Fundamentals of Microbiology

Lesson 5. PREPARATION FOR LIGHT MICROSCOPY

Module 2. Microscopy

Lesson 5

PREPARATION FOR LIGHT MICROSCOPY

5.1 Introduction

Microscopy is one of the most important techniques use in biological sciences. Owing to their minute size, microbial cells need to be given special treatments for their visualization under microscope. This chapter describes a number of such techniques used in light microscopy for preparation of microbial specimens.

5.2 Methods for Studying Microbes with a Compound Microscope

Two methods are generally used, 'wet method', and 'dry and fix method'.

5.2.1 Wet method

There are two primary methods generally used for studying microorganisms in wet conditions (i) wet mount method and (ii) hanging drop method.

5.2.1.1 Wet mount method

It is the most widely used method. A drop of fluid containing microorganisms to be-examined put on a glass slide and a coverslip made of thin glass is placed on it (Fig. 5.1). The fluid spreads out in a thin layer between coverslip and slide. The mount is now examined under the microscope. For higher magnifications (e.g. with 100 X objective) the oil-immersion technique is employed.

Fig. 5.1 Wet mount slide

A drop of immersion oil is put between the objective lens and cover slip before the microorganisms are examined under the microscope. The immersion oil fills the space between the specimen and the objective lens and thus replaces the air present between the specimen and the objective lens. The result is that the numerical aperture (NA) is improved and the level of magnification is increased.

A drop of immersion oil is put between the objective lens and coverslip before the microorganisms are examined under the microscope. The immersion oil fills the space between the specimen and the objective lens and thus replaces the air present between the specimen and the objective lens. The result is that the NA is improved and the level of magnification is increased (Figure 5.2).

Fig. 5.2 Wet mount method

5.2.1.2 Hanging drop method

Microscopic examination of live bacteria in wet mounts reveals whether the bacteria are motile or non-motile. Motility is an inheritable phenotype and is a useful criterion for identification and classification of bacteria. Because unstained transparent cells are examined, more examination time is usually needed to visualize and locate the cells than for stained preparations. This is particularly true because 400X rather than 1,000X magnification is used to see bacterial cells in this type of preparation, and therefore examination is critical. Due to these limitations, special techniques are used in order to prevent the wet mount from drying during the time required for microscopic examination. The hanging drop technique is a method in which a drop of bacterial suspension, preferably in mid-logarithmic phase, is enclosed in an air-tight chamber prepared in a special depression slide having a concave depression in the center (Fig. 5.3). The technique is done by applying petroleum jelly to all sides of a cover glass. It is a ‘hanging drop’ slide because the droplet remains untouched due to the concave shape of the cover glass and it just hangs from the cover glass.

Many bacteria show no motion and are termed non-motile. However, in an aqueous environment, these same bacteria appear to be moving erratically. This erratic movement is due to Brownian movement. Brownian movement results from the random motion of the water molecules bombarding the bacteria and causing them to move. True motility (self-propulsion) has been recognized in other bacteria and involves several different mechanisms. Bacteria that possess flagella exhibit flagellar motion. Helical-shaped spirochetes have axial fibrils (modified flagella that wrap around the bacterium) that form axial filaments. These spirochetes move in a corkscrew and bending-type motion. Other bacteria simply slide over moist surfaces in a form of gliding motion. The above types of motility or non-motility can be observed over a long period in a hanging drop slide (Fig. 5.3). Hanging drop slides are also useful in observing the general shape of living bacteria and the arrangement of bacterial cells when they associate together. A ring of Vaseline around the edge of the coverslip keeps the slide from drying out.

Fig. 5.3 Concave slide used in hanging drop method

Procedure for Hanging Drop Method (Fig. 5.4)

• With a toothpick, spread a small ring of Vaseline around the concavity of a depression slide. Do not use too much Vaseline.

• After thoroughly mixing one of the cultures, use the inoculating loop to aseptically place a small drop of one of the bacterial suspensions in the center of a coverslip.

• Lower the depression slide, with the concavity facing down, onto the coverslip so that the drop protrudes into the center of the concavity of the slide. Press gently to form a seal.

• Turn the hanging drop slide over and place on the stage of the microscope so that the drop is over the light hole.

• Examine the drop by first locating its edge under low power and focusing on the drop. Switch to the high-dry objective and then, using immersion oil, to the 90 to 100× objective. In order to see the bacteria clearly, close the diaphragm as much as possible for increased contrast. Note bacterial shape, size, arrangement, and motility. Be careful to distinguish between motility and Brownian movement.

• Discard the coverslip and any contaminated slides in a container with disinfectant solution.

Fig. 5.4 Hanging drop method

5.3 Dry and Fix Method

Microorganisms, particularly bacteria, being too small need their permanent preparation be made by drying and fixing them on clean slide with or without staining. For preparing a dry mount, a drop of distilled water with a small amount or culture is spread as a thin smear on a clean slide. The smear is allowed to dry and it is then 'fixed' by passing it through a flame two to three times with the smeared slide away from the flame. If desired, this dried and fixed amount may be stained and the preparation dried again for observation under the microscope.