General Microbiology LaboratoryGeneral Microbiology Laboratory

Bacterial StainsBacterial Stains

OverviewOverview

In our laboratory, bacterial morphology (form and structure) may be examined in two ways:

1. by observing living unstained organisms (wet mount).2. by observing killed stained organisms. Besides being very small, bacteria are also almost

completely transparent, colorless and featureless in their natural states.

Microscopy solve the size issue. Staining can make the structures of bacteria more

pronounced. Since bacteria are almost colorless and therefore show

little contrast with the broth in which they are suspended, they are difficult to observe when unstained. Staining microorganisms enables one to:

1: see greater contrast between the organism and the background,

2: differentiate various morphological types (by shape, arrangement, gram reaction, etc.),

3: observe certain structures (flagella, capsules, endospores, etc.).

What is a stain?

A stain (or dye) usually consists of a chromogen and an auxochrome. Reaction of a benzene derivative with a coloring agent (or chromophore) forms a chromogen. The auxochrome imparts a positive or negative charge to the chromogen, thus ionizing it. The ionized stain is capable of binding to cell structures with opposite charges.

Example of a dye:Methylene Blue ChlorideMBCl MB+ + Cl-

Basic stains (positive stain) are cationic; when ionized, the chromogen exhibits a positive charge. Basic stains bind to negatively charged cell structures like nucleic acids. Methylene blue, crystal violet and carbolfuchsin are common basic stains.

Acidic stains (negative stain) are anionic; when ionized, the chromogen exhibits a negative charge. Acidic stains bind to positively charged cell structures like proteins. Picric acid, eosin and nigrosin are common acidic stains.

Positive stains: Dye binds to the specimen Negative stains: Dye does not bind to the specimen, but rather around

the specimen.

Type of staining in Micro labType of staining in Micro lab

1. Simple stain

2. Differential Stain• Gram stain

• Acid fast Stain

3. Special stain• Capsular stain

• Endospore stain

• Flagellar stain

Part OnePart One

Simple stainSimple stain

Simple stainSimple stain

In this exercise, we will use simple stains to show the general structures of some bacteria. Usually, a single basic stain is used in the procedure. Simple stains do not usually provide any data for identification of the bacterium; they simply make the bacterium easier to see. • To observe basic external structures of cell with

bright field scope (cellular morphology)

MethodMethod

1. Obtain broth cultures of the bacteria.2. Using an inoculating loop, remove a loopful of suspension

from one of the tubes. Remember to use sterile technique.

3. Smear the bacteria across the center of the slide with the loop. If the bacterial suspension is very thick, add a drop of water and mix the bacteria and the water on the slide.

MethodMethod

4. Allow the smear to completely air dry.• Air dry first to prevent lysis (boiling)

5. Heat-fix the smear by quickly passing the slide through a Bunsen burner flame three times. This causes partial melting of the cell walls and membranes of the bacteria, and makes them stick to the slide. Do not overheat the slide as this will destroy the bacteria.

Heat Fixing• Kill.• Stops autolysis.• Adherence to slide.• Dye taking

6. Cover the smear with a few drops of one of the stains. Allow the stain to remain for the following periods of time:Carbolfuchsin- 15-30 seconds.Methylene blue- 1-2 minutes.Nigrosin- 20-60 seconds.

7. Gently rinse the slide by holding its surface parallel to a gently flowing stream of water.

8. Gently blot the excess water from the slide with bibulous paper. Do not wipe the slide. Allow the slide to air dry.

Observe the slide under the microscope with air and oil lenses.

A coverslip is not required. Repeat this process with the other bacteria and stains. Note the differences between the various types of stains and their appearances

Summary of simple stainSummary of simple stain

What to do???What to do???

DRAW what you see under the microscopeTake not of

• Shape• Color• Arrangement• Size of single cells

The simple staining makes it possible to see bacteria clearly, but it does not distinguish between organisms of similar morphology.

Part twoPart two

Differential stainDifferential stain

1.1. gram staingram stain

2.2. Acid fast stainAcid fast stain

Gram stainGram stain

Differential stain (Hans Christian Gram, a Danish doctor ). He developed a new method to stain bacteria so they can be visible in specimen samples.

The most important stainDifferentiate bacteria into two large groups (the Gram

Positive and the Gram negative)

Almost all bacteria are described by their Gram stain characteristics.

