{"id":741,"date":"2025-06-14T07:34:35","date_gmt":"2025-06-14T07:34:35","guid":{"rendered":"https:\/\/pharmacoguide.com\/?p=741"},"modified":"2025-06-14T07:34:35","modified_gmt":"2025-06-14T07:34:35","slug":"sop-title-serial-dilution-preparation-of-microbial-cultures","status":"publish","type":"post","link":"https:\/\/pharmacoguide.com\/?p=741","title":{"rendered":"SOP Title: Serial Dilution Preparation of Microbial Cultures"},"content":{"rendered":"

SOP Title:<\/u><\/strong> Serial Dilution Preparation of\u00a0 Microbial\u00a0 Cultures<\/u><\/strong><\/p>\n

    \n
  1. Objective:<\/strong><\/li>\n<\/ol>\n

    To lay down the Procedure for Serial Microbial Cultures Dilution.<\/p>\n

     <\/p>\n

      \n
    1. Scope<\/strong><\/li>\n<\/ol>\n

      This procedure is applicable for Serial Microbial Cultures Dilution in the Microbiology Section of Quality Control Department.<\/p>\n

        \n
      1. Responsibility:<\/strong><\/li>\n<\/ol>\n

        3.1 \u00a0\u00a0\u00a0\u00a0Quality Control: To prepare and review the SOP. To follow the procedures for Serial Dilution Preparation of Microbial Cultures as per this SOP.<\/p>\n

          \n
        • \u00a0Quality Assurance Department: To review and approve the SOP and Annexure.<\/li>\n<\/ul>\n

           <\/p>\n

            \n
          1. Accountability:<\/strong><\/li>\n<\/ol>\n

            Head Quality Control Department, Head Quality Assurance Department<\/p>\n

             <\/p>\n

              \n
            1. Procedure:<\/strong><\/li>\n<\/ol>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
              \u00a0<\/strong><\/td>\n\u00a0<\/strong><\/td>\n\u00a0<\/strong><\/td>\n<\/tr>\n<\/thead>\n
              \u00a0\u00a0 \u00a05.1<\/strong><\/td>\nSafety Precautions:<\/strong><\/td>\n<\/tr>\n
              5.1.1<\/td>\nWear personal protective equipments such as protective hand gloves, nose masks and over gown during operation.<\/td>\n\u00a0<\/strong><\/td>\n<\/tr>\n
              5.1.2<\/td>\nMicrobiologist should disinfect the hand gloves with 70% v\/v IPA before carrying out operation.<\/td>\n <\/td>\n<\/tr>\n
              \u00a0\u00a0 \u00a05.2<\/strong><\/td>\nProcedure for Serial Dilution: <\/strong><\/td>\n<\/tr>\n
              5.2.1<\/td>\nRemove the slants of the Working Cultures from the refrigerator and allow to stand for at least 2-3 hrs to attain room temperature.<\/td>\n <\/td>\n<\/tr>\n
              5.2.2<\/td>\nRefer Procedure for Media Preparation and Sterilisation as per SOP for Media preparation.<\/td>\n <\/td>\n<\/tr>\n
              5.2.3<\/td>\nInoculate a loopful of the bacterial cultures from working culture slants in test tubes containing 10 ml of Soyabean Casein Digest Medium and fungal cultures in Saborauds Dextrose Broth. Both SCDM and SDB are considered as stock cultures for dilution (100<\/sup>). Refer Annexure for List of cultures. Refer SOP for maintainance of cultures.<\/td>\n <\/td>\n<\/tr>\n
              5.2.4<\/td>\nIncubate the bacterial cultures at 30-35\u00b0C for 18-24 hours, fungal cultures at 20-25\u00b0C, Candida albicans for 48hrs and Aspergillus brasilliensis for 72 to 120 hours or until good sporulation.<\/td>\n <\/td>\n<\/tr>\n
              5.2.5<\/td>\nUse Buffered Sodium Chloride Peptone Solution to make the culture dilutions.<\/td>\n <\/td>\n<\/tr>\n
              5.2.6<\/td>\n\u00a00.05% polysorbate 80 may be added to the buffered solution to suspend spores of Aspergillus brasilliensis<\/td>\n <\/td>\n<\/tr>\n
              5.2.7<\/td>\nSwitch \u2018ON\u2019 the LAF and proceed for operation.<\/td>\n <\/td>\n<\/tr>\n
              \u00a0 \u00a0\u00a05.3<\/strong><\/td>\nA)\u00a0 Small Volume Serial Dilutions:<\/strong><\/td>\n<\/tr>\n
              5.3.1<\/td>\nLabel four screw-capped tubes as 1:<\/strong>100, 1:<\/strong>10,000, 1:<\/strong>1,00,000 and 1:1,000,000.<\/td>\n <\/td>\n<\/tr>\n
              5.3.2<\/td>\nIn scientific notation this would be 10–2<\/strong><\/sup>, 10–4<\/strong><\/sup>, 10–5<\/strong> <\/sup>and 10–6<\/strong><\/sup>. For details, refer Annexure.<\/td>\n <\/td>\n<\/tr>\n
              5.3.3<\/td>\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/strong>Using a sterile 10 ml pipette, add 9.9 ml of sterile BSCP aliquot into first, second and fourth tubes and add 9.0 ml in third tube.<\/td>\n <\/td>\n<\/tr>\n
              5.3.4<\/td>\nUsing a 100\u03bcL micropipette and sterile tip, transfer 0.1 ml of broth culture into the 10-2<\/sup> tube and Cap the tube. Glass pipette of 0.1 ml can also be used.<\/td>\n <\/td>\n<\/tr>\n
              5.3.5<\/td>\n\u00a0Mix it for a few seconds vigorously flick the tube to adequately disperse the bacteria evenly throughout the tube and break up bacterial clumps.<\/p>\n

