Arrange the following steps of applying immersion oil in the correct sequence.

Objectives

• Prepare a smear from a bacterial specimen.
• Prepare a simple stain of the smear.
• Use the microscope to identify features (shape, arrangement, size) of the bacterium.

In order to stain the bacterial specimen for microscopy one must first prepare the smear on the slide. This basically involves three steps----transferring a liquid suspension of the bacterium on the slide, drying the smear, and then heating slightly to firmly attach the smear to the slide. Once this is done, the staining procedure begins.

Note

Do NOT make your smear suspensions too thick. The dye will not penetrate well, and there will be far too many bacterial cells to see individual shapes and arrangements. One needs to be careful about thick smears when taking the specimen from an agar medium.

RECOMMENDATION: You may find it helpful to draw a circle (wax pencil is best) on the opposite side of the slide where you will spread your smear. This will help you later in locating the smear, sometimes a problem when moving the slide back and forth looking for your bacteria. The wax pencil is better than a marker because it will not wash off easily from the glass.

MATERIALS NEEDED

• NB culture of Staphylococcus epidermidis
• NA plate of E. coli
• dye tub
• dye reagents
• clean microscope slides
• wax pencil
• bibulous blotting paper
• immersion oil

THE PROCEDURES

1. First, make sure that you have a few clean slides. We recycle our microscope slides and the smears have to be removed from the glass. You will use a cleansing powder liquid that works well to remove the smear and the dye from the glass.
   • At the sink, pick up the bottle of the slide cleansing liquid , pouring some of the thick suspension onto the slide.
   • Use your finger to spread the suspension over each side of the slide, and then wash WELL with tap water.
   • Dry the slide.
2. You will make smears using 2 different types of cultures, a broth and an agar culture.
   • Label the 2 slides with the names of your bacteria.
   • Shaking the culture first, aseptically transfer a drop of the broth culture using an inoculating loop to a clean slide.
   • From the agar plate culture, remove a small inoculum (use only a small amount of large colonies) with a loop and suspend it in a small drop of distilled water. Mix the culture well into the water drop.
3. Label 2 slides---Staph and E. coli.
4. 
   Spread the smear suspensions over the glass slide so that it forms a thin layer and it is the size of nickel or larger. NO COVER SLIP USED!
5. Let the slide air-dry—totally. This step facilitates the fixing of the smear to the slide.
6. Heat-fix the dry smear by running the slide quickly through the flame a few times. If your fingers get hot, you have heat-fixed TOO MUCH. The heat-fixing will coagulate some of the protein material and cause the suspension to adhere to the glass slide.
7. A different dye will be used for each bacterium.
   • E. coli and crystal violet
   • Staphylococcus and safrinin
8. Staining the smear
   • Place the slide on the wire mesh sitting over the dye tub.
   • Flood the smear with the dye: let sit for 1 minute.
9. Wash the slide WELL with distilled water. Blot the smear slide with your bibulous paper pad.
10. Refer to the lab exercise on MICROSCOPY for help with the microscope.
11. Focus on the sample using the 10X lens (Be SURE that you are on brightfield microscopy).
12. Focus on the smear using the 100X oil immersion lens. Be sure to read the directions in the MICROSCOPY exercise so that you know how to move to the 100X objective lens using oil.
13. Identify the various shapes and arrangements of bacteria using the fields below.

1. CLEAN YOUR SLIDES using the slide cleanser at the sinks.

QUESTIONS

1. What is the disadvantage of having a really thick smear when staining?
2. Why are basic dyes more useful than acidic dyes when staining bacteria?
3. Why heat-fix the smear?
4. What names are given to the shapes seen in the 2 bacteria used in lab?

Contributors and Attributions

• Jackie Reynolds, Professor of Biology (Richland College)