BIOD171 Microbiology Lab Notebook (1)

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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: JG01 Introduction to Medical Microbiology Objective: To learn lab safety, how to maintain a lab notebook as well as identification and function of general lab equipment. Cultivation of samples (growth conditions) - equipment used Identification of samples (biochemical assays) - tests available Evaluation of samples (microscopy) - visualization and recognition of key characteristics Procedure: Basic Equipment Cleaning ● Autoclave ○ Heats up to 125 ℃ ○ Uses heat, pressure and steam to sterilize the environment. ○ Steam is more efficient to sterilize an environment compared to dry air. Growing ● Fixed incubator ○ Maintained around 37 ℃ ○ Able to hold petri dishes as well as tubes for liquid cultures. ○ Door is air tight and also can be covered by a metal door to control the light conditions throughout the experiment ● Shaker incubator ○ Maintained around 37 ℃ ○ Utilized when using a growth media, a liquid nutrient broth, to inoculate bacteria. ○ Shakes the culture in a swirling fashion to help aerate the bacteria,which in turn helps it to grow

Visualizing ● Microscopy Storing ● Refrigerator ○ Maintained around 4 ℃ ○ This temperature slows the bacteria's growth to preserve the samples that are being tested ○ Used when a study cannot be completed in one day Lab Safety ● Never eat or drink in the lab ○ contamination risks ● Use Personal Protective Equipment (PPE) ○ latex or thermal gloves (application dependent) ○ eyewear protection ○ lab coat ● Never leave the lab while wearing PPE ○ public hallway, bathroom, cafeteria Lab Notebook Title: First Name, Last Name Initial Exp# Title of Experiment Objective: Display purpose of the experiment Procedure: Each step of a given protocol is recorded ● Temperature/chemical used/growth conditions ● Clearly indicate any deviations that occur during experiment Notes: Additional information placed here. Results: A summary of final outcome of the experiment Notes: Remember to use lab notebook to take notes while watching lab videos. Results: Learned some functions of general lab equipment, general lab safety, and how to maintain a lab notebook.

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Lab 2 Notebook Back to Home Page Title: JG02 Basics of 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: Basic Parts: Standard light microscope ● Eyepiece (oculars): use fingers to grab and separate the pieces to fit the width of the eye. Must use both eyes. Will see a single circle in the viewfinder. Not two overlapping. ● Arm/neck: when moving microscope, hold the arm with one hand and use the other hand to carry the base. ● Objectives: provide the samples magnification. Can use arms to switch the magnification by rotating the lenses (will click when locked in place). The shortest has the least magnification. The magnifications are: 4x (shortest), 10x (yellow), 40x, 100x (largest). ● Revolving nosepiece ● Stage: flat surface where you will place your sample. Two basic ways: clamp holder (pinch 2 metal bars to hold the slides), stage clips (metal clips that rotate on the stage). Ensures the sample isn’t moving around. Has a stage guide which can help to control vertical access and horizontal access. ● Stage controls ● Stage clips ● Coarse/Fine focus: has outer which is coarse adjustment and inner knob which is fine focus (used to finetune). ● Iris diaphragm: controls how much light is let into the microscope which will affect the eye piece. Can be used to restrict how much light goes through to the sample. ● Light Source: can be adjusted using a knob to dim or brighten the light let into the sample you are trying to view. ● Base: make sure it is always steady to avoid vibrations into samples or imaging.

Visualization ● Types of objective: Dry vs Oil ● Intensity of light source: Too bright = saturation, too dark = low visibility ● Total magnification = (power of objective) x (power of eyepiece) ○ If a cell is 15mm in diameter, using a 40x objective and 5x eyepiece the cell will now appear to the eye 200x large or ~3000mm in diameter Results: Now able to identify the parts of a microscope, how it can be used to load and view a sample, how to use the functions, how to adjust magnifications and light, and how to calculate total magnification.

