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Veterinary Bacteriology and
Mycology




Target Group: Year III Vet. Med. Students

Ac a d e m ic Ye a r:

2 0 2 3 /2 4

1
 Course objectives
Upon completion of this course the students would be able to:
Describe general features of pathogenic bacteria and fungi
Identify laboratory hazards and strictly follow laboratory safety guidelines/bio
-security protocols, and conduct the respective procedures effectively
Identify pathogenic bacteria and fungi of veterinary and zoonotic importance
Describe specific features of pathogenic bacteria and fungi
Acquire comprehensive knowledge of major bacterial and fungal superf icial
and systemic diseases of domestic and wildlife animals
Select, collect, transport and preserve samples so as to diagnose and treat
specific animal diseases of bacterial and fungal origin
Carry out independently laboratory works like biochemical tests, gram
reactions, inoculation, isolation and identif ication techniques and become
capable of interpreting laboratory findings
Conduct laboratory diagnosis of specif ic animal diseases of bacterial and
fungal infections with sound recommendations
Give emphasis on major animal skin and hide diseases caused by bacteria
2
and fungi
Chapter 1: Introduction to veterinary microbiology

 Definition and importance of microbiology

 History of microbiology

 The scope and relevance of microbiology

3
 INTRODUCTION:

I. Historical development of Microbiology

Microbiology is defined as the study of organisms and agents

too small to be seen clearly by the unaided eye.

The subject began with the discovery of microorganisms in

1674 by Antonie van Leeuwenhoek, using a microscope of his
own design.

A microorganism or microbe is a microscopic living organism,

which may be single-celled or multicellular.

Microorganisms are distinct form macro organisms. 4

Microorganisms are very diverse and include all bacteria,
Virus, archea, most protozoa, some Fungi, Algae.

Some organisms studied by microbiologists CAN be visualized

without the aid of amplification [e.g. bread molds (fungus) and
filamentous algae].

These organisms are included in the discipline of microbiology

because of similarities in properties and techniques used to
study them

Microorganisms live in every part of the biosphere, including soil,

hot springs, "seven miles deep" in the ocean, "40 miles high" in
the atmosphere and inside rocks far down within the Earth's
5
crust etc.
 The scope of microbiology

Modern microbiology is a large discipline with many different

specialties.

It has a great impact on fields such as:

Medicine Genetics

Agricultural Biochemistry

Food sciences/industries Pathology

Ecology Pharmacology

Molecular biology etc. 6

 Relevance of Microbiology

T hose m ic rob es that inhab it hum ans also p lay

important roles; including helping the body digests
food and producing vitamins B and K.

I n a d d i t i o n , s o c i e t y i n g e n e ra l b e n e f it s f r o m
m ic roorganism s, as they are nec essary for the
production of:
Bread, Cheese, Beer, Antibiotics, Vaccines, Vitamins,
Enzymes and many other important products.

7
 Branches of Microbiology
Agricultural microbiology
Aquatic microbiology
Industrial microbiology
Food microbiology
Medical microbiology
Space microbiology
Environmental microbiology
Veterinary microbiology etc
8
Veterinary microbiology is concerned with microbial
agents affecting animals as well as humans and
animals (zoonotic microbes).

9
 Origin of microorganisms
At one time, commensal organisms in association with hosts
lost the ability to carry out key processes, thus coming to
depend on the host to provide essentials for growth and
survival.

With increased dependence, some organisms “defected,”

replicating at the host’s expense, using host cell machinery and
in some cases causing what we recognize as disease.

10
 The Discovery of Microorganisms

Ev en before m ic roorganism s were seen, som e

i n v e s t i g a t o rs s u s p e c t e d t h e i r e x i s t e n c e a n d
responsibility for disease.

Among others, the Roman philosopher Lucretius

(98–55 B.C.) and the physician Girolamo Fracastoro
(1478–1553) suggested that disease was caused by
invisible living creatures.
11
 Spontaneous Generation Debate
Abiogenesis Vs Biogenesis

Abiogenesis (Spontaneous generation) claims that life

can originate from non-living matter.

Biogenesis states that living cells originate from living cells.

The belief in the spontaneous generation of life from

nonliving matter was introduced by Aristotle, who
lived around 350 BC.
12
 Contrasting theories about the origins of microbes
led to experiments which greatly impacted the
methodologies of microbiology

This belief remained unchallenged for more than

2000 years.

Until….

13
 Francesco Redi - Experiments on Flies
First to formally challenge the accepted belief of spontaneous
generation.
Redi's Question: Where do maggots come from?
Hypothesis: Maggots come from flies.

Experiment: Redi put meat into three separate jars.

Jar-1
• Left open
• Maggots developed
• Flies were observed laying eggs on the meat in the open jar

Jar-2
• Covered with netting
• Maggots appeared on the netting
• Flies were observed laying eggs on the netting
Francesco Redi, Italian
physician, naturalist &
poet, 1626 – 1697.
Jar-3
• Sealed
• No maggots developed 14
 Flask covered

Flask unsealed Flask sealed
with gauze

15
 The Controversy Over Spontaneous Generation


John Needham & Lazzaro Spallanzani
The Question:
What causes tiny living things to appear in decaying broth?

Needham’s Hypothesis : Spontaneous generation.

Spallazani’s Hypothesis: Microbes come from the air. Boiling

will kill them.

Needham >

1713 - 1781

Spallazani >
1729 - 1799

16
Experiment: Heat broth to kill all life within, then see if, after heat
is removed, microbes reappear.

Needham and Spallanzani did almost identical experiments.

However, there was one key difference that critically impacted
their results. Needham didn’t cover his flasks, so material
(including microbes) from the surrounding air could drift into his
flasks.

Spallanzani’s flasks were sealed. This difference in experimental

design accounted for their opposing conclusions:

Needham: Life re-emerged due to spontaneous

generation.

Spallanzani: Heat killed the microbes, and, since the

flasks were sealed, microbes did not reappear. 17
 French chemist Louis Pasteur’s design of this experiment settled
the argument.

Louis Pasteur's disproved spontaneous generation.

18
Pasteur’s Swan Neck Experiment

19
20
21
 Pasteur’s Conclusions

The bended neck allowed air to enter the bottle and the liquid
but trapped any particulates including microorganisms.

No microbial growth as long as the liquid broth did not come

in contact with the microbes.

Hence air alone was not sufficient to generate life.

22
Contribution of Louis Pasteur to Microbiology

1. Finally disproved Spontaneous Generation

- Boiled broth in long-s-shaped necked flasks (unsealed)
Remained sterile
Proved that microorganisms are present in air, but air
does notcreate microbes

2. Beginning of the golden age of microbiology

3. Proved Microorganisms are present in nonliving matter

23
 4. Microbes can be destroyed by heat
- Aseptic Technique

5. His work led to the development of methods for

controlling the growth of microorganisms.

6. Fermentation mediated by yeast, not air

7. Pasteurization to prevent wine and beer spoilage (by

bacteria)

8. He hypothesized in 1857 that microorganisms are

responsible for infectious diseases

24
 Louis Pasteur & Industrial Microbiology

- Q: What is fermentation?

< yeast + grapes = yummy wine  (ethanol)

- What causes fermentation?
Some scientists thought that air caused bacteria + grapes = spoiled wine  (lactic
fermentation acid) >
Others thought that microbes caused fermentation .

- Q: What is pasteurization?

Fermentation = breakdown of sugar, resulting in either the

waste product of ethanol or lactic acid

Pasteurization is the process of heating a food, usually a

liquid, to a specific temperature for a certain amount of time,
and then immediately cooling it. Slows spoilage due to
microbial growth.
25
 Germ Theory of Disease

Recognition of microbes as disease

agents

The philosopher Lucretius (98–55 B.C.), the physician Girolamo

Fracastoro (1478–1553) and others suggested that disease
was caused by invisible living creatures (microorganisms).

L a t e r o n , s c i e n t i s t s b e g a n c o n f ir m a t o r y
investigations on the involvement of microbes 26in
 But, the first direct demonstration of the role of bacteria in
causing disease came from the study of anthrax by the German
physician Robert Koch (1843–1910).

Koch used the criteria proposed by his former teacher, Jacob

Henle (1809–1885), to establish the relationship between
Bacillus anthracis and anthrax.

Koch injected healthy mice with material from diseased

animals, and the mice became ill.

After transferring anthrax by inoculation through a series of 20

mice, he incubated a piece of spleen containing the anthrax
bacillus in beef serum.
The bacilli grew, reproduced.
When the isolated bacilli or spores were injected into mice,
anthrax developed.
27
The criteria for proving the causal relationship
between a microorganism and a specif ic disease
are known as Koch’s postulates and can be
summarized as follows:

28
1. The microorganism must be present in every case of the
disease but absent from healthy organisms.

2. The suspected microorganism must be isolated and grown in

a pure culture.

3. The same d isease must result when the iso lated

microorganism is inoculated into a healthy host.

4. The same microorganism must be isolated again from the

diseased host.
These postulates are still used today to conf irm
the cause of an infectious disease. 29
The study of microbial
structure

30
Indeed, numerous methods have been
developed to identify specif ic microbes, cellular
structures, DNA sequences, or indicators of
infection in tissue samples.

31
 The light microscope

A microscope (from the Ancient:Mikrós , "small" and,

Skopeîn , "to look" or "see") is an instrument used to
see objects that are too small for the naked eye.

M i c r o s c o p y i s t h e t e c h n i c a l f ie l d o f u s i n g
microscopes to view objects and areas of objects that
cannot be seen with the naked eye (objects that are
not within the resolution range of the normal eye).

Microscopic means invisible to the eye unless aided

by a microscope. 32
The invention of the microscope has opened up a whole new
dimension in science.

By using microscopes scientists were able to discover the

existence of microorganisms, study the structure of cells, and
see the smallest parts of plants, animals, and fungi.

Today, the microscope is still a commonly used tool to

diagnosis illness in hospitals and clinics all over the world.

Since their original invention, microscopes have moved beyond

the simple visible light refracting lenses.

Electrons, x-rays, and infrared rays are used by far more

sophisticated microscopes to detect even smaller and smaller
structures.
Scanning electron microscopes are able to resolve viruses, 33
which are far smaller than any cell.
Microscopes can be separated into several different
classes.

One grouping is based on what interacts with the sample to

generate the image, i.e., light or photons (optical microscopes),
electrons (electron microscopes) or a probe (scanning probe
microscopes).

Alternatively, microscopes can be classed on whether they

analyze the sample via a scanning point (confocal optical
microscopes, scanning electron microscopes and scanning
probe microscopes) or analyze the sample all at once (wide
f ie l d o pt i c a l m i c r o sc o pe a n d t r a n sm i ssi o n e l e c t r34o n
Even before microorganisms were seen, some investigators
suspected their existence and responsibility for disease.

Among others, the Roman philosopher Lucretius (98–55 B.C.)

and the physician Girolamo Fracastoro (1478–1553) suggested
that disease was caused by invisible living creatures.

The earliest microscopic observations appear to have been

made between 1625 and 1630 on bees and weevils by the Italian
Francesco Stelluti, using a microscope probably supplied by
Galileo.
35
H o w e v e r, t h e f ir s t p e r s o n t o o b s e r v e a n d d e s c r i b e
m i c r o o r ga n i s m s a c c u r a t e l y w a s t h e D u t c h a m a t e u r
microscopist Antony van Leeuwenhoek (1632–1723).

Leeuwenhoek observed both bacteria and protozoa.

Major contributions to the development of microbiology was

the invention of the microscope by Anton von Leuwenhoek and
the implementation of the scientific method.

The discovery and characterization of viruses was aided by

electron microscope, invented in 1931.

Many more viruses were then discovered and even yet new
viruses are being discovered.
36
Magnif ication is the apparent increase in size of an object and
is indicated by a number followed by an “× ” which is read
“times.”

Resolution (also calledresolving power ) is the ability to

distinguish between objects that are close together.

The better the resolution, the better the ability to distinguish

two objects that are close to one another.
37
The light microscope: uses visible light to observe
specimen

38
ELECTRON Microscope: uses electrons instead of visible
light to observe specimen

39
I n t h e i r n a t u ra l s t a t e , m o s t o f t h e c e l l s a n d
microorganisms that we observe under the microscope
lack color and contrast.

This makes it dif fic ult, if not impossible, to detect

important cellular structures and their distinguishing
characteristics without artificially treating specimens.

40
Preparing Specimens for Light Microscopy
In clinical settings, light microscopes are the most commonly
used microscopes.

The re are two basic type s o f pre paratio n use d to v ie w

specimens with a light microscope: Wet mounts and Fixed
specimens

The simplest type of preparation is the wet mount, in which the

specimen is placed on the slide in a drop of liquid.

Some specimens, such as a drop of urine, are already in a liquid

form and can be deposited on the slide using a dropper.
41
Solid specimens, such as a skin scraping, can be placed on the
slide before adding a drop of liquid to prepare the wet mount.

Sometimes the liquid used is simply water, but often stains are
added to enhance contrast.

Once the liquid has been added to the slide, a coverslip is

placed on top and the specimen is ready for examination under
the microscope.

42
The se c o nd me t ho d o f pre pari ng spe c i me ns fo r l i ght
microscopy is fixation.

The “f ixing” of a sample refers to the process of attaching cells

to a slide.

Fixation is often achieved either by heating (heat f ix ing) or

chemically treating the specimen.

43
In addition to attaching the specimen to the slide, f ixation also
kills microorganisms in the specimen, stopping their movement
and metabolism while preserving the integrity of their cellular
components for observation.

In addition to fixation, staining is almost always applied to color

certain features of a specimen before examining it under a light
microscope.

44
 Why stain cells?

 C e l l s a r e s t a i n t o r e v e a l t h e s i z e a n d s h a pe o f
microorganisms.

 The most basic reason that cells are stained is to

enhance visualization of the cell or certain cellular
components under a microscope.

 C e lls may also be staine d to highlight me tabo lic

processes.

 To differentiate between live and dead cells in a sample.

45
  Cells may also be enumerated by staining cells to
determine biomass in an environment of interest.

 Cells are stained to demonstrate the presence of internal

and external structures.

 Cells are stained to distinguish between different types of

organisms.

The preparatory steps involved depend on the type of

analysis planned

46
Bacterial staining is the process of coloring of
colorless bacterial structural components using stains
(dyes).

47
 Types of Staining Methods in Microbiology

1. Simple staining method

2. Differential staining method

3. Special staining method

48


Simple staining method

It is type of staining method in which only a single dye is used.

Usually used to demonstrate bacterial morphology and
arrangement.

Two kinds of simple stains

1. Positive staining: The bacteria or its parts are stained by the

dye.
Eg. Carbol fuchsin stain
Methylene blue stain
Crystal violet stain

2. Negative staining: The dye stains the background and the

bacteria remain unstained.
Eg. Indian ink stain 49

Negrosin stain
 Differential Stains

Staining procedure which differentiates or distinguishes
between types of bacteria is termed as differential staining
technique.

Methods for simple staining impart same colour to all bacteria

and other biological material, may be slight variation in shade.

On the other hand, differential staining methods impar t

distinctive color only to certain types of bacteria.
50
The basic principle underlying this differentiation is due to the
different chemical and physical properties of cell and as a
result, they react differently with the staining reagents.

Differential staining procedure utilizes more than one stain.

In some techniques the stains are applied separately, while in

other as combination.

There are two most important differential stains, namely, (A)

Gram stain and (B) Acid-fast stain.

51
 (A) Gram Stain

Gram stain is one of the most important and widely used

differential stains.

It has great taxonomic signif icance and is often the f irst step in
the identification of an unknown prokaryotic organism.

This technique divides bacteria into two groups:

(i) Gram positive those which retain primary dye like crystal
violet and appear deep violet in colour and

(ii) Gram negative, which lose the primary dye on application of

decolourizer and take the colour of counterstain like safranin or
basic fuchsin. 52
 Acid-Fast Stains
Acid-fast staining is another commonly used, differential
staining technique that can be an important diagnostic tool.

An acid-fast stain is able to differentiate two types of gram-

positive cells:
Those that have waxy mycolic acids in their cell walls, and
Those that do not.
Two different methods for acid-fast staining are the Ziehl-
Neelsen technique and the Kinyoun technique.

Both use carbolfuchsin as the primary stain.

The waxy, acid-fast cells retain the carbolfuchsin even after a
decolorizing agent (an acid-alcohol solution) is applied.
A secondary counterstain, methylene blue, is then applied, which
renders non–acid-fast cells blue.
53
 Special Stain
Capsule Staining

Certain bacteria and yeasts have a protective outer structure

called a capsule.

Since the presence of a capsule is directly related to a microbe’s

virulence (its ability to cause disease), the ability to determine
whether cells in a sample have capsules is an impor tant
diagnostic tool.

54
Capsules do not absorb most basic dyes ; therefore, a negative
staining technique (staining around the cells) is typically used
for capsule staining.

The dye stains the background but does not penetrate the
capsules, which appear like hales around the borders of the cell.

The specimen does not need to be heat-f ixed prior to negative

staining.
55
 Endospore Staining

Endospores are structures produced within certain bacterial

cells that allow them to survive harsh conditions.

Gram staining alone cannot be used to visualize endospores,

which appear clear when Gram-stained cells are viewed.

Endospore staininguses two stains to differentiate endospores

from the rest of the cell.

56
 Flagella Staining

Flagella (singular: flagellum) are tail-like cellular structures used

for locomotion by some bacteria, archaea, and eukaryotes.

Because they are so thin, f la gella typically cannot be seen

under a light microscope without a specialized f lagella staining
technique.

57
Flagella staining thickens the f lagella by f irst applying mordant
(generally tannic acid, but sometimes potassium alum), which
c o a t s t he f la ge l l a ; t he n t he spe c i m e n i s st a i ne d w i t h
pararosaniline (most commonly) or basic fuchsin

58
A f la gella stain of Bacillus cereus, a common cause of
foodborne illness, reveals that the cells have numerous f lagella,
used for locomotion.

59
Prokaryotic/bacterial cell structures and
function

An over view of prokaryotic cell structure

- Size

- Shape

- Arrangement

60
61
Morphology and structure of bacteria

Gross Morphology (The Size, Shape & Arrangement)

of Bacterial Cells

Bacteria are very small, most 0.5-1 µm in diameter

(width).

They display a variety of shapes, arrangements &

sizes, called morphology of the organism.
62
The majority of bacteria are found in three basic shapes:

1. Cocci (round or spherical).

2. Bacilli (rod)

3. Spirilli (spiral or helical)

63
64
 The bacterial cells appear in different arrangements

Most cocci are approximately 0.5 - 1.0 micrometer (µm) in diameter &
may be seen, based on their planes of division and tendency to remain
attached after replication, in one of the following arrangements:

1. Division in one plane produces either a diplococcus or streptococcus

arrangements.
-Micrococci: single cells
-Diplococcus: a pair of cocci
-Streptococcus: a chain of cocci, e.g.,Streptococcus pyogenes .

2. Division in two planes produces a tetrad arrangement.

-Tetrad: a square of 4 cocci.

3. Division in three planes produces a sarcina arrangement.

-Sarcina: a cube of 8 cocci.

4. Division in random planes produces a staphylococcus arrangement.

-Staphylococcus: cocci in irregular, often grape-like clusters, e.g.,Staph.
aureus 65
66
Bacilli or Rods Cocci in chain form
67
 Bacterial cell organization

 Structurally, a bacterial cell has three

structural regions:

1. Appendages

2. Cell envelope

3. Cytoplasmic region

68
Structures commonly found in a bacterial cell.

69
 Appendages
1. Flagella

 Are f il amentous protein structures attached to the cell

surface that provide the swimming movement for most
motile bacteria.

 Flagella propel the microorganism away from harm and

towards food in a movement known as taxis.
 It is originated / projected from plasma membrane

 Their presence in bacterial cell is detected by: Motility media,

Special staining methods, Dark - f ield microscopy & Electron
70

i
 Found in most Gm-b, many Gm+ bacilli & cocci includingE.
coli , shigella, salmonella, Listeria, pseudomonas, bacillus,
clostridium

 It also act as chemotaxis (able to sense the environment

and responding to some chemical food stuffs or toxic
substances by moving away)

71
Flagellar arrangements

1. Monotrichous: Bacteria with single polar flagellum.

2. Lophotrichous: Bacteria with bunch of flagella

at one pole

3. Amphitrichous: Bacteria with flagella

at both poles

4. Peritrichous: Bacteria with flagella

all over their surface

72
73
 2. Pili
Are cellular appendages, slightly larger than fimbriae, which
can transfer genetic material between bacterial cells in a
process called conjugation.

Two types of pili (Based on their function)

1. Common pili: The structure for adherence or attachment to

cell surface.

Attachment pili are sometimes called fimbriae - Neiseseria

gonorrhoeae

2. Sex pili: The structure for transfer of genetic material from the
donor to the recipient bacteria during the process of conjugation
74
75
3. Fimbriae

Are proteinaceous, sticky, hair-like projections used by cells to

attach to each other, substrates and other cells or tissues in
nature.

76
 The cell envelope




Is a descriptive term for the several layers of
materials that envelope or encloses the protoplasm of
the cell.

The cell protoplasm (cytoplasm) is surrounded by:

1. Plasma membrane
2. Cell wall and
3. Capsule.
 From inside to out side 77
Bacterial cell

78
 Capsules
Capsules or slime layers are produced by many bacteria to
surround their cells.
 Capsules have multiple functions in a particular organism.
1. Often mediate adherence of cells to surfaces.

2. Protect bacterial cells from engulfment by WBC.

3. Protect from attack by antimicrobial agents.

4. Capsules contain a great deal of water and can protect

bacteria against desiccation. 79
 Cell Wall
 Is located outside the plasma membrane.

 Gives the cell its shape and provides rigid structural support.

 protects the cell protoplast from mechanical damage and

osmotic rupture or lysis.

 The wall can protect a cell from toxic substances and is the
site of action of several antibiotics.

 Contains toxic components to host

 Provides staining characteristics to the bacterium

80
 Peptidoglycan layer
Peptidoglycan, also known as murein, is a polymer consisting of
sugars and amino acids that forms a mesh-like layer outside
the plasma membrane of most bacteria, forming the cell wall.

The peptidoglycan layer is substantially thicker in gram-positive

bacteria (20 to 80 nanometers) than in gram-negative bacteria
(7 to 8 nanometers), with the attachment of the S-layer.

81
Peptidoglycan forms around 90% of the dry weight of gram-
positive bacteria but only 10% of gram-negative strains.

Peptidoglycan, a polymer unique to prokaryotic cells, ‘imparts

rigidity to the cell wall’.

82
 Gram positive cell wall

Components of cell wall of Gram positive bacteria

Peptidoglycan and Teichoic acid

 Differences in the structure & chemical composition of the

cell walls of bacterial species account for:

Variation in their pathogenicity &

Influence other characteristics including staining properties.

83
 Gm+b which stain blue have a relatively thick uniform cell wall
which is composed mainly of peptidoglycan & teichoic
acids.

