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Home , Microscope slide, Microscopy

Unit 1 and Techniques

General and Medical Lab ■ 1

Lecture 1 Tasks for the day: ■ Cover Lecture 1 material. Vodcast found at: https://youtu.be/kszwtP3Bw2w ■ Perform Experiment 1A: Ubiquity of , Lab Period One ■ Perform Experiment 1B: Microscopy

I. ______is the study of ______. Special techniques are required to isolate, grow, and/or visualize these agents and II. Ubiquity and Importance of Microorganisms A. Where are microorganism 1. ______2. Food 3. 4. ______5. Clothing 6. ______7. Air 8. ______9. Acid 10. ______11. Feces B. Where aren’t they? Remember: Pure cultures are very 1. ______uncommon in nature. When working 2. In the interior of a healthy human body, excluding the digestive tractwith (e.g. sterile ______media and______, pure cerebral spinal fluid, ______, bone marrow, while it’s in the bladder). C. Without microbes: cultures, it is important to use 1.______sterile technique. _____ and dead and animals would not even decompose. 2. Without photosynthetic microorganisms, ______. 3.______. D. Despite the ubiquity of microorganisms, they______. Sterile media stays sterile until inoculated and then, if inoculated with a single microorganism, the culture is a ______.

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Parts of A

I

B J K C L D M N E O F G P H

A ______: I Oil-immersion objective : Magnify 10 Magnifies _____ B Nosepiece J High-dry objective lens: Magnifies _____ C ______: K Low-power objective lens: Holds specimen Magnifies ______D : ______L Power switch ______onto the specimen. E ______: M Knob to control the ______of Controls the amount of light that enters the objective. F Field iris diaphragm: Controls the amount N Coarse adjustment focusing knob: Makes of light leaving the source ______in focus. G Light source O Fine adjustment focusing knob: Makes ______in focus. H X-axis control knob: Moves specimen P Y-axis control knob: Moves specimen ______III. Microscopy General and Medical Microbiology Lab ■ 3

A. Types of microscopes: 1. ______a. Forms ______. 2. The ______a. In this type of , a hollow cone of light is focused on the specimen in such a way that only light reflected or refracted by the specimen forms an image. The image appears as ______. b. Allows for visualization of considerable ______in larger eukaryotic microorganisms. Also better for visualizing ______. 3. The phase-contrast microscope a. Converts slight differences into and density into ______. b. Often used to observe ______. 4. ______a. Exposes a specimen to UV, violet, or blue light and forms an image of the object with the resulting fluorescent light. 5. a. ______is focused on the specimen using magnetic in a vacuum. The ______passing through it and the beam is focused by magnetic lenses to form an enlarged visible image of the specimen on a fluorescent screen. b. The resolution is ______. c. Capable of well over ______.

How Does It Work? Light Microscopes A light microscope is a collection of mirrors and lenses. Microscope lenses act like a ______.

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Brief Recap: Types of Microscopes

Dark Field Microscope Phase Contrast Microscope A hollow cone of light hits the specimen and only Changes small differences in refractive index and cell refracted and reflected rays enter objective. Image is density into large variations in light intensity (enhances bright against a dark background. contrast). For an image, go to For an image, go to http://www.microscopy- http://microbewiki.kenyon.edu/index.php/File:Diato uk.org.uk/intro/illu/phase.html ms_in_dark_field_lighting.jpg.

Fluorescence Microscope Electron Microscope Short (UV, Blue) light is focused on the A beam of electrons hits specimen and is scattered. specimen and emission forms the image. Image forms on a fluorescent screen.For an image, go Can be used to view cells attached to an opaque to http://remf.dartmouth.edu/ surface. Staphylococcus_aureus_TEM For an image, go to http://www.nobelprize.org/educational/ physics/microscopes/fluorescence/gallery/index.html

C. Magnification and Resolution 1. Magnification a. The process of ______as an optical image. b. The shorter the ______, the greater the ______. (See How Does It Work? Focal points, right) c. The total magnification of a microscope is the ______of the magnifying power

Important formula to remember: Objective lens x Ocular lens = Total magnification

For example: Low power: (10X)(10X) = 100X High dry power: (40X)(10X) = 400X Oil immersion power: (100X)(10X) = 1000X

For example: What is the total magnification of our light microscopes if the 40X objective lens is being used? ______

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How Does It Work? Resolution ______The distance between the front surface of the lens and the surface of the cover or The diameter of the specimen when it is in focus. field of view at 100X total magnification for our microscopes has already been measured.

If the same wavelength of light is being used, which lens (a or b) has the greater resolution? ______

2. Resolution a. The ______objects that are ______. The better the resolving power, the closer the objects can be and still be seen as ______. b. Resolution is ______when ______wavelengths of light are used. c. Lenses with ______have shorter ______and better resolution. (See How Does It Work? Resolution, above) d. Immersion oil i. Oil has the______ii. Rays that would not enter the objective in air, due to and , can do so in oil. This effectively ______. e. Parfocal i. In a microscope that is parfocal, the image should______when the objective lens is changed. f. Determining the ______of objects i. Size can be estimated by first calculating the ______of the field of view at 100X total magnification. This can be done by viewing a ruler etched on a glass slide. ii.Now, by estimating ______by a microorganism, size can be estimated.

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iii. There is an ______relationship between the ______and the ______. The greater the magnification, the closer the object appears and the smaller the size of the field.

