3-11 Practice staining

3-11 Practice staining

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Table of Contents| Chapter Article List| Printable Version | Printable Chapter[Prev] | [Next]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 epidermidisSimple 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 epidermidisPseudomonas fluorescensAn unknownRecord 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.

Spore stain:1. Make a heat fixed smear of Bacillus as done previously.

2. Cover the smear with a small piece of paper towel, not hanging over the edges of the slide.

3. Place the slide on top of a small beaker containing about 1-2 inches of boiling water, being certain that the slide is horizontal. Instructor will demonstrate the ring stand set up. Be careful of burning yourself or knocking down the ring stand.

4. Cover the smear and paper towel with malachite green, and steam. Don't let the slide get dry--keep adding stain it is appears to approach dryness. Steam gently for about 5-10 minutes. REASON: You are cooking the malachite green into the endospore wall, as they are very resistant to staining.

5. Remove the slide from the steaming beaker, turn off the flame, and dispose of the green paper in the wastebasket, using your forceps. Wash the slide gently in running water about 20 seconds, getting all the extra green off the slide.

6. Counterstain with safranin for one minute, at the staining sink. Gently rinse with water, and shake off excess water into the sink. REASON: The running water washed the green stain out of the vegetative cells and sporangia, and they became colorless. The counterstain now dyes them red.

7. Gently blot the slide dry, no rubbing, and let it air dry. Examine with oil immersion optics. Observe red vegetative cells and sporangia, and green endospores and free spores.

Spore stain of a overnight culture of Bacillus subtilis.Note: The all red cells are vegitative cells. The free green "cells" are free spores. The cells that are red with a green interior structure are the cells with endospores.

Capsule Stain:(Negative stain)1. Place a drop of congo red on a clean slide. Mix a loop of Klebsiella culture into the stain drop.

2. Using a second slide, spread the drop of stain/microbe across the first slide, making a smear, and feathering the end.

3. Allow slide to air dry and then view under oil immersion.

Congo Red Capsule stain of Klebsiella pneumonia.Note: The red interior is the bacterial cell and the clear zone around the cell is the area where the capsule has excluded the congo red stain.

Questions:

1. What is the clinical value of the acid-fast stain? the capsule stain?

2. Why must the spore stain include a heating step? What would happen if this were omitted?

3. Describe the different arrangements of flagella. What is their importance?

4. Why is staining bacterial components useful in strain identification?

Capsule Stain

Purpose: The capsule stain is a differential stain which selectively stains external capsules surroundingbacterial cells. How it works: Capsules are highly ordered polymers of sugars and proteins that surround some bacterial cells, and can be easily dislodged by heat or water. Accordingly, capsule stains are not heat-fixed, and water is never used to rinse. The primary stain applied is crystal violet, which stains both the bacterial cell and the surrounding capsule. A 20% copper sulfate solution is then applied, which serves a dual function as both decolorizer and counterstain. It removes and replaces the crystal violet in the capsule only. At the end of the staining procedure, the capsule appears as a faint blue or white halo around a purple cell.Overview of capsule-staining process:Crystal Violet

20% Copper Sulfate

No Capsule

Capsule

Results:

Simple Stain

Simple stains provide a quick and easy way to determine cell shape, size, and arrangement.

1. Perform a bacterial smear, as discussed in Figure 3-35 on page 82 of your lab manual.

2. Saturate the smear with basic dye for approximately 1 minute. You may use crystal violet, safranin, or methylene blue.

3. Rinse the slide gently with water.

4. Carefully blot dry with bibulous paper.

5. Observe the slide under the microscope, using proper microscope technique.

Negative Stain

Negative staining is an excellent way to determine an organisms cellular morphology. Since the cells themselves are not stained, their morphology is not distorted in any way. The nigrosine provides a dark background against which the shapes of the unstained cells are clearly visible. This method provides a high degree of contrast not available in most other staining procedures.

1. Place a single drop of nigrosin on the left-hand end of a clean microscope slide.

2. Using a flamed loop and sterile technique, remove some organism from your slant and mix

it into the drop of nigrosin. Be sure there are no large clumps of organism, but try to avoid

spreading the drop.

