Chapter 3 - Microscopy and staining

3 - 1 Use of the microscope

The microscope, as shown in Figure 3-1, is one of the most important instruments utilized by the microbiologist. In order to study the morphological and staining characteristics of microorganisms such as bacteria, yeasts, molds, algae and protozoa, you must be able to use a microscope correctly.

The compound microscope used in microbiology is a precision instrument; its mechanical parts, such as the calibrated mechanical stage and the adjustment knobs, are easily damaged, and all lenses, particularly the oil immersion objective, are delicate and expensive. Handle the instrument with care and keep it clean.

The microscope is basically an optical system (for magnification) and an illumination system (to make the specimen visible). To help understand the function of the various parts of the microscope, we will follow a ray of light as it works its way through a microscope from the light source, through the lenses, up to the eye. Figure 3-8 traces the path of light through the parts of the microscope

First let us consider a primary feature of all microscopes, the light source. Proper illumination is essential for effective use of a microscope. A tungsten filament lamp usually serves as the source of illumination. If reflected illumination is used, a separate lamp provides a focused beam of light which is reflected upward through the condenser lenses by a mirror.

The light from the illuminating source is passed through the substage condenser. The condenser serves two purposes; it regulates the amount of light reaching the specimen and it focuses the light coming from the light source. As the magnification of the objective lens increases, more light is needed. The iris diaphragm (located in the condenser), regulates the amount of light reaching the specimen. The condenser also collects the broad bundle of light produced by the light source and focuses it on the small area of the specimen that is under observation.

Light then passes up through the slide and into the objective lens where the first magnification of the image takes place. Magnification increases the apparent size of an object. In the compound light microscope two lenses, one near the stage called the objective lens and another in the eyepiece, enlarge the sample. The magnifying power of an objective lens is engraved in the lens mount. Microscopes in most microbiology laboratories have three objective lenses: the low power objective lens (10X), the high-dry objective lens (40X) and the oil-immersion objective lens (100X). The desired objective lens is rotated into working position by means of a revolving nosepiece.

On both sides of the base of the microscope are the course and fine adjustment knobs, used to bring the image into focus. Rotation of these knobs will either move the specimen and the objectives closer or farther apart. The coarse adjustment moves the nosepiece in large increments and brings the specimen into approximate focus. The fine adjustment moves the nosepiece more slowly for precise final focusing. In some microscopes, rotation of the fine and course adjustment knobs will move the stage instead of the nosepiece.

Magnification alone is not the only aim of a microscope. A given picture may be faithfully enlarged without showing any increase in detail. The true measure of a microscope is its resolving power. The resolving power of the lens is its ability to reveal fine detail and to make small objects clearly visible. It is measured in terms of the smallest distance between two points or lines where they are visible as separate entities instead of one blurred image. The resolving power of the objective lens, engraved on the lens, allows us to predict which objective lens should be used for observing a given specimen. However, having good resolution in the microscope does not guarantee a visible image, the resolving power of the human eye is quite limited. Often further magnification is needed to obtain a good image.

When the oil-immersion objective lens is in use, the difference between the light-bending ability (or refractive index of the medium holding the sample) and the objective lens becomes important. Because the refractive index of air is less than that of glass, light rays are bent or refracted as they pass from the microscope slide into the air, as shown in Figure 3-9. Many of these light rays are refracted at so great an angle that they completely miss the objective lens. This loss of light is so severe that images are significantly degraded. Placing a drop of immersion oil, which has a refractive index similar to glass, between the slide and the objective lens decreases this refraction, and increases the amount of light passing from the specimen into the objective lens. This results in greater resolution and a clearer image.

The image of the specimen continues on through a series of mirrors and/or prisms that bend it toward the eyepiece. A further magnification takes place at the eyepiece producing what is called a virtual image. Total magnification is equal to the product of the eyepiece magnification and the objective magnification. Most often eyepiece lenses magnify 10-fold resulting in total magnifications of 100, 400, or 1000X, depending upon which objective is in place. Many modern microscopes will also have focusable eyepieces to compensate for differences between individuals and even between individual's eyes. The adjustment of these is important and is described below.

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3 - 2 Operating procedure

Below we describe detailed directions for the use of a microscope. This will give you an appreciation of their operation. These directions have been written as generally as possible, but it may be necessary for your instructor to make modifications for the exact microscopes you are using. Light microscopes used in teaching laboratories are designed for ease of use and with some practice should become automatic.

