Objectives:
- To present an important aspect in the study of bacteria.
- To familiarize the students with the techniques of staining.
- To introduce concept as to structure and differential characteristics of bacteria.
Methodology:
A. Preparation of bacterial smear
1. Aseptically transfer a loop of sterile seawater at the center of a clean glass slide. Take a loopful of your bacterial isolate culture, emulsify it with sterile seawater and spread out over an area. Be sure to flame sterilized the loop every time it is used.
2. Carefully dry the smear by holding the slide high over the flame to avoid steaming. Hold the slides between the fingers. Label each smear properly as to bacteria studied and stain used.
B. Application of Simple Stain
1. Apply a few drops of simple stain on the smear. Let it dry for a minute and wash off with tap water. Drain and blot dry with chamois paper. Observe demonstration of simple stains. Label slides as to stain used.
a. methylene blue
b. crystal violet
c. carbol fuchsin
2. Examine stained smears microscopically under oil immersion. Record your observation. Check the light source and alignment of the micoscope to ensure efficient illumination and better visualization of the specimen. Note color, shape and arrangement. Make illustrative drawings.
C. Application of Differential Stains.
1. Gram Stain
Prepare bacterial smears of your bacterial isolate and follow the following steps in Gram staining:
a. Stain smear with crystal violet for 1 min. Drain and blot dry to remove excess stain.
b. Cover smear with Gram iodine for 1 min. Drain and blot dry. Both Gram(+) and Gram (-) are now stained violet or purple.
c. Decolorize with ethyl alcohol for about 5-10 sec. Drain and blot dry. All G(-) are now completely decolorized and G(+) not affected.
d. Cover smear with Safranin, a stain that give contrast in color (counterstain) for 1min.
3. Examine under oil immersion objective lens. Note color, shape and arrangement. Make illustrative drawings.
D. Application of special staining procedures
1. Spore staining.
a. Prepare a smear of Bacillus subtilis
b. Flood the smear with malachite green and heat over the flame for 3 min. Keep the smear covered with the dye and don’t allow to boil
c. Wash with water
d. Apply Safranin for 30 sec then rinse with tap water.
e. Examine under the oil immersion objective and note the spores stained. Make illustrative drawings.
2. Capsule staining
a. Place a loopful of Staphylococcus aureus broth culture. Do not spread into a thin film.
b. Add a drop of nigrosin solution to the culture. Mix thoroughly and spread into a thin film.
c. Let it dry in air. Do not fix.
d. Apply safranin for 1 min then drain the stain. Do not wash the slide with water.
e. Examine under the oil immersion objectives. Bacterial ells are colored red or pink and the capsules appear as clear, unstained zones surrounding them, their outline demarcated against the black background provided by the nigrosin. Make illustrative drawings.
GUIDE QUESTIONS FOR DISCUSSION:
- List the stains use and the color imparted by each.
- Explain why loop is repeatedly flame-sterilized in the preparation of bacterial smear.
- Give the reasons for passing bacterial smear through the flame before staining.
- Give the reasons for flaming the mouth of cultured tubes after the cotton plug has been removed and just before it is reinserted.
- Why are the tubes held slantwise during manipulation?
- Comment on the use of mordant. Give examples.
Laboratory Exercise 6 Microscopic appearance of bacteria
Date Performed:
Rating
RESULTS
Simple stain
Bacteria | Stain | Color | Shape | Arrangement | Drawing |
Isolate | Methylene blue | ||||
Crystal violet | |||||
Safranin | |||||
Bacteria provided by the instructor | Methylene blue | ||||
Crystal violet | |||||
Safranin |
Gram Staining
Bacteria | Color | Shape | Arrangement | Drawing |
Isolate | ||||
Bacteria provided by the instructor |
