Introductory Bacteria Morphology

Introduction

How can one type of bacteria be distinguished from another type? Examine some representative bacteria and learn the basic morphology (size and shape) of different types of bacteria.

Concepts

• Bacteria

• Cocci
• Spirilli
• Bacilli

Background

Bacteria are mostly too small to be seen without the aid of a microscope. Some bacteria can be seen with 200–400X magnification. Most bacteria, however, can only be seen clearly at 1000X magnification and using an oil immersion, light-capturing technique.

The density of most bacteria is only slightly greater than that of water. As a result, bacteria are almost transparent and thus difficult to see in liquid suspension using conventional bright field microscopy. To more clearly visualize bacteria, dyes or stains are usually used. Staining not only makes bacteria more visible, but also allows their morphology (size and shape) to be easily distinguished.

To prepare bacteria for staining, a sample of bacteria in liquid is spread in a thin layer or smear on a clean glass slide. The thin bacterial film is allowed to dry and is then fixed to the glass to prevent it from washing off during the subsequent staining procedure. The bacteria are fixed by briefly passing the slide through a flame. This treatment sticks the bacteria to the glass. (The fixing process is similar to frying an egg—the egg-protein sticks firmly to the bottom of the frying pan.) The fixed bacteria are then exposed to the specific stain or staining procedure. Some staining procedures are considered general or universal stains and can be used to visualize many different types of bacteria. Other staining procedures, however, are very specific and have been developed to help distinguish one bacteria type from another. These more specific staining procedures are often referred to as differential staining procedures and are used to classify bacteria. The commonly used Gram stain is an example of a differential staining procedure.

Microbiology work requires specific knowledge, techniques, and training. This kit assumes a working knowledge of: (1) aseptic sterile transfer techniques; (2) streak-plating techniques; (3) sterile agar plate preparation; (4) nutrient broth tube preparations; (5) oil immersion microscope technique. Review these procedures or get training before attempting work with microbes.

Materials

Safety Precautions

Wear chemical splash goggles and chemical-resistant gloves when working in the laboratory. Wash hands and work areas after completing microbiological procedures. 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. All microbiology materials must be sterilized before disposal. Carefully clean immersion oil from microscope objectives immediately after use.

Prelab Preparation

Be sure to review the procedures identified in the Background section. This kit provides the basic materials needed to make slides of three morphologically different bacteria—bacilli (rods), cocci (spheres) and spirilli (spirals). The materials provided in the kit can be used as a classroom demonstration or as an all-class student activity.

In addition to a working knowledge of microbiological techniques, it is further assumed that the following materials are available:

1. Inoculating loops
2. Sterile Petri dishes
3. Nutrient agar or nutrient broth
4. Autoclave, pressure cooker or 10% bleach solution
5. Microscopes with oil immersion capabilities (1000X magnification)
6. Incubator

The procedures outlined below can be followed and completed as a teacher demonstration or students can make their own slides. The procedure is outlined working either from a streak plate culture or from a broth culture. At least two days before the lab, nutrient broth or solid agar plates of the bacteria cultures should be made and incubated into flourishing cultures. A culture tube or plate should be made for each working group following sterile techniques.

Procedure

Part I. Preparation of Smear

A. From a nutrient agar culture

1. Label a slide so that a specific side of the slide can be determined.
2. Place one drop of sterile water on the center of the top of the slide.
3. Use sterile technique and an inoculating loop to remove a small (nearly invisible) sample from a colony on the surface of the agar. Take only a barely visible amount of the bacterial colony. More is not better!
4. Dip the inoculating loop into the drop of water on the slide.
5. Disperse the bacterial cells in the water drop and spread the drop over about a ½" square in the center of the slide with the inoculating loop.
6. Allow the slide to dry.
7. Holding the slide with a slide holder, clothespin, or forceps, pass the slide slowly 3–4 times through the flame of a Bunsen burner on the bottom of the slide. This will fix the bacteria to the slide. Do not hold the slide directly in the flame.

B. From a broth culture

1. Label a slide so that a specific side of the slide can be determined.
2. Spread two loopfuls of broth culture over about a ½" square on the center of the slide with the inoculating loop. Be sure to use sterile technique if the culture is to be used again.
3. Allow the slide to thoroughly dry.
4. Holding the slide with a slide holder or forceps, pass the slide slowly 3–4 times through the flame of a Bunsen burner on the bottom of the slide. This will fix the bacteria to the slide. Do not hold the slide directly in the flame.

Part II. Staining the Smear

1. Place the slide on the top of a small beaker or other slide support device over a sink.
2. Cover the “fixed” bacteria with several drops of crystal violet stain.
3. Leave the stain on the slide for about one minute.
4. Briefly and gently rinse the slide with water. Use a water bottle or a trickle from a faucet.
5. Gently blot the slide dry with paper towels. Do not rub, as the entire preparation can be easily removed.

Part III. Viewing the Bacteria

1. Do not use a coverslip.
2. Place the slide on a microscope and begin to look for stained materials using 400X. Look for “clumps” of stained violet-colored material. Individual bacteria will not be visible at this magnification.
3. Center a stained area in the field of view. Rotate the nosepiece toward the 100X objective and place a drop of immersion oil on the top surface of the slide.
4. Gently move the 100X objective into the oil without moving the slide (1000X total magnification).
5. Focus on the stained materials using only the fine adjustment on the microscope.
6. Examine many fields of the bacteria until the bacteria size, shape, and morphology can be clearly discerned.
7. Record observations as appropriate.

Teacher Tips

• This kit contains enough bacteria and stain material for all students in a class of 30 to make their own set of bacterial slides. Careful slide preparation and examination will require 1–2 class periods.

• Pre-lab preparations are critical for the safe and organized execution of these lab procedures. Set up workstations where the procedures can be done safely and with minimal traffic in the class. Especially organize sink use if sink space is limited.
• Students should be able to make well-stained slides and be able to see the relative size and shape of the three morphological types provided.
• Pour the crystal violet stain into dropper bottles or use small beakers and disposable pipets to apply the stain.