Microbiology

SBT 1313
Coordinator: Asst. Prof. Dr. Nur Hafizah Binti Azizan
Co-instructor: Assoc. Prof. Dr. Zaima Azira Binti Zainal Abidin

1
SBT 1313
Sem 1, 2021/2022
Assessment weightage:

Methods Percentage

Quiz 10

Mid-Semester examination 20

Lab report 15

Assignment 15

Final examination 40

TOTAL 100

2
Basic Concept of Microbiology
SBT 1313 Fundamental of Microbiology

3
Outline
• Members of the microbial world
• The microscope
• Preparation and staining of specimens
• Electron microscopy
• Procaryotic cell structure and function
• Eucaryotic cell structure and function

4
Members of the Microbial
World

5
Members of the microbial world
• Microbiology – study of organisms and agents too small to
be seen clearly by naked eye.
• Objects less than about 1 mm in diameter cannot be seen
clearly, need microscope.
• But some microorganisms, particularly eucaryotic microbes
are visible to the naked eye.
• E.g. bread molds and filamentous algae.

6
Members of the microbial world
• Difficult to set boundaries of microbiology – led to the
suggestion of other criteria.
• Examples: important characteristic of microorganism – simple in
their construction, lacking highly differentiated cells, distinct tissue,
techniques to isolate.
• Microbes are diverse, and their classification has always
been a challenge for microbial taxonomists.
• Their early descriptions as either plants or animals were too
simple.
• Important breakthrough: prokaryotic cells, eukaryotic cells.

7
 Members of the microbial world
Prokaryotic cells Eukaryotic cells
• Greek: pro = before, karyon = • Greek: eu = true, karyon = nut
nut or kernel. or kernel.
• Their contents are not divided • Have a membrane-enclosed
into compartments by nucleus.
membrane.
• Have membrane-bound
• Lack a true membrane-delimited organelles.
nucleus.
• More complex morphology.
• Simple morphology.
• Larger in size.

8
Five kingdoms

9
Classification – great progress
• Much has been learned about the detailed structure of
microbial cells from the use of electron microscopy.
• Biochemical and physiological characteristics of many
different microbes have been determined.
• The sequences of nucleic acids and proteins from a wide
variety of organisms have been compared.

• Five-kingdom system is too simple!

10
 Three domains
• Cellular organisms was divided into 3 domains:
• Bacteria (the true bacteria or eubacteria)
• Archaea
• Eucarya (all eukaryotic organisms)

11
Taxonomic hierarchy
• Domain : Eukarya
• Kingdom : Animalia
• Phylum : Chordata
• Class : Mammalia
• Order : Primates
• Family : Hominidae
• Genus : Homo
• Species : H. sapiens

12
 Bacteria
• Procaryotes, usually single-celled.
• Most have cell walls that contain peptidoglycan.
• Habitat – soil, water, air, skin, mouth, intestines.

E. coli 13
Archaea
• Procaryotes.
• Distinguished from bacteria by many features, especially
• ribosomal RNA sequences,
• lack peptidoglycan in the cell wall, &
• have unique membrane lipids.
• Some are found in extreme environments.
• Methanogens – the methane-makers.
• Extreme halophiles.
• Extreme thermophiles.

14
Eucarya – 4 kingdoms
• PROTISTA - unicellular algae, protozoa, slime molds, and
water molds.
• FUNGI - unicellular yeasts, multicellular molds, mushrooms -
absorb organic matter through their plasma membranes.
• PLANTAE - macroscopic algae, mosses, ferns, conifers,
flowering plants - all are multicellular, all carry on
photosynthesis.
• ANIMALIA - sponges, worms, insects, vertebrates - all ingest
nutrients.

15
Viruses
• Acellular entities that must invade a host cell in order to
replicate.
• Virus classification is a mess – not composed of cells, so
there are arguments about whether they are even really
alive.
• Alive: They have either DNA or RNA, which must mean they
are living.
• Not alive:
• No cellular structure.
• No metabolism and no reproduction until they invade a
living cell.
• Do not have both DNA and RNA - living things have both.

