SOP Title: Identification and Purity Verification of Master Microbial Cultures
- Objective:
To lay down the procedure for Identification and Purity Verification of Master Microbial Cultures.
- Scope:
This procedure is applicable for Identification and Purity Verification of Master Microbial Cultures in Microbiology Section of Quality Control.
- Responsibility:
3.1 Quality Control Department: To prepare and review the SOP. To follow the procedure for Identification and Purity Verification of Master Microbial Cultures as per this SOP.
- Quality Assurance Department: To review and approve the SOP and Annexure.
- Accountability:
Head Quality Control Department, Head Quality Assurance Department.
- Procedure:
| 5.1 | Safety Precautions: | |
| 5.1.1 | Wear personal protective equipments such as protective hand gloves, nose masks and over gown during operation. | |
| 5.1.2 | Do not overheat the smear. | |
| 5.1.3 | Staining solution to be handled carefully. It should not fall on the floor. | |
| 5.1.4 | For oil immersion technique, use only one drop of immersion oil. Do not over flood the slide with immersion oil. | |
| 5.2 | Procedure for Identification and Purity: | |
| 5.2.1 | Media preparation for all the tests has to be performed by referring SOP, Media Preparation and Sterilization. | |
| 5.2.2 | Perform the Purity Check on receipt of Mother Culture by staining method by isolating the bacteria. | |
| 5.2.3 | The staining procedure differentiate bacteria into gram positive and gram negative based on their ability to retain the primary dye (Crystal violet ) or loose the primary dye and accept the colour of the counter stain (safranin). | |
| 5.2.4 | The gram positive bacteria produces blue/purple colour with this staining procedure while gram negative ones are pink or red in colour. | |
| 5.2.5 | The staining reagent are:
1) Crystal violet: A primary dye. 2) Grams iodine: A complexing agent. 3) Alcohol: A decoloriser. 4) Safranin: A Counter strain. |
|
| 5.2.6 | Prepare a thin smear of the culture to be examined on a glass slide. | |
| 5.2.7 | Air dry or heat fix the smear and flood the slide with Crystal Violet staining solution for 1 minute. Allow the slide to cool do not burn the slide. | |
| 5.2.8 | Wash the smear with 70% v/v Ethanol /Grams decolouriser till all the extra Crystal Violet stain is washed off. | |
| 5.2.9 | Wash with water and flood with Grams Iodine solution for 1 minute. | |
| 5.2.10 | Flood the smear with safranin solution for 1 to 2 minutes. Wash with water and Air dry the slide. | |
| 5.2.11 | Place a drop of cedarwood oil and examine under oil immersion lens 100X of the microscope. | |
| 5.2.12 | Gram positive organisms should observed blue in colour and Gram negative organisms should observe pinkish red colour. Record the results. | |
| 5.3 | Fungal Staining Procedure: | |
| 5.3.1 | Place a Lactophenol cotton blue reagent on a clean and dry slide and place a small loopful Aspergillus fungal culture into Lactophenol cotton blue reagent and tease the Aspergillus culture into thin preparation. | |
| 5.3.2 | Remove the excess stain using tissue paper. | |
| 5.3.3 | Wait for 15 minutes and observe under microscope. | |
| 5.3.4 | Place a coverslip on the slide before observing under the microscope.
Avoid air bubbles. |
|
| 5.3.5 | Observe Initially under low power for screening in low intensity and then under high power intensity. | |
| 5.3.6 | Aspergillus brasilliensis produce white or yellow growth colonies that are covered by Septate dark asexual fungal spores. Record the results. | |
| 5.4 | Procedure for Motility testing: | |
| 5.4.1 | Prepare suspension of the Bacterial culture in normal saline, with the help of sterile nichrome loop place a drop of suspension of Bacterial culture on the cover slip. | |
| 5.4.2 | Grease the four corners of the cover slip. | |
| 5.4.3 | Place a cavity slide on the top of the cover slip taking care that drop is not disturbed. | |
| 5.4.4 | Invert the slide so that the cover slip is on the top of the slide. | |
| 5.4.5 | Observe under the microscope, first under low power for screening in low intensity and then under high power. | |
| 5.4.6 | For motile organisms eg. S boydii, S.aureus, motility can be seen towards the edge of the drop and for non motile organisms eg. B. Subtilis, E. coli, S. abony. C albicans, P. aeruginosa, the cells will be evenly distributed. | |
| 5.4.7 | After confirmation of Purity Check of Culture from colony characteristic, staining and Motility, Carry out the Biochemical test for the Cultures and compare the results obtained, as per Annexure. | |
| 5.4.8 | Confirm the growth of cultures on selective media as per SOP – Procedure for Testing Growth Promotion, Inhibitory and Indicative Properties of Culture Media. | |
| 5.4.9 | The commonly used Biochemical tests are mentioned below.
