MCB2010L Practical 1 Study Guide completed

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2010

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Biology

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Feb 20, 2024

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MCB2010L Study Guide – Practical 1 NOTE: This is a GUIDE to what you need to study for the practical. It IS NOT a comprehensive list. To prepare for the practical, you should study all power points, lectures, and demonstrations. Ubiquity of Bacteria 1. Name four environmental locations where bacteria live. Glaciers Hotsprings Soil Water 2. Name two locations where bacteria live in humans. On the surface outer layer of Skin Inside our mouths 3. What is the difference between a chemically defined medium versus a complex medium? Chemically Defined mediums allows us to know the exact chemical composition being used. Complex mediums doesn’t provide the exact chemical composition being used. 4. What is “autoclaving”? Autoclaving is a process used to sterilize equipment through the use of steam and pressure that will kill all the bacteria. 5. Why is it important to autoclave? It’s important because without its numerous amounts of bacterial colonies could remain on the surfaces of equipment and when used could contaminate other microorganisms. 6. What are the parameters for autoclaving? The parameters for autoclaving includes setting the degrees at exactly 121C, adding pressure that is at a 15psi for a duration of 15 minutes. 7. Our of the areas that were swabbed in lab, was there an area that had more growth compared to the other areas? Yes, compared to the other environment swab the bottom of someone’s shoe showed more bacterial and colonial growth than that of others.  If yes, why do you think there was more growth? I believe this is the case because the shoe interacts with a variety of different environments and their corresponding bacterium.

Brightfield Microscopy 1. Label the parts of the microscope. 2. Define: a. Focus b. Resolution: is the ability to distinguish between two different specimen/object that are in close proximity. c. Contrast: is the difference in intensity between two separate specimen/object or an object/specimen and its background. d. Parfocal: is when an object/specimen is in partial focus when one is changing and increasing to a higher magnification. 3. How do you increase resolution? Increasing resolution can be done through increasing the amount of light being passed through the specimen or with a higher magnification. 4. How do you increase contrast? Contrast can be increased through the decreasing the amount of light passing through the specimen, by using a light phse contrast, or through the staining procedure. 5. Calculate the total magnification of a microscope and give the magnification of the ocular lense and the objective lense. 6. Calculate the magnification of the objective given the total magnification and the magnification of the ocular lens.

7. What type of tissue do we use to clean microscope lenses? Cleaning the microscope lenses requires the use of a special kind of tissue called Lens Paper. 8. Describe what the oil does to the light waves when placed on a slide. Upon reaching the 100x or Oil immersion lens there is little space for light to enter through the specimen which causes the light wave to refract decreasing the amount of light that reaches the specimen. The oil when used takes the refracted light and repositions it so that the light waves pass through the specimen allowing more light to hit the specimen. a. Why does oil immersion microscopy give higher resolution? Oil immersion provides for a higher magnification because it takes the refracted light and repositions the light wavelengths so that they enter and pass through the specimen which provides for more light to enter through the specimen which increase its resolution. 9. What is the total magnification of the following lenses? a. Scanning-4x: 4x*10x=40x b. Low-power-10x: 10x*10x=100x c. High-power-40x: 40x*10x=400x d. Oil immersion-100x: 100x*10x=1000x Bacterial Shapes 1. Name and draw the four basic bacterial shapes. Coccus Bacillus Spiral Vibrio

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2. Recognize the common bacterial arrangements Coccus Diplococcus Staphylococcus Streptococcus Bacillus streptobacillus 3. What does “ubiquity” mean? Describe how bacteria are ubiquitous. Ubiquity refers to the state or capacity of something to exist everywhere. A ubiquity of bacteria is notion of stating that bacteria can be found everywhere. 4. What organelles don’t bacteria have? Bacteria lack a nucleus and any membrane-bound organelles including mitochondria and endoplasmic reticulum.