16
 The scope and relevance of
microbiology
Basic Applied
Biology of the microorganisms Concern with practical problems
• Bacteriology • Disease
• Virology • Water and wastewater treatment
• Mycology (study of fungi) • Food spoilage
• Phycology or algology (study of algae) • Food production
• Protozoology • Industrial uses of microbes
• Microbial cytology and physiology
• Microbial genetics and molecular
biology
• Microbial taxonomy

17
The microscope

18
 Lenses and the bending of light
• Light microscope use glass lenses to bend and focus light
rays to produce enlarged images of small objects.
• When light passes from one medium to another, refraction
occurs.
• Refraction index – measure how greatly a substance slows
the velocity of light; the direction and magnitude of bending
is determined by the refractive indices of the two media
forming the interface.
Velocity = Distance travelled per unit time

19
Lenses and the bending of light
• Lens act like a collection of prisms operating as a unit.
• A convex lens will focus these rays to specific point – focal
point (F).
• Distance between the center of the lens and the focal point
– focal length (f).
• Lens with short focal length
will magnify an object more.

20
 The light microscope
"The rise of modern microbiology as one of the most important
sub-disciplines of biology is not imaginable without the discovery
of the modern light microscope. Since its introduction into
scientific investigation in the 17th century, the microscope
advanced to one of the single most important "experimental
tools" of modern microbiology and paved the way for the
formation of the cell theory of life as well as to the discovery of
numerous microbes, cells and cell structures."

21
Light microscope
• Can magnify cells up to a
thousand times, and resolve
details as small as 0.2 µm.
• A limitation is imposed by
the wavelike nature of light,
not by the quality of the
lenses.

22
Light microscope
• Three things are required for viewing cells in a light
microscope:
1) A bright light must be focused onto the specimen by
lenses in the condenser.
2) The specimen must be carefully prepared to allow light
to pass through it.
3) An appropriate set of lenses (objective and eyepiece)
must be arranged to focus an image of the specimen in
the eye.

23
The bright-field microscope
- Forms a dark image against a brighter background

24
The bright-field microscope
Objective lens
•4-times magnifying lens (= 4x)
•10-times magnifying lens (= 10x)
•40-times magnifying lens (= 40x)
•100-times magnifying lens (= 100x) - oil immersion lens

Eye pieces
•Binocular
•prisms and (usually) two ocular lenses

25
 The bright-field microscope
Condenser lens
•Focuses the light coming from the light source onto the specimen
which is placed on the stage.

Sub-stage condenser plate

•Houses the iris diaphragm which allows modification of the light
contrast.

Variable light source

•Light intensity can usually be regulated with the help of a dimmer
and a dimmer switch.

26
Total magnification power (Mtot)

Mtot = Mobj x Moc

27
Resolution
• The most important part of the microscope is the objective,
which must produce a clear image, not just a magnified one.
• Resolution – the ability of a lens to separate or distinguish
between small objects that are close together.
• Limit of resolution – how far apart adjacent objects must be
in order to be distinguished as separate entities.
• The smaller the limit of resolution, the greater the resolving
power.

28
Resolution
• Magnification (M) is achieved by the light microscope with
the help of glass lenses and their unique refraction
properties as well as resolution power.
• Unfortunately many (cheap) glass lenses magnify without
increasing the resolution of the object under observation.
• They give us an enlarged but very blurred view on the
object.
• The most important value of microscope lenses is indeed
their resolution.

29
30
Resolution
• To increase the cone angle, put a small drop of a highly pure
oil, referred to as immersion oil.
• The physico-chemical properties (refractive index) of the
immersion oil dramatically increase the cone angle of light
coming from the specimen à higher magnification.

31
To increase resolution
• Change 3 parameters :
• increasing the cone angle, q
• maximizing the refractive index, h
• decreasing the wavelength of the light source used to
observe the specimen

32
• Another important factor
which significantly
contributes to successful and
optimum light microscopy is
the proper alignment of the
path of light through the
microscope along the optical
axis of the microscope.

33
Preparation and staining of
specimens

34
Preparation and staining of
specimens
• Increase visibility
• Accentuate specific morphological features
• Preserve for further study

The stained cells should resemble living cells.

