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Mr. Pradeep Bawane

Assistant Professor
SVKM’S Institute of Pharmacy, Dhule
Introduction to Bacteria
• Bacteria (singular: bacterium) are relatively simple, single-
celled (unicellular) organisms. Because their genetic material is
not enclosed in a special nuclear membrane, bacterial cells are
called prokaryotes (pro-kar'e-ots), from Greek words meaning
prenucleus. Prokaryotes include both bacteria and archaea.

•Size: 0.75-4 microns.

•cylindrical (rod-shaped)
• spherical (coccoid)
•a single twist (vibrios)
•many twists like a corkscrew (spirochaetes).

• On the Basis of Gram Staining

• On the basis of spores
• On the basis of capsule
• On the basis of flagella
• On the basis of acid fast staining
• On the basis of biochemical reaction
• On the basis of environment
• On the basis of shape
Figure : Bacterial shapes. Most bacteria are (a) rod shaped (bacillus), (b)
spherical (coci) or (c) curved (vibro).
These basic shapes may join to form (d) pairs (diplococci), (e) -chains and f-
chain like a string of beads (streptococci), (g) sheets (sarcinae), (h) packets
like a bunch of grapes (staphylococci) or (i) irregular aggregates.
Three fundamental divisions of the bacterial cell occur in
all species: cell wall, cell or cytoplasmic membrane, and
Cell wall
• The cell wall of the bacterial cell is a complex, semirigid structure responsible for
the shape of the cell.

• The major function of the cell wall is to prevent bacterial cells from rupturing when
the water pressure inside the cell is greater than that outside the cell.

• It also helps maintain the shape of a bacterium and serves as a point of anchorage for

• The bacterial cell wall is composed of a macromolecular network called

peplidoglycan (also known as murein or mucopeptide), which is present either alone
or in combination with other substances.
• Peptidoglycan consists of a repeating disaccharide attached by polypeptides to form
a lattice that surrounds and protects the entire cell. The disaccharide portion is made
up of monosaccharides called N-acetylglucosamine (NAG) and N-acetylmuramic
acid (NAM) (from murus, meaning wall), which are related to glucose.
• The various components of peptidoglycan are assembled in the cell wall (Figure

• Alternating NAM and NAG molecules are linked in rows of 10 to 65 sugars to

form a carbohydrate "backbone" ( the glycan portion of peptidoglycan) .

• Adjacent rows are linked by polypeplides (the peptide portion of peptidoglycan).

Although the structure of the polypeptide link varies, it always includes
tetrapeptide side chains, which consist of four amino acids attached to NAMs in
the backbone.

• The amino acids occur in an alternating pattern of D and L forms (see Figure 2.
13, page 43)
Structure of the Gram-positive Cell
The Gram-negative outer membrane(1)
The Gram stain procedure
Developed in 1884 by the Danish physician
Hans Christian Gram

An important tool in bacterial taxonomy,

distinguishing so-called Gram-positive
bacteria, which remain coloured after the
staining procedure, from Gram-negative
bacteria, which do not retain dye and need to
be counter-stained.

Can be applied to pure cultures of bacteria

or to clinical specimens

Top: Pure culture of E. coli

(Gram-negative rods)
Bottom: Neisseria gonorrhoeae in a smear of urethral pus
(Gram-negative cocci, with pus cells)
The Gram Stain

Crystal iodine

Decolorise with

appear purple
Counterstain with
e.g. methyl red Gram-negatives
appear pink
Gram-positive cocci Gram-positive rods

Gram-negative cocci Gram-negative rods

Anaerobic Anaerobic
Gram-positive cocci Gram-positive rods

Anaerobic Anaerobic
Gram-negative cocci Gram-negative rods
Gram staining

• What was the primary stain used in gram

• How you can distinguish b/w two organism
• Which decolorizer used
• Give any two examples of gram positive and
gram negative bacteria.
Gram-Negative Rods

