Laboratory Exercises in Microbiology: Discovering the Unseen World PDF

City University of New York (CUNY) CUNY Academic Works Open Educational Resources Queensborough Community College 2016 Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation Joan Petersen CUNY Queensborough C...

City University of New York (CUNY) CUNY Academic Works Open Educational Resources Queensborough Community College 2016 Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation Joan Petersen CUNY Queensborough Community College Susan McLaughlin CUNY Queensborough Community College How does access to this work benefit you? Let us know! More information about this work at: https://academicworks.cuny.edu/qb_oers/16 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] Laboratory Exercises in Microbiology: Discovering the Unseen World through Hands-On Investigation By Dr. Susan McLaughlin & Dr. Joan Petersen Queensborough Community College Laboratory Exercises in Microbiology: Discovering the Unseen World through Hands-On Investigation Table of Contents Preface………………………………………………………………………………………i Acknowledgments…………………………………………………………………………..ii Microbiology Lab Safety Instructions…………………………………………………...... iii Lab 1. Introduction to Microscopy and Diversity of Cell Types……………………......... 1 Lab 2. Introduction to Aseptic Techniques and Growth Media………………………...... 19 Lab 3. Preparation of Bacterial Smears and Introduction to Staining…………………...... 37 Lab 4. Acid fast and Endospore Staining……………………………………………......... 49 Lab 5. Metabolic Activities of Bacteria…………………………………………….…....... 59 Lab 6. Dichotomous Keys……………………………………………………………......... 77 Lab 7. The Effect of Physical Factors on Microbial Growth……………………………... 85 Lab 8. Chemical Control of Microbial Growth—Disinfectants and Antibiotics…………. 99 Lab 9. The Microbiology of Milk and Food………………………………………………. 111 Lab 10. The Eukaryotes………………………………………………………………........ 123 Lab 11. Clinical Microbiology I; Anaerobic pathogens; Vectors of Infectious Disease….. 141 Lab 12. Clinical Microbiology II—Immunology and the Biolog System………………… 153 Lab 13. Putting it all Together: Case Studies in Microbiology…………………………… 163 Appendix I. Information About Lab Practical Exams…………………………………….. 165 Appendix II. Scientific Notation and Serial Dilution …………………………..………… 177 Appendix III. Introduction to Micropipetting …………………………………………….. 181 Laboratory Exercises in Microbiology McLaughlin and Petersen Preface Dear Student: Welcome to the world of Microbiology! Although invisible to the unaided eye, microbes play many important and beneficial roles in nature as well as within the human body. For example, they are critical for processes of decomposition and nutrient cycling, produce natural antimicrobial compounds, and help protect our bodies from dangerous pathogens (disease-causing microorganisms). However, not all microbes are “good guys”-pathogenic bacteria, viruses, and parasites continue to challenge health care providers in numerous ways. The evolution of antibiotic resistance among bacteria, as well as emerging and re-emerging infectious diseases, are just two examples of the significant challenges that we are currently facing. In recent years we have become increasingly aware of the critical roles of the human microbiome (all the microbes we carry within and on our bodies) on human health and disease, yet there is still so much that remains unknown. Health care providers need to have a full understanding of the microbial world to properly detect, diagnose, treat and prevent infectious diseases, as well as to know how to protect themselves and others from harm. We have designed the laboratory exercises in this book around a few major concepts-proper use of aseptic techniques, bacterial staining and microscopy, bacterial metabolism, and control of microbial growth. As you read through each exercise, perform the experiments, and interpret your results, try to always remember the “big picture”—how your knowledge of the microbial world will help you in your future career. Each laboratory exercise begins with objectives and key terms, and review questions are found at the end of each chapter. Be sure to familiarize yourself with the key terms—they are bolded within the text of each exercise. Review questions will help ensure that you understand the “big picture” as well. In addition, there are appendices that provide supplementary information to help you understand the material found in some of the exercises. The creation of this manual involved several semesters of testing out new laboratory exercises, modifying experiments and revising write-ups based on feedback from other instructors and students. We welcome additional comments and suggestions as to how we can improve the manual in the future. Best of luck with your studies—we hope that you will enjoy learning about and exploring the wonderful world of microbes as much as we have. Always remember- you are never alone because your microbes are always with you! A note to instructors: At Queensborough Community College, Lab 13 (Case studies in Microbiology) is not included in the manual but provided to our students as a handout in the last class—this is done so that the students can answer the questions based on what they observe in the lab rather than on prior preparation. A copy of this exercise, as well as answers to review questions can be requested by emailing either of the authors. Please feel free to contact us with questions or comments on the manual. Dr. Joan Petersen: [email protected] Dr. Susan McLaughlin: [email protected] i Laboratory Exercises in Microbiology McLaughlin and Petersen Acknowledgments The authors would like to thank the following people who contributed to the preparation of this manual:  Our College Laboratory Technician, Ms. Laura Rachiele, for her tireless dedication and willingness to try out numerous experiments with us, and for providing the photographs used in the manual  Our two student readers, Bharti Kumari and Stephanie Solomon, for the great effort they put into reading, evaluating, and providing the student perspective on the exercises  Our colleague Dr. Monica Trujillo for providing the Streptomyces cultures and for her expertise in helping to develop this exercise  Mr. Adam Morgenstern & Antonios Tsimounis for providing many of the illustrations  Our fellow Microbiology instructors at QCC for their helpful comments and suggestions on previous versions of the manual  QCC’s library for providing funding for us to complete this project and for their helpful guidance about publishing open educational resources  And finally, to all Microbiology students who have used this manual in its earlier versions for providing us with their comments and suggestions during the revision process ii Laboratory Exercises in Microbiology McLaughlin and Petersen Microbiology Lab Safety Instructions The instructions below are designed to keep you safe in the laboratory. Please read them carefully. If you have any questions about safe laboratory practices, ask your instructor. 1. No eating, drinking or smoking at any time. Do not bring food or drink items into the lab. Avoid all finger/hand-to-mouth contact. 2. A lab coat must be worn at all times in the microbiology laboratory. You will not be allowed to participate in the lab without one. 3. Follow all directions given by the instructor. Bring any safety concerns to the attention of the instructor. 4. Come to lab on time and prepared for that day's experiments. 5. Wash hands, and wipe down bench area with disinfectant prior to working. Before you leave the lab for the day wipe down your bench area with disinfectant and then wash your hands. Wash your hands at any time during the lab if you think you may have contaminated them. Wipe any surfaces or equipment with disinfectant immediately if you suspect contamination with living cultures. 6. Loose clothing and long hair must be tied back while working to avoid burning with open flames or inadvertent contamination. 7. Open-toed shoes (sandals, flip-flops etc.) cannot be worn in lab. 8. Use care with the Bunsen burners. Keep paper, alcohol and other flammable items away from the open flame. 9. Treat all living cultures of microorganisms (bacteria, yeast, etc.) as potential pathogens. Avoid spilling or spreading the microorganisms. Place all used materials in the appropriate waste containers designated for cultures (to be autoclaved). Use the techniques specified by the instructor for handling microorganisms. If there is a spill notify your instructor immediately. 10. Know where fire extinguishers and safety equipment are located in the lab. 11. To prevent contamination of these articles, books, coats, backpacks, etc. (anything you do not need for the Micro lab) must be placed in the designated area and should not be kept at the laboratory table. 12. Make sure to carefully read through the entire procedure before beginning an experiment in the lab. This will help prevent you from making mistakes that could compromise your safety. Notes:  Since many laboratory procedures are carried over to the next week, make sure you bring previous lab write-ups with you to the following lab.  