2022 BIO 171- Microbiology Portage Learning Lab notebook New updated Exam GRADED A+, Exams of Nursing

Download 2022 BIO 171- Microbiology Portage Learning Lab notebook New updated Exam GRADED A+ and more Exams Nursing in PDF only on Docsity!

Lab notebook New updated Exam GRADED A+

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

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Lab 1 Notebook

Back to Home Page Title: KD01 Introduction to medical microbiology & lab notebook procedures Course Objectives: Cultivation of samples: growth conditions and equipment used Identification of samples: biochemical assays & tests available Evaluation of samples: microscopy- visualization and key characteristics of specimens Basic Equipment: Cleaning: Autoclave 125°C- uses heat, pressure, & steam to sterilize; autoclave will take minutes to sterilize & hot dry air which will take hours; before opening make sure it is depressurized and will have very hot air. Growing (bacteria/pathogens) – Fixed incubator 37°C is airtight, shelving for petri dishes

• Shaker incubator 37°C shakes culture & rotates to aerate- liquid nutrient broth Visualizing: microscopy Storing: refrigerator @ 4°C to stop the growth and helps preserve samples & keeps long term Lab Safety

1. Never eat or drink in the lab: contamination risks
2. Use PPE (personal protection equipment): gloves- latex, nitrile (purple, blue,

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

green),thermal, or cold gloves (liquid nitrogen): eyewear: lab coat- chemical spills

3. Never leave lab while wearing PPE: including bathroom, hallway, or cafeteria Outline of how to set up Notebook Objective: To establish an organized template for keeping experimental records, procedures, and results. Procedure: 1. This is where each step of a given protocol is recorded 2. Be sure to clearly indicate any deviations that may occur during the experiment- put them in red Notes: Additional (helpful) information placed here Results: A summary of the final outcome of the experiment should be described here

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Lab 2 Notebook

Back to Home Page Title: KD02 Introduction to Microscopy Objective: To learn basic parts of microscope and how to view a sample Procedure:

**1. Review the parts of microscope

2. Load sample to be viewed
3. Choose magnification
4. Adjust microscope so the sample is clear Notes: Parts of microscope:
5. eye pieces (ocular lens): can be pulled apart; should be able to use both eyes and see one circle if not adjust
6. arm/neck: if moving this is what you will grab with one hand and hand under base
7. Objective lenses: this provides the magnification of the sample; shortest has the least magnification (4x, 10x, 40x,100x)
8. Stage: the 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 coverslip**

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

**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 (the lever slides)
7. Base- the bottom of the microscope and should be steady and on flat surface Load sample to be viewed:
8. Put sample on stage and make sure it is secured
9. Turn on. Left hand side you can dim or brighten the light. You can use either the knob or the diaphragm to control the light. Types of Objectives: dry vs oil (required on higher magnification) add a drop of oil onto slide- helps with light refraction and lense will be embedded in the oil
10. Intensity of light source: too bright = saturation (can’t see); too dark = low visibility. Start midway leave iris open
11. Stage guides below stage 2 knobs: top controls movement of the stage forward/backward. Lower know moves left and right. These put specimen in range of viewing.
12. if unsure of magnification: start on lowest power 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 removable Coarse adjustment will raise or lower the stage- do not touch the sample with the lenses. Look through eyepiece and slowly make adjustments. There will be a pointer inside the eye piece you can spin the eye piece and the line will point to your sample Results: By watching the video, I can identify the parts of the microscope and know their*

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

basic function and how to load a sample and view it.

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Lab 3 Notebook

Back to Home Page Title: KD03 Lab Mounting Techniques Objective: Examination of bacterial samples through different staining techniques. Identify samples based on color and shape Procedure: Dry Mount:

1. Clean slide (70% ethanol)- if it’s not clean hard to determine if you are looking at contaminant or the pathogen
2. Circle area on slide for easy location of specimen (optional)- draw circle on bottom of slide
3. Apply organism to slide: -If from culture, use sterile loop to apply to slide -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:
5. Clean slide (70% ethanol)
6. Circle area on slide for easy location of specimen using a wax hydrophobic pen – keeps water inside ring
7. Apply organism to slide: -If from culture, use sterile loop to apply to slide

