BIOD171 Microbiology Lab Notebook-3

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San Joaquin Valley College, Visalia *

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171

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Biology

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Dec 6, 2023

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docx

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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 Title: PN01: Introduction to Medical Microbiology Objective: To establish an organized template for keeping experimental records, procedures, and results. - Cultivation of samples (growth conditions) o Equipment used - Identification of samples (biochemical assays) o Tests available - Evaluation of sample (microscopy) o Visualization and recognition of key characteristics Procedure: 1. This is where each step of a given protocol is recorded 2. Be sure to clearly indicate any deviation that may occur during the experiment. Notes: Additional (helpful) information placed here Basic Equipment - Cleaning o Autoclave 125 degrees Celsius o Uses heat, pressure, and steam to clean its environment

o You obtain well sterilization with hot steam - Growing o Fixed incubator 37 degrees Celsius o Shaker incubator 37 degrees Celsius - Visualization o Microscopy (microscope) - Storing o Refrigerator 4 degrees Celsius o General Lab Safety - Never eat or drink in the labs o Contamination risks - Use Personal Protective Equipment (PPE) o Latex or thermal gloves (application dependent) o Eyewear protection o Lab coat - Never leave the lab while wearing PPE o Public hallway o Bathroom o Cafeteria Results: Safety in lab is probably the most important step when working in a laboratory. Eating in the lab is not allowed and a PPE should always be worn when being present in a lab to prevent contamination. There are different ways growing techniques are done by the use of Fixed and Shaker incubators. Microscopy is how visualization is done

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Lab 2 Notebook Back to Home Page Title: PN02: Introduction to Microscopy Objective: To become familiar with the basic components of a light microscope as well as how to load a sample for viewing. Procedure: 1. Review parts and components 2. Load sample slide onto microscope 3. Select magnification 4. Make necessary adjustments to optimize sample visualization. Notes: Parts of microscope: 1. Eye piece/ocular: adjustable for the fit of your eyes, You should be able to see one circle through both eyes 2. Arm/Neck: if moving the microscope one hand on the neck and one on the base 3. Objective lens: Lens that provide magnification (4x, 10x, 40x, 100x), has a revolving nose 4. Stage: Surface where you place your samples, there’s a clamp holder 5. Focus knob: Located on the side, it helps make the image clearer 6. Iris diaphragm: Determines how much light passes through the sample 7. Base: The bottom of the microscope, make sure that it is steady. NO SHAKING 8. Fine knob: moves the slide up and down 9. Top knob: moves the slide left and right

- Light microscopes require to be turned on. - Control light by the diaphragm or the dial on the side - Too much light can be bad for opaque slides which will not show the image. - Start mid way with light power than adjust. - Higher magnification since we are working with bacteria - Do not hit the glass side to the objective lens Results: After watching the video, I am now able to identify the parts of the microscope and also the function of what each part does.

Lab 3 Notebook Back to Home Page Title: PN03 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 mi wit a drop of distilled water 4. Air dry at room temperature until all moisture ha evaporated. Wet Mount 1. Clean slide (70% ethanol) 2. Circle area on slide for easy location of specimen using a wax hydrophobic pen – keeps water inside ring. 3. Apply organism to slide: a. If from culture, use sterile loop to apply to slide b. If from plate, use sterile loop to pick colonies and mix with a drop of distilled water. 4. View under microscope

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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 approx. 1-3 drops onto slide. b. Spread into a thin film 3. Allow to air ray 4. Fix organism to slide by passing 3 times through a flame.(Heat fixing). Do NOT overheat slide! 5. Flood the slide with crystal violet for 30-60 seconds. 6. Rinse slide with water. 7. Cover the gram’s iodine for 30-60 seconds 8. Rinse with water 9. Decolonize with alcohol 10. Counter Stan with Safranin for 30 seconds 11. Rinse with water 12. Blot dry and examine under microscope

