M Powell microbiology notebook

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Portage Learning *

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171

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Biology

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Jan 9, 2024

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Portage Learning BIO 171- Microbiology Lab Notebook 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 MP Exp#1 Title: Introduction to Medical Microbiology Objective: Cultivation of Samples (growth conditions) - Equipment used Identification of samples (biochemical assays) - Test available Evaluation of samples (microscopy) - Visualization and recognition of key characteristics Procedure: Basic equipment: Cleaning: autoclave 125 degrees Celsius - Uses heat, pressure, and steam to sterilize the environment and tools - Takes minutes to sterilize and utilizes hot air which takes hours - All bacteria, viruses, fungi, and spores are inactivated by autoclave Growing: fixed incubator 37 degrees Celsius - Is hermetically sealed, presence of shelving for petri dishes
shaker incubator 37 degrees Celsius - Shakes the culture, rotates to aerate liquid nutrient broth Visualizing: microscopy Storing: refrigerator 4 degrees Celsius - Slows bacteria growth and helps preserve sample Lab Safety: - Never eat or drink in the lab - Use PPE o Latex or thermal gloves o Eyewear o Lab coat - Never leave the lab while wearing PPE o Public hallway o Bathroom o Cafeteria Notes: - Make sure to keep lab notebook—can use on test so detailed notes would be good Results: General overview of what is used in Microbiology lab as well as how to properly set up lab notebook.
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Lab 2 Notebook Back to Home Page MP EX2 Title: Introduction to Microscopy Objective: To become familiar with the basic components of a light microscope as well as how to load as a sample for viewing. Procedure: 1. Review the parts of microscope. 2. Load sample to be viewed. 3. Choose magnification. 4. Adjust microscope to optimize sample visualization. Notes: Parts of microscope: 1. Eye piece – ocular lens—can be pulled apart; should be able to use both eyes and see one circle if not adjust 2. Arm/neck: if moving this is what you will grab with one hand and hand under base 3. Objective lenses: this provides the magnification of the sample; shortest has the least magnification: 4x / 10x / 40x / 100x 4. Stage: flat surface that you place your sample; holds the sample via a clamp (squeeze together to place sample and release); stage clips raise up and lay on the glass cover slip 5. Focus knobs: located on the side of the arm/neck; outside ring is the coarse adjustment – makes large steps in focus; inner ring is the fine focus—you can see the specimen but need more detail
6. Iris diaphragm: located below the stage determines how much light passes through the sample 7. Base- bottom of microscope, always on a flat surface Load sample to be viewed: 1. Put sample on stage—and secure 2. Turn on. Left hand side can dim or brighten light. You can use the knob or the diaphragm to control light Type of objectives: drive vs oil (high magnification) add a drop of oil onto slide—helps with light refraction and lense will be embedded in the oil. 3. Intensity of light source: too bright (can’t see: saturation); too dark, low visibility. Start midway leave iris open 4. Stage guides below stage 2 knobs: top controls movement of the stage forward/backward. Lower knob moves left and right. These put the specimen in range of viewing. 5. *start on lowest power of magnification if magnification is not given* Power of objective x Power of eyepiece = total magnification: 15 mm diameter object & total magnification is 200x larger and the diameter is 3000mm Eye pieces are labeled with magnification on them and are removeable Coarse adjustment will raise or lower stage—do not touch the sample with the lenses. Look through eyepiece and slowly adjust. There will be a pointer inside the eye piece you can spin the eye piece and the line will point to your sample. Results: After watching this video, I can identify each part of the microscope. I also know the basic function of each part of the microscope. This video also taught me how to load a sample and view it in the microscope.
Lab 3 Notebook Back to Home Page Title: MP EX3 Introduction to Gram Staining (Mounting Techniques) Objective: Microscopic examination of bacterial samples through various staining techniques. Identify color and shape of given samples Procedure: Dry Mount 1. Clean slide (70% ethanol) 2. Circle area on slide for easy location of specimen (optional) 3. Apply organism to slide: a. If from culture, use sterile loop to spread onto slide b. If from plate, use sterile loop to pick colonies and mix with a drop of distilled water 4. Air dry at room temperature until all moisture has evaporated. Wet Mount 1. Clean slide (70% ethanol) 2. Circle area on slide with a wax/hydrophobic pen 3. Apply organism to slide: a. If from culture, use sterile loop to spread onto slide b. If from plate, use sterile loop to pick colonies and mix with a drop of distilled water
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4. View under microscope a. Wet mount is ideal for viewing the motility of an organism. Do not dry out. Gram Staining 1. Clean slide (70 % Ethanol) 2. Apply organism to slide: a. Use sterile loop to spread 1-3 drops onto slide b. Spread into a thin film 3. Allow to air dry 4. Fix organism to slide by passing 3 times through a flame. Do NOT overheat slide! 5. Food the slide with crystal violet for 30-60 seconds 6. Rinse slide with water 7. Cover with Gram’s iodine for 30-60 seconds 8. Rinse with water 9. Decolorize with alcohol 10. Rinse with water 11. Counter with Safranin for 30 seconds 12. Rinse with water 13. Blot dry and examine under microscope Notes: Gram Positive bacteria = Purple - Thick peptidoglycan layer, retains crystal violet stain 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) Stain and Shapes: Acid Fast Stain: - Strong resistance to decolorization - Very few structures are acid-fast - Commonly used to identify mycobacterium - Carbolfuchsin dye retained (red dye) Negative Staining - Commonly used to identify organisms with opaque structures - Dark background via nigrosine dye o Negatively charged, repelled from membrane Results: I can identify bacteria under a microscope after using various staining techniques.
