3-11 Practice staining

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In the study and identification of bacteria, the microscope is indispensable! The series of micro-scopic observations in this exercise is designed to illustrate how bacteria may be viewed individually in their basic form, the cell. The second and third periods herein coincide with those of Experiment 1 where organisms isolated by the student are examined microscopically (and could be found to be more interesting than those provided in this exercise!).

Period 1

Materials

Hay infusions and various other items from nature

Slide with smears of Bacillus cereus and Staphylococcus epidermidis

Simple stain.

  1. You are provided with a microscope slide with two smears. Following the directions for microscopy and staining, heat-fix the slide, making sure the slide goes through the flame smear-side up.
  2. Gloves are available for the staining procedure. Placing the slide on the staining rack in the sink, cover the slide with crystal violet for one minute. For a review, look at the directions for the simple stain.
  3. Carefully rinse off the dye with tap water and blot the slide dry with paper towel or blotting paper.
  4. With both hands, obtain the light microscope from the cabinet (corresponding to your desk number). This is the type of microscope which we will always use to observe stained smears.
  5. Unless the instructor has other directions more directly applicable to the microscope you are using, use the simple procedure described in the operating procedure. Refer to this procedure as you study the cells in the two smears in the following steps.
  6. Place the slide on the stage such that it is oriented as illustrated above. Make sure the clips on the stage hold the slide securely.
  7. Begin your observations with the Bacillus cereus smear. (See figure below) When observing this organism with the oil-immersion objective, you will notice that the cells are relatively large and rod-shaped (bacilli) and are usually in chains. Record your observations on the next page.
  8. Repeat this procedure with the Staphylococcus epidermidis smear. Cells of this organism are spheres (cocci) which are usually arranged in clusters (staphylococci) and pairs.
  9. When you are through, be sure the microscope is put away properly (i.e., all oil wiped off, 10X objective lens in place, stage centered). It is recommended that you keep the slide. (To remove immersion oil from smears, place a few pieces of lens paper on the slide to absorb the oil. Then, add several drops of xylol to the lens paper. Peel the paper, now soaked with xylol, off the slide. Xylol is flammable! Keep it away from flames!)

Figure 3-13 Simple stain

A simple stain of S. epidermidis and B. cereus. S. epidermidis (A), B. cereus old (B), B. cereus young (C)

Wet Mount

  1. For observation of living microorganisms, various samples including a hay infusion are available. To study the microorganisms in the aqueous materials available, it is necessary to make wet mounts. The procedure is relatively simple:
  2. Using a capillary pipette or inoculating loop, pick up some of the material from around the surfaces of grass and leaves and from the bottom of the sample. Place a drop of suspended material on a clean microscope slide.
  3. With a toothpick, spread a very thin layer of vaseline over a small part of the palm of your hand. Take a clean coverslip (always held by the edges) and gently scrape all four edges along your palm, picking up a thin line of vaseline along each edge.
  4. Place the coverslip directly onto the drop on the slide in such a manner that some air bubbles are trapped. Place a small, multilayered piece of paper towel over the coverslip and press down. Discard the piece of paper towel into the disinfectant.
  5. Examine the wet mount with your light microscope or a phase CONTRAST microscope set up by the instructor at a special station in the back or side of the lab.
  6. Without removing the coverslip, discard the slide into the disinfectant container on the stage. (Refer to page viii for cleanup directions.)

If you haven't already, Figure 1-2 presents a movie of the types of life forms found in a hay infusion.

Period 2

Materials

Bacterial cultures growing either in a liquid medium (Heart Infusion Broth) or on a slant of an all-purpose medium followed by suspension in saline:

Escherichia coli - young culture, incubated 12-15 hours

Bacillus cereus - young culture, incubated 12-15 hours

Bacillus cereus - old culture, incubated 2-3 days

Figure 3-14 Gram stains

Gram stains of demonstration species. Below are shown typical Gram stain reactions of two species. E. coli (A), B. cereus old (B), B. cereus young (C). The images are slightly larger than what would be visible in a light microscope to improve clarity.

  1. On one clean glass slide, prepare smears of the three cultures. Go to smear prepapration if you need a refresher. Only when the smears have dried completely should the slide be heat-fixed.
  2. Perform the Gram stain procedure as described.
  3. As with any stained smear, definitive observations are made with the 100X, oil-immersion objective. Refer to the microscope directions already given, remembering to focus the slide initially with the 10X objective, moving then to the oil immersion objective.
  4. Keep in mind that the young cultures of B. cereus and E. coli are your positive and negative control cultures, respectively, for the observation of probable gram-variability of the older B. cereus culture.
  5. Using the figures below, record your observations in your notebook, noting the Gram reaction (positive if purple, negative if red) and the cellular shape. Is there any difference seen between the two cultures of Bacillus cereus? Is gram-variability evident for the older culture? Recall from the introduction to Experiment 1 that we can refer to old and young cultures but should not do so for individual cells. (Remember to discard the tubes and slides properly)

Period 3

Materials

This experiment will be done in class.

