Lab 3 – The Gram Stain Ninoska Garcia-Ortiz 063 053 2 Lab date: Tues, Sept 4, 2007 Objective

: To recognize the advantages of a differential gram stain. Learn how to do gram staining and understand the principle behind it. Theory Summary: • Named after Dr. Christian Gram • Widely used differential stain in microbiology • Usually involves 2 different dyes, allowing to determine if bacterium belongs to group A or B • Gram positive bacteria stain violet (purple • Gram negative bacteria stain pink • This is due to differences between their cell walls • Gram stain requires 4 different reagents: o Crystal violet (Gentian violet) is the primary dye. Purpose is to impart color to all organisms in smear. o Gram’s iodine (Lugol’s iodine) enhances binding between a dye and substrate by forming complex with dye which is relatively insoluble in alcohol. Said to act as a mordant o 95% ethanol (ethyl alcohol) a decolorizer removing primary stain from cells. Gram + bacteria lose primary stain at slower rate than Gram – bacteria. If done properly, Gram + will retain crystal violet, and stain violet. Gram – bacteria will lose all crystal violet, and be colorless o Safranin, a red dye, applied as a counterstain. Only colorless Gram – bacteria will pick it up, appearing pink under the microscope. Staining procedure pitfalls include: o Decolorizing to long; resulting in both Gram + and – bacteria losing primary dye, making all cell walls appear Gram -. o Decolorizing to briefly; both cell types will retain primary dye, appearing Gram +. Age of culture also affects results of Gram stain. Gram stain only valid with cultures 24 hours old, or less.

Questions 2A differential stain uses more than one dye and stains different kinds of organisms different colors. A cell stain uses only a single dye that does not differentiate between different types of organisms. Crystal violet: impart color to all organisms in the smear Gram’s iodine: acts as a mordant by enhancing binding between a dye and its substrate. 95% ethanol: decolorizer. Gram + retains crystal violet, Gram – will lose it. Safranin: counterstain. Colorless Gram – will pick it up, making it appear violet under microscope.


Conclusion In the medical field, the use of Gram stains can be very advantageous. The identification of a bacteria as being Gram positive or Gram negative would enable the appropriate antibiotic to be administered. Gram positive bacteria are particularly susceptible to Penicillin and Lysozyme. The expected results of the experiment would be for the Staphylococcus aureus smear to turn purple, indicating it to be Gram positive, and for the Escherichia coli smear to turn pink, meaning it is Gram negative.