microLAB 2: Bacterial Morphology and Staining

Bacteria are ___. what size? are their organelles membrane bound? what kind of chromosome?

prokaryotes.

• smaler in size ~ 1mm

• no membrane bound organelles

• Single circular chromosome

Bacteria basic shapes

• Bacilli (rod- shaped): streptobacilli (chains)

• cocci (sphere-shaped): streptococci (chains): straphylococci (clusters)

• spirilla (spiral-shaped)

• spirochaete (corkscrew)

• vibrio (comma-shaped)

Arrangement of cocci

• Diplococcus: cocci in pairs

• Streptococcus: cocci arranged in chains

• Streptococcus: cocci in chain

• Staphylococcus: grape like clusters

Why Do Staining?

• Most bacteria are colorless and provide minimal contrast

• Staining makes bacteria visibly observable

• To determine the morphology (form and structure),

arrangements and size of bacteria

types of staining:

• Negative staining: background is stained

• Simple staining: only 1 dye used

• Differential staining: to differentiate different

types of cells: eg. Gram stain, acid fast stain,

endospore Staining

• Special staining- more than one dye used-

special structures are seen. Eg. capsule

staining, flagella staining

NEGATIVE STAINING

• Negative, indirect or background staining is achieved by mixing bacteria

with an acidic stain or dye called a chromogen, such as Nigrosin, India

ink, or eosin.

• Spread of the mixture over a slide to form a film.

• No stain penetrates bacterial cell due to repulsion between the negative

charge of the stain and the negatively charged bacterial wall.

• Useful in observing bacterial morphology shape and size

negative staining procedure:

• No heat fixing required.

• Mix bacterial suspension bacteria in liquid media with acidic

stain (Nigrosin) & spread mixture on slide.

• Bacterial cells are not penetrated by stains.

• Air dry and focused with 4x, 10x, 40x & 100x

• Produces image of light bacteria against dark background.

what staining is this?

negative stain

Basic initial steps before staining:

- To observe more details about cell shape, size and arrangements.

- Bacterial smear preparation

 place a drop of bacterial cells on the central portion of a slide

in a circular fashion

 Air dry, Heat fix the slide –we use hot plates/not Bunsen

burners

 Bacteria attaches/adhere to the slide – appears dull white

SIMPLE STAINING

• Simple to perform – only one basic stain used. Eg.

Crystal Violet, Methylene blue, Carbolfuchsin..

• Then add dye to create a contrast between

bacteria & background the bacteria cell wall is (-)

• the simple stain is (+), it attracts to the (-)

bacterial cell wall

simple staining procedure:

• A bacterial smear is prepared , air dried and heat fixed.

• A heat fixed smear is flooded w methylene blue for 45-60

seconds, then washed w water

• Blot dry w bibulous paper and examine under the

microscope with 4x, 10x, 40x & 100x

GRAM STAIN

• Hans Christian Joachim Gram, a Danish scientist & physician (1853-

1938)

• The most widely used staining procedure in microbiology is the Gram

stain.

• Used to differentiate between Gram(+) and Gram (-) bacteria

gram stain procedure:

1. Primary stain -basic dye Crystal violet

2. Treat with iodine solution (Mordant) increases interaction

between bacterial cell & dye

3. Decolorize smear with 95% ethanol

4. Counterstain with safranin- a basic dye that stains-

- Gram negative pink/red (E. coli)

- Gram positive purple/blue (S. Aureus)

ACID FAST / NON ACID FAST STAINING

(Ziehl-Neelsen and Kinyoun)

Acid Fast microorganisms: high lipid contents in cell wall

- To stain Mycobacterium species especially M. tuberculosis

• Primary stain-Carbolfuchsin, counterstain-Methylene blue.

• Acid Fast microorganisms require heat that the stain can penetrate.

• Not easily decolorized, after primary stain with carbolfuchsin (red).

Non Acid Fast microorganisms: low lipid content in cell wall

• Easily decolorized and appear blue/brown after counterstain with

Methylene blue.

ENDOSPORE STAINING (Schaffer-Fulton or Wirtz-Conklin)

New structure that is highly resistant & capable of surviving in

unfavorable conditions or environment.

• Once conditions are favorable a new bacterial cell arises. This

structure is called an endospore, since it develops within the bacterial

cell.

• Endospores do not stain easily, but once they are stained, they strongly resist decolorization