CHAPTER 2 Morphological and Tinctorial Characteristics I.H.Siddique

2.1 Chapter review:

This chapter describes the morphological and tinctorial characteristics of bacteria. For identification of bacteria the morphology and staining techniques are given. In addition, the structure of a typical bacterial cell as well as the characteristic staining reactions are also profiled.

Lecture 2 Outline 

2.2 Introduction

An essential feature in the scientific study of microbes was their demonstration by optical systems. Leeuwenhoek (1623-1723) is credited with the discovery of the microbial world. Since then, great strides have been made in the construction and development of microscopes (compound, electron, etc.). In addition to the magnification values, the estimation of resolving power of an optical combination in a microscope is important (Resolving power is the distance separating 2 point sources of light if they are to be seen as 2 distinct images). The magnification of a microscope is that magnification that makes visible the smallest resolvable particle. A microscope with a 90-power objective lens and with a 10 power ocular lens magnifies the specimen 900 times.

The resolving power of a light microscope is about 2000 A=0.2 u.

The highest power of an ordinary compound microscope (oil-immersion lens) magnifies about 1,000 diameters. An object 0.5 um in diameter when magnified 1000 X appears about the size of a period on this page. Most viruses cannot be seen with ordinary microscopes. Another type of magnifying instrument called the electron microscope gives clear images of bacterial structures at enlargements up to one million and more diameters. Some useful relationships of measurements are:

 2.3 Importance of Morphology and Staining

In order to classify an unknown organism, one has to study the characteristic morphology (both internal and external structures), colony appearance (size, consistency, texture, and color, etc), specific staining, biochemical reactions, antigenic structure and pathogenicity.

2.4 Morphology of the Bacterial Cell

2.4.1 Shape.

There are three morphological types: spherical or coccus; rod-shaped or bacillus, and the spiral forms. (Fig. 1 & 2).

2.4.1.1 Cocci.

The cocci are usually spherical but variations may occur. For example, pneumococcus is lanceolate shaped, the gonococcus and meningococcus has a coffee bean shape. Slightly elongated forms, however, are referred to as coccobacilli. The subtypes are differentiated on the basis of the arrangement of the individual cells with respect to another and the plane or planes in which cell division occurs. The cocci which are single and scattered, as seen on microscopic examination, are called micrococci; when in pairs, diplococcus, and when in chain-like structures, streptococci. When cocci have a tendency to remain adjacent to each other, with cell division in two or more planes, they form an irregular, grape-like cluster known as staphylococci. When the tendency to remain together is not marked, and the cocci are found in a group of four (tetrads) or in cubical packets of eight cells (sarcina). Keep in mind that during the various procedures in preparation these organisms may not be seen in the characteristic forms described above.

 

2.4.1.2 Bacilli.

There are considerable variations in shape and size of bacteria in this group. When the cells are thicker in the center and taper towards the ends, they are known as fusiform bacilli (as in Fusobacterium). In other situations, the ends may be squared, B. anthracis; rods with round ends (as in Salmonella typhosa); club-shaped organisms (as in Corynebacterium diphtheria). In some cases, a tendency of the cells to remain attached after cell division results in streptobacilli.

2.4.1.3 The curved and the spiral forms.

In this group either the curved forms and the spiral shaped organisms are included. The curved forms are known as vibrios. The spiral forms may be of two kinds, a spring-like axial filament upon which there is a layer of contractile protoplasm enveloped in a delicate periplast (as in genus Treponema and genus Borrelia). In the other form, the spirals are very fine and close and one or both ends of the cell may have hooks. The protoplasm is spirally wound about a delicate, but firm elastic smooth axial filament enclosed within a well-defined cell wall (as in genus Leptospira).

2.4.1.4 Involution forms.

These include baloon-like structures, Y shaped bacillary forms, and cells containing large amounts of granular material. These degenerative forms result when organisms are grown under adverse conditions.

 

2.4.2 Size.

There is considerable variation in the size of different organisms. Cocci may vary from 0.15u to 2u in diameter. The influenza bacillus ranges from 0.2u to 0.5u while tetanus bacillus, 3-5u. The spirals measure 3-50u. Measurements for all types of bacteria must be based upon the average of normal cells as there is always a variation.

2.5 The Structure of the Bacterial Cell.

Bacterial cell structures may be divided into two groups: those external to the cell proper and those which occur within the cell.

