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The structure of the microbial cell. Control questions  1.⁠ ⁠The structure of the microbial cell. Morphology of bacteria, size of bacterial  2.⁠ ⁠What are the differences between prokaryotic and eukaryotic cells? (cell wall, cytoplasmic membrane, cytoplasm, ribosomes, mesosomes, intracytoplasmic inclutions, nucleus, slime layer and capsule, flagella, plasmids etc.)  3.⁠ ⁠Preparation for microscopy of living microorganisms.  4.⁠ ⁠The crushed drop. Hanging drop.  5.⁠ ⁠Drying and fixing the smear.  6.⁠ ⁠Dyeing techniques the smear.  7.⁠ ⁠A simple staining method.  8.⁠ ⁠Negative staining.  9.⁠ ⁠Silver impregnation 10.⁠ ⁠Differential staining methods (Gram staining, Staining of acid-proof bacteria.) 11.⁠ ⁠Intracellular structures, Staining of cytoplasmic inclusions. 12.⁠ ⁠Capsules and its staining methods 13.⁠ ⁠Spore formation, and its staining methods 14.⁠ ⁠Mobility of bacteria, study of motility In about 4 pages

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This covers the basic morphology, ultrastructure, and laboratory microscopy of bacteria.
  1. Bacterial cell structure and morphology
    Shapes, arrangements, dimensions, and the general organization of a bacterial cell.
  2. Prokaryotic versus eukaryotic cells
    Comparison of cell wall, plasma membrane, cytoplasm, ribosomes, nucleus, organelles, and genetic material.
  3. Bacterial surface structures
    Slime layer, capsule, flagella, and their functions.
  4. Internal bacterial components
    Nucleoid, plasmids, mesosomes as described in some teaching texts, storage inclusions, and ribosomes.
  5. Living-microorganism microscopy
    Preparation and examination of wet mounts, including crushed-drop and hanging-drop preparations.
  6. Smear preparation
    Making, air-drying, and heat or chemical fixation of bacterial smears.
  7. Basic staining methods
    Simple staining, negative staining, and silver impregnation.
  8. Differential staining
    Gram stain and acid-fast staining, including their principles and interpretation.
  9. Special-structure staining
    Demonstration of cytoplasmic inclusions, capsules, and bacterial spores.
  10. Bacterial motility
    Mechanisms of movement and laboratory methods for observing motility.
Bacteria are prokaryotes: they lack a membrane-bound nucleus and organelles, but possess a plasma membrane, cytoplasm, 70S ribosomes, and a nucleoid containing their chromosome. Their cell envelope and optional structures, especially capsules, spores, and flagella, affect survival, pathogenicity, identification, and staining behavior.
Microscopy depends on suitable specimen preparation. Living preparations show true motility and cell appearance, while fixed smears can be stained safely and consistently. Different stains exploit differences in cell-wall composition or highlight otherwise poorly visible structures.
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Which topic should I expand into a detailed 4-page study note?