35
Fixation
• A process by which the internal and external structures of
cells and microorganisms are preserved and fixed in
position.
• Inactivate enzymes that might disrupt cell morphology and
toughens cell structures so that they do not change during
staining and observation.
• A microbe usually is killed and attached firmly to the
microscope slide during fixation.

36
Heat fixation
• Usually for procaryotes.
• Heat preserves overall morphology, but not structures within cells.

Chemical fixation
• Protect fine cellular structure and morphology of larger and delicate
organism.
• Chemical fixatives penetrate cells and react with cellular
components, usually proteins and lipids, to render them inactive,
insoluble and immobile.
• Common fixative mixtures contain ethanol, acetic acid, mercuric
chloride, formaldehyde, and glutaraldehyde.

37
Dyes and simple staining
• Dyes have two common features:
• Chromophore groups – gives color and bind with cells by
ionic, covalent or hydrophobic bonding.
• Negative staining – give color to the background instead
of the objects.
• Simple staining – used a single dye (simple and easy to use).
• To determine the size, shape, and arrangement in
procaryotic cells.

38
 Dyes and simple staining
The most commonly used dyes: ionizable dyes.
Basic dyes Acidic dyes
•Methylene blue, basic fuchsin, •Easin, rose bengal, acid fuchsin –
crystal violet, safranin, malachite have negatively charged groups
green – have positively charged such as carboxyls (-COOH)
groups. and phenolic hydroxyls (-OH).
•Bind to negatively charged •Bind to positively charged cell
molecules like nucleic acids, structures.
many proteins, and the surfaces
of procaryotic cells.

39
 Differential staining
• Used to distinguish organisms based on their staining properties.

Gram staining
• Divides bacteria into 2 classes.
• Observation – purple cells are gram positive.
– red cells are gram negative.

40
41
Differential staining
Acid fast staining
•Commonly to identify Mycobacterium tuberculosis and M. leprae
(tuberculosis and leprosy) – have cell walls with high lipid content.
•Use harsh method – Ziehl-Neelsen method.
• Use heat and phenol to drive basic fuchsin into the cells.
•Once basic fuchsin has penetrated, these bacteria are not easily
decolorized by acidified alcohol (acid-alcohol) à acid-fast.
•Non-acid-fast bacteria are decolorized by acid-alcohol and thus are
stained by methylene blue counterstain.
•Observation: Red cells are acid-fast; blue cells are non-acid-fast.

42
Staining specific structures
Capsule staining – negative staining
•Reveals the presence of capsules, a network usually made of
polysaccharides that surrounds many bacteria and some
fungi.

Endospore staining – acid fast staining

•Requires harsh treatment to drive dye into endospore.

Flagella staining – simple staining

•Provides taxonomically valuable information about the
presence and distribution pattern of flagella on procaryotic
cells. 43
44
45
Electron microscopy

46
Transmission electron microscope
(TEM)
• Uses magnetic lenses to form an image from electrons that
have passed through a very thin section of a specimen.
• Resolution is high because the wavelength of the electrons
is very short.
• Thin section contrasts can be increased by treatment with
solutions of heavy metals like osmium tetroxide, uranium,
and lead.

47
Transmission electron microscope
(TEM)

48
49
50
Scanning electron microscope (SEM)
• Produces an image from electrons released from atoms on
an object’s surface.
• Used to examine the surfaces of microorganisms in great
detail; many SEMs have a resolution of 7 nm or less.
• Creates striking images of 3D objects.

51
52
53
Newer techniques in microscopy
Confocal scanning laser microscopy
•Use a laser beam to illuminate a specimen, usually one that
has been fluorescently stained.
•Aperture placed above the objective lens – focus the light.
•Normal light microscope – light from all areas of the object
enter the microscope are used to create an image.
•Has numerous applications – the study of biofilms, which can
form on many surfaces including in-dwelling medical devices
such as hip-joint replacements.