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

-if from plate, use sterile loop to pick colonies and mix with a drop of distilled water

4. View under microscope Main reason for using wet mount is to be able to view motility of organism. Do NOT let dry out- reapply distilled water if needed to keep organism viable. Allows to see flagella- some are not motile and stay in a fixed location. Can be a key to diagnosing pathogen. Gram Staining:
5. Clean slide (70% ethanol)
6. Apply organism to slide: -use sterile loop to put 1-3 drops/slide -spread into a thin film
7. Allow to air dry
8. Fix organism to slide by passing through flame 3 times; Do not overheat slide (takes very little heat) *series of dyes- depends on the bacteria it will either be absorbed or washed out
9. Rinse with crystal violet for 30 -60 seconds- dark purple dye
10. Rinse slide with water- (stains may wash out)
11. Cover with Gram’s iodine for 30-60 seconds
12. rinse with water
13. decolorize with alcohol
14. rinse with water
15. counterstain with safranin (red/pink) for 30 seconds
16. rinse with water
17. blot dry and examine

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Notes: GRAM POSITIVE : purple -thick peptidoglycan layer; keeps crystal violet color GRAM NEGATIVE : pink -thin peptidoglycan layer and is damaged by alcohol rinse & crystal violet rinsed away; pink color is from counterstain (Safranin) Once stained you can identify based on color/shape: Gram positive: Purple- bacteria in chains/clusters (cocci shaped) Gram negative: Pink- bacteria in rod shaped Gram staining isn’t always guaranteed to identify samples ACID FAST STAINING: -strong resistance to decolorization -very few structures are acid fast -commonly used to identify mycobacterium (TB) -carbolfuchsin dye retained (red); bacteria will remain red on blue background NEGATIVE STAINING: -used for organisms with opaque structures -dark background (Nigrosin) both dye and cell membrane negatively charged so dye is repelled

• will be able to see the clusters and shapes of bacteria WET LAB:

1. put on PPE ( gloves, eye wear, lab coat)

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

2. put samples in tube and ran the top of tubes through flame to sterilize

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

3. cleaned slides with chem wipe
4. draw circle on slide with wax pen to keep bacteria in circle
5. flame top of tube again
6. put loop in tube and swirl to collect sample
7. spread thin layer inside circle on slide (wax keeps liquid inside)
8. let air dry
9. heat fix by running slide over flame 3 times – should be no liquid left
10. repeat with remaining 4 samples *sterile loop should be disposed of in biohazard waste bin as well as anything else and put in autoclave to be sterilized before disposal. APPLYING STAIN TO EACH SAMPLE: Supplies needed: 5 specimens, tray to dye slides in, distilled water, chem wipes, clips, timer, crystal violet, alcohol, iodine, safranin
11. Cover entire circle with crystal violet dye for 1 min (all slides done simultaneously)
12. rinse off with water until no more color comes off
13. cover slides with iodine- apply with eye dropper don’t touch specimen (sit for 1 min)
14. rinse thoroughly with water
15. decolorization with alcohol can see some losing dye while others retain it
16. rinse thoroughly with water
17. counterstain with safranin thoroughly let sit for 45 seconds
18. final rinse –thoroughly
19. blot dry with wipe and then let air dry *make sure to throw wipes and everything in biohazard waste bin

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

10. gently use wipe to make sure slide is dry –remark if wax pen came off EXAMINE SLIDES UNDER MICROSCOPE: -40x magnification; stage lowered all the way -open clips and place slide on stage to secure -illumination set at 50% -diaphragm open all the way -no longer any liquid- take off eye wear -Focus microscope:
    1. raise sample to come into focus using coarse adjustment; make sure sample is in light field
    2. dark image in focus use fine focus to make image clear; move stage if needed Results: Organism 1: ( Gram +); Purple color; round cluster- Staphylococcus Aureus

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Organism 2 : (Gram -); Pink color; rod shaped; E-Coli Organism 3 : (Gram +); dark purple; rod shaped- Bacillus Subtilis

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Organism 4 : (Gram -); pink color; rod shaped; Pseudomonas Aeruginosa Organism 5 : (Gram +); purple color; spherical shaped w/chain structure; Steptococcus

