ACTIVITY 16 , 17 AND 18

The incubator, Different Methods

of Incubation and the Effects of
Temperature on the Viability and
Growth of Bacteria
 INCUBATOR
3 types of incubator :
CO₂ incubators- is an incubator in which a cylinder of carbon
dioxide is attached; carbon dioxide is periodically introduced into
the incubator to maintain a carbon dioxide concentration of
about 5-10%; it contains 15-20% oxygen in addition to carbon
dioxide; this is used to isolate capnophiles.

Non-CO₂ incubators- is an incubator containing room air and

thus contain about 20-21% oxygen

Anaerobic incubators- is an incubator containing an atmosphere

devoid of oxygen.
• Mesophiles
 optimum temperature between 30-40 ⁰C
• Thermophiles
 optimum temperature above 50⁰C
• Hyperthermophiles
 optimum temperature of 80 ⁰C or higher and a maximum temperature as
high as 115 ⁰C
• Psychrophiles
 grow below 20 ⁰C
 Psychrotroph- grows at 0 ⁰C but displays an optimum temperature
in the mesophile range, nearer room temperature
Minimum, maximum and optimum temperature
for growth of certain bacteria
                                       

Bacterium Minimum Optimum Maximum

Listeria
1 30-37 45
monocytogenes
Vibrio marinus 4 15 30
Pseudomonas
4 35 41
maltophilia
Thiobacillus novellus 5 25-30 42
Staphylococcus
10 30-37 45
aureus
Escherichia coli 10 37 45
Clostridium kluyveri 19 35 37
Streptococcus
20 37 40
pyogenes
Streptococcus
25 37 42
pneumoniae
Optimum growth temperature of some prokaryotes. 
Genus and species Optimal growth temp (degrees C)
Vibrio cholerae 18-37
Photobacterium phosphoreum 20
Rhizobium leguminosarum 20
Streptomyces griseus 25
Rhodobacter sphaeroides 25-30
Pseudomonas fluorescens 25-30
Erwinia amylovora 27-30
Staphylococcus aureus 30-37
Escherichia coli 37
Mycobacterium tuberculosis 37
Pseudomonas aeruginosa 37
Streptococcus pyogenes 37
Treponema pallidum 37
• Most bacteria cultures are incubated at 35°C- 37°C
• Increased concentration of CO₂:
 candle jar
 CO₂ incubator
 jar
 Bag
• Achieve an anaerobic environment thru:
 anaerobic chambers
 anaerobic jars
 anaerobic bags or pouches
 Anaerobic chambers
• Ideal anaerobic incubation system
• Contains a catalyst, a dessicant, an oxidation-
reduction indicator and anaerobic gas
• The catalyst (palladium-coated alumina pellets)-
removes residual oxygen from the atmosphere within
the chamber
• Dessicant - silica gel is used to absorb the water
formed when hydrogen combines with free oxygen in
the presence of the catalyst
• Carbon dioxide is required for the growth of many
anaerobic organisms
• Methylene blue or resazurin are used as oxygen-
reduction indicator
• Methylene blue remains white in the absence of
oxygen and turns blue in the presence of oxygen
• Resazurin goes from colorless in the absence of
oxygen to pink in the presence of oxygen
Palec, Maria Benneth MT8A, 2015 9
Alternative

Anaerobic jars Anaerobic bags

• use an envelope gas • 1 or 2 inoculated plates are
generator placed into a bag, an
• 2 gases are generated: oxygen-removal system is
carbon dioxide and activated and the bag is
hydrogen sealed and incubated
• Also useful transport
devices
Palec, Maria Benneth MT8A, 2015 11
 On the basis of oxygen requirements, bacteria
can be divided into following different categories
•:

• Aerobes
Grow in ambient air, which contains 21% oxygen and small amount of (0.03%)
of carbon dioxide
Bacillus cereus
• Obligate aerobes
They have absolute requirement for oxygen in order to grow
Pseudomonas aeruginosa; Mycobacterium tuberculosis
• Anaerobes
Usually can not grow in the presence of oxygen.
• Obligate anaerobes
 These bacteria grow only under condition of high reducing
intensity and for which oxygen is toxic.
 Clostridium perfringens , Clostridium botulinum
• Facultative anaerobes
  They are capable of growth under both aerobic and anaerobic
conditions
 Enterobacteriaceae group, Staphylococcus aureus 
• Aerotolerant anaerobes
 Are anaerobic bacteria that are not killed by exposure to oxygen
• Capnophiles
 require increased concentration of carbondioxide (5% to 10%)
and approximately 15% oxygen
  Haemophilus influenzae, Neisseria gonorrhoeae
• Microaerophiles
 can grow under reduced oxygen (5% to 10%) and increased
carbondioxide (8% to 10%).
 Higher oxygen tensions may be inhibitory to them
 Campylobacter jejuni, Helicobacter pylori 
Anaerobic incubation

Application
 for the production of an anaerobic
environment in the anaerobic jar
 for the cultivation of obligatory
and facultative anaerobes

• Anaerobic jar
 Hermetically sealed metallic or plastic jars from which it is possible to pump out
oxygen and replace it by special gases (helium, nitrogen, argon )

Principle
 Anaerocult contains components which chemically bind oxygen quickly and
completely, creating an oxygen-free (anaerobic ) environment and CO₂ atmosphere
Anaerobic Gaspak method

• Gaspak is commercially used and never reused

• Gaspak is a disposable containing pellets of sodium
borohydride, cobalt chloride, citric acid and sodium
bicarbonate
• These chemicals generate hydrogen and carbon dioxide when
water is added
• Hydrogen combines with oxygen in the presence of a catalyst
• After the inoculated plates are placed inside an air tight jar, the
packet of “gas-pak “ with water added is kept inside and the
lid is tightly closed
Quality assurance

• Chemical indicator:
 Turns white when reduced
• Biological indicator:
 Pseudomonas aeruginosa ( aerobic bacteria ) will
grow if anaerobiosis is not maintained
Capnophilic incubation
Principle:
 inoculated plates are placed inside a large air-tight
container and a lighted candle kept in it before the lid is
sealed- provides a concentration of carbon dioxide which
stimulates the growth of most bacteria