DCM 1.

BASIC MICROBIOLOGY

MUSAME GERALD
 INTODUCTION

 Microbiology is the study of microorganisms.

 Organisms that are invisible to the unaided eye.
 Hence can only be seen with the help of a microscope.
 Thus they are also referred to us microscopic organisms.

 Examples include bacteria, protozoa, certain algae and certain

fungi .

 Also includes viruses, which are microscopic but not cellular and
infectious agents like prions.
Characteristic Features Of Microbes
 Microorganisms are ubiquitous.
Found virtually everywhere.

 Most microorganisms are free-living and perform useful activities that benefit
animal and plant life.

 Some are parasitic, saprophytic and some are commensals, where they form
normal microbiota in man and other mammals.

 Microorganisms that have the ability to cause disease are called pathogens.
 DISCOVERY OF MICROORGANISMS

 Bacteria and protozoa were the first microorganisms to be observed by

humans.

 Over the past 400 years, many individuals contributed to our present
understanding of microorganisms. Contributions of some early microbiologists
are discussed below:

1. Antony van Leeuwenhoek (1632–1723)

 First person (1673) to observe bacteria and protozoa using a simple
microscope.
 Hence referred to as the "Father of Bacteriology,” and the “Father of
Protozoology.

1590 Zaccharias Janssen, a Dutch spectacle maker, invented the first

compound microscope.

Contributions of Antony van Leeuwenhoek

i. He constructed the first simple microscope.

ii. The first person to observe microorganisms (1673).

iii. Provided accurate description of bacteria.

2. Spontaneous Generation (Abiogenesis)
•John Needham (1713–1781), the English priest in 1745 purported
spontaneous generation (abiogenesis) of microorganisms in his experiments.

•Lazzaro Spallanzani (1729–1799), an Italian priest and naturalist opposed

this view who boiled beef broth for an hour, sealed the flasks, and observed
no formation of microbes.

• Franz Schulze (1815–1873), Theodore Schwann (1810–1882), Georg

Friedrich Schroder and Theodor von Dusch attempted to counter such
arguments
 John Tyndall (1820–1893), the English physicist finally in 1877 proved and
was able to explain satisfactorily the need for prolonged heating to
eliminate microbial life from infusions.

 Proposed tyndallization;

• A process that kills both heat-stable form and a heat-sensitive form of

bacteria.
3. LOUIS PASTEUR (1822–1895)
 Coined the term ‘Microbiology’: For the study of living organisms of
microscopic size.

 Louis Pasteur is known as ‘Father of Microbiology’ because his contribution

led to the development of microbiology as a separate scientific discipline.

Disapproved theory of spontaneous generation

 In a series of classic experiments, Pasteur proved conclusively by demonstrating the
ubiquity of microorganisms that all forms of life, even microbes, arose only from their like
and not de novo.
 The idea of abiogenesis vs biogenesis.
 Developed sterilization techniques.
 Steam sterilizer
 Hot-air oven and
 Autoclave

 Proposed germ theory of disease.

 Developed methods and techniques for cultivation of microorganisms.

 Devised Pasteurization , a process of destroying bacteria in foods (e.g. milk).

 Discovered the process of attenuation and the development of live
vaccines. e.g. developed live attenuated anthrax vaccine and rabies
vaccine.

An accidental observation that chicken cholera bacillus cultures left on the bench for
several weeks lost their pathogenic property but retained their ability to protect the birds
against subsequent infection

 Pasteur discovered forms of life that could exist in the absence of

oxygen. He introduced the terms “aerobes” and “anaerobes”
(fermentation)
4. Robert Koch (1843–1910)
Contributions of Robert Koch:
Koch developed methods of fixing, staining, and photographing bacteria.

Koch developed methods of cultivating bacteria on solid media.

 He devised a simple method for isolating pure cultures of bacteria by plating out mixed material
on a solid culture medium.

Discoveries of the causal agents of anthrax (1876), tuberculosis (1882) and

cholera (1883).
 Koch discovered that Bacillus anthracis produces spores, capable of resisting adverse
conditions
 Koch’s postulates
 According to Koch’s postulates, a microorganism can be accepted as the
causative agent of an infectious disease only if the following conditions are
satisfied:

Postulate 1:
 The microorganism should be found in abundance in all organisms suffering
from the disease.