Lab 4 Notebook Back to Home Page Title: MP04 Growth Media Objective: To understand the types and uses of growth media for the isolation and identification of unknown bacterial samples. Procedure: 4-Phase Dilution Streaking: Clonal isolation 1. Using sterile loop spread culture in area #1 2. Using a NEW sterile loop drag through the end of area #1 ONCE 3. Using a NEW sterile loop drag through the end of area #2 ONCE 4. Using a NEW sterile loop drag through the end of area #3 ONCE *Using a back and forth pattern to dilute the culture in each zone. *Invert plate and incubate overnight at 37 degrees Celsius (Non-selective agar) Quadarant 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 a 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 37 degrees Celsius
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Notes: - Non-selective media: important for the expansion of unknown bacteria - Selective media: Used to eliminate irrelevant bacteria from mixed cultures - Differential Media: Used to distinguish between species of the same group Different types of plating: Non-Selective Plates: 1. Lfl: most common; multipurpose 2. Fllood agar: red blood cells- important nutrients for growth 3. TSAYE: removed red blood cells; general purpose; yeast & trypticase soy Selective Media: 1. MacConkey: red; selects for GRAM NEG prohibits gram + 2. SMAC: pink; sorbitol instead of lactose; Selects for GRAM NEG; differential because it distinguishes between pathogenic and non pathogenic E. coli 3. EMfl: dark purple; selects for GRAM NEG; has eosin/methylene blue; differential because based on ability to ferment lactose: a. If ferments: colonies turn green b. If partially ferments: colonies turn pink c. If does not ferment: colonies stay original color *WHEN GROWING SAMPLES WRITE ON BOTTOM OF PLATE WITH MARKER- date, description, experiment number* WET LAB: (wear PPE: gloves, lab coat, goggles, gloves & tie hair back if long) 1. 4 Phase dilution streaks a. Individual packed sterile loops each time streaked b. Cultures in sterile tubes – don’t have the flame
c. Mark bottom & invert & place in incubator @ 37 degree Celsius overnight 12-14 hours 2. Quadrant Growth (divide into 4 quad on bottom; date, label 1-4 in each quadrant) a. Culture are growing in tubes b. Sterile loop to extract sample & streak quadrant, invert dispose loop in biowaste bin c. Repeat with remaining samples d. Place in incubator @ 37 degrees Celsius overnight *cap samples are use to make sure not to spill 3. Blood agar: non- selective & differential media (shows hemolysis state of bacteria) a. Divide plate in ½, looking at 2 cultures: one lyses blood cells the other doesn’t b. Following same process- streak each half with different samples & invert & incubator 4. EMB agar: look at gram negative bacteria; 1 ferments lactose the other doesn’t a. Divide plate in 1/2 ; streak each side with sample; invert; incubator overnight 37 degrees Celsius Results: - Phase four dilution streaking o Zone out has the most growth until zone three where clonal isolates o Can choose a single colony (round circle on plate) and can reinoculated in standard growth media to blow it up on the large scale. - Quadrant non-selective media o Each organism can grow at different rates. E. coli grew quickly, zone 2 has staph aureus with uniform growth pattern, zone three strep sample with less growth four has pseudomonas strain with considerably less growth - Blood Agar non-selective media
o E. coli and Staph aureus o Staph aureus exhibits beta hemolysis which is seen as a white film on the plate meaning the red agar has changed color because of the lysis of the red blood cells. o E. coli does not have the lysis properties while staph aureus has beta hemolysis qualities. - EMB Differential Media o Used to differentiate between Gram-negative bacteria that can either ferment lactose or those that cannot. o The EMB plate was streaked with E. coli which can ferment lactose causing growth and a metallic green color (key characteristics of E. coli on EMB)
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Lab 5 Notebook Back to Home Page Title: MP 05 Testing bacteria for antibiotic sensitivity Objective: To determine the threshold of antibiotic sensitivity across bacterial strains using the Kirby-Baur method (aka Standardized Disc Susceptibility Test) Procedure: 1. Streak Bacteria A across an LB agar plate for confluent growth using a sterile L-spreader 2. Evenly place the paper antibiotic discs on the plate 3. Invert plate and incubate at 37 degrees Celsius overnight 4. Measure zones of inhibition (diameter) 5. Compare results with sensitivity chart Notes: Results: List diameter of zone of inhibition for each disc Reference chart to determine sensitivity level: Resistant > Intermediate > Susceptible
Antibiotic Measurement Vancomycin 30 susceptible Clindamycin 16 intermediate Oxacillin 17 susceptible Tobramycin 22 susceptible Erythromycin 24 susceptible Penicillin 36 susceptible Gentamicin 10 resistant Cefazolin 19 susceptible
Lab 6 Notebook Back to Home Page Title: MP06 Enzymatic Assays Objective: To profile bacterial populations based on key enzymatic reactions and determine growth/metabolic characteristics.
