Differential Staining Techniques

By Elif TEKİN

Types of Staining

• Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope.

• Simple staining colors all cells of specimen in one type. Only one

stain is used and the aim is creating contrast for examination of cell morphology

• Differential staining colors cells by showing their different composition or

structures.

Differential Staining

– use two or more stains and allow the cells to be categorized into various groups or types.

– at least 2 types of stains are used the first stain is called the primary stain and the

second stain is called counterstain.

– are used for identification or structure examination of bacteria in addition to general morphological examination

Differential Staining

• Gram Staining• Endospore Staining• Capsule Staining

Gram Staining

• Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups.

Gram positive and Gram negative• Basic classification of bacteria is based on the cell

wall structure.• The amount of peptidoglycan, the characteristic

compound found in all true bacterial cell walls, is among one of the differences between the Gram Positive and Gram Negative cell walls.

Gram Staining

• Thick peptidoglycan• 90% peptidoglycan• Teichoic acids• Not many polysaccharides

Gram-positive cell walls Gram-negative cell walls

• Thin peptidoglycan• 5-10% peptidoglycan• No teichoic acids• Outer membrane has

lipids, polysaccharides

Gram StainingThere are 4 conditions to be followed for a valid Gram staining procedure:

Young cultures - must be young within 18-24hrs old (older cultures lose their Gram staining properties due to changes in the CWs as the cells get older)Thin smearthicker or uneven smears will result in uneven stainingand decolorizationFresh reagents - of proper strengthControl cultures - for a known GP bacterium and GN culture (S.aureus & E.coli)

Remember!Too thin smears are obtained usually with NB cultures. Few cells on

the slide to observe and it would be difficult to find them. Also you may lose them easily during staining. It happens when:- The culture is taken from NB or NA that is not dense enough- The culture is taken from upper part of NB tube without shaking - Heat fixation performed less than enoughcells are washed away.

Too thick smears are obtained usually with NA cultures. Too many bacterial cells on the slide. This prevents proper light penetration so lessening visibility. Also it would be difficult to discriminate individual cells and determine their sizes, shapes and structures. It happens when:- More than enough cells are taken from colonies of NA. (less than a single colony is OK)- Culture is taken from bottom part of NB tube without shaking

.

Remember!

• Distorted cells can be observed if heat fixation is done more than enough (3-4 times are OK). So the cells are seen different than their original sizes and shapes. If the smear is heated much more, all the cells coagulates and form a mass of molecules not single cells

Gram Staining ProcedurePreapare smears then;1. Apply few drops of crystal violet to cover the smear for 1 min2. Pour off excess dye and Rinse with dH2O3. Then cover the smear with Gram’s Iodine and leave it 1,5 min.4. Rinse with dH2O.5. Wash with 95% Ethanol (decolorizing agent) until fluid flows

off the smear is colorless 6. Rinse immediately with dH2O.7. Apply safranin to smear and wait 1 min.8. Wash with dH2O and Air-dryObserve under100x objective and Note colors, shapes,sizes and

staining properties of cells.

Gram Staining

Neisseria

Pseudomonas

Streptococcus

Gram StainingGram (-) cell are always seen as Gram (-) but Gram (+) cells can

be seen as Gram (-) due to some experimental errors. This unexpected result is called Gramvariable reaction.

Reasons for Gram-variable reaction:1. Age of the culture stained with Gram staining should be

smaller than 24 h. It should be not damaged by chemicals or not treated by antibiotics.

2. Too thick smears may cause improper decolorization. So smear thickness should be proper.

3. Too much heat fixation causes cells to rupture. In this way Gram (+) cells may lose CV-I and can take Safranin color.

Gram Staining4. Fresh chemicals should be used in proper concentrations.

Applications of low amount or concentrations of Crystal violet, iodine solution or safranin may cause Gram (+) cells stained as Gram (-).

5. Nature, concentration and amount of decolorizing agent is very important. If you apply too much, you cause Gram (+) cells to lose CV-I complex and appear in pink..Also prolonged washing between each step may cause over-decolorization

Endospore Staining

• Spore: structure that can survive for long periods of time in a metabolically inert state

• Produced when environmental conditions are poor

• Do not stain well with simple stains harsher protocol required (i.e. heat)

Endospore Staing (Schaffer and Fulton staining method)

• Malachite Green is used to stain the spore as primary stain. • Steam is applied to enhance penetration of dye to relatively

impermeable spore coat. It has low affinity to ordinary dyes.• After that slides are cooled to trap stain inside, resisting

decolorisation. Rinsing with water decolorizes MG from vegetative cells.

