Bacteria & Fungi

Bacterial classification depends on the following characteristics.

1. Morphology and arrangement
2. Staining
3. Cultural characteristics
4. Biochemical reactions
Fungi
1. Definition of fungi
2. Structure of fungal cell
3. Fungal specie identification
4. Fungal diseases and causative agent
5. Examples of economically important fungi
Morphology of bacteria
Bacterial cells are between 0.3 and 5µm in size.
They have three basic forms: cocci, straight rods, and curved or spiral
rods.
When bacteria are visualized under light microscope, the following
morphology are seen.
1. Cocci (singular coccus): Round or oval bacteria measuring
about 0.5-1.0µm in diameter. They are found in single, pairs,
chains or clusters.
Morphology of bacteria
2. Bacilli (singular bacillus): Stick-like bacteria with rounded, tapered,
square or swollen ends; with a size measuring 1-10μm in length by 0.3-
1.0μm in width.
3. Coccobacilli (singular coccobacillus): Short rods.
4. Spiral: Spiral shaped bacteria with regular or irregular distance
between twisting.
Morphology of bacteria
Morphology of bacteria
Staining of bacteria
Bacterial staining is the process of coloring colorless bacterial
structural components using stains (dyes).

The principle of staining is to identify microorganisms selectively

by using dyes, fluorescence and radioisotope emission.

Staining reactions are made possible because of the physical

phenomena of capillary osmosis, solubility, adsorption, and
absorption of stains or dyes by cells of microorganisms.
Staining of bacteria
• Individual variation in the cell wall constituents among different
groups of bacteria will consequently produce variations in colors
during microscopic examination.
• Nucleus is acidic in character and hence, it has greater affinity
for basic dyes.
• Whereas, cytoplasm is basic in character and has greater
affinity for acidic dyes.
Factors controlling selectivity of microbial cells
These factors include
1. Number and affinity of binding sites
2. Rate of reagent uptake
3. Rate of reaction
4. Rate of reagent loss (differentiation or regressive staining)
Factors controlling selectivity of microbial cells

Dyes absorb radiation energy in visible region of electromagnetic

spectrum i.e., “light”(wave length 400-650).
Absorption of anything outside this range is colorless.
Types of staining methods

1. Simple staining method

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

Procedure:
• Make a smear and label it.
• Allow the smear to dry in air.
• Fix the smear over a flame.
• Apply a few drops of positive simple stain like 1% methylene blue,
1% carbol fuchsin or 1% gentian violet for 1 minute.
• Wash off the stain with water.
• Air-dry and examine under the oil immersion objective.

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

bacteria remain unstained. Eg. Indian ink stain, Negrosin stain
Differential staining method

Multiple stains are used in differential staining method to

distinguish different cell structures and/or cell types. Eg. Gram
stain and Ziehl Neelson stain
A. Gram staining method
Developed by Christian Gram.
Most bacteria are differentiated by their gram reaction due to
differences in their cell wall structure.
Gram-positive bacteria are bacteria that stain purple with crystal
violet after decolorizing with acetone-alcohol.
Differential staining method

Gram-negative bacteria are bacteria that stain pink with the

counter stain (safranin) after losing the primary stain (crystal
violet) when treated with acetone-alcohol.
Required reagents:
• Crystal violet
• Gram’s Iodine
• Acetone-Alcohol
• Safranin
Gram staining method

Procedure:
1. Prepare the smear from the culture or from the specimen.
2. Allow the smear to air-dry completely.
3. Rapidly pass the slide (smear upper most) three times through
the flame.
4. Cover the fixed smear with crystal violet for 1 minute and wash
with distilled water.
5. Tip off the water and cover the smear with gram’s iodine for 1
minute.
Gram staining procedure cont…
6. Wash off the iodine with clean water.
7. Decolorize rapidly with acetone-alcohol for 30 seconds.
8. Wash off the acetone-alcohol with clean water.
9. Cover the smear with safranin for 1 minute.
10. Wash off the stain wipe the back of the slide. Let the smear to
air-dry.
11. Examine the smear with oil immersion objective to look for
bacteria.
Interpretation:
. Gram-positive bacterium ……………Purple
. Gram-negative bacterium …………..Pink
What are fungi?

