EXPERIMENT Bacterial Morphology and Staining Techniques Hands-On Labs, Inc. Version 42-0240-00-02 Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise. Experiment Summary: You will describe bacterial staining techniques and the differences between a Gram-positive and Gramnegative bacteria. You will identify the morphology and arrangements of bacterial species. You will prepare bacterial smears using direct, negative, and differential staining techniques and observe the results under a microscope. www.holscience.com 1 Hands-On Labs, Inc.
Objectives Upon completion of this laboratory, you will be able to: Discuss the morphology and arrangements of bacterial cells. Describe direct, indirect, and Gram staining techniques. Summarize the differences between Gram-positive and Gram-negative bacteria. Create tooth plaque smears using direct and negative staining techniques. Prepare Gram stains from cultured microbes. Compare morphologies of Gram stained cells using microscopy. Time Allocation: 3 hours + 48 hours incubation* *Incubation period is only required for those without active broth cultures of E. coli, S. cerevisiae, and S. epidermidis. www.holscience.com 2 Hands-On Labs, Inc.
Materials Student Supplied Materials Quantity Item Description 1 Active culture broth-e. coli 1 Active culture broth-s. cerevisiae 1 Active culture broth-s. epidermidis 1 Bleach 1 Camera, digital or smartphone 1 Clothespin 1 Disposable cup 1 Hand soap 1 Isopropyl alcohol (rubbing) 1 Matches or lighter 1 Pair of scissors 1 Permanent Marker 1 Roll of paper towels 1 Source of tap water HOL Supplied Materials Quantity Item Description 1 Apron 6 Blank slides 1 Chem bag-gram stain kit: 1 - Gram stain solution #1, Crystal Violet, 15 ml 1 - Gram stain solution #2, PVP Iodine, 15 ml 1 - Gram stain solution #3, Decolorizer, 15 ml 1 - Gram stain solution #4, Safranin, 15 ml 1 Congo red pipet-1ml 1 Face mask with ear loops 3 Inoculation Loops 1 Lens paper (50 sheets) 1 Pair of gloves 1 Safety goggles 1 Slide cover glass cube 1 Staining Tray 1 Sterile swabs (2 per pack) 1 Student microscope with 100x oil immersion lens* 1 - Immersion oil www.holscience.com 3 Hands-On Labs, Inc.
1 Tea candle 1 Test tube clamp *Microscopes are purchased separately from the LabPaq kit. Note: To fully and accurately complete all lab exercises, you will need access to: 1. A computer to upload digital camera images. 2. Basic photo editing software, such as Microsoft Word or PowerPoint, to add labels, leader lines, or text to digital photos. 3. Subject-specific textbook or appropriate reference resources from lecture content or other suggested resources. Note: The packaging and/or materials in this LabPaq kit may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List included in your LabPaq kit. www.holscience.com 4 Hands-On Labs, Inc.
Background Bacterial Morphology A bacterial cell can be classified into three general groups by its shape: coccus, bacillus, and spirilla. See Figures 1, 2, and 3. Coccus (cocci, plural) - a spherical or round-shaped bacterium with a diameter of about 0.5 µm Bacillus (bacilli, plural) - a cylindrical or rod-shaped bacterium about 0.5 µm to 20 µm in length Spirilla (spirillum, plural) - a helical or spiral-shaped bacterium about 15 µm in length Figure 1. Coccus (sphere-shaped), Staphylococcus aureus. Centers for Disease control and Prevention, William A. Clark. Figure 2. Bacillus (rod-shaped), Bacillus brevus. Centers for Disease control and Prevention, William A. Clark. www.holscience.com 5 Hands-On Labs, Inc.