• Enteric Bacteria
– E. coli
– Salmonella
– Shigella
– Yersinia
– Pseudomonas
– Proteus
– Vibrio cholerae
– Klebsiella pneumoniae
Gram-Negative Rods

• Fastidious GNRs
– Bordetella pertussis
– Haemophilus influenzae
– Campylobacter jejuni
– Helicobacter pylori
• Anaerobic GNRs
– Fusobacterium
Gram-Negative Cocci

• Neisseria gonorrhoeae
– The Gonococcus
• Neisseria meningitidis
– The Meningococcus
• Both Gram-negative
intracellular diplococci
Gram-positive Cocci

• Staphylococci
– Catalase-positive
– Gram-positive cocci in
• Staphylococcus aureus
– coagulase-positive
• Staph. epidermidis
– and other coagulase
negative staphylococci
Gram-Positive Cocci

• Streptococci
– Catalase-negative
– Gram-positive cocci in
chains or pairs
• Strep. pyogenes
• Strep. pneumoniae
• Viridans-type streps
• Enterococcus faecalis
Gram-Positive Rods

• Clostridia
– Anaerobes
– C. tetani
– C. botulinum
• Bacillus cereus
– Aerobe
Non-Gram-stainable bacteria

• Unusual gram-positives
• Spirochaetes
• Obligate intra-cellular bacteria
Unusual Gram-positives

• Mycoplasmas
– Smallest free-living
– No cell wall
– M. pneumonia, M.
• Mycobacteria
– Acid-fast bacilli, stained
by Ziehl-Neelsen stain
– M. tuberculosis
– M. leprae
– M. avium
1. flagella
Some rods and spiral form have this.
a). function: motility
b). origin : cell membrane flagella attach to the cell
by hook and basal body which consists of set(s) of rings
and rods
Gram - : 2 sets of ring and rods, L, P, S, M rings
and rods
e.g. E. coli
Gram + : S, M rings and rods
e.g. B. megaterium
Organ of bacterial locomotion
Structure of the flagellum
Flagella movement(1)
Flagella movement(2)
Flagella movement(3)
b).Origin (continued)
– The structure of the bacterial flagella allows it to spin
like a propeller and thereby propel the bacterial cell;
clockwise or counter clockwise ( Eucaryotic , wave
like motion.
– Bacterial flagella provides the bacterium with
mechanism for swimming toward or away from
chemical stimuli, a behavior is knows as
CHEMOTAXIX, chemosenors in the cell envelope
can detect certain chemicals and signal the flagella to
c). position




d). structure

protein in nature: subunit flagellin

2. Pili or Fimbriae
• Shorter than flagella and straighter , smaller.
• Only on some gram- bacteria.
a). function:
• adhere.
• One of the invasive mechanism on bacteria.
• Some pathogens cause diseases due to this. If
mutant (fimbriae) not virulent.
• Prevent phagocytosis.
pili - sex factor. If they make pili, they are + or donors
of F factor.

• It is necessary for bacterial conjugation resulting in

the transfer of DNA from one cell to another.

• It have been implicated in the ability of bacteria to

recognize specific receptor sites on the host cell

• In addition, number of bacteria virus infect only

those bacteria have F pilus.
b). Origin: Cell membrane

c). Position: common pili , numerous over the cell,

usually called fimbriae sex pile, 1-4/cell

d). Structure: composed of proteins which can be

dissociated into smaller unit

Pilin . It belongs to a class of protein Lectin which

bond to cell surface polysaccharide.
Cytoplasmic Membrane
• The bacterial cytoplasmic membrane is composed of a
phospholipid bilayer which is 5 to 10 nm in thickness and has all the
general functions of a cell membrane such as acting as a
permeability barrier for most molecules and serving as the location
for the transport of molecules into the cell.

• It acts as a semipermeable membrane controlling the inflow and

outflow of metabolites to and from the protoplasm.