(a) Catalase test, (b) Coagulase test ,(c) Oxidase test,(d) Sugar fermentation test ,(e) Indole test ,(f) Citrate test and (g) Urease test. |
|
| 5.5 | Biochemical tests for Escherichia coli: | |
| 5.5.1 | Procedure for Acid and Gas Test: | |
| 5.5.2 | Transfer a loopful of culture in 5 ml of sterile MacConkey Broth containing durhams tube. Incubate the test tube at 42 to 44°C for 24 hrs. | |
| 5.5.3 | Observation: Change in colour of MacConkey Broth from purple to yellow indicates the production of acid and formation of bubble in Durham’s tube indicates the production of gas. | |
| Procedure for Indole Test: | ||
| 5.5.4 | Transfer a loopful of culture in 5 ml of sterile Peptone water. Incubate the test tube at 42 to 44°C for 24 hours. | |
| 5.5.5 | Add a few drops of Kovacs reagent in the test tube and mix the content and allow the test tube to stand for about 2 minutes. | |
| Observation: Formation of red ring in the reagent layer indicates the production of Indole. | ||
| 5.6 | Biochemical Tests for Salmonella: | |
| 5.6.1 | Procedure for acidic butt and alkaline slant on Triple Sugar Iron (TSI) Agar. | |
| 5.6.2 | Streak a loopful of culture on the slant with sterile nichrome loop and stab the butt with straight sterile nichrome loop. | |
| 5.6.3 | Incubate at 30 to 35°C for 24 hours. | |
| 5.6.4 | Observation: Acidic butt should show Yellow colour and alkaline slant should show pink colour on TSI without production of H2S gas. | |
| 5.6.5 | Transfer a loopful of culture in 10 ml of RVSEB. Incubate at 30 to 35°C for 24 hrs. | |
| Observation: Change in colour of the medium from blue to colourless. | ||
| 5.7 | Biochemical test for Pseudomonas aeruoginosa | |
| 5.7.1 | Procedure for Oxidase Test: | |
| 5.7.2 | Place 2 or 3 drops of freshly prepared 1% w/v solution of N,N,N’,N’, Tetramethyl-4-phenylene-diamine dihydrochloride on a filter paper and smear with P. aeuroginosa colonies. For solution Preparation, refer SOP, Preparation and Handling of Laboratory reagents. | |
| 5.7.3 | Observation: Development of pink colour should change to purple colour. | |
| 5.8 | Biochemical test for Staphyloccocus aureus: | |
| 5.8.1 | Procedure for Coagulase test: | |
| 5.8.2 | Transfer a loopful of culture to individual tube containing 0.5 ml of coagulase rabbit or horse plasma. Incubate in water bath at 37°C, examine the tubes after 3 hours and subsequently at a 3 hour interval up to 24 hours. | |
| 5.8.3 | Observation: Formation of Clot. | |
| 5.9 | Biochemical Tests for Shigella boydii: | |
| 5.9.1 | Procedure for acidic butt and alkaline slant on Triple Sugar Iron (TSI) Agar. | |
| 5.9.2 | Streak a loopful of culture on the slant with sterile nichrome loop and stab the butt with straight sterile nichrome loop. | |
| 5.9.3 | Incubate at 30 to 35°C for 24 hours. | |
| 5.9.4 | Observation: Acidic butt should show Yellow colour and alkaline slant should show pink colour on TSI without production of H2S gas. | |
| 5.9.5 | Transfer a loopful of culture in 10 ml of GN Broth. Incubate at 30 to 35°C for 24 hrs. | |
| 5.9.6 | Observation: Change in colour from colourless to turbid. | |
| 5.10 | KOH String test or Gram Staining Alternative test | |
| 5.10.1 | Place a drop of 3% potassium hydroxide (KOH) on a glass slide. For solution Preparation, refer SOP, Preparation and Handling of Laboratory reagents. | |
| 5.10.2 | By using a nichrome loop, place a visible loopful of cells from a single, well isolated colony is mixed into the drop. | |
| 5.10.3 | Mix with Nichrome loop for about 60 seconds. | |
| 5.10.4 | Observation: If the mixture becomes viscous and a string is formed on lifting the nichrome loop (KOH positive) than the colony is considerd as gram negative. | |
| 5.10.5 | Note: This test is simple and can be done effectively on old cultures. This method can be done as a backup to gram staining. | |
| If Purity Check and Identification of any culture is not satisfactory, repeat the test. If Fails after rechecking, procure a new culture. | ||
| 5.10.6 | After Completion of the activity, decontaminate all the cultures and media by referring to SOP for Decontamination of microbial Waste. | |
- Definitions / Abbreviations:
- Definitions:
- Abbreviations:
| Abbreviation | Expansion |
| °C | Degree Centigrade |
| % | percent |
| v/v | Volume by volume |
| No. | Number |
| TSI | Triple Sugar Iron |
| IPA | Isopropyl Alcohol |
| RVSEB | Rappaport Vasiliadis Salmonella Enrichment Broth |
*Note – Ready to use SOP available in “DOWNLOAD” Section.