 Normally the thick, homogeneous cell wall of Gm+b is

composed primarily of peptidoglycan which often contains a
peptide interbridge

 They usually contain large amounts of teichoic acids w/c are

to either the peptidoglycan itself or plasma membrane lipids

84
Teichoic acid are polymers of either glycerol phosphate or
ribitol phosphate, with various sugars, amino sugars, and amino
acids

Te i c ho i c ac i d s - appe ar t o e x t e nd t o t he surfac e o f

peptidoglycan & are negatively charged

 They may be important in maintaining the structure of the

wall

85
The periplasm is an area of considerable enzymatic 86
87
 Gram negative cell wall

Components of cell wall of Gram negative bacteria

Peptidoglycan (murein),

Lipoprotein,

Phospholipid and

Lipopolysaccharide

88
• Gm-b have cell walls with a more complex structure, consisting
of an outer membrane & a periplasmic space containing a
comparatively small amount of peptidoglycan.

• The outer membrane is a protein-containing asymmetrical lipid

bilayer.

• The structure of the inner surface of the m/m resembles that of

the cytoplasmic membrane, whereas that of the outer surface is
composed of lipopolysaccharide (LPS) molecules.

89
• Low M.wt substances such as sugars & AAs enter through
specialized protein channels (porins) in the outer membrane.

• The thin peptidoglycan layer next to the plasma m/m may

constitute < 10% of the wall weight

• The outer membrane lies outside the thin peptidoglycan layer

• The LPS that contains lipid is called endotoxin w/c is toxic to

animals & human beings

• Appears similar to a cell membrane when viewed by electron

microscopy
Lipoprotein - the most abundant membrane protein
90
The Gram-Negative Envelope 91
92
93
Gram-Positive and Gram-Negative Cell Walls
M= peptidoglycan or murein layer; OM= outer membrane;
PM= plasma membrane; P= periplasmic space;
W= gram-positive peptidoglycan wall. 94
The mechanisms of gram
staining

95
Gram stain of Gram positive Staphylococcus cell Gram stain of Gram negative E. coli

96
Prokaryotic cell membrane

- Plasma membrane

- Mesosomes

97
 Cytoplasmic Membrane

Forms the outer structure of the cell and separates the cell's
internal structure from the environment.

It accounts for 30% of the dry weight of bacterial cell.

It is composed of 60% protein, 20-30% lipids (phospholipids) &

10-20% carbohydrate.

98
This membrane is selectively permeable because it permits
the transport of some substances and inhibits the transport of
others.

The membrane regulates the f lo w of molecules (such as

nutrients) into the cell and removes waste from the cell by
opening and closing passages called channels.

99
Mesosomes

Convoluted invagination of cytoplasmic membrane

often at sites of septum formation.

It is involved in DNA segregation during cell division

and respiratory enzyme activity.

100
 Cytoplasmic Matrix

Is the substance where the nucleoid, ribosomes,

plasmids and inclusion bodies are suspended.

101
 Ribosome

 Ribosomes are cytoplasmic granules composed of RNA and

protein, at which protein synthesis takes place.

 May be loosely attached to the plasma membrane.

Cytoplasmic ribosomes – synthesize protein which remain

within the cell.

Plasma membrane ribosomes- make proteins for transport to

outside.
102
 Neucleoid

Bacteria do not have a membrane-bound nucleus.

The i r ge ne t i c mat e ri al i s t ypi c al l y a si ngl e c i rc ul ar

chromosome.

It is located in the cytoplasm in an irregularly shaped body

called the nucleoid.

103
Bacteria may have extra chromosomal genetic material named
as plasmids.

Plasmids do not play any role in the normal function of the

bacterial cell but may confer certain additional properties (Eg.
Virulence, drug resistance) which may facilitate survival &
propagation of the MO.

104
 Plasmid

Is small fragment of extra chromosomal DNA.

Usually circular that replicates independently of the main

chromosome.

Plasmids make up about 5% of the DNA of many bacteria but

are relatively rare in eukaryotic cells.

Resistance plasmids enable bacteria to degrade or inactivate

antibiotics.

Other plasmids enable bacteria to produce chemicals that are

toxic to other organisms, including insects, humans, and other
bacter 105
 Chemotaxis
Chemotaxis is a fundamental sensory phenomenon by which
biological cells translate environmental chemical information
into motile behavior. (is the movement of an organism in
response to a chemical stimulus).

Bacteria, in particular, use chemotaxis to position themselves

within the optimal portion of their habitats by monitoring the
environmental concentration gradients of specif ic chemical
attractant and repellent ligands

106
Somatic cells, bacteria, and other single-cell or multicellular
organisms direct their movements according to cer tain
chemicals in their environment.

This is important for bacteria to f ind food (e.g., glucose) by

swimming toward the highest concentration of food molecules,
or to flee from poisons (e.g., phenol).

107
Positive chemotaxis occurs if the movement is toward a higher
concentration of the chemical in question;

Negative chemotaxis if the movement is in the opposite

direction.

C he mic ally pro mpte d kine sis (rand o mly d ire c te d o r

nondirectional) can be called chemokinesis

108
Spores/endosopres

• These are small, dehydrated and metabolically inactive

forms/resting or dormant cells that are produced by few
Bacillus anthracis &
Gm+b like Clostridium tetani.

• Spore is produced in response to nutrient limitation

• These struc tures are extraord inarily resistant to

environmental stresses: heat, ultraviolet radiation, gamma
radiation, toxic chemicals, disinfectants, desiccation &
dehydration.

• Sporogenesis (sporulation): spore is formed inside the cell

and c ontains bac terial DNA, c ytoplasm, c ytoplasmic
membrane. 109
It is made of mainly keratin like coat called calcium dipicolinate
(remarkable resistant factor), Peptidoglycan & water.

It is also important for classif ic ation and identif ic ation of

bacteria.

These spore forming bacteria can undergo

germination/cultivation by releasing the spore components due
to high amount of amino acids & sugars.

Endospores can be examined by light & electron microscopes &

by special (spore) staining
110
 Spore position in the mother cell or sporangium

• Differs among species, making it of considerable value in

identification

Location: Spores may be located

• Centrally
• Close to one end (subterminally)
• Terminally

111
 Spore formation - sporogenesis or sporulation

Commences when growth ceases due to lack of nutrients

It is a complex process and may be divided into seven stages

stage I - An axial filament of nuclear material forms

stage II - an inward folding of the cell membrane to enclose part of the DNA and produce
the forespore septum

stage III - the membrane continues to grow and engulfs the immature spore in a second
m/m

stage IV - cortex is laid down in the space b/n the two membranes, & both calcium &
dipicolinic acid are accumulated

stage V - coat synthesis, Protein coats then are formed around the cortex, & maturation
of spore

stage VI – completion of coat synthesis

stage VII - lytic enzymes destroy the sporangium releasing the spore
112
Sporulation requires only about 10 hours inBacillus megaterium
113
The transformation of dormant spores into active vegetative cells seems
almost as complex a process as sporogenesis

It occurs in three stages:

(1) Activation
(2) Germination
(3) Outgrowth

Often an endospore will not germinate successfully, even in a nutrient-rich medium,

unless it has been activated

Activation is a reversible process that prepares spores for germination

usually results from treatments like heating, or during suitable env‘t like enough nutrient

It is followed by germination - the breaking of the spore’s dormant state

This process is characterized by spore swelling, rupture or absorption of the spore coat,
loss of resistance to heat and other stresses, loss of refractility, release of spore
components, and increase in metabolic activity

114
Many normal metabolites or nutrients (e.g., amino acids and sugars) can
trigger germination after activation

Germination is followed by outgrowth

The spore protoplast makes new components, emerges from the remains of
the spore coat, and develops again into an active bacterium

115
Veterinary Bacteriology and Mycology




Target Group: Year III Vet. Med. Students

Semester I


Academic Year: 2023/24

1
 BACTERIAL NUTRITION & GROWTH


Bacterial growth and growth requirements

 Bacterial growth refers to an increase in the number of bacteria

(reproduction) rather than an increase in size of the bacteria.

The chemicals and elements that are utilized for bacterial

growth are referred to as nutrients or nutritional requirements.

2
  Organisms use a variety of nutrients for their energy
needs and to build organic molecules & cellular structures.

 Macronutrients needed in larger amounts: C, H, N, P, and S.

 Macronutrients needed in smaller amounts: Mineral salts

such as Ca , Fe , Mg , K
+2 +3 +2 +

 Micronutrients (trace elements); needed in very tiny

amounts; e.g. Zn , Mo , Mn .
+2 +2 +2

3
 Growth requirements

 Are physical and chemical requirements.

 The physical factors include: the pH, Temperature,

Osmotic pressure, Hydrostatic pressure, and Moisture
content of the medium in which the organism is growing.

 Chemical requirements include: Sources of carbon,

nitrogen, oxygen, hydrogen, sulfur, phosphorous and
trace elements. 4
 Physical requirements
1.Temperature
 Every bacterial species grows at a particular minimum,
optimum and maximum growth temperature.

Minimum Temperature: Temperature below which growth ceases.

Optimum Temperature: Temperature at which its growth rate is the

fastest.

Maximum Temperature: Temperature above which growth ceases.

5
Temperature and Microbial
Growth

6
 Temperature Con…
 Bacteria can be divided in to three different groups
according to their temperature requirement.

Psychrophiles: Cold loving bacteria.

Mesophiles: Moderate-temperature-loving bacteria.

Thermophiles- Heat loving bacteria. 7

 PH
 Each organism has a PH range and a PH optimum.

 Most organisms grow best between PH 6 and 8.

 But some organisms have evolved to grow best at low

or high PH.

 The internal PH of a cell must stay relatively close to

neutral even though the external PH is highly acidic or
basic.

8
 PH Con…

Acidophiles: Organisms that grows best at low PH (eg

Helicobacter pylori ).

Alkaliphiles: Organisms that grows best at high PH (eg

Vibrio cholera ).

Neutrophiles: Grow best at neutral PH. Most of

pathogenic bacteria are neutrophiles.
9
10
 Osmotic Pressure

 Microbes need a certain osmotic pressure to maintain

integrity and get nutrients.

 The pressure depends on the surrounding solute

concentration and water availability.

If a microbe is in a hypertonic solution, water will leave

the cell and the cell will shrink.
11
 Moisture

 Bacteria are about 80-90% water.

 They require moisture to grow because they obtain

most of their nutrients from their aqueous environment.

12
 Chemical Requirements
1. Carbon
Is the structural backbone of living matter and necessary
for all organic compounds.

2. Nitrogen, sulfur and Phosphorous

DNA synthesis needs nitrogen and phosphorous.
Protein synthesis needs nitrogen and some sulfur.
ATP needs Phosphorous.

Trace elements are very small amounts of certain

elements that are needed. Eg Iron, copper, and zinc.
13
 Bacterial Culture Media
 Culture is the term given to MOs that are cultivated in
the lab. for purpose of studying them

 Medium is the term given to the combination of

ingredients that will support the growth and cultivation
of MOs by providing all the essential nutrients required
for the growth (multiplication) in order to cultivate these
MOs in large numbers to study them.

 A culture media is special medium used in

microbiological laboratories to grow different kinds of
microorganisms.
14
 Bacterial Culture Media Cont.…

A growth or a culture medium is composed of different

nutrients that are essential for a microbial growth.

Since there are many types of microorganisms, each

having unique properties and requiring specif ic
nutrients for growth, the culture media are of many
types based on what nutrients they contain and what
function they play in the growth of microorganisms.

15
 Bacterial Culture Media Cont.…
 There are various reasons why bacteria have to be
grown (cultured) in the laboratory on artificial culture
media.

1. Diagnosing infectious diseases.

2. Studying its morphology and its identification.

3. To obtain antigens for vaccine development.

4. Certain genetic studies and manipulations of the

cells

5. Convenient way of separating bacteria in mixtures.16

 Bacterial Culture Media Cont.…

When culturing bacteria, it is very important to provide

similar environmental and nutritional conditions that
exist in its natural habitat.

Hence, an artif icial culture medium must provide all

the nutritional components & physical requirements
that a bacterium gets in its natural habitat.

Some of the common ingredients of culture media

include water, agar, peptone, carbohydrates, meat
extract, yeast extract and malt extract, buffer

17
 Types of culture media
Classification based on consistency

Liquid

Semi-solid

Solid

18
 A. Liquid Media

Often called broths

A liquid medium is usually prepared in tubes, f lasks, or bottles

and consists of various solutes dissolved in distilled water.

Once inoculated, microbial growth can occur throughout this

liquid, transforming a transparent medium into a cloudy (turbid)
suspension.

19
 B. Solid Media

Usually prepared in tubes or Petri plates

Provide a firm surface for microbes to grow on or within

Unlike a liquid medium, bacteria dispersed on a solid medium

can grow as a continuous layer or as separate colonies.

A solid medium is typically prepared by adding a solidifying

agent to a liquid medium.

20
 C. Semisolid Media

A medium having more of a “jellylike” consistency

Unlike a liquid medium that f lows freely, this medium cannot

be poured.

However, to give it less body or f irmness than a solid medium,

the solidifying agent used in its preparation is added in smaller
amounts.

One of the useful features of a semisolid medium is it can be

used for determining motility.

21
 Classification based on chemical composition

A. Synthetic Media

Whenever media are prepared to exact specifications, they

are known as synthetic or chemically defined media.

They are composed of highly purified organic & Inorganic

substances of known amounts and molecular composition.

Since these media are standardized and highly reproducible,

they are generally used in research labs.

These media can be very tedious to prepare and very expensive

since they require the purchase of highly purified compounds.

22
 B. Non synthetic Media

Contain one or more ingredients of imprecise composition

They are also known as complex media

A complex medium serves as a rich mixture of compounds that

is likely to supply the general nutritional requirements for a wide
variety of microbes

Used routinely in microbiology labs, these media are much

cheaper and less time-consuming to prepare than synthetic
media.

23
 Classification based on function

I. General Purpose Media

These media contain a diverse mixture of nutrients that can

support the growth of a broad range of bacteria

Non synthetic (complex) media like nutrient agar, nutrient

broth, and trypticase soy agar are good examples of general
purpose media.
24
 II. Selective Culture Media

Is special type of media which allows the growth of certain

microorganisms while inhibits the growth of the others.

Example: MacConkey agar medium.

25
 III. Differential Culture Media

Is a media that is used for differentiating between bacteria by

usi ng a n i d e nt i f ic a t i o n m a r k e r fo r a spe c i f ic t ype o f
microorganism.

Does not kill the others like selective media but only highlights
one type.

Eg. Blood agar is a common differential culture medium used

to identify bacteria that causes haemolysis in blood.

26
 IV. Isolation Culture Media

Is a simple agar containing solid medium that allows the

growth of microorganisms in the direction of the streaks.

For example the bacteria will only grow on the pattern made on
the solidified agar during the streak plate method.

This is the most commonly used medium in microbiological

labs.

27
 V. The Enrichment Culture Media

Is a liquid medium which allows the microorganisms to

multiply and has the essential nutrients that are required for it.

VI. Reducing medium

It is employed for growing obligate anaerobes.

Reducing medium particularly contains chemicals (reducing

agents) that deplete molecular oxygen.

28
 VII. Resuscitation Culture Media

I s a spe c i a l t ype o f m e d i a w hi c h i s use d fo r gr o w i ng

microorganisms that are damaged and have lost the ability to
produce due to certain harmful environmental factors.

This culture allows the organisms to regain their metabolism by

providing the nutrients that the organisms had been deprived of.

29
 VIII. The Preservation Culture Media

I s used to preserve a spec if ic type of mic roorganism,

preferably bacteria or a set of different microbial entities for a
long period of time.

The basic purpose of this culture is to let such microorganisms

grow safely in an ensured environment that has all the
i mpo r t ant nut ri e nt s and t o pro t e c t t he m agai nst any
environmental damage so these organisms can be used when
needed.

30
 Isolation of Pure Culture

Most specimens (from human tissue, animal tissue or

environmental samples) will be mixed, with a variety
of bacteria (or other microorganisms).

A single gram of feces, for example, has over 10 10

bacteria and that gram would have over 20 different
bacterial species.

To study microorganisms in the laboratory, we must

have them in the form of a pure culture; that is, one in
which all organisms are descendants of the same
organism.
31
 Isolation of Pure Culture

Two major steps are involved in obtaining pure

cultures from clinical samples:
1. Obtaining isolated bacterial colony

The most common way of separating bacterial cells on

the agar surface to obtain isolated colonies is the
streak plate method.
It provides a simple and rapid method of diluting the
sample by mechanical means.

As the loop is streaked across the agar surface, more

and more bacteria are rubbed off until individual
separate organisms are deposited on the agar. 32
 Isolation of Pure Culture cont.…

After incubation, the area at the beginning of the streak

pattern will show conf luent growth, while the area near
the end of the pattern should show discrete colonies.

2. Aseptically ‘’pick off’’ each isolated colony from the

isolation plate according to the colonial morphology of
all isolated colonies.

After incubation, all organisms in the new culture will

be descendants of the same organism; that is, a pure
culture.
33
 Isolation of Pure Culture cont.…

To supplement mechanical techniques of isolation such

as the streak plate method, many special-purpose
media are available to the microbiologist to aid in the
isolation and identification of specific microorganisms.

34
 Bacterial Growth
When the bacteria reach a certain size, they divide by
binary fission in a geometric fashion.

The number of cells arising from a single cell is 2n

after n generations.

Generation time is the time it takes for a single cell to

grow and divide.
35
Most bacteria rely on binary fission for propagation.
Conceptually this is a simple process; a cell just needs to grow
to twice its starting size and then split in two.

But, to remain viable and competitive, a bacterium must divide

at the right time, in the right place, and must provide each
offspring with a complete copy of its essential genetic material.

Understanding the mechanics of this process is of great

interest because it may allow for the design of new chemicals
or novel antibiotics that specif ic ally target and interfere with
cell division in bacteria.
36
Before binary f ission occurs, the cell must copy its genetic
material (DNA) and segregate these copies to opposite ends of
the cell.

As division occurs, the cytoplasm is cleaved in two, and in

many bacteria, new cell wall is synthesized.

37
Binary Fission

38
Generation Time Under Optimal Conditions (at 37oC)

Organism Generation
Time
Bacillus cereus 28 min

Escherichia coli 12.5 min

Staphylococcus aureus 27-30 min

Mycobacterium tuberculosis 18 – 24 hrs

Treponema pallidum (agent of Syphilis) 30 hrs

39
Some Unusual Forms of Reproduction in Bacteria:

There are groups of bacteria that use unusual forms or patterns

of cell division to reproduce.

Some of these bacteria grow to more than twice their starting

cell size and then use multiple divisions to produce multiple
offspring cells.

Some other bacterial lineages reproduce by budding.

Still others form internal offspring that develop within the

cytoplasm of a larger "mother cell".

Reading assignment: Budding in bacteria

40
 Stages of bacterial growth

Under ideal conditions, the growth of a population of

bacteria occurs in several stages termed:
1. Lag

2. Exponential or Logarithmic (log) phase

3. Stationary

4. Decline or Death phase.

41
Bacterial growth curve

42
 1. Lag phase

When a microorganism is introduced into the fresh
medium, it takes some time to adjust with the new
environment.
 This phase is termed as Lag phase.

The length of the lag phase depends directly on the

previous growth condition of the organism.

When the microorganism growing in a rich medium is

inoculated into nutritionally poor medium, the organism
will take more time to adapt with the new environment.
43
D/t microbial medias for bacterial
growth

44
 1. Lag phase Con…
The organism will start synthesizing the necessary
proteins, co-enzymes and vitamins needed for their
growth and hence there will be a subsequent increase in
the lag phase.

Similarly when an organism from a nutritionally poor

medium is added to a nutritionally rich medium, the
organism can easily adapt to the environment, it can
start the cell division without any delay, and therefore
will have less lag phase.

45
 2. Exponential or Logarithmic (log) phase

 During this phase, the microorganisms are in a rapidly

growing and dividing state.

Their metabolic activity increases and the organism

begin the DNA replication by binary fission at a constant
rate.

The growth medium is exploited at the maximal rate.

The culture reaches the maximum growth rate and the

number of bacteria increases logarithmically
(exponentially) 46
2. Exponential or Logarithmic (log) phase Con…

Finally the single cell divide into two, which replicate

into four, eight, sixteen, thirty two and so on (That is 20,
21, 22, 23.........2n, n is the number of generations).

This will result in a balanced growth.

47
48
 3. Stationary phase

As the bacterial population continues to grow, all the
nutrients in the growth medium are used up.

This results in the accumulation of waste materials,

toxic metabolites and inhibitory compounds such as
antibiotics in the medium.

This shifts the conditions of the medium such as PH

and temperature, thereby creating an unfavorable
environment for the bacterial growth.

49
 3. Stationary phase Con…
The reproduction rate will slow down.

The cells undergoing division is equal to the number of

cell death.

Finally bacterium stops its division completely.

The cell number is not increased and thus the growth

rate is stabilized.

50
 4. Decline or Death phase

The bacteria move on to the Death phase b/c of:

1.The depletion of nutrients

2. Accumulation of metabolic waste products

3. Accumulation of other toxic materials in the media.

During this time, the bacterium completely loses its

ability to reproduce.

51
4. Decline or Death phase Con…

Individual bacteria begin to die due to the unfavorable

conditions.

The number of dead cells exceeds the number of live

cells.

Some organisms which can resist this condition can

survive in the environment by producing endospores.

52
 Nutrient uptake

Every cell in every organism has a system for bringing

in nutrition and expelling waste.

Some are through pore-like protein structures called

transporters, which reside at the surface of the cell's
outer membrane.

Each pore is capable of transporting nutrients

individually.

53
 Passive Transport
A few molecules that are small can pass through the
phospholipid bilayer by the process of diffusion.

Passive transport does not require an energy input.

The process of diffusion occurs when molecules or ions move

from an area of greater concentration to an area of lower
concentration.

The movement is down the concentration gradient

Very small molecules such as O2 and CO2 move in this manner.

.
54
Rate of diffusion: is determined by the difference
in concentration of the nutrients or molecules on
the outside and the inside of the membrane.

55
 Osmosis
Is the movement of water from an area of higher
concentration to an area of lower concentration.

Hypertonic – refers to an environment with higher salt

concentration and lower water potential.

Hypotonic – higher water concentration (water potential)

and lower solute.

Isotonic – equal concentration of solute on both sides of

a membrane.

56
 Facilitated diffusion
Is a type of diffusion which uses a transport molecule.

Permease are proteins which are embedded in the cell

membrane.

They assist in the movement of other molecules across

the membrane.

This is faster process than regular diffusion, but again

is highly specific.

No energy is spent in the process. 57

Facilitated Diffusion

58
 Aquaporins

A major permease in bacteria is an aquaporin that

helps to move water in and out of the cell.

59
 Active Transport

Is transport of molecules and ions against the

concentration gradient from low concentration to high
concentration.

This requires the input of energy in the form of ATP.

ATP → ADP + P + energy.

60
ATP Binding Cassette Transporters (ABC Transporters)

Transporters are proteins that span the

membrane.

They bind to the ATP and hydrolyze or break

down ATP to produce energy.

61
 Symports
Move two molecules or a molecule and an ion in the
same direction.

Both can move in the same direction.

Usually one moves with the concentration gradient.

The other is pulled across the membrane against the

concentration gradient by the free energy of the
molecules that are moving.

