Question

You have been told to obtain a sputum sample and to perform microbiological staining in order to determine the identity of the pathogen causing a patient’s illness. You first perform a Gram stain, but upon microscopic analysis you visualize a mixture of pink and purple bacilli. Explain the results you have just observed and discuss what you may now do in order to identify the pathogen.

Answer 1

Bacterial identification can be primarily done on the basis of gram staining. Gram positive bacteria are Staphylococcus, Steptococcus, Bacillus, Enterococcus and Listeria which stained as purple color.

Gram negative bacteria are E.colo, proteus bacteria,Psuedomonas aeruginosa etc, it stained as pink in colour.

In the present situation the observed slide has both pink and purple colonies are observed. Hence the sample has both the bacterial infection is present. For further observation the microscopic slide is observed under high resolution (under oil immersion lens) to identify whether the present bacteria are cocci or bacilli.

In our case the colonies are bacilli hence probable gram positive bacteria are: clostridium, Listeria or Corynebacterium

probable Negative bacilli are: Escherichia coli, pseudomonas, proteus and klebsiella

now we can performe PCR to identify the pathogen, for performing PCR we first choose the sequence specific primer for each bacteria and run the reaction in thermocycler. only those bacteria which is present in sputum sample may bind to their specific primer and gives amplification.

amplification is observed by agarose gel electrophoresis, amplified bacterial DNA gives band.