Total Magnification 100X 400X 1000X Field 2000 μm/4 = 2000 μm/ 10 = diameter 2000 μm 500 μm 200 μm

Test your understanding: 1. Using what you just learned about determining the size of a microorganism, approximately how long is this specimen? ______

2. How much of the field of view would the microorganism above take up if the total magnification was 400X? ______

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Experiment 1A: Ubiquity of Microorganisms Microorganisms are ubiquitous; that is, they are present nearly everywhere. In this Terms and experiment, you will try to isolate and other microorganisms from various Definitions sources using different types of media. Agar: A polysaccharide derived from red algae Lab Period One used to solidify a liquid medium. Procedure Colony: A visible Students will work individually. population of From the side bench, collect: 2 TSA plates, and 1 TSB tube microorganisms Inoculation originating from a 1. Moisten a sterile swab with sterile water, using the on your bench. Using this single swab, collect a sample from any surface or object, such as cellphones, shoes, drinking parent cell and growing fountain, a strand of hair, various body parts, etc. Try whatever interests you, and be on a solid medium. creative. In theory, because a colony is derived from 2. After the sample has been collected, inoculate a Tryptic Soy Agar (TSA) plate by a single parent cell, gently rolling the swab over the surface of the agar. Discard the used swab into a it represents a pure biohazard container. culture. 3. Label the bottom of the plate with your name, lab section number, date, and the Culture media, source of the sample. Write on the outer edge so that the markings do not interfere or the singular with observing the colonies growing on the plate. “medium”: A 4. Inoculate a second TSA plate by the following procedure: solution of nutrients a. Open the lid of the plate. required for the growth b. Place it close to your mouth and cough hard three times onto the plate of bacteria Tryptic Soy Agar c. Place the lid back on. (TSA): A rich solid d. Correctly label the plate. medium that contains a 5. Inoculate a tube of Tryptic Soy Broth (TSB) by removing the cap, putting your digest of casein — the thumb over the top of the tube and inverting the tube several times. Replace the cap. principal in milk Label the tube with your name and lab section using a piece of tape. Do not write — and soy products. directly on the cap or tube. It is an all-purpose Incubation medium that supports 1. After inoculating culture media with microorganisms, place the plates into the the growth of many diverse organisms. incubation bucket in an inverted position — with the agar side up. There are only a Tryptic Soy Broth few exceptions to this rule of inversion that you will see later in the course. Place the (TSB): A rich liquid tubes into a rack in the incubation bucket. The incubation bucket will then be placed medium that contains a into the ; your TAs or instructor will generally do this. digest of casein — the principal protein in milk — and soy products. Lab Period Two It is a general-purpose Procedure medium that supports Students will work individually. If you are the growth of 1. Collect your plates from the trays on the side bench. interested in organisms Observe the TSA plates for colonies of various sizes, seeing the that are not exacting in incubator their food shapes, and colors. Each bacterial or fungal species where requirements. gives a characteristic colony color and morphology. microorganis Draw the colonies observed on both TSA plates in the ms are placed spaces provided in the results section for this to mature and experiment. Pick three colonies from either of the TSA grow, please ask your TA if plates and describe the colony color and morphology. you can help Also observe the cloudiness, or turbidity, of your TSB with this tube and estimate the number of bacteria per mL. process.

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Experiment 1A: Results 1. Draw the colonies observed on the TSA plates. Note: You will be able to make these observations only during the next lab period — after the plates have been incubated.

TSA Swab Plate TSA Cough Plate

2. In your own words, describe the colony morphology and color of three different colonies from either of the TSA plates. Colony 1: ______Colony 2: ______Colony 3: ______3. Does the nutrient broth tube show growth of bacteria? ______4. Based on the amount of turbidity, estimate the number of bacteria/mL present in the nutrient broth using the following table. ______Turbidity Bacteria per mL None 0-106 Light 107 Moderate 108 Heavy 109 Turbidity and bacteria count Note: Bacterial populations grown in liquid medium usually do not exceed 3x109 bacteria/mL.