5. What chemical is in the cell wall of all bacteria? Cell walls consist of a glucose combination that is called Peptidoglycan. 6. Which is bigger, a eukaryote or a prokaryote? Comparing the size of a prokaryote and eukaryote it is found that a eukaryote has a larger diameter than a prokaryote. 7. What is aseptic technique? Describe steps you take to make sure your cultures don’t get contaminated. Aseptic techniques are procedures that are used to sterilize and reduce contamination among various equipment used. Tools like the loop and needle are required throughout a procedure to be constantly made sterile, before starting these tools are placed through/above the incinerator and are held until the full metal portion show yellow flams and after each step either touching water or bacteria they are to be placed through once again, and after use should be placed through once again before putting it away. Plates and mediums should be kept tightly sealed until ready for use and after use should be sealed once again. After use of the bacteria its placed through the autoclave which will rid of the bacteria. If contamination happens or is suspected everything should be made sterile and procedure should be restarted. 8. Bacteria divide by what process? Bacteria divide through a process called binary fission and within a mater of 24hrs one bacteria would evolve into a colony viewed by the eye. Doubling Time is the time it takes a single bacterium to 9. Name 3 ways we can characterize bacteria. Bacteria can be characterized by its shape, smell, and Gram-stain. 10. Be able to identify the different shapes of bacteria when looking at a slide under a microscope. a. Identify what organism you are looking at ( S. aureus or B. subtilis )

Darkfield and Phase-Contrast Microscopy 1. Identify if the image is using Darkfield or Phase microscopy (use my PowerPoint) 2. What is the purpose of using darkfield or phase microscopy to view microorganisms? 3. What are advantages of darkfield microscopy? What are the disadvantages? Darkfield microscopy allows us to view living organisms, acquire a more detailed view of external structures, and with the use of fluorescent dyes an increase image of the specimen. However, darkfield microscopy requires a dark room, more intense amount of light, and dust can be mistaken for a specimen. 4. What is a “phase shift”? What does it result in? Phase shift is the action of shifting the slowness of the light wavelengths by ¼. This action results in a difference in refractive indexes between the specimen and the background causing a halo around the specimen and a bluish background. Motility 1. Draw and label the different types of arrangements of flagella around a bacillus cell. Monotrichous Amphitrichous Peritrichous Lophotrichous 2. Define: a. Taxis: a movement caused by a stimuli within an organism’s environment and can either be a movement towards or away from the specific stimuli. b. Chemotaxis: A chemical stimuli c. Phototaxis: a light stimuli d. Aerotaxis: an oxygen stimuli

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3. What is Brownian motion? A false movement caused by the bombardment of molecules of the solvent and is distinguished through the actions of a shake or jiggle. 4. What is Water movement? Another false movement that is caused by the water movement under the slide. 5. What is true motility? True motility is a rapid swimming movement caused by abrupt changes in direction. 6. How does one differentiate between Brownian movement, water movement, and true motility? 7. What type of agar did we use to determine motility of bacteria? Describe this agar. Sulfide indole Motility is a semi-soft agar that allows us to determine whether a bacteria has motility or not. If a bacteria consists of motility, it will present a cloudy tube and this is because the bacteria is growing on and away from the inoculation line. For bacteria with no motility they only grow along the inoculation line. 8. Of the two tubes, which one shows motility? 9. Name another way (besides the SIM agar) that you could determine motility. Wet mount 10. Which organism is motile, P. aeruginosa or S. aureus ? 11. What cellular structure do bacteria need to have in order to be motile? Bacteria must posses flagella in order to be able to perform true motility. Smear Preparation and Simple Staining