Figure 3. Spirillum (spiral-shaped), Rhodospirillum rubrum. Centers for Disease control and Prevention Bacterial Arrangement Bacteria can exist as single cells or they may naturally group into colonies, clusters, or chains. Their arrangement is determined by the way the cells divide and attach to other cells in the colony. Thus, cells can be categorized according to their arrangement. Only cocci and bacilli form organized arrangements; spirillum remain as single cells and do not form clusters or chains. Cocci have greater variation in their arrangement than bacilli. When a coccus bacterial cell divides but the daughter cells fail to fully separate after cell division, a pair of cocci is formed and the pair is called a diplococci. When cocci fail to separate and remain in groups of four, they form squares called tetrads. Cocci can also form cubical packets of eight cells, sixteen cells, and even greater numbers of cells, which are all called sarcina. Cocci can also aggregate into hundreds of cells in continuous, bead-like chains called streptococci and into grape like clusters and irregular clumps called staphylococci. Bacilli have fewer arrangements but can pair together end-to-end forming diplobacilli or link together forming a chain of cells called streptobacilli. Bacilli that line up side-by-side at angles in an arrangement or in stacks are called palisades. See Figure 4 for examples of bacterial morphologies. www.holscience.com 6 Hands-On Labs, Inc.
Figure 4. Examples of bacterial arrangements and morphologies. Alila Medical Images Staining Techniques The morphology of a bacterial cell is defined by its shape, size, and arrangement. However, viewing bacteria through a microscope is often challenging as the cell s cytoplasm is colorless and lacks contrast to the surrounding medium or environment. A single bacterium is not visible to the naked eye, and bacteria are on average 0.5 µm in diameter, ranging in length from 2 8 µm. Stains, also called dyes, are often used to observe cell morphology with a microscope. Staining increases the contrast between the cells and their background. A variety of staining techniques exist, including simple stains (direct stains and negative stains) and differential stains. Simple stain - One dye is used to directly stain the bacterial cell or the background. Cell shape, size, and arrangement may be determined with simple staining techniques. Direct stain - A type of simple stain; only the bacterial cell becomes colored, leaving the background or external environment clear and colorless. Negative stain - A type of simple stain; the cell is unstained and appears as a bright, often colorless object against a dark-stained environment. Differential stain - Two or more dyes are used. Cell morphology can be described and identification of the structural components (composition of the cell wall and other cell features like flagella, capsules, or endospores) is possible. Figure 5 shows one type of differential stain called a Gram stain. www.holscience.com 7 Hands-On Labs, Inc.
Figure 5. Gram stained bacteria (purple) and human cheek cells (pink). The Staining Process Stains are salts dissolved in a liquid and are composed of positive (+) and negative (-) ions. One of the ions is colored depending on the chemical composition of the solution. The colored ions are called chromophores. Chromophores have a positive or negative charge depending on whether the dye is basic (ph>7) or acidic (ph<7). The charge of the chromophores controls how the dye interacts with bacterial cells during the staining process. A basic stain contains positively charged (+) chromophores. Because the cell membrane is slightly negative (-) in charge and opposite charges tend to attract, basic dyes (+) have a high affinity for the cell s surface. Basic dyes (+) are used for direct stains which only color the cell s cytoplasm and leave the background outside of the bacterial cell colorless. Examples of basic dyes are crystal violet, methylene blue, safranin, basic fuchsin, and malachite green. An acidic stain contains negatively charged (-) chromophores. Acidic dyes (-) are used for negative stains in which the dye is repelled from the cell s negatively charged (-) surface. Negative stains color only the background or environment outside the cell. Congo red, india ink, and nigrosin are examples of acidic dyes. See Figure 6. www.holscience.com 8 Hands-On Labs, Inc.
Figure 6. Direct and negative staining. During staining, a thin film of cells called a smear is applied to a blank microscope slide. Often the cells are preserved on the slide through a fixation process, whereby a flame is used to kill the bacteria and firmly affix the cells to the slide. It is important to note that heating causes the cell to shrink in size and some cell features may become distorted. Overheating can cause the cells to deteriorate, removing them from the surface. In negative staining, the bacteria are not affixed by heating. In fact, if the bacteria is still alive it can act as a potential contaminant or pathogen. The Gram Stain Theory All bacteria cells can be classified into two groups based on differences in the structure and composition of the cell wall. A widely used form of differential staining, the Gram stain, was developed in 1884 by the physician Hans Christian Gram. The Gram stain uses two dyes to distinguish Gram-positive and Gram-negative bacteria. Like simple stains, differential stains can provide information on cell morphology such as shape, size, and arrangement. Gram-positive cells have a thick outer cell wall composed of a mesh-like polymer called peptidoglycan, adjacent to an interior plasma membrane. See Figure 7. Teichoic acids are only found in the cell wall of Gram-positive bacteria. The teichoic acids are a group of molecules that run perpendicular to the peptidoglycan sheets and provide structural support. The size of the cell wall of Gram-positive cells ranges from 20 80 nm in thickness and is cross-linked in two dimensions making a strong, more rigid framework. www.holscience.com 9 Hands-On Labs, Inc.