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Experiment 1B: Microscopy In this exercise, you will become familiar with a bright field microscope that you Terms and Definitions will be using throughout the semester. Every student will have received five slides Immersion oil: The refractive to place in a slide box for future use. When you receive your slides, select a box index is a measure of the relative from the large drawer in your lab bench and use the colored tape on the lab amounts by which light bends, or bench to label your box with your name and lab section. The primary refracts, when passing from one objective of this exercise is to gain experience in using the bright field medium to another. Light emitted microscope. You will observe the microbial life present in a provided water by the microscope light source will sample or an already prepared Winogradsky column as you practice working refract upon hitting the glass and with the microscope. specimen; it will then bend again Procedure when passing from the glass and Students will work individually. specimen back into air. Thus, if air 1. Place a slide on the lab bench frosted side up. The frosted section should exists between the front surface of the specimen or cover glass and feel rough. the lens, certain rays of light will 2. Draw a line on the slide with a Sharpie marker. This line will be used to be lost due to refraction. This help you focus. decreases resolution. Oil has the 3. Choose to observe some of the water from one of the provided water or same refractive index as glass. Winogradsky column samples. Collect some of the chosen sample using a Thus, placing oil between the transfer ; be sure to stir the sample first and try to pick up some of the front surface of the specimen and “gunk” from the mud/water interface. Put a drop of the sample next to the the lens stops light rays from line of the slide. being lost and effectively increases 4. With your forceps, pick up a coverslip and place it on top of the sample. Avoid resolution. bubbles by putting the cover slip down at an angle. Magnification: The process of 5. View the sample using the microscope by the following procedure: enlarging, as an optical image, the 6. Lower the microscope stage a little in order to secure the prepared slide onto size of an object the stage using the spring-loaded slide holder. Microscope: A device for magnifying objects that are too 7. Turn on the main power switch and adjust the light until a visible circle of small to be seen with the naked light shines up through the slide. Be certain both the aperture iris diaphragm eye are open and that the condenser is set to 0. Microscope, compound: A 8. Position the 10X objective lens directly microscope that employs two or above the focus line on the slide. Use the Remember: If more lenses coarse adjustment knob to bring the stage as you see more Microscope, simple: A single- close to the 10X lens as possible. Now, use than one circle of lens magnifier light when the fine adjustment knob to back the stage looking in the Parfocal: The objective lenses are away until the line comes into focus. mounted on the microscope so oculars, move 9. Using the x- and y-axis controllers, move the eyepiece they can be interchanged without the slide to view the sample. Try to identify having to appreciably vary the lenses until only some of the organisms. You may move to focus one circle of light Resolution: See resolving power. the high dry objective lens (40X) into place is present. Resolving power: The ability to to get a better look at the tiny microbes, but distinguish objects that are close please do not use the oil immersion lens together. The better the resolving (100X) at this point. power the closer the objects can be and still be seen as separate.

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10. Draw some of the organisms you observed in the results section for this experiment. When you have finished observing Microscope care the slide, remove it from the mechanical stage and discard the • Never slide a microscope across a bench surface. coverslip in the glass disposal container. Wash and save your Always carry a microscope with both hands; one microscope slide. hand should be placed on the arm and the other 11. Wash the slides under tap water and store them in the slide should support the base. box provided in your drawer. Do not place used slides back • Microscopes should be cleaned before and after into the original box. use. Use ONLY lens paper and lens cleaner. 12. Correctly prepare the microscope for storage and put it Kleenex, paper towels, and even Kimwipes can away scratch lenses. • Only use oil when using the 100X oil immersion lens. DO NOT get oil on the other objective lenses, as this can ruin them. • Store microscopes with the 10X (low power) objective lens in position or such that the region lacking a lens is in position. Turn the light intensity all the way down. • Do not wrap the cord around the microscope. Instead, fold the cord and place it between the arm and the stage or beneath the stage. • Use the course adjustment focusing knob to lower the stage towards the light source. Do not crank down on the knob! • Replace the dust cover before putting the microscope away. • Store the microscope in the cabinet with the arm facing out. ALWAYS FOLLOW THESE INSTRUCTIONS WHEN PUTTING AWAY THE

MICROSCOPE: 1. Turn off the power and place the 10X objective lens or region lacking a lens into position. Turn the

light intensity all the way down. 2. Clean each and every lens — all objective and ocular lenses — with lens paper and cleaning solution. Never ever ever use any other kind of tissue or towel. Always clean starting with the 10X and ending with the 100X; this prevents oil from being transferred to lenses other than the oil immersion lens. 3. If using oil, clean up any spilled oil present on other parts of the microscope. 4. Unplug the microscope, fold the electric cord and place it behind the stage. Never wrap the cord around the arm of the microscope. Replace the dust cover on the microscope and carefully put the microscope in the cabinet with the arm facing out.

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Experiment 1B: Results 1. Draw a few of the organisms that you observed in the water sample.

2. Use the method presented in the lecture to determine the approximate size of at least two organisms viewed. ______3. Complete the following table: Microscope type Function Bright field Microscope part Function Aperture iris diaphragm Condenser Low-power objective lens High-dry objective lens Oil immersion lens Ocular lens Mechanical stage Coarse adjustment knob Fine adjustment knob

4. Differentiate between resolving power and magnifying power of a lens. Resolving power: ______Magnifying power: ______5. What is meant by the term parfocal? ______

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6. What factors are influenced by the iris diaphragm? ______7. Which objective lens focuses closest to the slide? ______8. How is total magnification of a compound microscope determined? ______9. Describe the purpose of using immersion oil with the 100X objective lens? ______

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Lecture 2 Tasks for the day: ■ Cover Lecture 2 material. Vodcast found at: http://youtu.be/R1ErtvqVlXQ ■ Perform Experiment 1A: Ubiquity of Microorganisms, Lab Period Two ■ Perform Experiment 2A: Staining Microorganisms ■ Perform Experiment 2B: Morphological Unknown, Lab Period One

I. Simple Stains: Direct and Negative In the last lab we viewed samples (______) under the microscope. This is a fast way to view ______that is ______. We were able to make true assessments of ______. However, these wet mounts are ______and can be a potential ______. II.______samples (smear preparations) A. 1. ______fixation: simultaneously ______. This is the ______fixation method. 2. ______fixation: has the same results as the heat fixation. Examples of chemical fixatives are alcohol and formaldehyde. B. Disadvantages of a fixed sample Can’t observe specimen ______Causes a slight ______C. Advantages of a fixed sample 1. ______- can be used for long-term study. 2. The preparations ______(below) to enhance contrast and reveal specialized cell structures (e.g. flagella, endospores, capsules, cell walls etc..) III. Bacterial Cell Shapes We will deal mainly with the two most common shapes: ______