Based on differences of Cell wall structures

Importance of Gram StainImportance of Gram Stain

This staining method is still valuable today. • It is used in bacterial identification. • It is of great importance in diagnosis of infectious diseases

in culture and directly from clinical samples. • For instance, the majority of Gram-positive organisms are

susceptible to penicillin, while gram-negative bacteria are resistant to this antibiotics.

• It is also valuable to microbiologists, who can plan their culture procedures based on their knowledge of the bacterial forms they have seen in the specimen.

The numerous modifications of Gram’s original method are based on the concentration of the dyes, length of staining time for each dye, and composition of the Decolorizer.

Theory behind Gram stainTheory behind Gram stain

Reagents for Gram StainReagents for Gram Stain

Crystal Violet (purple).• Primary stain; positive stain• Stains cell wall purple

Iodine• Mordant• Combines with primary stain to form an insoluble complex that gets

trapped in thicker peptidoglycan layers Ethanol

• Decolorizer• CV-I complex washed out of Gram negative organisms because it

cannot be trapped by peptidoglycan layer; flows right through outer membrane

Safranin (pink)• Counterstain• Simple positive stain that provides contrasting dye for decolorized cells

(Gram negative)• Stains all cells, but only the negative ones actually appear pink.

Procedures and eventsProcedures and events

When slides are dry, label them as shown:

Examine all slides under oil with the oil-immersion objective.

Gram positive bacilliGram positive bacilli

Gram Positive cocciGram Positive cocci

Gram negative CocciGram negative Cocci

YeastYeast

ArtifactsArtifacts

Crystal violet precipitate on epithelial cell:

May be confused with Gram positive cocci

Crystal violet precipitate crystal on gram stain

Gram stainGram stain

Errors during stainingErrors during staining

Never ever used old culture.Time of Decolorizer:

• Over: G + see as G -.• Low: G- see as G +.

Time of fixation:• Over: G + see as G -.• Low: no sample on slide.

The acid-fast stain (modified Ziel-Neelsen method). The acid-fast stain (modified Ziel-Neelsen method).

The acid-fast stain is another differential

staining method. In this case, the target cells are usually members of

the genus Mycobacterium. The cell walls of these bacteria contain an unusually

high concentration of waxy lipids, thus making conventional simple stains and Gram stains useless.

The genus Mycobacterium contains two important human pathogens, M. tuberculosis and M. leprae, which cause tuberculosis and leprosy, respectively.

Acid Fast ReagentsAcid Fast Reagents

Carbolfuchsin (red), a phenolic stain: is the primary stain in the acid-fast test. It is soluble in the lipids of the mycobacterial cell wall.

Heating the specimen, or adding a wetting agent such as Tergitol,

increases the penetration of the carbolfuchsin.

Following application of the carbolfuchsin, the specimen is cooled and decolorized with a solution of 3% hydrochloric acid and 95% ethanol (acid-alcohol).

Since carbolfuchsin is more soluble in waxy cell lipids than in acid-alcohol, the acid-alcohol removes the carbolfuchsin from non-acid-fast organisms, but not from acid-fast organisms. Following decolorization, the sample is counterstained with methylene blue which Cannot penetrate mycolic acid; provides contrast to non acid fast cells.

Procedures Procedures

1. Prepare a smear organism and a on glass slides.

2. Allow the slides to air dry, and then heat fix the organisms.

3. Apply enough of carbolfuchsin with Tergitol to cover the bacteria. Allow it to set for five minutes. (Kinyoun stain)

ProceduresProcedures

4. (Alternate) zielh nelson If Tergitol is not available, apply enough carbolfuchsin to cover the bacteria. Place the slide on a pre-warmed hot plate set on low for 8 minutes. Do not allow the stain to evaporate or Boil. Add additional stain, if necessary. Remove the slide and allow it to cool.

5. Rinse the slide with acid-alcohol (15-20 sec), drop by drop, just until the alcohol runs clear.

6. Gently rinse the slide with water.

7. Apply enough methylene blue to cover the bacteria. Allow it to set for 30 sec.

8. Gently rinse the slide with water.

9. Blot (don't wipe) the slide dry with bibulous paper. Allow the slide to air dry.

10. Examine the slide under oil immersion. Positive organisms will appear pink or red; negative organisms will appear blue.

