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Microscope Slide Techniques - Bacterial Morphology

Slide negative staining technique spreader slide

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The nine exercises in this unit include the procedures for ten slide techniques that one might employ in morphological studies of bacteria.

A culture method in Exercise 18 also is included as a substitute for slide techniques when pathogens are encountered.

These exercises are intended to serve two equally important functions:
  1. to help you to develop the necessary skills in making slides and

  2. to introduce you to the morphology of bacteria.

Although the title of each exercise pertains to a specific technique, the organisms chosen for each method have been carefully selected so that you can learn to recognize certain morphological features.

For example, in the exercise on simple staining (Exercise 13), the organisms selected exhibit metachromatic granules, pleomorphism, and palisade arrangement of cells.

In Exercise 15, (Gram Staining) you will observe the differences between cocci and bacilli, as well as learn how to execute the staining routine.

The importance of the mastery of these techniques cannot be overemphasized. Although one is seldom able to make species identification on the basis of morphological characteristics alone, it is a very significant starting point. This fact will become increasingly clear with subsequent experiments.

Although the steps in the various staining procedures may seem relatively simple, student success is often quite unpredictable. Unless your instructor suggests a variation in the procedure, try to follow the procedures exactly as stated, without improvisation.

Photomicrographs in color have been provided for many of the techniques; use them as a guide to evaluate the slides you have prepared. Once you have mastered a specific technique, feel free to experiment.

The simplest way to make a slide of bacteria is to prepare a wet mount, much in the same manner that was used for studying protozoa and algae. Although this method will quickly produce a slide, finding the bacteria on the slide may be difficult, especially for a beginner.

The problem one encounters is that bacteria are quite colorless and transparent. Unless the diaphragm is carefully adjusted, the beginner usually has considerable difficulty bringing the organisms into focus.

A better way to observe bacteria for the first time is to prepare a slide by a process called negative, or background, staining. This method consists of mixing the microorganisms in a small amount of nigrosine or india ink and spreading the mixture over the surface of a slide.

(Incidentally, nigrosine is far superior to india ink.) Since these two pigments are not really bacterial stains, they do not penetrate the micro organisms Instead they obliterate the background, leaving the organisms transparent and visible in a darkened field.

Although this technique has limitations, it can be useful for determining cell morphology and size. Since no heat is applied to the slide, there is no shrinkage of the cells, and consequently, more accurate cell-size determinations result than with some other methods.

This method is also useful for studying spirochaetes that don't stain readily with ordinary dyes.

THREE MICROSCOPE SLIDE STAINING METHODS

Negative staining slide method

Negative staining can be done by one of three different methods. Figure 11.1 illustrates the more commonly used method in which the organisms are mixed in a drop of nigrosine and spread over the slide with another slide.

The goal is to produce a smear that is thick at one end and feather-thin at the other end. Somewhere between the too thick and too thin areas will be an ideal spot to study the organisms.

Figure 11.2 illustrates a second method, in which or of a full drop. In this method the organisms are spread over a smaller area in the center of the slide with an inoculating needle. No spreader slide is used in this method.

The third procedure (Woeste-Demchick's method), which is not illustrated here, involves applying ink to a conventional smear with a black felt marking pen.

If this method is used, it should be done on a smear prepared in the manner described in the next exercise. Simply put, the technique involves applying a single coat of felt-pen ink over a smear.

Note in the procedure below that slides may be made from organisms between your teeth or from specific bacterial cultures.

Materials:
  • microscope slides (with polished edges)

  • nigrosine solution or india ink

  • slant cultures of S. aureus and B. megaterium

  • inoculating straight wire and loop

  • sterile toothpicks

  • Bunsen burner

  • china marking pencil

  • felt marking pen

  1. Swab down your tabletop with disinfectant in preparation for making slides.

  2. Clean two or three microscope slides with Bon Ami to rid them of all dirt and grease.

  3. By referring to figure ll.I or ll.2, place the proper amount of stain on the slide.

  4. Oral Organisms: Remove a small amount of material from between your teeth with a sterile straight toothpick or inoculating needle and mix it into the stain on the slide.

    Be sure to break up any clumps of organisms with the wire or toothpick. When using a wire, be sure to flame it first to make it sterile.

    CAUTION: If you use a toothpick, discard it into a beaker of disinfectant.

  5. From Cultures: With a sterile straight wire, transfer a very small amount of bacteria from the slant to the center of the stain on the slide.

  6. Spread the mixture over the slide according to the procedure used in figure 11.1 or 11.2.

  7. Allow the slide to air-dry and examine with an oil immersion objective.

Save

If slide is of oral organisms, look for yeasts and hyphae as well as bacteria, Spirochaetes may also be present.

Smear Preparation

While negative staining is a simple enough process to make bacteria more visible with a brightfield microscope, it is of little help when one attempts to observe anatomical microstructures such as flagella, granules, and endospores.

Only by applying specific bacteriological stains to organisms can such organelles be seen. However, success at bacterial staining depends first of all on the preparation of a suitable smear of the organisms.

A properly prepared bacterial smear is one that withstands one or more washings during staining without loss of organisms, is not too thick, and does not result in excessive distortion due to cell shrinkage.

The procedure for making such a smear is illustrated in figure 12.1. The first step in preparing a bacteriological smear differs according to the source of the organisms.

If the bacteria are growing in a liquid medium, (broths, milk, saliva, urine, etc.), one starts by placing one or two loopfuls of the liquid medium directly on the slide.

From solid media such as nutrient agar, blood agar, or some part of the body, one starts by placing one or two loopfuls of water on the slide and then uses a straight inoculating wire to disperse the organisms in the water.