              \u00a0Note: Do not<\/strong> shake the test tube.<\/strong><\/td>\n

              		 <\/td>\n<\/tr>\n
              5.3.6<\/td>\nWith new sterile tip, transfer 0.1ml from first tube (1:100 or 10-2<\/sup>) and add it to the tube labeled as 1:10,000 or 10-4.<\/sup>\u00a0 Repeat the tube mixing procedure.<\/td>\n <\/td>\n<\/tr>\n
              5.3.7<\/td>\nWith new sterile tip, transfer 1.0 ml broth culture from the second tube (1:10,000 or 10-4<\/sup>) and add it to third tube labeled as 1:100,000 or 10-5<\/sup><\/p>\n

              Repeat the tube mixing procedure.<\/td>\n

              		 <\/td>\n<\/tr>\n
              5.3.8<\/td>\nWith new sterile tip, transfer 0.1 ml from the second tube (1:10,000 or 10-4<\/sup>) and add it to fourth tube labeled 1:1000,000 or 10-6.<\/sup> Repeat the tube mixing procedure.<\/td>\n <\/td>\n<\/tr>\n
              5.3.9<\/td>\nPlate out 0.1 ml and 1.0 ml of each dilution on sterile Petri plates in duplicate.<\/p>\n

              Also plate out stock cultures (100<\/sup>) in duplicates.<\/td>\n

              		 <\/td>\n<\/tr>\n
              5.3.10<\/td>\nPour about 20 ml of\u00a0\u00a0 Soyabean Casein Digest Agar cooled to 45\u00b0C in to petri plates for bacterial culture and Saborauds Dextrose Agar with or without antibiotic for fungal cultures.<\/td>\n <\/td>\n<\/tr>\n
              5.3.11<\/td>\nRotate clockwise and anticlockwise for uniform mixing. Repeat this procedure for all the cultures.<\/td>\n <\/td>\n<\/tr>\n
              5.3.12<\/td>\n\u00a0 \u00a0 \u00a0 \u00a0Incubate the plates at 30-35\u00b0C for 48 hrs except plate containing dilutions of Candida albicans which is incubated at 20-25\u00b0C for 48 hours and Aspergillus Brasilliensis for 72 to 120 hours.<\/td>\n <\/td>\n<\/tr>\n
              5.3.13<\/td>\nAfter completion of incubation period observe the plates and note down the colonies and select the dilutions which show colonies NMT 100 CFU(approximately 10\u2013100 CFU). If the dilutions are showing more than 100 CFU then further dilution to be done.<\/td>\n <\/td>\n<\/tr>\n
              5.3.14<\/td>\nRecord the results of Serial Culture Dilution in Annexure.<\/td>\n <\/td>\n<\/tr>\n
              \u00a0 \u00a0\u00a05.4<\/strong><\/td>\nB) Large Volume Serial Dilutions:<\/strong><\/td>\n<\/tr>\n
              5.4.1<\/td>\nSelect four sterile bottles and labeled as 1:100, 1:10,000,100,000 and 1:1,000,000.<\/td>\n <\/td>\n<\/tr>\n
              5.4.2<\/td>\nIn scientific notation this would be 10-2<\/sup>, 10-4,<\/sup> 10-5<\/sup> and 10-6<\/sup>. For details refer Annexure No. 01.<\/td>\n <\/td>\n<\/tr>\n
              5.4.3<\/td>\nAdd measure volume of sterile buffer solution in to the bottle as follows.<\/p>\n

              Labeled Bottle as. 1:100(10-2<\/sup>), 1:10,000 (10-4<\/sup>) and 1:1,000,000.(10-6<\/sup>)\u00a0 add 99 mL and for 100,000 (10-5<\/sup>) add 49 ml sterile buffer solution respectively.<\/td>\n

              		 <\/td>\n<\/tr>\n
              5.4.4<\/td>\nUsing a sterile 1mL pipette, transfer 1mL of broth culture into the 10-2<\/sup> bottle Repeat the tube mixing procedure as per 5.3.5.<\/td>\n <\/td>\n<\/tr>\n
