SIMPLE STAINING

SIMPLE STAINING 

AIM

            To determine the shape and arrangement of bacteria by using single stain.

PRINCIPLE

            Simple staining is the process of preliminary staining of bacteria used for the determination of shape and arrangement of bacteria. The process of visualizing the morphology of microbial population by using simple stain is called Simple staining. The Simple staining can be used as a quick and easy way to determine cell shape, size and arrangements of bacteria. In this staining, bacterial smear is stained with a single stain. Basic stains are usually preferred to determine the single and arrangement of bacteria, since bacteria nucleic acid. Cell wall compounds carry negative charge that strongly attracts cationic Chromogen and imparts some color to the bacteria. Most commonly used basic dyes are Methylene blue, Crystal violet or Safranine.

MATERIALS REQUIRED

i.       Bacterial culture

ii.     Microscopic slide

iii.   Inoculation loop

iv.   Methylene blue, Crystal violet or Safranine

v.     Bunsen Burner

vi.   Microscope

PROCEDURE

a)     Clean and dry the microscopic slide thoroughly.

b)     Prepare the bacterial smear on a clean slide.

c)     Dry the smear with air and fix with heat prior to staining.

d)     Flood the smear with Crystal violet (20 – 60 seconds) or Methylene blue (1 – 2 minutes) or Safranine or (1 – 2 minutes).

e)     Wash the stained smear with distilled water to remove excess stain.

f)      Dry the smear using a blotting paper (do not wipe to slide).

g)     Examine the smear under 100 X oil immersion field.

h)    Identify the various shapes and arrangements of bacteria.

Figure – 1: Steps involved in Simple staining (Source: quizlwt.com)

 OBSERVATION AND RESULTS

a)     Cocci – Spherical shape

b)     Spirilla – Spiral shaped

c)     Bacilli -  Rod shape

            The colour of the bacteria will be based on the selection of Stains. 

ü  Crystal violet (Violet colour)

ü  Methylene blue (Blue colour)

ü  Safranine or (Red or Pink colour)

                      A                                              B

A – Cocci stained with Crystal violet

B – Bacilli stained with Safranine