Course: MCB 103 Lab

Section: 1
Name: Noor-E-Khadiza Shama
ID: 1921168
Date: 25/02/20

Experiment 6

ENDOSPORE STAINING

PURPOSE
1. It is a form of differential staining so it is used to distinguish vegetative cells and endospores
2. Endospores have tough covering around it as a result they cannot be stained using basic
staining, so endospore staining process is particularly helpful

PRINCIPLE
Endospores are tough protein coats made of keratin, so the spores are highly resistant to normal
staining procedures. A endospore staining technique (the Schaeffer Fulton method) is a differential
staining method used to distinguish between the vegetative cells and the endospores. A primary stain
(malachite green) is used to stain the endospores. Because endospores have a keratin covering and
resist staining, the malachite green will be forced into the endospores by heating. In this technique
heating acts as a mordant. Water is used to decolorize the cells; as the endospores are resistant to
staining, the endospores are equally resistant to de-staining and will retain the primary dye while the
vegetative cells will lose the stain. The addition of a counterstain or secondary stain (safranin) is used to
stain the decolorized vegetative cells so that they can be distinguished from the endospores.
MATERIALS & EQUIPMENT
1. Bacillus cereus sample
2. Malachite green powder
3. Safranin
4. Bunsen burner
5. Staining tray
6. Inoculating loop
7. Glass slides
8. Microscope
9. Foil paper
10. Bibulous paper

PROCEDURE
1. 5.5g of malachite green powder is measured using balance and is put in a reagent
bottle.
2. Using measuring cylinder, 50ml distilled water is added in the bottle.
3. It is then mixed with a vortex mixer.
4. Prepare a heat-fixed smear on a clean microscope slide of bacteria Bacillus cereus.
5. Flood the smear with malachite green and to heat the smear,keep adding stain every
time it dries
6. Place the smear on the top of a beaker of water using gauge and place the beaker on a
warm hot plate
7. The smear is allowed to steam for 2 to 3 minutes.
8. The slide is removed from the hot plate, cooled and wash using water
9. Then the counter stain which is Safranin is added and kept for 30 seconds and rinsed
with water.
10. The smear is blot dry with bibulous paper and examine under 100X power lens with
immersion oil.
RESULT & OBSERVATION
The smear was observed under microscope wand the result is shown below.

DISCUSSION
After staining, the smear is observed under microscope. The red stained cells which were seen was
vegetative cells, the red color was due to Safranin which was the counter stain. This shows that these
cells did not have spores and so they couldn’t retain the primary stain which was Malachite green. The
green stained cells are endospores, they retained the primary stain which was Malachite green and so
they did not absorb red Safranin.

PRECAUTION
1. Prevent the stain from evaporating and adjust the hot plate temperature to prevent boiling
2. The smear should be heated carefully as hot water may burn skin