1. Raise the nosepiece using the course adjustment knob. This provides greater access for positioning the slide on the stage.

2. Rotate the nosepiece so that the 10X objective lens is in operating position.

3. Open the iris diaphragm approximately half way.

4. Turn on the in-base illuminator by depressing the push-type switch.

5. Place a stained specimen slide on the stage and with the naked eye position the specimen directly above the center of the condenser.

6. Use the thumbwheel below the eyepieces to adjust the interpupillary distance between the two eyepieces. This is important to be able to view specimens with both eyes, maximizing the quality of the image and preventing fatigue from prolonged use of one eye.

7. Move the microscope condenser by means of the condenser adjustment knob until the top of the condenser is almost at the highest position. There should be enough room to slide a piece of paper between the stage and the condenser, but no more. This will focus the light onto the slide.

8. Rotate the coarse adjustment knob in a clockwise direction to bring the 10X objective closer to the slide. View through the eyepieces and, without disturbing the coarse adjustment setting, slowly rotate the fine adjustment knob until the specimen is in the sharpest possible focus.

9. The left eyepiece tube is focusable to compensate for refraction differences of the eyes. The correct procedure is to bring the specimen into sharp focus looking though the right eyepiece only. Then focus for the left eye by turning the left eye tube collar fully counter-clockwise. Next, while viewing the specimen with the left eye only turn the knurled collar clockwise until the specimen is in sharp focus. Do not adjust the fine adjustment knob during this procedure.

10. Remove an eyepiece to view the back aperture of the objective lens. Close the condenser iris diaphragm, then re-open until the leaves of the diaphragm just disappear from view. Replace the eyepiece and view the specimen. The iris diaphragm may be closed slightly to enhance contrast, especially when viewing unstained specimens.

    Unstained specimens have only minimal contrast with their surrounding environments. As a result they can usually be viewed more effectively by setting the diaphragm at or near minimum opening. Reducing the diaphragm setting increases definition, contrast, and depth of focus but introduces diffraction problems and sacrifices resolution. Play with the diaphragm setting and select the best compromise by trial and error.

11. Once the specimen is in sharp focus using the 10X objective lens, it is then possible to rotate the nosepiece to the 40X objective lens without changing the position of the coarse adjustment knob. Very little refocusing with the fine adjustment knob is required since most light microscope objective lenses are parfocal. Remember that the iris diaphragm setting must be changed to allow more light to pass though the sample as the magnification increases.

12. If the specimen is to be viewed using the 100X oil immersion lens, immersion oil must be applied to the slide.

13. Rotate the 40X objective lens slightly to the side so that a drop of immersion oil may be placed on the specimen without getting it on the 40X lens.

14. Place a drop of immersion oil in the center of the circle of light formed on the specimen slide.

15. Carefully turn the nosepiece until the 100X objective lens snaps into place. The objective lens should be in the oil but must not touch the slide.

16. Increase the light intensity as required and rotate the fine adjustment knob to obtain a sharp focus of the specimen. If necessary make further adjustments to obtain optimal illumination.

If the microscope is not parfocal, it will be necessary to lower the objective lens as close to the slide as possible without touching it. This is done only while looking at the lens and slide from the side of the microscope. Bring the specimen into view by slowly raising the objective lens with the coarse adjustment knob. Next, focus with the fine adjustment knob and adjust the illumination as necessary. If this is not successful the first time, repeat the entire procedure.

In many cases, a preparation needs to be observed only under the oil immersion lens. In this case, first locate the specimen and center it in the field with the low power objective lens. Then add oil and rotate the oil immersion objective lens into position.

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3 - 3 Common Problems

Certain problems real or apparent, may be encountered while operating your microscope. Here is a trouble shooting guide to help you if you are having difficulty focusing a sample.

The sample can be focused at 10X, but it is difficult to find or blurry at 40X.

This is often caused by immersion oil on the 40X lens. Wipe the 40X lens with lens paper to remove the oil and refocus. This can be prevented by never viewing a specimen with the 40X objective after adding immersion oil to a slide.

The sample can be focused at 10X but when the 40X lens is rotated in place it contacts the slide.