1. Describe the procedure for making a smear from an agar plate (bacterial culture growing on a solid agar plate). Clean the slide with ethanol Sterilize all tool Add water to the slide After sterilizing the sterile inoculating loop, pick one colony and lightly touch it to the agar plate to gain some bacteria. Bring the bacteria over to the slide and smear with it the water spreading it out into a thin smear. After this let it air dry until there is no more glossy texture and sterilize the inoculation loo. Once the glossy texture is gone, heat fix the smear by placing it on the hotplate at a temperature of 70C until the bottom of the slide is hot. After this the smear is prepared and staining begins Flood the stain with whatever dye is required Let the dye sit for its designated time After the set time rinse the slide with water To set and dry the dye and prevent the oil from the oil immersion to harm the smear blot with bulbous paper. Once you blot the slide it is ready to be viewed. 2. What type of paper do we use to dry the slide? Bulbous Paper is used to dry the slide 3. Describe the procedure for making a smear from a liquid culture. Clean the slide with ethanol Sterilize all tools After sterilizing the inoculation loop dip it into the broth and take 1-2 loopfuls. Taking these 1-2 loopfuls place them onto the slide and spread the thinnest smear possible. Let this smear air dry until all glossy spots are gone and sterilize the loop through the Bunsen burner.

After the glossy texture is gone, heat fix it by placing it above and through the Bunsen burner a few times until the bottom of the slide has a hot texture. Once the heat fix is set, the staining procedure can occur. Flood the slide with the required dye and let it sit for its designated time. Once the stain has sat rinse it with water Blot the slide with bulbous paper to dry and remove any access dye. Once the slide is dried it can then be viewed through the microscope. 4. Why must a slide be air-dried and heat-fixed before staining? If you stain bacteria without air-drying or heat fixing the bacteria will first lose its shape and then once the dye hits it the microorganism will be washed away. 5. Define simple stain. How many stains are used. Is a basic dye used or an acid dye? Why? Simple stain is the staining technique that uses one dye to stain the cells in order to distinguish its overall morphology and cell structures.

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6. What is the difference between a simple stain and a differential stain? What can each of these tell us? Simple stain stains all the cells one color and allows a view of the microorganism’s overall morphology and structure. However, Differential Stain is the use of two or more dyes that allows us to differentiate between the different structure and morphology of the different cells. 7. Give an example of a dye we used to perform a simple stain Crystal Violet 8. Why are basic stains used more successfully on bacteria than acidic dyes? Basic dynes are more successful when staining bacteria because it is a cationic chromosphore which carries a positive charge which targets the cells which carry a negative charge. 9. What do basic dyes stain? Why? Basic Dyes stain the cells themselves this is because the basic dyes carry a cationic chromosphore or positive charge which when added to the bacteria target the cells which carry a negative charge. 10. What do acidic dyes stain? Why? Acidic Dyes stain the background leaving the cells colorless, this is because the acidic dyes carry an anionic or negative charge which when added to the slide targets the positive charged background which carries a positive charge. 11. What can a simple stain determine? Simple stain can be used to determine the overall morphology of the cells structure. 12. A student makes a smear by applying a bacterium onto the slide. A simple stain is performed, but when the student looks under the microscope, no organisms are seen. What did the student do wrong when making the smear? The student forgot to perform the preparation in order to stain a slide and because of this there was no adherent to stick the bacteria from sliding off the slide due to the dye or water.

13. A student makes a smear by applying a LARGE amount of bacteria onto the slide. A simple stain is performed, but when the student looks under the microscope, individual organisms can’t be distinguished. What did the student do wrong when making the smear? The student applied to much bacteria onto the slide and when smeared the bacteria were applied onto one another creating a thick smear. The student should have picked up a little dip of the bacteria and applied it in a thin smear. 14. A student makes a smear by applying a bacterium onto the slide. A simple stain is performed, but when the student looks under the microscope, the organisms are misshapen and look weird. What did the student do wrong when making the smear? The student did not let the bacteria air dry before heat fixing it causing the flames from the heat to evaporate or misshape the bacteria. Gram Stain 1. Define differential stain. Give two examples we talk about in the course. Differential stain is a staining technique that uses two or more dye that allows us to compare the difference between the cells. Gram Stain Acid-Fast Stain 2. What morphological difference does the Gram stain distinguish bacteria based on? Gram stain distinguishes the cell types, structure, or certain chemical components. 3. Draw the structure of a Gram positive cell wall. 4. Draw the structure of a Gram negative cell wall. 5. Describe the step by step procedure for performing a gram stain. Assume smear preparation and heat fixing has already been done. By sure to name the dyes used.

