Stain
Exercise 3.9 Endospore Stain (Differential Stain)
Eula Lewis
Bio 2921
February 27, 2013
Zere Ezaz, Ph. D.
Objective: 1. To learn to endospore stain 1. To distinguish endospores, free spore, and vegetative cells
Theory:
A dormant form of bacterium that allows it to survive poor environmental conditions are called endospores. Their tough outer covering is made of keratin and is also the reason they are resistant to heat and chemicals. The strength of keratin also makes it resist staining. Due to this drastic measures like steaming the bacterial emulsion is used to force primary stains such as malachite green into endospores in the Schrieffer Fulton method. This stain can be left on the slide for 15 minutes or more to stain the spore. Vegetative cells and spore mother cells can be decolorized with water because malachite green is water -soluble and has a low affinity. After that they are counter stained with safranin. Spores can be differentiated by location and shape. The can be located in the central area of the cell which is the middle. They can be located in the terminal region of the cell which is the end. They can also be located between the central and terminal area which is called the sub-terminal. Also the spore can be either spherical or elliptical shaped. Prediction: We will create a smear on a slide using B. cereus culture #1 or #2. The smear will be heat fixed. Then the primary stain malachite green will be added to the slide. Cover the slide with a strip of bibulous paper. Place the slide over steam for ten minutes. Keep slide moist by continuing to apply malachite to the slide. Remove the paper and gently rinse the slide with water. The spore cells should remain green but malachite should wash away from the vegetative and spore mother cells. Then I will counter-stain with safranin. The vegetative cells will appear red. I will apply immersion oil and view the slide using the microscope. I should view free spore cells appearing
Eula Lewis
Bio 2921
February 27, 2013
Zere Ezaz, Ph. D.
Objective: 1. To learn to endospore stain 1. To distinguish endospores, free spore, and vegetative cells
Theory:
A dormant form of bacterium that allows it to survive poor environmental conditions are called endospores. Their tough outer covering is made of keratin and is also the reason they are resistant to heat and chemicals. The strength of keratin also makes it resist staining. Due to this drastic measures like steaming the bacterial emulsion is used to force primary stains such as malachite green into endospores in the Schrieffer Fulton method. This stain can be left on the slide for 15 minutes or more to stain the spore. Vegetative cells and spore mother cells can be decolorized with water because malachite green is water -soluble and has a low affinity. After that they are counter stained with safranin. Spores can be differentiated by location and shape. The can be located in the central area of the cell which is the middle. They can be located in the terminal region of the cell which is the end. They can also be located between the central and terminal area which is called the sub-terminal. Also the spore can be either spherical or elliptical shaped. Prediction: We will create a smear on a slide using B. cereus culture #1 or #2. The smear will be heat fixed. Then the primary stain malachite green will be added to the slide. Cover the slide with a strip of bibulous paper. Place the slide over steam for ten minutes. Keep slide moist by continuing to apply malachite to the slide. Remove the paper and gently rinse the slide with water. The spore cells should remain green but malachite should wash away from the vegetative and spore mother cells. Then I will counter-stain with safranin. The vegetative cells will appear red. I will apply immersion oil and view the slide using the microscope. I should view free spore cells appearing