Antibody ➢ TBST/BSA for ~45 minutes at RT ➢ Place on rocker ➢ Secondary antibody is now binding primary antibody, which will be specific for your protein or antigen of choice 7: Membrane Development ➢ BCIP/NBT at RT, covered ➢ Place on rocker ➢ Closely monitor ▪ If overdevelop your block • Get a dark background that will cover entire membrane ➢ Dark bands showing up
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▪ Intensity of bands dependent on actual amount of protein that is present or how long you develop your blot ➢ Know where protein of interest lies ❖ Agglutination Tests Procedure: ❖ Can be done in 96-well plate if coated ❖ Negative results for agglutination ➢ No clumping ❖ Positive results for agglutination ➢ Clumping reaction ➢ Reacting with antibodies Notes: ❖ Agglutination tests: a rapid assay to determine if antibodies to a specific pathogen are present. ➢ Combining antigen (patient) with antibodies (kit) cause agglutination (clumping) ❖ Western blot: Separation of proteins based on size ➢ Western Blot ▪ Detecting protein in your samples ❖ ELISA (Enzyme-Linked Immunosorbent Assay): utilizes antibodies and colorimetric readout to indicate an antigen of interest ➢ Sandwich ELISA (STEPS A-D are colorless) Results:
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❖ ELISA Assay Results: ❖ Sample 1: nice protein expression ➢ Well 1: no protein ➢ Well 2: a bit of protein ➢ Well 3: absent ➢ Well 4, 5, 6: express protein ➢ Has multicolor ladder ❖ Western Blot Results: ❖ Sample 2: ➢ A lot of protein smearing in some of lanes ➢ Some absence of proteins ➢ Last blot has two nice bands of protein without background ❖ Agglutination Test Results: ❖ Staph Kit ➢ Q1: negative control ➢ Q2: E. coli reagent= would expect to not react ➢ Q3: Staph aureus= Positive for agglutination ➢ Q4: Staph epidermidis= positive for agglutination
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Lab 9 Notebook Back to Home Page Title: Unknown Pathogen Analysis Objective: To identify an unknown pathogen based on the given developed during this course Procedure: Observe results from 2 cultures shown on the powerpoint for information in identifying sample. Notes: Two samples were brought to the lab from the hospital for diagnosis. After culturing the samples on LB agar plate we began the analysis. Due to an emergency our co-worker has left the remaining work to us. The co-worker informs us the 2 liquid culture samples were accidentally combined before the Gram stain was started. The 2 cultures remain separated on LB plates for future tests. Results: Gram Stain Results: - Color: o Purple (Gram + bacteria) o Pink (Gram – bacteria) - Shape: o Gram + bacteria: Clusters (cocci family) o Gram – bacteria: Rods (capsule shape)
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- Gram Positive Cocci bacteria cluster o Possibly Staph aureus? - Gram Negative Rods bacteria o E. Coli or Pseudomonas? MacConkey Plates Results: - Culture A: no growth o Gram (+) bacteria - MacConkey inhibits Gram (+) growth - Culture B: o Gram (-) bacteria- MacConkey only grows Gram (-) o Slight cloudiness, red o Possible E. Coli BAP Plates Results: - Culture A: beta hemolysis. o Complete lysis of RBCs, which cleared blood from medium - Culture B: gamma hemolysis o No hemolysis of RBCs, so no change in medium EMB Plates Results: - Culture A: no growth o Gram (+) bacteria - EMB inhibits Gram (+) growth - Culture B: o Gram (-) bacteria- EMB only grows Gram (-)
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Catalase Test Results: - Culture A: coagulase positive= bubbles - Culture B: coagulase positive= bubbles Oxidase Test Results: - Culture A: Negative - Culture B: Negative Coagulase Test: - Culture A: Positive= clotted - Culture B: Negative= liquid FINAL RESULTS: - Culture A: Staph aureus - Culture B: E. Coli
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