Stains and Microscopy

Principle stain used for microscopic examination of bacteria

Nearly all clinically important bacteria can be detected by this method

Considered a differential stain because it separates species into two large groups (Gram positive and gram negative)

Gram Stain

Organisms that take up the crystal violet

Have a thick peptidoglycan layer

Gram-Positive

Organism that allow the crystal violet to wash out easily with the decolorizing step

Cell walls have a thinner peptidoglycan layer and an outer lipid bilayer that is dehydrated during the decolorization process

Gram-negative

Gram Stain primary stain left on for 10-30 seconds

Crystal violet

Mordant in Gram stain

Gram’s iodine

Decolorizer (gram stain)

Alcohol or actetone

Secondary or counter stain in gram stain

Safranin

Cocci

• Clusters, gram positive

• Clusters, gram negative

• Chains

• Pairs

• Diplococci

Rods

• Gram negative

• Gram negative coccobacilli

• Gram positive

Gram stain morphology

Specifically designed for a group of bacteria whose cell walls contain long chain fatty acids call mycolic acids

Mycobacteria are the most encountered

Acid Fast Stain

Classic method of acid-fast stain that requires heat to allow the primary stain, carbolfuchsin to enter the mycolic acid

Ziehl-Neelsen

Modified version of acid-fast stain that does not require heat for the primary stain to enter the mycolic acid

Kinyoun acid fast method

Primary stain (ZN AF)

Carbofuschsin

Decolorizer (ZN AF)

3% HCL in 95% ethanol

Counterstain (ZN AF)

Methylene blue

Primary stain (K AF)

Carbofuschsin

Decolorizer (K AF)

1% sulfuric acid

Counterstain (K AF)

Methylene blue

Stain that binds with nucleic acid and fluoresces orange under ultraviolet light

Metachromatic stain that will stain RNA orange and DNA green

Key stain for organisms with no cell wall

Bacteria and yeasts will fluoresce bright orange against a green- fluorescing or dark background

Acridine Orange Stain

The waxy mycolic acids have an affinity to the fluorochromes

Mycobacterial cells will glow bright yellow or orange against a dark or greenish background

The stain is typically used in conjunction with a traditional acid-fast stain

Auramine Rhodamine

Method is used to directly examine the patient specimens for bacteria that are difficult and slow to grow

• Legionella species and Chlamydia trachomatis

Fluorescent-tagged antibodies are targeting a specific antigen on a particular organism

Immunofluorescence staining

Not made on throat, nasopharyngeal, or stool specimens because of the abundance of normal flora

The most common stain is the gram stain

All appropriate specimens should have this prepared

• Test the quality of the specimen

• Give an early indication of what may be wrong with the patient

• Guide the microbiologist on what to look for in a particular specimen

Direct Smear