Lab 3 Notebook Back to Home Page Title: JG03 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: - if from culture, use sterile loop to spread onto 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 1. Clean slide (70% ethanol) 2. Circle area on slide with a wax/hydrophobic pen 3. Apply organism to slide: - if from culture, use sterile loop to spread onto slide - if from plate, use sterile loop to pick colonies and mix with a drop of distilled water 4. View under microscope - 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: - use sterile loop to spread ~1-3 drops onto slide - 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. Flood the slide with crystal violet for 30-60 seconds

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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. Counterstain 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: Escherichia coli, staphylococcus aureus Acid Fast Stain: - strong resistance to decolorization - very few structures are acid-fast - commonly used to identify mycobacterium - carbolfuchsin dye retained (red eye) Negative Staining: - commonly used to identify organisms with opaque structures - dark background via nigrosin dye - negatively charged, repelled from membrane Results:

Lab 4 Notebook Back to Home Page Title: JG04 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: Clonial 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 We use a back and forth pattern to dilute the culture in each. Invert plate and incubate overnight at 37 D Celsius Notes: ● Non-Selective Media important for the expansion of unknown bacteria. Anything will grow. ● Selective Media - used to eliminate irrelevant bacteria from mixed cultures. May inhibit one from growing. ● Differential Media- used to distinguish between species of the same group.

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● Nonselective plates: LB plate is a nonselective plate put at 37 degrees. Plate should be inverted. Blood Agar is red in color and it contains RBCs the yeast abstract is and RBCs are important ● Selective Media: red and Has a slight cloudiness MacConkey Algar is Selective media because only gram negative bacteria grows on it. Inhibits gram positive bacteria growing. ● Selective media is used for e Coli ● EMB also selective and grows for gram negative. Dark red but looks bluish green. Can also be used for differential media. One ferments lactose and one does not. Bottom quadrant does not ferment lactose. E Coli showed color stain Results: 4 Phase dilution streaking: Colonial Isolation: - Zone 1 growth Zone 2 and 3 has nice colony isolates. The culture was not as concentrated. Non Selective Agar: - Divided into a 4th. Zone 1 E coli strain grew rapidly Zone 2 staph aureus sample Uniform growth pattern Zone 3 Strep sample not as much growth Zone 4 conservatively less growth. Blood Alger is divided into equal quadrants. First was E Coli Second was Staph aureus exhibits a beta hemolysis with a white film. Red part of Algar turned clear. Bottom quadrant is much darker. E Coli does not. Non Selective plate EMB top quadrant ws streaked with E Coli and gave a partial green metallic color. Bottom quadrant was gram negative pseudomonas strain has no color change.

Lab 5 Notebook Back to Home Page Title: JG05 Testing bacteria for antibiotic sensitivity Objective: To determine the threshold of antibiotic sensitivity across bacterial strains using the Kirby- Bauer method. 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 37C overnight 4. Measure zone of inhibition 5. Compare results with sensitivity chart Notes: Non selective plate. Pipet is used to spread the bacteria. L spreader moves the culture around. When mixing you will feel resistance which means the culture is being mixed in the agar. Using sterile forceps removes antibiotic disc from the top and places it a finger width away from the edge. The discs have a letter and the number the letter shows the antibiotic, and the number is the dosage. Once discs are added, invert, and store overnight in 37C. Zone of inhibition is how much zone how much bacterial killing occurred in each disk.

Results:

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Lab 6 Notebook Back to Home Page Title: JG06 Enzymatic Assays Objective: To profile bacterial populations based on key enzymatic reactions and determine growth characteristics. Procedure: - Oxidase Test: qualitative test to determine presence or absence of cytochrome c oxidase activity in bacteria 1. Using sterile loop pick isolated colony from Bap 2. Smear directly onto the reaction area of slide 3. Examine test area for color change within 20 seconds - Catalase Test: qualitative test to determine presence or absence of catalase, enzyme used to break down hydrogen peroxide 1. Using sterile loop pick colony from plate 2. Smear colony directly onto the glass slide 3. Add 2 drops of hydrogen peroxide to each smear - Coagulase Test: qualitative test to determine presence or absence of coagulase, enzyme plays a role in formation of blood clots 1. Using sterile loop carefully pick colony from plate 2. Add colony to tube of rabbit plasma, incubate overnight at 37C 3. Next day, examine samples for precipitant - Lipase Test: qualitative test to determine presence or absence of lipase, enzyme that hydrolyzes triglycerides (fatty acids/lipids) 1. Using sterile loop unknown culture onto spirit blue plates 2. Streak colony across plate, incubate overnight at 37C 3. Next day, examine samples for precipitants Notes: - Oxidase test - negative - no color change - positive - purple color - Catalase test - negative - no bubble