Postulate 2:
 It should be possible to isolate the microorganism from the diseased
organism and grown in pure culture.
Postulate 3:
 The cultured microorganism should cause the disease when Inoculated into a
healthy organism.

Postulate 4:
 It should be possible to re-isolate the microorganism from the inoculated
diseased experimental host/animal and be identified as being identical to the
original specific causative agent.
 Limitations of Koch’s postulates

 To fulfill Koch’s Postulates, it is necessary to culture the pathogen in the laboratory ( in

vitro) or on artificial culture media.

 However, certain pathogens donot grow on artificial media (viruses, rickettsias ,

chlamydias , Mycobacterium leprae and Treponema pallidum.

 Viruses, rickettsias, and chlamydias are obligate intracellular pathogens

 Can only survive and multiply within living host cells.

 Hence can only be grown in cell cultures (Human or animal cells , embryonated chicken eggs, or
certain animals referred to as laboratory animals e.g, Mycobacterium leprae is propagated in
armadillos, Treponema pallidum grows well in the testes of rabbits and chimpanzees..
 Another difficulty that is sometimes encountered while attempting to
fulfill Koch’s Postulates is that certain pathogens become altered
when grown in vitro.

 Some become less pathogenic, while others become nonpathogenic. Thus, they will no longer
infect animals after being cultured on artificial media.
5. JOSEPH LISTER (1827–1912)
•Called the ‘Father of Modern Surgery’.

•He applied Pasteur’s work and introduced aseptic techniques in surgery

(1867).

•The approach was remarkably successful effecting a pronounced drop in

mortality and morbidity due to surgical sepsis.

• He established the guiding principle of antisepsis for good surgical practice

• Paul Ehrlich is known as ‘Father of Chemotherapy’.
• ™. Edward Jenner is known as the ‘Father of Immunology’.
Areas Exploiting Practical Uses Applications of Microbiology

 Immunology
 Public health microbiology and epidemiology .
 Aim to monitor and control the spread of diseases in communities.

 Food microbiology, dairy microbiology, and aquatic microbiology.

 Examine the ecological and practical roles of microbes in food and water Agricultural
microbiology is concerned with the relationships between microbes and crops, with an
emphasis on improving yields and combating plant diseases.

 Biotechnology.
 Includes any process in which humans use the metabolism of living things to arrive at a
desired product, ranging from bread making to gene therapy
 Industrial microbiology.
 Concerned with the uses of microbes to produce or harvest large quantities of
substances such as beer, vitamins, amino acids, drugs, and enzymes

 Genetic engineering and recombinant DNA technology

 Involve techniques that deliberately alter the genetic makeup of organisms to mass-
produce human hormones and other drugs, create totally novel substances, and
develop organisms with unique methods of synthesis and adaptation. This is the most
powerful and rapidly growing area in modern microbiology .
 RAT I

Describe briefly, giving specific examples, the Applications/roles of Microbes,

in the following fields:
• Ecology
• Food industry.
• Medicine and pharmaceutical industry
• Biotechnology/Genetic engineering

HAND IN PRINTED COMPLETED ASSIGNMENT BY FRIDAY 13th

/10/2021 BEFORE 6.00PM.
Note: Any form of Xeroxing/duplicating work shall lead to loss of marks to all
parties involved.
 Useful references:
1.Essentials of Microbiology
Surinder Kumar
First Edition: 2016

2. Foundations in Microbiology
4th Edition
Kathleen Park Talaro and Arthur Talaro

Online versions available.

 BACTERIA

Bacterial structure
STRUCTURE OF BACTERIA
Bacteria have a simple cell structure consisting of:

Cytoplasm containing the bacterial chromosome (genome),

ribosomes, stored energy inclusions and often plasmids.

Cytoplasmic membrane and mesosomes.

Cell wall (except bacteria with deficient cell walls).

External structures, including (depending on species) a

capsule, fimbriae (pili),and flagella.
Genome:
 Bacteria are prokaryotes,

 i.e. their genetic material is not organized into chromosomes

inside a nuclear membrane

 it consists of a single usually circular chromosome of dsDNA

which lies coiled in the cytoplasm, attached to a septal
mesosome.
Plasmids:
 Are small, self-replicating, ds circular DNA molecules.