Figure 7. Cell wall structure of Gram-positive bacteria. Alila Medical Images. Gram-negative cells have a thin cell wall comprised of a single layer or sheet of peptidoglycan sandwiched between an outer and inner cell membrane. The thin cell wall is only cross-linked in one dimension and is flexible but more susceptible to lysis. The outer cell membrane also contains specialized molecules or proteins unique to Gram-negative cells called lipopolysaccharides (LPS) which are endotoxins that produce reactions such as fever or shock in Gram-negative infections. The size of the cell wall of Gram-negative cells ranges from 2 8 nm in thickness. See Figure 8. www.holscience.com 10 Hands-On Labs, Inc.
Figure 8. Cell wall structure of Gram-negative bacteria. Alila Medical Images. The cell membrane of bacteria acts as a barrier that is selectively permeable to certain substances. Furthermore, the morphology of the cell membrane can exclude certain drugs and antibiotics from penetrating the cell. Gram-negative bacteria are more resistant to antibiotics than Gram-positive bacteria. In humans, most pathogens are Gram-negative organisms. www.holscience.com 11 Hands-On Labs, Inc.
The Gram Stain Procedure For any bacterial sample, the process for Gram staining includes applying a series of four chemicals: primary stain (crystal violet, a purple dye), mordant (iodine), decolorizer (alcohol and acetone), and counterstain (safranin, a red dye). Study the steps of the Gram staining procedure outlined in Figure 9. Figure 9. Process of Gram staining using crystal violet, iodine, decolorizer, and safranin. Cells that retain the primary stain (crystal violet) throughout the Gram stain procedure are Gram-positive cells. Gram-positive bacteria have cell walls that are thick and extensively crosslinked, allowing the dye complex to stay bound and fixed in the decolorization step. Cells that are colorless after the decolorization step but counterstain with a second dye (red safranin) are Gram-negative. Gram-negative bacteria have an outer cell membrane which is adjacent to and covers the cell wall and is composed of lipids or mostly water. During the decolorization step, the cell membrane is dissolved, exposing the thin cell wall and allowing the crystal violet dye to be washed away. Colorless Gram-negative cells are counterstained in the final step to increase contrast for visualization and identification. As Gram-positive cells age (24-48 hours old), their cell wall degrades causing the peptidoglycan layer to become weak and the crystal violet dye to leak and wash away during the decolorization step. These Gram-positive cells are known as Gram-variable cells and may appear Gram-negative or may appear as both types of cells when stained. www.holscience.com 12 Hands-On Labs, Inc.
Exercise 1: Direct and Negative Stains. In this exercise, you will use simple staining techniques with direct and negative stains to prepare smears. You will identify basic morphology including bacteria shapes and arrangements. 1. Clear a work area and gather all materials listed for this experiment. 2. Wash your hands thoroughly with soap and warm water. 3. Put on the safety gloves, face mask, apron, and goggles. 4. Disinfect the work surface by wiping the work surface with a 10% bleach solution. 5. Label the end of a blank slide 1-Direct-M with a permanent marker. The number 1 indicates that this is the first slide, Direct indicates that the procedures will involve a direct stain, and M indicates that the sample will be taken from the mouth. See Figure 10. Figure 10. Labeled Slide. 6. Flip the slide over and draw a dime-sized circle on the underside of the slide. See Figure 11. Figure 11. Drawing a circle underneath the slide. www.holscience.com 13 Hands-On Labs, Inc.
