Microbiology Staining Techniques and Bacterial Identification

Simple Staining

Conventional staining technique using basic dyes in alcoholic or aqueous solutions (1-2% dilution).
Dyes release OH- and accept H+, resulting in a positive charge, thus classified as positive or cationic dyes.
Colors most bacteria by adhering to their negative charge, leaving the background colorless.
Examples: safranin, methylene blue, crystal violet.
A direct staining method using a single stain to make microorganisms visible.
Used to examine organisms' shape, size, and arrangement, aiding in distinguishing groups.
Process: smear preparation, heat fixing, and staining.

Exposure Time of Basic Stains

Methylene blue: 1-2 minutes
Crystal violet: 20-60 seconds
Carbol fuschin: 15-30 seconds
Safranin: 30-60 seconds

Principle of Simple Staining

Basic dye (positive chromophore) attracts to negative cell components like nucleic acids and proteins.
Results in a colored bacterial cell against a colorless background.

Procedure of Simple Staining

Smear preparation
Heat fixing
Staining

A. Smear Preparation

Creating a thin film of bacterial culture on a glass slide.
1. Clean grease-free slide.
2. Add a drop of distilled water at the center.
3. Add inoculum from bacterial culture using a sterilized inoculating loop.
4. Mix inoculum with water to form a thin film by uniformly rotating the loop.

B. Heat Fixing

Moving prepared slides over a Bunsen burner's flame three times, then air dry.
Reasons for heat fixing:
- Fixation of specimen to the glass slide.
- Helps the stain penetrate the smear.

C. Staining of Bacteria

Identifying morphological characteristics through microscopic examination.
1. Add stain to the heat-fixed smear.
2. Allow the stain to stand for 1 minute for penetration.
3. Wash off the glass slide carefully.
4. Blot dry with absorbent paper (do not wipe).
5. Examine under a microscope from low to high power; add oil immersion for 100X objective.

Advantages of Simple Staining

Simple method using a single reagent.
Rapid method, taking 3-5 minutes.
Helps examine bacterial shape, size, and arrangement.
Differentiates bacterial cells from non-living structures.
Useful in preliminary study of bacterial morphological characteristics.

Disadvantages of Simple Staining

Limited information beyond morphological characteristics.

McFadyen Stain (Polychrome Methylene Blue)

Used for capsule staining of Bacillus anthracis.

Composition

Methylene blue 95%: 0.300 gm
Ethyl alcohol: 30.000 ml
Potassium hydroxide: 0.010 gm
Distilled water: 100.000 ml

Directions

Create a thin smear from a small drop of culture on a slide.
Air dry and fix by dipping in absolute alcohol for 30-60 seconds.
Apply a large drop of Polychrome methylene blue stain, spreading with a loop, and leave for 30-60 seconds.
Wash off with water, blot dry, and observe under oil immersion (100X objective).

Principle and Interpretation

Simple and reliable method to confirm the presence of poly-D-glutamyl capsulated B. anthracis.
Polychrome methylene blue contains homologs like azure A and azure B, produced by oxidation during ripening.
Oxidation of methylene blue forms a violet compound.

Differential Stains

Stains that react differently with different cell types, important in taxonomic identification.
Gram stain is the most important and widely used differential stain for bacteria.
Bacteria divided into Gram-positive and Gram-negative based on their reaction to the Gram stain.
Differential response is based on cell wall structure and composition differences.
Acid-fast stain differentiates mycobacteria based on lipid content in their cell wall.
Gram stain indicates Gram-positive (purple) or Gram-negative (pink) cell walls.
Gram-negative organisms lose primary stain after decolorization and appear pink after counterstaining.
Developed by Hans Christian Gram in 1884, cornerstone of bacterial identification.

Gram Stain Groups

Gram-positive bacteria: thick peptidoglycan layer (90% of cell wall), stains purple.
Gram-negative bacteria: thin peptidoglycan layer (10% of cell wall, high lipid content), stains red/pink.

Exceptions for clinically important bacteria

Intracellular bacteria (e.g., Chlamydia).
Bacteria lacking a cell wall (e.g., Mycoplasma).
Bacteria too small for light microscopy (e.g., Spirochetes).

Steps of Gram Staining

Fixation of clinical materials to the slide by heating or methanol (methanol preserves host cell morphology).
Application of primary stain (crystal violet): all cells stain blue/purple.
Application of mordant (iodine): forms crystal violet-iodine (CV-I) complex; cells remain blue.
Decolorization: Gram-positive and Gram-negative cells are differentiated.
Organic solvent extracts blue dye complex from lipid-rich Gram-negative bacteria.
Application of counterstain (safranin): stains decolorized Gram-negative cells red/pink; Gram-positive remain blue.