Young bacterial cultures growing on slants of Heart Infusion Agar:

Staphylococcus epidermidis

Pseudomonas fluorescens

An unknown

Record the number of your unknown!

Figure 3-15 Typical reactions of example strains for test

The classic Gram reactions for Staphylococcus epidermidis (A) and Pseudomonas fluorescens (B). From this, determine whether they are Gram (+) or Gram (-). Note we do not show an unknown as this must be done in class. The images are slightly larger than what would be visible in a light microscope to improve clarity.

  1. On a clean glass slide, prepare heat-fixed smears of the three cultures, noting that these cultures are growing on a solid medium. Therefore the cells must be dispersed in a drop of water when preparing the smears, as a smear is always a dried suspension of cells. Take care not to make the smears too thick! S. epidermidis and P. fluorescens are your positive and negative control cultures (respectively) for your unknown.
  2. Perform the Gram stain procedure and note the Gram reaction and cellular shape. Record your results. Fill out and turn in your description of your unkonwn. Save your slide until your graded unknown is returned.

Period 4

Materials

For the capsule stain:

36-48 hour culture of Klebsiella pneumoniae growing on a slant of EMB Agar (a high-sugar medium)

Dropper bottle of filtered India ink

For the acid-fast stain:

3-day culture of Mycobacterium smegmatis growing on a slant of Trypticase Soy Agar plus 1% glycerol

18-24 hour culture of Micrococcus luteus (the negative control culture) growing in Nutrient Broth

Dropper bottles of carbol fuchsin (freshly-made), acid alcohol and methylene blue

Capsule stain

Figure 3-16 The capsule stain

A capsule stain using India ink at 1000x magnification. The cells of Klebsiella pneumoniaeare surrounded by a dark background. The capsule is the clear area surrounding the cells. The photomicrographs is slightly enlarged for clarity.

capsule stain

  1. Using the culture of Klebsiella pneumoniae, Place one loopful of water on a slide and emulsify in it a bit of growth from the slant or plate culture of the designated organism. Add a drop of filtered India ink to the cell suspension. It often works out well to place the drop of India ink adjacent to the cell suspension on the glass slide.
  2. Obtain a clean coverslip (no fingerprints, smudges, dirt, etc.) and rim it lightly with vaseline; the vaseline can be gently scraped from a thin layer applied to the palm of the hand. Place a small, multi-layered piece (about 1-2 cm2) of paper towel over the coverslip and press down firmly; discard the paper towel into the disinfectant.
  3. Using the regular light microscope, focus initially with the 10X objective, switching to the 45X objective and then - if needed - the 100X, oil-immersion objective. Adjust the light intensity as required with the iris diaphragm. The outline of the cell can be seen within the area of the clear capsule.
  4. Alternatively, the phase microscope can be used. Heed the precautions regarding use of this microscope. Excellent observations can be made with just the 40X objective lens (which takes no immersion oil).
  5. When finished, without removing the coverslip, discard the slide directly into the disinfectant. Never discard capsule stains and other wet mounts with the stained smears, as viable cells are still present and the slides must be disinfected!. Record your observations below.
Acid-fast stain

Figure 3-17 The acid fast stain

A photomicrograph of Mycobacterium smegmatis (pink) and Micrococcus luteus (blue) at 1000x magnification. M. smegmatis is acid-fast, retaining the carbol fuchsin dye, thus appearing pink. M. luteus is not acid-fast, loses the carbol fuchsin during decolorizaiton, and is counter-stained with methylene blue.

acid fast stain.

  1. Prepare a mixed smear of two organisms as follows: Place a drop of the Micrococcus luteus broth culture on a slide. Into this drop, add cells from the Mycobacterium smegmatis culture. Disperse the cells as much as you can (the Mycobacterium cells tend to clump), and prepare a smear about the size of a nickel. Let it air-dry completely, and then heat-fix it well, passing the slide through the flame an extra one or two times.
  2. Perform the acid-fast procedure (page 148, observing the slide with the regular light microscope) and record your observations below.
  3. As with all stained smears, discard the slide in the appropriate container.
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