Summary of Acid fast stainSummary of Acid fast stain

Under the microscopeUnder the microscope

Acid Fast bacilli (red) mixed with non acid fast (blue cocci

Acid Fast bacilli (red)Non Acid Fast bacilli (blue)

Special stainsSpecial stains1.1. Capsular stainCapsular stain2.2. Flagellar stainFlagellar stain

3.3. Endospore stainEndospore stain

Part 3Part 3

Special stainSpecial stain

Emphasize certain cell parts

Some bacteria have characteristic surface structures (such as capsules or flagella) and internal components (e.g., endospores) that may have taxonomic value for their identification. When it is necessary to demonstrate whether or not a particular organism possesses a capsule, is flagellated, or forms endospores, special staining techniques must be used.

Bacterial endosporesBacterial endospores

Resting structures formed by some bacteria for survival during adverse environmental conditions (nutrient limitation or extreme environments)

The endospore is a highly resistant differentiated bacterial cell that are highly resistant to heat, boiling and drying out and are difficult to destroy

Endospores can remain dormant indefinitely ((not reproductive), but germinate quickly when the appropriate trigger is applied

Metabolically inactive Stable for years Endospores differ significantly from the vegetative , or

normally functioning, cells Formed by Gram-positive bacteria (e.g. Bacillus, Clostridium)

Endospore structureEndospore structure

Medically significant spore formersMedically significant spore formers

Bacteria disease

Bacillus anthracis anthrax

Clostridium botulinum botulism

Clostridium perfringens gas gangrene

Clostridium tetani tetanus

Staining proceduresStaining procedures

1.1. Malachite greenMalachite green is the primary stain .which is placed on blotting paper over the smear gently heating over a warm water bath to penetrate the spore coat.

2. The bacteria are decolorized with water.water. leaves the endospores green as the stain is driven into the endospore . The malachite green is washed out of the vegetative cells with the water.

3. It is then counterstained with safranin safranin.

Do not allow the stain evaporate. to prevent formation of metallic sheet

IllustrationIllustration

Under microscopeUnder microscope

Problems Interpreting Endospore StainProblems Interpreting Endospore Stain

It should be noted that any debris on the slide can also take up and hold the green stain. Everything that ends up green on the slide is not necessarily an endospore. Endospores are small and typically oval. Large or irregular globs of green on the slide may be artifacts.

Acid-fast cells, such as members of Mycobacterium and Nocardia have waxy molecules in their cell wall that will take up and retain the malachite green stain when subjected to the endospore staining process.

Endospores killed when dry heat is applied at high temperatures or for long periods, by steam heat under pressure (in the autoclave), or by special sporicidal (endospore-killing) disinfectants.

Capsular stainCapsular stain

What is Capsule?What is Capsule?

Capsules are structures composed of carbohydrate or glycoprotein that lay outside of an organism's cell wall and thus are in direct contact with the environment. Many bacteria produce capsules under the right conditions.

Functions of a capsuleFunctions of a capsule

1. Protect the cell from desiccation (drying)2. Protect the cell from phagocytes (being

engulfed by white blood cells)3. Provide a food reserve when certain organic

compounds are in excess. 4. A virulence determinant of pathogenic

microbes5. They serve as binding or adhesion agents for

sticking cells together and/or to a surface such as a rock in flowing stream or a tooth

Theory behind capsule stainTheory behind capsule stain

Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces

Because most capsule materials are water soluble, simple stains will not adhere to them.

In this stain we use acidic and basic dyes: Acidic dye as India Ink and Nigrosen use to stain the

background of the slide but basic dye as methylene blue and crystal violet use to stain the cell

ImportantImportant

Older cultures are more likely to exhibit capsule production. 

When performing a capsule stain on your unknown, be sure the culture you take your sample from is at least five days old.

This stain is used for direct microscopic examination of capsules of microorganisms.

The India ink gives a semi opaque background against which the clear capsules can be easily visualized.

ProceduresProcedures

1. Use an inoculating needle to suspend the organism in a drop of India Ink at one end of the slide.

2. Place the short end of a clean microscope slide into the suspension and spread the mixture across the slide to form a thin layer.

3. Allow to air dry. Do not heat fix.4. Cover the smear with methylene blue for 2-3

minutes. 5. Rinse gently with water and allow to air dry.6. Examine with oil immersion.7. Diagram the appearance of the organism.

Interpretation

Capsules appear as clear zones (halos) around the refractile organism.Examples: Bacteria with capsules: Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas .

End of lecture