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LAB REPORT NUMBER TWO

DATE: 3/25/2010

inal attachment
Lab Experiment number 11 
PURPOSE:
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. 
MICROORGANISMS USED:
E. coli, B. cereus, S. aureus & E.coli (mixture) 

RESULTS/DATA USED:
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. 

CONCLUSIONS
E. coli is Gram negative, B. cereus is Gram positive, S. aureus & E. coli mixture is Gram positive.

REVIEW QUESTIONS:
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
PURPOSE:
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...
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