______

Bacterial Cell Arrangements

Strepto- chain of cells Diplo- Tetrad- Sarcina- Staphylo-Irregular clusters of cells

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IV. The composition of a stain A. Solvent How Does It Work? B. A solute contains Acidic A- A- A- ______, A- A- which are highly conjugated and give A- the its A- ______. A- 1. ______A- ______dyes A- a. Contain A- A- ______Cell with a negative Positively charged chromophores charged groups, which bind ______the cell to ______surface. The entire cell is stained ______. the color of the dye. b. Direct dyes are the ______and Basic Dyes examples include methylene blue, basic fuchsin, crystal C+ violet, safranin and . c. Applied to bacterial smears that have been ______. 2.______Cell with a negative Negatively charged chromophores are ______dyes ______the negative charge a. Possess______of the cell surface. They ______, leaving the cell uncolored. such as carboxyls (-COO-) and hydroxyls (-OH-). b. Can be used to determine morphology and cellular arrangement in bacteria that are ______to withstand heat-fixing. V. Staining categories A. ______(today) 1. Uses a ______(acidic or basic) and all organisms stain the ______. 2. Is a ______method to determine cell size, shape and arrangement. B. ______(Labs 3 and 4) 1. Divides bacteria into ______based on staining properties. 2. Is ______but the color of staining gives information ______in addition to size, shape and arrangement. VI. Some processes used in the identification of bacterial unknowns: A. ______(staining)

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B. ______(e.g. type of colony and time it takes to grow) and ______(e.g. carbohydrate fermentation and production of virulence factors) C. Results can be coupled with a ______.

Colony morphology terms When recording colony morphology, it is important to also record color, optical properties (translucence, sheen) and texture (moist, mucoid, dry). However, remember that color is often influenced by environment.

Shape: Margin (edge): Elevation:

Circular Entire (smooth) Flat

Irregular Undulate (wavy) Raised

Punctiform (tiny) Rhizoid Convex

Lobate Pulvinate

Filamentous Umbonate

New tools you will encounter in this lab course:

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Experiment 2A: Staining Microorganism

Terms and Definitions I. Smear Preparation Chromophores: Groups with The first step in most bacterial staining procedures is the preparation of a smear. conjugated double bonds that In a smear preparation, cells from a culture are spread in a thin film over a small give the dye its color. area of a microscope slide, dried, and then fixed to the slide by heating or other Colony morphology: chemical fixatives. A good smear preparation should be: Characteristics of a colony 1. A thin layer of cells so that individual cells can be observed. such as shape, edge, elevation, 2. Fixed appropriately to allow repeated washings during staining. color and texture. Colony: A single cell divides Procedure exponentially forming a small, Students will work individually visible collection of cells. Colonies are observed when Note: A good smear preparation is the key to a high-quality stain. Care bacteria are grown on a solid taken when creating a smear will allow for accurate observations. medium. Each colony usually IMPORTANT TIME-SAVING TIP: Prepare this smear at the same time as contains 107-108 bacteria. you prepare the smear of your unknown (See Experiment 4: Morphological Direct, cationic, basic or positive Unknown). dyes: These contain positively 1. Use a slide from your slide box. If necessary, clean the slide using soap and charged groups. Examples water. Dry the slide using a KimWipe. Place the frosted side of the slide facing include methylene blue, basic up and draw a circle (about the size of a nickel) on the bottom, unfrosted, side fuchsin, and . of the slide. Place 2-3 loopfuls of water on the frosted side of the slide within These dyes directly bind to and stain the negatively the area demarked by the circle. Don’t forget to draw a focus line on the charged surface of bacterial frosted side of the slide. cells. 2. Flame an inoculating needle and allow it to cool. Pick up a tiny amount of Fixation, heat or chemical: an Escherichia coli colony and mix it into the drop of water on the slide. Application of heat or a 3. Flame the needle and transfer a small amount of a Saccharomyces cerevisiae chemical (95% Ethanol with colony in the same manner to the same drop. You now will have a mixture of 3% HCl) to a bacterial smear E. coli (bacteria, prokaryotic cell) and S. cerevisiae (, eukaryotic cell) in the preparation. This procedure same smear preparation. simultaneously kills and 4. Air-dry the slide completely. Heat fix the slide by passing it over the flame 3 attaches the bacteria to the times. The slide should be uncomfortable to the skin but not painful. The slide slide. Negative, anionic, or acidic dyes: Contain functional is now ready to be stained as described in the next part of the procedure. groups that have a negative II. Simple Stains charge. Examples include A. Direct Stains eosin, nigrosin and Congo B. Negative stains red. These dyes are repelled by the negatively charged A: Direct Stains surface of bacterial cells. The cell wall of most bacteria has an overall net negative charge and thus can be Thus, they stain the stained directly with a single basic (positively charged) stain or dye. This type of background, leaving the stain allows us to observe the shape, size and arrangement of bacteria. bacterial cells clear and bright against a dark background. Turbidity: The cloudy appearance of a liquid medium due to the presence of bacteria.

