Microbial analysis of oral suspension.

      1.0       Objective

     To lay down a procedure for microbial analysis of oral suspension.

      2.0        Responsibility

      Microbiologist/QC-Executive.

      3.0        Accountability

      Head-QC executive

                   Microbial Limit

                   Total bacterial count:100cfu/gm/ml

                   Total bacterial count:10cfu/gm/ml

      4.0         Procedure

          Total microbial count:

         Dissolve 10 g or dilute 10 ml of the preparation being examined, unless otherwise specified, in                     

          buffered sodium chloride-peptone solution pH 7.0 or any other suitable medium shown to have no              

          antimicrobial activity under the conditions of the test and adjust the volume to 100 ml with  

          the same medium. If necessary, adjust the pH to about 7.  

       4.1   Procedure for pathogen testing of oral Suspension

4.1.1    Transfer 10 mL oral suspension sample in 100 mL soyabean casein digest medium (SCDM).

4.1.2    After incubation, observe for growth in terms of turbidity, if it is observed perform pathogen testing.

         4.2     Tests for Escherichia coli (E. coli).

4.2.1    Transfer 1 mL of 24 hour old culture] suspension (SCDM) in 5 ml MacConkey’s Broth (MB) tube containing Durham’s tube and  incubate it at 42° to 44° for 24 to 48 hours.

4.2.2    After incubation, observe for purple to yellow color change of the MacConkey’s broth   with gas production in the Durham’s tube. If so, proceed with the following steps.

4.2.3    Subculture 1 mL of broth on a plate of MacConkey’s agar (MA) at 35° to 37° for 18 to 72 hours.

4.2.4    If growth of red, non-mucoid colonies is observed after incubation, then transfer 1 mL MacConkey’s broth in 5 mL peptone water (PW) tube and incubate at 42° to 44° for 18 to 24 hours.

4.2.5    After incubation, add 0.5 mL Kovack’s reagent into the above peptone water and if red color ring

                        appears the Indole test indicates the possible presence of E.coli.

4.2.6             Perform Gram staining of the colonies observed on MA .

4.2.7             Observe for presence of Gram negative rod.

 4.2.8         If Gram negative rod is present, streak loop full colony on Levin Eosin-Methylene Blue Agar   

                   (LEMBA) plate.

       4.2.9       Incubate this LEMBA plate at 35-37° C for 18 to 72 hours.

      4.2.10      After incubation, observe the plate and  if none of the colonies exhibits a characteristic  

                        metallic  sheen under reflected light, and if none exhibits a blue-black appearance under  

                       transmitted light, the test specimen meets the requirement for the absence of E. coli.

        4.3        Test for Salmonella Spp.

 4.3.1    Transfer 1 mL of 24 hour old culture] suspension (SCDM) in 10 mL Fluid Selenite Cystin Medium (FSCM) and        Tetrathionate Broth (TB) and incubate at 42° to 44° C for 18 to 24 hours.

4.3.2      Subculture on at least two different agar media chosen from agar media, Bismuth Sulphite Agar (BSA),                Deoxycholate Citrate Agar (DCA) and Brilliant Green Agar (BGA). Incubate at 35° to 37° C for 18 to

             72 hours.

4.3.3      The probable presence of salmonella is indicated by the growth of cultures having the  following  

              appearance:

                 BSA: well-developed, colour less colonies.

                 DCA: well-developed, red colonies, with or without black centers.

    BGA: small, transparent, colourless or pink opaque-white colonies often surrounded by a pink or

    red zone.

4.3.4     Perform Gram staining according to SOP  if the above characteristic colonies are observed.

4.3.5     If Gram negative rod is present, streak loop full culture from suspected colonies on Triple Sugar Iron (TSI) agar slant in tubes, using surface and deep  inoculation (stabbing) and incubate at 41° to 43° C for 18 to 24 hours.

4.3.6    The presence of salmonella spp. is confirmed if there is a change of color from red to yellow and usually a formation of gas, with or without production of hydrogen sulphide in the agar in the deep inoculation but not on the surface culture.

          4.4      Test for Pseudomonas aeroginosa

   4.4.1     Strick loop full of 24 hour old culture suspension (SCDM) on a plate of Cetrimide Agar (CA).

         4.4.2      Incubate this plate at 35 - 37° C for 18 - 72 hours.

  4.4.3     Perform Gram staining of the colonies observed on CA.

  4.4.4      Observe for presence of Gram negative rod.

  4.4.5     If Gram negative rod is present, streak representative suspect colonies from the CA plate on the surface   

               of  Pseudomonas fluorescin agar(PFA) medium for detection of fluorescin and Pseudomonas pyocyanin  

               agar (PPA)for detection of pyocyanin.

        4.4.6      Incubate these plates at 35° - 37° C for 48 to 72 hours.

  4.4.7     Examine the streaked surface of the both agar media under UV light, and interpret the results with  

               reference to the following table.

Selective Media	Characteristics	Fluorescence in UV light	Oxidase Test	Gram Staining
Cetrimide Agar	Generally Greenish	Greenish	Positive	Gram Negative rods
Pseudomonas Agar for detection of Fluorescin	Generally Colourless to yellowish	Yellowish	Positive	Gram Negative rods
Pseudomonas Agar for detection payocynin	Generally Greenish	Blue	Positive	Gram Negative rods

4.4.8     Oxidase disc test:

4.4.9    Transfer the colony from the PFA medium or PPA medium to disc that previously impregnated with N, N- dimethyl-p-phenylenediamine dihydrochloride.

4.4.10   If the disc colour is changed (from white to purple) within five to ten seconds it confirms the presence of

                 Pseudomonas aeroginosa.

        4.5       Test for Staphylococcus aureus:

4.5.1     Strick a loop full of 24 hour old culture suspension (SCDM) on a plate of Mannitol Salt Agar (MSA) (VPA) and Agar.

4.5.2       Incubate the plate at 35° to 37° C for 48 to 72 hrs.

4.5.3     After incubation, examine the streaked plates and interpret the results with reference to the following   table.

Selective Media	Characteristics	Gram Staining
VJA	Black colony surrounded by yellow zone	Gram positive cocci in cluster
BPA	Black, shiny colony surrounded by clear zone of 2 to 5 mm.	Gram positive cocci in cluster
MSA	Yellow colony surrounded by yellow zone	Gram positive cocci in cluster

       4.5.4     Perform Gram staining of the suspect colonies observed.

4.5.5    Observe for presence of Gram positive cocci in cluster.

4.5.6     If Gram positive cocci in cluster are present, perform coagulase test.

4.5.7     Take sterile test tubes each containing 0.5mL of mammalian, preferably rabbit or horse, plasma with or               without suitable additives.

4.5.8      Transfer representative suspect colonies from the surface of the VJA or MSA or BPA plate to the above               individual tubes and incubate in a water bath at 35° to 37° C.

4.5.9     Examine the tubes after 3 hours and at suitable intervals up to 24 hours. Compare the negative and                 positive controls.

4.5.10    If coagulation is observed, the specimen confirms the presence of Staphylococcus aureus.