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Lab 4 Notebook

Back to Home Page Title: KD04 Growth Media Objective: Understanding types/uses of growth media for isolation and identifying unknown bacterial samples. Procedure: 4 - Phase dilution streaking: Clonal Isolation- purpose is to isolate individual bacterial colonies

1. Use sterile loop spread culture in area #
2. Use NEW sterile loop drag through end of area #1 ONCE
3. Use NEW sterile loop drag through end of area #2 ONCE
4. Use NEW sterile loop drag through end of area #3 ONCE

• Use the back and forth motion to dilute culture in each zone.
• Invert plate & incubate at 37 degrees Celsius overnight. [Non - selective agar] Quadrant Growth: Rapid test - multiple isolates; purpose is to grow bacteria separately without cross contaminating

1. Use sterile loop spread unknown culture A in area #
2. Use NEW sterile spread unknown culture B in area #
3. Use NEW sterile loop spread unknown culture C in area #
4. Use NEW sterile loop spread unknown culture D in area #

• Use the back and forth motion to dilute culture in each zone.
• Invert plate & incubate at 37 degrees Celsius overnight. Notes: Non- Selective Media: important for the expansion of unknown bacteria Selective Media: eliminates irrelevant bacteria from mixed cultures

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Differential Media: distinguishes between species of the same group

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

DIFFERENT TYPES OF PLATING: (they have different nutrient selections) once streaked: invert plate to keep contaminates out NON-SELECTIVE PLATES:

1. LB: most common; multipurpose
2. Blood agar: red blood cells- important nutrients for growth
3. TSAYE: removed red blood cells; general purpose; yeast & tripticase soy SELECTIVE MEDIA:
4. MacConkey: red; selects for GRAM NEG prohibits gram +
5. SMAC: pink; sorbitol instead of lactose; Selects for GRAM NEG; differential because it distinguishes between pathogenic and non pathogenic E.coli
6. EMB: dark purple; selects for GRAM NEG; has eosin/methylene blue; differential because based on ability to ferment lactose: -if ferments: colonies turn green -if partially ferments: colonies turn pink -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 -individual packed sterile loops each time streaked -cultures in sterile tubes – don’t have to flame -mark bottom & invert & place in incubator @ 37 degrees Celsius overnight 12-14hrs

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

2. Quadrant Growth (divide into 4 quad on bottom; date, label 1-4 in each quadrant)

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

-culture are growing in tubes -sterile loop to extract sample & streak quadrant, invert, dispose loop in biowaste bin

• repeat with remaining samples -place in incubator @ 37 degrees Celsius overnight *cap samples after use to make sure not to spill 3. Blood agar: non-selective & differential media (shows hemolysis state of bacteria) -divide plate in ½; looking at 2 cultures: one lyses blood cells the other doesn’t -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 -divide plate in ½; streak each side with sample; invert; incubator overnight 37°C Results: 1. Phase 4 Lab : easy to see that you have individual colonies in P3 zone. Now you would be able to pick the colony and grow to a large scale. 2. Quadrant Growth: (zone 1) E. coli grew rapidly; (zone 2) S. aureus- good growth; (zone 3) strep- not as heavy growth; (zone 4) pseudomonas- formed a colony similar to diluted plate growth 3. Blood agar: (1)E. coli doesn’t have lyses properties therefore it grows but it isn’t clear or white (2)Staph aureus- beta hemolysis ( red agar becomes clear has the bacteria lyses the blood cells) 4. EMB agar: (1) E. coli which ferments lactose and will change color to a metallic green on the EMB plate (2) pseudomonas which does not ferment lactose so there was no color change and not as strong of growth.

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Lab 5 Notebook

Back to Home Page Title: KD05 Testing bacteria for antibiotic sensitivity Objective: To determine the threshold of antibiotic sensitivity across bacterial strains using the Kirby-Bauer method (AKA Standardized Disc Susceptibility Test) Procedure:

**1. Streak the 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°C overnight
4. Measure zones of inhibition (diameter)
5. Compare results with sensitivity chart Notes:** White zones: Susceptible to the antibiotic and the bacteria has been killed Susceptible: means the antibiotic would work for treating the bacterial infection Non-white zones (no clearing): completely resistant to the antibiotic and no change- the bacteria grows all the way up to the disk Zone of inhibition: this is where there is a white/clearing around the antibiotic disc after being left in the incubator overnight; measure the area in millimeters and compare to the chart *used for any type of

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

bacteria* Wet Lab: *use PPE ; non selective agar; 8 different antibiotics on agar plate; coat surface of agar plate with bacteria creating lawn growth & then placed disks.