For supplementary color pictures of media, microorganisms, videos, etc., see the Blackboard Review site. Instructions for signing up for this site are provided in the syllabus. iii Laboratory Exercises in Microbiology McLaughlin and Petersen Instructions for Good Laboratory Practice and Care of Laboratory Equipment Correct use and care of the laboratory equipment is considered a fundamental part of good laboratory technique. All students working in the microbiology laboratory are responsible for maintaining equipment and materials in proper working condition. Please read over and follow the instructions listed below: Microscopes The most critical (and most expensive) piece of equipment in the microbiology laboratory is the microscope. If you expect to see specimens through the microscope, it must be kept clean and in good condition. You must use the microscope assigned to your seat. Instructions for the use and care of the microscope can be found in Lab 1 of the lab manual. Report any problems with your microscope to your instructor. Inoculating loops and inoculating needles Inoculating loops and needles are used to transfer bacteria into and from culture media. Inoculating loops have a loop at the end, while inoculating needles end in a point. Inoculating loops are the most common method of transferring bacteria. Inoculating needles are used when stabbing into a medium during specific inoculation procedures, or when it is necessary to pick up a small amount of bacteria from one colony on an agar plate without contacting bacteria in other colonies. Bunsen burners A Bunsen burner is a source of open flame that is used to sterilize loops and needles, as well as flaming the lips of test tubes during inoculations. You must always take great care when operating a Bunsen burner! To light the Bunsen burner, turn the handle of the valve so it is in line with the tubing connecting the Bunsen burner to the gas. Using a striker or a BBQ lighter, light the Bunsen burner. If the Bunsen burner does not immediately light, turn off the gas and determine the cause of the problem. NEVER leave the gas on if the Bunsen burner is not lit. DO NOT lean over the Bunsen burner while lighting it. Once the Bunsen burner is lit, be careful to keep all flammable items, including lab coats, hair, shirt sleeves, scarves, tissues, alcohol, etc. away from the flame. When the Bunsen burner is not actively in use it should be kept in the pilot setting for safety. If you smell gas at any time, check to make sure that the Bunsen burner is still lit. If the flame goes out at any time, TURN OFF THE GAS. When you are finished using the Bunsen burner, be sure to return it to the pilot setting before turning off the gas. Before leaving the lab, make sure that the gas is off (handle of the valve perpendicular to the tubing connected to the Bunsen burner). Microscope slides Any disposable glass slides should be discarded in the sharps container. Do not discard glass slides in the waste cans. Petri dishes and test tubes All materials used for handling or culturing microorganisms are to be disposed of as follows: test tubes placed in racks in a bin for autoclaving; petri dishes in the other bin for autoclaving and disposal. iv Laboratory Exercises in Microbiology McLaughlin and Petersen Spillage Any living culture material that is spilled, either on tables or on the floor, is to be treated immediately with disinfectant and cleaned up with paper towels. Notify the instructor of any spills. The paper towels that you use to clean up the spill should be placed in the bin with the petri dishes for autoclaving. Prepared slides Prepared slides that are used during the semester must be returned clean to the trays from which they were taken. Cleanliness of the room Any papers on the floor at the end of the laboratory period are to be picked up and discarded in the wastebasket. The same is true for your laboratory bench area. VERY IMPORTANT! DO NOT throw plates, tubes, swabs, slides, pipets, pipet tips, broken glass, etc. into the regular garbage. These items need to be disposed of properly. Throwing potentially contaminated items into the regular garbage is a safety issue for students, instructors, lab techs and the cleaning staff. If these items are found in the regular garbage the ENTIRE BAG OF GARBAGE must be autoclaved before disposal. If you are unsure about where an item should go, always ask your instructor. v Laboratory Exercises in Microbiology McLaughlin and Petersen vi Laboratory Exercises in Microbiology McLaughlin and Petersen Laboratory Exercise 1: Introduction to Microscopy and Diversity of Cell Types Objectives 1. Review the principles of light microscopy and identify the major parts of the microscope. 2. Learn how to use the microscope to view slides of several different cell types, including the use of the oil immersion lens to view bacterial cells. 3. Learn about the shapes and arrangements of some common types of bacteria. 4. Review the taxonomic classification system used in scientific nomenclature. Key Terms: microorganism, magnification, resolution, working distance, parfocal, parcentric, prokaryotic, eukaryotic, bacillus, coccus, spirillum, spirochete, morphology, bacterial arrangements, depth of field, field of view, taxonomic classification Introduction The first microscope was developed in 1590 by Dutch lens grinders Hans and Zacharias Jansen. In 1667, Robert Hooke described the microscopic appearance of cork and used the term cell to describe the compartments he observed. Anton van Leeuwenhoek was the first person to observe living cells under the microscope in 1675—he described many types of cells, including bacteria. Since then more sophisticated and powerful scopes have been developed that allow for higher magnification and clearer images. Microscopy is used by scientists and health care professionals for many purposes, including diagnosis of infectious diseases, identification of microorganisms (microscopic organisms) in environmental samples (including food and water), and determination of the effect of pathogenic (disease-causing) microbes on human cells. This exercise will familiarize you with the microscopes we will be using to look at various types of microorganisms throughout the semester. The Light microscope What does it mean to be microscopic? Objects are said to be microscopic when they are too small to be seen with the unaided eye—they need to be magnified (enlarged) for the human eye to be able to see them. This includes human cells and many other types of cells that you will be studying in this class. 1 Laboratory Exercises in Microbiology McLaughlin and Petersen The microscope you will be using uses visible light and two sets of lenses to produce a magnified image. The total magnification will depend on which objective lens you are using—the highest magnification possible on these microscopes is 1000X—meaning that objects appear 1000X larger than they actually are. Resolution vs. magnification: Magnification refers to the process of making an object appear larger than it is; whereas resolution is the ability to see objects clearly enough to tell two distinct objects apart. Although it is possible to magnify above 1000X, a higher magnification would result in a blurry image. (Think about magnifying a digital photograph beyond the point where you can see the image clearly). This is due to the limitations of visible light (details that are smaller than the wavelength of light used cannot be resolved). The limit of resolution of the human eye is about 0.1 mm, or 100 microns (see Table 1 for metric review). Objects that are smaller than this cannot be seen clearly without magnification. Since most cells are much smaller than 100 microns, we need to use microscopes to see them. The limit of resolution of the light microscope you will be using today is about 0.1 m, or 100 nm. This means that we can view objects that are 1000X smaller than what we can see with our eyes alone. Biologists typically use microscopes to view all types of cells, including plant cells, animal cells, protozoa, algae, fungi and bacteria. The nucleus and chloroplasts of eukaryotic cells can also be seen—however smaller organelles and viruses are beyond the limit of resolution of the light microscope (see Figure 1). Table 1: Metric units commonly used in Microbiology The basic unit of measurement of length in the metric system is the meter. There are 1000 millimeters (mm) in one meter. 1 mm = 10-3 meter There are 1000 micrometers (microns, or µm) in one millimeter. 1 µm = 10-6 meter There are 1000 nanometers in one micrometer. 1 nm = 10-9 meter 2 Laboratory Exercises in Microbiology McLaughlin and Petersen Figure 1. Procedures A. Use and Care of the Microscope When instructed to do so, obtain the microscope that corresponds to your seat number from the cabinet (power cords are in a plastic box in the drawer near your seat). Familiarize yourself with the major parts and their functions—you may use the information found at the end of this chapter to guide you. Basic Guidelines for Using the Microscope 1. Always carry the microscope with two hands. 2. Always use the microscope that is assigned to your seat number. 3 Laboratory Exercises in Microbiology McLaughlin and Petersen 3. Clean the lenses with lens cleaner (Windex) and lens tissue before and after use. 4. Report any problems with the microscope to your instructor immediately. 5. Oil must be cleaned off completely before returning the microscope to the cabinet. If you accidentally get oil on the 40X objective, clean it immediately. Microscopes must always be returned to the cabinet clean. 6. Microscopes should always be put away with a low power objective (4X or 10X) over the stage. 7. Always lift the microscope to reposition it—do not drag it across the surface of the table! Total Magnification: The microscope you are using has two sets of lenses that both contribute to the total magnification of the image. The ocular lenses magnify your image 10X. There are 4 different objective lenses—each with a different magnification. The total magnification is calculated as follows: Total magnification= ocular magnification x objective magnification Since the ocular magnification of our microscope is 10X, determining the total magnification of an object with this microscope simply requires multiplying the objective magnification by 10. (Note: other microscopes may have ocular lenses with a different magnification, for example 12X.) Fill in the chart below Name of lens Objective magnification Total magnification Scanning Low High-dry Oil immersion Diversity of Cells: There are many types of cells found among the diverse forms of life on the planet. All cells have certain features in common, such as a plasma membrane surrounding the cell, cytoplasm within the plasma membrane, and DNA as the molecule that stores genetic material. However, there is a great deal of diversity among the cells that make up living organisms. Some living organisms are composed of one cell (unicellular); others are composed of many cells (multicellular). 