Examination of Unstained Bacteria

Bacteria can be best examined and studied under the microscope. It may be desirable to examine unstained bacteria to determine their biologic grouping, motility, and reaction and chemicals or specific sera. These properties may be determined in a hanging drop preparation or in a wet mount. A few species of bacteria that cannot be stained by the methods to be discussed are often examined by darkfield illumination.

Hanging drop preparation

To examine bacteria using the microscope in a hanging drop method, one must use an inoculating loop for transferring the material to be examined to, a cover glass to fit over and a hanging drop slide.

The inoculating loop is made by a piece of fine wire about 3 inches in length. One end is fastened in a handle and the other end is fashioned into a loop about ahs inch in diameter. Platinum, Nichrome V, or tungsten alloy are used for making inoculating loops because repeatedly heating these metals in a flame to sterilize them does not at the same time destroy them, and the wire loop cools quickly after being heated.

A hanging drop slide on the other hand is a thick glass slide with a circular concavity or depression as its center. A cover glass or cover slip is a piece of very thin glass about 7/8 inch square.

To make the preparation, the first thing to do is to spread a small amount of petroleum jelly around the concavity of the slide. If the specimen to be examined is a culture growing on a solid medium or material such as thick pus, take up a loopful of specimen with the wire loop and mix thoroughly with a drop of sterile isotonic saline solution placed in the center of the cover glass. If bacteria growing in a liquid medium are to be examined, transfer a drop of the fluid to the cover glass by means of the wire loop. Place the hanging drop slide over the cover glass in such a way that the center of the depression lies over the drop. The petroleum jelly seals the cover glass to the slide, holds it in place, and prevents evaporation. Invert the slide now so that the drop to be examined hangs from the bottom of the cover glass but does not touch the surface of the concavity at any point. The preparation is ready for examination under a microscope. Examine with the 4 mm. high dry lens, and reduce the amount of light passing through it by partly closing the diaphragm of the substage condenser of the microscope. Examine all parts of the drop, but the best areas for microscopic study are usually near the edges where cells in a single layer are more evenly dispersed in the fluid medium. When hanging drop preparations are observed, brownian motion and flowing of organisms in currents in the microscope should not be mistaken for true motility. Care must be taken when one is viewing a hanging drop with the microscope’s oil-immersion objective that the contamination from the specimen is not spread and that the microscope objective is not soiled either from the specimen or from the ring of petroleum jelly.

Another thing to remember is that the inoculating loop must be sterilized immediately before and after each transfer of material containing bacteria for examination under a microscope. Since hanging drop preparations contain living bacteria, discard the slide and cover glass into a suitable container of disinfectant after the microscope examination is finished.

Wet Mount

Another method in used in the examination of unstained bacteria with the use of the microscope is the wet mount method. It is similar to the hanging drop preparation except that an ordinary microslide is used instead of the thick hanging drop slide with its central depression, and the fluid specimen spreads to fill the narrow space between cover glass and microslide. Many of the applications are the same.

Darkfield Illumination

To examine certain delicate bacteria that are invisible in the living state in the light microscope, dark-field illumination is the best method to use. Dark field illumination is ideal in the study of delicate bacteria that cannot be stained by standard methods or bacteria that are so distorted by staining as to lose their identifying characteristics. Its greatest usefulness is in the demonstration of Treponema pallidum in chancres and other syphilitic lesions, but it is of value in the examination of many other organisms as well.

The suitably prepared specimen to be examined under the microscope is placed on a microslide and covered with a cover slip. Sealing the cover glass to the slide with a ring of melted paraffin prevents the cover glass from slipping and accidental infection of the fingers. Dark-field illumination depends on the use of a substage condenser so constructed that the light rays do not pass directly through the object being examined, as is the case with an ordinary condenser, but strike it from the sides at almost a right angle to the objective of the microscope. The microscopic field becomes a dark background against which bacteria or other particles appear as bright silvery objects. A similar effect is seen when a beam of light enters a darkened room and renders visible particles of dust that cannot be seen in a better lighted room.