Preparation of Gram Stain Reagents

Requires simultaneous use of chemical reagents and washing.
Primary stain: crystal violet
Mordant: iodine
Decolorizer: ethanol or acid-alcohol
Counter stain: safranin or dilute carbol-fuchsin
Stained slide is observed under oil immersion (100x) using a bright field microscope.

Crystal Violet Preparation

Dissolve 2.0 g certified crystal violet in 20.0 ml of 95% ethyl alcohol.
Dissolve 0.8 g ammonium oxalate in 80.0 ml distilled water.
Mix the two solutions and let stand overnight at room temperature ( $25°C$ ).
Filter through coarse filter paper before use.
Store at room temperature ( $25°C$ ).

Gram's Iodine Preparation

Grind 1.0 g iodine (crystalline) and 2.0 g potassium iodide in a mortar.
Add to 300.0 ml distilled water.
Store at room temperature ( $25°C$ ) in a foil-covered bottle.

Decolorizer

Acetone, 95% ethanol, or ethanol-iodine can be used.

Acetone-Alcohol Decolorizer

Mix distilled water with absolute ethanol or methanol.
Transfer to a screw-cap bottle.
- Caution: Ethanol and methanol are flammable.
Measure and add acetone to the alcohol solution.

Caution: Acetone is highly flammable.

Label as Highly Flammable and store safely at room temperature.

Counterstain

Safranin and dilute carbol-fuchsin are common.

Principle of Gram Stain

Differences in cell wall composition account for Gram staining differences.
Gram-positive cell wall: thick peptidoglycan layer with teichoic acid cross-linking, resists decolorization.
Crystal violet dissociates into CV+ and Cl- ions, penetrate cell walls of both types, staining cells purple.
Iodine interacts with CV+ to form large CV-I complexes.
Decolorizing agent interacts with lipids in cell membranes.
Gram-negative outer membrane (lipopolysaccharide) is lost, exposing thin peptidoglycan layer.
Ethanol makes Gram-negative cell walls leaky, washing out CV-I complexes.
Gram-positive cell walls: thick, dehydrated by ethanol, trapping CV-I complexes.
After decolorization, Gram-positive cells are purple, Gram-negative are revealed by safranin counterstain and stain pink.

Procedure of Gram Staining

Smear Preparation: Fix material on a slide with methanol or heat; cool if heat-fixed.

Gram Staining Protocol

Flood air-dried, heat-fixed smear with crystal violet for 1 minute; smear quality affects results.
Wash gently with tap water for 2 seconds.
Flood with Gram’s iodine (mordant) for 1 minute.
Wash gently with tap water for 2 seconds.
Flood with decolorizing agent (acetone-alcohol) for 10-15 seconds, drop by drop until running clear.
Flood with safranin (counterstain) for 30 seconds to 1 minute.
Wash gently until no color in effluent, then blot dry.
Observe under oil immersion (100x) using a bright-field microscope.

Results

Gram-negative bacteria stain pink/red.
Gram-positive bacteria stain blue/purple.

Reporting Gram Smears

Numbers of bacteria: many, moderate, few, or scanty.
Gram reaction: Gram-positive or Gram-negative.
Morphology: cocci, diplococci, streptococci, rods, or coccobacilli; intracellular organisms noted.
Presence and number of pus cells.
Presence of yeast and epithelial cells.

Variations in Gram Staining Results

Limitations: Mycobacteria stain weakly, and Mycoplasma, Rickettsiae, and Chlamydiae may not take up dyes or are too small.
Sensitivity: Detection requires $10^4$ to $10^5$ organisms per milliliter.

Variations in Gram Reaction

Gram-positive bacteria may lose crystal violet retention due to:
- Cell wall damage from antibiotics or heat fixation.
- Over-decolorization.
- Old iodine solution (yellow instead of brown).
- Old culture.
Thick smears may prevent full decolorization of Gram-negative bacteria.

Importance in Anaerobic Bacteriology

Same procedure and reagents, but safranin counterstain is left on for 3-5 minutes; 0.5% aqueous basic fuchsin can also be used.

Basic Classification of Medically Important Bacteria

Ziehl-Neelsen (ZN) method of acid-fast staining is used to stain Mycobacterium species including M. tuberculosis, M. ulcerans, and M. leprae and nontuberculous mycobacteria (NTM).
Acid-fast bacilli (AFB) in stained smears provide initial evidence of mycobacteria.
Smear microscopy is the quickest and easiest procedure.