62
 Antiports

Two molecules are moving across the membrane.

One molecule is moving against the concentration

gradient in one direction.

The other is moving with the concentration gradient

63
 Siderophores
Iron is a very important nutritional ion for the bacterial
cell.

Siderophores are small molecules that are able to form

a complex with iron to bring it into the cell.

64
 Porins

Are proteins located in the outer cell membranes (the

outer lipid membrane).

They are responsible for moving molecules through the

outer membrane in Gram negative bacteria.

The molecules are moved into the periplasmic space

and then into the cell.

65
 Group Translocation
In this process the molecules are modified as they
are transported across the cell membrane or into the
cell.

Group Translocation is a type of active transport.

Many vital molecules are brought into the cell in this

manner.

Involves the transfer of a phosphate group.

This energizes the molecule so that it can be

transported into the cell. 66
Reading Assignment:
Nutrient uptake

67
 Microbial growth measurements

 For unicellular organisms such as the bacteria, growth

can be measured in terms of two different parameters:

1. Changes in cell mass

2. Changes in cell numbers per milliliter.

 Methods for measurement of the cell mass involve

both direct and indirect techniques.

68
1. Direct physical measurement of dry weight (Organism
is dried and then weighed.), wet weight, or volume of
cells after centrifugation.

2. Direct chemical measurement of some chemical

component of the cells such as total N, total protein, or
total DNA content.

69
3. Indirect measurement of chemical activity such as
rate of O2 production or consumption, CO2 production or
consumption etc.

4 . Turb i d i ty m e asure m e nts e m p l o y a v ari e ty o f

instruments to determine the amount of light scattered
by a suspension of cells.

70
 Particulate objects such as bacteria scatter light in proportion to
their numbers.
The turbidity or optical density of a suspension of cells is directly
related to cell mass or cell number, after construction and
calibration of a standard curve.

Turbidity:- uses a spectrophotometer to estimate the number of

cells.
A spectrophotometer sends a beam of visible light through a culture
and measures how much light is scattered.

Scales read in either % absorbance or % transmission.

Measures both live and dead cells

71
Spectrophotometer

72

of Cell Numbers
Methods for Measurement

Measuring techniques involve direct counts, visually or

instrumentally, and indirect viable cell counts.

1. Direct microscopic counts are possible using special

slides known as counting chambers.

Dead cells cannot be distinguished from living ones.

73
74
2. Electronic counting chambers count numbers and
measure size distribution of cells.

The suspending medium must be very clean.

Such electronic devices are more often used to count

eukaryotic cells such as blood cells.

75
3. Indirect viable cell counts, also called plate counts,
involve plating out (spreading) a sample of a culture on a
nutrient agar surface.
The sample or cell suspension can be diluted in nontoxic
diluents (e.g. water or saline) before plating.

Dilutions are done to count cells more easily.

If plated on a suitable medium, each viable unit grows
and forms a colony.

Each colony that can be counted is called a colony

forming unit (cfu) and the number of cfu's is related to
the viable number of bacteria in the sample.
76
77
Calculation: Number of colonies on plate × reciprocal of
dilution of sample = number of bacteria/ml.
Example, if 32 colonies are on plate of 1/10,000 dilution,
then the count is 32 × 10,000=32,000/ml in sample.

78
 Cultural Characteristics of bacteria
Bacterial medium contains all the food and nutrients that the
bacteria need to grow.
As the bacteria consume the nutrients, they begin to grow &
multiply.
This generates thousands to millions to billions of cells that
begin to pile up, becoming visible to the naked eye.

This pile of cells originates from one cell and is called a bacterial
colony.A bacterial colony consists of numerous bacterial cells
derived from one parent.

Each species of bacteria produces a colony that looks different

from the colonies produced by other species of bacteria.
79
Examination of the form and structure of bacterial
colonies is termed colony morphology and is:

One of the first steps in characterizing and identifying a

bacterial culture.

80
 Colony Morphology
Colony morphology is cultural characteristics of a bacterium on
an agar Plate. The following are the characteristics used to
accurately and consistently describe the morphology of a
bacterial colony:
Size
Shape
Color (also known as pigmentation)
Texture
Height (elevation)
Edge (margin) and Smell
Each of these categories has its own vocabulary, allowing other
scientists reading your description to paint an accurate picture
81

of the colony
82
83
An agar plate that has been exposed to the air or cultured with a specimen
might grow many bacteria. These bacteria have different colony morphologies.

84
Nine obviously different colonies are numbered: some colony
types recur in various areas of the plate (note # 3 and # 4).

Not only are pigment differences seen, but also size, edge,
pattern, opacity, and shine.

Two circles have been drawn around merging colonies, where

the species of the 2 colonies are different.

Trying to pick a bit of one of those adjacent colonies increases

the chances of picking up another mixed culture, consisting of
the 2 species that were merged together.

ALWAYS pick a well-isolated colony when sub culturing

85
 Size of the bacterial colony

The size of the colony can be described in two ways.

The more accurate technique would be to measure the diameter

of the colony with a ruler and report the size in millimeters.

The second technique would simply be to describe the colonies

as punctiform (tiny pinpoints), small, medium, or large.

Shape of the bacterial Colony

Shape refers to the overall appearance of the colonies.

The descriptors here are circular, irregular, f il amentous (has

individual thin projections), or rhizoid (has thin, branching
projections).
86
 Color/Pigmentation of bacterial colony
Some bacteria produce pigments, giving the colony a distinct
color.

Pigments span the entire color spectrum.

Recording the color is the first step.

Opacity of colony:
Is the colony transparent (clear), opaque (not transparent or
clear), translucent (almost clear, but distorted vision–like
looking through frosted glass),

87
 Texture of bacterial colony
Texture: Refers to the characteristics of the colony surface.
Colonies can be dry, mucoid (thick, stringy and wet), moist,
smooth, rough, rugose (wrinkled) or contain concentric rings.

Height of bacterial colony

The colony height, or elevation, is a description of how the
colony grows vertically.
To see the elevation of the colonies it may be helpful to look
through the side of the petri dish.
The descriptors here are flat, raised, convex (sloping up from the
edges), pulvinate (sloping steeply from the edges and very in the
center), and umbonate (has a raised center)
88
 Edge/Margin of bacterial colony

Edge, or Margin, describes the borders of the colony. The edge

can be entire (smooth, with no projections), undulate (wavy),
lobate (lobed), filamentous or rhizoid

Being able to visibly differentiate bacteria based on the

appearance of their colonies is a crude, but essential f irst step in
isolating the different types of bacteria in the sample.

Colony morphology alone is not a reliable way to identify

bacteria, as many different types of bacteria have similar colony
89
morphology.
 Large Colonies, Opaque, Round, Entire
Small Colonies, Red Pigment,
Round, Entire, Opaque

Filamentous, Irregular
Opaque

90
Punctiform, Yellow, Round, Opaque Brown Pigment, Irregular, Translucent

91
 Bacterial Pathogenecity
The bacteria and other microbes that are consistently
associated with an animal/man are called the ‘’normal flora’’, or
more properly the "indigenous microbiota" of the animal or man.

Although most bacteria are saprophytes which grow on organic

matter in the environment, a small number, referred to as
bacterial pathogens, produce infection and disease in animals
and humans.

Infections with some bacteria, such as anthrax caused by

Bacillus anthracis , are invariably fatal.
92
 Pathogenicity and Virulence

A pathogen is a microorganism that is able to cause disease in a

man, animal, plant, or insect.

Pathogenicity is the ability of a microorganism to produce

disease in a host organism.

Pathogenesis deals with origin, development & effects of a

disease.

Microbes express their pathogenicity by means of their virulence,

a term which refers to the degree of pathogenicity of the microbe.
93
The virulence of a bacterium relates to its ability to
invade and produce disease in a normal animal.

Highly virulent organisms produce serious disease or

death in many affected animals whereas bacteria of low
virulence rarely produce serious illness.

Hence, the determinants of virulence of a pathogen are

any of its genetic or biochemical or structural features
that enable it to produce disease in a host.
94
 Obligate and opportunistic pathogens

An ‘Obligate pathogen’ can be found in the host animal only in

connection with disease.

Microorganisms, which are found in healthy host animals, but

which may cause a disease in certain circumstances are known
as ‘Opportunistic pathogens’.

Environmental saprophytic bacteria which cause a disease in a

compromised host which typically would not occur in a healthy
(non-compromised) host are acting as opportunistic pathogens.
95
Opportunistic infection can also result from by the commensals
which normally colonize epithelial surfaces without deleterious
effects.

A member of the normal f lora such asStaphylococcus aureus

E. coli
or can cause an opportunistic infection.

Pasturella multocida in cattle andP. haemolytica in small

ruminants cause pneumonia during stress (e.g. transportation).

Dermatophylus congolensis (in ruminants) is a normal f lora of

the skin and cause skin disease in stressed and weak animals.
96
Infectious diseases are those caused by living organisms
(bacteria, fungi, protozoa, viruses etc.)

Non-infectious diseases are those caused by non-living agents

such as chemical and physical agents, deficiency diseases,
genetic disease, etc.

To cause disease a pathogen must:

1. Gain access to the host

2. Adhere to host tissues

3. Penetrate or evade host defenses

4. Damage the host, either:- directly or accumulation of microbial
97

wastes
Outcomes of Infection and factors which determine the
outcome of infection:

Infection does not necessarily lead to infectious

disease.

In fact, infection probably rarely leads to infectious

disease.

Some bacteria rarely cause disease if they do infect;

some other bacteria will usually cause disease if they
infect.
98
The following factors determine whether or not over t
disease will result after microbial entry to the host organism:

Pathogen-related determinants factors

 Virulence of the organism (virulent & avirulent)

 Stability in the environment (stable Vs susceptible)

 Infective dose (low dose Vs high dose)

 Route of entry (appropriate site)

 Tissue tropism (affinity to specific organ, tissue, cell)

 Species of the host (some host specific & wide host

99
range)
Host-related determinants
 Breed, Sex & age of the host

 Genetic factors
 Physiological factors like pregnancy, lactating,
Metabolic dysfunction

 Immune status of the host

 Other factors such as stress, nutritional status,
infection with other bacteria or virus which renders
the host susceptible.

100
Mechanisms of bacterial pathogenicity

Microbes express their pathogenicity by means of their virulence.

Hence, the determinants of virulence of a pathogen are any of

its genetic or biochemical or structural features that enable it to
produce disease in a host.

101
Certain qualities of pathogenic bacteria underlie the
mechanisms by which they cause disease:

i. Invasiveness
Invasiveness is the ability to invade tissues.
It encompasses mechanisms for:

Colonization (adherence and initial multiplication)

Production of extra cellular substances which facilitate invasion

(invasins)

Ability to bypass or overcome host defense mechanisms.

102
Colonization and Adherence
The first stage of microbial infection is colonization: the
establishment of the pathogen at the appropriate portal of entry.

Pathogens usually colonize host tissues that are in contact with

the external environment.

They are known as routes of infection (portal of entry).

Sites of entry (portal of entry) in animal hosts include:

The urogenital tract

The digestive tract
The respiratory tract
The conjunctiva, skin, and cornea.
103
Adhesins / Microbial adherence factors

A key step in the host-pathogen interaction is adherence of the

pathogen to host surfaces.

These surfaces include skin, mucous membranes (oral cavity,

nasopharynx, urogenital tract), and deeper tissues (lymphoid
tissue, gastric and intestinal epithelia, alveolar lining, endothelial
tissue).

Numerous mechanical forces produced by the host act to wash

microbes from these surfaces: saliva secretion, coughing,
sneezing, mucous flow, peristalsis, and blood flow. 104
A common trait of microbial pathogens is the expression of
factors that bind to molecules on various host tissue cells and
render the microbe resistant to these mechanical washing forces.

Once bound or “adhered” to a specif ic host cell surface, the

pathogen is then able to initiate its specif ic biochemical
processes that will result in disease including proliferation, toxin
secretion, host cell invasion, and activation of host cell signalling
cascades.

INVASION
Once adhered to a host surface, some pathogens gain deeper
access into the host to perpetuate the infection cycle. 105
Organisms that infect these regions have usually developed
tissue adherence mechanisms and some ability to overcome or
withstand the constant pressure of the host defenses at the
surface.

In its simplest form, bacterial adherence or attachment to an

eucaryotic cell or tissue surface requires the participation of two
factors: a receptor and ligand.

The receptors so far def in ed are usually specif ic

carbohydrate or peptide residues on the eucaryotic cell
surface. 106
 The bacterial ligand, called an adhesin, is typically a
macromolecularcomponent of the bacterial cell
surface which interacts with the host cell receptor.

A d h e si ns a nd re c e p t o rs usua l l y i nt e ra c t i n a
complementary and specific fashion.

107
Terms used to describe adherence factors in host-parasite
interactions
A D H E R E N C E DESCRIPTION
FACTOR
Adhesin A surface structure or macromolecule that binds a bacterium to a
specific surface

Receptor A complementary macromolecular binding site on a (eucaryotic)

surface that binds specific adhesins or ligands

Ligand A surface molecule that exhibits specif ic binding to a receptor

molecule on another surface

Fimbriae and Filamentous proteins on the surface of bacterial cells that may
common pili behave as adhesins for specific adherence

S-layer Proteins that form the outermost cell envelope component of a

broad spectrum of bacteria, enabling them to adhere to host cell
membranes and environmental surfaces in order to colonize.

Capsule A detectable layer of polysaccharide (rarely polypeptide) on the

surface of a bacte rial ce ll w hich may me diate spe cif ic or
nonspecific attachment 108
A bacterial "adhesin" attaches covalently to a host "receptor" so
that the bacterium "docks" itself on the host surface.

The adhesins of bacterial cells are chemical components of

capsules, cell walls, pili or fimbriae.
The host receptors are usually glycoproteins located on the cell
membrane or tissue surface. 109
Examples of specific attachments of bacteria to host cell or
tissue surfaces

Bacterium Adhesin Receptor Attachment Diseas

site e
Streptococcus Protein F Amino terminus P h a r y n g e a l Sore throat
pyogenes of fibronectin epithelium

Streptococcus C e l l - b o u n d N - M u c o s a l pneumonia
pneumoniae protein acetylhexosami epithelium
ne -ga la ctose
disaccharide

Staphylococcus C e l l - b o u n d Amino terminus M u c o s a l Various

aureus protein of fibronectin epithelium

110
Examples of specific attachments of bacteria to host cell or tissue surfaces Cont…

Bacterium Adhesin Receptor Attachme Disease

nt site
Enterotoxigenic Type-I fimbriae Species-specif ic I n t e s t i n a l Diarrhea
E. coli carbohydrate(s) epithelium

Uropathogenic Type I fimbriae C o m p l e x U r e t h r a l Urethritis

E. coli carbohydrate epithelium

Uropathogenic P-pili (pap) Globobiose Upper urinary Pyelonephriti

E. coli linked to tract s
ceramide lipid
Vibrio cholerae N - Fucose and I n t e s t i n a l Cholera
methylphenylalan mannose epithelium
ine pili carbohydrate

111
 Production of extra cellular substances (invasins) which
facilitate invasion:

The invasion of a host by a pathogen may be aided by the

production of bacterial extracellular substances which act
against the host by breaking down primary or secondary defenses
of the body.

These substances are referred to as invasins.

Most invasins are proteins (enzymes) that act locally to damage

host cells and/or have the immediate effect of facilitating the
growth and spread of the pathogen.
112
 Invasins usually act at a short range (in the immediate vicinity of
bacterial growth) and may not actually kill cells as part of their
range of activity.

Exotoxins are often cytotoxic and may act at remote sites

(removed from the site of bacterial growth).

Also, exotoxins typically are more specif ic and more potent in

their activity than invasins.

Bacterial Invasins are grouped into two: Spreading Factors and

Enzymes that Cause Hemolysis and/or Leucolysis

113
 Spreading Factors:
"Spreading Factors" is a descriptive term for a family of bacterial
enzymes that promote the spread of the pathogen.

An example of bacterial invasions which are spreading factors

include:

Hyaluronidase : It is produced by streptococci, staphylococci, and

clostridia. The enzyme attacks the interstitial cement ("ground
substance") of connective tissue by depolymerizing hyaluronic
acid.

Collagenase: It is produced byClostridium histolyticum &

Clostridium perfringens .
It breaks down collagen, the framework of muscles, which
114
Neuraminidase : It is produced by intestinal pathogens such as
Vibrio cholerae Shigella dysenteriae .
and

It degrades neuraminic acid (also called sialic acid), an

intercellular cement of the epithelial cells of the intestinal
mucosa.

Streptokinase and staphylokinase: are produced by streptococci

and staphylococci, respectively.

115
Enzymes that Cause Hemolysis and/or Leucolysis:
These enzymes usually act on the animal cell membrane by
insertion into the membrane (forming a pore that results in cell
l ysi s), o r by e nz ymat i c at t ac k o n pho spho l i pi d s, whi c h
destabilizes the membrane.

They are referred to as leukocidines, lecithinases, phospholipases,

and hemolysins.

Hemolysins, notably produced by staphylococci (i.e., alpha toxin ),

streptococci (i.e., streptolysin) and various clostridia, may be
channel-forming proteins or phospholipases or lecithinases that
destroy red blood cells and other cells (i.e., phagocytes) by lysis.

116
Beta-hemolytic Streptococcus . This is the characteristic
appearance of a blood agar plate culture of the bacterium.

N o t e t he t r a nsl uc e nc y a r o und t he ba c t e r i a l c o l o ni e s,
representing hemolysis of the red cells in the culture medium
due to production of a diffusible hemolysin (streptolysin).
117
Evasion of host defenses
It is the ability to bypass or overcome host defense mechanisms.
Some pathogenic bacteria are inherently able to resist the
bactericidal components of host tissues.
For example, the poly-D-glutamate capsule of Bacillus anthracis
protects the organisms against cell lysis by cationic proteins in
sera or in phagocytes.
The outer membrane of Gram-negative bacteria is a permeability
barrier that is not easily penetrated by hydrophobic compounds
such as bile salts which are harmful to the bacteria.
Pathogenic mycobacteria (e.g.Mycobacterium tuberculosis ) have
a waxy cell wall that resists attack or digestion by most tissue
bactericides.
Intact lipopolysaccharides (LPS) of Gram-negative pathogens
may protect the cells from complement-mediated lysis or the
action of lysozyme. 118
ii. Toxigenesis

Toxigenesis is the ability to produce toxins. Bacteria may

produce two types of toxins: Exotoxins and Endotoxins.

Toxins are analogous to biological weapons in that these are

proteinaceous or non-proteinaceous molecules produced by
bacteria to destroy or damage the host cell.

Examples of non-proteinaceous toxins are LPS (endotoxin) for

Gram negative organisms and teichoic acid for Gram positive
organisms.

Proteinaceous toxins (exotoxins) are generally enzymes which

are delivered to eukaryotic cells 119
Exotoxins are released from bacterial cells and may act at
tissue sites remote from the site of bacterial growth.

Endotoxins are cell-associated substance. (In a classic sense,

the term endotoxin refers to the lipopolysaccharide component
of the outer membrane of Gram-negative bacteria).

Hence, bacterial toxins, both soluble and cell-associated, may

be transported by blood and lymph and cause cytotoxic effects
at tissue sites remote from the original point of invasion or
growth.

Both Gram-positive and Gram-negative bacteria produce

120
soluble protein exotoxins.
A few protein toxins obviously bring about the death of the host
and are known as "lethal toxins“.
As "foreign" substances to the host, most of the protein toxins
are strongly antigenic.

In vivo, specif ic antibody (antitoxin) neutralizes the toxicity of

these bacterial proteins.

Toxoids are detoxif ie d toxins which retain their antigenicity

and their immunizing capacity.

The formation of toxoids can be accelerated by treating toxins

with a variety of reagents including formalin, iodine, pepsin,
ascorbic acid, ketones, etc.
Toxoids are the immunizing agents against diphtheria and
tetanus that are part of the dpt vaccine.
121
 Sources and activities of bacterial toxins
NAME OF TOXIN BACTERIUM ACTIVITY
INVOLVED
Anthrax toxin (EF) Bacillus anthracis  Edema Factor (EF) is an adenylate cyclase that causes
increased levels in intracellular cyclic AMP in phagocytes
and formation of ion-permeable pores in membranes
(hemolysis)

Cholera enterotoxin Vibrio cholerae  ADP ribosylation of G proteins stimulates adenylate

cyclase and increases cAMP in cells of the GI tract,
causing secretion of water and electrolytes

E. coli LT toxin Escherichia coli  Similar to cholera toxin

Shiga toxin Shigella dysenteriae  Enzymatically cleaves rRNA resulting in inhibition of
protein synthesis in susceptible cells
Botulinum toxin Clostridium botulinum  Zn++ dependent protease that inhibits neurotransmission
at neuromuscular synapses resulting in flaccid paralysis

Tetanus toxin Clostridium tetani  Zn++ dependent protease that inhibits neurotransmission
at inhibitory synapses resulting in spastic paralysis

Staphylococcus Staphylococcus  Massive activation of the immune system, including

enterotoxins* aureus ly mph ocy t es a n d ma cr oph a ges, lea ds t o emesis
(vomiting)
Toxic shock syndrome S t a p h y l o c o c c u s  Acts on the vascular system causing inflammation, fever
toxin (TSST-1)* aureus and shock
122
 Characteristics of bacterial endotoxins and exotoxins
PROPERTY ENDOTOXIN EXOTOXIN

1. Chemical nature Lipopolysaccharide (mw = Protein (mw = 50-1000kDa)

10kDa)

2. Relationship to cell Part of outer membrane Extracellular, diffusible

3. Denatured by boiling No Usually

4. Antigenic Yes Yes

5. Form toxoid No Yes

6. Potency Relatively low (>100ug) Relatively high (1 ug)

7. Specificity Low degree High degree

8. Pyrogenicity Yes Occasionally

123
 MEKELLE UNIVERSITY
COLLEGE OF VETERINARY SCIENCE
Department of Veterinary Basics and Diagnostic Science

Veterinary Bacteriology and Mycology

Year-III DVM

Biruk Mekonnen (DVM, MSc)

1
Laboratory Diagnosis of Bacterial and Fungal Diseases

 students should be able to:

– acquire knowledge and skills in applying veterinary

laboratory techniques for:
• Bacterial and fungal Diseases diagnosis

• Research undertakings

 It will also help for:

• Day-1 Competencies: (OIE, International standard)
• Exit Exam: > 90-100%
2
 Day-1 Competencies: (OIE, International standard)

– Students are expected to acquire the Lab competence:

• to select appropriate diagnostic tests/techniques

• able to collecting, preserving and safely transporting

clinical samples

• to apply/ perform diagnostic tests/techniques.