In most cases this is caused by the slide being place on the stage upside down †with the smear facing the stage. Check your slide carefully to make sure it is placed on the stage correctly.

The fine adjustment knob does not turn in the direction required for sharp focusing.

This indicates that it has been turned to the limits of its threads, either upward or downward, as the case may be. Screw it back to about one-half the thread distance (About four turns), use the coarse adjustment to raise or lower the objective lens sufficiently to bring the specimen into view; then refocus with the fine adjustment.

What I am viewing does not look like bacteria.

Check to make sure you are in the correct focal plane that you are focusing on the smear and not dust on the lenses. To verify this, move the slide while looking at it. Anything in the smear should move in the field of view.

What I am viewing does not look like bacteria. Part II

If you are in the correct focal plane, there may be problems with smear preparation. Did you heat fix too much? Was the amount of culture applied sufficient? Did you stain the slide correctly? Many apparent microscope problems can be attributed to poor slide preparation.

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3 - 4 Proper care of the microscope

The following rules, cautions and maintenance hints will help keep your microscope in good operating condition.

1. Use both hands when carrying the microscope: one firmly grasping the arm of the microscope; the other beneath the base. Avoid jarring your microscope.

To keep the microscope and lens systems clean:

2. Never touch the lenses. If the lenses become dirty, wipe them gently with lens tissue.

3. If blurred specks appear in the field of view this may be due to lint or smears on the eyepiece. If the specks move while rotating the eyepiece, the dust is on the eyepiece and cleaning the outer lens of the eyepiece is in order. If the quality of the image is improved by changing objective lenses, clean the objective lens with lens paper.

4. Never leave a slide on the microscope when it is not in use.

5. Always remove oil from the oil-immersion objective lens after its use. If by accident oil should get on either of the lower-power objective lenses, wipe it off immediately with lens tissue.

6. Keep the stage of the microscope clean and dry. If any liquids are spilled, dry the stage with a piece of cheesecloth. If oil should get on the stage moisten a piece of cheesecloth with xylol and clean the stage, then wipe it dry.

7. When not in use, store your microscope in its cabinet. Put the low power objective lens into position at its lowest point above the stage. Be sure that the mechanical stage does not extend beyond the edge of the microscope stage. Wrap the electrical cord around the base.

   To avoid breaking the microscope:

8. Never force the adjustments. All adjustments should work freely and easily. If anything does not work correctly, do not attempt to fix it yourself, immediately notify your instructor.

9. Never allow an objective lens to jam into or even to touch the slide or cover-slip.

10. Never focus downward with the coarse adjustment while you are looking through the microscope. Always incline your head to the side with eyes parallel to the slide and watch the objective as you move it closer to the slide. This will prevent you from smashing the objective into the slide.

11. Never exchange the objective or eyepiece lenses of different microscopes, and never under any circumstances remove the front lenses from objective lenses.

12. Never attempt to carry two microscopes at one time

If you follow these rules, you will never have trouble with your microscope.

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3 - 5 Staining microorganisms

Preliminary identification of bacteria is usually based upon their cell morphology and grouping and the manner in which they react to certain staining procedures. The purpose of this section is to demonstrate some common staining reactions used to categorize microorganisms.

Unstained bacteria are practically transparent when viewed using the light microscope and thus are difficult to see as shown in Figure 3-2. The development of dyes to stain microorganisms was a significant advance in microbiology. Stains serve several purposes:

• Stains differentiate microorganisms from their surrounding environment

• They allow detailed observation of microbial structures at high magnification

• Certain staining protocols can help to differentiate between different types of microorganisms.

Most dyes consist of two functional chemical groups as shown in Figure 3-3. The chromophore group, which give dyes their characteristic color; and the auxochrome group, containing an ionizable chemical structure, which helps to solubilize the dye and facilitates binding to different parts of microorganisms. Previously, dyes were classified as acidic or basic, depending upon whether the pigment was negatively or positively charged at neutral pH. More accurately, dyes can be referred to as anionic (-) or cationic (+) and this is the convention that will be used in this manual. Cationic dyes (crystal violet, methylene blue) will react with groups on bacteria that have a negative charge. Anionic dyes (eosin, nigrosine) will react with groups that have a positive charge. Since most bacteria have many positive and negative groups in their cell walls and other surfaces, they will react with both cationic and anionic dyes.

