Materials Included In Kit

Additional Materials Required

Prelab Preparation

Be sure your microscopes are in good working condition prior to this lab. Oil immersion capabilities are necessary for success with this laboratory activity. Students will need instruction on the proper use of immersion oil and oil immersion lenses prior to this lab. A pre-lab practice session would be very useful for students.

Safety Precautions

All the stains in this lab are permanent and will stain the skin and clothing. Students should wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Methylene blue, safronin and crystal violet solutions can be disposed of following Flinn Suggested Disposal Method #26b. Iodine solution should be disposed of following Method #12a. Bacteria cultures should be sterilized before disposal. They can be autoclave sterilized or flooded with bleach solution. The solutions can then be flushed with volumes of water down the drain and the solid disposed of in normal trash in plastic bags.

Teacher Tips

• Part I of this lab can be completed in half a class period on the first day. Parts II and III can be completed in 1–2 class periods depending upon the number of slides and drawings students prepare and their success in finding bacteria.

• Enough materials are supplied to provide five complete sets of cultures (15 in all). Student groups sharing the drops out of these cultures can vary from 4–6 students depending upon your class size. The slide work in the lab can be done individually or in teams of two depending upon your preference.
• The kit provides extra slides if slides are cleaned and reused during the observation phases of the laboratory.
• Slide staining trays are convenient for this laboratory if they are available. The use of trays avoids congestion at sink areas during staining and destaining activities.
• Once the bacterial smears have been air dried, the use of a coverslip is not necessary. In fact, it is better without a coverslip. Images will be clearer without a potentially dirty coverslip in the way.
• Tap water can be used during all parts of the lab or staining and cleaning.
• Cultures should be kept at room temperature or warmer, in a safe place and out of direct sunlight.

Sample Data

Students should have little difficulty finding bacteria in their cultures. With the three different culture media, they should see a variety of types and shapes. The Gram-stained slides should reveal at least two different varieties. Results will likely depend on students’ ability to use a microscope and their ability to be patient and observe in a systematic fashion.

Introduction

“Though my teeth are kept usually very clean, nevertheless, when I view them in a magnifying glass, I find growing between them a little white matter as thick as wetted flour; in this substance, I judged there might probably be living creatures.”

—Anton von Leeuwenhoek, 1632–1723

Welcome to the world of microorganisms! Bacteria and other microorganisms are nearly everywhere.

Concepts

• Gram stain

• Cocci
• Bacteria morphology
• Bacilli
• Spirilla
• Bacteria arrangement

Background

Part I. Culturing Bacteria

What conditions are necessary for bacteria to grow? Bacteria and other microorganisms can be found in nearly any moist environment that has the minimal required nutrients for life—an energy source and basic elemental building blocks such as carbon, hydrogen, oxygen and nitrogen. Bacteria can be found in very common places, such as on food, or in unusual places such as hot volcanic springs.

Microbiologists often create solutions rich in nutrients to encourage the rapid growth of bacteria and other microorganisms. These mixtures are referred to as culture media. The nutrients present in culture media provide necessary building blocks for bacteria to grow and reproduce very quickly. Culture media can be made in a semi-solid “Jello-like” state (agar) or it can be in liquid form (broth). Bacteria are easy to grow. When conditions are right they grow and reproduce very rapidly! In this part of the activity three different food sources for growing a variety of bacteria will be tested.

Part II. Invisible Organisms

Living bacteria are almost colorless and lack sufficient contrast with the liquid in which they are suspended to be clearly visible. Staining bacteria increases their contrast with their surroundings so that they are more visible. The staining process kills the bacteria, however, and living processes such as movement are no longer visible.

Most bacterial stains are salts. When salts are dissolved they separate into positive and negative ions. Salts used as stains have one ion that appears colored in the visible spectrum. The stain used in this part of the lab is methylene blue. It is the salt methylene blue chloride. When it is dissolved it dissociates as follows:

Methylene blue chloride → methylene blue⁺ + chloride^–

The color of the stain is in the positively charged methylene blue ion.

Bacterial cells have a slight negative charge when the pH of their surroundings is near neutral, which it generally is when they are actively growing. The negatively charged bacterial cell combines with the positively charged methylene blue ion, with the result being that the cell is stained and looks blue. The opposite charges of the cell and the stain ion attract each other and the methylene blue “sticks” (bonds) to the cell.

Part III. Classifying Bacteria

In 1885, Hans Christian Gram was working in a lab, staining bacteria. He was not satisfied with the staining technique he was using because not all bacteria seemed to retain the stain equally. What he considered to be a defect in the staining technique eventually led to one of the most widely used tests for distinguishing bacteria from one another. The differential staining method became known as the Gram Stain.

Bacteria can be classified as either Gram-positive and stain purple, or Gram-negative, and stain reddish–pink. The distinction between the two is based upon the inability of the purple stain (crystal violet) to be flushed out of the cell during the Gram staining procedure. This seems to be related to the different chemical and/or structural makeup of the cell walls of gram-positive and gram-negative bacteria. Because their structures are different they accept and retain stains in different ways. Gram staining is generally the first step in identifying an unknown bacterial species and is routinely used in even the most sophisticated microbiological laboratories.

In addition to classifying bacteria as Gram-positive or Gram-negative, they can also be classified by their shape and groupings. There are three basic shapes for bacteria: (a) round or cocci (coccus, singular), (b) rod-shaped or bacilli (bacillus, singular) and (c) spiral-shaped or spirilla (spirillum, singular). Bacteria can be found in different groupings: (a) singly (one single bacterium), (b) diplo (a pair of bacteria joined together), (c) strepto (a chain of bacteria) and (d) staphylo (a cluster of bacteria).