54
Procaryotic cell structure and
function

55
Shape, arrangement and size

56
Procaryotic cell

57
58
Plasma membrane
Functions:
•Retain cytoplasm.
•Selective permeable barrier (only certain particles can move
in and out).
• nutrient uptake, waste excretion, protein secretion
•Location of variety of crucial metabolic processes.
• respiration, synthesis of lipids, photosynthesis
•Receptor molecule – to detect and respond to chemicals in
their surroundings.

59
Bacterial plasma membrane structure

60
Chemical nature of membrane lipids
• Membrane-associated lipids are structurally asymmetric,
with polar and nonpolar ends = amphipathic.
• Polar ends interact with water = hydrophilic.
• Polar ends insoluble in water = hydrophobic.

61
Bacterial membranes
• Many of amphipathic lipids are phospholipids.
• The lipid composition varies with environmental
temperature so that the membrane remain fluid during
growth.
• Differ from eucaryotic – lack sterols (steroid-containing
lipids) such as cholesterol, but have many sterol-like
molecules called hopanoids.

62
 Archeal membranes
• Differ from bacteria and
eucarya – have branched
chain hydrocarbons attached
to glycerol by ether links,
rather than fatty acids
connected by ester links.

63
Bacterial cell wall

Gram-positive cell wall Gram-negative cell wall

•20-80 µm peptidoglycan layer. •2-7 µm peptidoglycan layer.
•Plasma membrane. •7-8 nm outer membrane.
•Thicker and more resistant to •Plasma membrane.
osmotic pressure.
64
Peptidoglycan
• Enormous, meshlike polymer
composed of many identical units.
• The polymer contains 2 sugar
derivatives:
• N-acetylglucosamine (NAM)
• N-acetylmuramic acid (NAG)
• and several different amino acids.

65
 Peptidoglycan
• The backbone of a peptidoglycan
strand composed of alternating NAG
and NAM residues.
• Chains of peptidoglycan are joined
together by cross-links à strong.
• Directly: carboxyl group of the
terminal D-alanine & amino
group of diaminopimelic acid.
• Peptide bridge.
• Most gram negative cell wall lacks
peptide interbridge.

66
 58 Chapter 3 Procaryotic Cell Structu

Gram-positive cell walls

O
• Thick, composed primarily of peptidoglycan.
O P O-
• Usually contain large amounts of secondary cell wall O
polymers, including teichoic acids. CH2
• Polymers of glycerol or ribitol joined by phosphate H C O R
groups. CH2

• Lipoteichoic acids – those covalently connected to O

membrane lipids. O P O-
O
• Teichoic acids extend to the surface of peptidoglycan à CH2
Gram-positive CW = negative charge.
H C O R
• Absent from Gram negative bacteria. CH2
O
O P O-
O
67
 Gram-positive cell walls
• Periplasmic space – smaller than gram-negative bacteria. Have
relatively few proteins.
• Peptidoglycan sac is porous and any protein secreted by the wall
usually pass through it.
• Enzyme secreted by Gram-positive bacteria = exoenzymes.
• Degrade polymeric nutrients.

68
69
 Gram-negative cell walls
• More complex.
• Periplasmic space (1-71 nm) ~ 20-40% of total cell volume.
• Periplasmic proteins:
• Nutrient acquisition – hydrolytic enzymes, transport proteins.
• Energy conservation.
• Modification of toxic compound.
• Peptidoglycan synthesis.
• Outer membrane & peptidoglycan is linked by 2 ways:
• Braun’s lipoprotein
• Adhesion sites joining the outer membrane & the plasma
membrane – may be regions of direct contact or possibly true
membrane fusions.
70
71
 Gram-negative cell walls
• Most unusual constituents
of outer membrane:
Lipopolysaccharide (LPSs):
• Complex molecules
containing lipid and
carbohydrate.
• Have 3 parts:
• lipid A
• core polysaccharide
• O side/O antigen

72
Function of LPSs
• Negative charge on bacterial surface.
• Stabilize outer membrane structure.
• Surface attachment and biofilm formation.
• Create a permeability barrier to restrict entry of toxic
substances.
• Protect pathogenic Gram-negative bacteria from host
defenses.
• Act as an endotoxin and cause some of the symptoms that
arise in Gram-negative bacterial infections.