4 Laboratory Exercises in Microbiology McLaughlin and Petersen Cells that have a nucleus and other organelles are eukaryotic; bacterial cells do not have these intracellular structures—they are prokaryotic. Some types of cells (plants, algae and some bacteria) are photosynthetic (capable of using light energy to make organic compounds from inorganic materials.) Others take in organic molecules as a source of energy. There are several other similarities and differences between cell types that you will learn about throughout the semester. In today’s lab you will use the microscope to look at various types of cells that are often studied in a Microbiology course. B. Examination of different cell types under the microscope In this activity you will look at some prepared slides, as well as make a few of your own. Materials:  Prepared slides: Blue-green algae, mixed protozoa, bacterial types  Slides and cover slips  Pond water sample (if available)  Live Saccharomyces culture  Methylene blue stain  Prepared slides (demos): may include Pinworm (Enterobius), pinworm eggs, mosquito, yeast cells (Saccharomyces), bacterial slides showing varying morphologies and arrangements Instructions: Use the spaces provided at the end of this exercise to draw pictures as you are viewing the slides. 1. Place your first slide (blue-green algae) on the mechanical stage and make sure that it is level and held firmly in place. Your sample /smear should be facing upwards. 2. Use the knobs located below the stage to move the slide left and right, and up and down until the stained area of the slide is centered over the light source. 3. Position the scanning power objective (4X) in place over the slide. (Note: objective will “click” into place). 5 Laboratory Exercises in Microbiology McLaughlin and Petersen 4. Use the course adjustment knob to bring the stage up as far as it will go.  When using the scanning or low power objectives the working distance (the distance between the lens and the slide) is large enough so that the slide will never make contact with the lens. This is not the case when using the high-dry and oil immersion lenses, where the working distance is significantly less. This is why the coarse adjustment knob can only be used with the two low-power lenses. 5. Rotate the coarse adjustment knob away from you until the image comes into focus.  You will not be able to make out much detail at this power—the purpose is to find where your specimen is on the slide so that it is easier to locate when you switch to high power. Low power objectives have a large field of view (the circular area seen when looking through the microscope) and a large depth of field (the thickness of a specimen that is in sharp focus). As magnification increases, both the field of view and depth of field decrease, which is why it is easier to locate your specimen using a low power objective. 6. If needed, use the fine adjustment knob to improve the clarity of the image. 7. After viewing the slide at scanning power, move the slide so that the area you want to focus on is in the center of the field of view.  Since your microscope is parcentric, when you increase magnification you will be zooming in at the center of the field of view. Objects that are not centered at low power may be out of the field of view at high power. 8. Rotate the objective lens nosepiece so that the 10X objective is in place over the slide. Re-focus and adjust the light (if needed) under the 10X objective.  The microscope you are using is parfocal—this means that when it is in focus with one lens in place the same stage position will be in focus with all other lenses. Therefore when switching objectives, DO NOT change the position of the stage—just click the objective you wish to use into place. Note the differences in the appearance of the cells from when you were using the 4X objective. 6 Laboratory Exercises in Microbiology McLaughlin and Petersen 9. After focusing at 10X, rotate the objective lens nosepiece so that the 40X objective is positioned over the slide. Re-focus using the fine adjustment knob and adjust the light if needed.  Note: Remember that you CANNOT use the coarse adjustment knob at high power (40X or 100X objectives). When you are using the high-power lenses the lens is very close to the slide (small working distance) therefore using the coarse adjustment knob at high power could result in damage to the lens, damage to the slide, or both. Remember to sketch what you are looking at in the spaces provided. 10. When you are finished looking at the blue-green algae slide, return it to the slide box and proceed to the next slide—Mixed protozoa. Follow the instructions above to view the slide with the 4X, 10X and 40X objectives in place. Draw diagrams of each.  When viewing the mixed protozoa slide, you should scan different areas of the slide, as there are many types of protozoans and algae present on this slide. Some organisms you might see include Volvox, Amoeba, and Paramecium. Note that in most of these cells the nucleus is clearly visible. 11. Sketch a few areas of this slide with the 10X or 40X objective in place. Use the charts in the lab to identify some of the organisms you see. 12. Return the mixed protozoa slide to the slide box and use a clean slide and cover slip to prepare a wet mount of a live pond sample (see instructions below). View this slide at 4X, 10X and 40X magnification. C. Preparation of Wet Mounts These slides will be prepared with live microbes on them. Since the liquid is left on the slide, cover slips are needed to prevent this liquid from touching the lenses. 7 Laboratory Exercises in Microbiology McLaughlin and Petersen a. Live protozoa: use a dropper to place 1-2 drops of a pond water sample onto a clean microscope slide. Take a cover slip and place it down over the water as shown in the diagram below (try to avoid air bubbles). b. Preparation of a live yeast culture: place one drop of the yeast (Saccharomyces) suspension and one drop of methylene blue onto a clean microscope slide, and prepare a wet mount as shown. Figure 2. Preparation of a wet mount Coverslip Lower slowly Observe these slides under the microscope (use the magnifications suggested by your instructor). As time permits, your instructor may have you examine other types of cells that are set up on demonstration scopes. D. Observing bacterial cells under the microscope. In this exercise you will learn how to use the oil immersion lens, and observe some common morphologies and arrangements of bacteria. 1. Obtain a bacterial types slide from the slide box and place it on your microscope. Since bacterial cells are very small, you will need to use the highest magnification (100X objective, or 1000X total magnification) to see them clearly—however, as with all slides, you should use a low-power objective lens to focus on the slide before moving to high power. The bacterial types slide may be viewed with all 4 objectives—alternatively, your instructor may ask you to skip some (for example, just use the 10X and 100X objectives). Follow the instructions you receive in class. 8 Laboratory Exercises in Microbiology McLaughlin and Petersen  There are three areas of bacteria on the slide. The area closest to the slide label is the darkest and therefore the easiest to find: the area on the rightmost side of the slide is very faint and you may not be able to detect any stain with your eyes. 2. Focus with a low-power objective on the first area of the slide.  The cells will still be very small at this magnification and you will not see any detail. 3. Once in focus, switch the objectives to 10X and then to 40X (this step may be skipped). Adjust light and focus as needed. 4. You are now ready to use the oil immersion lens. This lens requires the use of immersion oil, which has the same index of refraction as glass, to prevent light from scattering and focus it on your specimen (we need a lot of light to see clearly at this high magnification).  Immersion oil MUST be used with the 100X lens.  Immersion oil should NEVER be used with the high dry lens. 5. Rotate the nosepiece so that there is no objective over the stage. Add 1-2 drops of immersion oil to the slide right above where your sample is. 6. Click the 100X objective into place. If done correctly, you should only need to fine focus a little bit to bring the cells into view.  Remember to NEVER use the coarse adjustment knob when focusing under 40X or 100X objectives.  If you “get lost,” it’s better (and faster) to go back to low power and refocus, then switch back to 100X.  Once there is oil on the slide you CANNOT use the 40X lens—rotate the nosepiece the other way to use 4X or 10X to refocus. 7. Use the spaces provided to draw the cells. At this point, your instructor will discuss the morphology (shape) and arrangements of the cells you are looking at. (See Figure 3. at the end of the exercise for more information.) 9 Laboratory Exercises in Microbiology McLaughlin and Petersen 8. When you are finished, move the slide to the right to find the second area of cells (middle of the slide), and then to the third area of cells (right side of the slide).  The third area of cells is very faint and the cells are more spread out. If you “get lost”—try going back to find the second area and then carefully move the slide across to the third area while keeping the slide in focus. 