This covers the basic morphology, ultrastructure, and laboratory microscopy of bacteria.
  1. Bacterial cell structure and morphology
    Shapes, arrangements, dimensions, and the general organization of a bacterial cell.
  2. Prokaryotic versus eukaryotic cells
    Comparison of cell wall, plasma membrane, cytoplasm, ribosomes, nucleus, organelles, and genetic material.
  3. Bacterial surface structures
    Slime layer, capsule, flagella, and their functions.
  4. Internal bacterial components
    Nucleoid, plasmids, mesosomes as described in some teaching texts, storage inclusions, and ribosomes.
  5. Living-microorganism microscopy
    Preparation and examination of wet mounts, including crushed-drop and hanging-drop preparations.
  6. Smear preparation
    Making, air-drying, and heat or chemical fixation of bacterial smears.
  7. Basic staining methods
    Simple staining, negative staining, and silver impregnation.
  8. Differential staining
    Gram stain and acid-fast staining, including their principles and interpretation.
  9. Special-structure staining
    Demonstration of cytoplasmic inclusions, capsules, and bacterial spores.
  10. Bacterial motility
    Mechanisms of movement and laboratory methods for observing motility.
Bacteria are prokaryotes: they lack a membrane-bound nucleus and organelles, but possess a plasma membrane, cytoplasm, 70S ribosomes, and a nucleoid containing their chromosome. Their cell envelope and optional structures, especially capsules, spores, and flagella, affect survival, pathogenicity, identification, and staining behavior.
Microscopy depends on suitable specimen preparation. Living preparations show true motility and cell appearance, while fixed smears can be stained safely and consistently. Different stains exploit differences in cell-wall composition or highlight otherwise poorly visible structures.Please choose one section for the detailed 4-page note:
  1. Bacterial cell structure and morphology
  2. Prokaryotic versus eukaryotic cells
  3. Preparation of specimens for microscopy: wet mounts, crushed drop, hanging drop, smear fixation
  4. Staining methods: simple, negative, silver impregnation, Gram, acid-fast
  5. Cytoplasmic inclusions, capsules, spores, and motility

Write the complete four-page note covering all 14 questions

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Structure of the Microbial Cell and Basic Bacteriological Microscopy

1. Structure, morphology, and size of bacterial cells

Bacteria are unicellular prokaryotic microorganisms. Most medically important bacteria are approximately 0.5 to 3 μm in size, although their length and width vary with species, culture conditions, and age. Their small size requires a compound light microscope, usually with the oil-immersion objective, for routine examination.
The main bacterial shapes are:
  • Cocci: spherical or oval cells. They may occur as single cells, pairs (diplococci), chains (streptococci), clusters (staphylococci), tetrads, or packets.
  • Bacilli: rod-shaped cells. They may be short, long, straight, curved, single, paired, or in chains. Very short rods are termed coccobacilli.
  • Vibrios: curved comma-shaped rods, for example Vibrio cholerae.
  • Spirilla: rigid spiral-shaped bacteria.
  • Spirochetes: thin, flexible, corkscrew-shaped bacteria, for example Treponema.
  • Filamentous or branching bacteria: for example Actinomyces and Nocardia.
Cell shape and arrangement are useful initial clues to bacterial identification. Microscopy commonly assesses cell size, shape, arrangement, and Gram-stain reaction. Staphylococcus aureus appears in grape-like clusters, Streptococcus pyogenes in chains, and Neisseria or Streptococcus pneumoniae may appear as diplococci. [Medical Microbiology 9e, p. 138-139]

2. Prokaryotic and eukaryotic cells: main differences

FeatureProkaryotic cell, bacteriaEukaryotic cell
SizeUsually 0.5-3 μmUsually >5 μm
NucleusNo true nucleus or nuclear membraneTrue membrane-bound nucleus
ChromosomeUsually one circular, haploid chromosome in nucleoidMultiple linear chromosomes, commonly diploid
Membrane-bound organellesAbsentPresent: mitochondria, ER, Golgi apparatus, lysosomes
Ribosomes70S, made of 50S and 30S subunits80S, made of 60S and 40S subunits
Cytoplasmic membraneUsually lacks sterols, except MycoplasmaContains sterols
Cell wallPeptidoglycan is characteristic of bacteriaAbsent in animals; fungi have chitin-containing wall; plants have cellulose
Cell divisionBinary fissionMitosis and meiosis
Gene transferConjugation, transformation, transductionSexual reproduction and meiosis
[Medical Microbiology 9e, p. 139]

