Diagnosis of Mycobacterum tuberculosis

         I  Direct method

A ..  Microscopical examination

      (a  A portion of mucopurulent material will be taken on a grease-free glass slide and with the help of platinium wire with loop , the smear is prepared and it is stained b e ziehl-neelsen technique .

(b)    Antiformin method ( concentration technique ) 

               Composition of Antiformin –

i)                    Liquid sodium chlorinate – 1 part

ii)                  15% sodium hydroxide -  1 part

iii)                Distilled water – 8 part .

 Advantage of Antiformin –

i)                    It dissolves the mucous

ii)                  It kills the other group of organisms .

   Techniques …..

i)                    One part of sputum is added with three part of Antiformin  (20%) and mixed thoroughly .

ii)                  The mixture is inoculated at 37*c for 30 minutes .

iii)                Then it is centrifuged for 30 minutes . The deposit is used for smear and staining iis done by Ziehl-Neelsen technique .

iv)                Petroff’s method ( concentration technique )  …..

     Technique -----

i)                    one volume of sputum is added with 4 volume of 4% Caustic soda and mixed thoroughly .

ii)                  The mixture is kept in incubator at 37*c for 30 minutes and ashaken from time to time .

iii)                Then the mixture is centrifuged at 3000 r.p.m

iv)                The supernatant fluid will be poured off

v)                  The deposit will be treated with normal saline and neutralized with 8% hydrochloric acid or dilute aceticacid solution . Then it will be centrifuged .

vi)                The deposit is used for smear and stained by ziehl-neelsen technique .

Advantages of Petroff’;s method

i)                    Caustic soda will kill the other organism and liquefy the mucous .

ii)                  The deposit may be used for staining by ziehl neelsen technique , for culture in Lowenstein Jensen’s medium and for inoculation in guinea pig .

B…    Culture -  Lowenstein jensen’s medium and Dorset’s egg medium are used for the culture .

C..  Animal pathogenicity test – rabbit and guineapig are the animals of choice . here concentrated                                                           material ( Antiformin method or petroff method ) is inoculated to the                                                     animal of choice . The animal will die in 12 weeks ( in case of bivine                                                     type ) and 3 to 9 months ( in case of human type ) . Lungs and                                                               kidney will be affected in case of rabbit and spleen and liver will be                                                       affected in case of guinea-pig .

II  .. Indirect nmethod ----

i)                    Allergic reaction like mantoux test

ii)                  Complement fixation test

iii)                Increase in sedimentation of blood corpuscles .

iv)                Laevodeviation of Arneth index .