Notes: - Gram positive bacteria = Purple o Thick peptidoglycan layer, retains crystal violet stain. - - Gram negative bacteria = Pink o Thin (single) peptidoglycan layer, damaged by alcohol rinse step and crystal violet stain washed away o Pink color derived from Safranin (secondary countestain) - Stains and shape: - Gram (-) Rods - Gram (+) Chains

- Gram (+) Clusters Acid Fast Stain - Strong resistance to decolonization - 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 nigrosin dye o Negatively charged, repelled from membrane Wet lab 1. Put on PPE 2. Put samples in tube and run the top of the tubes through the flame for sterilization 3. Clean slides with a chem wipe

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4. Draw a circle with a wax pen in order to keep the bacteria in the area. 5. Run the top of the tube the flame again 6. Put loop in the tube and stir it around 7. Spread a layer inside the wax pen circle. 8. Air dry it 9. Heat over flame 3 more times 10. Repeat for the other 4 samples 11. Dispose loop in biohazard waste Staining each sample 1. Cover the entire circle with crystal violet dye for 1 min. 2. Rinse off with water until no more color is present 3. Cover slide with iodine through an eye dropper and let it sit for 1 min. 4. Rinse off with water 5. Decolonization with alcohol 6. Rinse off with water 7. Counterstain with safranin thoroughly and let it sit for 45 seconds 8. Final rinse 9. Blot dry with a wipe than let it air dry 10. Use wipes to completely dry the slide. Examine under a microscope 1. 40x magnification with the stage lowered all the way 2. Place the slide on the stage by opening the stage clips 3. Turn the microscope on

4. Illumination set at 50% 5. Diaphragm open all the way. 6. Focus microscope a. Raise the sample so that it focuses while using the coarse knob b. Dark image in focus uses the fine focuses to make it clearer. Results: Sample #1: Staphylococcus aureus (gram +, purple color round cluster like) Sample #2: Escherichia coli (gram -, pink color, rod shape, capsule like)

Sample #3: Bacillus subtilis (gram +, dark purple color, rod shape) Sample #4: Pseudomonas aeruginosa (gram -. pink color, rod shape)

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Sample #5: Streptococcus (gram +, purple color spherical shape, chain structure)

Lab 4 Notebook Back to Home Page Title: PN04 Introduction to 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 loo 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 the area#3 ONCE - Use a back and forth pattern to dilute the culture in each zone. - Invert plate and incubate overnight at 37 degrees Celsius [Non-selective agar] 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 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. (DO NOT CROSS INTO OTHER QUADRANTS) - Invert plate and incubate overnight at 37 degrees Celsius

Notes: - Non-selective Media : Important for the expansion of unknown bacteria - Selective Media : Used to eliminate irrelevant bacteria from mixed cultures (May only grow Gram-positive or Gram-negative) - Differential Media : Used to distinguish between species of the same group Results: - 4 phase o Zone 1 growth o Zone 2 growth o Zone 3 has a single colony isolated - Quadrant growth (Non-selective) o Zone 1 E-coli strand grew rapidly. o Zone 2 Staphorsius grew. o Zone 3 Streptococcus grew but not that heavy o Zone 4 Pseudomonas strand in Zone 4 has less growth - Blood agar divided into two quadrants (Non-selective) o E-coli darker and no lysis property o Staphorsius: Betahemolysis - EMB plate (Differential Media) o Top quadrant is Ecoli is able to ferment lactose gave partial green metallic color o Gram-negative Pseudomonas does not ferment lactose so no color change or rapid growth

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Lab 5 Notebook Back to Home Page Title: Objective: Procedure: Notes: Results:

Lab 6 Notebook Back to Home Page Title: Objective: Procedure: Notes: Results:

Lab 7 Notebook Back to Home Page Title: Objective: Procedure: Notes: Results:

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Lab 8 Notebook Back to Home Page Title: Objective: Procedure: Notes: Results:

Lab 9 Notebook Back to Home Page Title: Objective: Procedure: Notes: Results:

BIOD171 Microbiology Lab Notebook-3

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