Bacterial cell envelope

The bacterial cell envelope includes the cytoplasmic membrane and, in most bacteria, the cell wall.
Cytoplasmic membrane:
This is a phospholipid-protein bilayer that controls transport of nutrients and waste products. It is also important for energy production, secretion, synthesis of cell-wall components, and maintenance of ion gradients. Because bacteria do not contain mitochondria, much of their respiratory energy generation occurs at the cytoplasmic membrane.
Cell wall:
The cell wall gives bacteria their shape and protects against osmotic lysis.
  • Gram-positive bacteria have a thick peptidoglycan layer and often contain teichoic acids.
  • Gram-negative bacteria have a thin peptidoglycan layer, a periplasmic space, and an outer membrane containing lipopolysaccharide (LPS).
  • Mycoplasma lack a cell wall and therefore are not reliably Gram stained.
Cytoplasm:
The cytoplasm contains water, enzymes, metabolites, ribosomes, nucleoid material, and storage inclusions. It has no membrane-bound mitochondria, Golgi apparatus, or endoplasmic reticulum.
Ribosomes:
Bacterial ribosomes are 70S particles. They synthesize proteins and are selective targets of several antimicrobial drug classes.
Nucleoid and plasmids:
The nucleoid is the irregular region containing the bacterial chromosome. It is not enclosed by a nuclear membrane. Plasmids are small, extrachromosomal DNA molecules that replicate independently. They may carry genes for antimicrobial resistance, toxins, metabolic functions, or virulence factors.
Mesosomes:
Older microbiology texts described mesosomes as infoldings of the cytoplasmic membrane involved in respiration and cell division. They are now generally considered artifacts produced during chemical fixation rather than true normal bacterial organelles.

3. External structures: slime layer, capsule, pili, and flagella

Some bacteria have an external glycocalyx, usually composed of polysaccharide and less often polypeptide.
  • A slime layer is loose, unorganized, and easily removed.
  • A capsule is well organized, firmly attached, and usually not easily removed.
Capsules can help bacteria resist phagocytosis, adhere to host tissues or surfaces, and form biofilms. Colonies of heavily encapsulated bacteria may appear mucoid. The capsule is a significant virulence factor in organisms such as Streptococcus pneumoniae, Klebsiella pneumoniae, and Haemophilus influenzae.
Pili or fimbriae are short protein appendages. Ordinary fimbriae promote attachment to host cells. Sex pili participate in conjugation and transfer of plasmid DNA.
Flagella are long helical protein appendages that produce bacterial movement. They rotate by a membrane-powered motor. Flagella may be:
  • Monotrichous: one flagellum at one pole
  • Lophotrichous: tuft at one pole
  • Amphitrichous: flagella at both poles
  • Peritrichous: flagella distributed around the surface
Flagella can support movement toward favorable conditions and away from harmful ones, a response termed chemotaxis. [Sherris & Ryan’s Medical Microbiology, 8th ed., p. 810]

4. Intracytoplasmic inclusions and spores

Cytoplasmic inclusions are deposits of reserve substances. They may contain glycogen, lipids, polyphosphate, sulfur, or other materials. Metachromatic or volutin granules are polyphosphate-containing inclusions, classically demonstrated in Corynebacterium diphtheriae.
Endospores are dormant, highly resistant survival structures formed inside certain bacteria, especially species of Bacillus and Clostridium. A spore is not a reproductive form: one vegetative bacterial cell produces one endospore, which later germinates into one vegetative cell.
Spores resist drying, heat, radiation, and many chemicals because of their multilayered coat, low water content, calcium-dipicolinate, and metabolically inactive state. Their position may be central, subterminal, or terminal and can assist identification.

5. Preparation for microscopy of living microorganisms

Living bacteria may be examined in a wet mount to observe size, shape, grouping, and motility. A wet mount is best prepared from a young culture because old cultures can show reduced motility and irregular morphology.
Basic method:
  1. Place a small drop of sterile saline, broth culture, or water on a clean glass slide.
  2. Add a very small amount of bacterial culture.
  3. Apply a coverslip carefully to avoid air bubbles.
  4. Examine promptly, initially with low power and then with high dry power or phase-contrast microscopy.
Living preparations should be handled with appropriate laboratory precautions. Wet mounts of potentially pathogenic organisms should only be undertaken in a properly equipped laboratory and according to local biosafety procedures.