3. Place the end of another, clean microscope slide at an angle to the end of the slide

containing the organism and spread the drop out into a film. This is done by contacting the

drop of nigrosin with the clean microscope slide and using the capillary action of the

dye/microscope slide to spread the nigrosin across the smear:

4. Allow the film to air dry.

5. Observe the slide under the microscope, using proper microscope technique.

Gram Stain

1. Perform a bacterial smear, as discussed in Figure 3-35 on page 82 of your lab manual.

2. Saturate the smear with crystal violet for 1 minute.

3. Rinse the slide gently with water.

4. Saturate the smear with iodine for 1 minute.

5. Rinse the slide gently with water.

6. Decolorize with Gram decolorizer (acetone/alcohol) for 3-5 seconds ONLY; if you leave the

decolorizer on too long, it will bleach the crystal violet out of a gram positive cell!

7. Rinse the slide gently with water.

8. Counterstain with safranin for 1 minute.

9. Rinse the slide gently with water.

10. Carefully blot the slide dry with bibulous paper.

11. Observe the slide under the microscope, using proper microscope technique.

With proper staining technique

Gram positive bacteria will stain purple.

Gram negative bacteria will stain red/pink.

TIP: When making a smear of an unknown organism to gram stain, place three

organisms on your slide: a known gram positive, a known gram negative, and your

unknown (and LABEL which is which!). That way, if theres a problem with your

staining technique, it will be reflected in the known organisms, and youll know you

need to do the stain again to get accurate results.

Capsule Stain

1. Place a single drop of India ink on the left-hand end of a clean microscope slide.

2. Using a flamed loop and sterile technique, remove some K. pneumoniae (or the organism you

want to stain) from your slant and mix it into the drop of India ink. Be sure there are no large

clumps of organism, but try to avoid spreading the drop.

3. Place the end of another clean microscope slide at an angle to the end of the slide containing the

organism. Spread out the drop out into a film. This is done by contacting the drop of India ink

with the clean microscope slide and using the capillary action of the dye/ slide to spread the

India ink across the smear. Refer to the Negative Stain portion of this handout for a diagram.

4. Allow the film to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule!

5. Saturate the slide with crystal violet for 1 minute.

6. Rinse the slide gently with water.

7. Allow the slide to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule!

8. Observe the slide under the microscope, using proper microscope technique.

The background will be dark.

The bacterial cells will be stained purple.

The capsule (if present) will appear clear against the dark background.

background

capsule

bacterium

TIP: If you are staining an unknown organism, be sure your culture is several days

old. Young, fresh cultures wont have developed capsules yet.

Acid Fast Stain

1. Perform a bacterial smear, as discussed in Figure 3-35 on page 82 of your lab manual.

2. Place a small piece of bibulous paper over the smear and saturate the paper with carbolfuchsin.

3. Heat the slide gently over the bunsen burner for 5 minutes. Be sure to keep the bibulous paper

saturated with carbolfuchsin during heating; if the slide is steaming, youre okay; if it stops

steaming, add more carbolfuchsin!

4. Rinse the slide gently with water and dispose of the used bibulous paper in the trash. DO NOT

leave the bibulous paper in the sink or drain!

5. Decolorize the slide with acid-alcohol until the rinsate runs clear.

6. Rinse the slide gently with water.

7. Counterstain with methylene blue for 2 minutes.

8. Rinse the slide gently with water.

9. Carefully blot the slide dry with bibulous paper.

10. Observe the slide under the microscope, using proper microscope technique.

Acid-fast cells will stain fuchsia. Non-acid-fast cells will stain blue.

Endospore Stain

1. Perform a bacterial smear of Bacillus or your unknown, as discussed in Figure 3-35 on page 82

of your lab manual.

2. Place a small piece of bibulous paper over the smear. Saturate the paper with malachite green.

3. Heat the slide gently over the bunsen burner for 5 minutes. Be sure to keep the bibulous paper

saturated with malachite green during heating; if the slide is steaming, youre okay; if it stops

steaming, add more malachite green!

4. Rinse the slide gently with water and dispose of the used bibulous paper in the trash. DO NOT

leave the bibulous paper in the sink or drain!

5. Counterstain with safranin for 2 minutes.

6. Rinse the slide gently with water.

7. Carefully blot the slide dry with bibulous paper.

8. Observe the slide under the microscope, using proper microscope technique.

Endospores will stain green. Parent cells will stain red.