Lecture #1: BACTERIAL CYTOLOGY

The morphology of the bacterial cell constitutes the size, shape, structure and arrangement. The unit of bacterial measurement is the micrometer. The bacteria most frequently studied in the laboratory measure approximately 0.5 to 1.0 by 2.0 to 5.0 um.

Morphologic types according to shape and arrangement

1. Spherical or ellipsoidal: The Cocci

Round cells which multiply by binary fission and their arrangement depends on the plane of division

Diplococci – occur in pairs of cells

Streptococci – cells arranged in bead or chains

Staphylococci – irregular clusters resembling bunches of grapes

Sarcinae – cuboidal arrangement of usually 8 or more cells along three dimensions.

2. Cylindrical: The Bacilli

They do not have the variety of patterns exhibited by the cocci. Occasionally, they maybe:

Diplobacilli – occurs in pairs

Streptobacilli – occur in chains

Most bacilli, however, occur as single, unattached cells. Some are only slightly longer than they are wide; others are several times as long as they are wide.

3. Spiral–shaped: The Spirilla 

These occur predominantly as unattached individual cells. Bacteria exhibit differences in:

a.      Length

b.      Number of amplitude of spirals

c.       Rigidity of cell walls

Short, incomplete spirals are known as comma bacteria or vibrios

Most of the bacteria seen in the laboratory exhibit these three main morphologic types. Other common types seen are:

a.      Filamentous – like the tubercle bacilli

b.      Mycelial forms – such as the fungus like bacteria

c.       Fusiform with a stalk protruding from one pole

Approximate composition of the bacterial cell:

1.      Water                                                             70%

2.      Dry weight                                                   30%

Protein                       70%

RNA                           12%

DNA                           3%

Lipids                         6%

Polysaccharides       5%

Phospholipids          4%

EXTERNAL STRUCTURES OF THE CELL

                                                                         

1. Flagella – are thin hair–like appendages protruding through the cell wall. It consist of three parts:

a. Basal or granular body attached to the cytoplasmic membrane

b.  Hook – like axial structure attached to the basal body

c.   Long thin untampered filament

According to location and number of flagella, bacteria are classified as:

a.   Monotrichous – with one polar flagellum

b.   Lopotrichous – with two or more polar flagella

c.   Ampitrichous – with tufts of flagella at both poles

d.  Peritrichous – eight or more flagella distributed over the surface

Atrichous – the term give to those bacteria which do not possess flagella

            Chemical composition: Protein (flagellin)

            Functions

a. Motility – move at high speed

b. Antigenicity – the protein composition serves as the antigen and therefore elicit antibody specific for flagella. This is the basis of the Immobilization Test for Syphilis wherein the flagella are agglutinated or rendered immobile by antiserum (antibody) specific for Treponema pallidium.

Methods for detection of flagella

a.  Motility test

1.  Directly observed by microscopic examination

a. Wet mount

b. Hanging drop

True motility – if the bacteria seem to be going in a definite direction.

Brownian movement – if the bacteria are bouncing back and forth rapidly due to the bombardment from molecules of water.

2.  Indirectly observed thru growth of bacteria in a semisolid medium

a. Motile – growth of bacteria is away from the inoculation line

b. Non–motile – growth of bacteria is confined along the inoculation line

Flagella are very difficult to demonstrate microscopically because of their very small size and their fragile state. Ordinary staining methods do not demonstrate them. A mordant must be used, which is a special reagent that deposit on the flagellum, making it appear thicker.

b. Direct visualization of flagella

1. In unstained preparations – using Brightfield microscopy and mordanting

2. In stained preparations – using Phase contrast, Darkfield or Electron microscopy

c.   Indirect evidence of Serologic Testing

“H” antigen is the flagellar antigen used

            Not all motile bacteria possess flagella

            Not all bacteria have flagella. They are present in many species of bacilli, rare

            in cocci

2. Pili

They are shorter and finer than the flagella. They are seen both in motile and non–motile bacteria, therefore they are not concerned with motility.

Chemical composition:      Protein

Types of pili

a.  Ordinary pili (colonization antigen) – plays a role in the adherence of symbiotic bacteria to host cells.

b. Sex pili – responsible for the attachment of the donor cell and recipient cell in conjugation.