• In this function, plasma membrane has selective permeability.

• (Note: Staining of bacteria depends upon the chemical and

physical nature of cytoplasmic membrane and cytoplasm.)
a. control permeability
b. transporte’s and protons for cellular metabolism
c. contain enzymes to synthesis and transport
cell wall substance and for metabolism
d. secret hydrolytic enzymes
e. regulate cell division.
• Fluid mosaic model. phospholipid bilayer and
protein (structure and enzymatic function). Similar
to eukaryotic cell membrane but some differs. e.g.
sterols such as cholesterol in Euk not in Prok.
The cytoplasmic membrane
Functions of
the cytoplasmic membrane(1)
Transport proteins
III. Cytoplasm
80% water, nucleic acids, proteins, carbohydrates, lipid and inorganic ions etc.
contains enzymes that generate ATP, enzymes involved in the
synthesis of peptidoglycan
1. Bacterial chromosomes
• a single large circular double stranded DNA no histone proteins.
• The only proteins associated with the bacterial chromosomes are the ones
for DNA replication, transcription etc.

2. Ribosome:
• Bacteria contain a group of ribosomes called polyribosomes present
in the cytoplasm of the cell. It is 70S type in bacterial cell having two
• the large unit is 50S and the smaller unit 30S. Ribosomes help in
protein synthesis.
Function: protein synthesis
The bacterial chromosome and
4. Mesosomes

A large invaginations of the plasma membrane,

irregular in shape.

a. increase in membrane surface, which may be

useful as a site for enzyme activity in respiration
and transport.

b. may participate in cell replication by serving as a

place of attachment for the bacterial chromosome.
4. Inclusions
Not separate by a membrane but distinct.
Granules of various kinds:
* glycogen,
*polyhydroxybutyric acid droplets (PHB)
i.e. fat droplets
* inorganic metaphosphate (metachromatic granules) - in
general, starvation of cell for almost any nutrients
leads to the formation of this to serve as an
intracellular phosphate reservoir.
IV. Special Structure

* Endospores
Spore former: sporobactobacilli and sporosarcinae - no
medical importance. bacillus and clostridium have
medical importance.

* Position: median, sub-terminal and terminal have

small water, high calcium content and dipicolinic acid
(calcium dipicolinate)
extremely resistant to heat, UV, chemicals etc. may be
due to many S containing A.A for disulfide groups.

• Bacterial diversity can be seen in terms of variation in

cell size and shape (morphology), adaptation to
environmental extremes, survival strategies and
metabolic capabilities.
• Such diversity allows bacteria to grow in a multiplicity
of environments ranging from hot (65°C) to deep
freezers (–20°C), from high (pH 1) to low (pH 13)
acidity and high (0.7 M) to low osmolarity (water).
• In addition, they can grow in both nutritionally rich
(compost) and nutritionally poor (distilled water)

• Thin spiral bacteria

• Viewable by phase-
contrast microscopy or
silver stain
– Leptospira
Obligate intracellular bacteria

• Rickettsia
• Coxiella burneti
• Chlamydias
– C. pneumoniae

• Importance of bacteria
• Nature of bacteria
• Classification of bacteria
– Gram-positive versus Gram-negative
– Rods and Cocci
– Aerobic versus anaerobic
Monochromatic staining

• Principle:
• Staining in single dye solution contains salts.
• Auxochrome and chromophore.
• Responsible for dissociation and colour resp.
• In basic dye chromophore is positively charge
and the cell have negative charge.
• Ex. Methylene blue, crystal violet etc.

• Smear preparation
• Crystal violet for 1 min
• Excess stain was washed with d. water
• Drying of slide
• Observe under oil immersion lens
• This is used to study morphological
• What do you mean by monochromatic
• What is the role of auxochrome and
• Which stain you have used?
• How chromophore will colour the cell.

• Bacteria may be either motile or non motile.

