1.0 OBJECTIVE :
- To provide a procedure for testing the Microbiological Cultures for their Purity & Identity.
2.0 SCOPE :
- This procedure applies for the cultures used in Microbiology Section.
3.0 RESPONSIBILITY :
- Microbiologist
4.0 ACCOUNTABILITY:
- Head of Department
5.0 PROCEDURE :
5.1 Procure the standard stock cultures needed during analysis of various tests from the institution with due authorization from the Department Head. Check the received stock of cultures for intactness & correctness of the cultures. Preserve copy of delivery challan of the culture. Preserve the culture under refrigeration at a temperature between 2°C to 8°C.
5.2 The lyophilized ampoules/culture slants/ culture sticks shall be removed from the refrigerator and shall be allowed to acclimatize to the ambient temperature.
5.3 In case of mother cultures in lyophilized ampoules, the ampoule shall be surface disinfected with 70% IPA and allow it to dry under LAF.
5.4 Mark at the middle of the ampoule with a sterilized ampoule cutter & break it carefully at the marked area.
5.5 Carefully remove the cotton plug and add about 0.3 to 0.4 ml of SCDM medium with a sterile pipette.
5.6 Mix well and inoculate the contents into 10 ml of sterilized SCDM in a test tube.
5.7 In case of mother cultures in slants, the transfer shall be made by the inoculation loop from the culture and shall be inoculated in 10 ml of sterilized SCDM in a test tube.
5.8 In case of cultures in sticks, the stick shall be removed and inoculation shall be done by immersing the stick in 10 ml of sterilized liquid medium in a test tube. The stick shall be manually rotated in the medium.
5.9 The disinfected lyophilized ampoules/culture slants/ culture sticks shall be decontaminated in the autoclave at 121°C for 30 minutes.
5.10 During the incubation period, the enriched tubes shall be checked for any growth in terms of turbidity for bacterial and yeast culture and growth of mycelium in case of fungal cultures.
5.11 During observation of enriched tubes, if growth is observed, the cultures shall have then proceeded to the next stage of purity check.
5.12 After the growth observed in the enriched medium, purity check shall be done for all the cultures and simultaneously shall be streaked in the maintenance medium.
5.13 Observe the slants or stabs for uniformity of growth and for contamination by other Micro organisms before starting analysis and record the observation.
5.14 Use SCA for Candida albicans, observed the morphological characters of the colonies like size, shape, margin, colour, opacity, consistency etc.
5.15 Pick up a single isolated colony from the plate or take a loop full of culture directly from the suspension, in sterile saline and perform the other tests.
5.16 GRAM STAINING:
- Prepare a smear of the culture under test. Dry and fix with gentle heat.
- Flood the smear with Gram’s crystal violet for 1 minute.
- Wash with Distilled Water and flood with Gram’s Iodine for 1 minute.
- Wash with distilled water and decolourize with 95 % v/v ethanol till the dye stops coming out.
- Wash gently with distilled water.
- Counter stain with Safranine 0.5 % w/v for 1 minute.
- Wash with distilled water, dry and examine under oil immersion objective microscope.
5.17 MOTILITY :
- Motility caused by movement of flagella is easily seen by direct microscopic observation of motile organisms like hanging drop method.
- Aseptically transfer one drop of the culture to the centre of the cover slip.
- Invert the slide and centre its cavity over the drop of the culture. Press down on the edge of the cover slip. Quickly and carefully turn the slide so that the hanging cavity.
- First locate its edge in the centre of the microscopic field with the low power objective & markedly reduce the light.
- The edge will be seen as a bright way line against the gray background.
- Switch to the high power objective & refocus on the edge of the drop.
- Streak the pathogenic cultures on their selective media.
5.18 CHARACTERSTICS OF CULTURES:
- Escherichia coli:
- Morphological Characters on SCDA – 1-3 mm, smooth, low convex, translucent, colorless to pink colonies.
- Gram Staining – Gram Negative (Pink coloured) short rods.
- Motility – Motile.
- Growth characters on selective media.
| Medium Used | Observation on specified Medium |
| Macconkay Agar | Pink coloured, short rods |
- Salmonella abony :
- Morphological Characters on SCDA – 2-3 mm, Circular, Moist, smooth, convex, translucent, colorless, or white colonies.
- Gram Staining – Gram Negative (Pink coloured) rods.
- Motility – Motile.
| Medium Used | Observation on specified Medium |
| Xylose Lysine Deoxycholate Agar | Red colonies, with or without black center |
- Pseudomonas aeruginosa:
- Morphological Characters on SCDA – 2-3 mm, low convex, rough, oval opaque musty smell, confluent growth showing iridescent sheen in reflected light producing diffusible pigment pyrocyanin.
- Gram Staining – Gram Negative (Pink coloured, rods shaped)
- Motility – Motile.
| Medium Used | Observation on specified Medium |
| Cetrimide Agar | Green colonies |
- Staphylococcus aureus:
- Morphological Characters on SCDA – 1-2 mm, Circular, low convex butyrous, opaque with yellow zones.
- Gram Staining – Gram Positive cocci in cluster.
- Motility – Non Motile.
| Medium Used | Observation on specified Medium |
| Mannitol Salt Agar | Yellow colour colonies with yellow zones. |
- Bacillus subtilis :
- Morphological Characters on SCDA, 3-5 mm, Circular to regular with cerrated margins, opaque flat dry, off white colonies.
- Gram Staining – Gram Positive spore bearing thick rods.
- Motility – Motile.
| Medium Used | Observation on specified Medium |
| Soyabean Casein Digest Agar | White coloured colonies |
- Candida albicans:
- Morphological Characters on Sabouraud Chloramphenicol Agar,2-3 mm entire, Circular, convex, opaque, paste like, white in colour yeast.
- Microscopic Observation : Oval to circular cell with 3-5 m diameter and budding cells are observed.
| Medium Used | Observation on specified Medium |
| Sabouraud Chloramphenicol Agar | Oval to circular shape |
- Shigella boydii :
- Morphological Characters on SCDA, small often 2-3mm in diameter, circular, convex, colorless, smooth and translucent.
- Gram Staining – Gram negative short rods.
- Motility – Non motile
| Medium Used | Observation on specified Medium |
| Xylose Lysine Deoxycholate Agar | Red colonies,without black center |
5.19 Frequency : Every Six month
6.0 ABBREVIATIONS:
| Abbreviation | Expanded form |
| SCDA | Soyabean Casein Digest Agar |
| SCDM | Soyabean Casein Digest Medium |
| SCA | Sabouraud Chloramphenicol Agar |
| SOP | Standard Operating Procedure |
| mm | Millimeter |
| % | Percentage |
| °C | Degree Celsius |
| w/v | Weight / Volume |
| v/v | Volume / Volume |
7.0 ANNEXURES:
| Annex. No. | Title |
| 01 | Purity Testing Of Microbiological Cultures |
8.0 SOP REFERENCES
- Indian Pharmacopoeia
END OF THE SOP
ANNEXURES :
Annex. No. 01 Purity Testing Of Microbiological Cultures
