MORPHOLOGICAL AND TINCTORIAL PROPERTIES OF BACTERIA - E. L. Zaytseva - 2015

CHAPTER IV. MICROSCOPY OF LIVING BACTERIA

The examination of microorganisms in a living state is used primarily to study bacterial Morphology and motility, as well as to identify intracellular reserve inclusions. Determining the shape and motility of Bacteria from a freshly isolated culture is performed using "crushed drop" or "hanging drop" preparations, i.e., a droplet placed on a Microscope slide and covered with a coverslip.

Methods FOR PREPARING NATIVE PREPARATIONS

I. The "crushed drop" method. To prepare a crushed drop mount, it is essential to use thoroughly degreased slides, since microbial Cells will not adhere to The surface of dusty or greasy Glass.

Materials:

1) microscope slide;

2) coverslip;

3) isotonic sodium chloride solution;

4) test tube or Petri dish with a bacterial culture grown on solid or liquid nutrient media;

5) bacteriological loop or sterile Pasteur pipette;

6) spirit lamp.

Preparation technique:

1. Using a bacteriological loop or a sterile Pasteur pipette, place a drop of broth culture in the center of a degreased microscope slide and spread it evenly over an area of 1 cm².

To prepare a smear from an Agar culture, place a drop of physiological saline on the slide and emulsify a loopful of microbes taken from the medium within it.

2. Cover with a coverslip, the edges of which have been smeared with vaseline oil, and press it gently onto the slide to avoid trapping air bubbles and to prevent the liquid from spilling past the edges of the glass. The droplet is thus "crushed" between them. Use a glass rod to press the coverslip down onto the slide.

3. Apply a drop of immersion oil onto the coverslip and examine under a x90 objective and x7 eyepiece with the condenser slightly lowered.

When working with relatively large bacteria, dry objectives (x40 or even x8) can be used, omitting the immersion oil.

The main disadvantage of the crushed drop method is the rapid drying of the preparation; therefore, it must be examined microscopically immediately after preparation.

II. The "hanging drop" method for determining bacterial motility follows the same general principle, but differs in the assembly of the chamber.

Materials:

1) depression microscope slide;

2) coverslip;

3) isotonic sodium chloride solution (NaCl);

4) test tube or Petri dish with a bacterial culture grown on solid or liquid nutrient media;

5) a bacteriological loop or sterile Pasteur pipette;

6) an alcohol burner.

Preparation technique:

1. Take a coverslip (not a microscope slide) and place a drop of the microbial suspension under study onto it.

2. Cover it with an inverted depression slide, positioned so that the drop is centered within the well. To seal the slides, apply a thin layer of petroleum jelly around the edges of the depression. Gently press the microscope slide onto the coverslip so that they adhere to each other. Then, flip the slide over so the coverslip faces upward. The droplet will now be suspended directly over the well, creating a hermetically sealed chamber that prevents the drop from drying out for a long time.

3. Perform Cell/15.html">Microscopy in the same manner as described in the previous method.

Evaluation of results: in both "crushed" and "hanging" drop preparations, non-motile microbes exhibit Brownian motion, whereas motile microorganisms traverse large distances at a uniform speed, occasionally rotating around their own axis as they cross the entire field of view.

III. Vital staining of bacteria. This method involves The Use of highly diluted dye solutions that do not exert toxic effects on the bacteria.

Materials:

1) microscope slide;

2) coverslip;

3) 0.001% methylene blue solution;

4) test tube or Petri dish with a bacterial culture grown on a solid or liquid nutrient medium;

5) bacteriological loop or sterile Pasteur pipette;

6) alcohol burner.

Preparation technique:

1. Place a drop of 0.001% methylene blue solution in the center of a microscope slide, and introduce the bacterial suspension into it.

2. Next, prepare a "crushed drop" slide and examine it under the microscope.