Bacterial staining

Made by

Sameh aly gad

Demonstrator of microbiology and immunology

College of pharmacy

Minia university

outlines

Dyes(stains)

Negative staining

Structural staining

Spore stain

Capsule stain

Simple staining

Differential staining

Gram stain

Acid-fast stain

Dyes

Why we need to stain bacteria?

Bacteria are transparent and colorless , so they would be invisible to naked eye if observed under a microscope

thus

bacteria should be stained with certain dyes in order to

visualize bacterial cell or their internal structures using the light microscope.

Dyes (continued)

Dye (stain):

colored organic compound in the form of salt, composed of positive and negative ion, one of these ions

is responsible for colour called chromogen.

Types of Dyes:

Basic dyes

Acidic dyes

Dyes (continued)

Basic dyes:

In which chromogen is the positive ion (cation).

Basic dye has the form: dye+Cl

-

examples include crystal violet, methylene blue and safranin.

Acidic dyes:

In which chromogen is the negative ion (anion).

Acidic dye has the form: Na+dye-

Examples include nigrosin and India ink.

Simple staining

Simple staining is carried out to visualize bacteria and to compare morphological shapes and arrangements of bacterial cells.

In simple stain, the bacterial smear is stained with a single basic dye.

Bacterial cell surface is slightly negative so it tends to bind strongly to the cationic chromogen of basic dyes.

Thus , basic dyes are used to stain bacterial cells

Simple staining procedure

1. Add one loopful of the sample onto a glass slide.

2. Allow it to air-dry.

3. Heat-fix the specimen on the glass slide ,unless the specimen is heat-fixed ,the bacterial smear will wash away during the staining procedure.

4. Staining: crystal violet and wait for 1 min. or safranin and wait for 3-4 min.

5. Wash the smear with tap water to remove the excess stain.

6. Blot dry, then add cedarwood oil and examine under a microscope. Sameh aly gad

Bacterial shapes and arrangement

Bacillus subtilis stained with crystal violet Saccharomyces stained with crystal violet

Staphylococci stained with crystal violet

Negative staining

It is a type of simple staining in which acidic dyes such as India ink or nigrosin are used.

The acidic dye with its negatively charged chromogen will not penetrate the cell due to the repulsion with the negatively charged bacterial surface.

As a result the bacterial cells remain unstained but easily discernible against the coloured background so it is also called indirect stain.

Since no heat is used, negative stain is used to visualize cells that are too delicate to be heat-fixed.

Negatively stained staphylococci

Differential staining

b) Acid fast staina) Gram stain

Gram stain

Most bacteria are classified into two main groups: gram positive and gram negative bacteria.

Comparison between gram positive and gram negative bacteria.

Gram stain (cont…)

It is a differential stain which distinguishes between gram positive and gram negative bacteria depending on the

difference in the cell wall structure.

Principle:

• Gram positive bacteria when treated with a basic dye such as crystal violet , then with iodine (will fix the stain to the cell)

and then subjected to a subsequent treatment with a decolourizing agent such as alcohol/acetone mixture remain

stained with the basic dye and the decolourizer doesn,t

remove the dye ,

Gram stain (cont…)

however, gram negative bacteria when treated similarly will be readily decolourized with the alcohol acetone mixture and

become invisible , thus to render the unstained gram negative bacteria visible, a counterstain is applied.

Why gram positive bacteria resist decolourization by alcohol/acetone mixture but the gram negative ones can not?

Because the cell wall of gram negative bacteria has high lipid content and low peptidoglycan content so when treated with acetone/alcohol mixture dissolves their lipids creating pores

Gram stain (cont…)

In the cell wall through which crystal violet dye will diffuse, and gram negative bacteria become unstained.

On the other hand, gram positive bacteria has high peptidoglycan content and low lipid content so when

treated with the decolourizer, it will dehydrate the cell wall causing it to shrink and the pores to close and thus

the diffusion of the dye will be blocked

Gram stain procedure

Prepare The Smear On a Glass Slide , Air Dry

Heat Fixation

Flood With Crystal Violet (primary stain)

Washing with water

Flood with gram‘s iodine, Washing with water

Decolorization with acetone/alcohol mixture

Washing with water

Flood with safranin(2 ry

stain), washing with water

Blot dry using paper, Examine with the microscope

Gram stain (cont…)

If the specimen contains both gram positive and gram negative bacteria, gram positive ones appear violet while

gram negative ones appear pink to red

Gram stained staphylococci

Type of stain: differential stain

Name of stain: gram stain

Gram reaction: positive gram

Colour: violet

Size: small

Shape: spherical or cocci

Arrangement: grapelike clusters

Name of m.o: Staphylococcus sp.

