4. Bacterial Smears and Simple Stains Introduction The refractive index of most bacteria is only slightly greater than that of water; therefore bacteria are difficult to see in liquid suspension because they are almost transparent. To visualize bacteria, stains are used. Bacteria usually carry a negative charge (-). Most stains are either positively (+) or negatively (-) charged. Some stains are neutral. Positive or basic stains are attracted to the negatively charged bacteria (crystal violet, safranin, methylene blue) whereas negative stains are repelled from bacteria. A few stains (e.g. India ink) are composed of particles too large to enter the cells and are also referred to as negative stains. Staining allows observation of cell morphology (e.g. size and shape) and arrangements (clusters, chains, strands). Relationship to Class Instruction: Chapters 3 and 4. Lab report due on: _______________________________________
Simple stain of Staphylococcus with methylene blue http://phil.cdc.gov/phil/home.asp
Purpose of Laboratory ď‚ˇ
Use aseptic techniques to remove a bacterial sample from a culture
Prepare bacterial smears from solid cultures
Perform a simple stain
Materials and Methods Organisms Escherichia coli: rod-shaped bacteria, usually single cells Bacillus megaterium: rod-shaped bacteria, arranged in strands Staphylococcus epidermidis: round-shaped bacteria, arranged in clusters Candida sp.: yeast cells 4-1
Supplies Microscope slides
Lens paper and immersion oil
Preparation of Bacterial Smears Special Safety Precautions Discard all biohazard material appropriately: Slides go in the metal tray.
Cleaning of Slides Pre-clean a slide by passing it briefly through the flame of a Bunsen burner. Use a clothespin. Let the slide cool before proceeding. Handle the slide only from edges.
General Instructions A loopful of bacteria in liquid culture or a sample of bacteria from a solid culture dispersed in a drop of water is spread as a thin layer or smear on a clean glass slide. The thin bacterial film is allowed to air dry. It is fixed to the glass to prevent it from washing off during the subsequent staining procedure. Fixing is performed by passing the slide briefly through a flame; this treatment sticks (glues) bacteria to the glass. The bacterial film should not be too thin or too thick. If it dries as a thick opaque layer, it is too thick and should be discarded and repeated. If there is no cloudiness on the dried slide, it may be too thin. Further, the slide must be clean and free of oil from fingers or the bacterial film will not stick. Once the bacterial film is fixed, it is flooded with stain and allowed to sit for approximately one minute, during which time the stain penetrates the cells. The stain is then washed off with a gentle stream of water and blotted dry. View the dried, stained smear under the microscope. In this exercise, each student will prepare 2 slides: 1 slide with smears of either E. coli or B. megaterium. 1 slide with smears of either S. epidermidis or Candida. Choose one of the three simple stains available. Donâ€™t forget to record which stain was chosen in the experiment.
Smear Preparation from Cultures on Solid Media loop. The drop should look cloudy, not milky.
1. Assemble the materials necessary for making the smears: glass slides, paper towels, culture tubes, sharpie, inoculating loop and a Bunsen burner.
7. Flame the loop by placing it in the flame at the hub of the holder where the wire is attached and then gradually moving the wire through the flame, until the wire glows. Finally the loop itself is flamed until it is glowing. In this way the broth dries out gradually before incineration. If the wet loop is put immediately in the flame, it will create aerosols and contaminate the surrounding area.
2. Mark glass slides with a sharpie on the back of the slide. On each slide, draw 1 large circle. Write the initials of the culture used (EC, SE, BM, or C) on the very top edge making sure that you can read them when looking at the slide right side up. Turn the slide over, so that the sharpie marks are on the underside.
8. Repeat steps above for all the samples. 9. Air-dry the slide. Speed up the drying process by placing the slide on a warm plate. The surface of the slide will be dull and not shiny. DONâ€™T OVERHEAT.
3. Deposit a small drop of water from a wash bottle in the center of the circle.
10. Fix the bacteria onto the slide by passing the slide, smear side up, quickly through the flame of the burner two or three times.
4. Sterilize your loop (from the hub of the handle along the entire length) in the Bunsen burner flame until it glows.
11. Avoid getting the slide too hot. This fixation process coagulates the proteins and fixes the bacteria onto the slide so they will not get washed off. The smear is now ready to be stained(see next steps)
5. Cool the loop by touching lightly the agar surface and pick a small amount of bacteria from a single colony. 6. Disperse the bacteria in the drop by mixing in a circular motion with the
Pitfalls and Troubleshooting Too much material - suspension should be barely cloudy So much liquid that it takes forever to dry Heating the smear before letting it air dry, boiling the bacteria instead of attaching them Overheat the smear, melting cell walls and possibly breaking the slide Forgetting to fix the smear all together. Bacteria will wash off.
NOTE: When the slide is dry, the specimen may be hardly visible especially if the sample was taken from a liquid culture; however, the surface of the slide will be dull and not shiny.
Simple Stain 12. Place the slide on a bench towel and cover each smear with one or two drops of basic stain. After one minute, wash off the stain with water. Use a wash bottle and rinse gently. No squirting.
15. Examine with the microscope, starting with the scanning objective (lowest magnification) followed by the 20X and to the 100X oil immersion lens. (Final magnification is 1000X for oil immersion objective)
13. Drain off excess water by tapping gently on the towel and blot dry around smear with a paper towel or Kimwipe. Be careful not to blot your sample away.
16. Record your observations in your notes. Draw cell shape (cocci, rods) and cell arrangement (single, chains, clusters) in the lab results worksheet while in class. Repeat staining procedure for second slide.
14. Air-dry. the slide
Lab Report for Simple Stains The lab report on simple stains will be corrected extensively, but not graded. It is an opportunity for you to know what is expected. In the future, I will not correct as extensively, but grade your work. For this lab report, I use three assessment levels or rubrics: check means that your lab report is good and you just have to work on some details, check with mark means that the lab report is OK, but there are quite a few mistakes to correct, an X means that the lab report needs a lot of work. The assessments are not grades, they are meant to help you write a solid lab report. Introduction: Goal of experiment and reason to perform the experiment. Materials and Methods: briefly tell what you did. Refer reader to “Microbiology Manual lab 4” for experimental details. Don’t forget to record which stain you actually used and which organisms were stained. Results: attach lab drawings. Describe in words cell morphology (spheres, rods) and arrangements (group, single cells). Include the name of the organism if it is not printed and the total magnification. Discussion: Compare the shape and arrangement of the different cultures. You stained only two bacterial cultures, but you also observed the slides of your lab partner. Conclusion: General comments about the whole exercise. What did you learn? How would you improve your results, if they needed improvements? Don’t forget to include final magnification for each drawing. Use scientific style for the names of bacterial cultures: Escherichia coli or Escherichia coli. Abbreviate the genus after the first use: e.g. E coli. For more details on how to write a lab report, refer to “How to write a lab report” in the lab manual. 4-4
Bacterial Smears and Simple Stains Escherichia coli