Chapter 4

Staining methods

Bacteria are nearly transparent and almost the same refractive index as the medium around them. Everything on this page exists to fix that — by adding contrast where there was none.

What each one leaves on the slide

Fig. 1The five fields you are asked to recognise. Colour in each is the stain itself, not a legend.

Before any stain: the smear

Three steps, in order, and skipping any of them ruins the slide.

01
Spread
A thin film of culture in a small drop of water. Thick smears will not decolourise evenly and cannot be read.
02
Air dry
Completely. Heat applied to a wet smear boils the cells and distorts them.
03
Heat fix
Pass through a flame two or three times. This kills the cells and glues them to the slide so they survive the washing.

One exception: a negative stain is never heat fixed. That is the point of it — the cells keep their true size and shape, which is why it is the honest way to measure a capsule.

The methods

Grouped by kind. Which kind a stain belongs to is itself an exam question.

Simple

One dye, one colour. Tells you the shape and arrangement, and nothing else.

Stain	Reagents	Shows	Reads as	Note
Simple stain	One basic dye — methylene blue, crystal violet or safranin	Shape, size and arrangement	Every cell the same colour	Tells you what the cells look like, not what they are.

Negative

The dye is repelled by the cell and stains the background instead. No heat fixing, so nothing shrinks.

Stain	Reagents	Shows	Reads as	Note
Negative stain	An acidic dye such as nigrosin or India ink	Capsules, and cell shape without heat distortion	Dark background, clear cells	The dye is repelled by the cell, so it stains around it. No heat fixing, so nothing shrinks.

Differential

More than one dye, and the cells respond differently. This is where identification starts.

Stain	Reagents	Shows	Reads as	Note
Gram stain	Crystal violet, iodine, alcohol, safranin	Cell wall type	Purple is positive, pink is negative	The single most useful stain in clinical microbiology.
Acid-fast stain	Carbolfuchsin, acid-alcohol, methylene blue	Waxy mycolic acid in the wall	Red is acid-fast, blue is not	Identifies Mycobacterium — tuberculosis and leprosy — which the Gram stain cannot stain reliably.

Structural

Aimed at one part of the cell rather than the cell as a whole.

Stain	Reagents	Shows	Reads as	Note
Endospore stain	Malachite green with heat, then safranin	Endospores inside or outside the cell	Green spore, pink cell	Heat is needed to drive the dye through the spore coat.
Capsule stain	Negative stain plus a simple counterstain	The capsule	Clear halo around a stained cell on a dark field	—
Flagella stain	A mordant that builds up on the flagellum, then a stain	Number and placement of flagella	Flagella thick enough to see by light microscope	—

Choosing one

Exam questions phrase this as a scenario. Work backwards from what you need to see.

You need the wall type

Gram stain. Purple is positive, pink is negative — unless the organism is Mycobacterium, in which case the Gram stain will not take.

You suspect tuberculosis

Acid-fast stain. Mycolic acid in the wall holds carbolfuchsin through an acid-alcohol wash, so the cell stays red where everything else turns blue.

You need to see a capsule

Negative stain, or a capsule stain. Heat fixing shrinks a capsule away, so any method that heat fixes will under-report it.

You suspect a spore-former

Endospore stain. Malachite green driven in with heat, then safranin: green spore inside a pink cell means Bacillus or Clostridium.