Yeasts, such as brewers’ yeast, and moulds, such Penicillium

chrysogenum which produces the antibiotic penicillin, are classified as
fungi. Yeast cells tend to grow as single cells that reproduce asexually in
a process known as budding, although a minority of species (e.g.
Schizosaccharomyces pombe) reproduce by fission. Many yeast species
are capable of sexual reproduction and the formation of spores. In
contrast, moulds grow as masses of overlapping and interlinking hyphal
filaments and reproduce by producing masses of spores in a variety of
structures. This division between yeasts and moulds based on growth
morphology is not clear-cut, as some yeasts can produce hyphae under
specific conditions (e.g. Candida albicans), while many normally
filamentous fungi possess a yeast-like phase at some point in their life
cycle.
What are fungi?

Fungi are eukaryotic organisms, i.e. their cells possess a nuclear

membrane, consequently there are many similarities between the
biochemistry of fungal cells and human cells.
Fungi are widely distributed in nature, occurring as part of the normal
flora on the body of warm-blooded animals, as decomposers of organic
matter and as animal and plant pathogens.
Medically, fungi are an extremely important group of microbes, being
responsible for a number of potentially fatal diseases in humans, but a
significant number of fungi are of great benefit to humanity in terms of
the production of alcoholic beverages, bread and antibiotics. Fungi have
also been utilized for a range of molecular biological applications.
What are fungi?

From a taxonomic point of view the Kingdom fungi can be subdivided

into six Classes. The Class Oomycetes contains the mildews and water
moulds, the Class Ascomycetes contains the mildews, some moulds and
most yeast species (including Saccharomyces cerevisiae), the Class
Basidiomycetes contains the mushrooms and bracket fungi, the Class
Teliomycetes contains the rust fungi (plant pathogens), the Class
Ustomycetes contains the smuts (plant pathogens) and the Class
Deuteromycetes contains species such as Aspergillus, Fusarium and
Penicillium.
The structure of the fungal cell……………

The typical yeast cell is oval in shape and is surrounded by a rigid

cell wall, which contains a number of structural polysaccharides
and may account for up to 25% of the dry weight of the cell wall
Glucan accounts for 50–60%, mannan for15–23% and chitin for
1–9% of the dry weight of the wall, respectively, with protein and
lipids also present in smaller amounts.
The thickness of the cell wall may vary during the life of the cell
but the average thickness in the yeast C. albicans varies from 100
to 300 nm.
The structure of the fungal cell………….

Glucan, the main structural component of the fungal cell wall, is a

branched polymer of glucose which exists in three forms in the
cell, i.e. b-1,6-glucan, b-1,3-glucan and b-1,3,-b-1,6 complexed
with chitin.
Mannan is a polymer of the sugar mannose and is found in the
outer layers of the cell wall.
The third principal structural component, chitin, is concentrated in
bud scars which are areas of the cell from which a bud has
detached.
The structure of the fungal cell………….

Proteins and lipids are also present in the cell wall and under
some conditions may represent up to 30% of the cell wall
contents. Mannoproteins form a fibrillar layer that radiates from
an internal skeletal layer, which is formed by the polysaccharide
component of the cell wall.
The innermost layer is rich in glucan and chitin, which provides
rigidity to the wall and is important in regulating cell division.
Enzymatic or mechanical removal of the cell wall leaves an
osmotically fragile protoplast that will burst if not maintained in an
osmotically stabilized environment
The structure of the fungal cell………….

Incubation of protoplasts in an osmotically stabilized agar growth

medium will allow the re-synthesis of the cell wall and the
resumption of normal cellular functions.
The periplasmic space is a thin region that lies directly below the
cell wall. It contains secreted proteins that do not penetrate the
cell wall and is the location for a number of enzymes required for
processing nutrients before entry into the cell.
The structure of the fungal cell……………

The cell membrane or plasmalemma is located directly below the

periplasmic space and is a phospholipid bilayer that contains
phospholipids, lipids, protein and sterols.
The plasmalemma is approximately 10nm thick and in addition to
being composed of phospholipids also contains globular proteins.
The dominant sterol in fungal cell membranes is ergosterol,
which is the target of the antifungal agent amphotericin B.
Sterols are important components of the plasmalemma and
represent regions of rigidity in the fluidity provided by the
phospholipid bilayer.
The structure of the fungal cell……………

Most of the cell’s genome is concentrated in the nucleus, which is

surrounded by a nuclear membrane that contains pores to allow
communication with the rest of the cell.
The nucleus is a discrete organelle and in addition to being the
repository of the DNA, also contains proteins in the form of
histones.
Yeast chromosomes vary in size from 0.2 to 6 Mb and the number
per yeast is also variable, with S. cerevisiae having as many as
16 while the fission yeast Sch. pombe has as few as 3
The structure of the fungal cell…………..

