NEGATIVE CAPSULE STAINING

 NEGATIVE CAPSULE STAINING

AIM

            To observe the Capsule present in bacteria under dark background.

PRINCIPLE

       A Capsule is a gelatinous outer layer secreted by bacterial cell (Klebsiella pneumoniae and Streptococcus pneumoniae) and that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides.  

         Negative staining requires the use of an acidic stain such as India ink or Nigrosin. The acidic stain, with its negatively charged Chromogen, will not penetrate the cells because of the negative charge on the surface of bacteria. Therefore, the unstained cells are easily discernible against the colored background.

            Heat fixation is not required and the cells are not subjected to the distorting effects of chemicals and heat, their natural size and shape can be seen. It is possible to observe bacteria that are difficult to stain, such as some Spirilla. Because heat fixation is not done during the staining process, keep in mind that the organisms are not killed and slides should be handled with care.

           Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with India ink or Nigrosin. India ink is difficult to obtain nowadays; however, Nigrosin is easily acquired.

MATERIALS REQUIRED

i.       Glass slide

ii.     Inoculation loop

iii.   Microscopic slide

iv.   Microscope 

v.     Marker pen

vi.   Bacterial culture – Klebsiella pneumoniae/Streptococcus pneumoniae

vii. Nigrosin or Indian Ink

PROCEDURE

a)     Clean and dry the microscopic slide thoroughly.

b)     Place a small drop of Nigrosin near one end of a well-cleaned and flamed slide.

c)   Using aseptic technique, place a loopful of inoculum from the bacterial culture in the drop of Nigrosin and mix.

d)    Place a slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide.

e)     Allow the smear to dry without heating (Do not heat fix the slide).

f)     Dry the slide (air dry) and observe under Low power objectives (10 x or 40 x) or High power Oil immersion (100 x) of the Microscope.

OBSERVATION AND RESULTS

            Transparent colourless capsules were observed in the dark background under the microscope.

Figure – 1: Negative capsule staining of Bacteria (Source: pinterest.com)