1.0
OBJECTIVE
1.1
2.0
SCOPE
2.1
3.0
Microbiologist
ACCOUNTABILITY
4.1
5.0
This SOP covers the procedure of stain techniques and is applicable to
Microbiology Labs –I and II, Unit -I
RESPONSIBILITY
3.1
4.0
To lay down a procedure for staining the microorganisms in both vegetative and
spore form.
Head - Quality Control
PROCEDURE
5.1
Gram Staining
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
5.1.6
5.1.7
5.1.8
5.1.9
5.1.10
5.1.11
5.1.12
5.1.13
5.1.14
5.1.15
5.1.16
5.1.17
5.2
Ensure that the laminar airflow is working.
Place a drop of sterile water/purified water on the grease free glass slide.
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.
Allow to air dry the smear and heat fix it by passing the slide gently
through a flame.
Flood/cover the smear with Gram’s Crystal violet solution for 1 min
exactly.
Rinse the stained slide with tap water and drain off excess.
Flood the smear with Gram’s Iodine solution for 1 min.
Rinse the slide with tap water and drain off excess.
Decolorize the smear with Gram’ decolourizer for 15 to 30 seconds by
placing the slide on the staining stand.
Let the decolorizer flow across the stained smear until solvent flows
colorlessly from the slide.
Do not over decolorize the stained smear.
Immediately wash the slide with tap water and drain off excess.
Counter stain the slide with Gram’s safranin for 45 seconds.
Wash the slide and blot dry/air dry.
Examine the slide under oil immersion objective/lens.
Gram-positive organisms are Violet.
Gram-negative organisms are red/pink.
Spore Staining
5.2.1
5.2.2
5.2.3
Ensure that the laminar airflow is working.
Place a drop of sterile water/purified water on the grease free glass slide.
Scrap a colony (if growth is on solid media) take a loopful with the help
of inoculating wire loop (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.
5.2.4 Place the slide over a beaker of boiling water, with bacterial film (smear)
upper side.
5.2.5 When the large droplets are condense on lower side of the slide, flood the
smear with schaeffer and fultons spore stain A for 1 min.
5.2.6 Wash the slide with cold water.
5.2.7 Flood the slide with Schaeffer and Fulton’s stain B for 30 secs.
5.2.8 Wash the slide and blot dry/air dry.
5.2.9 Observe the slide under oil immersion objective.
5.2.10 Spores are green in colour.
5.2.11 Vegetative cells are pink in colour.
5.3
Fungal Staining
5.3.1
5.3.2
5.3.3
5.3.4
5.3.5
Ensure that the Laminar airflow is not working.
Place 1-2 drops of Lacto phenol cotton blue stain on a grease free glass
slide.
Take a small amount of fungal culture with a needle from the slant and
place in the stain solution on the glass slide and gently scrap it.
Wait for 5 minutes and place a cover slip with out air bubble.
Observe the slide under 10 X / 40 X objective /Lens.
END OF DOCUMENT