Staining of microorganisms in both vegetative and spore form procedure

1.0 OBJECTIVE

To describe a procedure for staining the microorganisms in both vegetative and spore form.

2.0 SCOPE

This procedure is applicable to the procedure of stain techniques in Microbiology Lab

3.0 RESPONSIBILITY

3.1 Microbiologist /Officer is responsible for the execution.

3.2 HOD or designee is responsible for review, effective implementation and compliance to the stain techniques procedures for execution.

4.0 PROCEDURE

4.1 Gram Staining

4.1.1 Ensure that the laminar airflow is working.

4.1.2 Place a drop of sterile water/purified water on the grease free glass slide.

4.1.3 Scrap a colony (if growth is on solid media) or take a loopful from the suspension with the help of inoculating wire loop (nichrome/platinum) and Prepare a smear on the grease free glass slide by rotating the loop.

4.1.4 Allow to air dry the smear and heat fix it by passing the slide gently through a flame.

4.1.5 Flood/cover the smear with Gram’s Crystal violet solution for 1 min exactly.

4.1.6 Rinse the stained slide with tap water and drain off excess.

4.1.7 Flood the smear with Gram’s Iodine solution for 1 min.

4.1.8 Rinse the slide with tap water and drain off excess.

4.1.9 Decolorize the smear with Gram’ decolourizer for 15 to 30 seconds by placing the slide on the staining stand.

4.1.10 Let the decolorizer flow across the stained smear until solvent flows colorlessly from the slide.

4.1.11 Do not over decolorize the stained smear.

4.1.12 Immediately wash the slide with tap water and drain off excess.

4.1.1 Counter stain the slide with Gram’s safranin for 45 seconds.

4.1.14 Wash the slide and blot dry/air dry.

4.1.15 Examine the slide under oil immersion objective/lens.

4.1.16 Gram-positive organisms are Violet.

4.1.17 Gram-negative organisms are red/pink.

4.2 Spore Staining

4.2.1 Ensure that the laminar airflow is working.

4.2.2 Place a drop of sterile water/purified water on the grease free glass slide.

4.2.3 Scrap a colony (if growth is on solid media) take a loopful with the help of Inoculating wire loops (nichrome/platinum) and Prepare a smear on the grease free slide by rotating the loop. Allow to air dry and heat fix it by passing the slide gently through a flame.

4.2.4 Place the slide over a beaker of boiling water, with bacterial film (smear) upper side.

4.2.5 When the large droplets are condense on lower side of the slide, flood the smear With malachite green 1 min. Steam the slide for five minutes on the steam bath in the fume hood.

4.2.6 Wash the slide with cold water.

4.2.7 Counter stain the smear with safranin for 30 secs.

4.2.8 Wash the slide and blot dry/air dry.

4.2.9 Observe the slide under oil immersion objective.

4.2.10 Spores are green in colour.

4.2.11 Vegetative cells are pink in colour.