Question

my unknown is B.megaterium.

If im going to take a sample from my Indole test and Litmus milk test(litmus acid,coagulation test) which is both negative and my urease,catalase test which is positive result. What could be the Gram or Acid fast result if im going to take a loopful from each tube(Litmus milk, Indole test, urease and catalase) and perform gram staining or acid fast staining?

Answer 1

Acid fast Staining:

• Main principle of Acid-fast staining is to distinguish between acid fast and non-acid-fast bacteria.
• Ziehl-Neelsen staining is the classic method of Acid-fast staining.
• Due to acid fastness, the acid-fast bacteria resist decolorization by acids or acid alcohols.
• During acid fast staining primary stain used are acid fast stains which are phenol-based stain and can stain acid-fast bacteria cell wall. Example Carbolfuchsin.
• However, water-based stains like methylene blue cannot be used.
• The non-acid-fast bacterial call also take up the primary stain carbolfuchsin.
• The cells are then washed with acid-alcohol during decolorization step.
• The acid-fast cell prevents entry of acid-alcohol, due to hydrophobic mycolic acid and do not decolorize ad retain the primary stain.
• The non-acid-fast bacteria are decolorized.
• The counterstain enters the non-acid fast call wall and stains them. Like counter stain Methylene blue stains non-acid fast bacteria.
• Thus, acid-fast bacteria appear pink due to carbolfuchsin and non-acid-fast bacteria stains blue.

Bacillus megaterium does not have mycolic acid in the cell wall.

Thus, they will take up counter stain and will appear blue/purple.

B. megaterium will be acid fast negative.

Gram Staining:

• Gram staining is one of the principal techniques to observe and differentiate specific bacteria.
• It was devised Hans Christian Gram in 1884.

• Certain bacteria designated as Gram positive bacteria, have thick peptidoglycan layer.
• While, Gram negative bacteria have thin peptidoglycan layer surrounded by lipopolysaccharide layer.
• Gram staining procedure uses stains to distinguish Gram positive bacteria from Gram negative.
• The thicker peptidoglycan layer forms complex with primary stain crystal violet and mordant Gram’s Iodine, along with heat fixing. This stain is retained even during the decolorization phase with alcohol.
• While cells with thinner peptidoglycan layer cannot retain primary stain, after decolorization and take up the secondary stain Safranin.

B. megaterium has thick peptidiglycal layer.

It is Gram positive bacteria.

Thus, it will appear violet with crystal violet stain.

B. megaterium will represent Gram positive bacilli