Crushed drop or simple wet mount

In the crushed drop preparation, a drop of bacterial suspension is placed on a slide and covered with a coverslip. The liquid layer is thin because the coverslip presses the drop flat. It is quick and simple, but evaporation and heating from the microscope lamp can limit observation time.

Hanging-drop preparation

The hanging-drop method is more suitable for observing motility over a longer period.
  1. Put a small drop of bacterial suspension in the center of a coverslip.
  2. Apply petroleum jelly to the corners of a cavity slide.
  3. Invert the cavity slide over the coverslip so the drop hangs into the cavity.
  4. Turn the preparation over carefully and observe microscopically.
The suspended drop evaporates more slowly and provides space for motile organisms to move.

6. Motility of bacteria and its study

True bacterial motility is usually produced by flagella. In a wet mount or hanging drop, motile bacteria show purposeful directional movement across the field. Nonmotile bacteria may still exhibit Brownian movement, which is random vibration caused by collisions with water molecules. Brownian movement does not result in net movement from one location to another.
Motility can be studied by:
  • Wet mount or hanging-drop preparation
  • Phase-contrast or dark-field microscopy
  • Semisolid motility agar, in which motile bacteria spread away from the stab line
  • Flagellar staining, which makes thin flagella visible by coating or thickening them
Do not mistake liquid currents, drifting, or Brownian movement for genuine motility.

7. Preparing, drying, and fixing a bacterial smear

A smear is a thin film of bacteria spread on a glass slide for staining.

Preparation

  1. Use a clean, grease-free labeled slide.
  2. For culture from solid medium, place a small drop of sterile water or saline on the slide.
  3. Transfer a tiny amount of culture into the drop and spread into a thin, even film.
  4. For broth culture, place a small loopful directly on the slide.
  5. Allow the film to air dry completely.
A smear must be thin. A thick smear causes poor staining, clumping, faulty decolorization, and difficulty recognizing morphology.

Fixation

Fixation attaches bacteria to the slide, kills most organisms, preserves morphology, and allows repeated washing during staining.
  • Heat fixation: Pass the fully air-dried slide rapidly through a flame several times, avoiding overheating.
  • Chemical fixation: Methanol may be used for some specimens or procedures.
Slides must be air dried before heat fixation. Heating a wet smear may cause splattering, distortion, and aerosol formation. Modern laboratory safety guidance often favors a controlled slide warmer rather than an open flame, especially for specimens that might contain mycobacteria. The CDC safe-work guidance advises treating acid-fast smears as potentially contaminated even after fixation.

8. Dyeing techniques for bacterial smears

Bacteria are nearly transparent in ordinary bright-field microscopy. Staining increases contrast and makes cellular morphology visible. Common dyes include methylene blue, crystal violet, carbolfuchsin, and safranin. [Sherris & Ryan’s Medical Microbiology, 8th ed., p. 128]
Staining methods are classified as:
  1. Simple staining: one dye is used.
  2. Negative staining: the background is stained instead of the cell.
  3. Differential staining: distinguishes types of bacteria, for example Gram and acid-fast stains.
  4. Special staining: demonstrates structures such as capsules, spores, flagella, and intracellular inclusions.

9. Simple staining

A simple stain uses one basic dye, such as methylene blue, crystal violet, or safranin. Basic dyes have a positive charge and bind to negatively charged bacterial components.
Procedure:
  1. Prepare, dry, and fix a smear.
  2. Flood with stain for an appropriate time.
  3. Rinse gently with water.
  4. Blot dry.
  5. Examine under oil immersion.
Simple staining demonstrates bacterial shape, size, and arrangement, but it does not distinguish Gram-positive from Gram-negative bacteria or identify special structures.