9. When you are finished, clean all oil off of the slide and return it to the slide box. Results Blue Green Algae Low power High power ____________ Total Magnification ______________ Mixed Protozoa Low power High power ______________ Total Magnification ______________ 10 Laboratory Exercises in Microbiology McLaughlin and Petersen Bacterial Types Bacilli Low power 1000X (oil immersion) ____________ Total Magnification ____________ Cocci Low power 1000X ____________ Total Magnification ____________ Spirilla Low power 1000X ____________ Total Magnification ____________ 11 Laboratory Exercises in Microbiology McLaughlin and Petersen Use the page below to draw additional sketches of the organisms you observe. Name of organism ________________ Name of organism ________________ Magnification _______ Magnification _______ Name of organism ________________ Name of organism ________________ Magnification _______ Magnification _______ Name of organism ________________ Name of organism ________________ Magnification _______ Magnification _______ 12 Laboratory Exercises in Microbiology McLaughlin and Petersen Review Questions 1. Why is it important that health care professionals know about microorganisms? 2. What is the main difference between prokaryotic and eukaryotic cells? 3. Why is it important to use immersion oil when using the 100X objective? 4. Are there things that are too small to be seen with a light microscope? Explain. 5. Of the organisms you have looked at today, which are unicellular and which are multicellular? Unicellular: Multicellular: 6. Fill in the blanks: As magnification increases, the area of the field of view _________________, the depth of the field of view _______________________, the working distance __________________, and the amount of light required ____________________. 13 Laboratory Exercises in Microbiology McLaughlin and Petersen 7. Distinguish between morphology and arrangement. 8. What do you think would happen if you tried to view a slide using the oil immersion lens but forgot to add the oil? 9. Use the information below to label the parts of the microscope on the figure provided. Parts of the Microscope a) Ocular (eyepiece): what you look through to view your slide. Our microscopes are binocular, which means they have two eyepiece tubes and both eyes are used (monocular microscopes have only one tube). Typically ocular lenses magnify an image 10X, although some have other magnifications (ex: 12X). Binocular microscopes allow for adjustment of the distance between pupils so that both eyes can be used to observe one image. b) Objective lenses are the lenses close to the stage. Usually microscopes will have 3-5 lenses of different magnifications (ours have 4—4X, 10X, 40X and 100X magnifications). Objective lenses are located on a rotating turret to allow for changes in magnification. c) Coarse adjustment knob: the larger (and outermost) of the two focusing knobs—moves the stage toward or away from the objectives to bring the image into focus at low power. d) Fine adjustment knob: smaller knob within the coarse adjustment knob—used for “fine-tuning” an image. Only fine focus can be used when the 40X and 100X objectives are in place. 14 Laboratory Exercises in Microbiology McLaughlin and Petersen e) Stage: the platform where the slide to be viewed is placed. A mechanical stage holds the slide in place and allows for the movement of the slide to view different areas. f) Illuminator (light source): found at the base (bottom) of the microscope below the stage. g) Condensor/iris diaphragm assembly: found directly beneath the stage. This assembly can be raised and lowered using a knob at the side of the microscope. For our purposes, the assembly should be positioned very close to the bottom of the stage. The condenser is a lens that focuses the light from the illuminator onto the specimen. The iris diaphragm controls the amount of light that passes through the specimen. The iris diaphragm can be opened and closed by twisting the ridged ring of the assembly. h) Base: the bottommost part of the microscope that contains the illuminator. i) Arm: positions the objective lenses and the oculars above the stage. When moving the microscope, the base should be supported with one hand, while the arm is grasped with the other hand. 15 Laboratory Exercises in Microbiology McLaughlin and Petersen Figure 2. Label the Microscope Diagram 16 Laboratory Exercises in Microbiology McLaughlin and Petersen Figure 3. Common shapes and arrangements of bacteria Note: There are other less common bacterial morphologies (e.g., filamentous, squares, etc.) that are not shown here. Spirals Cocci Coccus Diplococci Tetrad Sarcina Spirochete Streptococci Spirillum Staphylococci Vibrio Bacilli Bacillus Diplobacilli Streptobacilli Coccobacillus Palisades 17 Laboratory Exercises in Microbiology McLaughlin and Petersen Scientific Nomenclature Scientific nomenclature is based on a taxonomic classification system. Taxonomic classification is a hierarchical system used to classify and compare organisms. There are 8 ranks in this system, listed below in the order of the most general (broadest) to the most specific: 1. Domain 2. Kingdom 3. Phylum 4. Class 5. Order 6. Family 7. Genus 8. Species Prokaryotic organisms like bacteria may have classifications below the species level, including strain, subspecies, serotype, morphotype or variety. Taxonomic classification indicates how closely organisms are related. For example, two organisms sharing the same Class are more closely related than two organisms sharing the same Phylum. Binomial nomenclature: The scientific name of an organism consists of two words: the genus name and the specific epithet. The genus name comes first and is always capitalized; once identified it can be abbreviated to a single letter. The second word is known as the specific epithet and is not capitalized. The two words together make up the scientific name or species name. The genus can be used alone (you can refer to the genus Staphylococcus or the genus Bacillus) but the specific epithet without the genus name has no scientific significance. Scientific names in print should always be either italicized or underlined and should always be underlined when written. For example, the scientific name for human beings is Homo sapiens or H. sapiens. The scientific name of a bacterium is Staphylococcus aureus or S. aureus. The scientific name often includes a description of the characteristics of an organism. The scientific name Staphylococcus aureus tells you the morphology and arrangement of the individual cells belonging to this bacterial genus (staphylococcus = spheres in clusters) and also tells you that S. aureus often grows in colonies with a golden color (“aureus”). Although scientific names are often descriptive occasionally these descriptions can be deceiving. For example, Haemophilus influenza is a bacterium (not a virus), and does not cause influenza. 18 Laboratory Exercises in Microbiology McLaughlin and Petersen Laboratory Exercise 2: Introduction to Aseptic Techniques and Growth Media Objectives 1. Learn how to inoculate growth media using proper aseptic procedures 2. Learn how to streak for single colonies 3. Understand the uses of selective and differential growth media 4. Determine the properties of some common bacterial types when grown on selective and differential growth media Key Terms: agar, broth, general purpose medium, selective medium, differential medium, colony, aseptic techniques, inoculation, streak-plate technique, contamination, sterile, pure culture, mixed culture Introduction Growth Media To study bacteria and other microorganisms, it is necessary to grow them in controlled conditions in the laboratory. Growth media contain a variety of nutrients necessary to sustain the growth of microorganisms. There are two commonly used physical forms of growth media: liquid media and solid growth media. A liquid medium is called a broth. Solid growth media usually contains agar, which is a mixture of polysaccharides derived from red algae. It is used as a solidification agent because it (1) is not broken down by bacteria, (2) contains no nutrients that can be used by bacteria and (3) melts at high temperatures, and yet is solid at temperatures used for most bacterial growth. Solid growth media is used in the following forms: agar plates, agar slants and agar deeps. To make agar deeps or agar slants, melted agar is poured into a test tube and then allowed to solidify vertically (agar deep), or at a slant (agar slant). Agar plates are made by pouring melted agar into a petri dish. Agar slant Agar deep Agar plate Figure 1. 19 Laboratory Exercises in Microbiology McLaughlin and Petersen Broths can be used to determine growth patterns in a liquid medium, and for certain types of inoculations and metabolic tests that you will be doing later in the semester. They are also the method of choice for growing large quantities of bacteria. Agar slants are commonly used to generate stocks of bacteria. Agar plates can be used to separate mixtures of bacteria and to observe colony characteristics of different species of bacteria (you will perform an experiment in this lab to illustrate this). Deeps are used for several different types of differential metabolic tests (e.g., the gelatinase test, which you will perform in Lab 5). Growth media can be categorized based on their chemical constituents, or the purpose for which they are used.  Complex growth media contain ingredients whose exact chemical composition is unknown (e.g. blood, yeast extract, etc.).  Synthetic (also called chemically defined) growth media are formulated to an exactly defined chemical composition.  A general purpose growth medium (e.g. tryptic soy agar (TSA) or Luria broth (LB) is used to grow a wide variety of non-fastidious bacteria. This type of medium is often a complex growth medium.  A selective growth medium contains chemicals that allow some types of bacteria to grow, while inhibiting the growth of other types. An example of a purely selective growth medium is PEA, phenylethyl alcohol agar, which allows Gram positive bacteria to grow while inhibiting the growth of Gram negative bacteria.  