7. Vigorously scrape the inside of your teeth and gums with a sterile swab. 8. Set the slide so that the label is legible and facing upward with the drawn circle facing downward. Placing the swab only inside the area of the drawn circle, apply the smear to the slide using a circular motion as shown in Figure 12. 9. Allow the slide to COMPLETELY air dry. Figure 12. Smearing the slide. 10. Heat fix the sample with the following steps. a. Light the tea candle. b. Orient the slide so the smear and label face upward. Hold the slide at its edge with a clothespin or test tube clamp. c. Pass the slide over the flame 5 times for no more than a few seconds. See Figure 13. Figure 13. Heat fixing a sample. Note: Do not hold the slide directly in the flame, too much heat will destroy the sample and potentially break the slide. 11. Allow the slide to cool for 10 minutes. 12. Place the slide in the staining tray, smear side up. Locate the Crystal Violet (Gram Stain # 1) provided in the experiment bag. Add 1-2 drops of Crystal Violet stain to the center of the drawn circle, as shown in Figure 14. Allow the stain to sit for 60 seconds. www.holscience.com 14 Hands-On Labs, Inc.
Figure 14. Applying Crystal Violet stain. 13. Gently rinse the slide with tap water by holding it at an angle over the staining tray and allowing water to flow over the stain on the slide and into the tray. Do this until the liquid stain is removed from the slide and the water runs clear. 14. VERY GENTLY blot the slide with a paper towel to remove excess liquid. See Figure 15. Figure 15. Drying a slide with paper towels after staining. Note: Do not wipe the slide or it will remove the smear. Do not apply excessive pressure or slide will break. www.holscience.com 15 Hands-On Labs, Inc.
15. Place the dried slide label-side up on the microscope stage and view at low, medium, and then high magnification. 16. Use a digital camera to take a photograph of the slide at the power that best distinguishes the stained cells. Refer to the appendix entitled Taking Microscope Photos for guidance with taking microscope photos with a digital camera. 17. Resize and insert the photograph in Data Table 1 of your Laboratory Report Assistant. Refer to the appendix entitled Resizing an Image for guidance with resizing an image. 18. Record the total magnification and your observations into Data Table 1. 19. Label the end of a blank slide 2-Neg-M with a permanent marker. The number 2 indicates that this is the second slide, Neg indicates that the procedure will involve a negative stain, and M indicates that the sample will be taken from the mouth. 20. Use the scissors to open the pipet of Congo red stain over a trash can. Use a wet paper towel to wipe scissors after cutting the pipet. Throw the used wet paper towel in the trash. 21. Place a single drop of Congo red on the slide just inside of the labeled edge. See Figure 16. Figure 16. Placement of Congo red on labeled slide. 22. Gather the second sterile swab and vigorously scrape the inside of your teeth and gums. Place the swab into the drop of Congo red stain and agitate the sample to transfer cells from the swab to the stain. 23. Locate a clean blank slide. As shown in Figure 17, place this slide at a 30 degree angle to contact and spread the dye across the slide. www.holscience.com 16 Hands-On Labs, Inc.
Figure 17. Spreading Congo red across the slide. 24. Allow the slide to COMPLETELY air dry. Liquid should NOT be visible on the slide. See Figure 18. Figure 18. Dry slide prepared with Congo red. 25. With the slide label facing upward, place the slide on a microscope and view at low, medium, and high magnification. 26. Take a photograph of the slide at the power that best distinguishes the stained cells and insert the image into Data Table 1 of your Laboratory Report Assistant. 27. Record the total magnification of the image and your observations of cell shape and arrangement into Data Table 1. 28. Soak the stained slides and swabs in undiluted bleach, then wrap in paper towels and dispose of in the garbage. 29. Pour excess dyes that remain in the staining tray down the center of a drain with copious amounts of tap water. 30. Retain other items for use in Exercise 2. www.holscience.com 17 Hands-On Labs, Inc.
Questions A. What is the purpose of heat fixing? Why did the slides need to be heat fixed during the direct stain but not during the negative stain? B. Define the three major types of bacterial shapes. Which of these did you observe in the exercise? C. Is Crystal Violet a basic substance or an acidic substance? Is Congo red a basic or an acidic substance? Describe how you were able to arrive at this conclusion and relate your findings to your experimental observations. www.holscience.com 18 Hands-On Labs, Inc.