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Procedure Students will work individually. 1. Use the smear prepared in the previous procedure. Staining is done at the sink. 2. Add several drops of methylene blue, enough to cover the smear, and wait 1 minute. 3. Rinse the slide with water from the squirt bottle and blot the slide with bibulous paper. There is no need to remove paper from the bibulous booklet; simply place the slide into the booklet and gently blot. 4. Redraw the focus line on the top of the slide, if necessary. 5. Focus on the line with the 10X objective (refer to the microscope focusing procedure in Lab 1 if you are unsure how). Once you have focused on the specimen using the 10X objective, move the 40X objective lens into position. Use the fine adjustment knob to bring In a direct stain, organisms are stained directly with a single the specimen into focus. Now, use the basic dye, while the background appears unchanged. following procedure to view the specimen using the 100X (oil-immersion lens): a. Rotate the nosepiece to the empty slot between the 40X and 100X objectives. b. Add a drop of oil to slide where the light passes through. The oil has the same refractive index as the glass slide and thus prevents light loss. c. Move the 100X objective lens into position. The lens will be immersed in oil. d. The specimen will be out of focus, but you will probably see a blurry image. Focus using the fine focus knob. Turn — slowly — ½ a turn toward you. If the specimen does not come into focus, turn back to the approximate starting position and then turn a ½ turn away from you. If the specimen is still not in focus, return to the l0X (low power objective) and begin again. NEVER use the Coarse Focus knob when using the 100X objective. e. Once the specimen is in focus, find a field that has isolated organisms. Then, while viewing the organisms, fine-tune the image by gently adjusting the condenser diaphragm to give the best light. If necessary, adjust the fine focus to give the sharpest image. If you have difficulty in bringing the image into view, move the stage adjustment back and forth while focusing. 6. After examining the slide, move the oil immersion objective away from the slide. Clean the objective thoroughly with lens paper and lens cleaning solution. 7. In the results section for this experiment, draw the organisms observed in the microscopic field. Note: Saccharomyces cerevisiae is a species of yeast. It is a relatively large single-celled eukaryotic organism. Escherichia coli is a tiny, rod-shaped, prokaryotic bacterium.

B. Negative Stains In contrast to direct stains that bind to bacteria directly, a colors the background of a smear rather than the bacteria. These stains have negatively charged functional groups, so they will not bind directly to negatively charged bacteria. The advantages of negative staining are: 1. Bacteria are not heat fixed so morphology changes are not problematic, and 2. Some bacterial species resist basic stains (Mycobacterium) and one way they can be visualized is with the negative stain

Like basic staining, negative staining does not differentiate bacteria; one can only determine morphology.

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Procedure: Students will work individually. 1. Using a flamed inoculating loop, place 2-3 loopfuls of in two separate circles on a clean slide. There is no need to add water to the Congo Red. 2. Using a flamed inoculating needle, pick up a small amount of Bacillus subtilis and stir it into the left drop of Congo Red. 3. Use a toothpick to scrape material from your teeth near the gumline and stir this into the second drop of Congo Red. Be sure to keep the two drops separate. 4. Air dry — do not heat fix. 5. Flood the slide with acid-alcohol (95% ethanol, 3% HCl) until it turns blue. This generally takes about 2 seconds. Drain the excess acid-alcohol into the appropriately labeled waste container, but do not wash the slide. In a negative stain, organisms appear white or 6. Allow the slide to air dry; do not blot. colorless against a blue-stained background, 7. Examine both smears. First focus using the 10X objective. For best viewing, locate an un-cracked region You will not be able to see individual organisms, but you of dye on the edges of the smear before should be able to focus on the stain. Then move to 40X moving to the 100X objective lens. and finally to the oil immersion lens with oil. Draw a typical microscopic field for each smear in the results section. Note: Organisms appear white, or colorless, against a blue stained background. For best viewing, locate an un-cracked region of dye on the edges of the smear before moving to the 100X objective lens.

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Experiment 2A: Results II. Simple stains 1. Direct Stain: Draw and label examples of E. coli and S. cerevisiae. Be sure to illustrate the relative sizes of each microorganism.

2. Negative Stain: Draw and label examples of B. subtilis and of the performed tooth scraping. Be sure to illustrate the relative sizes of each microorganism.

3. What is the purpose of simple staining? ______4. Differentiate between basic and acidic dyes. ______5. What is the purpose of heat fixation? ______6. Is heat fixation done for all stains? Explain. ______

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Experiment 2B: Morphological Unknown The staining procedures introduced in Experiments 3-8 are commonly used by to help characterize and identify bacteria. These stains often make it possible to determine the group of organisms to which an unknown isolate belongs. With few exceptions, staining is the first step in identifying a bacterial unknown. Although staining alone does not give sufficient information about the organism to make a definitive identification, it will give some important clues. You will be given an unknown pure culture on which you will perform the various stains as you go through the next few lab periods. The Morphological Unknown Table, found in the results section for this experiment, is where you can record the findings from these experiments on your unknown. Lab Period One Procedure: Students will work individually. 1. Collect an unknown from the side bench. Record the number of your unknown in the Morphological Unknown Table in the index. Your TA will also record the number of your unknown. It is important that the same unknown number is used throughout the identification process. 2. Perform a direct stain (methylene blue) on your unknown. Use the same staining procedure as was used for the E. coli and S. cerevisiae mixed smear in Experiment 3. Determine the shape of your unknown and any distinctive arrangements of the cells. Record your observations. Lab Period Two This procedure is meant to continue your investigation of your morphological unknown. In this lab period, you will perform a Gram-stain and an acid-fast stain on your unknown microorganism as described in Experiment 5. 1. Collect your unknown from the side bench. Verify that the number matches the one used for your direct stain. 2. Perform a Gram stain and an acid-fast stain on the unknown microorganism. 3. Record your observations. Lab Period Three This procedure is meant to continue your investigation of your morphological unknown. In this lab period, you will perform an endospore stain and attempt to identify your unknown microorganism. 1. Collect your unknown from the side bench. Verify that the number matches the one used in previous labs. 2. Perform an endospore stain as described in Experiment 4A. 3. Record your observations. 4. Based on the data you have acquired on your morphological unknown, identify the organism using the dichotomous keys provided on pages xxxv-xxxviii in the appendix. Verify the identity of your unknown with a TA or instructor. Be sure that you are provided with a species name in addition to your determined genus name. This will enable you to further investigate the organism. Please, once you know the genus and species name of your unknown, use your laptop or mobile device to research one interesting thing about the bacterium. When you engage in your daily authentic conversation with your TA, please tell her or him what you learned!