A differential growth medium is formulated such that different types of bacteria will grow with different characteristics (e.g. colony color). An example of a differential growth medium is blood agar, which differentiates among bacteria based on their ability to break down red blood cells and hemoglobin. Blood agar is also a complex growth medium because it contains blood. A growth medium can be both selective and differential. For example, EMB (eosin methylene blue agar) inhibits the growth of Gram positive bacteria. Gram negative bacteria that grow on this medium are differentiated based on their ability to ferment the sugars lactose and sucrose. (Note: the Gram staining procedure divides bacteria into 2 main groups: Gram-positive bacteria and Gram-negative bacteria, based on their cell wall structure. You will be doing Gram staining in the next lab period.) 20 Laboratory Exercises in Microbiology McLaughlin and Petersen Characteristics of Bacterial Growth Even on general purpose growth media, bacteria can exhibit characteristic growth patterns. On agar plates, bacteria grow in collections of cells called colonies. Each colony arises from a single bacterium or a few bacteria. Although individual cells are too small to be viewed, masses of cells can be observed. Colonies can have different forms, margins, elevations and colors. Observing colony characteristics is one piece of information that microbiologists can use to identify unknown bacteria. Some examples of growth characteristics on different forms of growth media are shown at the end of the lab. Aseptic Technique and Inoculation Inoculation is the purposeful introduction of bacteria into a sterile growth medium. A material is sterile when it has no living organisms present; contamination is the presence of unwanted microorganisms. Aseptic techniques are practices that prevent the contamination of growth media. When working in a microbiology laboratory, you must always remember that bacteria are present on all surfaces in the lab, as well as on your own hands and clothing. Aseptic techniques are designed to prevent the transfer of bacteria from the surrounding environment into a culture medium. These techniques require care and concentration. Pay attention to what you are doing at all times! Aseptic techniques include the following practices: 1. Minimize the time that cultures and growth media are open to the environment. 2. Disinfect the work area before and after use. 3. Do not touch or breathe into the sterile culture media or the stock cultures. 4. Loops, needles, pipets, etc. should be sterilized before they are used. 5. When working with tubes, the tube caps should not be placed on the table top; they should be held in your hand while inoculating. 6. When removing the caps from test tubes, flame the lip of the test tube after the cap is removed. This heats the air inside the tube, so the air moves out of the tube, preventing contaminants from entering the tube. 7. Information about the use of the Bunsen burner can be found in the General Introduction in the Lab Manual. 21 Laboratory Exercises in Microbiology McLaughlin and Petersen General Procedure for inoculating media Note: See figure on next page 1. Sterilize an inoculating loop or needle in the flame of a Bunsen burner. The portion of the loop or needle that will contact the stock culture or the growth medium must turn bright orange for effective sterilization. For the most rapid sterilization, place the loop at the top of the inner blue cone of flame—this is where the temperature of the Bunsen burner is the hottest. Remove the loop from the flame after it is properly heated- keeping the loops in the flame for too long will eventually cause them to crack. 2. If you are picking a colony from a plate, cool the inoculating loop on agar that does not contain any bacterial colonies. 3. Pick a small amount of bacteria (you do not need much). If you are inoculating a tube of broth or an agar slant, remove the cap of the tube (do not set the cap down on the table) and flame the lip of the tube. Throughout the procedure, hold the tube at an angle to reduce the probability of particles entering the opening. Insert the loop into the tube and transfer bacteria to the growth medium. Be careful that only the sterilized part of the loop touches the tube or enters the growth medium. 4. Flame the lip of the test tube before replacing the cap. 5. Sterilize the inoculating loop again. Streaking for single colonies In the real world outside the laboratory, bacteria grow in communities made of many bacterial species. If you need to identify the types of bacteria present in environmental or medical samples, you must have a way to separate out the different types and produce pure cultures. A pure culture contains a single bacterial species, whereas a mixed culture may contain many different types of bacteria. The process described in Procedure B (the streak plate method) describes the method that you will use to separate different types of bacteria in a mixture (see pg. 29). 22 Laboratory Exercises in Microbiology McLaughlin and Petersen Inoculating a Plate from a Broth Culture 1. Sterilize the inoculating loop. 2. Remove the cap from tube. Do NOT put the cap of the tube down on the lab bench—hold it 1 3 in your hand. 2 3. Flame the lip of the tube. 4. Place sterile portion of inoculating loop into broth, then remove. 5. Flame the lip of the tube 4 5 6. Replace the cap. 7. Gently streak the surface of an agar plate with the inoculating loop. 8. Sterilize the inoculating loop. 6 7 8 Notes about Labeling and Incubating Plates 1. Always label your plates/tubes BEFORE you do your inoculations. You can use Sharpies on the plates, but wax markers ONLY on tubes. When labeling tubes, label the tube itself—don’t label the cap! 2. Make sure you label the bottom of the plates (the part of the plate that holds the agar). 3. Place plates inverted (upside down) for incubation. This prevents condensation from falling on the surface of the agar and disrupting the streaking pattern. 23 Laboratory Exercises in Microbiology McLaughlin and Petersen Media Used in This Lab Exercise Note: See the Microbiology Review site for color pictures of media. Tryptic soy agar (TSA): General purpose complex growth medium. Mannitol-salt agar (MSA): Differential and selective growth medium. This medium contains 7.5% NaCl, the carbohydrate mannitol and the pH indicator phenol red (yellow at pH 8.4). It is selective for staphylococci due to the high concentration of NaCl, and differentiates based on the ability to ferment mannitol. Staphylococci that ferment mannitol produce acidic byproducts that cause the phenol red to turn yellow. This produces a yellow halo in the medium around the bacterial growth. MS Agar Selectivity Interpretation Identification Growth Organism not inhibited by NaCl E.g., Staphylococcus, Micrococcus No growth Organism inhibited by NaCl Not Staphylococcus Differentiation Yellow halo Organism ferments mannitol Probable S. aureus No yellow halo Organism does not ferment Staphylococcus species (other than S. mannitol aureus); Micrococcus (yellow colonies) Eosin-methylene blue agar (EMB): Differential and selective growth medium. This medium contains peptone, lactose, sucrose and the dyes eosin Y and methylene blue. Gram positive organisms are inhibited by the dyes, so this medium is selective for Gram negative bacteria. The medium differentiates based on the ability to ferment lactose (and/or sucrose.) Organisms that cannot ferment either of the sugars produce colorless colonies. Organisms that ferment the sugars with some acid production produce pink or purple colonies; organisms that ferment the sugars and produce large amounts of acid form colonies with a green metallic sheen. This medium is commonly used to detect the presence of fecal coliforms (like E. coli)—bacteria that grow in the intestines of warm-blooded animals. Fecal coliforms produce large amounts of acid when fermenting lactose and/or sucrose; non-fecal coliforms will produce less acid and appear as pink or purple colonies. 24 Laboratory Exercises in Microbiology McLaughlin and Petersen EMB Agar Result Interpretation Identification No or poor growth Organism inhibited by dyes Organism is Gram-positive Good growth Organism not inhibited by Organism is Gram-negative dyes Colorless growth Organism does not ferment Non-coliform sucrose or lactose Growth is pink and Organism ferments lactose Coliform bacteria mucoid and/or sucrose with some acid production Growth is dark (purple Organism ferments lactose Possible fecal coliform to black with or without and/or sucrose, with large (E. coli) green metallic sheen) amounts of acid production Procedures Make sure you follow aseptic procedures and label everything carefully! A. Observation of bacterial growth characteristics on selective and differential media Each pair of students: 1 MSA and 1 EMB plate Bacteria used: Staphylococcus aureus, Escherichia coli, Micrococcus luteus, and Pseudomonas aeruginosa Inoculation of selective and differential media 1. Divide the MS and EMB agar plates into 4 areas. Label each area with the type of bacteria that will be streaked there. 2. Use the inoculation loop to make a single streak of S. aureus, E. coli, M. luteus, and P. aeruginosa in each of the 4 areas (see below). 3. Incubate the plate until the next lab period. 25 Laboratory Exercises in Microbiology McLaughlin and Petersen B. Inoculation for single colony isolation Each pair of students: 4 TSA plates 1. Sterilize your inoculating loop. 2. Use your loop obtain some bacteria from your sample. 3. Streak the bacteria on a small portion of the plate. 4. Sterilize your loop. 5. Cool the loop on an uninoculated part of the agar surface, and then pass it once through the already-streaked region of the plate. Streak another small portion of the plate. 6. Sterilize your loop. Cool the loop, then pass it once through the second streak, and make a third streak. Depending on the density of the original sample, single colonies will usually appear by the second or third streak. 