BACTERIAL GROWTH CURVE

Lag phase – Period between inoculation and beginning of multiplication of bacteria.

After inoculating into a culture media, bacteria do not start multiplying immediately, but take
some time to build up enzymes and metabolites.
 Bacteria increase in size due to accumulation of enzymes and metabolites
 Bacteria reach maximum size at end of lag phase
 The duration of lag phase varies with species, size of inoculum, nature of culture medium
and temperature

Log phase/ Exponential phase – In this phase, bacteria divide exponentially so that the growth
curve takes shape of straight line. The bacterium is:
 Smaller in size
 Biochemically active
 Uniformly stained

Stationary Phase – cell division stops due to depletion of nutrients and accumulation of toxic
products.
 Number of progeny cells formed is just enough to replace the number of cells that die.
 Viable count stationary but total count keeps raising.
 Bacterium become gram variable.
 Sporulation occurs in this phase
 More storage granules formed.

Phase of Decline – population of bacteria decrease due to cell death.

 Cell death occurs due to nutritional exhaustion and toxic accumulation and even by
autolytic enzymes.
 Decline in viable count not total count.
 Involution forms seen.
 BACTERIAL FLAGELLA
 Organ of locomotion of bacteria
 Flagella are long, unbranched, filamentous appendages
 composed of protein flagellin
 arise at the level of the cell membrane and extend through the cell wall in the
surrounding medium.

Arrangement of Flagella
 Monotrichous – Single polar flagellum at one pole
Ex. Vibrio cholerae, Pseudomonas, Campylobacter
 Lopotrichous – Tuft of flagella at one pole
Ex. Spirillum
 Amphitrichous – Single polar flagellum at both poles
Ex. Alcaligenes fecalis
 Peritrichous – Flagella distributed over the entire cell surface
Ex. Salmonella typhi, Escherichia coli

Structure of Flagella
Composed of 3 parts
 Filament
 Composed of protein flagellin, arranged in parallel subfibrils
 Longest portion of flagella
 Filament is semi-rigid
 Extends from cell surface to tip
 Hook - Connects filament to the basal body
 Basal Body – composed of complex rings embedded in the cell
Gram Positive Bacteria Gram Negative Bacteria
1. Outer ring  Outer rings
P-peptidoglycan layer 1) L - lipopolysaccharide
outer membrane
2) P - peptidoglycan layer

2. Inner ring  Inner rings

M-plasma membrane 3) M - plasma membrane
4) S - periplasmic space
 BACTERIAL CAPSULE
A layer of amorphous viscid material lying outside the cell in a well organized
manner and not easily washed off is called capsule.
Most bacterial capsules are polysaccharide in nature, except Bacillus anthracis has
capsule polypeptide in nature.

Examples of capsulated bacteria

Pneumococcus, Hemophilus influenza, Meningococcus, Klebsiella pneumoniae,
Bacillus anthracis

Uses
 Contributes to bacterial virulence - Protects bacteria from phagocytosis, helps in
biofilm formation and adhesion
 Source of nutrition and energy
 Capsules as vaccines – have antigenic property, capsular vaccines available for
pneumococcus, Hemophilus influenza serotype – b.

Detection of capsule
 Negative staining – by India ink & Nigrosin staining
Capsule appears as clear refractile halo around the bacteria whereas bacteria
and background appear black
 M’ Faydean Capsular stain – used to demonstrate capsule of Bacillus anthracis
by using polychrome methylene blue stain.
 Serological Test
 Quellung Reaction – capsular serotypes of Streptococcus pnuemoniae are
detected by adding antisera mixed with methylene blue. Capsule becomes
swollen refractile and delineated.
 Capsular Antigen – detected in a sample of CSF by latex agglutination test
by using specific anticapsular antibodies coated on latex particles.
Ex. Pneumococcus, meningococcus, Hemophilus influenza
 McIntosh and Filde’s Anaerobic Jar
It consists of a 8*5 inch (20*12.5 cm) jar of stout glass or metal with a tight fitting metal
lid. The lid can be clamped airtight with a screw and is fitted with two tubes with taps, one
for introduction of gas inside (inlet)and the other as outlet for vacuum valve.
The lid also contains two terminals that can be connected to an electric supply. A
capsule containing alumina pellets coated with palladium (palladinished alumina) is suspended
under the lid by stout wires which are connected with the terminals to heat the catalyst for its
activity. Nowadays, catalyst active at room temperature is also available.
Principle:

McIntosh and Fildes’ anaerobic jar works on

the principle of evacuation and replacement,
where the air inside the chamber is evacuated
and replaced with mixture of gases (consisting
of 5%CO2, 10%H2 and 85%N2) .

Removal of Residual Oxygen – Done by using

a catalyst (sachet containing aluminum pellets
coated with palladium) The catalyst acts as a
catalyzing agent causing slow combination of
hydrogen and oxygen to form water. Reduced
methylene blue is generally used as indicator
(mixture of NaOH, methylene blue, and
glucose). It becomes colorless anaerobically but regains blue color on exposure to oxygen.

Procedure

1. Keep the inoculated culture plates inside the jar along with an indicator.
2. Screw tight the lid
3. Close the inlet tube and connect outlet tube to a vacuum pump ( at least three quarters of
the air of the jar can be removed).
4. Note the pressure on a vacuum gauze and when the pressure is reduced to 100 mm Hg
(i.e., 600 mm below atmospheric), tightly close the outlet tap.
5. Connect the inlet tap is to a hydrogen supply and then open it. Hydrogen is passed
through a small wash bottle.
6. Bring the reduced pressure up to 760 mm Hg (i.e., atmospheric) by monitoring on the
vacuum gauze as 0.
7. Switch on the electric terminals for heating the palladinised crystal (When room
temperature catalyst is used heating is not required).
 The catalyst helps the combination of hydrogen and residual oxygen to form
water. This process is allowed to continue for 20 minutes.
8. Incubate the McIntosh and Fildes’ jar in an incubator at 37°C for 48 hours.

Monitoring efficacy of anaerobiosis:

Reduced methylene blue indicator is used to check the efficacy of anaerobiasis. A tube
containing reduced methylene blue solution had to kept inside the jar along with the culture
plates. Methylene blue is colorless in reduced conditions and turns blue when oxidized.
 CANDLE JAR
 Inoculated media are placed in a jar with lighted candle and then jar is sealed.

 Burning candle reduces Oxygen to the point where the flame goes off.

 Provided an atmosphere of approximately 3-5% CO

 Useful for – Capnophilic bacteria – Brucella abortus

Pneumococcus, gonococcus

 Microaerophilic bacteria require 5% of O for optimum growth.