Lab Experiment number 11
To learn the Gram stain technique, the reason for the stain, and how to identify the results of the organisms stained. MATERIALS:
Bunsen burner, inoculating loop, staining tray, glass slides, bibulous paper, lens paper, oil, and microscope METHODS:
Apply Crystal Violet (Primary stain) for 1 minute.
Rinse with D-water
Apply Iodine (Mordant) for 1 minute.
Rinse with D-water.
Apply Alcohol (Decolorize) for 30 seconds.
Rinse with D-water.
Apply Safarin (Counterstain) for 1 minute.
Blot dry with bibulous paper.
E. coli, B. cereus, S. aureus & E.coli (mixture)
E.coli cell shape was bacilli (rod) with a diplobaccillus arrangement. The color was pink because it was Gram negative. B.cereus cell shape was bacilli (rod) with a diplobacillus arrangement. The color was purple because it was Gram positive. S.aureus & E.coli (mixture) cell shape was cocci (spherical) with a staphylococcus arrangement. The color was mostly purple with some noticeable pink but the mixture was Gram positive.
E. coli is Gram negative, B. cereus is Gram positive, S. aureus & E. coli mixture is Gram positive.
Question 1: What are the advantages of differential staining procedures over the simple staining technique? Answer: Simple stains are used to just give color to microbes on slides. Differential stains tell the chemical composition of organisms. Source: http://www.bmb.psu.edu/courses/micro107/notes/staining.htm
Question 2: Cite the purpose of each of the following reagents in a differential staining procedure. Answer:
a. Primary stain: Passes the color of the stain to all of the cells. b. Counterstain: Used to stain red the cells that have been decolorized (Gram – cells). c. Decolorizing agent: removes the primary stain so that the counterstain can be absorbed. d. Mordant: Increases the cells’ affinity for a stain by binding to the primary stain. Source: Microbiology – A Laboratory Manual 4th Edition/ James G. Cappuccino, Natalie Sherman/ 2008/ Pages 73 & 74 Question 3: Why is it essential that the primary stain and the counterstain be of contrasting colors? Answer: Cell types or their structures can be distinguished from one another on the basis of the stain that is retained. Source: Microbiology – A Laboratory Manual 4th Edition/ James G. Cappuccino, Natalie Sherman/ 2008/ Pages 73 Question 4: which is the most crucial step in the performance of the Gram staining procedures? Explain. Answer: Decolorization is the most crucial step of the Gram stain. Over-decolorization will result in lost of the primary stain causing Gram positive organisms to appear Gram negative. Under-decolorization will not completely remove the CV-I (crystal-violet-iodine) complex, causing Gram negative organisms to appear Gram positive. Source: Microbiology – A Laboratory Manual 4th Edition/ James G. Cappuccino, Natalie Sherman/ 2008/ Pages 74 Question 5: Because of a snowstorm, your regular laboratory session was cancelled and the Gram staining procedure was performed on cultures incubated for a longer period of time. Examination of the stained Bacillus cereus slides revealed a great deal of color variability, ranging from an intense blue to shades of pink. Account for this result. Answer: The organisms lost their ability to retain the primary stain and appear to be gram-variable. Source: Microbiology – A Laboratory Manual 4th Edition/ James G. Cappuccino, Natalie Sherman/ 2008/ Pages 74
LAB EXPERIMENT NUMBER 12
The purpose of the Acid fast stain is to identify the members of the genus Mycobacterium, which represent bacteria that are pathogenic to humans. Mycobacteria has a thick, waxy wall that makes penetration by stains extremely difficult so the acid fast stain is used because once the primary stain sets it cannot...