Gram Stain Procedure Chemical applied Its purpose Primary Stain: Crystal Violet Flood the slide with crystal violet and let it stand for 1 minute before rinsing. Mordant: Iodine is added and let it stand for 1 minute in order to adhere to the crystal violet to for the chemical complex of Crystal Violet- Iodine after 1 minute rinse. This dye is used to stain the Gram- Positive Bacteria. However, it will stain both the Gram-Positive and Negative. Decolorizer (95% Ethanol) is added and let it sit for 1-2 second than wash immediately after. Decolorizer removes the outer membrane of the Gram-Negative Bacteria along with the Crystal Violet iodine complex, which leaves the Gram-Negative Cells clear. Counterstain: Safranin is added and let sit for 1 minute before rinsing any excess dye. This stains the Gram-Negative Bacteria cell wall red. Dry and Observe dry using the hot plate or bulbous paper and observe under oil immersion. Adheres the crystal violet to the slide which prevents the crystal dye from being removed from the Gram-Positive Bacteria. Both Gram-Positive and Gram-negative still purple

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6. Explain how the gram stain works. Do NOT just repeat the procedure. Tell me why it works the way it does. The Gram stains are designed to attach to the peptidoglycan layer of the bacterial cell wall. Since the Gram-Positive don’t consist of a outer layer the Crystal Violet can easily attach to the wall. However, since the Gram-Negative posses an outer layer outside the peptidoglycan layer it requires a second stain in order to stain fully. Due to this it requires double staining but also ways to prevent a mixture. Through the use of the iodine mordent and decolorizer the staining process can prevent the lost of color from one wall over the other. a. What is the mordant? What does the mordant do? The mordant is an Iodine solution that adheres the crystal violet to the bacterial cell wall preventing it from being washed away during the following steps. b. What is used for decolorization? What happens during the decolorization? The decolorizer is Ethanol 95% which when applied removes the outer membrane layer of the Gram-Negative bacteria and leaches out the crystal violet-mordant complex. This leaves the cells of the Gram-negative bacteria clear. c. What does the counter-stain do? The counter-stain (Safranin) allows for the peptidoglycan layer of the Gram-Negative bacteria to be stained and later for both Gram-positive and Gram-negative bacteria to be compared, 7. What color are Gram positive organisms? Gram-Positive organisms are purple 8. What color are Gram negative organisms? Gram-negative organisms are red 9. A student mixes a known Gam positive organism onto the same slide to make a smear, then performs a gram stain on the slide. When she observes it under the microscope, only Gram negative organisms are

seen. What did the student do wrong? (she did not forget the primary stain!) The student forgot to add the mordant iodine to the slide before continuing on in doing so once she rinsed or added dye this rinsed the Crystal violet dye off the slide. 10. Be able to differentiate between a Gram stain and an acid fast stain under the microscope. a. Read the stain (Positive/negative, color and shape of each type of bacteria) Staphylococcus aureus Gram + coccus Pseudomonas aeruginosa Gram – rod Bacillus subtilis Gram + rod Acid-Fast stain 1. What 2 genus’s (types) of bacteria is the acid-fast stain used to detect? Staphylococcus aureus Acid Fast - coccus Mycobacterium smegmatis Acid Fast + rod 2. For the diagnosis of what diseases is the acid-fast stain performed? TBS and Leprosy 3. What is the morphological difference between acid-fast organisms and non-acid-fast organisms (what chemical is found in the cell wall of acid- fast organisms)? Acid Fast bacteria contain a waxy membrane that is composed of mycolic acid. 4. What color are acid-fast positive organisms? Acid-Fast Bacteria Positive organisms are colored pink. 5. What color are acid-fast negative organisms? Acid-Fast Negative organisms are stained blue.

6. Be able to differentiate between a Gram stain and an acid fast stain under the microscope. a. Be able to read the stain (Positive/negative, color and shape of each type of bacteria) Staphylococcus aureus Acid Fast - coccus Mycobacterium smegmatis Acid Fast + rod

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MCB2010L Practical 1 Study Guide completed

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