• They can interpreting diagnostic results

3
Day-1 Competencies: (OIE, International standard)

– Example: Suspected of Bovine Mastitis: Economic important

– (to treat, control and prevention: Lab Knowledge and skill essential)

• Appropriate diagnostic techniques. Parasitological Vs Bacteriological

• Appropriate sample: Milk, Blood, Urine

• How to collect specimen (Milk).. collecting milk sample sterile procedure

• How to transport safely from field to Lab (protect the sample, environment)

• Apply the test and interpreting diagnostic results

• Control and prevention of Bovine Mastitis

4
 Bacteriological tests: Outline
1. Isolation of Bacterial Pathogens

2. Identification of Bacterial pathogen

2.1. Primary Identification

Cultural characteristics
Staining the isolated bacteria
 the Gram-stain
 the Acid-Fast stain
 The Special Stain (Capsule & Spore)
Catalase and Oxidase tests
Motility test
Oxidation-Fermentation (O-F) test
Potassium Hydroxide (KOH) test

5
 Outline
2.2. Secondary Identification
Coagulase test
CAMP test
Triple Sugar Iron (TSI) Slant agar test
IMViC Tests:-
 Indole test
 Methyl Red (MR) test
 Voges-Proskauer test
 Citrate test

3. Antimicrobial sensitivity test

6
 Isolation and Identification of Microorganisms

 There are several methods to identify the different type of

bacteria:

Isolation in pure form

Staining reaction

Cultural characteristics/Morphology of colony

Biochemical properties

 Serology and Molecular tests

7
Isolation of Bacterial in pure form: Why Isolation?

 Studies on the biochemical, antigenic and other characters of bacteria can

be done only if the organism available in the pure form.

 Isolation refers to the separation of a bacterial strain from a mixed

population of living microbes in order to identify the microbe(s)
of interest.

 Naturally Microbes are present in the environment as mixed population

 For example: In water, skin flora, oral flora or gut flora

 Example: Interest of isolating S. aures from milk sample
(Mixed population= S. aures, Streptococci, E. coli, Lactobacilli…..)

 A pure culture theoretically contains a single bacterial species.

8
Technique for isolation:

 There are a number of procedures available for the isolation of

pure cultures from mixed populations.

 A pure culture may be isolated by:

1. Plating on solid culture media:

 Purpose of plating is to isolate individual bacterial cells (colony-forming units)

 use spread plating with a glass spreader or streak plating with a loop

 Clinical sample is streaked onto a solid medium (like: MacConkey agar,

nutrient agar or blood agar) in such a way can
ensure isolated distinct colonies. 9
2. Use of selective growth condition

 Most important example is the growth of anaerobic

bacteria

 They will not take place in an environment having oxygen.

10
 Identification of Bacteria

 The most important task of a bacteriology is:

• to identify the pathogens from the clinical sample so
that appropriate treatment can be introduced.

 Definitive identification of bacteria is also essential for:

• Epidemiological purposes: trace-back of disease outbreaks

associated with microbial infections: so that, proper control,
prevention, and eradication measures:
– Example: Anthrax eradication

• Research purposes;
11
 Identification of Bacteria

 Challenges in Bacterial Identification

• Traditional/Conventional methods of bacterial identification rely

on mutual similarities of their phenotypic identification of the
causative organism using:
Bacterial staining,
Culture and
Biochemical methods.

 However, these methods of bacterial identification suffer from two

major drawbacks:
1). First, they can be used only for organisms that can be
cultivated in vitro/lab.
Example: M. leprae has never been cultured in vitro
12
 2). Second, some strains show unique biochemical
characteristics that do not fit or beyond a characteristic of any
known genus and species.

 To overcoming these limitations, in the past decade, molecular

techniques have been introduced

 Several non-culture based methods have emerged in the past

decades . Example: PCR: detect its genome

13
 The isolated bacteria are needs to be further identified so
as to classify the bacteria to genus and species level.

 Commonly used Methods:

1. Staining of the isolated bacteria: to look at the

• staining reactions, shape and arrangement of bacteria

• example:
– Grams staining,
– Acid fast staining,
– Special staining

14
2. Biochemical testing
• The staining is followed by use of various biochemical
tests to get closer to the identification of bacteria.

• looking for metabolic properties of the bacteria.

• The commonly used biochemical tests are:

(a) Catalase test;
(b) Coagulase test;
(c) Oxidase test;
(d) Sugar fermentation test;
(e) Indole test;
(f) Citrate test;
(g) Urease test
15
3. Motility testing
• to distinguish motile bacteria from non-motile bacteria
• Can be tested by:
o Preparing a wet mount and is then observed under the
microscope.
o Inoculating the bacteria in the semi-solid motility medium.

4. Serological tests
• Looking for antigen-antibody reaction
• Several testes;
ELISA……. Identify E. coli
Rose Bengal test .. Screening of Brusella

16
5. Molecular techniques:
• looking for the gene sequence

• PCR

17
 Identification of bacterial pathogens

 In clinical bacteriology, bacteria can be identified to its

species level using the two main identification tests.

 These are:
1. Primary Identification Tests
2. Secondary Identification Tests

1. Primary Identification Tests

 After a pure culture has been obtained, few relatively simple
tests known as collectively primary tests will be
used to identify the bacterium to a generic level.
18
 Identification of bacterial pathogens

 These (Primary Tests) includes:

– Cultural characteristics;

– Staining (the Gram-stain, Acid-Fast stain, Special stain);

– Catalase tests;

– Oxidase tests;

– Motility test;

– Oxidation-Fermentation (O-F) test;

– Potassium Hydroxide (KOH) test

19
 1. Cultural characteristics (colony morphology)

 It is the characteristics of an individual colony of bacteria

growing on solid media.

 These provide additional information for the identification of a

bacterium.

 Colonies differ in their morphology which is helpful to

identify bacteria.

i. Shape: circular, irregular, radiate or rhizoid.

ii. Size: diameter in mm (The diameter of a representative colony may be measured).

iii. Elevation: flat, raised, low convex, dome shaped

20
 1. Cultural characteristics (colony morphology)

iv. Margin: Entire, wavy, lobate, filiform

v. Surface: smooth, wavy, rough, granular, papillate, glistening etc.

vi. Texture: dry, moist, mucoid, brittle, viscous, butyrous (buttery).

vii. Color: colorless, pink, black, red, bluish‐green.

21
 Shape of the colony

Elevation of the colony

Margin of the colony

22
 Bacterial colonies should be examined in a good light and, a
low power magnifying lens can help to see
morphological details.

1. When viewed form above: Colonies may appear:

• round, irregular or branching;
• transparent or opaque and
• their surface may be smooth or rough, dull or shiny.

2. When viewed form the side: Colonies may appear

• flat or raised in varying degrees
• some with a central elevation or depression.
3. When touched with wire loop:

• Some colonies are soft and easily emulsified such as S.aureus.

• Where as others are difficult to break up such as S. pyogens.;
M. tb

4. The colour of colonies:

• This also helps to identify bacteria, especially when using
differential media containing indicators.

• Example-
• V. cholera in TCBS agar appear Yellow
• Corny bacterium diphteriae in Tellurite agar appear black.
25
Staphylococcus Streptococcal colonies

o round, usually golden-yellow o vary in color from gray to

colonies, whitish and usually glisten
colonies.
o Soft/smooth
o Dry/rough 26
Staphylococcus aureus: Streptococcus pyogens
growth with golden-yellow growth with gray to
colonies whitish and usually glisten
colonies

27
 Mac Conkey Agar

Enterobacter cloacae Eschericia coli :

growth with pink colonies growth with pink colonies

28
Bacillus subtilis growth with Proteus spp. growth with……
………………

29
 Lowenstein-Jensen media

M. tuberculosis growth with pale- yellowish, rough colonies

30
Question: How do you appreciate the cultural characteristic of
bacteria in a liquid media?

 In a Fluid Medium following Characters are Observed

i. Degree of growth‐Absence, scanty, moderate, abundant etc.

ii. Present of turbidity and its nature.

iii. Presence of deposit and its character.

iv. Nature of surface growth.

v. Ease and disintegration and odor.

31
 2. Staining the isolated bacteria

Definition

 Stains (dyes) are coloured chemical compounds that are used

to selectively give colour to the colourless
structures of bacteria or other cells.

 Bacterial staining is the process of imparting colour to the

colourless structures (cell wall, spore, etc) of the
bacteria in order to make it visible under the
microscope.

32
 2. Staining the isolated bacteria

 Uses of staining:

• it is the main and most important step in the identification

of bacteria.

1. to observe the morphology, and arrangement of bacteria

2. to differentiate one group of bacteria from the other group.

33
 The isolated bacteria are stained by various methods
depending upon the bacteria in focus.

• Ex. Acid fast staining in case of suspected mycobacterium

infection

 The age of the culture is important.

• In older cultures, staining characteristics either vary or
are not brought out well.

 Type of staining methods

1. Simple staining method
2. Differential staining method
3. Special staining method
1. Simple staining method

• It is a type of staining method in which only a single dye

is used

• Simple stains bring-out the best morphology

• There are two kinds of simple staining methods

A. Positive staining
B. Negative staining

35
A. Positive staining:

• The bacteria or its parts are stained by the dye.

• Ex. Methylene blue stain, Crystal violent stain

B. Negative staining:

• The dye stains the background and the bacteria

component remain unstained.

• e.g. Indian ink stain.

36
2. Differential staining method

 A method in which multiple stains (dye) are used to

distinguish different group of bacteria.

 Example:
Gram’s stain,
Ziehl-Neelson stain.

37
3. Special staining method
These are stains, which are used to stain capsules and
spores.
There are two types of special staining methods
A. Capsule Staining method
B. Spore staining method

Note: Differential and special stains are necessary to bring

out characteristics like:
– Gram negative and gram positive bacteria,
– Acid fast and non acid fast ,
– spirochetes, capsule and Flagella, etc.
38
39
 Grams staining method
 Gram's stain is a laboratory staining technique that distinguishes
between two groups, Gram positive and Gram negative
bacteria, based on differences in the structure of their cell walls.

 It is named after its developer, Danish bacteriologist Christian

Gram in 1984.

Basic concepts:
 Most bacteria are differentiated by their gram reaction due to
differences in their cell wall structure.

 The surface of bacterial cell has got a negative charge due to the
presence of polysaccharides and lipids (PG) this has made the
surface of the bacteria to have affinity to cationic or basic dyes.
40
 The peptidoglycan itself is not stained; instead it seems to act as
a permeability barrier preventing get rid of crystal violet.

 During decolonization step, the alcohol is supposed

 To shrink the pores of gram-positive thick peptidoglycan. Thus, the
purple crystal violet-iodine complex is retained and the bacteria
remain purple.
 In contrast, gram-negative peptidoglycan is very thin with larger
pores,
 Alcohol treatment also may extract lipid from the gram negative wall
which increases its porosity further.

 Hence, purple crystal violet-iodine complex readily removes from

gram-negative bacteria and

 it appeared red bacteria due to stained by a red counterstain (safranin

or acid fuchsin)

41
Principle
 In the staining technique, the cells on microscopic slide are heat-fixed (killed)
and stained with a basic dye (e.g. crystal violate), which stains all bacterial cells
blue. Then, slides are treated with an Gram’s iodine to fix (mordant) the crystal
violet stain on the cell form a water-insoluble complex with the crystal violet
dye. Then, cells are decolorized with alcohol. Due to the differences in their cell
wall, the purple crystal violet-iodine complex is retained during the short
decolorization step and the Gram-positive bacteria remain purple. Whereas, the
purple crystal violet-iodine complex is removed from Gram-negative bacteria
and it takes the counter stain color, Red. Hence, Gram stain divides the bacteria
into Gram positive & Gram negative.

 Required reagents
 Crystal violet
 Gram’s Iodine
 Acetone-Alcohol
 Safranin

42
 The basic procedure of Gram stain goes like this:

i. Take a heat fixed bacterial smear.

ii. Flood the smear with CRYSTAL VIOLET for 1 minute, then wash with water.
[PRIMARY STAIN]
iii. Flood the smear with IODINE for 1 minute, then wash with water.
iv. Flood the smear with ETHANOL‐ACETONE, quickly, then wash with water.
[DECOLORIZER]
v. Flood the smear with SAFRANIN for 1 minute, then wash with water.
[COUNTERSTAIN]
vi. Blot the smear, air dry and observe.
vii. Examine under microscope

Result:
i. Gram positive bacteria‐violet
ii. Gram negative bacteria‐pink
43
Flow charts of procedures of Gram’s stains

44
Reporting of Gram’s stained smear

The report should include the following:

1. Gram reaction of the bacteria whether Gram positive or Gram negative
2. Morphology and arrangement of the bacteria whether cocci, diplococci,
streptococci, rods, coccobacili

Shape: Rod
Shape: Cocci Size: medium
Size: small Color: red
Color: purple Arrangement: singly or in pairs
Arrangement: Cluster or grapes like Gram reaction gram-negative
Gram reaction gram-positive 45
46
 i. Coccus (round)

Staphylococcusspecies
Streptococcus species

ii. Bacillus (rod, stick)

Clostridium spp.

47
What are the Factors which contribute to false Gram’s staining result? (reading
assignment)

I. False Gram positive reaction

 Preparation of too tick smear – the stain will not be fully washed.
 Application of crystal violet -presence of sediment in crystal violet -this can
be avoided by filtering the stain before use.
 Fixing before drying brings alteration of cell wall and morphology.
 Decolourizing time - when insufficient decolourization time is used or
(when not fully decolorized).
II. False Gram Negative reactions
 Making smear from old culture.
 Cell wall damage due to antibiotic.
 Excessive heat fixation of smear.
 Over decolourization of smear (decolourization for longer time).
 Use of an iodine solution which is too old (i.e. yellow in stead of brown in
colour) (its mordant effect will be decreased).
48
 The Acid-Fast stain (AFB) Ziehl-Neelson staining method

 Developed by Paul Ehrlich in1882, and modified by Ziehl and Neelson

 Acid fast stain:

 is used for staining mycobacteria which are hardly stained by
Gram‘s staining method.
 is performed in cases suspected of Mycobacterial infection.
Eg. Tuberculosis, leprosy, etc.

 Once the Mycobacteria is stained with primary stain (Carbol-

fuchsin) it can not be decolorized with acid, so named
as acid-fast bacteria.
Principle
The smear is heat-fixed, flooded with a carbol-fuchsin and heated until
steam arises. The heating allows melting of cell wall of mycobacteria
which facilitate entrance of the primary stain into the bacterium. After
washing with water, the slide is covered with 3% HCL (decolourizer). Then
washed with water and flooded with counter stain methylene blue ( M.
tuberculosis, M. bovis OR malachite green (M. leprae).

The ability of the bacteria to resist decolorization with acid alcohol confers
acid fastness to the bacterium.

Required reagents
 Carbol fuchsin
 Acid-Alcohol
 Methylene blue/Malachite green
50
51
Results
 Acid-fast bacilli (AFB) are :
 hot pink or red,
 straight or slightly curved rods,
 occurring single or in a small groups.

 Non acid-fast bacteria (background) are light blue/green.

AFB positive (arrow indicated) A= Non-acid fast bacteria

B= AFB positive
52
 Mycobacterium tuberculosis
Factors which bring false positive results why?
 Using scratched slides on which deposits of stain may look like bacilli.
 Using unfiltered carbol fuchsin which may contain crystals .
 Carelessness in heating the carbol fuchsine, allowing it to dry and crystallize on
the smear.
 Inadequate decolorizing of the smear.
 Special staining method (Capsule, Spore)

A. Capsule staining method (Indian ink)

 This technique is used for showing the presence of capsules around

bacteria

 The capsule stain employs an acidic stain and a basic stain to detect capsule
production.
 Capsules are formed by organisms such as Klebsiella pneumoniae.

 Most capsules are composed of polysaccharides, but some are composed of

polypeptides.

Required reagents
o Crystal Violet ………………………..10g/l (1% w/v) stain
o Copper sulphate………………………200g/l (20%w/v)
54
Procedure
1. Fix the direct smear using alcohol
2. Cover the smear with crystal violet stain and heat gently until the steam begins to raise
and leave to stain for one minute.
3. Wash off the stain with the copper sulphate solution (don’t use water WHY?).
4.Wipe the back of the slide and place it in a draining rack to air dry.
5. Examine the smear microscopically using oil immersion objective to look for capsulated
bacteria.

Result
• Bacterial cell -dark purple
• Capsule outline Pale blue
• Positives= clear "halos" around cells
when viewed under oil immersion
• Negative= no clear "halos" around cells
when viewed under oil immersion

55
56
B. Spore staining method

 The primary dye malachite green is a relatively weakly binding

dye to the cell wall and spore wall.

 In fact, if washed well with water, the dye comes right out of the
cell wall. That is why there does not need a decolourizer in this
stain.

 It is based on the binding of the malachite green and the

permeability of the spore Vs. cell wall.

 The steaming helps the malachite green to permeate the spore

wall.

57
Procedure
1. Prepare smear of the spore-forming bacteria and fix in
flame.
2. Cover the smear with 5% malachite green solution and
heat over steaming water bath for 2-3 minutes.
3. Wash with clean water.
4. Add1% safranine for 30 seconds.
5. Wash with clean water.
6. Dry and examine under the oil immersion objective.
58
Flow charts of procedures of spore stains

59
Principle:
 The primary stain in the endospore stain procedure, malachite green,
is driven into the cells with heat.

 Since malachite green is water-soluble and does not adhere well to

the cell, and since the vegetative cells have been disrupted by
heat, the malachite green rinses easily from the vegetative cells,
allowing them to readily take up the counterstain

Results
 Spores ……Light green
 Vegetative…..Red
 Catalase test
Purpose:
 to detect the presence of the enzyme catalase in bacteria.

 The morphologically similar Enterococcus or Streptococcus

(catalase-negative ) and Staphylococcus or micrococci (catalase-
positive ) can be differentiated using the catalase test.

Principle of Catalase Test:

 The enzyme catalase mediates (catalyze) the breakdown of hydrogen
peroxide into oxygen and water.
 The presence of the enzyme in a bacterial isolate is manifested when a
small inoculum is introduced into hydrogen peroxide, and the
rapid elaboration of oxygen bubbles occurs.

 The lack of catalase is marked by a lack of or weak bubble production.

61
 Catalase is present in most cytochrome containing aerobic and
facultative anaerobic bacteria (except streptococcus
spp).

 Hydrogen peroxide:
 It forms as one of the oxidative end product of aerobic carbohydrate
metabolism;

 If this is allowed to accumulate in the bacterial cells, it becomes

lethal to the bacteria.

 Thus,

o Catalase helps in converting H2O2 to H2O and O2

o Optimal pH for catalase action is 7.

62
 Material Required for Catalase Test
 Hydrogen peroxide, 3% H2O2
 Test tubes, slides
 Test organisms (18-24 hrs)

 Procedure of Catalase Test

1. Tube Method
 Pour/transfer1-2 ml of hydrogen peroxide
solution into a test tube.
 Using a sterile wooden stick or a glass rod,
take fresh colonies of test organism and
immerse in the hydrogen peroxide solution

 Observe for immediate bubbling.

63
2. Slide Method
 Use a loop or sterile wooden stick to transfer a small amount of
colony in the surface of a clean, dry glass slide.
 Place a drop of 3% H2O2 in the glass slide.
 Observe for the evolution of oxygen bubbles.

64
 Result Interpretation of the test
• Catalase Positive: Copious bubbles produced, active bubbling
• Catalase Negative: No or very few bubbles produced.

Quality Control of Catalase Test

Positive: Staphylococcus aureus– ATCC 33592
Negative: Enterococcus faecalis– ATCC 29212
65
Catalase Positive
• Micrococcus
• Staphylococcus
• Bacillus
• Listeria monocytogenes
• Enterobacteriacae
• Gonococcus &Meningococcus
• Vibrio cholerae
• Pseudo/Aero/Plesiomonas

Catalase Negative
• Streptococcus
• Clostridium

66
 False Positive and Negative:
• It is important not to contaminate the bacterial colony under test with
blood agar.
o Red blood cells contain catalase and their presence will give a
false positive result.

• Old cultures may loose their catalase activity, possible resulting in

a false negative result.

Precautions
 Avoid colonies taking from blood agar

 18 to 24 hrs colony only should be used

 Reagent to be fairly fresh as it is very unstable and easily breaks down

on exposure to light
 Reagent to be stored in brown colored bottle in refrigerator at 4°c 67
 Oxidase Test
Principle

 The test is used to detect bacteria that produce the enzyme

cytochrome oxidase which catalyze oxidation of
reduced cytochrome by oxygen molecule

 Cytochromes are iron containing hemoprotiens and in

aerobic respiration they transfer electrons(H) to
oxygen to form water.

 The reagent contains tetramethyl-p phenylenediamine which

serves as an alternate substrate for the cytochrome
oxidase reaction.

 In the reduced state, the reagent is colorless but when oxidized

it becomes dark purple.
 Purpose:
 is used to determine if an organism possesses the cytochrome oxidase enzyme.

 Used to assist in the identification of organisms which produce the enzyme

cytochrome oxidase

 Material Required
 Fresh Oxidase reagent (Tetramethyle-p-phenylenediamine dihydrochloride, 1%)

 Filter paper or oxidase regent strip or others

 Method
 Place a piece of filter paper in a clean petri dish

 Add 2 or 3 drops of freshly prepared oxidase reagent

 Smear a colony on filter paper using glass rod (not an oxidizing wire loop)

 Look for the development of a blue-purple color within a few seconds

69
 Result
 Positive Oxidase test …Color change to purple (Blue–purple color)
within-
– 10 seconds = positive
– 10-60 seconds = delayed positive
– >60 seconds = Negative

 Negative Oxidase test…..No blue –Purple color

 Quality controls
Positive control‐Pseudomonas spp
Negative control –E. coli

Note: Precautions in interpretation

 The test is only reliable as long as the time limit for a positive result is
adhered to (up to 60 seconds maximum) 70
Filter Paper Method Swab Method Test tube Method

71
Oxidase Positive
•Pseudomonas spp.
•Aeromonas spp.
•Vibrio spp.
•Alcaligenes spp.
•Neisseria spp.
•Haemophilussps

Oxidase Negative
Enterobacteriaceae
• Acenitobacter spp.

72
 Motility test
Principles
 It is not a biochemical test since we are not looking at
metabolic properties of the bacteria rather
 to check for the ability of bacteria to migrate away from a line
of inoculation.

Purpose:
It helps to differentiate species of bacteria that are motile
from non‐motile.

Method:
 Motility testing is performed by:
1. by inoculating the bacteria in the semi-solid motility medium
(Stabbing method).
2. by preparing a wet mount and then observed under the microscope.
73
 Stabbing motility media Method:
 Media and Reagents Used:
 Motility media: contains tryptose, sodium chloride, agar, and a
color indicator (make the results easier to see)
 How to Perform Test:
 Stab the motility media straight line with inoculating needle and
withdraw the needle very carefully to avoid destroying the straight line

 Observations can be made after incubating the sample for 24-48 hrs

 Reading Results:

1. Positive test:
 Growth spreads out (migrated) from the line of inoculation (motile).
 Highly motile organisms provide growth throughout the tube.

2. Negative test:
 Growth only along the stab line (non-motile bacteria) 74
 From left to right: From left to right:
+ – + – +

 Migration away from the line of inoculation = Motile (turbidity, cloudiness)

No migration away from the line of inoculation = Non motile (negative)

75
 Oxidation-Fermentation (O-F) test
Principle
 Bacteria utilize glucose and other carbohydrates using certain
metabolic pathways.
Some are oxidative routes but
others involve a fermentation reaction.