- positive - bubbles - Coagulase test - negative - no precipitant - positive - presence of fibrin aggregators (clots) - Lipase test - negative - no change - positive - reduction in color (zone of clearance) Results: Oxidase Test: E Coli is gram positive. But it did not turn color so it is Negative for oxidative activity. Positive for lactose fermentation. For Pseudomonas there is a bluish yellow color and it is positive for oxidative activity but negative for lactose fermentation. Catalase Test: Staph e is catalase positive as it has bubbles. Streptococcus catalase (no bubble) is active and is able to break down hydrogen peroxide. Coagulase Test: Both gram positive and both staph. Gram positive staph aureus is positive for coagulase but gram positive staph epidermis is coagulase negative. With negative control with no bacteria added we can see it is still liquid with no coagulation. Staph epidermis is still in liquid form, Staph aureus there is clouding and it does not move, it is positive for coagulation.

Lab 7 Notebook Back to Home Page Title: JG07 Secondary Assays for Bacterial Characterization Objective : To profile bacterial populations based on additional biochemical approaches to better determine growth/metabolic characteristics Procedure: Indole Test:

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TSI Test: API Test: Notes: Indole Test: ● Biochemical test to determine a bacterial species ability to convert tryptophan into indole. ● It has 4 quadrants where you can place your sample. ● Yellow color represents indole negative. Red or pinkish is indole positive so tryptophan is being broken down ● Indole test is important because its responsible for regulating various aspects of bacterial physiology: Spore formation, plasmids stability and drug resistance. TSI Test: ● Triple sugar iron agar is used to assess the ability of bacteria to ferment sugars and produce hydrogen sulfide. ● Used to test for intestinal bacteria, and they are gram negative examples are Salmonella and Shigella ● Slants contain PH indicator ● Red color one has no bacteria ● First yellow tube tells of fermentation of sugars. Empty space on bottom is because gas is produced but it is not hydrogen sulfide gas because it did not turn black

● Second tube is yellowish orange tells us only glucose fermentation It has no gas ● Third tube Black color means gas is being produced; it has hydrogen sulfide gas. ● Fourth tube is negative control and tells you how it works with no bacteria API Test: ● Analytical profile index ● Broad, multi panel test used for rapid characterization of bacterial species ● CIT and VP and gel wells should be filled entirely highlighted by red arrow. ● Arrows in blue indicate wells that need to be covered in mineral oil at the end. ● This creates anaerobic environment for bacteria H2S looking for production of hydrogen sulfide gas. Results: Indole Test: 1. covax Reagent grown overnight. 4 or 5 drops of reagent were added to the liquid culture. It turned pink and it is positive as it has the ability to break down tryptophan into indole. This is an E Coli culture gram negative. Positives for tryptophan break down into indole. 2. 5 more drops of reagent are added to each tube. No pink color for the second or third vulture. 2nd was Pseudomonas gram negative and negative for indole 3rd was Salmonella negative for both as well TSI test: 1. Slant agars used. Negative control was first and remained red which is regular color. First example E Coli gram negative and it has a solid yellow color all the way up the scent and it's able to ferment all three types of sugar. It also produced gas as it had space on bottom. 2. Last sample two discolorations. Gram negative salmonella sample fermented all three sugars and produced / fermented hydrogen sulfide gas API test: 1. 20 wells across the strip. 2. 3 wells CIT CP and GEL needs to be filled in entirety ADH, LDC, ODC, H2S, URE Are anaerobic tests filled almost to the top of the well and then mineral oil was added to make it anaerobic. 3. 5 mils of distilled water was added to the train and API strip was lowered in.