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Experiment 2B: Results 1. Number of Unknown: ______2. Colony color and morphology (growth on the agar slant): ______3. Staining characteristics:

Direct Stain Gram stain Color: ______Color: ______Shape: ______Shape: ______Arrangement: ______Arrangement: ______Gram reaction (G+ or G-)______

Acid-fast Stain Endospore Stain Color: ______Color: ______Shape: ______Shape: ______Arrangement: ______Arrangement: ______Acid-fast (yes/no): ______Endospore (yes/no): ______

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4. Use the key to determine which organism you might have: ______Although you will only be able to determine a possible genus, please verify this with either the instructor or TA. At this point, you will be given both a genus and species name to enable you to perform some additional research on the bacterium. •Please, once you know the genus and species name of your unknown, use your laptop or mobile device to research one interesting thing about the bacterium. Please write about this below.

•When you engage in your daily authentic conversation with your TA, please tell her or him what you learned!

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Lecture 3

Tasks for the day: ■ Cover Lecture 3 material. Vodcast found at: https://youtu.be/JtyV9QCP8OU ■ Perform Experiment 2B: Morphological Unknown, Lab ■ Perform Experiment 3A: Gram Stain ■ Perform Experiment 3B: Acid-fast Stain Period Two

I. Gram Stain A. Developed in 1884 by the Danish physician ______. . Used to ______between ______. C. The mechanism by which Gram-positive cell walls resist decolorization is not fully understood. However, it is thought that: 1. Gram-positive cell walls, upon alcohol treatment may become dehydrated and the ______behind the thick peptidoglycan layer. 2. In contrast, Gram-negative cell walls have ______and it is thought that the alcohol may extract these lipids, making the ______. 3. It is very important to perform the Gram stain on ______because, as Gram-positive cells age, they may lose their ability to retain the crystal violet dye. II. Acid-fast stain A. Used to detect cells capable of ______. 1. Cells that can retain this stain are termed ______. 2. Acid-fast cells have a ______; specifically, they contain a great deal of a waxy lipid called ______. B. Acid-fast stains are important for identifying bacteria in the genus ______(e.g. Mycobacterium leprae and Mycobacterium tuberculosis, which are thepathogens responsible for ______). C. The cell wall integrity is destroyed in dead cells, so it is important to perform this staining procedure on ______.

See the next few pages for diagrams on how Gram stains and acid-fast stains really work.

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Gram stain table Procedure Reagent Color: Gram-positive Color: Gram-negative Fixed cells on slide COLORLESS COLORLESS Primary stain Crystal violet PURPLE PURPLE Mordant Iodine PURPLE PURPLE Decolorizer Alcohol PURPLE COLORLESS Counterstain Safranin PURPLE RED Here’s an easy way to remember the procedure for a Gram stain:

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Acid-fast stain table Color: Acid-fast Color: Nonacid-fast Procedure Reagent bacteria bacteria Primary dye Carbolfuchsin RED RED Decolorizer Acid-alcohol RED COLORLESS Counterstain Methylene blue RED BLUE

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Experiment 3A: Gram Stain

In a gram stain, Gram-positive cell walls remain violet in color and Gram-negative cells turn bright pink.

The previous experiment introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. The Gram stain is a differential stain commonly used in the microbiology that differentiates bacteria on the basis of their cell wall structure. This is the most important staining technique in .

Procedure Students will work individually. 1.Using a sterile inoculating loop, add 1 loopful of sterile water to the slide. Prepare a mixed smear of Escherichia coli (a G- rod) and Staphylococcus epidermidis (a G+ coccus). 2.Air dry and heat fix. 3.Cover the smear with crystal violet — the primary stain — TIME SAVING TIPS for 1 minute. ■ You will need to turn in a Gram-stain 4.Gently wash off the slide with water. slide as part of the Gram Stain Report 5.Add Gram’s iodine — the mordant — for 1 minute. assignment (find more information on 6.Wash with water. this assignment on page iii in the 7.Decolorize with 95% ethanol. This is the tricky step. appendix). Therefore, if the above stain Stop decolorizing with alcohol as soon as the purple color or that of your unknown (performed has stopped leaching off the slide. The amount of time later in this lab period) are stains of this takes will vary depending on thickness of smear. which you are proud, keep them! Immediately wash with water. Be sure to dispose of all ■ One element of the first skills test is ethanol waste in the appropriately labeled waste container. the ability to accurately perform a Gram 8.Cover the smear with safranin for 30 seconds. stain. If you feel confident with your 9.Wash both the top and the bottom of the slide with water. technique, ask your teacher or TA if they have time to observe your work. 10. Blot the slide with bibulous paper. 11. Using the 10X objective lens, focus first on the focus line Don’t forget! already drawn on the slide, then on the smear. Then, proceed to If you need a reminder on how to focus the 100X objective lens. using the 10X objective lens, follow the Note: Escherichia coli is a tiny pink (Gram-) rod. focusing procedure in Lab 1. For a Staphylococcus epidermidis is a purple (Gram+) sphere or coccus. reminder on how to focus using the 100X lens, follow the focusing procedure in Lab 2.