 The O-F test is used to determine which route is used.

the oxidative or
fermentative metabolism of a carbohydrate or
 its non-utilization.

 Fermentation is a anaerobic process, and bacterial fermenters of

carbohydrates are usually facultative anaerobes
 Oxidation is a aerobic process, and bacterial oxidizers of
carbohydrates are usually strict aerobes (obligate aerobes)
76
 As the organism grows and uses the sugar, the fermentation will produce acid
which change the pH and the pH indicator show a color change.

 The pH indicator is bromothymol blue

77
Purpose
 The test is used to differentiate between species, particularly Gram-negative
rods.

 Those organisms that utilize

 Carbohydrates aerobically (Oxidation) such as Pseudomonas aeruginosa from

 those that utilize it anaerobically (Fermentation) such as members of the

Enterobacteriaaceae and

 those that do not utilize carbohydrates at all (Non fermenters) such as

Alcaligenes faecalis .

Note:
• A facultative anaerobe is an organism that makes ATP by aerobic respiration
if oxygen is present, but is capable of switching to fermentation or anaerobic
respiration if oxygen is absent.

• An obligate aerobe, by contrast, cannot make ATP in the absence of oxygen, and

• Obligate anaerobes die in the presence of oxygen. 78

Materials Required for O-F test
• Test organism
• Hugh Leifson medium (semi‐solid medium containing the carbohydrate
under test (usually glucose) and a pH indicator
• Sterile paraffin oil (liquid paraffin)

Procedure:

• Two tubes are inoculated (test organism) by stabbing and

• one is immediately sealed with paraffin oil to produce

anaerobic conditions and the second left uncovered
(aerobic)

• Both are incubated for up to 14 days at 35-37oC

• Examine daily for carbohydrate utilization.

79
Results interpretation:

1. Oxidizing organisms:
 eg Pseudomonas species, produce
Covered Open
an acid reaction in the open tube only (anaerobic) (aerobic)
 Can breakdown carbohydrate only aerobically

2. Fermenting organism:
 eg. Enterobacteriaaceae, produce acid reaction
throughout the medium in both tubes
 Can breakdown carbohydrate both aerobically Covered Open
(anaerobic) (aerobic)
and anaerobically
3. Non-fermenters:
 eg. Alcaligenes faecalis
 cannot breakdown carbohydrate aerobically or
anaerobically
Covered Open80
(anaerobic) (aerobic)
 Result:
Reaction Positive Negative
Oxidation Yellow Blue or Green
Fermentation Yellow Green
Inert (unreactive) Blue or Green

Reaction Open tube Sealed tube

Oxidation Yellow Green
Fermentation Yellow Yellow
Inert (unreactive) Green Green
Covered tube Open tube
(anaerobic) (aerobic)

Covered tube Open tube

(anaerobic) (aerobic)
Covered tube Open tube
82
(anaerobic) (aerobic)
 Potassium Hydroxide (KOH) Test

 It is a fast alternative method for differentiate gram-positive and

gram-negative organisms.

3% Potassium Hydroxide is a reagent recommended for use

 Principle:
 Gram-negative cell walls are broken down by 3% KOH and
 In turn release viscoid chromosomal material which causes the
suspension to become thick and stringy.
 This reaction characterizes gram-negative bacteria.

 Materials Required
(1) Loop sterilization device,
(2) Inoculating loop, swabs, collection containers,
(3) Supplemental media,
(4) Quality control organisms,
(5) Glass slides.
 Procedure
1. Place 2 drops of 3% Potassium Hydroxide on a microscope slide.

2. Stirring a colony (young) into the solution with a circular motion.

3. Pull loop gently from suspension and examine for stringy,

viscous material within the first 30 seconds of mixing.

 Result Interpretation

 Positive Test ( Gram negative bacteria)

 Stringy, viscous gel formation pulled up
from suspension within first 30 seconds

 Negative Test (Gram positive bacteria)

 No stringy, viscous gel material within first 30 seconds

84
 2. Secondary Identification Tests

 Once the bacterium has been identified to a generic level,

 Tests based on the type of organism can be carried out to identify the
species.

 These tests are called Secondary Identification Tests and includes:

 Coagulase test
 CAMP test
 IMViC Tests it is an abbreviation which stands for:
• Indole test
• Methyl Red (MR) test
• Voges-Proskauer (VP) test
• Citrate test
 Triple Sugar Iron (TSI) Slant agar test
85
Coagulase test (tests for the pathogenicity of staphylococci isolates)

Principle:

 This test is used to differentiate coagulase positive Staphylococcus (S.

aureus) from coagulase negative Staphylococci.

 The enzyme coagulase is a key feature of pathogenic Staphylococcus

 The enzyme creates coagulation of blood, allowing the organism to be safe

from the host's protective mechanisms rather effectively

 S. aureus produces two forms of coagulase enzyme:

 Bound coagulase: bound to the cell wall
 Free coagulase: Secreted

86
 Rabbit plasma contains fibrinogen

 If the test organism possesses coagulase enzyme, it converts fibrinogen

to fibrin and causes clumping of cells or fibrin clot

 Bound coagulase (clumping factor):

 When a bacterial suspension is mixed with plasma:

• bound coagulase adsorbs fibrinogen from the plasma and changes it

• So it precipitates on the staphylococci, causing them to clump resulting in

cell agglutination.

 Free coagulase:
 is produced extra‐cellularly by the bacteria

 It causes the formation of a clot when S. aureus colonies are

incubated with plasma.
87
Methods:

Reagent:
o rabbit plasma (fresh or reconstituted commercial freeze dried)
o the test organism:

 There are 2 methods:

1. Slide coagulase test
o It detects bound coagulase
o Faster

2. Tube coagulase test

o It detects free coagulase
o also can detects bound coagulase for confirmation.
o More accurate

88
a) Slide test:
• A loopful of colony is emulsified in a drop of water or saline on microscopic slide

• A drop of rabbit plasma is added and mixed well with the bacterial suspension

• The slide is gently shaked for 5 to 10 seconds.

b) Tube test:
• 0.5 ml of rabbit plasma is placed in a small test tube

• Add two drops of broth culture made from a colony on agar

• The tube is rotated gently to mix the contents

• Then, incubated at 37°C for 2 - 4 hrs

89
Reading results
1. Slide test:
Positive reaction: Macroscopic clumping in 10 seconds or less
Negative reaction: No clumping

Note: All negative slide tests must be confirmed using the tube test.

2. Tube test:

Positive: Clot of any size (a)

Negative: No clot (b)

Coagulase Positive : Staphylococcus aureus

a b
Coagulase negative: Staphylococcus epidermidis
90
 Coagulase test correlates with pathogenicity

 Pathogenic strains: coagulase-positive

o Staphylococcus aureus
o S. intermedius
o S. hyicus

 Caogualse negative: occur as commensals and in the environment

S. epidermidis
S. Saprophyticus

91
CAMP test (presumptive identification tests for streptococci)

Principle:

 Group B streptococci can be distinguished from other beta-hemolytic

streptococci by their production of a substance called the CAMP factor.

 CAMP: the names of the investigators (Christie, Atkins, and Munch-Petersen)

who first described the factor.

 This factor is a diffusible peptide that acts together/synergistically with

the beta hemolysin produced by S. aureus, enhancing the effect of the
latter on a sheep blood agar plate.

 i.e., the factor completes the lysis of the RBCs, only partially hemolyzed
by the beta hemolysisn of the staphylococcus
92
 Beta hemolysin
 It breaks down the red blood cells and hemoglobin completely.
 This leaves a clear zone around the bacterial growth.
 Such results are referred to as β-hemolysis (beta hemolysis).

 Alpha hemolysin:
 Partially breaks down the red blood cells and leaves a greenish color behind.

 Gamma hemolysis:
 is a lack of hemolysis in the area around a bacterial colony.
 A blood agar plate displaying gamma hemolysis actually appears brownish.

93
Procedure
 A culture of S. aureus, with a wide zone of partial hemolysis is streaked
across the center of a sheep blood agar plate

 A streak of the suspect Group B streptococcus is made at right angles to

the staphylococcus streak

 Incubation: at 37°C for 18—24 hrs

Result Interpretation:
 Positive CAMP test = an “arrow-head shaped” of complete hemolysis
(in the nearest proximity to the S. aureus and streptococcal growth)

 Negative CAMP test = No "arrowhead" shaped hemolysis.

94
 Figure: CAMP test for Group B streptococci.

S. agalactiae is causing the characteristic arrow-head clearing of the partial hemolysis of

S. aureus (vertical streak).

95
This test is used for S. agalactiae which has a factor that synergistically increases
S. aureus beta hemolysis

96
 IMViC Tests

 are frequently employed for identification of Enterobacteriaceae

group which includes Klebsiella, Enterobacter, Salmonella, and
Escherichia coli.

 These IMViC tests consists of :

Indole test
Methyl Red (MR) test
 Voges-Proskauer (VP) test
Citrate test

97
Indole Test

Principle:
 Indole test is performed to determine the ability of the organism to split
the amino acid tryptophan into Indole.

 Bacteria that possess the enzyme tryptophanase are capable of

hydrolyzing tryptophan with the production of Indole, Pyruvic acid and
ammonia

Purpose:
 It helps to differentiate species of the family Enterobacteriaceae.

Media and Reagents used:

 Tryptone broth contains tryptophan.
 Kovac’s reagent:
o contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol

98
Procedure:
 inoculate tryptone broth with the test organism
 incubate for 18 to 24 hrs at 37c
 add 15 drops of Kovac’s reagent down the inner wall of the tube

Interpretation:
Positive = bright red ring at the interface of the reagent and the broth (in seconds)
Negative = bright red ring not developed
Indole Positive:
E.coli
Proteus vulgaris

Indole Negative:
Salmonella spp.
Klebsiella spp.
Enterobacter aerogens

99
Methyl Red/Voges‐Proskauer (MR/VP) test

 The methyl red (MR) and Voges-Proskauer (VP) tests

are read from a single inoculated tube of MR-VP broth.

 After 24-48 hours of incubation, the MR-VP broth is split into two
tubes.
one tube is used for the MR test;
the other is used for the VP test.

 MR-VP media contains glucose and peptone.

 All enteric bacteria utilize glucose for energy; however the end
products vary depending on bacterial enzymes.

 Both the MR and VP tests are used to determine what end products
result when the test organism degrades glucose.

100
1. Bacteria (E. coli) that produce acid as end product from glucose:
 causing the pH to drop below 4.4 (acidic) and
 When the pH indicator methyl red is added to this acidic broth
 it will be cherry red (a positive MR test).
2. Bacteria (Klebsiella and Enterobacter) that produce more neutral
products from glucose (e.g. acetyl methyl carbinol).
 In this neutral pH, the growth of bacteria is not inhibited

 thus, the bacteria begin to attack the peptone in the broth, causing
the pH to rise above 6.2.

 At this pH, methyl red indicator is a yellow color (a negative MR

test)

 When VP reagents added to a broth in, they turn to a pink-color (a

positive VP test).
101
 MR-VP
Purpose:
 Both tests are used to differentiate species of the family Enterobacteriaceae.

Media and Reagents used:

o Glucose broth (glucose and peptone)
o Methyl Red indicator for MR test
o Voges Proskauer reagents: alpha‐naphthol & potassium hydroxide

MR test
Principle:
 To test the ability of the organisms to produce acid end product from
glucose fermentation

102
Procedure:
o Inoculate the MR/VP broth with a pure culture of test organism
o Incubate at 35°for 48 to 72 hrs.
o Add 5 drops of MR reagent to the broth

Result interpretation:
– Positive result is red (indicating pH below 6)
– Negative result is yellow (indicating no acid production)

MR Positive: E. coli

MR Negative:
Enterobacter aerogenes
Enterobacter cloacae
Klebsiella spp.

103
Left: Negative/Right: Positive
104
 VP Test (acetoin production)
Principle:
 To determine the ability of the organisms to produce neutral end product
acetyl methyl carbinol (acetoin) from glucose fermentation
Procedure:
o Inoculate pure culture of the test organism into MR/VP broth
o Incubate for 24 hrs at 37°c
o Add VP reagent (alpha-napthol and potassium hydroxide) to the broth
Interpretation:
Positive : Pinkish red color at the surface of the medium
Negative : Yellow color at the surface of the medium

Positive
Klebseilla pneumoniae
Enterobacter
Negative
E.coli

105
VP: left + and right –
106
Citrate utilization Test

 This test is one of several techniques used to assist in the identification

of enterobacteria.

 The test is based on the ability of an organism to use citrate as its only
source of carbon and energy.

Principle:

 The test organism is cultured in Simmon's citrate media which contains

sodium citrate, an ammonium salt and indicator bromothymol blue.

 Growth in the medium is shown፡

 by turbidity and
 a change in color of the indicator from light green to blue, due to alkaline
reaction following citrate utilization.
107
Procedure:
o Inoculum is streaked over the slant of Simmon’s citrate agar
o incubated for 24‐48 hrs.

Result interpretation:
o Positive = Green → Deep blue.
o Negative = No color change

Positive: Klebsiella pneumoniae

Negative: E. coli

Left‐Positive: Right‐Negative
108
 IMViC Tests
Indole Test Citrate Test
MR test VP Test

Citrate Test
Indole Test MR test VP Test

E.coli + + - -
Proteus vulgaris +
Salmonella spp. -
Enterobacters +
Enterobacter aerogens - -
Enterobacter cloacae -
Klebsiella spp. - -
Klebsiella pneumoniae + +
Klebseilla pneumoniae
Indole Test MR-VP Citrate Test
MR test VP Test

Citrate Test
Indole Test MR test VP Test

E.coli + + - -
Proteus vulgaris +
Salmonella spp. -
Enterobacters +
Enterobacter aerogens - -
Enterobacter cloacae -
Klebsiella spp. - -
Klebsiella pneumoniae + +
Klebseilla pneumoniae
 MEKELLE UNIVERSITY
COLLEGE OF VETERINARY SCIENCES
Department of Veterinary Basics and Diagnostic Science

Systemic Bacteriology

1
 Part II. Systemic Bacteriology
Outline

5. Major Pathogenic Bacteria

5.1. Staphylococcus species
5.2. Streptococci
5.3. Bacillus
5.4. Clostridium
5.5. Listeria, Erysipelothrix
5.6. Corynebacterium species
5.7. Actinomyces andActinobacillus
5.8. Brucella
5.9. Mycobacterium
2
 Outline

5.10. Enterobacteriaceae

5.11. Pasturella species andMannheimia hemolytica

5.12. Campylobacter Species

5.13. Mycoplasma

5.14. Chlamydia andRickettsial Species

3
 Bacteria

Gram-positive Gram-negative
Bacteria Bacteria

Gram-positive Gram-positive Gram-negativ

e Gram-negative
Cocci Bacilli
Cocci Bacilli

 Bacillus,
 Non-enteric Bacilli
 Clostridium
 Staphylococcus
 Listeria,  Enterobacteriacea
 Micrococcus,
 Erysipelothrix
 Enterococcus,  Neisseria e
 Corynebacteriu
and  Moraxella
m,  Pasteurella and
 Streptococcus
 Mycobacterium,
 Actinomyces,
 Haemophilus
 Nocardia
4
 Gram positive cocci:
 Staphylococcus, Micrococcus,
 Streptococcus and Enterococcus

 Gram negative cocci:

Neisseria,
Moraxella

 Gram-Positive Bacilli
 Endospore-formers: e.g.Bacillus, Clostridium
 Non-endospore-formers: e.g.Listeria, Erysipelothrix
 Irregular shaped and staining properties: e.g.Corynebacterium,
Mycobacterium, Actinomyces, Nocardia

 Gram-Negative Bacilli
 Aerobic Gram-Negative: Non-enteric Bacilli
 Enterobacteriaceae Family 5

 Oxidase-Positive Nonenteric Pathogens:Pasteurella andHaemophilus

 The Spirochetes:
Leptospira ,
Treponema,
Borrelia
 Curviform Gram Negative Bacteria:
Vibrio,
Campylobacter ,
Helicobacter
 Rickettsia

 Chlamydia

 Mycoplasma and L-Form

6
 I. Gram-positive Bacteria

 Family: Micrococcaceae (G+ cocci)

 3 Genera:
 Staphylococcus (pathogenic)
 Micrococcus (non-pathogenic)
 Planococcus (marine habitats)

 Genus: Staphylococcus
General characteristics:
 Gk, “Staphyle” = bunch of grapes ” “kokkos”-grain
 Gram positive cocci, occurring in cluster or bunches of grapes
 Facultative anaerobic (fermentative), ferments sugars but produces no
gas

7
 Non-motile, Non-spore forming, 1μ dia.
 Non-capsulated with the exception of few strains
 Catalase positive, Oxidase-negative
 Resist dehydration, relatively heat resistant and tolerate many
common disinfectants.
 Hallmark clinical manifestation is an abscess
 Growth:
o on nutrient and blood agars,
o but not on MacConkey agar

 Staphylococci can be differentiated from Streptococci by

testing for the enzyme catalase:
o Staphylococci catalase positive while streptococci do not have
it.

8
 Colony morphology ofStaphylocucus
Gram reaction: gram-positive
o round, usually golden-yellow
Shape: Cocci
colonies,
Size: small
o Soft
Color: purple
o often with hemolysis, when grown on
Arrangement: Cluster or grapes
blood agar plates.
like
9
 Grouping for Clinical Purposes:

 The staphylococci are divided in two groups based on the

presence or absence of the enzyme coagulase.

1. Coagulase positive Staphylococci (pathogenic)

• Staphylococcus aureus
• Staphylococcus intermidius

2. Coagulase negative Staphylococci (non-pathogenic)

• Staphylococcus epidermidis
• Staphylococcus saprophyticus

10
 Staphylococcal Species

S. aureus : principal pathogenic species

– Named because of yellow/gold color of colonies
– Due to production of β-carotene pigment (a virulence factor)
– Coagulase positive, ferments mannitol
– Causative agent of mastitis in cows, abscess
– Food poisoning in humans

S. epidermidis :
– Named because of its prevalence on human skin
– Found on skin, teats, hair and superficial mucosa of many animals but in
less numbers than seen in humans
– Colonies are non-pigmented and non-hemolytic
– coagulase negative
– an opportunistic invader of low virulence

11
S. Intermedius
– Most prevalent disease-producing species in dogs and other
carnivores
– colonies are non-pigmented,
– Coagulase positive
– vary in hemolytic pattern (often β-hemolytic)

Staphylococcus hyicus

– Exudative Epidermitis of Swine (Greasy Pig Disease)

– Coagulase variable

12
 Pathogenicity and Virulence factors

 Pathogenicity: refers to the absolute ability of an infectious agent to

cause disease/damage in a host; an infectious agent is either
pathogenic or not.

 Virulence is an ability of an organism to infect the host and cause

disease

 Virulence factors:
 are molecules that assist the bacterium colonize the host at cellular
level.

 These factors are either secretory, membrane associated or cytosolic in

nature.

 It includes: 13
o bacterial toxins,
o cell surface protein that mediate bacterial attachment,
 Virulence Factors
• Cell-Associated Virulence Factors
– Capsule or slime layer (glycocalyx)
– Teichoic acid is covalently linked to PG
– Protein A
– Peptidoglycan (PG)
– Clumping factor (bound coagulase)
• Extracellular toxins(Exotoxins)
– Hemolytic toxins (α lysin, β lysin, γ lysin and δ lysin)
– Leukocidin
– Enterotoxin
– Exfoliative toxin (epidermolytic toxin)
– Toxic Shock Syndrome toxin
• Extracellular enzymes
– Coagulases - Hyaluronidase
– Nuclease - Staphylokinase
– Lipases - Phospholipases

14
 Cell-Associated Virulence Factors

 Capsular polysaccharides:
– Few strains ofS. aureus are encapsulated and these strains tend
to be more pathogenic
– It prevents ingestion of the organism by polymorphonuclear
leukocytes

 Teichoic acid
– is a major antigenic determinant of all strains of S. aureus
– It facilitates adhesion of cocci to host cells and
– protects them from complement mediated opsonization
– It covalently linked to PG and is species specific:
• S. aureus : ribitol teichoic acid (polysaccharide A)
• S. epidermidis : glycerol teichoic acid (polysaccharide B)

15
 Peptidogycan
– Provides rigidity to the cell wall of staphylococci
– Stimulates both humeral and cell mediated immune response in the host

 Protein A
– Group specific antigen found in >90% of S. aureus
– It is chemotactic, anticomplementary, antiphagocytic, etc

 Clumping factor (bound coagulase)

– It is a surface component that causes the organisms to clump
when mixed with plasma

– This factor reacts directly with fibrinogen in plasma, causing rapid

cell agglutination

16
 Extracellular toxins
 Hemolysins
– Almost every strain ofS. aureus produces one or more of four
hemolytic, memberane damaging exotoxins known as α, β, γ and δ
lysins)
– Α lysin is the most important in pathogenicity

 Leukocidin
– Damage polymorphonuclear leukocytes and macrophages and
produce dermonecrosis

 Epidermolytic toxins (Exfoliative toxins) – 2 types

– Epidermolytic toxin A is heat stable and its production is
controlled by chromosomal gene
– Epidermolytic toxin A is heat labile and plasmid-controlled

17
 Enterotoxins

– About 40% all clinical isolates ofS. aureus produce

exotoxins that cause food poisoning in man

– There are about nine antigenic types (A, B, C, D, E, G, H, I and J).

– Enterotoxin F is know known as Toxic shock syndrome toxin

1(TSST-1)

– Some strains may produce toxins of more than one type

18
 Extracellular Enzymes

 Coagulases
– It activates a coagulase-reacting factor (CRF) normally present
in the plasma, causing the plasma to clot by the conversion of
fibrinogen to fibrin

 Hyaluronidase (“spreading factor” ofS. aureus)

– > 90% strains ofS. aureus produce hyaluronidase but the amount
varies
– It hydrolizes hyaluronic acid present in the intercellular ground
substance of connective tissue, thus facilitating spread of the
organism to adjacent areas

 Staphylokinase (fibrinolysin)- has fibrinolytic activity 19

 Susceptibility to physical and chemical agents

 Staphylococci are usually killed by a temperature of 60oC for 30

min

 Disinfectants such as:

 Chlorhexidine, Hexachlorophane, and phenol kill staphylococci rapidly.

 Mercuric chloride is poor disinfectant to staphylococci

 They are very sensitive to aniline dyes; thus they are inhibited on
blood agar medium containing 1 in 500,000 crystal violet

 Staphylococci are resistant to β-lactam antibiotics due to the

presence of β-lactamase (penicillinase) enzyme
20
 Streptococci/Enterococci –
General Description
 Family: Streptococcaceae
 Genus: Streptococcus, Enterococcus
 Pyogenic pathogens :-
 Gram positive cocci in chains
 Non-motile,
 Catalase negative, Oxidase-negative

 Heterogeneous group that cause a diversity of different

diseases

 Enterococci, formerly streptococci, established as separate

species based on DNA homology studies
21
 Classification Systems for Streptococci

 Hemolysis on blood agar plates

–S. pyogenes is β hemolytic (complete)
– Viridans (oral) streptococci are α hemolytic (incomplete)
– Enterococci are γ hemolytic (no hemolysis)

 Lancefield grouping based on group specific carbohydrate antigens.

