SOP For Spore Staining Technique

by

SOP For Spore Staining Technique

1.0 Objective

1.1 To establish a procedure for performing spore staining to differentiate bacterial endospores from vegetative cells.

2.0 Scope

2.1 This SOP is applicable to spore staining procedures carried out in the Microbiology Laboratory for identification and characterization of spore-forming bacteria.

3.0 Responsibility

3.1 Microbiologist or above – Microbiology Laboratory:

  • Preparation and execution of the staining procedure.

  • Microscopic examination and interpretation of results.

3.2 Head – Microbiology Section / Nominee:

  • Review and approval of the SOP.

  • Ensuring compliance with the procedure.

4.0 Accountability

4.1 Head – Quality Control / Nominee:

  • Overall compliance with this SOP.

5.0 Procedure

5.1 Preparation of Smear

5.1.1 Prepare a thin smear from an isolated pure culture on a clean glass slide.

5.1.2 Allow the smear to air dry completely.

5.1.3 Heat-fix the smear gently by passing the slide over a flame.

5.2 Primary Staining

5.2.1 Place the prepared slide over a small beaker containing boiling water positioned on a hot plate to generate steam.

5.2.2 Cover the smear with a small piece of filter paper.

5.2.3 Saturate the filter paper with 5% aqueous malachite green solution.

5.2.4 Maintain steaming conditions and keep the filter paper moist with stain for approximately 30 minutes.

5.3 Washing and Counterstaining

5.3.1 Remove the filter paper carefully and allow the slide to cool.

5.3.2 Rinse the slide gently with water to remove excess malachite green.

5.3.3 Counterstain with safranin solution for approximately 30 seconds.

5.3.4 Wash gently with water and blot dry using Whatman filter paper.

5.4 Microscopic Examination

5.4.1 Observe the stained smear under a microscope using 100X objective with immersion oil.

5.4.2 Interpret the staining results as follows:

  • Endospores appear green.

  • Vegetative cells appear pink/red.

5.4.3 Observe and record the location of spores within the bacterial cell, such as:

  • Central spore

  • Subterminal spore

  • Terminal spore (with or without swollen sporangium)

SOP For Viable Particle Monitoring of Drain Points – Sterile Product Manufacturing Facility

Post Views: 28

Leave a Comment