 Important in transfer of genetic material within and between

bacterial species through specialized sex pili.

 Depending on the genes contained in the plasmid, one

bacterium may confer on another, properties such as
antimicrobial resistance or toxin production.

 Different plasmids can be found in the same bacterium

 Bacterial structure…

Ribosomes:
 These are sites of protein production distributed in the cytoplasm.

Inclusion granules:
 Composed of volutin, lipid and polysaccharide.

 These inclusions are sources of stored energy.

CYTOPLASMIC MEMBRANE:
• Acts as a semi-permeable membrane, controlling the movement of
water, nutrients, and excretory substances in and out of the cell.

• It also secretes extracellular enzymes and toxins.

MESOSOMES:
• They are sites of respiratory enzyme activity and assist with cell
reproduction.
CELL WALL:
This provides the bacterial cell with rigidity and protects
against osmotic damage.

Helps maintain the shape of the bacteria.

Protects plasma/cytoplasmic membrane & the cell contents

Provides anchorage for the flagella

Antigenic Determinant
Provides ability of some species to cause disease

 It is the site of action of some antibiotics

Based on differences in the composition of bacterial cell walls,
revealed by the Gram staining technique bacteria can be divided
into those that are Gram positive, and those that are Gram
negative.

The cell wall of Gram negative bacteria contains a smaller

amount of peptidoglycan.

•Gram-negative bacteria have an outer membrane covering the

peptidoglycan. The outer membrane contains the endotoxin,
LPS.
•LPS is comprised of lipid A, the toxic portion
Peptidoglycan/murein is composed of:
Sugars;

N-acetylglucosamine (NAG)

N-acetymuramic acid (NAM)

Amino acids cross link NAG & NAM

Gram + VS Gram –VE CELL WALL
Acid-Fast Cell Wall

 Acid fast bacteria (Mycobacterium & Norcadia) contain high

concentrations waxy lipid called mycolic acid in their cell wall that
prevents the uptake of dyes.

 The mycolic acid forms a layer outside of a thin layer of

peptidoglycan.

 Mycolic acid & peptidoglycan are held together by a

polysaccharide
Flagella –

 They are for locomotion.

 The arrangement of flagella may be as :

(i)Monotrichous – single flagella on one side

(ii) Lophotrichous – tuft of flagella on one side

(iii) Amphitrichous – single or tuft on both sides

(iv) Peritrichous – surrounded by lateral flage

 Monotrichous Vibrio cholerae

Lophotrichous Bartonella bacillifornis

Amphitrichous Spirillum serpens

Peritrichous Escherichia coli

 Bacterial structure…

Fimbriae/pili
 They enable organisms to adhere to host cells and to one another.

 Specialized sex fimbriae enable genetic material to be transferred

from one bacterium to another, a process called conjugation.

Capsule
 Possessed by some bacteria. It is protective and usually increases
the virulence of an organism.
 Bacterial structure…

Spores:
 formed by bacteria of the genera Bacillus and Clostridium.

 Spores may be terminal sub-terminal or central.

 Spores are protective structures enabling the bacterium to

survive adverse conditions.

 They are thick-walled and able to withstand dehydration,

heat, cold, and the action of disinfectants.
CLASSIFICATION OF BACTERIA
 CLASSIFICATION OF BACTERIA

Bacteria are classified based on the following criteria :

Morphology
Staining reactions
Cultural characteristics
Biochemical reactions
Antigenic structure
Growth requirements(Energy Source and Nutrient Source)
cellular respiration
Classification of bacteria based on morphology

Morphologically bacteria can be classified as:

 Cocci (Singular: coccus)
• Round/oval bacteria.

• May form pairs (diplococci) or chains (streptococci) or

irregular groups (staphylococci )

Bacilli/rods (Singular: rod, bacillus)

• Rod-shaped/cylindrical cells
Vibrios (Singular: vibrio)
• Curved or comma shaped rods.

Spirilla (Singular: spirillum)

• Regularly coiled

Spirochaetes (Singular: spirochaete).