• Motility may be due to flagella or flexible
nature of organism.
• Cocci are generally non motile but they
exhibit Brownian motion.

• Loopful of bacterial culture was place on

centre of cover slip.
• A light layer of Vaseline applied around top
edges of slide.
• The slide was place inverted on cover slip
• The slide was inverted again to original
• Observe under high power.
• What do you mean by motile bacteria
• Which method can be used to determine
motility of bacteria.
• Give the ex. Of motile bacteria.
Negative staining
• Principle
• It involves staining of background and not the cell.
• Acidic dye are used which on ionization yields anion
having coloring power.
• The bacterial cell have slightly negative charge thus
acidic dye does not stain the cell but forms a deposit
around the cell.
• The cell appears light areas in dark field
• This is useful for microbes such as spirochetes which
can not stain by ordinary method.

• Loopful suspension on slide stain with equal

amount of nigrosin solution.
• With another slide a thin smear is made.
• The dye was allowed to dry.
• Observe under oil immersion lens
• What do you mean by negative staining.
• How background colour will form by acidic
• Which dye you have used for negative
Cell wall staining
• Principle
• Bacterial cell wall is strong rigid structure that support
the weaker biochemically more active parts.
• It measure about 10-25 micrometer.
• The main component of cell wall is peptidoglycan.
• Tannic acid is mordant to fix crystal violet.
• Congo red decolorized the protoplasm
• Cell wall once stain does not loose the stain.

• Smear preparation
• Add drop of tannic acid and warm gently
• Add 5 % solution of crystal violet (1 min)
• Wash the slide with water
• Add 5% congo red solution
• Dry and observe under oil immersion lens.
• What is the main component of bacterial cell
• Which mordant you have used in cell wall
• Which primary stain you have used in cell
wall staining.
• What was counter stain.
Preparing Specimens for Viewing – Differential Stains

m u s
Acid-Fast Stain
This stains the cells of the genera
Mycobacterium and Nocardia, which
cause many diseases in humans,
including tuberculosis, leprosy, and Create a smear of the organism that
you are testing. Cover the smear
other lung and skin infections. with a strip of blotting paper.

Saturate paper with Ziehl’s

carbolfuchsin and heat for 3 – 5
Cells of these bacteria have large amounts minutes. Remove blotting paper.
of waxy lipid in their cells walls, so the
Gram stain and other water-based stains Wash slide with tap water and then
don’t work well on them. decolorize the smear for 10 - 15 seconds
with acid alcohol. Rinse again with tap

Cells are determined to be acid-fast or not,

Apply crystal violet for 1 minute,
based on whether they retain their wash, blot dry.
primary stain after decolorization.

Blotting paper : Ziehls carbolfuchsin (3 – 5 min heat) : rinse: Acid Alcohol (10 – 15 sec) : rinse : crystal violet (1 min)
Differential Stains

Acid-Fast Stain
Distinguishes between two large groups of

Acid-fast (AF) and Nonacid-fast (NAF)

How do AF and NAF cells differ?

What was the primary stain?

Is there a mordant?

What was the decolorizer?

What was the counterstain?

Preparing Specimens for Viewing – Differential Stains

Endospore Stain
Some bacteria produce endospores, dormant, highly-
resistant cells inside the cytoplasm of the bacteria, that
can survive environmental extremes (desiccation, heat,
harmful chemicals)

Most notable genera: Bacillus and Clostridium

Endospores cannot be stained by normal staining procedures

because their walls are practically impermeable to all

Endospore stain uses heat to drive the primary stain,

malachite green into the endospore.

After cooling, the sample is decolorized with water and counter

stained with safranin.

Results in green stained endospores and red-colored

vegetative cells.
b w e

Malachite Green (5 min heat) : rinse : safrinin (20 sec)

Differential Stains

Endospore Stain

Distinguishes between what two


What was the primary stain?

Is there a mordant?

Is there a decolorizer?

What was the counterstain? E