2.5.1.1 Flagella.

flagella or organs of locomotion. Flagellae are long filamentous appendages located in or just inside the cell wall. They occur most commonly in rod-shaped bacteria. The bacteria having a single polar flagellum are designated as monotrichous; those having two or more flagella at one end of the cell, lophothrichous; those having flagella at both ends of the cell, amphitrichous; and those having flagella usually 8-12, distributed over the cell surface are peritrichous. The vigor of bacterial movement depends on the number of flagella. Certain species (e.g., Proteus) may produce a huge number, especially when cell division is slowed; such cells spread in a thin film ("swarm") on the surface of moist agar media. This is known as "swarming phenomenon." ( Fig. 3) 

2.5.1.2 Pili (Fimbriae).

The electron microscope has revealed a group of still finer filamentous appendages, called pili (L., hairs) or fimbriae. (L., fringe). They are shorter, thinner, and straighter than flagella and on the same cell they vary in thickness (about 7.5-10nm) and length (up to several micrometers). They consist of a protein (pilin) and are non-motile.

2.5.1.3 Capsule.

Many bacteria under suitable growth conditions, are surrounded by a layer of gelatinous material known as slime layer or capsule. The thickness of the capsule varies with growth conditions. Among the pathogenic bacteria, the presence of capsule is associated with virulence as it interferes in the phagocytosis of bacteria. Some bacteria (pneumococcus) have a thick capsule. The chemical composition of capsules varies with the type of bacteria; it is antigenic and reacts with homologous antibody. This results in the swelling of the capsule, known as the Quelling reaction observed by Neufield in 1902.

2.5.1.4 The Cell Wall.

he outer structure of the bacterial cell consists of the cell wall proper and the plasma membrane which lines the inner surface of the cell wall. The cell wall is 10-20u in thickness and is 20% of its dry weight. It is rigid and relatively non-elastic but highly ductile in the intact cell. The cell wall substance consists of peptide, polysaccharide, and lipid. Cell walls are antigenic and consist of peptide, polysaccharide, and lipid. Cell walls are antigenic and are of value in serological identification of the bacteria. Electron microscopy reveals the walls of gram-positive organisms as a relatively thick (15-80 nm) with the cytoplasmic membrane closely apposed to its inner surface. This dense material is peptidoglycan: a network of polysaccharide chains cross-linked by an oligopeptide. Gram-negative cells have a much thinner peptidoglycan layer, covered by a closely apposed outer membrane. The two layers are not usually distinct on electron micrographs. The cell wall of gram-negative organisms are more complex and have more lipids than gram-positive organisms.

2.5.1.5 The plasma

or cytoplasmic membrane of the bacterial cell lies on the inner surface of the cell wall, separating it from the cell protoplasm. This material can be isolated. The studies on its chemical analysis have shown that it is a complex lipoprotein, containing 40% protein and 20-25% lipid, and makes up about 10% of the dry weight of the cell. The plasma membrane plays an important role in selective permeability and osmosis regulation.

Protoplasts and Spheroplasts- When the peptidoglycan layer of the cell wall is digested by lysozyme or when its synthesis is specifically blocked, the cell ordinarily lyses. However, in a hypertonic medium (e.g., 20% sucrose or 0.5 M KCL), the cell survives as an osmotically sensitive sphere. With gram-positive organisms, this product is free of wall constituents and is called a protoplast. With gram-negative organisms these osmotically sensitive spheres retain much of the outer membrane and are called spheroplasts. If a bacterial cell is placed in a hypertonic solution, it will swell and burst; in hypotonic solution,it will shrink.

 

2.5.2 Internal structures

Although no line of demarcation exists between external and internal structures, the following structures are described under this heading.

2.5.2.1 The Bacterial Endospore-

This occurs only in genus Bacillus and genus Clostridium. The bacterial spore is a refractile oval body formed within the bacterial cell and found both intracellularly and extracellularly. During examination the size and location of the spore in a bacterial cell are important. The spore may be smaller, larger or of equal size of the bacteria. It is said to be central when it is located in the center of the vegetative cell, subterminal when located between the center and the end of the cell, and terminal when it occurs at the end of the cell. In shape, the spores may be round or elliptical. Spores are resistant to physical and chemical environmental conditions otherwise fatal to vegetative forms. The presence of dipicolinic acid in a spore is associated with heat resistance.

