LAB 5: Differential Stains II

Gram Staining and Acid Fast Staining

What are the two main groups?

gram+ and gram-

Gram staining

A differential staining technique that provides an easy differentiation of bacteria into one of two groups

Gram + cell

• thick peptidoglycan

• 90% peptidoglycan

• teichoic acid

• 1 layer

• not many polysaccharides

• In acid fast cells, contains mycolic acid

• stains purple/violet

Gram- cell

• thin peptidoglycan

• 5-10% peptidoglycan

• no teichoic acid

• 3 layers

• outer membrane as lipids, polysaccharides

• no acid fast cells (mycolic acid)

• stains pink

Gram staining includes several items, which are?

• a primary stain (crystal violet)

• a mordant (helper) iodine solution

• a decolorizer (95% ethanol)

• a counterstain (safranin)

What does ethanol cause to the cells?

denaturation of the proteins in the outer membrane

Function of Safranin

make the cells visible

Mordant (iodine) function. 

Strengthens the dye binding, making gram+ cells retain the purple stain. 

4 conditions that need to be followed for a valid gram staining procedure.

1. young cultures

2. thin smear

3. fresh reagents

4. control cultures

Young cultures

must be young within 18-24 hours old.

Older cultures will lose their gram staining properties due to changes in the cell wall as the cells get older.

Thin smear

thicker or uneven smears will result in uneven staining and decolorization

Fresh reagents

proper strength

Control cultures

for a known gram+ and gram- culture

Endospore

dormant and highly resistant cell which contains a copy of cellular DNA. Endospores will germinate into the vegetative cells when nutrients are around

Endospore are resistant to?

• drying/desiccation

• high temp

• radiation

• chemical damage

What stain is used for endospore staining?

malachite green

Mycobactera

These are acid fast, cell wall is thick (~60% lipid, much less peptidoglycan). Cell wall is rich in mycolic acid. 

Mycolic acid

Waxy, located in mycobacteria

Acid Fast Staining Purpose?

It is to see the presence of Mycobacterium which are resistant to simple or gram staining methods and can only be visualized by acid fast staining.

Steps to Acid fast staining

1. Primary stain: carbolfushin

2. decolorizer: acid-alcohol

3. counterstain: methylene blue

Carbolfuschin

• has to be cooked into the cell

• All cells will take this stain

• steaming over the bunsen burner,

Acid alcohol

Decolorizer; mycolic acid holds carbolfuschin in acid fast cell

other non acid fast cells would lose the stain and become colorless.

Methylene Blue

• acid fast do not absorb methylene blue so it will stay red/pink

• “normal“ gram+ and gram- absorbs this stain, appearance will change to blue