Page 81 - MNUmicrobiology practical 2025

General Microbiology & Immunology (PM 401) second level Semester 4 2024/2025

• An endospore is a mainly inactive version of a cell that is analagous to a survival

bunker. When environmental conditions are poor, or even deadly, bacteria that are

able to will produce endospores to survive.

• When environmental conditions improve, endospores can produce the active cells

again (active cells are called vegetative cells.)

• Bacterial endospores are the most resistant structures of all living organisms, and

they can live in this dormant dehydrated state for hundreds of years (even some

documented at thousands of years).

• The environmental conditions that trigger sporulation (stimulate the formation of

spores) can vary and may include nutrient depletion, desiccation, chemicals, heat,

etc.

Principle
• The Modified Ziehl Neelsen stain endospore stain is a differential stain that enables

visualization of endospores and differentiation of spores from vegetative cells.

• The primary stain, carbol fuchsin, attaches to the cell wall of vegetative cells and the

spore wall of endospores and mature spores. In fact, if washed well with weak

decolorizer (0.25%- 0.5% H2so4), the stain comes right out of the cell walls of

vegetative cells. In contrast, carbol fuchsin will not wash from the spore wall. Once

the stain is in the spore wall, it is locked in and will not wash out.

• When the carbol fuchsin is washed out of the vegetative cells they become

transparent. A counterstain, methylene blue, is used to stain the vegetative cells

blue.

Modified Ziehl Neelsen staining requirements

1- Stock culture containing spore forming organism #1

2-Stock culture containing non spore forming organism #2

General Microbiology & Immunology (PM 401) Practical note Page 70