Acid Fast Bacillus (AFB) in Z.N Smear

The cell wall of Mycobacteria contains high concentrations of lipid, making them waxy, hydrophobic, and impermeable to routine stains. They are resistant to acid and alcohol, described as acid-fast bacilli (AFB).

Procedures for Acid-Fast Staining:

Carbolfuchsin methods: Ziehl-Neelsen and Kinyoun methods (light /bright field microscope)
Fluorochrome procedure: Auramine-O or auramine-rhodamine dyes (fluorescent microscope).

Principle of Ziehl-Neelsen Method of Acid-Fast Staining

Mycobacteria, which do not stain well by Gram stain, are stained with carbol fuchsin combined with phenol.

In the ‘hot’ ZN technique, the phenol-carbol fuchsin stain is heated to enable the dye to penetrate the waxy mycobacterial cell wall.
In the ‘cold’ technique known as Kinyoun Method, stain are not heated but the penetration is achieved by increasing concentration of basic fuchsin and phenol and incorporating a ‘wetting agent’ chemical.
The stain binds to the mycolic acid in the mycobacterial cell wall.
After staining, an acid decolorizing solution is applied. This removes the red dye from the background cells, tissue fibers, and any organisms in the smear except mycobacteria which retain the dye and are therefore referred to as acid-fast bacilli (AFB).

Following decolorization, sputum smear is counterstained with malachite green, or methylene blue which stains the background material, providing a contrast color against which the red AFB can be seen.

Among the Mycobacterium species, M. tuberculosis and M. ulcerans are strongly acid fast. When staining specimens for these species, a 3% v/v acid alcohol is used to decolorize the smear, where as M. leprae is only weakly acid fast. 0.5-1% v/v decolorizing solution is therefore used for M. leprae smears and also different staining and decolorizing time.

Note: 0.5% Acid alcohol or 5% Sulphuric acid is used for Atypical AFB because they (eg. Mycobacterium leprae, Nocardia asteroides) are much less acid and alcohol fast than Mycobacterium tuberculosis bacilli.

Sample Collection & Preparation:

Due to overnight accumulation of secretions, first morning specimens are more likely to yield better recovery of AFB.

Direct Smear: Smear prepared directly from a patient specimen prior to processing.
Indirect Smear: Smear prepared from a processed specimen after centrifugation (is used to concentrate the material)

Reagents Required:

Carbol fuchsin stain (filtered)
Acid alcohol 3% v/v (or 20% sulfuric acid)
Malachite green 5 g/l (0.5% w/v) or Methylene blue, 5g/l

Procedures

Spread the sputum evenly over the central area of the slide using a continuous rotational movement. The recommended size of the smear is about 20 mm by 10 mm.
Place slides on the dryer with smeared surface upwards, and air dry for about 30 minutes.
Heat fix dried smear.
Cover the smear will carbol fuchsin stain
Heat the smear until vapor just begins to rise (i.e. about 60 degree Celsius). Do not overheat (boil or dry). Add additional stain if necessary. Allow the heated stain to remain on the slide for 5 minutes.
Wash off the stain with clean water.
Cover the smear with 3% v/v acid alcohol for 2-5 minutes (or 20% sulfuric acid) or until the smear is sufficiently decolorized, i.e. pale pink. Note: Check to see that no more red color runs off the surface when the slide is tipped. Add a bit more decolorizer for very thick slides or those that continue to “bleed” red dye.
Wash well with clean water
Cover the stain with malachite green stain for 1-2 minutes
Wash off the stain with clean water
Wipe the back of the slide clean, and place it in a draining rack for a smear to air dry (DO NOT BOLT DRY).
Examine the smear microscopically, using the 100x oil immersion objective (10X eyepiece for a total of 1000X magnification) and scan the smear systematically.

Procedural Note:

Heating carbol fuchsin during the staining process involves working with flammable or caustic reagents. Take appropriate measures, such as using PPE and a well-ventilated area to minimize the risk of chemical exposure or fire hazard.

*Heat fixation of untreated specimen may NOT kill *M. tuberculosis* (exercise care when handling slides) whereas alcohol fixation is bactericidal.*
Acid alcohol is flammable, therefore use it with care.
Take great care while heating carbol fuchsin (as the staining rack may contain flammable chemicals) to reduce the fire risk.
Slides must not touch each other when placed on staining rack to prevent the transfer of material from one slide to another.