10. Negative staining

Negative staining stains the background while the bacteria remain unstained or lightly stained. Acidic dyes such as India ink, nigrosin, or eosin are used. Because the dye is repelled by the negatively charged bacterial surface, it forms a dark background around the cells.
Advantages:
  • No heat fixation is required.
  • Cells are not distorted by heat.
  • Cell size and capsules are more easily appreciated.
Negative staining is especially useful for demonstrating a capsule as a clear halo surrounding the bacterial cell against a dark background. It is also historically associated with India-ink visualization of the capsule of Cryptococcus in cerebrospinal fluid.

11. Silver impregnation

Silver impregnation methods deposit metallic silver on very thin microorganisms or structures, making them thick and dark enough to be seen with the light microscope. They have been used for delicate organisms such as spirochetes and for flagella.
In silver staining, silver salts are reduced and deposited on the organism. The stained cells or structures appear brown to black. The method requires careful technique because excessive silver deposition can produce background artifacts.

12. Differential staining methods

Gram staining

The Gram stain is the most widely used differential bacterial stain. It differentiates bacteria according to cell-envelope structure.
Reagents and sequence:
  1. Crystal violet: primary stain
  2. Gram iodine: mordant
  3. Alcohol or acetone-alcohol: decolorizer
  4. Safranin: counterstain
Results:
  • Gram-positive bacteria retain the crystal violet-iodine complex and appear purple.
  • Gram-negative bacteria are decolorized and then take up safranin, appearing pink-red.
The thick peptidoglycan layer of Gram-positive cells retains the dye complex, whereas Gram-negative bacteria have thin peptidoglycan and an outer membrane, so they lose the primary stain during decolorization. [Medical Microbiology 9e, p. 138-139]
Important errors include using an old culture, preparing an excessively thick smear, over-decolorization, under-decolorization, or overheating during fixation. Old or damaged Gram-positive bacteria may become Gram variable or falsely Gram negative.

Acid-fast staining

Acid-fast staining is used mainly for organisms with lipid-rich, mycolic-acid-containing cell walls, especially Mycobacterium species.
The principal methods are:
  • Ziehl-Neelsen method: uses heat to drive carbolfuchsin into the cell.
  • Kinyoun method: “cold” method, using more concentrated reagents without heating.
  • Fluorochrome method: uses auramine-based stains and fluorescent microscopy.
Basic principle:
Carbolfuchsin stains cells red. Acid-alcohol decolorizes non-acid-fast organisms, but acid-fast bacilli retain carbolfuchsin and remain red. A counterstain, commonly methylene blue, colors the background or non-acid-fast cells blue. [Sherris & Ryan’s Medical Microbiology, 8th ed., p. 129]

13. Staining of intracellular inclusions, capsules, and spores

Cytoplasmic inclusions

Metachromatic granules can be demonstrated with special stains such as Albert stain or Neisser stain. In Corynebacterium diphtheriae, they may appear as dark bluish-purple granules, often near the poles of the bacillus.

Capsule staining

Capsules are poorly stained by ordinary methods because they are usually nonionic and water soluble. Common methods include:
  • Negative staining with India ink or nigrosin
  • Anthony method
  • Welch method using crystal violet followed by copper sulfate
Water should not be used aggressively because it can dissolve or disrupt the capsule. In the Welch method, copper sulfate removes excess dye and helps preserve the capsule. [Jawetz, Melnick & Adelberg’s Medical Microbiology, 28th ed., capsule-stain section]

Spore staining

Endospores resist ordinary stains. In the Schaeffer-Fulton method:
  1. Apply malachite green to a heat-steamed smear.
  2. Wash with water.
  3. Counterstain with safranin.
Endospores appear green, whereas vegetative bacterial cells appear red or pink. [Jawetz, Melnick & Adelberg’s Medical Microbiology, 28th ed., endospore-stain section]

Summary

The bacterial cell is a prokaryotic cell with a cytoplasmic membrane, cell wall, cytoplasm, 70S ribosomes, nucleoid, and sometimes plasmids, capsule, pili, flagella, inclusions, or endospores. Bacterial morphology and staining are fundamental to identification. Wet mounts and hanging-drop preparations help demonstrate living cells and true motility. Fixed smears permit simple, negative, differential, and special staining. Gram staining classifies bacteria as Gram positive or Gram negative, while acid-fast staining identifies bacteria with mycolic-acid-rich cell walls. Capsule, spore, inclusion, and flagellar stains reveal specialized structures relevant to bacterial survival, virulence, and laboratory diagnosis.