 Most β and some α hemolytic streptococci can be typed by this
method

 Biochemical properties
– Catalase negative, facultative anaerobes
22
 α hemolytic
(incomplet
e)

γ-hemolysis (non-hemolytic)
23
 Streptococcal groups (Lancefield Groups)

 On the basis of group specific carbohydrate (C) antigens in the cell wall,
β-hemolytic streptococci are divided into 21 serological groups from A – W
except I and J.

Group-A streptococci
• S. pyogenes
Group-B streptococci
• S. agalactia –causative agent of bovine mastitis
Group-C streptococci
• S. equi, S. equisimilis, S. dysgalactia, & S. zooepidermicus
Group D streptococci
• Enterococci, S. bovis andS. equinus
Group G streptococci
• Normal flora of the skin
Viridans streptococci are not parts of the lancefield classification
because they don’t have C carbohydrate
24
 Cultural characteristics

Aerobes and facultative anaerobes

Temperature range is 22 – 40C, o

optimum temperature being 37C o

Growth:
They are fastidious – require the addition of blood or serum to media
for growth
oFastidious: will grow only if special nutrients are present in their
culture medium.

They tolerate the bile salts in MacConkey agar - appear as small pin
point colonies

Edwards medium is selective for streptococci

Mucoid colonies are formed by strains which produce large capsule

Cause hemolysis on blood agar

25
Virulence factors

 Enzymes:

– Hyaluronidase (spreading factor):

• destroys connective tissue and aids in spreading infecting bacteria.
– Streptokinase (fibrinolysin)
• Can lyse blood clots and may be responsible for the rapid spread of
the organism.
– C5a peptidase
• Prevents streptococci from C5a-mediated recruitment and
• activation of phagocytes, and is important for survival of
S. pyogenes in tissue and blood. HOW??

 Exotoxins:
– Pyrogenic exotoxins A-C - function as super-antigens producing a
sepsis syndrome 26
 Common diseases include:
 Pneumonia - St. pneumoniae
 Strangles – contagious equine rhinopharyngitis (St. equi )
 Bovine mastitis; St. agalactiae ,St. dysg .,
St.uberis, St. pyogenes
 Supportive lesions
 Metritis
 Arthritis
 Endocarditis
 Identification of Gram-positive cocci

None

28
 Family Neisseriaceae

 Gram-negative cocci
 Residents of mucous membranes of warm-blooded animals
 Genera include:
Neisseria, Moraxella, Acinetobacter.

 GenusNeisseria
– Gram-negative, bean-shaped, diplo-cocci
– None develop flagella or spores, Capsules on pathogens
– Aerobic or micro-aerophilic
– Strict parasites, do not survive long outside of the host
– Produce catalase and cytochrome oxidase
– Pathogenic species require enriched complex media and CO2 - Chocolate
Agar
29
 Other Gram-negative Cocci and Coccobacilli

 GenusBranhamella
– Branhamella catarrhalis –
found in naso-pharynx:
significant opportunist in cancer, diabetes, alcoholism

 GenusMoraxella
– M. bovis – causative agent of pink eye in bovines
– Coccobacilli; found on mucous membranes

 Genus Acinetobacter
– Gram-negative coccobacilli;
– Non-living reservoir;
– source of nosocomial infections
30
 Gram-Positive Bacilli
Three general groups:

1. Endospore-formers
Bacillus,
Clostridium

2. Non-endospore-formers (regular shaped and staining properties)

Listeria,
Erysipelothrix

3. Irregular shaped and staining properties

(Non-endospore-formers)
Corynebacterium,
Mycobacterium,
Actinomyces,
Nocardia
31
32
 I. Spore-forming Bacilli

 GenusBacillus

 GenusClostridium

33
 General Characteristics of the GenusBacillus

 Gram-positive, endospore-forming rods

 Motile exceptBacillus anthracis

 Aerobic and catalase positive (difference from clostridium)

 They can be grown readily on general purpose media

 Source of antibiotics –B. polymyxa (Source of polymyxin)

 Bacillus cereus causes gangrenous mastitis and abortion in

animals and food poisoning in humans

 Bacillus anthracis – causative agent of Anthrax

34
 Bacillus anthracis

 Differs from other species

 It is non-motile
 Capsulated
 Most of the time, it produces rods in very long chains - (20-30) rods in chain

 It is non-hemolytic on 5% sheep blood agar.

 Even if some strains are hemolytic, they cause weak
hemolysis

 Capsulation depends on CO2 concentration of the medium.

 When CO2 and bicarbonate ions are in higher concentration there will
be capsule formation
35
36
 Bacillus anthracis

 In ordinary growth condition (aerobic-nutrient) medium the

organism produces:
 Rough, medusa head (abnormally dilated) type colony

 In nutrient broth, the organism shows cottony wool growth

37
 The Spores are:

 ellipsoidal, centrally placed and don’t bulge the bacilli

but clostridium spores bulge the bacilli.

38
 B
A

Bacillus (a)B. anthracis , spores elliptical and central

(b)B. subtilis , spores elliptical and central.
39
 Bacillus anthracis’ s pores are not formed within the body during life and
also after death until it is opened.

 Sporulation is enhanced in the absence of nutrients and

presence of plenty of O .2

 That is why we don’t open the carcass of animal died of

anthrax – because sporulation occurs in the presence of O 2

 Virulence factors:
 Polypeptide capsule (antiphagocytic) and

 Exotoxins

 Anthrax infections are classified by route of entry;

 Cutaneous anthrax
 Inhalation anthrax
 Gastrointestinal anthrax
40
Anthrax in animals

41
Anthrax in humans

42
 Bacillus cereus

 Common air-borne and dust-borne

Bacillus cereus g rows in foods, spores survive cooking and reheating

 Ingestion of toxin-containing food causes:

Nausea, vomiting,
abdominal cramps and
diarrhea; 24 hour duration

 Usual methods of disinfection and antisepsis are ineffective

 No effective treatment

 Increasingly reported in immunosuppressed

43
 B. cereus are:

 Predominantly responsible for food poisoning in humans

and canine.

 produce enterotoxins that causes food poisoning

 Three exotoxins (all of which are required for virulence) include:

 Edema factor

 Protective antigen factor

 lethal factor

44
 The GenusClostridium

 Gram-positive, spore-forming rods

 Unlike genus bacillus, anaerobic and catalase negative
 Anaerobic condition
– Cl. Novyi – strictly anaerobic
– Cl. perfringens – somewhat aero-tolerant

 Of some 80 species of clostridia, perhaps 10 are of veterinary importance

 Motile exceptCl. Perfringens

 Of the pathogenic species, onlyCl. perfringens is encapsulated.

45
 Oval or spherical spores produced only under anaerobic conditions

 Causes:
wound infections,
tissue infections, and
food intoxications

 Growth conditions

– Most require enriched media – include amino acids, carbohydrates,

vitamins and blood or serum
– Optimum growth occurs at 37°C

– Strictness of anaerobic requirements varies among clostridial

species

46
 – Clostridia prefer 2% to 10% CO2 in their environment

– These conditions can be produced in a jar in which the oxygen is

reduced catalytically by hydrogen, generated along with CO2 from a
commercially available packet
Anaerobic jar

 Incubation:
 under strict anaerobic conditions

 with the atmosphere containing 2- 10% CO2

47
 Appearance and positions of spores

C. perfringenes :–
Spores are oval, subterminal or terminal and non-bulging
C. tetani :–
Spores are spherical, terminal and twice the size of vegetative
cells giving them typical drumstick appearance
C. septicum: –
Spores are oval, central or subterminal and bulging
C. botulinum: –
Spores are oval, subterminal and bulging
C. novyi:–
large oval and subterminal spores
48
49
50
 Clostridia can be grouped into two groups based on disease
– Histotoxic :–
C. perfringenes type A,
C. chauvoei,
C. novyi,
C. septicum,
C. hemolyticum,
C. sordelli

– Enterotoxic :–
C. perfringens (C. welchii ) A-E, type A – most common cause of gas
gangrene
C. perfringenes – ………Enterotoxemia, food poisoning, gas gangrene
C. chauvoei – ………….Blak leg, septisemia
C. tetani – ……………….. Lockjaw (tetanus)
C. septicum – …………..malignant edema
C. botulinum –…………..botulism
C. hemolyticum –……….. red water 51

C. novyi
 Virulence Factors

 Pathogenic species produce one or more exotoxins, which

account for their pathogenicity
 Virulence factors
– toxins –
• alpha toxin – causes RBC rupture, edema and tissue
destruction
– collagenase
– hyaluronidase
– Dnase
– C. tetani produces an oxygen labile hemolysin
(tetanolysin) and potent neurotoxin (tetanospasmin)
52
 Tetanus

 Caused byClostridium tetani

 Common resident of soil and GI tracts of animals

 Causes: tetanus or lockjaw, a neuro-muscular disease

 Horses are most susceptible

 Spores usually enter through:

 accidental puncture wounds,

 burns, and

 crushed body parts. 53

 Anaerobic environment is ideal for vegetative cells to grow
and release toxin.

 Tetanospasmin:– neurotoxin causes

 paralysis by binding to motor nerve endings;
 blocking the release of neurotransmitter for muscular
contraction inhibition; muscles contract uncontrollably

 Death most often due to paralysis of respiratory muscles

54
 Clostridial Food Poisoning
 Clostridium botulinum :–

 rare but severe intoxication usually from home canned food

 intoxication associated with inadequate food preservation

 commonly inhabits soil and water

 Clostridium perfringens :–

 mild intestinal illness;

 second most common form of food poisoning worldwide

 Spores contaminate food that has not been cooked thoroughly

enough to destroy spores.

 Spores germinate and multiply (especially if unrefrigerated).

 When consumed, toxin is produced in the intestine; acts on

epithelial cells, acute abdominal pain, diarrhea, and nausea 55

 Rapid recovery
 Common Disease caused byClostridium

1. Clostridium botulinum : botulism

2. Clostridium tetani: tetanus

3.Clostridium perfringens: Gas Gangrene

56
 Gram-Positive Regular Non-Spore-Forming Bacilli

• Listeria

• Erysipelothrix

57
 Listeria
 The Genus Listeria:
 there are 8 species,
 Of this, Listeria monocytogenes is important as a cause of a
wide spectrum of diseases in animals and humans
 Non-spore-forming Gram-positive coccobacilli or rod-shaped
measuring 1-3µm in length
 None acid fast and Motile at 20-25oC by means of peritrichate
flagella and show characteristics ‘tumbling’ (end-over-end) motility.

 They are also non-motile at 37oC

 None capsulated
 Resistant to cold, heat, salt, pH extremes and bile
 Virulence attributed to ability to replicate in the cytoplasm 58of
cells after inducing phagocytosis; avoids humoral immune
 Listeria monocytogenes:

 is a Gram-positive rod-shaped bacterium.

 It is a facultative anaerobic bacterium, capable of surviving in the

presence or absence of oxygen.

 It is the agent of listeriosis, a fatal infection caused by eating food

contaminated with the bacteria.

 Listeriosis has been recognized as an important public health

problem in the United States.

59
 Cultural characteristics
– Listeriae are aerobes and facultative anaerobes
– They can grow over a temperature range of 2- 43oC,
optimum temperature 35-37oC
– They can grow on ordinary media containing fermentable
carbohydrates but growth is better on blood agar.

– Colonies ofL.monocytogenes produce a narrow zone of

slightly hazy β-hemolysis

 Biochemical reactions
– Listeria are oxidase, urease, indole and H2S production
negative and
– Catalase, Voges-Proskauer and methyl red positive

60
61
 Erysipelothrix
 Erysipelothrix rhusiopathiae
 is non-motile, non-sporing, non-capsulated, straight or slightly
curved, gram-positive rod
 Aerobic and facultative anaerobe and growth is improved by
5-10% CO2

 It can grow in the temperature range of 15-44oC, optimum

temperature for growth being 30-37oC
 Produces H2S in kligler iron agar,

 ferment sugars with the production of acid without gas

 Enters through skin abrasion, multiples to produce erysipeloid, dark
red lesions
 Primary reservoir – tonsils of healthy pigs
62
 Gram-Positive Irregular Non-Spore-Forming Bacilli

 There are 20 genera but medically and veterinary important genera include:

– Corynebacterium, Mycobacterium

– Actinomyces, Nocardia

 Pleomorphic

 All produce catalase, possess mycolic acids, and a unique peptidoglycan.

 The familyMycobacteriaceae are:

 Gram-positive,
 Non-motile,
 Catalase-positive and
 have a rod like to filamentous morphology (Corynebacteria are often
pleomorphic)
 As a group, they produce characteristic long chain fatty acids termed as mycolic acids

63
 Genus Corynbacterium

 Corynbacteria are:

 belong to the familyMycobacteriaceae and are part of the

CMN group (Corynebacteria, Mycobacteria and Nocardia ).

 Pleomorphic, gram positive bacilli arranged in V forms or criss-

crossing cells (“Chinese letter appearance”) or in packets of
parallel (“palisades”)

 They frequently show club shaped swellings and hence

named corynebacteria (coryne means club)

 Another characteristic of this organism is its granular and

uneven staining

 Virulence factors assist in attachment and growth.

64
65
Corynebacterium diphtheriae
 non-capsulated,
 Gram-positive bacillus,
 Pleomorphic
 V forms or criss-crossing cells (“Chinese letter
appearance”)
 Cultural characteristics
– Aerobic and facultative anaerobe
– Optimum temperature for growth is 37oC
– It can grow on ordinary nutrient agar, but growth is
improved by the presence of animal proteins such as serum
or blood

 Biochemical characteristics

– C. diptheriae ferments glucose, maltose and on rare

occasions sucrose with the production of acid without gas
– Don’t ferment lactose, mannitol and trehalose

– C. diptheriae is readily killed by moist heat in 10 minutes and

the commonly used disinfectants

67
 Important species

 Corynbacterium diptheriae causes diphteria in humans

 C. pseudotuberculosis causes caseous abscess in ruminants and horses

 C. equi (Rodhococcus equi) causes a supportive pneumonia of young

foals

 C. renale colonizes the uro-genital tract

 Common diseases include:

Summer mastitis –C. pyogens in cattle & sheep
Lymphadenitis –C. ovis in sheep & goat
Bovine mastitis –C. bovis
Supportive lesions -C. pyogens
Ulcerative Lymphangitis - C. equi in equine
Pyelonephritis & Cystitis –C. renalae
68
 GenusMycobacterium

 Mycobacteria are slender rods with lipid-rich cell walls that are
resistant to penetration by chemical dyes, such as those used in
the gram stains.

 they stain poorly but, once stained, cannot be easily decolorized

by treatment with acidic solvents. Hence, they are termed
acid-fast

 Complex cell wall [high lipid content (25% by weight)]

 Gram-positive irregular bacilli, Strict aerobes, Produce catalase

 produce characteristic long chain fatty acids termed mycolic

acids.

 Do not form capsules, flagella or spores, No toxins (exceptionM.

ulcerans )
69

 Grow slowly (slow generation time 18-24 hrs)

 Distinctive Cell Wall
High lipid content (25% by weight)
 The cell wall

 Resistant to acid decolorization, (acid-fast )

 Resistant to degradation

 Inhibits phagosome/lysosome fusion

 Induces TNF-alpha

 Scavenges reactive oxygene intermediates (ROI)

 The very lipid nature of the cell wall provides some resistance to:
o drying,
o acid or alkaline conditions.
 Pathogenic mycobacterial species

 Mycobacterium tuberculosis – causes human tuberculosis

(TB)

 M. bovis –causes:
• Bovine TB,
• Human TB

 M. avium ssp. paratuberculosis – John’s disease in animals

 M. leprae – the causative agent of leprosy

71
• The main signs of Johne's disease in cattle
are
• progressive weight loss and
• chronic diarrhoea
72
• Clinical pictures of Leprosy
• skin lesion
• deformities

73
Acid fast bacilli, occurring single or in a small groups.

74
 Mycobacterium species:

are very slow growers.

grow in a complex media which contain lipids, and other possible

additional substances like egg + basal media

mostly used type of media is Lowenstein-Jensen media

Colonies ofM. tuberculosis (buff/

pale yellow color, rough)
on LJ culture medium

Day 28

Day 21

 M. leprae has never been culturedin vitro

but can be grown on the footpads of armadillos. 75
 Actinomyces

 Gram positive, non-motile, non-spore forming and

 pleomorphoic which can occur as

• rod,

• coccobacilli or

• branching filamentous.

 Have distinctive/ distinguishable/typical cell wall

constituents

 Some species are anaerobic, while others are facultative

anaerobic 76
 Actinomyces
 Animal actinomyces spp. are capnophilic or facultative
anaerobes

o Capnophilic are microbes that thrives/grow-well in the

presence of carbon dioxide

 All require rich media, preferably containing serum or blood

 No growth occurs in Sabouraud’s agar or at room

temperature

 Catalase negative with some exception (A. viscosus)

 Killed rapidly by heat and disinfectants and require frequent

passage to survive in culture 77
Genus: Actinomyces

Species: A. bovis, A. israel

 They are commensals of the oral cavity and cause diseases under
conditions such as damage to the oral mucosa.

 So, they are opportunistic pathogens.

 these bacteria can cause actinomycosis, a disease characterized

by the formation of abscesses in the mouth, lungs, or the GIT.

 A. bovis causes “lumpy jaw” (rarefying osteomyelitis) in cattle

78
 Actinomyces bacteria under the microscope

Clinical picture: Actinomycosis 79

 Nocardia

 Nocaria are aerobic, non-motile, saprophytic bacteria present in

most environments
 Generalized supportive and pyo-granulomatous process occur in:

Immuno- compromized and massively exposed individuals

 Of the main three species, accounts for most cases are:

N. asteroids, N. brasiliensis, and N. otitidiscaviarum

 All animal species are susceptible but dogs are most commonly
affected

 In dairy cattle, nocardial mastitis is economically important

 Pathogenic nocardia are obligate aerobes growing on simple

media (e.g. Sabouraud’s agar)
80
 Gram-Negative Bacilli/Rods

 Large, diverse group of non-spore-forming bacteria

 Wide range of habitats:–

• Large intestines (enteric),
• zoonotic,
• respiratory,
• soil, Water

 Most are not medically/clinically important;

 some are true pathogens,

 some are opportunists.

 All have a lipopolysaccharide outer membrane of cell wall – endotoxin.
81
82
 Aerobic Gram-Negative Non-enteric Bacilli

 Pseudomonas and Burkholderia – an opportunistic

pathogen

 Brucella andFrancisella – zoonotic pathogens

 Bordetella andLegionella – mainly human pathogens

 Alcaligenes – opportunistic pathogen

83
 Pseudomonas:

 Small Gram-negative motile aerobic rods with a single polar

flagellum
– Monotrichous – single flagellum at one end, e.g.P. aeruginosa
– Lophotrichous – small bunches arising from one end of cell, e.g.P.
fluorescens
 Free living

– Primarily in soil, sea water, and fresh water and vegetation;

– also colonize human and animals intestine

 Frequent contaminants in homes and clinical settings

84
 Use aerobic respiration (oxidative);
 Produce oxidase and catalase
 Many produce water soluble pigments.

 Species of clinically important:

P. aeruginosa
P. pseudomallei

85
1. Oxidizing organisms:
 eg Pseudomonas species, produce
an acid reaction in the open tube only Covered Open
 Can breakdown carbohydrate only aerobically (anaerobic) (aerobic)

2. Fermenting organism:
 eg. Enterobacteriaaceae, produce acid reaction
throughout the medium in both tubes
 Can breakdown carbohydrate both aerobically
and anaerobically Covered Open
(anaerobic) (aerobic)
3. Non-fermenters:
 eg.Alcaligenes faecalis
 cannot breakdown carbohydrate aerobically or
anaerobically

Covered Open86
(anaerobic) (aerobic)
 Pseudomonas aeruginosa

The common name of the organism is Bacillus of green pus

Found in human and animal intestine, water, soil and moist environment in
hospitals.

Frequent contaminant of ventilators, IV solutions, anesthesia equipment

Primarily a nosocomial pathogen.

Opportunistic pathogen

Invasive and toxigenic, produces infections in patients with abnormal host defenses

Clinical features:
Pathogenic only when introduced into areas devoid of normal defenses
eg.
oBreached mucus membrane or skin,
ouse of IV line or urinary catheterization,
oneutropenia of any cause
87
 Responsible for abscess and wound infection.
 It cause mastitis in cattle,
 abortion in horse,
 otitis and dermatitis in dogs and
 pyogenic infections in humans

 Resistant to soaps, dyes, quaternary ammonium

disinfectants, drugs, drying

 Produce grapelike odor and greenish-blue pigment

(pyocyanin)
88
 Burkholderia

 Formerly the organisms under this genus were recognised

under genus pseudomonas but due to DNA homology
studies currently separated from this genus

 Burkholderia mallei (former namePseudomonas mallei )–

causative agent of glanders in equines

 B. pseudomallei (former nameP. peudomallei )– causes:

pseudoglanders in horses and

meloidosis (characterized by respiratory tract infection)

89
 Glanders:

 is an infectious disease that is caused by the bacterium

Burkholderia mallei .
 While people can get the disease, glanders is primarily a disease
affecting horses.
 It also affects donkeys
 The types of infection include:
o Localized, pulmonary (lung) infections,
o bloodstream infections, and
o chronic infections.
 The symptoms of glanders depend upon how the organism enters
the body.
 Common symptoms of glanders include:
o fever with chills and sweating,
o muscle aches,
o chest pain, muscle tightness, and headache.

90
91
 GenusBrucella

 The genusBrucella :
 is composed of gram negative coccobacilli, non-motile and
non-spore forming.
Brucella are intracellular parasites.

Cultural and staining characteristics

 Most are aerobic butB. abortus andB. ovis grow best in a

5-10% CO2--enriched environment.

 Growth on laboratory media is slow and often not visible till

48hrs of incubation at 37oC;

92
 It needs enriched media because they are fastidious.
 On Nutrient agar, colonies are:
 very small, raised, convex and translucent.

 Selective media for Brucella include:

– Serum Dextrose agar
– Treptose agar
– Dextrose glycerol potato media

 There is a stain known as Brucella differential staining or

Modified Ziehl Neelsen stain which involves staining with carbon
fuchsin, sulfuric acid (acetic acid) & methylene blue as a
counterstain.

 The organism appears pink against a blue background.

93
 Species of medical and veterinary importance include:

– TheBrucellae are generally associated with animal infections but most

are also pathogenic for humans.

– All human infections come from animals; Such diseases are called
"zoonoses".

– there is no human to human transmission.

–Brucella abortus (cattle),B. suis (pigs),

–B. melitensis (goats),B. ovis (sheep),
–B. canis (dogs)

 The organisms under this genus are also used as potential bio-weapon

 Disease conditions caused by the above species include:

 Abortion in females, Not in women WHY?
 Orchitis and epididymitis in males,
 Poll evil or fistullous wither in horse,
 Malta fever or undulant fever in human 94
95
 Gram- negative rods bacteria

Oxidase negative bacteria/ Oxidase positive

Entero-bacteriaceae bacteria

Lactose-Ferme  Pseudomoas
None-Lactose
nters Fermenters  Vibrio
 Campylobacter
 Escherichia spp.  Salmonella  Helicobacter
 Klebsiella spp. spp.
 Enterobacter  Shigella spp.
spp.  Proteus spp.
 Citrobacter spp.