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Procedure: Oxidase Test: Qualitative test to determine the presence or absence of cytochrome c oxidase activity in bacteria. 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
Note: oxidase negative = no color change Oxidase positive = purple color Catalase Test: Qualitative test to determine the presence or absence of catalase, an enzyme used to break down hydrogen peroxide 1. Using a sterile loop carefully pick colony from plate 2. Smear colony directly onto the glass slide 3. Add 2 drops of hydrogen peroxide to each smear Note: Catalase negative = no bubbles Catalase positive = bubbles Coagulase Test: Qualitative test to determine the presence or absence of coagulase, an enzyme that plays a role in the formation of blood clots. 1. Using a sterile loop carefully pick colony from plate 2. Add colony to tube of rabbit plasma, incubate overnight at 37 degrees Celsius 3. (next day) examine samples for precipitants Note: Coagulase negative = no precipitant Coagulase positive = presence of fibrin aggregates (clots) Lipase Test: Qualitive test to determine the presence or absence of lipase, an enzyme that hydrolyzes triglycerides (fatty acids/lipids) 1. Using a sterile loop unknown culture onto spirit blue plates 2. Streak colony across plate, incubate overnight at 37 degrees Celsius 3. (next day) examine samples for precipitants
Note: Lipase negative = no change Lipase positive = reduction in color (zone of clearance) Notes: - Oxidase Test - Catalase Test - Coagulase Test - Lipase Test Results: Oxidase test: (differently tested 2 gram negative bacteria E. coli – oxidase negative (no change) Pseudomonas- oxidase positive activity (turned colors-purplish, yellow, green) Catalase Test: (differentially tested 2 gram positive bacteria) S. aureus: catalase positive – (formed bubbles) Streptococcus: catalase negative – (no bubbles formed) Coagulase Test: (differentially tested 2 gram positive, staph bacteria) S. aureus: coagulase positive- cloudiness, no movement (clotted) S. epiderminus: coagulase negative – no change, liquid did not clot
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Lab 7 Notebook Back to Home Page Title: MP 07 Secondary Assays for Bacterial Characterization Objective: To profile bacterial populations based on additional biochemical approaches to better determine growth/metabolic characteristics. Procedure Test Number 1: Indole Test: Biochemical test to determine a bacterial species ability to convert (breakdown) tryptophan into indole. 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 seconds Notes: INDOLE NEGATIVE= YELLOW COLOR INDOLE POSITIVE= RED/PINK-ORANGE COLOR Procedure Test Number 2: 1. Using a sterile loop pick an isolated colony from BAP 2. Inoculate bacteria in media broth ‘ 3. Grow at 37 degrees Celsius overnight 4. Add ~5 drops of Kovac’s reagent to culture
Notes: INDOLE NEGATIVE = YELLOW COLOR INDOLE POSITIVE = RED/PINK-ORANGE COLOR TSI Test: (Triple Sugar Ion) agar is used to assess the ability of bacteria to ferment sugars and/or produce hydrogen sulfide. 1. Use a sterile loop to inoculate slant from liquid culture 2. Press loop into the agar slant 3. While removing loop, streak slanted side of agar with loop 4. Grow at 37 degrees Celsius overnight 5. (next day) examine samples API Test: Broad, multi-panel test used for rapid characterization of bacterial species 1. Fill at tubes with bacteria culture a. Fill CIT, VP, an GEL entirely b. Add mineral oil to ADH, LDC, ODC, H25, URE = anaerobic 2. Add 5 mL distilled water into tray, add API strip and cover with lid 3. Incubate at 37 degree Celsius overnight Notes: -indole test -TSI test -API test Results:
Indole test # 2: e. coli culture = indole positiveti pseudomonas = indole negativeti salmonella = indole negative TSI test: e. coli- yellow =able to ferment all 3 types of sugarti salmonella- black= ferment all 3 sugars and hydrogen sulfide gas API Test: ONPG – positive (yellow / ADH – negative (yellow) / LDC – negative (yellow)/ OCD – positive (bright red)/ CIT – negative (pale green)/ URE- negative (yellow)/ TDA – negative (yellow)/ IND – positive (pink)/ GLU- positive (yellow)/ MAN – positive (yellow)/ INO – negative (blue)/ SOR – positive (yellow)/ RHA – positive (yellow/ SAC – negative (blue)/ MEL – positive (yellow)/ AMY – negative (blue)/ ARA – negative (yellow) Sample 1: ONPG- positive, ADH- positive, LDC-positive, ODC- negative, CIT-negative, H2s- negative, URE- negative, TDA-negative, IND- positive, VP- negative, GEL- negative, GLU-positive, MAN- positive, INO- negative, SOR- positive, RHA- positive, SAC- negative, MEL- positive, AMY- negative, ARA- negative Sample 2: ONPG- negative, ADH- negative, LDC- negative, ODC- negative, CIT- positive, H2s- negative, URE- negative, TDA- negative, IND- negative, VP- negative, GEL- positive, GLU-positive, MAN- negative, INO-negative, SOR- negative, RHA- negative, SAC- negative, MEL- negative, AMY- positive, ARA- positive
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Lab 8 Notebook Back to Home Page Title: MP08 Biochemical Assays for Bacterial Antigen Detection Objective: To understand the assays available for detecting bacterial or viral antigens Procedure: ELISA: Enzyme Linked Immunosorbent Assay utilizes antibodies and a colorimetric readout to indicate an antigen of interest Notes: - Color intensity = abundance of antigen - Light yellow = low amounts of antigen present - Dark yellow = high amounts of antigen present
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Western Blot: Separation of proteins based on size, utilizes antibodies and a colorimetric readout to indicate an antigen of interest. Western Blot Part 1: Loading the gel Part II: Running the gel (35 mAmps/gel) Part III: Transferring the gel (25V for ~75 Minutes) Part IV: Blocking the gel (2% BSA for ~45 Minutes at RT/ Place on Rocker) Part V: Primary Antibody (TBST/BSA for ~45 minutes at RT/ Place on Rocker) Part VI: Secondary Antibody (TBST/BSA for ~45 minutes at RT/ Place on rocker) Part VII: Membrane development
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BCIP/NBT at RT, covered Place on rocker Closely monitor Agglutination Test: A rapid assay to determine if antibodies to a specific pathogen are present. Most commonly tested against patient serum or blood. Notes: - Combining antigen (patient) with antibodies (kit) cause agglutination (clumping) Notes: - ELISA - Western blot - Agglutination tests Results:
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1 st membrane (nice protein expression): well1- no protein/ well 2-quite a bit of protein/ well 3- absent/ well 4- express protein/ well 5- express protein/ well 6- express protein Blot 2: protein smearing in 4 lanes there’s absence of proteins and at the end a blot has 2 nice bands of protein without all of the background Agglutination Test: quadrant 1- negative reagent control/ quadrant 2- negative bacterial control (e. coli)/ quadrant 3- positive result for agglutination (staph aureus)/ quadrant 4- positive result of agglutination (staph epidermidis)
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Lab 9 Notebook Back to Home Page Title: Unknown Pathogen Analysis Objective: To identify an unknown pathogen(s) based on the given assays developed during this course. Procedure: Pink rods/clusters – negative – Pseudomonas or E.coli Purple dots/clusters- positive – Staph aureus or strep Culture A: no growth Culture B: red color- only gram negative will grow
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Culture A: Lysed- Staph aureus Culture B: Red- E. Coli Culture A: No growth Culture B: colonies with fluorescent green metallic sheen – gram negative – strong lactose fermentation – E.coli Culture A: Bubbles- positive- staph Culture B: Bubbles- positive- staph
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Culture A: negative- no change- E. Coli Culture B: negative- no change- E.Coli Coagulase Test: Culture A: light yellow- Coagulated (positive) – Staph Aureus Culture B: light yellow- No coagulation (negative)- staph epidermis Notes: Two samples from the hospital was brought to your lab for diagnosis. After culturing the samples on LB agar plates another co-worker began the analysis. However, due to an emergency your co-worker has left the remaining work to you. Before leaving your co-worker informs you the 2 liquid culture samples were accidentally combined before the Gram stain was started. However the 2 cultures remain separated on LB plates for future tests. Goal: To correctly Identify 2 pathogens. Results: Culture A: Staph Aureus Culture B: E. Coli
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