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Structure of the Microbial Cell and Methods of Studying Bacteria

1. Structure, morphology, and size of bacteria

Bacteria are unicellular prokaryotic microorganisms. Most bacteria are about 0.5-3 μm wide and 1-10 μm long. Their small size requires examination with a light microscope, usually using the oil-immersion objective.
According to shape, bacteria are classified as:
  • Cocci: spherical or oval cells. They may occur singly, in pairs (diplococci), chains (streptococci), clusters (staphylococci), tetrads, or packets.
  • Bacilli: rod-shaped bacteria. They may be short, long, straight, or arranged in chains.
  • Coccobacilli: very short rods, intermediate between cocci and bacilli.
  • Vibrios: curved, comma-shaped rods.
  • Spirilla and spirochetes: spiral or corkscrew-shaped bacteria.
  • Filamentous bacteria: branching forms, such as Actinomyces.
Cell shape, size, arrangement, and staining reaction are important early steps in identification.

2. Prokaryotic versus eukaryotic cells

Bacteria are prokaryotes, whereas fungi, protozoa, plants, and animals are eukaryotes.
FeatureProkaryotic cellEukaryotic cell
NucleusNo nuclear membrane; DNA lies in nucleoidTrue membrane-bound nucleus
ChromosomesUsually one circular chromosomeMultiple linear chromosomes
OrganellesNo mitochondria, ER, or Golgi bodiesMembrane-bound organelles present
Ribosomes70S80S
SizeUsually 0.5-3 μmUsually >5 μm
Cell divisionBinary fissionMitosis and meiosis
Cell wallPeptidoglycan in most bacteriaAbsent in animals; chitin in fungi; cellulose in plants
The bacterial cell wall maintains shape and prevents osmotic lysis. Gram-positive bacteria have thick peptidoglycan, while Gram-negative bacteria have thin peptidoglycan plus an outer membrane containing lipopolysaccharide.
The cytoplasmic membrane controls transport, secretion, respiration, and energy production. Unlike eukaryotic membranes, it generally lacks sterols, except in Mycoplasma.
The cytoplasm contains enzymes, water, metabolites, ribosomes, inclusions, plasmids, and the nucleoid. Mesosomes were historically described as membrane infoldings involved in respiration and cell division; they are now usually considered fixation artifacts.
The nucleoid contains bacterial chromosomal DNA. Plasmids are small extra-chromosomal DNA molecules that may carry genes for antimicrobial resistance or virulence.

3. Capsule, slime layer, flagella, and inclusions

Some bacteria produce a glycocalyx outside the cell wall.
  • A slime layer is loose and unorganized.
  • A capsule is organized and firmly attached to the cell.
Capsules are usually polysaccharide. They help bacteria attach to surfaces, form biofilms, and resist phagocytosis. Heavily encapsulated bacteria often form mucoid colonies.
Flagella are long protein filaments that rotate and propel bacteria through liquids. Flagellar arrangements may be monotrichous, lophotrichous, amphitrichous, or peritrichous. They allow true motility and chemotaxis.
Intracytoplasmic inclusions are storage deposits of glycogen, lipid, sulfur, or polyphosphate. Volutin or metachromatic granules are polyphosphate inclusions, particularly associated with Corynebacterium diphtheriae.