• Flexible and coiled.
• Spirochaetes are divided into three main groups:-
I. Treponemes, which are thin with regular tight coils.
II. Borreliae, which are large with irregular open coils,
III. Leptospires, which are thin with many tightly packed coils that are
difficult to distinguish.
 Classification of bacteria…

6. Pleomorphic:
Lacking a distinct shape e.g. mycoplasma.
Classification of bacteria…

Shape
Coccus – spherical, round
or ovoid

Bacillus – rod-shaped

Spirillum – spiral-shaped

Vibrio – comma-shaped
2. Classification of bacteria based on gram staining

This divides bacteria into 2 groups:

i.Gram-positive
ii.Gram-negative bacteria.

Gram positive bacteria stain purple while gram negative bacteria stain
red (see gram technique).

The different stains are due to differences in the cell walls of gram-
positive and gram-negative bacteria.
 Gram staining

Materials
Crystal Violet (Primary Stain)
Lugols Iodine (Mordant)
50% Acetone Alcohol (dicolouriser)
Dilute Carbol Fuchsin/ safranin (counter stain)
Specimen
Clean slide
Heat source
Water
Applicator stick
 Gram staining…..

Protocol;
 Prepare a smear on a glass slide.

 Heat fix the smear

 Add primary stain (crystal violet) & incubate for 1 minute. Rinse with clean
water.

 Add a mordant (Gram's iodine), incubate for 30 seconds.

 Decolourize with ethanol or acetone (critical stage)

 Add secondary stain (safranin/basic fuchsin) & incubate 1 min

 Gram staining…..

Gm +s have a thick cell wall made of peptidoglycan, which stain purple and Gm
-s have a thinner layer which stain pink/red.

Gm -s also have an outer membrane made up of lipids & separated from the
cell wall by a periplasmic space.

Crystal violet (CV) dissociates into CV+ and chloride (Cl–) ions

 These ions penetrate through the cell wall & cell membrane of the cells. The CV+ ion
interacts with -vely charged components of bacterial cells and stains the cells purple.

 Lugol’s Iodine (I– or I3–) interacts with CV+ and forms large CV–I complexes within the inner &
outer cell layers
Gram staining…..

Decolorizer interacts with the lipids of the cell membrane.

Gm -s cell loses its outer membrane plus the CV–I complex leaving the
peptidoglycan layer exposed.

Gm +s get dehydrated & the large CV–I complexes become trapped
within the cell due to the multilayered peptidoglycan.

Gm +s retains primary dye (purple) while Gm -s loses it.

Counterstain (+ charge) is applied to give decolorized Gm -s a pink color

3. Classifying Bacteria based on by Cellular Respiration

Aerobic bacteria, or strict aerobes - require oxygen.

Anaerobic bacteria, or strict anaerobes - cannot tolerate oxygen,

Facultative anaerobes – are generally aerobes, but have the

capacity to grow in the absence of oxygen
 4. Classifying Bacteria by Growth Factors

Under this scheme, they are generally classified according to:

Energy source

Nutrient source

Temperature; Mesophiles , Thermophile etc

PH; acidophiles,Alkalophile
Energy Source
Chemotroph – chemical compounds as an energy source (most
pathogenic bacteria are chemotrophs.)

Phototroph - light as energy source

Nutrient Source
Heterotroph – derive carbon from preformed organic nutrients such as
sugar (most pathogenic bacteria are heterotrophs.)

Autotroph – derive carbon from inorganic sources such as carbon dioxide

 .
5 Classification based on cultural characteristics

This is based on colony features e.g

Size (large/small)
Shape (circular/spindle/irregular/filamentous/rhizoid)
Elevation (raised/flat/convex/pulvinate)
Margins (entire/undulate/lobate/curled/filiform
Texture (smooth/rough)
Pigmentation (pigmented/nonpigmented)
Optical property (opaue.transparent)
Appearance (shinny/dull)
6. Classification based on Biochemical characteristics
Examples include:
Ability to ferment sugars.

Capacity to digest or metabolize complex polymers such as proteins

and polysaccharides.

Production of gas

Presence of enzymes such as catalase, oxidase and decarboxylases

Sensitivity to antimicrobial drugs.