1st streak 2nd streak 3rd streak Use the procedure outlined above to streak S. aureus, E. coli, M. luteus, and P. aeruginosa for single colonies onto individual TSA plates. Remember that you are trying to isolate single cells or a cluster of cells to form individual colonies. Do not pick up too much bacteria or spread it evenly all throughout the plate! Incubate until the next lab period. 26 Laboratory Exercises in Microbiology McLaughlin and Petersen C. Isolation of single colonies from mixed cultures using the streak plate method Each pair of students: 1 MSA and 1 EMB plate Mixed cultures: Unknown A and Unknown B 1. Streak Unknown A for single colonies on an MSA plate. 2. Streak Unknown B for single colonies on an EMB plate. 3. Incubate until the next lab period. D. Culturing Microorganisms from Environmental Samples Each pair of students: 1 MSA plate, 1 EMB plate, 1 TSA plate; 1 tube of sterile water; 1 sterile swab. 1. Pick one part of the environment that you would like to sample. Possibilities include: bottom of shoe, floor, cell phone, sponge on table, or the bottom of your handbag or backpack. Be inventive—pick an area you would find interesting! Communicate with your lab partners so that different environments are represented at your table. My environmental sample is from: ___________________________________ 2. Moisten a swab in the sterile water; before the removing the swab, press it against the side of the tube to remove excess moisture. 3. Firmly swab the region you would like to sample. 4. Use the swab to inoculate the TSA plate, streaking the entire surface of the plate (see below). Use the same swab to streak the MS plate and the EMB plate. 5. Dispose of the swab in designated plastic container at your lab table. (DO NOT throw swabs in the regular garbage!) 6. Incubate the plates until the next lab period. TSA Plate EMB Plate MS Plate 27 Laboratory Exercises in Microbiology McLaughlin and Petersen Results Record the results of your experiments in the tables below. A. Observation of growth characteristics on selective and differential media Mannitol salt Agar Bacteria Growth Describe growth Interpretation (+/-) characteristics S. aureus E. coli P. aeruginosa M. luteus EMB Agar Bacteria Growth Describe growth Interpretation (+/-) characteristics S. aureus E. coli P. aeruginosa M. luteus 28 Laboratory Exercises in Microbiology McLaughlin and Petersen B. Inoculation for single colony isolation Tryptic soy agar Bacteria Growth Describe growth characteristics (+/-) S. aureus E. coli P. aeruginosa M. luteus C. Isolation of single colonies from mixed cultures Unknown A: Describe growth characteristics on MSA and make a sketch in the circle below. Unknown B: Describe growth characteristics on EMB and make a sketch in the circle below. 29 Laboratory Exercises in Microbiology McLaughlin and Petersen Based on your results, answer the following questions: 1. How effective was the streaking procedure in separating the different types of bacteria? 2. What conclusions do you have regarding the types of bacteria in the unknowns? Unknown A Unknown B D. Environmental Sample Record the results of your own environmental sample and the other sample at your bench in the two tables below Your environmental sample from _______________________________________ Growth (+/-) Describe growth characteristics TSA EMB MSA 30 Laboratory Exercises in Microbiology McLaughlin and Petersen Lab partner environmental sample from ____________________________________ Growth (+/-) Describe growth characteristics TSA EMB MSA Answer the following questions and provide evidence to support your answers. 1. Did you detect any Staphylococcus species in your environmental sample? 2. Were Gram-negative bacteria present? 3. Were there any coliforms present? Was E. coli present? 4. Was S. aureus present? 5. Was there evidence for organisms other than bacteria? (Ask your instructor.) Your instructor may ask you to save some of your environmental samples for further analysis. 31 Laboratory Exercises in Microbiology McLaughlin and Petersen Review Questions 1. Based on your results and the information in the lab manual, fill in the table below. Ability to Ability to Predicted Gram ferment ferment Coliform or Reaction lactose/sucrose mannitol Non-coliform S. aureus E. coli P. aeruginosa M. luteus 2. What general type of growth medium would you use to: (a) grow one type of bacteria but inhibit the growth of another type? (b) discriminate between different types of bacteria? 3. Why is it necessary to sterilize the loop between streaks when streaking for single colonies? 32 Laboratory Exercises in Microbiology McLaughlin and Petersen 4. Define and/or explain the use of the following: (a) synthetic medium (b) agar (c) broth 5. A bacterial species is inoculated on EMB agar. (a) The bacteria do not grow. Why? (b) If the bacteria ferment lactose, what would you expect to see? (c) The bacteria produce clear colonies. Why? 33 Laboratory Exercises in Microbiology McLaughlin and Petersen 6. What medium would you use (TSA, EMB, MS) if you wanted to determine if a Staphylococcus isolate could ferment mannitol? Describe what you would see on this medium. 7. If you were testing water for the presence of fecal coliforms, what sort of medium would you use: TSA, EMB agar or MS agar? If fecal coliforms were present, what would their growth characteristics be on this medium? 34 Laboratory Exercises in Microbiology McLaughlin and Petersen Examples of Bacterial Growth Characteristics in Broths, Slants and Plates Even on general purpose growth media, bacteria can exhibit characteristic patterns of growth. Some examples are shown below. While these growth patterns are an important piece of information when identifying a bacterial species, they are not sufficient for a positive identification. Staining procedures and metabolic tests must be used for a definitive identification. Growth Characteristics in Broths Growth Characteristics on Slants Arborescent Filiform Echinulate Turbid Pellicle Sediment Flocculent Growth Characteristics on Plates Form Circular Irregular Filamentous Rhizoid Elevation Raised Convex Flat Umbonate Crateriform Margin Entire Undulate Filiform Curled Lobate 35 Laboratory Exercises in Microbiology McLaughlin and Petersen 36 Laboratory Exercises in Microbiology McLaughlin and Petersen Laboratory Exercise 3: Preparation of Bacterial Smears and Introduction to Staining Objectives 1. Learn the differences between simple staining and differential staining techniques. 2. Learn how to prepare a bacterial smear from cultured organisms. 3. Learn the differences between gram positive and gram negative bacteria. 4. Learn how to perform the gram stain procedure. 5. Use microscopy to examine gram stained cells. 6. Learn about some special staining procedures, and view examples of these under oil immersion. Key Terms: Gram stain, bacterial smear, simple stain, differential stain, Gram positive, Gram negative, Gram variable, capsule, spirochete, flagella, negative staining, silver stain Introduction Most types of cells do not have much natural pigment and are therefore difficult to see under the light microscope unless they are stained. Several types of stains are used to make bacterial cells more visible. In addition, specific staining techniques can be used to determine the cells’ biochemical or structural properties, such as cell wall type and presence or absence of endospores. This type of information can help scientists identify and classify microorganisms, and can be used by health care providers to diagnose the cause of a bacterial infection. One type of staining procedure that can be used is the simple stain, in which only one stain is used, and all types of bacteria appear as the color of that stain when viewed under the microscope. Some stains commonly used for simple staining include crystal violet, safranin, and methylene blue. Simple stains can be used to determine a bacterial species’ morphology and arrangement, but they do not give any additional information. Scientists will often choose to perform a differential stain, as this allows them to gather additional information about the bacteria they are working with. Differential stains use more than one stain, and cells will have a different appearance based on their chemical or structural properties. Some examples of differential stains are the Gram stain, acid-fast stain, and endospore stain. In this lab you will learn how to prepare bacterial cells for staining, and learn about the gram staining technique. 37 Laboratory Exercises in Microbiology McLaughlin and Petersen The Gram Stain This very commonly used staining procedure was first developed by the Danish bacteriologist Hans Christian Gram in 1882 (published in 1884) while working with tissue samples from the lungs of patients who had died from pneumonia. Since then, the Gram stain procedure has been widely used by microbiologists everywhere to obtain important information about the bacterial species they are working with. Knowing the Gram reaction of a clinical isolate can help the health care professional make a diagnosis and choose the appropriate antibiotic for treatment. Gram stain results reflect differences in cell wall composition. Gram positive cells have thick layers of a peptidoglycan (a carbohydrate) in their cell walls; Gram negative bacteria have very little. Gram positive bacteria also have teichoic acids, whereas Gram negatives do not. Gram negative cells have an outer membrane that resembles the phospholipid bilayer of the cell membrane. The outer membrane contains lipopolysaccharides (LPS), which are released as endotoxins when Gram negative cells die. This can be of concern to a person with an infection caused by a gram negative organism. Figure 1. shows the major differences between the Gram positive and Gram negative cell walls (also refer to your textbook for additional information). The differences in the cell wall composition are reflected in the way the cells react with the stains used in the Gram stain procedure. Gram stains are best performed on fresh cultures—older cells may have damaged cell walls and not give the proper Gram reaction. Also, some species are known as Gram-variable, and so both Gram positive and Gram negative reactions may be visible on your slide. Although the vast majority of bacteria are either Gram positive or Gram negative, it is important to remember that not all bacteria can be stained with this procedure (for example, Mycoplasmas, which have no cell wall, stain poorly with the Gram stain). 