Function

a. The F pilus (sex or fertility pilus) serves as a port of entry of genetic material during bacterial conjugation.

b.  Attachment site for bacterial viruses

c.  Facilitates adherence to mammalian surfaces

d. Antigenicity

3. Capsule or slime layer or glycocalyx

When bacteria synthesized a large amount of polymer, it forms a condensed, well defined layer surrounding the cell, it is called a capsule. When it forms a loose meshwork of fibrils extending outward from the cell, it is called glycocalyx. In some cases, masess of polymer are formed which appear to be totally detached from the cells but in which the cells may be entrapped is referred to as slime layer.

This structure gives a mucoid or viscous colony.

Chemical composition

a. Polysaccharide (e.g. Streptococcus pneumoniae)

b. Polypeptide (e.g. Bacillus anthracis)

c.  Nucleic acid admixed with polysaccharide (e.g. Mycobacterium tuberculosis)

Function

a.  Antiphagocytic and antibacteriophage

b.  Antigenicity

Significance of the capsule for bacteria

a.  Provides a protective covering to the cell

b.  Serves as a reservoir of food

c.  Site for disposal of wastes substances

d. For pathogenic bacteria, capsule increases infectivity, so loss of the capsule renders the bacteria avirulent

Significance of the capsule for man

a. Associated with virulence, bacteria makes the infection more difficult to combat because of its antiphagocytic property

b. Responsible for the slime encountered in industrial process, making it an ecological nuisance

c. Capsular material can be extracted and used in the preparation of dextran, cellulose and levans from sucrose

d. Some organisms like Streptococcus mutans owes its capacity to adhere to tightly to tooth enamel to glycocalyx

The size of the capsule depends relatively on the method used to demonstrate it. In wet preparation, this structure appears thick and in dry preparations, it tends to shrink.

            Detection of capsule:

a. Stained preparations – special dyes or reagents are used.

b. Relief or Indirect or Negative staining – India ink is the most commonly used.

4. Cell wall

In between the capsule, if present, and the cytoplasmic membrane lies the cell wall. It is rigid and elastic, retains its original shape even after being subjected to osmotic pressure changes or freezing followed by thawing.

Chemical composition:      Peptidoglycan and diaminopimelic acid

Peptidoglycan or murein or mucopeptide is a complex polymeric substances found in all bacterial cell walls. It provides strength and rigidity of the cell wall.

In addition to the peptidoglycan,

            The Gram positive cell wall contains

a. Teichoic acid – may be ribitol or glycerol type, confers antigenic property, binds and maintains supply of magnesium

b. Polysaccharide and peptides – responsible for endotoxic shock.

            The Gram negative cell wall contains

a. Lipoprotein

b. Protein – phospholipid bilayer

c. Lipopolysaccharide – confers toxicity of gram negative bacteria in the so called endotoxin which is released when bacterial cell lyse.

Functions

a. Protects the cell from mechanical damage and from osmotic rupture in dilute media.

b. Essential for bacterial growth and cell division

c. Responsible for the shape of the cell

d. Major distinction between gram (+) and gram (–) bacteria

e. Antigenic specificities

f.  Acts as a primer during cell division for its own biosynthesis

RELATIVE DIFFERENCES BETWEEN

CHARACTERISTICS                    GRAM (+)                              GRAM (–)

Cell wall composition                     low in lipids                                     high in lipids

Susceptibility to penicillin                         more susceptible                  less susceptible

Inhibition by basic dyes                  marked inhibition                less inhibition

Nutritional requirement                 relatively complex               relatively simple

Resistance to physical                     more resistant                       less resistant

            disruption

Hydrolytic action                             susceptible                            resistant

Protoplast, spheroplast and L–forms

Removal of the entire cell wall by both physical and chemical criteria will form Protoplast. A protoplast assumes a spherical shape has the following additional characteristics:

a.      Non–motile

b.      Does not divide

c.       Does nor form new cell wall

d.     Usually not susceptible to infection by bacteriophage

A protoplast may be produced by

a. Removal of the cell wall by treating the cells with an enzyme (lysozyme) which has selectively dissolves the cell wall material

b. Growing the organism in an environment which prevents the synthesis of cell wall substances but does not interfere with growth and reproduction.

Protoplasts are usually derived from Gram (+) bacteria.

Treatment of the Gram (–) bacterial cell wall under the same conditions as above may remove peptidoglycan but a major portion of the cell wall material or constituents will remain. These are called spheroplast.