Specific Stains

India Ink Method (Capsule Visualization)

Most bacterial capsules are composed of polysaccharide however some genera produce polypeptide capsules.
The polymers which make up the capsule tend to be uncharged and as such they are not easily stained. For this reason, we use a negative stain to visualize them. That is, we use a stain which stains the background against which the uncolored capsule can be seen.
Burri´s India ink method, uses India ink to color the background and crystal violet (or safranin) to stain the bacterial cell.

Capsule Stain Procedure

Place a single drop of India ink on a clean microscope slide, adjacent to the frosted edge.
Using a flamed loop and sterile technique, remove some K. pneumoniae (or the organism you want to stain) from your tube or plate and mix it into the drop of India ink.
Place the end of another clean microscope slide at an angle to the end of the slide containing the organism. Spread out the drop out into a film.
Allow the film to air dry.
Saturate the slide with crystal violet for 1 minute.
Rinse the slide gently with water.
Allow the slide to air dry.
Observe the slide under the microscope

Results:

The background will be dark. The bacterial cells will be stained purple. The capsule (if present) will appear clear against the dark background

Laboratory diagnosis of some pathogenic bacteria

1. Staphylococci

1.1 GRAM STAIN

Staphylococcus is a genus of bacteria that is characterized by a round shape (coccus or spheroid shaped), Gram-positive (purple), and found as either single cells, in pairs, or more frequently, in clusters that resemble a bunch of grapes.

1.2 STAPHYLOCOCCI - BLOOD AGAR CULTURE

On blood agar, S. aureus usually displays a light to golden yellow pigment, whereas S. epidermidis has a white pigment and S. saprophyticus either a bright yellow or white pigment. However, pigmentation is not always a reliable characteristic.
On blood agar, S. aureus is usually beta hemolytic, S. epidermidis and S. saprophyticus are almost always nonhemolytic.

1.3 Catalase Test

Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using a loop or sterile wooden stick
Place a drop of 3% $H2O2$ on to the slide and mix.
A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling.
A negative result is no bubbles or only a few scattered bubbles.

*Equation: $H2O2 \longrightarrow H2O + O2$

1.4 Mannitol Salt Agar Culture (MSA)

MSA is a selective and differential media used for the isolation of Staphylococci from mixed cultures.

MSA Components

5% NaCl – selects for species of Staphylococcus.
This concentration of salt is too high for most other bacteria to withstand and, therefore, inhibits their growth.
Mannitol fermentation produces acid end products which turn the medium yellow.
Yellow indicates mannitol positive and no color change indicates mannitol negative.
Phenol red pH indicator – yellow in acid pH (the same indicator that is used in phenol red carbohydrate fermentation broths).
On MSA, only pathogenic Staphylococcus aureus produces small colonies surrounded by yellow zones. The reason for this color change is that S. aureus have the ability to ferment the mannitol, producing an acid, which changes the indicator color from red to yellow.
The growth of other types of bacteria is usually inhibited. This growth differentiates S. aureus from S. epidermidis, which forms colonies with red zones.

Expected Results

On MSA, pathogenic S. aureus ferments mannitol, thereby changing the color of the medium from red to yellow
S. epidermidis grows on MSA but does not ferment mannitol (media remains light pink in color, colonies are colorless).

1.5. Baird Parker Agar culture

Baird Parker Agar medium is formulated on the principle that staphylococci are able to reduce tellurite to tellurium and to detect lecithinase from egg lecithin.
Lithium chloride and potassium tellurite acts as inhibitor agent for contaminating microflora. The tellurite additive is toxic to egg yolk-clearing strains other than S. aureus and imparts a black color to the colonies. Egg yolk tellurite enrichment is added as supplement.
On 18-24 hrs incubation, colonies of Staphylococcus aureus appear black and shiny, with a fine white rim, surrounded by a clear zone. Upon further incubation, for 48 hrs, an opaque zone is developed around colonies, which can be due to lipolytic activity.

1.6. Coagulase Test

Coagulases are enzymes that clot blood plasma by a mechanism that is similar to normal clotting.
This enzyme is a good indicator of the pathogenic potential of S. aureus.
Most strains of S.aureus produce one or two types of coagulase; free coagulase and bound coagulase.
Free coagulase is an enzyme that is secreted extracellularly and bound coagulase is a cell wall associated protein.
Free coagulase can be detected in tube coagulase test and bound coagulase can be detected in slide coagulase test. In the test, the sample is added to rabbit plasma and held at 37° C for a specified period of time.