Staining protocols can be divided into 3 basic types, simple, differential, and specialized. Simple stains react uniformly with all microorganisms and only distinguish the organisms from their surroundings. Differential stains discriminate between various bacteria, depending upon the chemical or physical composition of the microorganism. The Gram stain is an example of a differential stain. Specialized stains detect specific structures of cells such as flagella and endospores.

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3 - 6 Preparation of a Bacterial Smear for Staining

Before staining and observing a microbe under a microscope, a smear must be prepared. The goal of smear preparation is to place an appropriate concentration of cells on a slide and then cement them there so that they do not wash off during the subsequent staining procedure. Figure 3-4 demonstrates smear preparation.

The best smears are made from bacteria that have grown on a solid surface such as an agar slant or plate. A bit of growth from a culture is mixed with distilled or tap water to form a slightly turbid solution and this is spread on a clean grease free slide. When staining broth cultures, a drop of broth is transferred directly to a slide, using no extra water. The procedure for making a smear is as follows:

1. If more than one culture is to be examined using the same stain, it is possible to prepare up to 6 smears on the same slide. Before preparing the slide, divide it into the appropriate number of sections and clearly label each section on the underside of the slide.

2. If your culture has been grown on a agar slant or agar plate. Place a small drop of water on a clean, grease-free slide. Next, using a sterile loop or straight wire needle, transfer a bit of the growth to the drop of water and rub the needle around until the material is evenly emulsified. Spread the drop over a portion of the slide to make a thin film. The suspension should be only slightly turbid.

3. If you are using a broth culture, the broth culture must have clearly visible turbidity. Transfer a loopful of culture from the broth onto a clean grease free slide. Spread the drop over a portion of the slide to make a thin film.

4. Allow the film to air-dry. To get a good stain, it is important to let the smear dry completely. Excess water left on the slide will boil during the fixing stage, causing most microbe present to rupture. Rushing this step will result in a poor final stain.

5. Once dry, "fix" the smear to the slide by passing the bottom of the slide through the tip of the burner flame several times for a one second. After heat fixing, touch the heated portion of the slide to your hand. It should be comfortably warm, but not burning hot.

6. Take care not to under-fix (the smear will wash off) or over-heat (the cells will be ruptured or distorted) the slide. The correct amount of heat fixing is learned by experience.

7. Allow the smear to cool and apply the stain.

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3 - 7 The Simple Stain

In a simple stain, the smear is stained with a solution of a single dye which stains all cells the same color. Differentiation of cell types or structures is not the objective of the simple stain. However, certain structures which are not stained by this method may be easily seen, for example, endospores and lipid inclusions.

Simple stains are, well simple. One makes a smear and the applies a single stain to the slide. Below is a procedure for a simple stain.

1. Prepare and heat-fix a smear of the organism to be studied.

2. Cover the smear with the staining solution. If crystal violet or safranin is used, allow one minute for staining. The use of methylene blue requires 3-5 minutes to achieve good staining.

3. Carefully wash off the dye with tap water and blot the slide dry with blotting paper, an absorbent paper pad or a paper towel.

Three steps, now wasn't that easy?

The above movie demonstrates the simple stain.

Figure 3-10 shows a light micrograph of what a simple stain should look like.

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3 - 8 The Gram Stain

The Gram stain, performed properly, differentiates nearly all bacteria into two major groups. For example, one group, the gram-positive bacteria, include the causative agents of the diseases diphtheria, anthrax, tetanus, scarlet fever, and certain forms of pneumonia and tonsillitis. A second group, the gram-negative bacteria, includes organisms which cause typhoid fever, dysentery, gonorrhea and whooping cough. In Bacteria the reaction to Gram stain reagents is explained by different cell wall structures. Gram-positive microbes have a much thicker cell wall, while that found in Gram-negative microbes is thinner. Microbes from the Archaea domain contain different cell wall structures than that seen in microbes commonly found in the lab (Bacteria domain). However, they will still have a species specific Gram stain reaction, even though the underlying macromolecular structures are different.