96
 Enterobacteriaceae Family

Characteristics

 Named, as well coliforms or entero-bacilli (Enteric)

 Large family of small, non-spore-forming Gram-negative rods

 Many members inhabit soil, water, decaying matter, and

 Found as normal flora in intestinal tract of humans and

animals.

 Most frequent cause of diarrhea through enterotoxins

 Enterics, along withPseudomonas sp. , account for almost 50% of

nosocomial infections.
97
 Facultative anaerobes,

 All ferment glucose, reduce nitrates to nitrites,

 Oxidase negative, and catalase positive.

 Release endotoxin from their cell wall.

 Some release exotoxin

 They are divided into:

 Coliforms (lactose fermenters) and

 Non-coliforms (non-lactose fermenters)

 Enrichment, selective and differential media utilized for

screening samples for pathogens
98
99
100
101
102
 Antigenic Structures and Virulence Factors
 Complex surface antigens contribute to pathogenicity and
trigger immune response:
• H – flagellar Ag
• K – capsule and/or fimbrial Ag
• O – somatic or cell wall Ag – all have
• Endotoxin

103
 Escherichia coli : The most prevalent Enteric Bacillus

 Most common aerobic and non-fastidious bacterium in gut

 150 strains
 Most are motile; some are capsulated.
 Some have developed virulence through plasmid transfer, others
are opportunists.
 Some common diseases caused byE. coli include:
– Colibacillosis
– Bovine mastitis – Environmental type.
– Respiratory, genital and urinary tract infections.

 Treatment: Based on antibiotic sensitivity pattern

104
 Pathogenic Strains ofE. coli

 EnterotoxigenicE. coli
causes severe diarrhea due to heat-labile toxin and heat-
stable toxin – stimulate secretion and fluid loss;
also has fimbriae

 EnteroinvasiveE. coli
causes inflammatory disease of the large intestine.

 EnteropathogenicE. coli
linked to wasting form infantile diarrhea

 EnterohemorrhagicE. coli, O157:H7 strain,

causes hemorrhagic syndrome and kidney damage
105
Colibacillosis Bovine mastitis – Environmental
106type
 Other Coli-forms
Genus: Klebsiella

Characteristics:

 Non-motile, lactose-fermenting, capsulated, gram-negative rods.

 Clinically important mainly as opportunists

 Main species of medical importancce:

K. pneumoniae
K. rhinoscleromatis

 Klebsiella pneumoniae –

 normal inhabitant of respiratory tract,

 has large capsule,

107

 cause of nosocomial pneumonia, meningitis, bacteremia,

 Enterobacter sp. –

UTIs,

surgical wounds

 Citrobacter sp. –

 Opportunistic UTIs and

bacteremia

108
 Non-coliform Lactose-Negative Enterics

Proteus sp.

– organisms under this genus are characterized by

swarming type of growth (swarm on surface of moist
agar in a concentric pattern).

– They are motile by peritrichous flagella.

– Culture have a fishy odor

– involved in UTI, wound infections, pneumonia,

septicemia, and infant diarrhea

109
110
 Salmonella and Salmonellosis

GenusSalmonella

 The genus comprises a group of bacteria that consists of about

2500 serotypes in which some are host-specific and others are
non host-specific.

 Species:
– S. abortus bovis
– S. abortus equi they cause abortion in their respective
hosts
– S. abortus ovis
– S.typhi - cause typhoid in man
– S. paratyphi - Paratyphoid in man 111

– S. dubllin - salmonellosis in cattle

 – S. cholerae Suis – Septicaemia in pigs
– S. gallinarum - cause fowl typhoid in birds
– S. pullorum – cause Pullorum disease in birds
– S. Enteritidis andS. typhimurium –
cause Salmonellosis in domestic animals & man. Non host-specific

 Salmonellae possess 3 major antigens;

 the "H" or flagellar antigen,
 the "O" or somatic antigen and
 the "K" or capsular antigen.

 Most serotypes are motile (exception,S. gallinarum and S.

pullorum )

112
 Grow on Ordinary media, but best grow on Blood agar and
MacConkey agar at 37 C.o

 No haemolysis on Blood agar.

 On MacConkey agar:

o Colonies are pale because it is lactose non-fermentative →

Lactose-ve.
 Other Selective and Enriched media used to cultivate Salmonella include:
– Selenite Cystine (SC) Broth,

– Rappaport-Vassiliadis (RV) medium

– Tetrathionate(TT) Broth,

– BPLS Agar,

– XLD Agar,

– Salmonella Shigella (SS) agar

113
 GenusShigella

 Important species:
 S. dysenteriae,

 S. sonnei,

 S. flexneri and

 S. boydii

 The disease condition is collectively referred to as shigellosis

 S. dysenteriae causes bacillary dysentery

 Invades villus of large intestine, can perforate intestine or invade blood

114
 Oxidase-Positive Non-enteric Pathogens:
Pasteurella andHaemophilus
GenusPasteurella

 The namePasteurella was introduced by Pasteure after his work on

Fowl cholera.

 Pasturella are found as commensals in the URT of animals and man,

and cause disease when there is stress.

 P. multocida and P. hemolytica were the two main species responsible

for most human and animal infections.

– Pneumonic pasteurellosis(Shipping fever) – P.hemolytica

– Fowl cholera (Avian pasteurellosis) – P. multocida
– Haemorrhagic septicaemia– P. multocida
– Mastitis in ewes – P. hemolytica
115
 Genetic analysis ofPasteurella organisms has resulted in a new
classification for several strains of the organism commonly
involved in respiratory tract disease in cattle.
Mannheimia haemolytica (formerlyPasteurella haemolytica ) is the
new taxonomic classification, as suggested on the basis
of results from a study published in 1999 (Angen O, Mutters R,
Caugant DA, et al.Int J Syst Bacteriol 1999;49:67-86).
 Mannheimia haemolytica,
 Pasteurella trehalose and from pneumonic lungs of sheep and
 Pasteurella multocida goats.

Molecular characterization ofMannheimia haemolytica

116
Pneumonic Pasteurellosis

117
Pneumonic Pasteurellosis

118
GenusHaemophilus

 Tiny Gram-negative pleomorphic rods

 Fastidious,

 Sensitive to drying, temperature extremes, and disinfectants

 None can grow on blood agar without special techniques.

 They can grow best on chocolate agar (lysed blood cells) because
they require hemin, NAD or NADP

 Important species of veterinary importance is:

H. paragallinarum the causative agent of infectious coryza in
poultry.
119
120
 The Spirochetes

 the spirochetes are a large, heterogeneous group of spiral,

motile bacteria
 the family spirocheteacea consists of three genera of free-
living, large spiral organism

 the family Treponematacea includes three genera whose

members are human pathogen

 Gram negative human pathogens

– Treponema
– Leptospira
– Borrelia

121
GenusLeptospira :

 Tight, regular individual coils with a bend or hook at one or

both ends

 Species
L. biflexa – harmless, free-living saprobe

L. interrogans – causes leptospirosis, a zoonosis

 bacteria shed in urine;

 infection occurs by contact with contaminated urine;

 targets kidneys, liver, brain, eyes

122
123
 Curvi-form Gram Negative Bacteria

Three genera:

 Vibrio –
 Comma-shaped rods,
 Single polar flagellum
 Important species:V. cholera …………in humans

 Campylobacter –

 Slender, curved or spiral bacilli, often S-shaped or gull-winged pairs; one or more
flagella
 Most important:
• Campylobacter jejuni
• C. fetus

 Helicobacter –
 Spirochete with tight spirals and endo-flagella
 Important species:Helicobacter pylori - Gastric Pathogen
124
Medically Important Bacteria of Unique
Morphology and Biology

125
 Family Rickettsiaceae

 Contains about 23 species of pathogens, mainly in the

genusRickettsia

 Cause diseases – rickettsioses

 All are intracellular parasites requiring live cells for

cultivation.

 Spend part of their life cycle in arthropod vectors

 Rickettsioses are important emerging diseases.

126
 Rickettsia

 Obligate intracellular parasites

 Gram-negative cell wall
 among the smallest bacteria
 pleomorphic rods or coccobacilli
 Non-motile
 Ticks, fleas and lice are involved in their life cycle.
 Bacteria enter endothelial cells and cause necrosis of the
vascular lining:
 Vasculitis, vascular leakage and thrombosis.

 Treat with tetracycline and chloramphenicol.

127
 The Chlamydiaceae

 Obligate intracellular parasites

 Small, Gram-negative cell wall

 Alternate between 2 stages:

– elementary body –
• small metabolically inactive,
• extracellular,
• infectious form released by the infected host

– reticulate body –
• non-infectious,

• actively dividing form,

• grows within host cell vacuoles 128

Species:
 C. trachomatis: is responsible
 for human veneral (lymphogranuloma venereum),
 ocular (trachoma, inclusion conjunctivitis) and
 respiratory infections.
 In other words, oculo-urogenital infections

 C. psittaci produces systemic diseases including:

 psittacosis,
 ornithosis and
 pneumonitis in many mammals (including man) and birds129
 Molliculites and Other Cell-Wall-Deficient Bacteria
The mycoplasmas:
are essentially bacteria lacking a rigid cell wall during their entire life cycle,
although they are also much smaller than bacteria.

The first organism of this type was associated with pleuropneumonia of

cattle, and was originally called the pleuropneumonia organism (PPO).

Since that time, a number of organisms with similar morphological

characteristics and cultural properties have been isolated.

These are commonly referred to as pleuropneumonia-like organisms or

PPLO.

A certain group of mycoplasmas produce extremely tiny colonies on agar

plates, and are called the T-strains. 130
CBPP /CCPP

131
 Bacteria that have lost their cell walls

 Exposure to certain drugs or enzymes can result in cell wall-

deficient bacteria called

 L forms or L-phase.
– Induced or occur spontaneously

– May be involved in some chronic diseases

– L- phase variants of group A streptococci,

Proteus , andCorynebacterium ,

Mycobacterium paratuberculosis
132
 WOLLO UNIVERSITY
SCHOOL OF VETERINARY MEDICINE

Module: Veterinary Microbiology I

Year-III DVM

Mycology

Biruk Mekonnen (DVM, MSc)

WU-SVM
1
 Chapter 4- Veterinary Mycology
Outline of the Lecture

o General Mycology
✓ Introduction to Mycology
✓ Importance
✓ Nutrition and Metabolism
✓ Reproduction
✓ Structure and classification of fungi
o Systemic Mycology:
✓ Veterinary important fungi that can induce superficial and systemic
infection Mycosis (pathogenic and opportunistic) and
✓ Mycotoxicosis
2
 Objectives
➢ Upon the completion of this unit, the students will be
able to:
❖ Define and describe the general characteristics of fungi
❖ List the distinguishing characteristics of fungi from other
organisms like bacteria.
❖ Describe the importance of fungi: the beneficial and harmful
effects of fungi
❖ Explain the morphology, growth and nutrition of fungi.
❖ Describe the reproductive mechanisms of fungi
❖ Explain the major mycosis (disease caused fungi) and
Mycotoxicosis
3
 Introduction to Mycology: History of Mycology

➢ The term mycology is derived from the Greek words Mykes =

mushroom; Logos = study. Thus, Mycology is the study of
mushrooms.

➢ The mushrooms are among the largest fungi

➢ This terminology is applied for fungi for the first time in 1936
by M.J. Workei.

➢ Systemic study of fungi started after the invention of

microscope by Van Leeuulenhook in the 17th century

➢ Antonio Micheli, the Italian botanist is called the founder of the

science of mycology who, in 1729, published Nova Plantarum
Genera, in which his researches on fungi were included.
7/22/2022 4
 ……….History of Mycology
➢ Mycosis is any disease caused by a fungus
➢ A fungus is a member of a large group of eukaryotic organisms
such as Yeasts and Molds, as well as the more familiar
Mushrooms
❖ Fungi are commonly called yeasts, molds, mushrooms

➢ Why fungi classified under microbiology? Most of them are

macroscopic, CAN be visualized without the aid of
amplification

❖because of similarities in properties and techniques used to

study them,
❖ they are included in the discipline of microbiology
7/22/2022 5
 General Characteristics of Fungi
➢ Fungi are:
– Eukaryotic : it contains
✓membrane-bound nuclei with paired chromosome
✓membrane-bound organelles
– Spore producing organism
– Achlorophylous (luck chlorophyll) organisms with absorptive
nutrition [non-photosynthetic].
– Hence, they are heterotrophic organisms & found in a form of
tubular structures called hyphae
– Have filamentous, branched somatic structures, known as
hyphae which are surrounded by cell walls.
– Reproduce both sexually and asexually

7/22/2022 6
Heterotrophic organisms: saprobes and/or parasites

✓ Dependent on absorption of preformed organic carbon

compounds from their habitat for their nutrition that are
saprobes (hence their frequent association with decaying matter)
and/or parasites (utilizing living tissue).

✓ They secrete enzymes into the substratum and absorb the digested
compounds through their cell walls resulting in
o extracellular digestion and absorptive nutrition.

✓ Saprophytic: require a preformed organic source of carbon for

their nutrition (living on dead organic matter) and/or
✓ Parasitic: utilizing living tissue

7
➢ Most fungi are obligate aerobes (most molds); some are
facultative anaerobes (yeasts); but
➢ are not obligate anaerobes

➢ None of the pathogens are obligate intracellular

➢ Not motile, but few (e.g. Chytrids) have motile phase.

➢ Fungi generally like acidic pH - grow at lower pH of (pH- 5) than bacteria.

➢ Most fungi are multinucleated and multicellular organisms

➢ Fungal cell membrane has the ergosterol.

❖ Biosynthesis of ergosterol is inhibited by the azole antifungals.

➢ have a complex cell wall made up of chitin and glucan

❖ The antifungal compounds, polyoxins and echinocandins, inhibit the
biosynthesis of chitin and glucan, respectively.
8
 Similarities and differences of fungi with other cells
➢ The spores have the same purpose as the seeds which plants
produce & each one is capable of growing into a new
individual fungus under the right conditions.

➢ Fungi can’t make their food from sunlight, water & CO2 as
plants do, in the process called photosynthesis.
– because they lack the green pigment known as chlorophyll,
which plants use to capture light energy.

➢ Like animals, they must obtain their food from other organisms
– They may break down or ‘rot’ dead plants & animals – saprophytes.
– They may feed directly on living plants & animals – parasites
– They may associate with the roots of plants – Mycorrhizae

7/22/2022 9
➢ Since they have cell wall, fungi are more close to plants but they
are totally different from plants in that fungi are:
✓achlorophylous &
✓they store food in the form of glycogen but plants store in
the form of starch.

➢ Yeast cells are unicellular like bacteria

10
 Differences between Fungi and Bacteria
Characteristic Fungi Bacteria
Cell type Eukaryotic Prokaryotic
Cells nature Unicellular (yeast)/ Unicellular
multicellular (mold)
Sterol Present in yeast cell wall Absent except mycoplasma
Optimum pH 3.8-5.6 6.5-7.5
Optimum temperature 22-30oC (saprophytes),
30-37oC (parasites) 20-37oC (mesophiles)
Oxygen requirement Strictly aerobic (molds), Aerobic to anaerobic
facultative (some yeasts)
Light None Some photosynthetic groups occur
Sugar concentration High up to 4 - 5% 0.5-1%
in media
Carbon source Organic Inorganic and/or organic
Cell wall
structural components Chitin, cellulose, or hemicellulose Peptydogycan
Reproduction Sexual and asexual Binary fission or budding

Energy utilization Heterotrophy Either heterotrophy or autotrophy

7/22/2022 11
Importance of fungi
➢ Fungi are one of the most important group of organisms on this
planet.
➢ They are important in huge variety of ways:
1. Useful effects of fungi
– biodegradation & biodeterioriation (Recycling)
– industrial fermentation processes
– biochemical and Medicines
– Fungi as direct sources of food
– Mycorrhizae and plant growth
– Biocontrol
2. Harmful effects of fungi
– Animal and human Disease
– Food Spoilage
7/22/2022 12
 Fungi as agents of biodegradation & biodeterioriation
(Recycling):

➢ Biodegrade – to change back to a harmless natural state by

the action of bacteria & fungi
➢ Saprophytic fungi utilize dead organic materials as sources of
nutrients & are responsible for the biodegradation of
organic materials in our environment, particularly plant
materials in the form of leaf litter & other plant debris.
❖ Such fungi play a vital role in recycling essential elements,
particularly carbon
❖ Fungi, together with bacteria, are responsible for most of the
recycling which returns dead material to the soil in a
form in which it can be reused.

7/22/2022 13
 Involvement of fungi in industrial fermentation processes:

➢ Yeasts & mycelial fungi are used in a variety of industrial

fermentation processes:
– Saccharomyces cerevisiae are used extensively in brewing
beers & wines, as well as in bread making.
• Convert glucose sugar to ethyl alcohol & CO2.
• In the bakery, it is the small bubbles of CO2 during
fermentation that raise the bread
• In the brewery, it is the alcohol that is the important end
product.
➢ In the making of some dessert wines, grapes left in the field
purposely to become infected with Botrytis cinerea, the
“noble rot”, which enhance the sweetness of the grapes.

7/22/2022 14
 Use of fungi in the production of
commercial biochemical and Medicines

• E.g. Aspergillus niger is used in the large-scale commercial

production of citric acid.
• Penicillin is derived from a common fungus called
Penicillium (P. chrysogenum).
• Cephalosporins (3rd generation antibiotics) are produced by
Cephalosporium acremonium.

7/22/2022 15
Fungi as direct sources of food:

• Fungi are also important directly as food for humans.

✓ Many mushrooms are edible &
✓ different spp are cultivated for sale worldwide.

• Some yeasts & mycelial fungi are:

✓ cultured on a large scale & then
✓ undergo further processing to provide various protein rich
food products

7/22/2022 16
Mycorrhizae and plant growth:
• Fungi are virtually important for the good growth of most plants,
including crops, through the development of
Mycorrhizal associations (A mutually beneficial fungi
association with plant roots).

• As plants are at the base of most food chains, if their growth is

limited, all animal life, including human, would be
seriously reduced through starvation.
• Biocontrol:
• Fungi such as the Chinese caterpillar fungus, which parasitize
insects, can be extremely useful for controlling
insect pests of crops.
– The spores of the fungi are sprayed on the crop pests.

7/22/2022 17
 Harmful effects of fungi

Animal Disease:

• Fungi:
✓ can also parasitize domestic animals causing diseases, but

✓this is not usually a major economic problem.

✓A wide range of fungi also live on/in humans, but

most coexist harmlessly.

7/22/2022 18
Food Spoilage:

• It has already been noted that fungi play a major role in

recycling organic material.

• Fungi which make our bread go mouldy are only recycling

organic matter

• Fungal damage can be responsible for large losses of

stored food, particularly food which contains any moisture.

• Dry grains can usually be stored successfully, but the

minute they become damp, moulds are likely to render
them inedible.

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 Nomenclature
• Carolus Linnaeus (1732): Swedish scientist, developed
system of classification – binomial nomenclature

• The nomenclature of fungi is governed by the International

Code for Botanical Nomenclature (ICBN).

• At least 6 kingdoms are now recognized:

– Eubacteria: True bacteria
– Archaebacteria: Ancient bacteria
– Protista: (Eucaryotic, Protozoa, e.g. Amoeba)
– Animalia
– Plantae
– Fungi (Eumycota)
20
 Nomenclature: Hierarchical Classification
• Kingdom Fungi

– Phylum Basidiomycota

– Class Basidiomycetes

▪ Order Agaricales

▪ Family Agaricaceae

▪ Genus Agaricus

• Species: Agaricus campestris

21
 Morphology of Typical Fungal Cell

7/22/2022 22
 Morphology of Fungal Cell

Fungal cell walls

• Polysaccharides: (bacteria: peptidoglycan)
– glucans (polymer of glucose) and chitin provide tensile strength;

– mannans (polymer of mannose) are on cell wall surface and

covalently linked to protein (mannoproteins).
• Mannoproteins:
✓are gel like components from matrix throughout the wall
and

✓are immunogenic and allerogenic.

7/22/2022 23
• The fungal cell wall:
– provides certain antigenic properties

– functions in the recognition of events from the environment

– is a dynamic structure that is subject to change and

modification at different stages in the life of a fungus

• Melanins—dark brown to black pigments which confer:

✓resistance to enzyme lysis,
✓mechanical strength and
✓protect cells from UV light, solar radiation and desiccation

7/22/2022 24
➢ Fungal cell membranes:

✓ Similar to other eukaryotic membranes;

✓except fungal cell membrane has ergosterol which is

target of many antifungals

➢ Ribosomes:

✓ 80S=40S+60S; except in mitochondria.

❖Fungi are usually unaffected by antibacterial agents WHY?

7/22/2022 25
Nucleus:
• The nuclei of most fungi are quite small.

• The hyphae of fungi almost invariably contain large

numbers of nuclei.

• In aseptate forms, nuclei generally appear to be distributed

randomly throughout the cytoplasm of hypha.

• In septate forms, individual hyphal compartments may routinely

contain one, two or many nuclei depending upon the
species & phase of life cycle examined

7/22/2022 26
➢ There are two basic morphological forms of fungi: hypha and
yeast

1. Hypha (mold form):

✓ is the basic element of filamentous fungi with a branched,
tubular structure,
✓ 2-10 μm in width, usually colorless threads
✓ grow in a mass as mycelium
✓ Mycelium: the web or mat-like structure of hyphae
❖Vegetative /substrate mycelium:
oPortion that obtains nutrients (specialized for nutrition)
❖Reproductive or Aerial mycelium:
o Portion connected with reproduction.
o develop above the nutrient medium
27
Mycelium showing Reproductive &
vegetative structures

Single Hyphae

28
29
Septa: Regular cross-walls formed in hyphae.
✓ Hyphae with septa are septate,
✓ those lacking septa are called aseptate or coenocytic.

7/22/2022 30
 Hyphae/ Molds

➢ are classified and identified partially on the basis of whether

the hyphae are sepatate or aseptate.

➢ Branching patterns and diameters of hyphae are also often

diagnostic.
➢ Sepatate hyphae
✓ The hyphae with cross walls is termed as septate
✓ It partitioned hyphae in to individual cellular compartments.

➢ In aseptate hyphae –
✓ the hyphae without cross-walls (septa) termed as aseptate
✓ no physical boundaries to distinguish individual cells in this hyphae.

7/22/2022 31
2. Yeasts form

✓ Single cells (5 to 8 µm in diameter) fungus with single nucleus

✓ Generally, yeast cell are larger than bacteria
✓ Vary considerably in size, and
✓ are commonly spherical to oval shaped, rarely form filaments.
✓ they possess most of the other eukaryotic organelles
✓ Reproduces either:
❖ Asexually - by budding and transverse division or
❖The pattern of budding is often diagnostic
❖ Sexually - through spore formation

✓ Colonies are usually characterized by a smooth surface

7/22/2022 32
Pseudohyphae (yeast-like) :

✓ Several elongated yeast cells connected together and resembling

true hyphae

33
➢ Dimorphism in fungi: is the condition in which certain fungi may
show the two forms of growth; i.e.
✓as mold at room temperature and
✓as a yeast or spherule at body temperature

➢ Many pathogenic fungi are dimorphic: have two forms

➢ May convert from a hyphal or a spore form to a yeast form (and

vice versa) depending on environmental
conditions in those fungi called the dimorphic fungi.