1. spread the bacteria with pipet 150 microliters
2. placed drops all around the plate and discard the pipet
3. use sterile L spreader (looks like a rake) keeping lid above plate spread bacteria thoroughly over plate- discard spreader
4. use forceps to remove disk and place on plate a finger width apart from edge and plenty of room between each remaining disk (don’t break surface of the agar) *make sure there is equal spacing to see zones of inhibition; each disk is labeled
5. invert and place in incubator 37°C overnight *if there is any overlap, go to area where there isn’t overlap and measure radius and double for diameter Results: Looking for how much bacteria killing happened around each disk

• List diameter zone of inhibition for each disc
• Reference chart to determine sensitivity level Resistance > Intermediate > Susceptible (killed the bacteria) Antibiotic Resistance Intermediate Susceptible Erythromycin ≤^13 14-22^ ≥ 23 Gentamicin ≤ 12 13-14^ ≥^15 Oxacillin ≤ 10 11-12^ ≥^13 Penicillin ≤ 28 ---^ ≥^29

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

WET LAB RESULTS: On the back of the plate mark diameter of each sample to measure (measure in millimeters)

Antibiotic Diameter

Measurement

Results

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

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Title: KD06 Enzymatic Assays Objective: Profile bacterial populations based on key enzymatic reactions and determine growth/metabolic characteristics.

Procedures:

Oxidase Test: Qualitative test to determine the presence/absence of

cytochrome c oxidase activity in bacteria

1. Use a sterile loop pick an isolated colony from BAP (blood agar plate.)
2. Smear directly onto the reaction area of the slide.
3. Examine test area for color change within 20 seconds *cyto. C used in ETT during aerobic respiration (if oxygen is present turns purple) *pseudomonas and gonorrhea are positive for oxidase *differentiates gram neg bacteria

Catalase Test : Qualitative test to determine the presence/absence of

catalase, an enzyme used to breakdown hydrogen peroxide. Procedure:

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 *oxygen rich environment; hydrogen peroxide forms oxygen radicals which kills cells *catalase breaks down hydrogen peroxide *differentiates between staph/strep: staph is catalase positive *run experiments twice to avoid a false negative

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Coagulase test : Qualitative test to determine the presence/absence of

coagulase, an enzyme that plays a role in the formation of blood clots. Procedure:

1. Using a sterile loop carefully pick colony from plate.
2. Add colony to tube of rabbit plasma, incubate overnight at 37 ℃.
3. Next day examine sample for precipitants (cloudy) *Fibrin (blood clots) good for bacteria as it is a defense mechanism used to identify antibiotics by bacteria *fibrin coat protects from antibiotics and phagocytosis *staph aureaus is coagulase positive

Lipase test : Qualitative test to determine the presence/absence of lipase, an

enzyme that hydrolyzes triglycerides (fatty acids/lipids) Procedure:

1. Use a sterile loop unknown culture onto spirit blue plates.
2. Streak colony across plate, incubate overnight at 37 ℃.
3. Next day, examine sample for precipitants *look for zone of clearing- if there is lipase is present and is changing the pH *lipase positive b. subtillis Qualitative test is yes/no based on visual results; these tests rapidly identify bacteria

Notes:

Oxidase Test: Oxidase negative = no color change Oxidase positive = purple color Catalase Test Catalase negative = no bubbles Catalase positive = bubbles Coagulase test Coagulase negative = no precipitant Coagulase positive = presence of fibrin aggregates (clots)

2022 BIO 171- Microbiology Portage Learning

Lab notebook New updated Exam GRADED A+

Lipase test Lipase negative = no change Lipase positive = reduction in color (zone of clearance)

Results:

Oxidase Test: (differentially 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

2022 BIO 171- Microbiology Portage Learning