Spores are of tremendous significance in sterilization and surgery because of their resistance to chemical disinfectants, boiling and other destructive environmental conditions. It is important to learn what organisms produce spores and how to kill the spores and organisms.

2.5.2.2 Cytoplasmic Contents

• a. Mesosomes. Thin sections of bacteria often reveal one or more large, irregular invaginations of the plasma membrane called mesosomes. Additional membranes within these pockets may be lamellar or vesicular. These structures evidently compartmentalize the cell, somewhat like the endoplasmic reticulum in eukaryotes.

  b. Ribosomes. The cytoplasm of bacteria is thickly populated with ribosomes; roughly spherical, densely stained objects about 18nm in diameter. Ribosomes are mostly grouped in chains called polysomes. These cannot be recognized in a cell section because of the close packing.

  c. Cytoplasmic Granules. The bacterial cell contains a variety of large granules (as large as 0.6u) which when stained with certain basic dyes are known as metachromatic or Babes-Ernst granules. Other visible granules (polysaccharide or lipid in nature) can be stained. These granules are the sites of enzymatic activity and are prominent in some species of bacteria (like C. diptheria).

2.5.2.3 Nuclear Body (nucleoid)

In bacteria, unlike eukaryotic cells, the cytoplasm has a high concentration of RNA and hence is as basophilic at the nuclear region; accordingly, the latter cannot be revealed by basic dyes. However, discrete nuclear bodies in bacteria can be recognized under the light microscope through special procedures, such as the DNA-specific Feulgen (fuchsin sulfite stain) method, or selective hydrolysis of the RNA (in fixed cells) by HCL or RNAse, followed by a basic stain. Most bacilli contain two or more such bodies per cell, since cell division lags behind nuclear division. More detailed study of these bodies with the electron microscope established the concept of bacteria as prokaryotic cells, lacking the discrete chromosomes, mitotic apparatus, nucleolus and nuclear membrane of eukaryotic cells. Hence the nuclear region in bacteria is referred to as the nuclear body or nucleoid.

With most methods of preparing sections the nuclear region in bacteria appears as a compact, centrally located mass, less dense than the cytoplasm, with parallel strands in some areas and excluding ribsomes. This compact body can be seen without fixation, by fluorescence microscopyor by phase contrast in cells growing in a medium of high refractive index. Moreover, if cells are gently lysed in the presence of a high salt concentration (especially divalent cations), to neutralize electrostatic repulsion of the nucleic acid chains, the nucleoid can be recovered intact: when freed of membranes by detergents, it contains about 60% DNA, 30% RNA, and 10% protein. It is less dense than the cytoplasm, although it contains about 10% of the cell volume, DNA is only 2%-3% of the cell's dry weight. 

2.6 Staining Reactions

Intact bacterial cell stains readily with basic dyes such as crystal violet, methylene blue but relatively poorly with acidic dyes such as eosin. The affinity for basic dyes is probably due to acidic protaplasm or nucleoproteins. The following staining procedures are employed in identification of organisms:

2.6.1 The Gram-stain.

The procedure will be explained in the laboratory. On the basis of this method, bacteria are separated in 2 main groups. The gram-positive bacteria are those which retain the primary stain and are deep violet in color. The gram-negative bacteria are those which are decolorized and stained by a counterstain, safranin and is red in color.

Several theories have been proposed to explain this phenomenon

a) A. differential permeability to the alcohol-soluble dye-iodine complex.

b) A difference in the isoelectric point of the cell contents between the gram-positive and gram-negative bacteria ( the gram-positive organisms have lower IEP) and

c) the site of Gram reaction is in the outer layers and the internal material being gram-negative in both kinds of bacteria.

2.6.2 The acid-fast stain.

Some bacteria are resistant to decolorization with agents such as acid-alcohol and therefore, are termed as acid-fast. The members of the genus Mycobacterium are acid-fast. This characteristic may well be due to high lipid contents, wax and mycolic acid present in this genus.

2.7 Colonial Morpholoygy of Bacteria-

When bacteria are grown on solid media, they form masses of cells visible to the naked eye. They have characteristics which vary in relation to the size of the colony (1 mm to several mm), and shape of the colonies (smooth, irregular, raised, depressed, etc.). 

AV-AT Review - T/S #290

AV-AT Tape TV 136; TV 420 

Lecture 2 Assignments:

The Bacterial Cell, Part l & Part II

Structure and Function, TS-290