❖ In response to changes in various environmental factors

(nutrients, CO2 tension, oxidation-reduction potentials, temperature)
7/22/2022 34
(1) the yeast (Y) form:
✓ Found in the animal,
✓ parasitic (pathogenic) stage at 37 °C

(2) the mold or mycelial form (M)

✓ Found in the external environment
✓ Saprophytic stage
✓ at room temperature (25 °C)

35
 Morphologic characteristics of Dimorphic Fungi

7/22/2022 36
 Summary
Yeasts and Molds

➢ A mold (US) or mould (UK ) is a fungus that grows in the form of

multicellular filaments called hyphae (multicellular).

➢ In contrast, fungi that can adopt a single-celled growth habit are

called yeasts (unicellular)

➢ The network of these tubular branching hyphae, called a

mycelium, is considered a single organism.

• Yeasts (unicellular, budding)

• Molds (mycelial, spores)

• Dimorphs (both)
37
Fungal morphology – Yeast, hyphae

38
 Fungal growth, nutrition and metabolism
➢ Yeasts:

✓ growth curve similar to bacteria;

✓ generation times (GT) vary;

✓ some are rapid growers, such as Candida albicans (GT~45 min)

➢ Molds:
✓ Grow from apical tip;

✓ highly variable generation times;

❖Zygomycetes tend to be rapid;

❖ Dimorphic pathogens tend to be slow.

7/22/2022 39
Growth requirements

➢ Most are obligate aerobes,

– Most molds are aerobic

– Yeasts are facultative anaerobes

➢ they utilize glucose, require Fe, and a lot of water.

➢ growth at 37oC is a virulence factor.

➢ They require Acidic pH (5.0)

7/22/2022 40
Media: Medically important fungi are usually easy to grow, but
important points:
1. Tissue forms of dimorphic fungi may be difficult:
e.g., spherules of Coccidioides immitis,
yeast forms of Histoplasma capsulatum;
therefore must alert the lab as to these possibilities.
2. Fungal sporulation may require special conditions.
3. Sabouraud dextrose agar (SDA agar) is a common universal medium.
If chloramphenicol and cycloheximide are added, it will especially be good for:
a. dermatophytes, Candida albicans
c. primary systemic pathogens from contaminated specimens But not
for Cryptococcus neoformans, Candida spp. other than C. albicans,
or rapid growers
4. Blood agar or other non-selective media are ok for specimens from
sterile body sites.
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7/22/2022 42
7/22/2022 43
 Culture

Bird seed agar plate

Mucoid colonies of
showing the typical brown colour effect
C. neoformans on SDA. seen with C. neoformans.

7/22/2022 44
 Fungal Reproduction
➢ Many fungi have the ability to reproduce by asexual and
sexual means
– Anamorph= asexual stage
• Mitospore= spore formed via asexual reproduction
(mitosis), commonly called a conidium or
sporangiospore

– Teleomorph= sexual stage

• Meiospore=spore formed via sexual reproduction
(resulting from meiosis),
• type of spore varies by phylum

7/22/2022 45
➢ Asexual reproduction:
✓ Also called somatic or vegetative reproduction &
✓ doesn’t involve the union of nuclei, sex cells or sex organs

➢ It may be accomplished by
– fission of somatic cells yielding two similar daughter cells
– budding of somatic cells or spores, each bud (a small outgrowth of
the parent cell) developing into a new individual
– fragmentation or disjoining of the hyphal cells, each fragment
becoming a new organism; or
– spore formation.

➢ Asexual spores, whose function is to disseminate the species, are

produced in large numbers.
7/22/2022 46
Sexual reproduction:
➢ is carried out by fusion of the compatible nuclei of two parent
cells.
➢ The process of sexual reproduction has three stages
– Plasmogamy - the joining of two cells and fusion of their
protoplasts
– Karyogamy - the two haploid nuclei of mating cells fuse
together to form a diploid nucleus.
– Meiosis - which again reduces the number of chromosomes
to the haploid number.

7/22/2022 47
➢ The sex organelles of fungi,

✓ if they are present, are called gametangia (singular, gametangium).

✓ They may form differentiated sex cells (gametes)

✓ If the male and female gametangia are morphologically

different,

❖ the male gametangium is called the antheridium (plural, antheridia)

and

❖ the female gametangium is called the oogonium (plural, oogonia).

7/22/2022 48
 Generalized Life Cycle of a Fungus

• x

49
 Reproduction

Life cycle of the zygomycete Rhizopus 50

 Classification of fungi
➢ Broadly fungi are classified into two categories:

✓ Eumycota (true fungi) and

✓ Pseudomycetes (false fungi).

➢ Eumycota are again classified into four major phyla or divisions.

7/22/2022 51
 Classification of fungi
➢ Fungus classified in to five categories based on (reproductive structure)
o spore types
o morphology of hyphae,
o sexual cycles.

➢ These groups are:

1. Oomycetes,
2. Zygomycetes,
3. Ascomycetes,
4. Basidiomycetes
5. Deuteromycetes.

➢ Except for the deuteromycetes, all fungi produce sexual spores

❖Most fungi reproduce by sexual sporulation.

➢ Asexual spores that are generally responsible for initiating animal/

human infection.
52
1. Oomycetes (water mold)
✓ Characterized by:

✓ the production asexual spore (zoospores)

✓ Many are unicellular; others are composed of

asptate hyphae (single-celled filament).

✓ Cell wall is not chitin rather cellulose

✓ Water molds (saprobes or parasite of fish) and downy mildews (plant

parasite- potato blight)

✓ Eg. Phytophthora infestans- it is the etiologic agent of potato blight.

53
2. Zygomycetes
✓ Characterized by unenclosed zygospores produced at ends of hyphae

✓ aseptate hyphae

✓ asexual sporangiospores, and

✓ sexual zygospores.

✓ are usually harmless to:

❖ humans and animals

✓ For example, Rhizopus nigricans

(black bread mold)

54
3. Ascomycetes (sac fungi)
✓ Includes both yeasts and filamentous fungi
✓ the hyphae is septate
✓ Ascospores are enclosed in asci (sac-like structures) at the ends of
hyphae or yeasts
✓ Asexual spore are called conidia born on conidiophore

55
They include fungi as:

Genus Saccharomyces

✓ the yeasts
✓ beneficial to man, that leaven bread and ferment alcoholic
beverages.

Pencillum notatum
✓ mold that used to produces the antibiotic penicillin.

A mold Aspergillus
✓ common microbial contaminant
✓ causes a fatal pulmonary disease (Aspergillosis) in people with
lowered resistance.

Claviceps purprea (ergot fungus)

✓ Produces a potent toxin that causes spasm of the smooth muscle.

56
4. Basidiomycetes
✓ Septate hyphae

✓ Basidiospores are produced on a club-shaped structure called a basidium

✓ produce exogenous sexual basidiospores on a basiduim

✓ Example: Mushrooms,

57
5. Deuteromycetes (“Imperfect fungi”)
✓ No sexual stage is known for these fungi

✓ Since sex is either non existent or undiscovered in these organisms,

they are commonly called the imperfect fungi.

✓ Many parasitic fungi fall into this class

• Examples: Candida, Epidermophyton

58
Summary

59
7/22/2022 60
 Importance of fungi
Beneficial & Harmful Effects of Fungi
Beneficial Effects

➢ Fungi (saprobic) serve essential roles in the global ecosystem.

➢ Fungi have versatile biochemistry and produce myriads of chemical

compounds. (Used as medicines and other industrial chemicals).

1. Important part of food chain (Biological roles):

✓ Involved in decomposition (decomposers) and nutrient recycling (recyclers)
❖ Recycle nutrients back into the environment (sewage treatment plants)

2. Biosynthetic factories –
✓ The fermentation property is used for the industrial production of alcohol,
fats, citric, oxalic & gluconic acids

✓ Yeast is used in the production of wine, beer, bread, etc

61
 Harmful Effects of Fungi

1. they parasitize human, animal and plants,

✓ Human & animal diseases including allergies.

✓ Plant diseases: Over 70% of all plant diseases are caused by fungi

2. Toxins produced by poisonous fungi with food =Mycotoxicosis

3. Spoilage of agriculture/animal products

✓ Vegetables & cereals
✓ Dairy products

4. Damage the products:

✓ such as magnetic tapes and disks, glass lenses, marble and wax.
62
 Parasitic fungi
➢ Some fungi acquire nutrients by attacking live animals or plants.

➢ Of the 150,000 known species of fungi, about 50 have been

identified as primary human pathogens.

➢ The disorders are usually slowly progressing and range from

✓ very mild disease of the skin and hair that most people know
of as a ring worm to

✓ overwhelming invasion of the entire body

7/22/2022 63
 Fungi as animal parasite

➢ Animal/Human mycoses are caused by:

1. True / primary fungal pathogens

❖ they can invade and grow in a healthy, non-compromised host
2. Opportunistic pathogens

❖ they cause mycosis in immuno-compromized host

64
 Overview of Fungal disease

1. Fungal Allergies

❖ Fungal allergies are common;

❖ Molds grow on any damp organic surface, and spores are

constantly in the air.

7/22/2022 65
2. Mycotoxicosis

➢ Some fungi produce toxic substances that poison a person who

ingest them.

➢ These poisonous substance are collectively called mycotoxicosis

(myco= fungus, toxin= poison).

➢ Mycotoxcosis may result from ingestion of fungal contaminated

foods. Example – Poisonous mushroom

➢ Most fungal toxins are produced when the fungus grows in moist
environment at relatively high temperature.

7/22/2022 66
 Mycotoxicosis in Domestic animals
Disease Toxins Fungi or mold
Aflatoxicosis Aflatoxins Aspergillus flavus,
A. parasiticus
Ergotism Ergot alkaloids Claviceps purpurea
Estogenism and vulvo- Zearalenone Fusarium graminearum
vaginitis
Facial eczema Sporidesmins Pithomyces chartarum
Fescue foot Ergovaline Acremonium
coenophialum
Ochratoxicosis Ochratoxin, also Citrinin Aspergillus ochraceous,
Penicillium citrinum
Porcine pulmonary Fumonisin B1and Fusarium moniliforme and
oedema Fumonisin B2 F.proliferatum
Dicumarol Penicillium spp., Mucor
Sweet clover poisoning spp. and Asperillus spp.
 Mycotoxicosis

➢ In considering the effects of mycotoxins on animals, it is important

to distinguish between “mycotoxicosis” and “mycosis.”

➢ Mycotoxicosis:
✓is used to describe the action of mycotoxin(s) and

✓is frequently mediated through a number of organs, notably the

liver, kidney, lungs, nervous, endocrine, and immune
systems.

➢ Mycosis
✓refers to a generalized invasion of living tissue(s) by growing
fungi.
✓Example: Histoplasmosis…. Invade the lymphatic tissue
69
➢ Mycotoxin:

✓are toxins produced by some fungi (many fungi don't produce mycotoxins)

✓ Metabolic products or by-products of fungi (molds)

✓ are contaminants of agricultural commodities, foods, and feeds.

✓Contamination occurs through spores contaminating the grain.

✓Fungi that produce mycotoxins both :

❖Prior to harvest (Fusarium) and

❖during storage. (most common storage fungi are species

of Aspergillus and Penicillium.
70
➢ Storage conditions that favor production of mycotoxins:
✓ Temperature (40 - 90o F ; 4 - 32o C)

✓ Relative Humidity (> 70%)

✓ Moisture (22-23% in grain)

✓ Oxygen (1-2%)

➢ The toxicity of the mycotoxins varies considerably:

✓The type of toxin,

✓the animal species exposed to it,
✓ the extent of exposure,
✓ the age, (young Vs adult) and
✓Nutritional status.
71
Why Great Concern?
• Some mycotoxins are DEADLY at very small dosages.

• Some mycotoxins are carcinogenic.

• Some mycotoxins cause huge losses in productivity in animals.

72
AFLATOXIN

➢ is probably the worst common mycotoxin

➢ Produced by:
✓ Aspergillus spp. - especially A. flavus, so quite common in peanuts
✓ A. parasiticus and
✓ Penicillium puberulum.

➢ Can be deadly at low dosages

– In 1st outbreaks (~1960) 100,000 turkeys died + many ducks.

➢ Aflatoxin Effects:
❖ Inhibits protein synthesis
❖ Liver damage/Hepatic toxin
❖ Susceptibility to Infection
❖ Residues/Carcinogenic in chronic situations.
73
74
➢ Aflatoxin levels in cow milk and feed in the Addis Ababa milk shed

✓ a survey on aflatoxins in cow’s milk and dairy cattle feed in the

Addis Ababa area.

✓ The results showed levels of aflatoxin in some of the milk samples

significantly higher than that allowed by European Union (EU) and
USA standards (ILRI, 2015)

75
 Mycotoxins Produced by Other Fungi

1.Fumonison
✓ Produced by Fusarium moniliforme
✓ Deadly to horses
• equine leukoencephalomalacia
✓ Swine - pulmonary oedema
✓ Renal toxicity and hepatotoxic

2. Ergot
✓ Produced by claviceps africana, Claviceps purpurea
✓ produces ergotamine and other alkaloids.
✓ Psychoactive - convulsions, hallucinations, abortions
✓ Paralysis, GI disturbance, gangrene of extremities,
death. Sorghum Ergot 76
 Treatment and prevention
• Treatment of mycotoxin-induced disease

a. For most mycotoxins, there is no specific treatment or antidote

b. Supplement with vitamins & selenium may be helpful, and provision of

adequate high-quality protein

• Preventing Mycotoxins
1. Use “clean” procedures.
2. Prevent contamination
3. Inhibit mold growth
✓ Drying
✓ Refrigeration
✓ Mold inhibitors 77
3. Fungal infection
➢ Fungi cause some of the most persistent and disfiguring disease
still prevalent through out the world

➢ Diseases caused by fungi are collectively called mycoses

(singular, mycosis).

➢ The incidence of the disease (infection) is related to:

✓the degree of exposure to fungi in living conditions,
✓occupation,
✓to immune status.
➢ There are four general categories on the basis of the primary
tissue affinity of the pathogen.
7/22/2022 78
 Factors predisposing to fungal infections

Susceptibility to fungi
Factors predisposing to fungal infections

1. Antibiotic for long duration, especially broad spectrum

2. Burns & other conditions affecting skin: trauma, eczema, ulcers etc.

3. Catheter: Indwelling catheters

– Catheters (peripheral/ central/ urethral) - disrupt the non-specific
defenses (mechanical barrier).

79
 Factors predisposing to fungal infections

4. Prolonged treatment with corticosteroids.

5. Immunosuppressive drugs or Irradiation therapy

6. Surgical & other procedures: Transplants

7. Debilitating conditions of the host (HIV infection, cancer,

diabetes, etc).

8. Drug addiction

9. Age: old age

80
1. Superficial mycoses
➢ Are fungal infections confined to the outer most dead
layers of skin, hair and nails
➢ symptoms - discoloration, scaling, or de-pigmentation of the skin.

➢ Do not elicit a cellular response from the host.

➢ Infections are generally painless.

7/22/2022 81
2. Cutaneous Mycoses

➢ Restricted to keratinized layer of the skin (epidermis), hair, or

nails.

➢ Elicit an inflammatory response, resulting in:

✓ more serious skin diseases with uncomfortable painful and
✓ sometimes even crippling symptoms.
➢ Symptoms - include

✓Itching, Scaling or ring like patches of the skin;

✓Brittle or broken hairs;

✓Thick discolored nails.

7/22/2022 82
 Fungi in Cutaneous Mycoses

Trichophyton Microsporum Epidermophyton

Hair Hair Skin

-
Skin Skin

Nails - Nails
Clinical Manifestations: Tinea = ringworm
Ringworm infection in cattle

85
Cutaneous lesions on the face of a horse
suffering of epizootic lymphangitis
 3. Subcutaneous mycoses

➢ Are infections confined to :

❖the subcutaneous tissue

❖ the deeper layer of the skin

➢ Symptoms include:
❖ Ulcers, that progress and do not heal and the
presence of draining sinus tracts.

7/22/2022 87
4. Systemic mycoses

➢ Are caused by pathogenic and/or opportunistic fungi affect

internal organs or tissues of the body.
➢ Symptoms
• very general as fever, and fatigue;
• a chronic cough and chest pain.

➢ Are infections caused by a group of fungi which cause infections

in patients who are immunocompromised

7/22/2022 88
7/22/2022 89
 Primary pathogen

1. Histoplasmosis/Epizootic Lymphangitis

Etiologic agent: Histoplasma capsulatum

Host: Animal and human
Characteristics:
✓ is a granulomatous disease
✓ it forms intracellular mycosis
✓ is a dimorphic fungus:
• The tissue form appears small oval yeast cells in phagocytic cells,
• The mycelial form shows:
❖small microconidia &
❖the characteristic large, round macrocondidia with finger-like
projections, tuberculate conidia.

90
 tissue forms of small oval yeast cells
in phagocytic cells
Lesions of cutaneous EL on the horse from 91
which HCF was isolated
2. Blastomycosis.

Etiologic agent: Blastomyces dermatitidis

Host: most cases dog and human and rarely horse, cat
Characteristics:
• is a chronic disease characterized by formation of granulomatous and
supportive nodules
• is a dimorphic fungus (mold at 25 oC, and yeast at 37oC)
• The yeast phase appears as:
❖ large, round thick walled yeasts that show a single bud

• The mycelial or filamentous form appears as:

❖branching hyphae bearing round conidia.

92
3. Coccidioidomycosis

Etiologic agent: Coccidioides immitis

Host: dog, horse, cattle, swine, sheep ,cat and human
Characteristics:
• Mainly pulmonary infection
• is a dimorphic fungus (mold at 25 oC, and yeast at 37oC)

a. The yeast form looks as spherules & endospores

a. The filamentous form appears as hyphae/arthrospores

93
94
95
 Pathogen with intermediate virulence
1. Sporotrichosis

Etiologic agent: Sporothrix schenkii

Host: human, and some animals
Characteristics:
• It is a chronic, subcoutaneous granulomatous, generally self-
limited disease of the lymphatic vessels and lymph nodes.

• is a dimorphic fungus (mold at 25 oC, and yeast at 37oC)

• It is a small (3-5um) cigar shaped yeast in tissue and

• a slow-growing, grey colony in laboratory culture.

96
Cigar bodies
97
2. Dermatophytoses (ringworm) (keratinophilic fungi)

Etiologic agent: Genera of Dermatophytes

Host: human, and some animals
Characteristics:
• It affect the keratinized tissue (epidermis) such as hair, nails & skin

• causing an infection that does not spread into subcutaneous or deeper tissues.

• Dermatophytes are among the few fungi causing communicable diseases

• Dermatophytes, produce extracellular enzymes (keratinases) which are

capable of hydrolyzing keratin.

• The infection extends radially to form a circular lesion called ring worm,

• Most are characterized by the presence of clear (hyaline), septate hyphae

which is 5-6 m in diameter.

98
 CUTANEOUS MYCOSES

Dermatophytes

Dermatophyte Skin Hair Nails

Microsporum X X
Trichophyton X X X

Epidermophyton X X

99
 Opportunistic Mycoses

➢ Opportunistic fungal pathogens have little or no virulence;

host defenses must be impaired.

➢ Opportunistic mycoses are caused by fungi that ordinarily are

not considered pathogenic for animals/humans

➢ These organisms are common in all environments.

➢ They are monomorphic.

➢ Some genera of opportunistic fungi that cause systemic
mycoses include:
❖Candida, Cryptococcus,
❖Aspergillus, Pneumocystis, and Mucor. 100
1. Candidiasis

Etiologic agent: C. albicans

Host: human, cattle swine, horse. poultry, dog ,cat
Characteristics:
• Yeast-like ovoid fungus, 4-6 µm in diameter,

• facultative anaerobes

• is not a dimorphic fungus, as both yeast & hyphae are seen in

tissue.

• It grows as round to oval yeast-like cells with Pseudohyphae

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 Candidiasis

Candida albicans

102
2. Aspergillosis:

Etiologic agent: A. fumigatus is the major species.

Host: cattle swine, horse. poultry, dog ,cat, human,
Characteristics:
• causes aflatoxicosis
• Growth is rapid (1-3 days) and colonies are flat

Typical conidiophore of Aspergillus

A. Conidia B. Stesrigma C. Uesicle
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D. Conidiophore
• as

104
3. Cryptococcosis

Etiologic agent: Cryptococcus neoformans:

Host: human, cattle , horse. dog ,cat
Characteristics:
• is a subacute, or chronic infection
• frequently involves the central nervous system, respiratory system and
the eye
• facultative intracellular pathogen,

• It is characterized by feature of a thick capsule both in culture & tissue fluid.

• A true yeast, round 4 –10 µm,

• surrounded by mucopolysaccharide capsule.

105
 Cryptococcosis

Cryptococcus with India ink Cryotocaccus neoformans a

budding cell: (a).Capsule, (b) Yeast
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 Mycotic Abortion

• Common causes of abortion in cattle

• Usually sporadic, but can reach 10% in a herd
• Most common genera:
✓Aspergillus,Absidia,
✓Rhizopus, Mucor, and
✓others moulds
• Dams healthy

• Placentitis is consistent, pneumonia in about half of foetuses

• Occasionally skin lesions are present on the foetus

• No treatment is known but recovery of the dam appears to be

spontaneous
Mycottiic aborttiion, fetal dermattiittiis
Normal condition
Mycotic abortion, placentaitis
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 Antifungal agents: General considerations:

1. Antibacterial agents generally do not affect fungal growth.

2. The number of antifungal agents are limited, but options are increasing.

3. Antifungal agents have limited spectrum of activity, thus need to

know the etiologic agent.

4. It is difficult to treat fungal infections. Why?

5. Emergence of resistance and choices of antifungal therapy makes

microbiology knowledge desirable.

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Types of antifungal agents
1. Polyene macrolides: amphotericin B and nystatin (such as
Mycostatin)
Mechanism of Action (MA): Preferential affinity for
ergosterol in cell membrane; fungicidal
2. 5-fluorocytosine (5-FC): flucytosine
MA: Converted to 5-FU, fungus makes bogus RNA.
3. Azoles: ketoconazole (oral or topical); itraconazole (oral);
fluconazole (oral or i.v.)
MA: Blocks ergosterol synthesis; Fungistatic

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4. Griseofulvin
MA: Inhibits mitosis
5. New agents: Voriconazole:
✓ Caspofungin (Cancidas): inhibits β-1,3 glucan synthesis

✓ Terbinafine (Lamisil): inhibits ergosterol synthesis:

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 Antifungal Agents’ mode of actions

Sites of action of some Antifungal chemotherapeutic Agents 116

 Look how they help each other.
Why the one stands to support others does not refuse?
Think, Help others, and sacrifice your life to others-----

117