In addition to the genetic material in the nucleus the yeast cell

often has extra chromosomal information in the form of plasmids.
For example, the 2-mm plasmid is present in S. cerevisiae,
although its function is unclear, and killer plasmids in the yeast
Kluyveromyces lactis which encode a toxin.
Actively respiring fungal cells possess a distinct mitochondrion,
which has been described as the ‘power-house’ of the cell.
The enzymes of the tricarboxylic acid cycle (Kreb’s cycle) are
located in the matrix of the mitochondrion, while electron transport
and oxidative phosphorylation occur in the mitochondrial inner
membrane. The outer membrane contains enzymes involved in
lipid biosynthesis.
The structure of the fungal cell…………….

The mitochondrion is a semi-independent organelle as it

possesses its own DNA and is capable of producing its own
proteins in its own ribosomes, which are referred to as
mitoribosomes.
The fungal cell contains a vast number of ribosomes, which are
usually present in the form of polysomes (lines of ribosomes
strung together by a strand of mRNA). Ribosomes are the site of
protein biosynthesis.
The system that mediates the export of proteins from the cell
involves a number of membranous compartments including the
Golgi apparatus, the endoplasmic reticulum and the
plasmalemma.
The structure of the fungal cell……………

In addition, the vacuole is employed as a ‘storage space’ where

nutrients, hydrolytic enzymes or metabolic intermediates are
retained until required.
Diagrammatic representation of ‘typical’ yeast cell.
Fungal species identification methods

The first step in identifying a fungus in clinical samples is to

observe the tissue sample microscopically, often in conjunction
with specific staining procedures.
For instance, addition of potassium hydroxide to the specimen
facilitates any fungal elements present to be distinguished from
host structures.
Tissue samples routinely tested for fungi in the diagnostic
laboratory include blood, cerebrospinal fluid, bronchial aspirates
and biopsy material, all of which are usually sterile
Fungal species identification methods

While microscopic analysis of samples is certainly rapid and

inexpensive it is not very sensitive. Consequently, in the
diagnostic laboratory samples are routinely inoculated onto agar
plates that specifically allow the growth of fungi.
A wide variety of media have been developed for the selective
growth of fungal species. On these media filamentous fungi are
often identifiable to the species level based on their colony
morphology following macroscopic and microscopic analysis, with
many species having recognizable characteristic hyphal and
conidial structures.
Fungal species identification methods

As C. albicans and the recently discovered closely related

species C. dubliniensis can grow in the yeast or hyphal form, the
ability of these species to produce germ-tubes (i.e. hyphae) can
easily be tested in the laboratory by incubation of the cells in
serum.
Similarly, these species are also the only members of the genus
Candida to produce large spore-like structures called
chlamydospores on specific media. Identification of other
clinically important Candida species has been greatly facilitated by
the recent introduction of commercially available chromogenic
media, which allow the Candida species to be distinguished from
each other on the basis of colony colour
Fungal species identification methods

In addition, Candida species can also be identified on the basis of

their different nutrient (especially carbohydrate) requirements,
which can be assessed using commercial kits.
In addition, as in the case of direct microscopic analysis of
samples, the levels of fungal elements present in samples taken
are often very low and are not present at sufficiently high levels to
be detected using culture-based methods.
Therefore, in many cases the identity of the agent responsible for
causing a fungal infection is only determined once the infection
has been cured using empirical treatment. Indeed sometimes the
cause of disease is only identified at autopsy.
Fungal species identification methods

Clearly, to improve our ability to treat fungal diseases effectively

there is a great need to develop techniques that allow the
detection and identification of infecting fungal species more
quickly. Currently, two areas of research are endeavoring to
facilitate this goal.
Firstly, it has been proposed that rather than detecting the
infecting species it may be more appropriate to monitor the host
immune response to specific pathogens.
Fungal species identification methods

While both ELISA-based and PCR-based technologies have the

potential to revolutionize the diagnosis of fungal infections as they
are very rapid and accurate, they are still at the experimental
stage and are only in use in specialized laboratories.
Examples of fungal diseases and selected causative agents
Examples of economically important fungi