38 Laboratory Exercises in Microbiology McLaughlin and Petersen Figure 1. Gram Positive Cell Wall Teichoic acids Peptidoglycan Cell membrane Gram Negative Cell Wall LPS Porin Outer membrane Peptidoglycan Cell membrane Special Stains There are a variety of staining procedures used to identify specific external or internal structures that are not found in all bacterial species (see table at the end of this exercise for a comparison of staining procedures). You will do some of these staining procedures in the next lab (acid-fast staining and endospore staining). In today’s lab, you will observe prepared slides of special stains: a capsule stain (Klebsiella pneumoniae), flagella stain (Proteus vulgaris) and spirochete stain (Treponema pallidum). Capsule Stain Some bacteria secrete a polysaccharide-rich structure external to the cell wall called a glycocalyx. If the glycocalyx is thin and loosely attached, it is called a slime layer; if it is thick and tightly bound to the cell, it is 39 Laboratory Exercises in Microbiology McLaughlin and Petersen called a capsule. The glycocalyx can protect the cell from desiccation and can allow the cell to stick to surfaces like tissues in the body. They may also provide cells with protection against detection and phagocytosis by immune cells and contribute to the formation of a biofilm: in this way a glycocalyx can act as a virulence factor; (contributes to the ability of an organism to cause disease). Capsules can be detected using a negative staining procedure in which the background (the slide) and the bacteria are stained, but the capsule is not stained. The capsule appears as a clear unstained zone around the bacterial cell. Since capsules are destroyed by heat, the capsule staining procedure is done without heat-fixing the bacteria. Silver Stain Flagella (long whip-like structures used for bacterial motility) and some bacteria (e.g. spirochetes) are too thin to be observed with regular staining procedures. In these cases, a silver stain is used. Silver nitrate is applied to the bacteria along with a special mordant; the silver nitrate precipitates around the flagella or the thin bacteria, thus thickening them so they can be observed under the light microscope. Procedures A. Preparation of a Bacterial Smear and Gram Staining This semester you will be performing three staining procedures: Gram stain, acid-fast stain, and endospore stain. All three of these staining procedures begin with the preparation of a bacterial smear. Materials  Clean microscope slides  Staining trays and newspaper  Gram stain reagents: crystal violet, Gram’s iodine, safranin, 95% ethanol  Water bottle (for rinsing)  Bacterial cultures: Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa, Corynebacterium xerosis, and Neisseria sicca 40 Laboratory Exercises in Microbiology McLaughlin and Petersen How to make a bacterial smear 1. Label a clean glass slide as demonstrated by your instructor. 2. Add a small drop of saline to the slide (you will usually put two bacteria on one microscope slide- Follow your instructor’s specific instructions). This can be done by placing a drop of saline onto your inoculation loop and then transferring it to the slide. If you use the saline dropper directly on the slide, do not release a full drop. 3. With an inoculation loop or needle, pick up a small amount of bacteria. Mix it well with the saline and spread the mixture over a wider area of the slide.  Be careful not to have the two smears run into each other. 4. Air dry the bacterial specimen on the slide (slide warmers may also be used). 5. When slides are completely air-dry, heat fix the bacterial specimen by passing the slide slowly over the flame twice (your instructor will demonstrate this).  Heat fixing kills cells, and adheres them to the slide.  Cells will be rinsed off the slides if they are not heat fixed properly.  Be careful not to overheat the slides in this procedure After heat-fixing is complete, you are ready to gram stain your slide. Figure 2. Heat fixation 41 Laboratory Exercises in Microbiology McLaughlin and Petersen GRAM STAINING PROCEDURE  For all steps in the gram staining procedure, add enough of the solution to cover the areas of the slide that have bacteria on them. You do not need to flood the entire slide.  All staining should be done over a staining tray. Be sure to put newspaper under the tray in case of spillage.  Gloves should be worn while staining and removed before working with the microscope. STEPS EXPLANATION 1. Add a few drops of crystal violet (primary stain) to the smear and let it sit for 60 seconds. All cells are purple after this step. Stopping 2. Rinse the slide with water. here would be a simple stain. 3. Add a few drops of Gram's iodine (mordant) to Gram’s iodine forms a complex with crystal the smear and let it sit for 60 seconds. violet All cells are purple after this step. 4. Rinse the slide with water. 5. Decolorize with 95% ethanol: let the alcohol Gram positive cells retain crystal violet and run over surface of slide until no more crystal remain purple violet color comes out of the smear (time Gram negative cells lose crystal violet and varies—no more than 5-10 seconds). are now colorless 6. Rinse with water. Water rinse stops the decolorization process 7. Add a few drops of safranin (counterstain) and Safranin is a pink/red dye let it sit for 60 seconds. 8. Rinse with water. Blot dry on bibulous paper. Gram positive cells remain purple; gram Be careful not to wipe off the bacteria. negative cells are now pink/red 9. Observe your slide under the microscope. For each organism, determine morphology, arrangement and Gram reaction 42  Laboratory Exercises in Microbiology McLaughlin and Petersen Gram Stain Results Draw sketches for each type of bacteria that you observe. Identify its morphology, arrangement, and Gram reaction. Species ____________________________ Species ____________________________ Morphology ________________________ Morphology ________________________ Arrangement _______________________ Arrangement _______________________ Gram reaction ______________________ Gram reaction ______________________ Species ____________________________ Species ____________________________ Morphology ________________________ Morphology ________________________ Arrangement _______________________ Arrangement _______________________ Gram reaction ______________________ Gram reaction ______________________ 43  Laboratory Exercises in Microbiology McLaughlin and Petersen Species ______________________ Species ______________________ Species ______________________ Morphology __________________ Morphology __________________ Morphology __________________ Arrangement __________________Arrangement __________________ Arrangement __________________ Gram reaction _________________Gram reaction _________________ Gram reaction _________________ Species ______________________Species ______________________ Species ______________________ Morphology __________________Morphology __________________ Morphology __________________ Arrangement __________________ Arrangement __________________ Arrangement __________________ Gram reaction _________________ Gram reaction _________________ Gram reaction _________________ 44  Laboratory Exercises in Microbiology McLaughlin and Petersen Special Stain Results Observe the special stains set up at the demo microscopes at the back of the lab. Make sketches below. Species ____________________________ Species ____________________________ Staining technique Staining technique ___________________________________ ___________________________________ Comments: Comments: _____________________________________ _____________________________________ _____________________________________ _____________________________________ Species ____________________________ Staining technique ___________________________________ Comments: _____________________________________ _____________________________________ 45 Laboratory Exercises in Microbiology McLaughlin and Petersen Summary of Common Bacterial Staining Techniques Simple Stains  Used to provide color to otherwise transparent bacterial cells Crystal Violet, Methylene Blue, Safranin  Can be used to determine cell size, morphology and arrangement Gram Stain  Common differential stain  Gram reaction (positive or negative) reflects cell Primary stain – crystal violet wall properties Mordant – iodine; decolorizer- 95% Ethanol  Also used to determine cell size, morphology and arrangement Counterstain – Safranin Acid-Fast Stain  A differential stain used to detect bacteria with Primary stain – Carbol fuchsin mycolic acid cell walls (genera Mycobacterium and Nocardia) Decolorizer – acid alcohol  Developed to detect the bacterial species that causes tuberculosis Counterstain – Methylene blue  Acid-fast organisms resist decolorization with acid-alcohol Endospore Stain  Endospores resist staining with basic stains Primary stain: Malachite green  Endospores stain with malachite green; vegetative cells stain with safranin Counterstain: safranin Capsule Stain (Negative staining)  Negative stains are neither heat-fixed nor rinsed Uses an acidic stain: (Congo red or Nigrosin)  The background of the slide is stained by acidic stains (capsule remains unstained) and a basic stain: (crystal violet or safranin)  The cells within the capsule are stained with Basic stains  Examples of encapsulated cells: Bacillus anthracis, Streptococcus pneumoniae, and Klebsiella pneumonia Flagella Stain  Used to see bacterial flagella that are too slender Silver nitrate to be seen with other staining techniques  Silver nitrate makes flagella appear larger than they are  Can be used to determine arrangement of flagella for identification. o Ex: Proteus vulgaris has peritrichous flagella Spirochete stain  Used to visualize slender spirochetes like Silver nitrate Treponema pallidum 46 Laboratory Exercises in Microbiology McLaughlin and Petersen Review Questions 1. Explain the major differences between the gram positive and gram negative cell wall. 2. Salmonella typhi is a gram negative organism. a. What color will it appear when simple stained with crystal violet? b. What color would it be if it was gram stained correctly? 