Detection of Bound Coagulase - Slide Test

Divide the slide into two sections with grease pencil. One should be labeled as „test” and the other as „control”.
Place a small drop of distilled water on each area.
Emulsify one or two colonies of Staphylococcus on blood agar plate on each drop to make a smooth suspension.
The test suspension is treated with a drop of citrated plasma and mixed well with a needle.
Do not put anything in the other drop that serves as control. The control suspension serves to rule out false positivity due to auto agglutination.
Clumping of cocci within 5-10 seconds is taken as positive.
Some strains of S. aureus may not produce bound coagulase, and such strains must be identified by tube coagulase test

Detection of Free Coagulase Tube Coagulase Test

Most strains of S. aureus produce one or two types of coagulase; free coagulase and bound coagulase. The free coagulase secreted by S. aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma.

Three test tubes are taken and labeled “test”, “negative control” and “positive control”.
Each tube is filled with 1 ml of 1 in 10 diluted rabbit plasma.
To the tube labeled test, 0.2 ml of overnight broth culture of test bacteria is added.
To the tube labeled positive control, 0.2 ml of overnight broth culture of known S. aureus is added.
To the tube labeled negative control, 0.2 ml of sterile broth is added.
All the tubes are incubated at $37°C$ .
Positive result is indicated by gelling of the plasma, which remains in place even after inverting the tube.
If the test remains negative until four hours at $37°C$ , the tube is kept at room temperature for overnight incubation.

Samples must be observed for clotting within 24 hours. This is because some strains that produce coagulase also produce an enzyme called fibrinolysin, which can dissolve the clot. Therefore, the absence of a clot after 24 hours is no guarantee that a clot never formed. The formation of a clot by 12 hours and the subsequent disappearance of the clot by 24 hours could produce a so-called false negative if the test were only observed at the 24-hour time.

2. STREPTOCOCCI

2.1 GRAM STAIN

The genus Streptococcus is a diverse collection of Gram-positive cocci typically arranged in pairs or chains (in contrast to the clusters formed by Staphylococcus). Streptococcus pneumoniae are lancet shaped (ovoid) cocci in short chains, diplococci and single cocci

2.2 STREPTOCOCCI - BLOOD AGAR CULTURE

The classification of species within the genus is complicated because three different schemes are used: hemolytic patterns: complete (β) hemolysis, incomplete (α) hemolysis, and no hemolysis (γ);

Alpha-hemolysis

Streptococcus pneumoniae, Streptococcus salivarius, viridans are referred to collectively as viridans streptococci, a name derived from viridis, referring to the green pigment formed by the partial, α-hemolysis of blood agar. Encapsulated, virulent strains of S. pneumoniae often forming highly mucoid, glistening colonies (production of capsular polysaccharide) surrounded by a zone of α -hemolysis. When α-hemolysis is present, the agar under the colony is dark and greenish. Streptococcus pneumoniae and a group of oral streptococci (Streptococcus viridans or viridans streptococci) display alpha hemolysis. This is sometimes called green hemolysis because of the color change in the agar. Alpha hemolysis is caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin to green methemoglobin. Alpha-hemolytic colonies with depressions in their centers are characteristic of pneumococci. Some strains produce high amount of capsular polysaccharide which gives glistening appearance.

Beta Hemolysis

Streptococcus pyogenes, or Group A beta-hemolytic Streptococci (GAS), and Streptococcus agalactiae, or Group B beta-hemolytic Streptococci (GBS) blood agar cultures display beta hemolysis. Beta hemolysis (β-hemolysis),sometimes called complete hemolysis, is a complete lysis of red blood cells in the media around and under the colonies, the area appears lightened (yellow) and transparent.

Gamma Hemolysis

If an organism does not induce hemolysis, the agar under and around the colony is unchanged, and the organism is called non-hemolytic or said to display gamma hemolysis (γ-hemolysis). Enterococcus faecalis (formerly called Group D Streptococci) displays gamma hemolysis

2.3 STREPTOCOCCI - SEROLOGIC PROPERTIES TESTING

a. Bacitracin Sensitivity Test (β-hemolytic streptococci Group A, GAS)

Bacitracin is a polypeptide antibiotic interfering with the synthesis of peptidoglycan, a unique chemical fabric bacteria include in their cell walls.
Bacitracin is useful in helping identify Streptococci and other Gram-positive bacteria.
This test determines whether the bacterium is either sensitive (susceptible) to bacitracin or resistant to the drug.
Principle: Bacitracin test is used to determine the effect of a small amount of bacitracin (0.04 U 6) on an organism. Streptococcus pyogenes (GAS) is inhibited by the small amount of bacitracin in the disk (visible zone of inhibition of growth); while other beta-hemolytic streptococci usually are not.

b. CAMP Test (β-hemolytic streptococci Group B, GBS)

S. agalactiae is the only species that has the group B antigen.
CAMP - it is an acronym for