• Safranin is used as counterstain to stain the colorless vegetative part.

• At the end of the staining the spores appear in green and vegetative parts appear in red.

Endospore StainingPrepare smear!1. Put the slide to tripod places in staining rack.2. Cover the smear with Malachite Green and wait1 min.3. Pass flame under slide quickly for many times. It will take

about 3-5 min. Do not dry the smearPay attention to not to boil the smear. Heat it until you observe

vapor after removal of flame4. Cool the slide and Wash with dH2O.5. Apply Safranine and wait for 1 min.6. Wash with dH2O. Then perform blotting. Observe the specimen with 100x objective. Try to differentiate spores and vegetative parts.

Endospore Staining

Capsule StainingCapsule is a polysaccharide-containing material lying outside of

the bacterial cell. It is a kind of glycocalyx secreted by cytoplasm.

The traditional capsule is the one forming a kind of tightmatrix excluding large particles.

Its functions are related mostly with pathogenecity:• It aids attachment of certain pathogenic microorganisms to host.• Engulfment of pathogens by phagocytic cells of immune system

becomes difficult with presence of capsule.• It has role in preventing dessication by binding some water.

Capsule Staining

Negative Staining performed in order to observe capsule lying outside the cell.

Since most capsules are largely water soluble and nonionic, they do not bind ordinary stains.

Also heat application of ordinary smear preparation damages the capsule.

Capsule Staining• Indian ink is used to color the background. Its particles are too

large to pass from capsule so it is excluded.

• Safranin is used to color cells.

So the colorless uniform zone between pink cell and dark background is the capsule.

Not all clear zones are capsules. Shrinkage of cells, withdrawal of ink and cracks cause some clear

zones to form. Capsule is seen as a uniform clear zone.

Capsule Staining Protocol1. Place 1 drop of Indian ink near one edge of a clean slide.2. Aseptically get a loop of culture given to you and mix it with the ink drop on

slide.3. Use a second slide as spreader and spread the mixture first by touching the

short edge of spreader to the mixture.Let the mixture run across the edge and then spread the liquid across the

length of the slide by holding the spreader tilted. 4. Let it air dry completely,no heat fixation and no washing,Heat detoriates capsule5. Cover the smear with Safranin and wait for 1 min.6. Rinse with dH2O.7. Perform blotting and air dry if necessary.8. Observe with 100x objective

Capsule Staining

GRAM STAINING1.Apply few drops of crystal

violet to cover the smear for 1 min

2. Pour off excess dye and Rinse with dH2O

3. Then cover the smear with Gram’s Iodine and leave it 1,5 min.

4. Rinse with dH2O.5. Wash with 95% Ethanol

(decolorizing agent) until fluid flows off the smear is colorless

6. Rinse immediately with dH2O.

7. Apply safranin to smear and wait 1 min.

8. Wash with dH2O and Air-dry

ENDOSPORE STAINING1. Put the slide to tripod places in staining rack.2. Cover the smear with Malachite Green and wait1 min.3. Pass flame under slide quickly for many times. It will take about 3-5 min. Do not dry the smearPay attention to not to boil the smear. Heat it until you observe vapor after removal of flame4. Cool the slide and Wash with dH2O.5. Apply Safranine and wait for 1 min.6. Wash with dH2O. Air dry

CAPSULE STAINING1. Place 1 drop of Indian ink near one edge of a slide.2. Aseptically get a loop of culture given to you and mix it with the ink drop on slide.3. Use a second slide as spreader.

4. Let it air dry completely,no heat fixation and no washing,Heat detoriates capsule5. Cover the smear with Safranin and wait for 1 min.6. Rinse with dH2O.7. Perform blotting and air dry if necessary.

• Group 1,3Gram staining B. cereus, E.coliEndospore staining B. subtilis

Capsule Staining K. pneumonia • Group2

Gram staining B. cereus(old), E.coliEndospore staining B. subtilis

Capsule Staining K. pneumonia• Group 4

Gram staining Serratia marcescens, E.coliEndospore staining B. subtilis

Capsule Staining K. pneumoniae

• Group 5Gram staining K. pneumoniae, E.coli

Endospore staining B. subtilisCapsule Staining K. pneumonia