              5.4.5<\/td>\nUsing a new sterile 1ml pipette, transfer 1ml from the (1:100 or 10-2<\/sup>) and add it to the bottle labeled 1:10,000 (10-4<\/sup>). Repeat the bottle shaking procedure.<\/td>\n <\/td>\n<\/tr>\n
              5.4.6<\/td>\n\u00a0 \u00a0 \u00a0 \u00a0Using a new sterile 1 ml pipette, transfer 1 ml from the 1:10,000 (10-4<\/sup>) bottle and add it to the 1:1,00,000 (10-5<\/sup>) bottle. Repeat the shaking procedure.<\/td>\n <\/td>\n<\/tr>\n
              5.4.7<\/td>\n\u00a0 \u00a0 \u00a0 \u00a0 Using a new sterile 1 ml pipette, transfer 0.1 ml from the 1:10,000 (10-4<\/sup>) bottle and add it to the 1:1,000,000 (10-6<\/sup>) bottle. Repeat the shaking procedure.<\/td>\n <\/td>\n<\/tr>\n
              5.4.8<\/td>\n\u00a0 \u00a0 \u00a0 \u00a0 Follow the procedure as per 5.3.9 to 5.3.15.<\/td>\n <\/td>\n<\/tr>\n
              5.4.9<\/td>\n\u00a0 \u00a0 \u00a0After completion of incubation and result recording, the petriplates and test tubes shall be decontaminated as per define SOP.<\/td>\n <\/td>\n<\/tr>\n
              5.4.10<\/td>\n\u00a0 \u00a0 \u00a0 \u00a0 Store the test tubes or bottles containing 100 CFU culture at 2-8\u00b0C in refrigerator for its further use. Discard the rest of the dilution tubes.<\/td>\n <\/td>\n<\/tr>\n
               <\/td>\n <\/td>\n<\/tr>\n
              5.4.11<\/td>\n\u00a0 \u00a0 \u00a0 \u00a0 If the Serial dilution results are not proper, than the microbiologist should investigate the cause. The Serial Dilution has to be performed again to get count within range.<\/td>\n <\/td>\n<\/tr>\n
              5.5<\/strong><\/td>\nSerial Dilutions Preparations<\/strong><\/td>\n<\/tr>\n
              5.5.1<\/td>\n\u00a0 \u00a0 \u00a0 \u00a0Frequency<\/strong> : Monthly<\/td>\n <\/td>\n<\/tr>\n
              5.5.2<\/td>\n\u00a0 \u00a0 \u00a0 \u00a0Serial dilutions to be performed for all micro organisms.<\/td>\n <\/td>\n<\/tr>\n
              <\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
                \n
              1. Definitions <\/strong>\/ Abbreviations: <\/strong><\/li>\n<\/ol>\n
                  \n
                • Definitions:<\/strong><\/li>\n
                • Abbreviations:<\/strong><\/li>\n<\/ul>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
                  Abbreviation<\/strong><\/td>\nExpansion<\/strong><\/td>\n<\/tr>\n
                  LAF<\/td>\nLaminar Air Flow<\/td>\n<\/tr>\n
                  \u00b0C<\/td>\nDegree centigrade<\/td>\n<\/tr>\n
                  ml<\/td>\nmililiter<\/td>\n<\/tr>\n
                  \u00b5l<\/td>\nmicrolitre<\/td>\n<\/tr>\n
                  %<\/td>\npercentage<\/td>\n<\/tr>\n
                  v\/v<\/td>\nVolume by volume<\/td>\n<\/tr>\n
                  SCDA<\/td>\nSoyabean Casein Digest Agar<\/td>\n<\/tr>\n
                  SDA<\/td>\nSaborauds Dextrose Agar<\/td>\n<\/tr>\n
                  SDB<\/td>\nSaborauds Dextrose Broth<\/td>\n<\/tr>\n
                  BSCP<\/td>\nBuffered Sodium Chloride Peptone Solution<\/td>\n<\/tr>\n
                  SCDM<\/td>\nSoyabean Casein Digest Medium<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