By putting the shape and arrangement together, we can describe the morphology of the bacteria. For example, if a bacterium is round and is found in a chain we would call it a streptococcus. If it is a rod and is found in pairs, it is a diplobacillus.

Materials

Safety Precautions

Items in the laboratory are never considered to be food. Never eat any items found in the laboratory. All the stains in this lab are permanent and will stain your skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. When working with culture materials always wash your hands before leaving the laboratory.

Procedure

Part I. Culturing Bacteria

1. Boil enough tap water to fill three culture jars (approximately 200 mL). Remove from heat and let cool to room temperature. While the water is cooling go on to step 2.
2. Place dry materials in three labeled culture jars as follows:

Culture jar A—Place 10 peppercorns in the bottom of the culture jar.
Culture jar B—Cover the bottom of the culture jar with a single layer of bean seeds.
Culture jar C—Cut hay into small pieces (½" long) and add enough to fill the jar about ⅓ full.

3. Pour the cooled, boiled water into each jar so that it is about ¾ full.
4. Place the covers loosely on the jars. The jar needs to “breathe” so just have the cover loosely ajar on top of each container.
5. Allow the container to stand undisturbed for two days in a warm place (60–90 °F). When a scum (thin film) forms on the surface of your culture media, go on to Part II of the lab.

Part II. Invisible Organisms

1. A clean microscope slide is critical for examining bacteria with a microscope. If the slide is dirty, dirt will be examined and not bacteria. Thoroughly clean and dry three glass microscope slides.
2. Use a clean plastic toothpick to take a drop of scum from the surface of one of your culture jars from Part I of this lab. Place the drop in the center of a microscope slide and spread the drop into a thin film in the center of the slide.
3. Let this film air dry on the slide. Label the slide A, B or C depending on its culture source.
4. When the film is dry, pass the slide, film side up, through a Bunsen burner flame. Hold the slide with a test tube holder as the slide will get hot. Two or three deliberate, but quick, passes through the flame will “fix” the bacteria to the surface of the slide.
5. After the slide has cooled, cover the bacterial smear with 2–3 drops of methylene blue stain. Let the stain sit for about 2 minutes. (Do this over a staining rack or sink.)
6. Now wash the stain from the slide using tap water. Do this in a gentle washing fashion using a Beral-type pipet. Wash gently with water until no more color seems to be washing out.
7. Drain the slide by allowing water to run off while holding the slide vertically. Allow the slide to air dry.
8. Examine the stained smear with a microscope. A coverslip is not necessary. Pick a portion of the smear where the bacteria are well distributed and numerous. Bacteria cells, their shape, and their grouping pattern will be visible with high power magnification (400X). Oil immersion (970 to 1000X) should be used to examine the bacteria for more detail. Caution: Be sure to have proper instruction on the use of oil immersion and its cleanup before attempting to use the 100X objective on a microscope.
9. Make drawings of your observations on the Bacterial Study Worksheet.
10. Repeat steps 1–9 for the other two cultures from Part I. Record the results on the Bacterial Study Worksheet.
11. Thoroughly wash and dry used slides and toothpicks with soap and water. Thoroughly wash your hands with soap and water before leaving the laboratory.
12. Consult with your instructor if any materials will be disposed of after this part of the laboratory.

Part III. Classifying Bacteria

1. Use a clean plastic toothpick to take a drop of scum from the surface of one of your culture jars from Part I of this lab. Place the drop in the center of a clean microscope slide and spread the drop into a thin film in the center of the slide.
2. Allow the slide to air dry and then flame fix the smear in the flame of a Bunsen burner using a test tube holder to hold the slide. Be sure to pass the slide through the flame film side up.
3. After the slide has cooled, cover the bacterial smear completely with crystal violet solution and let stand for one minute.
4. Rinse the slide with tap water using a Beral-type pipet. Rinsing should be sufficient to remove excess stain.
5. Flood the smear on the slide with iodine solution. (The iodine serves as a mordant, or dye retainer).
6. Let the slide stand for one minute and then rinse with water using a Beral-type pipet as previously done.
7. Decolorize the smear using 95% ethyl alcohol. Use a Beral-type pipet and apply one drop at a time—just until no more color runs off—for about 30 seconds. (Be sure to do this over a staining tray or sink.) Note: Decolorization time is determined primarily by the thickness of the smear—shorter for thin smears, longer for thick smears. It is important to not over decolorize at this step.
8. Rinse with water as in step 4. This rinse step is necessary to stop the action of the alcohol decolorizer.
9. Apply safranin solution and let stand for about 45 seconds. (This step is where the Gram-negative bacteria will be stained.)
10. Rinse briefly with water as in step 4. Allow the slide to air dry.
11. Examine your Gram-stained smear with a microscope. A coverslip is not necessary. Pick a portion of the smear where the bacteria are well-distributed and numerous. Oil immersion (970X to 1000X) should be used to examine for detail. Note: It should be easy to distinguish Gram-positive bacteria from Gram-negative bacteria. The Gram-positives should appear to be bluish-black to purple in color while the Gram-negative will be reddish in color.
12. Make drawings of your observations on the Bacteria Study Worksheet. Use colored pencils or labels to indicate which bacteria are Gram-positive and which are Gram-negative. Try to further classify the bacteria based upon shape and grouping as described in the Background.
13. Repeat the Gram stain procedure on other samples from your cultures. Try to find as many bacteria types as possible.
14. Thoroughly wash and dry used slides and toothpicks with soap and water. Thoroughly wash your hands with soap and water before leaving the laboratory.
15. Consult with your instructor if any materials will be disposed of after this part of the laboratory.

Student Worksheet PDF

10258_Student1.pdf