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S1 API Number: 5046550 S2 API Number: 2206002

Lab 8 Notebook Back to Home Page Title: JG08 Biochemical Assays for Bacterial Antigen Detection Objective: To understand the assays available for detecting bacterial or viral antigens Procedure: Notes: ELISA ● Antigen is short for antibody generating pathogen. Antibody is the blue Y shaped structure. A shows the blue Y shaped structure known as the antibody. Add patient samples to each well. If the antigen is present and recognized by the antibody it will be bound (B). From B to C this is the wash step. Any antigen that is not bound will be washed to be removed C is the capture of the antigen to the antibody, followed by incubation with a secondary antibody that recognizes the antigen D has a second antibody with a tag. It makes it a sandwich enzyme with a substrate that will react once activated. A-D is a clear culture E is final step everything is activated and then you get a color change

Western Blot

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● Separation of protein based on size. Mix samples with a loading dye called STS - negatively charged dye. Dye binds to it. Red gel marked with M is called a marker. Sample runs from the negative terminal to a positive terminal. Purple smear called a dye front. Prevent any nonspecific antibody from bonding to the membrane. Add second antibody.

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Agglutination Tests ● Top two rows are negative. The bottom two rows are positive reactions with the antibodies due to clumping. ● Samples 1345 shows clumping ● Sample 2 shows weak if any ● Sample 6 shows no reactions Results: Western blot detects proteins based on its molecular mass. 1st Sample membrane nice protein expression 1. Well 1 no protein 2. Well 2 quite a bit of protein. 3. Well 3 absent 4,5,6 also expressed the protein. 2nd sample molecular standards had protein standards blot at the end and two nice bands of protein of protein and not a lot of background. But overall, it had a lot of background Agglutination Tests Used on a card each quadrant for a different sample. Specific kit is for Staph. Place a drop in the circle of each card. To determine id species or culture containing a staph sample grab a sterile wooden stick and use staph culture and mix it with the drop well. 1. Make sure it is not clumping in quadrant one as a negative reagent control. 2. For the second one on quadrant 2 it was negative bacterial control using E coli. 3. Quadrant 3 had dark blue clumps (agglutination) positive result for. 4. Quadrant 4 we see a positive agglutination in staph aureus. Quadrant 4 staph epidermis. Agglutination test can help you differentiate against a species that potentially staph in origin or something else like E Coli or pseudomonas

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Lab 9 Notebook Back to Home Page Title: JG09 Unknown Pathogen Analysis Objective: To identify an unknown pathogen based on the given assays developed during this course. Procedure: Notes: Two samples from the hospital were brought to your lab for diagnosis. After culturing the samples on a LB agar plate another co worker began to analyze. However, due to an emergency your co-worker has left the remaining work for you. Before leaving your co worker informs you the 2 liquid cultures were accidentally combined before the gram stain was started. However, the 2 cultures remain separated on the LB plates for future tests. Goal is to correctly identify the 2 pathogens 1st Assay: Gram stain: It is purple with clusters/pink rods. The purple indicates positive, and the pink indicates negative

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2nd Assay:

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Results: 1. Gram stain: It has purple cocci clusters that are gram positive and pink rods that indicate gram negative. 2. 2. MacConkey Plates: Culture A there is No growth observed Culture B there is Lactose fermentation and it is E.coli 3. Blood Agar Plates: Culture A has beta hemolysis and Culture B has no beta hemolysis 4. EMB Plates: Culture A No growth observed. Culture B Green metallic growth so its E. Coli 5. Catalase Test: Culture A Bubbles/positive/staph Culture B also Bubbles/positive/staph 6. Oxidase Test Culture: A has No color change/negative Culture B also has No color change/negative. 7. Coagulase Test: a. Culture A Positive (Coagulated) - staph aureus b. Culture B- Negative as it did not coagulate Final Result: Culture A is staph aureus and Culture B is E. Coli

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BIOD171 Microbiology Lab Notebook (1)

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