73
The mechanism of gram staining
• Gram stain reaction results – difference of cell walls.
• Peptidoglycan itself is not stained but act as a permeability
barrier preventing loss of crystal violet.
• During the procedure, the bacteria are first stained with
crystal violet and next treated with iodine to promote dye
retention.
• When gram-positive bacteria treated with ethanol, the
alcohol shrink the pores of the thick peptidoglycan.
• Dye iodine complex is retained during this short
decolorization step and bacteria remain purple.

74
The mechanism of gram staining
• Gram-negative peptidoglycan is very thin, not highly cross-
linked and has a larger pores.
• Alcohol treatment also extract enough lipid from the gram-
negative outer membrane to increase its porosity
• Alcohol more readily removes the purple crystal violet-
iodine complex from gram-negative bacteria.
• Gram-negative bacteria are easily stained red or pink by the
conterstain safranin.

75
Gram staining

76
 . Protoplast formation induced by incubation with penicillin in an
elling due
Archeal cell walls
H2O influx Lysis

ir plasma mem-
• Lack peptidoglycan. CW
an those of bacte-
• Cell wall structure and chemistry differ from those of the
CM
ot clear,bacteria.
although
any species may CPL
• Exhibit great diversity in their cell wall make-up: Some
all,
ced mycoplasmas
composed of heteropolysaccharides, some composed of
by incubation with penicillin in an isotonic medium.
glycoprotein, and some composed of protein.
(a)
• The most common type is an S-layer; 20-40 nm thick.

doglycan.Why does Polysaccharides 0.1 µm S-layer

SL
CW
nd glutamic acid Plasma membrane CM
Plasma membrane
CM
Cytoplasm Cytoplasm
CPL
CPL
teria and gram- 77
Eucaryotic cell structure and
function

78
Eucaryotic cell structure and function
• The most obvious difference between eucaryotic and
procaryotic cells is: has a true, membrane-delimited nucleus
and many membraneous organelles.
• Membraneous organelles compartmentalize the cytoplasm
of the cell and perform specific functions.
• Allows cell to carry out different biochemical and
physiological functions in separate compartments
simultaneously under independent control and proper
coordination.
• Also provides more surface area for membrane-associated
activities, e.g. respiration.

79
80
 Plasma membrane Mechanical cell boundary, selectively permeable barrier with transport systems, mediates cell-cell
interactions and adhesion to surfaces, secretion
Cytoplasmic matrix Environment for other organelles, location of many metabolic processes
Microfilaments, intermediate Cell structure and movements, form the cytoskeleton

Plasma membrane and membrane

filaments, and microtubules
Endoplasmic reticulum
Ribosomes
Transport of materials, protein and lipid synthesis
Protein synthesis

structure
Golgi apparatus
Lysosomes
Mitochondria
Packaging and secretion of materials for various purposes, lysosome formation
Intracellular digestion
Energy production through use of the tricarboxylic acid cycle, electron transport, oxidative phosphorylation,
and other pathways
Chloroplasts Photosynthesis—trapping light energy and formation of carbohydrate from CO2 and water

• Eucaryotic membranes are similar in structure and function

Nucleus
Nucleolus
Repository for genetic information, control center for cell
Ribosomal RNA synthesis, ribosome construction

to those of bacteria. The two differ in terms of their lipid

Cell wall and pellicle
Cilia and flagella
Strengthen and give shape to the cell
Cell movement

composition.
Vacuole Temporary storage and transport, digestion (food vacuoles), water balance (contractile vacuole)

• Contain lipid rafts – enriched with certain lipids and proteins

and participate in a variety of cellular processes.
O CH3
O
CH3
P N!
O O O CH3
Hydrophobic tails O"
O

(a) Phosphoglyceride O

O CH3
OH
CH3
P N!
O O CH3
O"
NH

(b) Sphingolipid O

OH

81
(c) Sterol
Sources
• Prescott, L.M., Harley, J.P., & Klein, D.A. (2008).
Microbiology, 7th Ed., McGraw-Hill.

82