3. Explain how bacterial cells would look in the gram staining procedure if the following mistakes were made: a. Decolorizer left on too long b. Decolorizer not left on long enough c. Slide not heat-fixed before staining 4. What is the difference between a simple stain and a differential stain? 47 Laboratory Exercises in Microbiology McLaughlin and Petersen 5. Explain why it is important to use only a small amount of bacteria when preparing a smear. 6. What are the two things that are stained in a capsule stain? What is NOT stained in a capsule stain? 7. Why would a health care provider be interested in knowing the gram reaction of a pathogenic bacterium? 48 Laboratory Exercises in Microbiology McLaughlin and Petersen Laboratory Exercise 4: Acid fast and Endospore Staining Objectives 1. Learn about microorganisms that have acid-fast cell walls. 2. Perform the acid-fast staining procedure and view cells under oil immersion. 3. Learn about some of the microorganisms that are endospore formers. 4. Perform the endospore staining procedure and view cells under oil immersion. Key Terms: mycolic acid, endospore, carbol fuchsin, methylene blue, malachite green, Ziehl-Neelson method, Kinyoun method, Schaeffer-Fulton method Acid-Fast Staining Introduction Most bacterial species are either Gram positive or Gram negative, however some organisms have different cell wall properties that make them difficult to stain with this method. For example, some species of bacteria have a waxy lipid (mycolic acid) in their cell walls. These organisms generally do not Gram stain very well (those that do would usually appear gram positive) and are more clearly visible with the acid-fast staining technique. Acid-fast staining was developed by Robert Koch in 1882 and later modified by other scientists. Koch used the method to observe the “tubercle bacillus”—what we now call Mycobacterium tuberculosis, in sputum samples. While acid-fast and gram staining are both differential stains, the acid-fast stain is much more specific. Many bacteria are either gram positive or gram negative, but very few are acid-fast. Two acid-fast genera that are important as human pathogens are Mycobacterium and Nocardia: Pathogenic species include M. tuberculosis, M leprae, M. bovis, M. avium, and N. asteroides. The protozoan parasite Cryptosporidium can also be stained using this procedure. There are 2 different methods of acid-fast staining—both involve techniques that make the cell wall more permeable to the primary stain. The Ziehl-Neelson method uses steam heat to allow stain to penetrate, whereas the Kinyoun (cold method) uses a wetting agent mixed in with the primary stain. In this lab we will be using the Kinyoun method. 49 Laboratory Exercises in Microbiology McLaughlin and Petersen Kinyoun Staining Procedure 1. Prepare a slide with Mycobacterium smegmatis on one side and Micrococcus luteus on the other side. (Alternatively, both bacteria may be mixed into one smear).  Be sure to break up clumps of M. smegmatis before staining. 2. Air dry and heat fix as usual. 3. Add carbol fuchsin (primary stain): leave on for 5-7 minutes. 4. Rinse with water.  Note: not all of the primary stain will be removed by water in this step 5. Decolorize with Acid-alcohol: 1-2 quick rinses 6. Rinse with water. 7. Add methylene blue (counterstain) and leave on for 2-3 minutes. 8. Rinse with water, blot dry, and look at beautiful bacteria. Acid-fast organisms retain the primary stain and will appear bright red: non acid-fast organisms are decolorized with acid-alcohol and pick up the methylene blue counterstain. Epithelial cells that may be present in a clinical sample will also appear blue. Endospore staining Introduction Endospores are the most resistant forms of life. They can resist desiccation (drying), boiling and radiation—in addition, disinfectants and antibiotics cannot penetrate an intact spore coat. For this reason they are difficult to eliminate from the environment with standard methods of disinfection, and they are difficult to treat in the case of an infection. Endospores are a survival mechanism for the bacterial species that produce them. When conditions are favorable, vegetative bacterial cells will continue to grow and divide; however when nutrients are depleted, cells will begin to form endospores. Endospores are not metabolically active, but contain all the materials needed by cells to survive. When conditions for growth are again favorable, the spore will germinate and form a cell that is identical to the cell that produced it. Endospores are produced by certain types of Gram positive 50 Laboratory Exercises in Microbiology McLaughlin and Petersen bacilli, like Clostridium and Bacillus, as well as other species. Endospore-forming pathogens include C. tetani, C. botulinum, C. difficile, and B. anthracis. In today’s lab we will use the Schaeffer-Fulton method (without heat) to view endospores. Since we are not using heat, it is important to leave the stain on for a long time to allow it to penetrate the spore coat. Schaeffer-Fulton Staining Procedure 1. Prepare a smear with Bacillus subtilis or Clostridium sporogenes on one side of the slide, any other bacteria on the other side 2. Air dry and heat fix as usual. 3. Add malachite green (stains spores) and leave on for at least 10 minutes. 4. Rinse briefly with water. 5. Stain cells with safranin (stains vegetative cells) for 1 minute. 6. Rinse with water, blot dry and look at beautiful bacteria If the bacterial species is an endospore former, you will see pink vegetative cells as well as green oval-shaped endospores: non-spore formers will appear only as pink vegetative cells. Note: Color pictures of organisms stained with the Ziehl-Neelson procedure and the Schaeffer-Fulton procedure can be found on the Blackboard Microbiology Review site. Additional Gram staining: if time allows, your instructor may have you prepare some additional slides with the Gram stain procedure. 51 Laboratory Exercises in Microbiology McLaughlin and Petersen Results Acid Fast Stain Species ____________________________ Species ____________________________ Morphology ________________________ Morphology ________________________ Arrangement _______________________ Arrangement _______________________ Acid fast result ______________________ Acid fast result ______________________ Endospore Stain Species ____________________________ Species ____________________________ Morphology ________________________ Morphology ________________________ Arrangement _______________________ Arrangement _______________________ Additional Gram Stains Endospore result ______________________ Endospore result ______________________ 52 Laboratory Exercises in Microbiology McLaughlin and Petersen Additional Gram Stains Species ____________________________ Species ____________________________ Morphology ________________________ Morphology ________________________ Arrangement _______________________ Arrangement _______________________ Gram reaction ______________________ Gram reaction ______________________ Species ____________________________ Species ____________________________ Morphology ________________________ Morphology ________________________ Arrangement _______________________ Arrangement _______________________ Gram reaction ______________________ Gram reaction ______________________ 53 Laboratory Exercises in Microbiology McLaughlin and Petersen Species ____________________________ Species ____________________________ Morphology ________________________ Morphology ________________________ Arrangement _______________________ Arrangement _______________________ Gram reaction ______________________ Gram reaction ______________________ Species ____________________________ Species ____________________________ Morphology ________________________ Morphology ________________________ Arrangement _______________________ Arrangement _______________________ Gram reaction ______________________ Gram reaction ______________________ 54 Laboratory Exercises in Microbiology McLaughlin and Petersen Review Questions 1. Distinguish between the Ziehl-Neelson and Kinyoun methods of acid-fast staining. 2. Why is it important to leave the malachite green on the slide for at least 10 minutes in the endospore- staining procedure? 3. Do you think you would find more endospores in a freshly prepared culture or in an older culture of Bacillus subtilis? Explain. 4. Why do you think an infection caused by an endospore former might be harder to treat than one caused by a non-spore former? 55 Laboratory Exercises in Microbiology McLaughlin and Petersen Review of Staining Procedures To help you review the staining procedures in Labs 3 and 4, fill out the table below with information about these staining procedures. This information should include (but not be limited to) the following:  What does the staining procedure tell you about bacterial cell structure, or the types of structures produced by bacteria?  Is the staining procedure used to detect specific types of cells? If so, what are they?  What do the positive and negative results look like at the end of the procedure?  Is there any clinical relevance to the results of the stain