Biology 1913 Laboratory

PREPARING A SMEAR
AND SIMPLE STAINING


In an Introductory Microbiology laboratory, live, potentially pathogenic, bacteria are usually never observed with the microscope. Instead, live microorganisms must first be killed to ensure that there is no spread of a [potential] disease but more importantly the M.O. (microorganism) does not wash off during staining. Preparing a live microorganism to be viewed with the microscope is called a SMEAR. This procedure involves aseptically transferring the bacterium onto a clean slide then killing the M.O. by HEAT FIXING. Once a smear has been prepared, the M.O. must be STAINED for microscopic viewing. The purpose of making a smear and simple staining is to study the MORPHOLOGY of the microorganism. MORPHOLOGY is the study of shape and size. Other simple characteristics such as the growth pattern of a microorganism (clusters, pairs, chains, etc...) can also be studied using the simple stain technique.

HEAT FIXING is the process by which a live microorganism has been aseptically transferred (smeared) onto a clean slide, letting the smear air dry, then the slide ran through flames several times. Heat fixing not only kills the bacterium but also "fixes" (adheres) the organism to the slide so that it will not wash off during the staining process. A thin smear must be made in order for the slide preparation to be successful. As the slide crosses through the flames, the smear forms a white ash. DO NOT OVER FLAME THE SMEAR!! If the smear is flamed too much, the organism will not properly stain because the cells were destroyed.

STAINING: Bacteria, like most individual cells, are basically transparent and therefore need some type of stain in order to view them microscopically. When only one stain is applied, this is called a SIMPLE STAIN. The most common stain used for smears (and wet mounts) is Methylene Blue. Other stains such as IKI, crystal violet, and safranin may also be used. After preparing the smear, the stain is applied for a specified amount of time. In simple staining, it is important that the stain is allowed to penetrate the cell wall. Usually this is accomplished within one minute.

SMEAR AND SIMPLE STAINING TECHNIQUES

1.

Label one edge of a clean slide with the name (abbreviation) of the bacterium to be smeared and your initials.

2.

Aseptically transfer a loop of a designated bacterium from a broth culture onto the center of a clean slide and make a thin smear.

3.

Allow the smear to air dry. (Do not wave the slide to speed up the process).

4.

Hold the slide with a clothes pin and run the slide through the flame several times until a thin white ash can be seen. (Do not over heat)! If you see a burnt edge or brown within the smear, then you have over heated.

5.

Place the slide onto the staining rack.

6.

Flood the slide (making sure the smear is coated) with Methylene Blue.

7.

Wait for ONE (1) minute.

8.

Rinse the slide with distilled water (wash bottle).

9.

Place the slide between two sheets of bilbilous paper or Kimwipes and lightly pat dry. (Any excess water should be allowed to air dry. Do not rub the slide to dry; this will remove the organism.

10.

Observe the slide with the microscope first with low (10X) power; switch to the 40X and observe; slightly rotate the 40X objective lens from the specimen and apply a small drop of immersion oil; view with the 100X (oil immersion) lens.

11.

If the slide is to be discarded, place the slide into the beaker of 10% Bleach solution found on the teacher=s table.

MAKE ONE SMEAR OF STAPHYLOCOCCUS EPIDERMIDIS (STAPH. EPI) AND ONE OF ESCHERICHIA COLI (E.COLI).

Make note of the morphology of each organism.

BACTERIUM FROM PLATE OR SLANT: Follow the above steps except in step # 2, first apply a small drop of distilled water onto the center of the slide then smear the [solid] bacterium into the water. 

DO NOT GATHER TOO MUCH BACTERIUM FROM THE SLANT OR PLATE! This will make it difficult to make a thin smear. The stained specimen will therefore be too thick to properly observe the morphology.

NOTE: Do not put stains in the sink. Do all staining and washing on the staining rack and tray.