                  \u00a0<\/strong><\/p>\n

                  \u00a0<\/strong><\/p>\n

                  List of Cultures for Serial Dilutions<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
                  Culture Name<\/strong><\/td>\nAbbreviation<\/strong><\/td>\n<\/tr>\n
                  Bacillus subtilis<\/em><\/td>\nB. subtilis<\/em><\/td>\n<\/tr>\n
                  Candida albicans<\/em><\/td>\nC. albicans<\/em><\/td>\n<\/tr>\n
                  Escherichia coli<\/em><\/td>\nE. coli<\/em><\/td>\n<\/tr>\n
                  Pseudomonas aeruginosa<\/em><\/td>\nP. aeruginosa<\/em><\/td>\n<\/tr>\n
                  Salmonella abony<\/em><\/td>\nS. abony<\/em><\/td>\n<\/tr>\n
                  Staphylococcus aureus<\/em><\/td>\nS. aureus<\/em><\/td>\n<\/tr>\n
                  Shigella boydii<\/em><\/td>\nS. boydii<\/em><\/td>\n<\/tr>\n
                  Aspergillus brassiliensis<\/em><\/td>\nA. brasilliensis<\/em><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

                  \u00a0<\/strong><\/p>\n

                  *Note – Ready to use SOP available in \u201cDOWNLOAD\u201d Section.<\/u><\/em><\/strong><\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n

                   <\/p>\n","protected":false},"excerpt":{"rendered":"

                  SOP Title: Serial Dilution Preparation of\u00a0 Microbial\u00a0 Cultures Objective: To lay down the Procedure for Serial Microbial Cultures Dilution.   Scope This procedure is applicable for Serial Microbial Cultures Dilution in the Microbiology Section of Quality Control Department. Responsibility: 3.1 \u00a0\u00a0\u00a0\u00a0Quality Control: To prepare and review the SOP. To follow the procedures for Serial Dilution […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"closed","sticky":false,"template":"templates\/template-full-width.php","format":"standard","meta":{"footnotes":""},"categories":[7],"tags":[],"class_list":["post-741","post","type-post","status-publish","format-standard","hentry","category-microbiology"],"_links":{"self":[{"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=\/wp\/v2\/posts\/741","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=741"}],"version-history":[{"count":1,"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=\/wp\/v2\/posts\/741\/revisions"}],"predecessor-version":[{"id":742,"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=\/wp\/v2\/posts\/741\/revisions\/742"}],"wp:attachment":[{"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=741"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=741"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/pharmacoguide.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=741"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}