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

2- A 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.

3- 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.