Gram stained bacillus sp.

Type of stain: differential stain

Name of stain: gram stain

Gram reaction: positive gram

Colour: violet

Size: intermediate

Shape: rod-shaped

Arrangement: chains

Name of m.o: Bacillus sp.

Type of stain: differential stain

Name of stain: gram stain

Gram reaction: positive gram

Colour: violet

Size: large

Shape: oval

Arrangement: single

Name of m.o: Saccharomyces sp.Gram stained saccharomyces

Gram stained Escherichia coli

Type of stain: differential stain

Name of stain: gram stain

Gram reaction: gram negative

Colour: pink

Size: small

Shape: rod shaped

Arrangement: single

Name of m.o: Escherichia coli

Mixture of gram positive staphylococci and gram negative E.coli

Acid-fast stain(Ziehl-Neelsen stain)

Members of the genus mycobacterium are resistant to simple and gram staining procedure due to their thick waxy (fatty) cell wall which makes the penetration of dyes extremely difficult and requires the application of heat.

But once the dye has penetrated, it can not be removed even with the vigorous use of acid alcohol as decolourizing agent, due to this property, these organisms are called acid fast.

The most important human pathogens of them are mycobacterium tuberculosis and mycobacterium leprae.

Acid-fast stain procedure

1. Prepare a smear of the microorganism on a clean slide.

2. Allow the smear to air-dry then fix it by flaming.

3. Place the slide over a steam bath and flood the smear with strong carbol fuchsin and keep it for 5 mins. caution :don

,t

allow stain to dry; replenish stain as needed. also, prevent stain from boiling by adjusting the temperature applied.

4. Allow to cool and then wash with tap water.

5. Decolourize with acid alcohol, till the effluent runs almost clear with a slight red tinge.

Acid-fast stain procedure

6. Wash with tap water 7. Counterstain with methylene blue for 2 mins.8. Wash smear with tap water.9. Blot dry and examine with a microscope.

Acid fast bacteria appear red while non acid fast bacteria appear blue.

Structural stain

b) Capsule staina) Spore stain

c) Flagella stain

Spore stain(Schaeffer-Fulton method)

Some gram positive bacteria especially members of the genera clostridium and bacillus have the ability to form spore(endospore) when exposed to drastic unfavorable conditions such as drying, heating, lack of nutrients and so on.

Spores formed by these bacteria are highly resistant to unfavorable conditions and can remain viable for several years, once the conditions become favorable the spore germinates.

Spore stain(cont..)

Spores don,t accept the stain easily due to their thick

impervious coat and require the use of heating during staining.

Procedure:

1. Prepare smear of the microorganism, allow to dry and the heat-fix to a slide.

2. Place the slide over a steam bath, flood the smear with malachite green dye and keep it for 3-5 mins, replenish smear with malachite green dye if some evaporates and don't allow the stain to boil.

Spore stain(cont..)3. Allow the slide to cool and wash with tap water to remove

excess stain.4. Counterstain with safranin for 2 min.5. Rinse the slide with running tap water to remove excess stain.6. Blot dry with bibulous paper and examine under oil

immersion.

Capsule stain

It is a well organized gelatinous layer that is secreted by the cell, surrounds and adheres to the cell wall.

Capsule is not common to all organisms and the organisms that have a heavy capsule are generally virulent and capable of producing disease.

It protects bacteria against the phagocytic activities of host phagocytes.

It also serves as a barrier against antimicrobials preventing them from entering the cell.

Capsule stain(cont..)

Most capsules are polysaccharides (ex: Streptococcus pneumoniae) in nature but some are glycoproteins or polypeptides.

Since capsule fails to retain standard dyes, it can't be stained by simple staining procedure or even gram stain, but it can be visualized indirectly using negative staining technique.

A basic dye is used to stain the bacterial cell and acidic dye is used to colour the background where the capsule appears as a clear halo zone between the coloured background and the stained cell.

Capsule stain(cont..)

Procedure

Prepare a smear from the encapsulated microorganism( ex. Klebsiella pneumoniae) without heat fixation.

Stain with safranin for 5-6 mins.

Wash with tap water(polysaccharide capsules are water-soluble thus gentle washing is required, otherwise the capsule will be removed with water).

Put a drop of nigrosin at one end of the slide and spread it with another slide.

Allow it to air-dry and examine under oil immersion.

Figure showing indirectly stained capsule (Klebsiella pneumoniae capsule)