Several species of bacteria have been observed which undergo a transition from their normal morphological forms to very small bodies the so called L–forms. They may rise spontaneously during some state in the culture of the bacterial form, as its formation may be induced by:

a.      Penicillin and other antibiotics

b.      Antibodies specific for the bacterial form

c.       Enzymes such as lysozyme

L–forms have little if any rigid cell wall layer, however, it is able to survive, grow and propagate. They grow best on semisolid media, producing very small colonies. Their cultural appearance closely resembles the Mycoplasma. They are capable of reverting to normal form because of its residual peptidoglycan when removed from the inducing medium.

            Significance of the L–forms:

a. They can produce chronic infection, the organisms persists and become sequestered in a protective medium

b. They are relatively resistant to antibiotic therapy

c. They can produce a relapse or a recent overt/acute infection

INTERNAL STRUCTURES OF THE CELL

1. Cytoplasmic membrane / protoplasmic membrane

This structure is a thin covering lying immediately beneath the cell wall.

Chemical composition: complex lipoprotein

This structure is destroyed by certain agents like detergents, polymyxin and other antibiotics. Damage to this membrane by physical and chemical agents may result in the death of the cell.

Functions:

a. Essential for viability

b. Selective permeability – controls the passage of nutrients and waste products into and out of the cell.

c.  It contains the enzymes that synthesize complex liquids as well as the components of the cell wall, enzymes involved in electron transport and oxidative phosphorylation.

2. Mesosomes

It is an extremely important structure associated with several vital processes of the cell since it is an extension of the cytoplasmic membrane.

Function:

a. They are involved in septum formation during the process of bacterial cell division

b. They are associated with bacterial nuclear material and its replication and certain enzymatic process like electron transport.

3.  Ribosomes

These are RNA–protein particles. It contains the enzymes, which function in protein synthesis.

4. Nuclear material

Bacterial cells do not contain the nucleus characteristic of the cells of the higher plants and animals. They do, however, contain “bodies” within the cytoplasm that are regarded as nuclear structure, and the DNA of the bacterial cell is confined to this area. The DNA carries within its molecules the messages that control the activities of the cell. It can be demonstrated by the use of Feulgen stain and electron microscope.

5.  Cytoplasmic inclusions

These are concentrated deposits of certain substances. They are also known as volutin granules or metachromatic granules. They are not equally prominent in all bacterial cells and their appearance is influenced by the age of the cells and the environment where they have grown. Granules serve the cells as a source of stored foods.

Chemical composition

a.  Metaphosphate and polyphosphate

b.  Polysaccharide granules

c.   Sulfur as globules in the cell.

Detection:

a. Identified by using stains which, because of a high degree of affinity for the chemical material of the granules make them stand out against the cytoplasm.

b. Treating the granulated cell with chemical solutions known to dissolve the material of the granules.

6. Endospores

These are thick–walled oval body produced by some bacteria. These oval bodies are commonly called spores. They are resistant to physical and chemical agents due to dipocolinic acid–calcium complex and in part to their dehydrated state.

As long as environment conditions are adverse to the growth of the bacterium, the endospore will remain a spore. However, if conditions become favorable for growth, it germinates and become vegetative cell. The spores represent a dormant (resting) phase of the bacterial cell.

The following bacteria are capable of producing endospores:

a.  Bacillus

b.  Clostridium

c.  Desulfotomaculum

d. Sporolactobacillus

e.  Sporosarcinae

Location of the spores within the cell:

a.   Central

b.   Terminal

c.   Subterminal

Detection

1. In unstained cell suspension, spores are observed as intracellular refractile bodies.

2. In stained preparations by conventional method, spores appear as colorless area in stained cell. The spores are commonly stained with malachite green.

MAJOR CHARACTERISTICS OF MICROORGANISM

The characteristic of the microorganism have to be determined in detail before one can identify and classify the microbes. Among the major characteristics which are observed and determined include the following:

1.  Morphological characteristics – the size of the cells, the shape and their arrangement, differentiation and identification

2. Cultural characteristics – the nutrients required for growth and the environmental conditions that will favor their growth

3. Metabolic (biochemical) characteristics – the manner in which the microorganisms carries out the chemical processes of life

4. Chemical composition characteristics – the identification of the major characteristics chemical constituent of the cell.

5.  Antigenic characteristic – the detection of the cell chemical components that provide evidence for similarities between species

6.  Genetic characteristics – the analysis of the deoxyribonucleic acid (DNA) and the determination of the reaction between DNA materials extracted from different species.