Bacteria growing on solid media tend to cling to each other and must be dispersed sufficiently by dilution in water; unless this is done, the smear will be too thick.

The most difficult concept for students to understand about making slides from solid media is that it takes only a very small amount of material to make a good smear.

When your instructor demonstrates this step, pay very careful attention to the amount of material that is placed on the slide. The organisms to be used for your first slides may be from several different sources.

If the plates from Exercise 9 were saved, some slides may be made from them. If they were discarded, the first slides may be made for Exercise 13. which pertains to simple staining. Your instructor will indicate which cultures to use.

making a bacteria smear microscope slide

FROM LIQUID MEDIA - (Broths, saliva, milk, etc.)

If you are preparing a bacterial smear from liquid media, follow this routine, which is depicted on the left side of figure 12.1.

Materials:
  • microscope slides

  • Bunsen burner

  • wire loop

  • china marking pencil

  • slide holder (clothespin ), optional

  1. Wash a slide with soap or Bon Ami and hot water, removing all dirt and grease. Handle the clean slide by its edges.

  2. Write the initials of the organism or organisms on the left-hand side of the slide with a china marking pencil.

  3. To provide a target on which to place the organisms, make a ½" circle on the bottom side of the slide, centrally located, with a marking pencil. Later on, when you become more skilled, you may wish to omit the use of this "target circle."

  4. Shake the culture vigorously and transfer two loopfuls of organisms to the center of the slide over the target circle. Follow the routine for inoculations shown in figure 12.2. Be sure to flame the loop after it has touched the slide.

    CAUTION

    Be sure to cool the loop completely before inserting it into a medium. A loop that is too hot will spatter the medium and move bacteria into the air.

  5. Spread the organisms over the area of the target circle.

  6. Allow the slide to dry by normal evaporation of the water. Don't apply heat.

  7. After the smear has become completely dry, pass the slide over a Bunsen burner flame to heat-kill the organisms and fix them to the slide.

Note that in this step one has the option of using or not using a clothespin to hold the slide.

microscope slide aseptic organism removal

FROM SOLID MEDIA

When preparing a bacterial smear from solid media, such as nutrient agar or a part of the body, follow this routine, which is depicted on the right side of figure 12.1. Materials:

  • microscope slides

  • inoculating needle and loop

  • china marking pencil

  • slide holder (clothespin), optional

  • Bunsen burner

  1. Wash a slide with soap or Bon Ami and hot water, removing all dirt and grease. Handle the clean slide by its edges.

  2. Write the initials of the organism or organisms on the left-hand side of the slide with a china marking pencil.

  3. Mark a "target circle" on the bottom side of the slide with a china marking pencil.

  4. Flame an inoculating loop, let it cool, and transfer two loopfuls of water to the center of the target circle.

  5. Flame an inoculating needle then let it cool. Pick up a very small amount of the organisms, and mix it into the water on the slide. Disperse the mixture over the area of the target circle.

    Be certain that the organisms have been well emulsified in the liquid. Be sure to flame the inoculating needle before placing it aside.

  6. Allow the slide to dry by normal evaporation of the water. Don't apply heat.

  7. Once the smear is completely dry, pass the slide over the flame of a Bunsen burner to heat-kill the organisms and fix them to the slide. Use a clothespin to hold the slide if it is preferred by your instructor.

    Some workers prefer to hold the slide with their fingers so that they can monitor the temperature of the slide (to prevent overheating).

Simple Staining

The use of a single stain to color a bacterial organism is commonly referred to as simple staining. Some of the most commonly used dyes for simple staining are methylene blue, basic fuchsin, and crystal violet.

All of these dyes work well on bacteria because they have color-bearing ions (chromophores) that are positively charged (cationic).

The fact that bacteria are slightly negatively charged produces a pronounced attraction between these cationic chromophores and the organism. Such dyes are classified as basic dyes.

The basic dye methylene blue (methylene+ chloride-) will be used in this exercise. Those dyes that have anionic chromophores are called acidic dyes. Eosin (sodium+ eosinate- ) is such a dye. The anionic chromophore, eosinate-, will not stain bacteria because of the electrostatic repelling forces that are involved.

The staining times for most simple stains are relatively short, usually from 30 seconds to 2 minutes, depending on the affinity of the dye.

After a smear has been stained for the required time, it is washed off gently, blotted dry, and examined directly under oil immersion.

Such a slide is useful in determining basic morphology and the presence or absence of certain kinds of granules.

An avirulent strain of Corynebacterium diphtheriae will be used here for simple staining. In its pathogenic form, this organism is the cause of diphtheria, a very serious disease.

One of the steps in identifying this pathogen is to do a simple stain of it to demonstrate the following unique characteristics: pleomorphism, meta-chromatic granules, and palisade arrangement of cells.

Pleomorphism pertains to irregularity of form:

i.e., demonstrating several different shapes. While C. diphtheriae is basically rod-shaped, it also appears club-shaped, spermlike, or needle-shaped. Bergey's Manual uses the terms "pleomorphic" and "irregular" interchangeably.

Metachromatic granules are distinct reddish-purple granules within cells that show up when the organisms are stained with methylene blue. These granules are considered to be masses of volutin, a polymetaphosphate.

Palisade arrangement pertains to parallel arrangement of rod-shaped cells. This characteristic, also called "picket fence" arrangement, is common to many corynebacteria.

proceedure for simple slide staining

PROCEDURE

Prepare a slide of C. diphtheriae, using the procedure outlined in figure 13.1. It will be necessary to refer back to Exercise 12 for the smear preparation procedure.

Materials:
  • slant culture of avirulent strain of

  • Corynebacterium diphtheriae

  • methylene blue (Loeffler's)

  • wash bottle

  • bibulous paper

microscope slide capsule presence