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Experiment 3A: Results 1. Draw and label examples of Escherichia coli and Staphylococcus epidermidis.

2. What is the difference between a simple and a differential stain? ______3. Describe the function of each of the following in the Gram stain: Mordant: ______Primary stain: ______Decolorizer: ______Counterstain: ______4. Which step in the Gram stain is most likely to cause poor results if done incorrectly? ______5. Why must fresh bacterial cultures be used in a Gram stain? ______6. Briefly describe the mechanism of Gram staining. That is, explain — based on cell structure — why Gram- positive cells are thought to stain purple whereas Gram-negative cells stain pink. ______

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Experiment 3B: Acid-fast stains

In an acid-fast stain, only bacteria that is acid-fast will retain the color of the primary stain.

Mycobacterium and many Nocardia species are called acid-fast because, during an acid-fast staining procedure, they retain the primary dye carbolfuchsin despite decolorization with the powerful solvent acid-alcohol (95% ethanol with 3% HCl). Nearly all other genera of bacteria are nonacid-fast. The acid-fast genera have the waxy hydroxy- lipid called mycolic acid in their cell walls. It is assumed that mycolic acid prevents acid-alcohol from decolorizing protoplasm.

Procedure Students will work individually. 1. Add one loopful of sterile water to a microscope slide. 2. Make a smear of Mycobacterium smegmatis. Mix thoroughly with your loop. Because this bacteriumis waxy and resistant to mixing with water, it will be necessary to mix a bit more than you may have previously done and to very thoroughly disperse with the loop. Next, transfer a small amount of Staphylococcus epidermidis to the same drop of water. You will now have a mixture of M. smegmatis and S. epidermidis. 3. Air dry and heat fix well. 4. Cover the smear with carbolfuchsin dye. Place a piece of paper Safety first! towel on top of the dye. Be sure the paper towel is saturated with the Carbofuchsin is a potential dye. 5. Place the slide on the rack over dry heat for 2 minutes. carcinogen. Please wear gloves 6. Cool and rinse with water. when working with this dye. 7. Decolorize by placing a drop of acid-alcohol (95% ethanol with 3% HCl) on the slide and allowing it to sit for 15 seconds. Discard the acid- alcohol in the labeled waste container. 8. Wash the top and bottom of slide with water and clean the slide bottom well. 9. Counterstain with methylene blue containing 1% acetic acid for 30 seconds to 1 minute. 10. Wash and blot the slide with bibulous paper. 11. When viewing the slide under the microscope, focus first on the 10X lens and then move to the oil immersion lens. 12. Draw a typical microscopic field and record in the results section of this experiment. Note: The acid-fast Mycobacterium retains carbolfuchsin and stains hot pink. The Staphylococcus epidermidis is decolorized and the counterstain colors them blue.

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Experiment 3B: Results 1. Draw and label examples of Mycobacterium smegmatis and Staphylococcus epidermidis.

2. What is the primary stain used in the acid-fast staining procedure? ______3. What is the purpose of the heat during the acid-fast staining procedure? ______4. In a clinical microbiology laboratory, the acid-fast stain would be used for diagnosis of what diseases? ______5. What makes a microorganism nonacid-fast? ______

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Lecture 4

Tasks for the day: ■Cover Lecture 4 material. Vodcast found at: https://youtu.be/BmCINUN9M58 ■Perform Experiment 2B: Morphological Unknown,Lab Period Three ■Perform Experiment 4A: Endospore Stain ■Perform Experiment 4B: Capsule Stain ■Perform Experiment 4C: Flagella Stain I. The Bacterial Endospore A. A ______that develops within vegetative cells of several genera 1. ______: rods, e.g. the pathogen Bacillus anthracis 2. ______: rods, e.g. the pathogens Clostridium botulinum and Clostridium tetani 3. ______: cocci B. Develop in nature to allow for survival ______C. Extraordinarily resistant to ______such as heat, UV light, chemical disinfectants, and desiccation. In fact, some spores have remained viable for 100,000 years or more! 1 D. ______in the mother cell frequently differs among species and can depend on the time lapsed after formation.

Terminal spore with ______swollen ______Subterminal ______sporangium E. Endospore structure ______Contains normal A thin, delicate covering cell structures, such as ribosomes and a nucleoid ______

______Several fairly thick regions ______of the protein keratin; Peptidoglycan region, impermeable to chemicals which can occupy as much as half of the spore volume 1. Cano, R.J. & M.K. Borucki (1995) Revival and Identification of Bacterial Spores in 20- to 40-Million-Year-Old Dominican Amber. Science, 268 1. The spore’s mechanism of resistance is ______. Following are some possibilities: i. ______have been found that protect the DNA from heat, radiation, and desiccation. ii. The cortex may ______, protecting it from heat and radiation. General and Medical Microbiology Lab ■ 34

iii. The high concentration of ______appears to play a role in DNA protection, thus enhancing spore resistance to heat, H2O2, and desiccation (Journal of Bacteriology, June 2006, p. 3740-3747, Vol. 188, No. 11 (Setlow et. al.)) II. ______(sporogenesis or sporulation) A. Steps 1. The cell stops growing and the ______. 2. The cell ______. A copy of the DNA is enclosed and the______.

