Gram Staining

I.  History

a.  Hans Christian Gram, Danish, late 1800s, early 1900s

A.  Published staining method in 1884

B.  “ … it is very defective and imperfect, but I hope .. it will turn out to be useful”

II.  Staining Basics

a.  Why Stain? – Enhance contrast, reveal properties

b.  To select or not to select? – Gram Staining is selective

c.  Positive or negative? – Gram is positive staining; Uses basic dye which is attracted to slightly negative charge of bacterium

III.  Gram Stain Theory

a.  Bacteria have a cell wall composed of peptidoglycan

A.  Gram Positive have thick, highly cross-linked peptidoglycan

B.  Gram Negatives have thin, less organized peptidoglycan

  1. They also have an outer membrane for further protection

b.  A solution of a basic dye, Crystal Violet, is first applied

A.  The positively charged CV attracted to negatively charged bacteria

c.  A mordant, Gram’s Iodine, is applied which fixes the CV between the peptidoglycan layers

A.  It’s thought that the Iodine cause the CV to precipitate and become trapped within the peptidoglycan cross-links

B.  Gram’s Iodine is a mixture of Iodine and Iodide

d.  A decolorizer, EtOH is applied

A.  This dissolves and washes away the CV … if it can reach it

B.  EtOH easily permeablizes the OM and washes out CV from Gram Negs

C.  But EtOH can’t reach all of the CV in Gram Pos and the crystals are retained in the cell wall

e.  A counter stain, safranin, is applied

A.  Another basic stain, loves Nucleic Acids, stains red

f.  Purple Color of CV dominates is still around so Gram + = purple

g.  With no CV around the red safranin shows through so Gram - = pinkish

IV.  Procedures – Remember Goldilocks?

a.  Smear

A.  Clean the slide

B.  Sterilize your loop (and let it cool)

C.  Take a small amount of liquid

  1. Too little = hard to find bacteria
  2. Too much = won’t stain properly

D.  Let it air dry … completely, be patient

b.  Fix

A.  Quickly and briefly heat – do not bake

  1. Too little = won’t fix to slide and will wash away
  2. Too much = morphology is ruined and won’t stain properly

c.  Stain

A.  How to mess up a Gram stain

  1. Mess up the smear to start with
  2. Apply stain to the wrong side of the slide
  3. Stain or rinse the smear incompletely
  4. Allow stain to dry up before rinsing
  5. Forget to decolorize or decolorize incompletely
  6. Too little = false +
  7. Too much = false negative
  8. Rub off the material when you blot it

d.  Look

A.  Use lower mags to find and focus bacteria. When found and focused THEN use oil immersion

B.  Remember:

  1. DO use drop of oil on illuminated portion of the slide
  2. DO NOT use coarse focus knob with oil immersion
  3. DO wipe the oil off the objective lens each time