Exercise 2: Differential Stains In this exercise, you will prepare bacterial smears via Gram stains using pure samples from broths inoculated in a previous experiment. Note: If you do not have active broth cultures of E. coli, S. cerevisiae, and S. epidermidis you must inoculate new broths a minimum of two days before beginning this exercise. 1. Label the end of a blank slide E. coli with a permanent marker. Flip the slide over and draw a dime-sized circle on the underside of the slide. 2. Gather the active E. coli broth and an inoculation loop. 3. Sterilize the inoculation loop by placing it in a cup of alcohol for 30 seconds. Remove the loop and shake to dry. 4. Remove the lid from an active E. coli broth, and pass the lip through the flame of your tealight candle to sterilize. 5. Carefully insert the loop into the broth. 6. Transfer the liquid adhering to the loop over to the dime-sized circle on the labeled side of the slide. See Figure 19. Figure 19. Transferring broth to slide. 7. Flame the lip of the broth tube before replacing the cap. 8. Place the inoculation loop into undiluted bleach prior to disposal. 9. Allow the slide to completely air dry. 10. Heat-fix the sample by holding it sample side up with a clothespin or test tube clamp while passing above the flame five times. See Figure 20. www.holscience.com 19 Hands-On Labs, Inc.
Figure 20. Heat fixing a sample. Note: Do not hold the slide directly in the flame. Too much heat will destroy the sample and potentially break the slide. 11. Allow the slide to cool. 12. Repeat steps 1-11 for S. cerevisiae and S. epidermidis. 13. Place a slide sample side up in the staining tray. See Figure 21. Figure 21. Slide positioned in staining tray. 14. Add 1-2 drops of Crystal Violet (Gram Stain # 1). Wait 60 seconds. Gently rinse the slide with tap water over the staining tray. 15. Pour the contents of the staining tray down the middle of a drain and return the slide to the tray. See Figure 22. www.holscience.com 20 Hands-On Labs, Inc.
Figure 22. Rinsed slide. 16. Add 2-3 drops of PVP Iodine (Gram Stain # 2). Wait 60 seconds. Gently rinse the slide, and return it to the staining tray. 17. Add 2-3 drops of Decolorizer (Gram Stain # 3). Wait 30 seconds. Gently rinse the slide, and return it to the staining tray. 18. Add 2-3 drops of Safranin (Gram Stain # 4). Wait 30 seconds. Gently rinse the slide. 19. Place the slide on paper towels and allow it to completely dry. 20. Repeat steps 14-20 for the remaining 2 slides. See Figure 23. Figure 23. Gram stained slides drying. 21. With the slide label and sample facing upward, place the E. coli slide on a microscope and view it at low, medium, and then high magnification. 22. Take a photograph of the slide at the power that best distinguishes the stained cells and insert the image into Data Table 2 of your Laboratory Report Assistant. www.holscience.com 21 Hands-On Labs, Inc.
23. Record the total magnification of the image and your observations of cell shape, arrangement, color and Gram + or Gram - into Data Table 2. 24. Repeat steps 21-23 for 2 remaining slides. 25. Once all procedures have been completed and all questions at the end of each section have been answered, clean all surfaces and equipment. 26. Place the stained slides and used inoculation loops in undiluted bleach for 20 minutes, then wrap them in paper towels and dispose of them in the garbage. 27. Discard the Congo red pipet in the garbage. 28. Return active culture broths to your incubation location. 29. Clean all other items with soap and water and return them to your LabPaq kit for future use. 30. Use a 10% bleach solution to clean all work surfaces. Questions A. Describe the difference between Gram-negative and Gram-positive bacteria. Which type of bacteria are most often treatable with antibiotics and why? B. List the steps of the Gram stain technique and describe the purpose of each. Be sure to include a description of the color of the bacteria during each step. C. What is the major advantage of a differential stain, such as the Gram stain, over a simple stain? Was this observed in your experiment? www.holscience.com 22 Hands-On Labs, Inc.