3. The immature spore is ______.

4. The ______is laid down in the space between the two membranes

5. ______are formed.

6. The spore ______and______.

Spore Coat

Exosporium

III. Transformation of dormant spores into active, vegetative cells. A. Stages 1. ______

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Some change in environment (usually exposure to ______or certain chemicals) causes the spore to prepare for germination. 2. ______a. Often triggered by the ______. b. The spore takes on water, ______. c. ______begins to increase. 3. Outgrowth d. The spore coat makes new components, emerges from the remains of the spore coat and______. IV. ______Endospores A. Only ______produce endospores so these should be the cells used for staining. B. In addition to resisting heat and chemicals, the protein coat of an endospore is also ______. Therefore, the staining procedure must be fairly ______.

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V. Bacterial ______A. A well-organized ______. B. Generally composed of ______. C. Help bacteria resist ______by host phagocytic cells, therefore increasing the ______of these bacteria (e.g. ______.) D. Contain a ______and can protect ______. E. Exclude bacterial ______and most hydrophobic ______. F. Can enhance the ability of certain bacteria to ______(e.g. Streptococcus mutans ______). VI. ______bacteria with capsules A. The polysaccharides in the capsule make staining difficult, therefore capsule staining procedures generally ______, leaving the capsule ______. It appears as a ______around the cell. B. Cells with capsules are not heated as heat treatment ______and the shrinkage of the cell can cause a ______.

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Experiment 4A: Endospore Stain

In an endospore stain, steam is used to enable a malachite green dye to penetrate the tough endospore coat. After washing, only the endospores retain the primary green stain.

The presence of endospores in a bacterial culture can be detected by staining with malachite green. Because the endospore coat is so tough, steam is used to enable dye penetration. After washing, only the endospores will retain the green primary stain. Safranin is then used as a counterstain for vegetative cells. The endospore stain is a differential stain because it differentiates spore-formers from non-spore-formers.

Procedure Students will work individually. 1. Prepare a smear of Bacillus subtilis using a darkly colored colony grown on nutrient sporulation media, or NSM. 2. Air dry the smear and heat fix it. 3. Cover the smear with the primary stain malachite green. Place a paper towel on top of the dye. Steam the slide for five minutes on the steam bath in the . 4. After the slide is cool, rinse both the top and the bottom well with water. 5. Counterstain with safranin for 30 seconds. 6. Rinse both the top and bottom of the slide well with water. Clean the bottom of the slide with a paper tower and blot dry. 7. Allow the preparation to air dry. Focus on the smear using the 10X objective and then observe the sample using the oil immersion. Record your observations in the results section of this experiment.

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Experiment 4A: Results 1. Draw and label examples of Bacillus subtilis and any endospores seen.

2. What is the primary stain used in the endospore stain? ______3. What special procedure is used in the endospore stain? ______4. What is the function of an endospore? ______5. List two genera of bacteria that produce endospores. ______

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Experiment 4B: Capsule Stain

A capsule stain allows observers to analyze bacteria for a slimy layer, called a “capsule” that usually consists of a highly hydrated layer of polysaccharide or, in a few cases, polypeptide. Capsules appear as light-colored halos around bacteria.

Many bacteria are surrounded by a slimy layer called a capsule that usually consists of a highly hydrated layer of polysaccharide. The capsule can have a number of different functions: helping bacteria escape phagocytic white blood cells; protecting against bacteriophage and dehydration; and facilitating the adherence of bacteria to surfaces. Staining of the capsule requires that the cells not be heat-fixed, since any exposure to heat destroys the capsule.

Procedure Students will work individually. 1. Make a light smear from the milk culture of Klebsiella pneumoniae. Take 1-2 loopfuls and spread it throughout the circle. Do not dilute the culture with water. 2. Air dry thoroughly. Make certain that there is not evidence of moisture on the slide when you proceed to the next step. DO NOT HEAT FIX! 3. Flood the slide with 1% crystal violet. Allow the stain to sit for 1 minute. 4. Rinse very gently with water. 5. Flood the slide with 20% CuSO4 (copper (II) sulfate) and allow this reagent to sit on the slide for 20 seconds. 6. Rinse very gently with water. 7. Air dry; do not blot. 8. Observe the stain using the 10X, 40X, and oil immersion lenses. Record your observations in the results section of this experiment.

Experiment 4C: Flagella Stain

Motile bacteria are characterized by propeller-like structures called flagella. These structures vary in distribution but allow equipped bacteria to move quickly — albeit via a convoluted path — through aqueous, sometimes even highly viscous, environments. The flagella stain is the only stain that we will not perform in lab; however, please take time to observe this stain in the demo microscope at the back of the room and draw this stain in the results section following this experiment.

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Experiment 4B: Results 1. Draw and label examples of Klebsiella pneumoniae and any capsules seen.

2. Which dye is used in the capsule stain? Is it acidic or basic? ______3. Capsules are composed of what types of molecules? ______4. What is the relationship between the presence of a capsule and bacterial pathogenicity? ______5. In the capsule stain, why is the bacterial smear not heat fixed? ______

Experiment 4C: Results 1. Draw the microscope field from the flagella stain demonstration.

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