4. Microscopy of living microorganisms

Living bacteria can be examined in a wet mount to study their morphology and motility. A drop of saline or fresh broth culture is placed on a clean slide, mixed with a small amount of bacteria, and covered with a coverslip. The preparation should be examined immediately.
A crushed-drop preparation is an ordinary wet mount in which the coverslip flattens the drop. It is simple and rapid but dries quickly.
In a hanging-drop preparation, a small drop of bacterial suspension is placed on a coverslip. The coverslip is inverted over a cavity slide, so the drop hangs inside the cavity. This prevents rapid drying and permits longer observation of movement.
True motility is directional movement of bacteria across the field. It must be distinguished from Brownian movement, which is random vibration caused by collisions with water molecules, and from passive drifting due to liquid currents.

5. Preparation, drying, and fixation of a smear

A bacterial smear is a thin film of bacteria on a glass slide for staining.
  1. Use a clean, labeled slide.
  2. Place a small drop of sterile water or saline on the slide when using culture from solid media.
  3. Mix a tiny amount of bacteria into the drop and spread it into a thin film.
  4. Allow it to air dry completely.
  5. Fix the smear.
Fixation attaches cells to the slide, kills most organisms, preserves morphology, and prevents the smear from washing away during staining. Heat fixation is done by passing the air-dried slide briefly through a flame. Excessive heating distorts bacteria. Chemical fixation, such as methanol fixation, may be used for selected specimens. Smears must never be heated while still wet because this can cause splashing and artifacts.

6. Staining techniques

Bacteria are almost transparent in ordinary light microscopy. Stains increase contrast and reveal shape, arrangement, and special structures.

Simple staining

A simple stain uses one basic dye, such as methylene blue, crystal violet, or safranin. The dye stains bacterial cells, allowing observation of their size, shape, and arrangement. It does not differentiate bacterial groups.

Negative staining

Negative staining uses an acidic dye such as India ink, nigrosin, or eosin. The background is stained dark, while bacterial cells remain clear or lightly stained. Heat fixation is avoided. This method preserves cell size and is useful for demonstrating capsules as clear halos around cells.

Silver impregnation

Silver impregnation deposits metallic silver on delicate structures, making them visible as dark brown or black. It can demonstrate very thin organisms, especially spirochetes, and may also be used for flagella.

7. Differential staining

Gram staining

Gram staining differentiates bacteria by cell-wall structure.
Steps:
  1. Crystal violet: primary stain
  2. Gram iodine: mordant
  3. Alcohol or acetone-alcohol: decolorizer
  4. Safranin: counterstain
Results:
  • Gram-positive bacteria retain crystal violet and appear purple because their thick peptidoglycan layer traps the dye-iodine complex.
  • Gram-negative bacteria lose crystal violet during decolorization and take up safranin, appearing pink or red.
Old cultures, overly thick smears, excessive decolorization, and overheating may give incorrect results.

Acid-fast staining

Acid-fast staining is used mainly for Mycobacterium species. Their cell walls contain mycolic acids, which resist decolorization by acid-alcohol.
Carbolfuchsin is the primary stain. Acid-fast bacteria retain it and remain red. Non-acid-fast cells are decolorized and take up a blue or green counterstain. Ziehl-Neelsen uses heat, while Kinyoun staining is a cold method.

8. Staining of inclusions, capsules, and spores

Metachromatic granules may be stained by Albert or Neisser staining methods.
Capsules may be demonstrated by negative staining or by special methods such as the Anthony or Welch method. Water should be avoided during capsule staining because it can dissolve or remove capsular material.
Some bacteria, particularly Bacillus and Clostridium, form endospores. Spores are dormant survival structures, not reproductive forms. They resist heat, drying, and chemicals. In the Schaeffer-Fulton spore stain, malachite green stains spores green, while safranin counterstains vegetative cells red.

9. Study of bacterial motility

Motility can be examined by wet mount, hanging-drop preparation, phase-contrast microscopy, or semisolid motility agar. In motility agar, motile bacteria spread away from the inoculation line, while nonmotile bacteria remain confined to the stab line.
Thus, bacterial morphology, cell structures, motility, and staining reactions provide the basic laboratory information required for identification and for understanding bacterial survival and pathogenicity.
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