The Gram stain is one of the most useful differential stains in bacteriology, including diagnostic medical bacteriology. The differential staining effect correlates to differences in the cell wall structure of microorganisms (at least Bacteria, but not Archaea as mentioned above). In order to obtain reliable results it is important to take the following precautions:

• The cultures to be stained should be young - incubated in broth or on a solid medium until growth is just visible (no more than 12 to 18 hours old if possible). Old cultures of some gram-positive bacteria will appear Gram negative. This is especially true for endospore-forming bacteria, such as species from the genus Bacillus. In this class, many of the cultures will have grown for more than 2 days. For most bacteria this is not a problem, but be aware that some cultures staining characteristics may change!

• When feasible, the cultures to be stained should be grown on a sugar-free medium. Many organisms produce substantial amounts of capsular or slime material in the presence of certain carbohydrates. This may interfere with decolorization, and certain Gram-negative organisms such as Klebsiella may appear as a mixture of pink and purple cells.

Gram stain procedure

Below is a procedure that works well in the teaching laboratories.

1. Cover the slide with crystal violet stain and wait one minute.

2. After one minute wash the stain off (gently!) with a minimum amount of tap water. Drain off most of the water and proceed to the next step. It may help to hold the slide vertically and touch a bottom corner to paper toweling or blotting paper.

3. Cover the slide with iodine solution for one minute. The iodine acts as a mordant (fixer) and will form a complex with the crystal violet, fixing it into the cell.

4. Rinse briefly with tap water.

5. Tilt the slide lengthwise over the sink and apply the alcohol-acetone decolorizing solution (dropwise) such that the solution washes over the entire slide from one end to the other. All smears on the slide are to be treated thoroughly and equally in this procedure. Process the sample in this manner for about 2-5 seconds and immediately rinse with tap water. This procedure will decolorize cells with a Gram negative type of cell wall but not those with a gram-positive type of cell wall, as a general rule. Drain off most of the water and proceed.

6. As the decolorized gram-negative cells need to be stained in order to be visible, cover the slide with the safranin counterstain for 30 seconds to one minute.

7. Rinse briefly and blot the slide dry. Record each culture as Gram positive (purple cells) or Gram negative (pink cells).

The above video demonstrates the Gram stain procedure, while Figure 3-11 shows the results of a Gram stain for gram-positive and gram-negative negative bacteria.

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3 - 9 The Endospore Stain

Cells of Bacillus, Desulfotomaculum and Clostridium (and several other, lesser-known genera--see Bergey's Manual) may, as a response to nutrient limitations, develop endospores that possess remarkable resistance to heat, dryness, irradiation and many chemical agents. Each cell can produce only one endospore. It is therefore not a reproductive spore as seen for some organisms such as Streptomyces and most molds. The endospore is essentially a specialized cell, containing a full complement of DNA and many proteins, but little water. This dehydration contributes to the spores resistance and makes it metabolically inert. The endospore develops in a characteristic position (for its species) in the vegetative cell. Eventually the cell lyses, releasing a free endospore.

Endospore Stain Procedure

Endospore stains require heat to drive the stain into the cells. For a endospore stain to be successful, the temperature of the stain must be near boiling and the stain cannot dry out. Most failed endospore stains occur because the stain was allowed to completely evaporate during the procedure.

1. Place the heat-fixed slide over a steaming water bath and place a piece of blotting paper over the area of the smear. The blotting paper should completely cover the smear, but should not stick out past the edges of the slide. If it sticks out over the edges stain will flow over the edge of the slide by capillary action and make a mess.

2. Saturate the blotting paper with the 5-6% solution of malachite green. Allow the steam to heat the slide for five minutes, and replenish the stain if it appears to be drying out.

3. Cool the slide to room temperature. Rinse thoroughly and carefully with tap water.

4. Apply safranin for one minute. Rinse thoroughly but briefly with tap water, blot dry and examine. Mature endospores stain green whether free or in the vegetative cell. Vegetative cells stain pink to red.

The above video demonstrates the endospore stain

Figure 3-12 shows a photomicrograph of an endospore stain.

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3 - 11 Practice staining

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!)

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

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!

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

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

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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3 - 12 Summary of Microscopy and Staining

Staining and viewing microbes under the microscope is often necessary in for their identification and classification. The identity of a microbe can help in determining the cause of a disease or the source of food spoilage. Microscopes also have important roles in genetics, cell structure, biochemistry and many other scientific disiplines. Hopefully, this short introduction has helped you to understand the visualization of microorganisms.

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