Identification of bacteria

Identification of bacteria

• Colonies from culture samples can be transferred to a microscope slide and stained for microscopic analysis

• Staining colours the cells improving visibility under the microscope

GRAM’s Stain

1 - Application of Crystal Violet

2 - Application of Gram Iodine

3 - Wash with Decolorising Agent

4 - Application of Safranin

Staining with a suitable stain, for example ..

Gram stain allows cell morphology to be observed

Bacteria can be classified by the thickness or composition of the cell wall structure

• Gram’s positive

• Gram’s negative

• Acid fast

Gram’s stain

• This is the most commonly used stain in microbiology that provides the first step in identifying bacteria

• Bacteria are categorised by the colour that they stain

Gram’s positive

Blue/Purple

Gram’s negative

Red/Pink

Gram stain process - Step 1

Crystal Violet - Primary stain added to specimen smear

Cell effects - Stains cell purple or blue

Gram stain process - Step 2

Iodine - Mordant makes dye less soluble so it adhere to cell walls

Cell effects - Cells remain purple or blue

Gram stain process - Step 3

Alcohol - Decolouriser washes away stain from gram-negative cell walls

Cell effects - Gram-positive cells remain purple or blue. Gram-negative cells are colourless

Gram stain process - Step 4

Safranin - Counterstain allows dye adherence to gram-negative cells

Cell effects - Gram-positive cells remain purple or blue. Gram-negative cells appear pink or red.

Alternative bacterial stains - Simple/structural stains (methylene blue)

Colours cells allowing shape, size and arrangement to be observed

Alternative bacterial stains - Differential stains (Gram’s stain, Ziehl-Neelsen)

This is a combination of fixative and 2 dyes, a primary and counter stain, that allows differentiation of cells

Alternative bacterial stains - Structural stains

This stains certain parts of the cell, e.g. Flagella, microspore and capsule also aiding in identification of the bacterium

Gram positive bacteria have a thick, porous peptidoglycan layer (outer layer of the cell) which absorbs both stains (will also absorb toxic substances so much easier to destroy), but the purple crystal violet stain is most visible. For example ..

Several staphylococcal and streptococcal spp.

Gram’s negative bacteria have a think peptidoglycan layer which is protected by a further impenetrable outer membrane. Have small holes ‘porins’ are contained within the outer membrane. The think peptidoglycan layer does not retain the crystal violet stain but absorbs the pink safranin stain. For example ..

Pseudomonus aeruginosa

Gram’s negative bacteria - Slime layer

• Have an outer membrane and ‘slime layer’ which repel the stain and disinfectant, therefore are much more difficult to destroy

• Slime layer also provide camouflage to the antigen, which is what is detected by the immune system to identify it as a foreign body

Gram-positive organisms stain violet / purple, for example ..

• Clostridia Spp

• Staphylococcus Spp

• Listeria

Gram-negative organism stain pink / red, for example ..

• Some species of Escherichia coli

• Salmonella

• Pseudomonas

Acid fast bacteria

• Rarely diagnosed in vet practice

• Have a waxy (mycolic acid) outer layer

• Highly resistant to staining and treatment

• Gram positive bacteria - do not stain well with this method

• Ziehl-Neelsen stain required (red on blue background)

• All other organisms stain blue

• Can be zoonotic and cause very significant illness

Acid fast bacteria examples

• Mycoplasma

• Mycobacterium tuberculosis

• M leprae

• Nocardia

Procedure for Ziehl-Neelsen Staining

1 - Apply primary stain of carbolfuchsin for 30 seconds

2 - Heat fix cells to the slide using flame

3 - Decolourise with acid alcohol for 15-20 seconds

4 - Apply counterstain of methylene blue tor 30 seconds then rinse excess stain