Environmental factors affecting microbial growth

Environmental factors affecting microbial growth

Contents

• Different physical/ environmental requirements for
bacterial growth

• Classification of bacteria depending on temperature
requirement

• Classification of bacteria based on oxygen requirement

• Classification of bacteria based on pH requirement

• Cultivation of anaerobic bacteria

Intended
Learning objectives

At the end of this lecture, the student will be able to:

• List the environmental factors affecting microbial growth

• Classify organisms based on their optimum temperature, pH
oxygen requirement for growth

• Explain the methods for the cultivation of anaerobic
bacteria

Introduction

• The growth of microorganisms also is greatly affected by
the chemical and physical nature of their surroundings

• An understanding of environmental influences aids in the
control of microbial growth and the study of the ecological distribution of
microorganisms

Environmental
factors affecting microbial growth

A. Water activity or osmotic pressure

• Microorganisms that grow over wide ranges of water
activity or osmotic pressure are called osmotolerant

• Staphylococcus aureus can be cultured in media containing
any sodium chloride concentration up to about 3 M

• The yeast Saccharomyces rouxii will grow in sugar solutions

• Alga Dunaliella viridis tolerates sodium chloride
concentrations from 1.7 M to a saturated solution.

• Halophiles require high levels of sodium chloride to grow

B. pH

• Acidophiles have their growth optimum between pH 0 and 5.5

• Neutrophiles, between pH 5.5 and 8.0

• Alkalophiles prefer the pH range of 8.5 to 11.5

• Extreme alkalophiles have growth optima at pH 10 or higher.

C. Temperature

• Most important factor influencing the effect of
temperature on growth is the temperature sensitivity of enzyme catalyzed
reactions

• Beyond a certain point further increases actually slow
growth, and sufficiently high temperatures are lethal.

• Microbial membranes are also disrupted by temperature extremes;
the lipid bilayer simply melts and disintegrates.

• At very low temperatures, membranes solidify and enzymes don’t
work rapidly

Classes of
bacteria based on their temperature ranges for growth

D. Oxygen
Concentration

• Organism able to grow in the presence of atmospheric O2 is
an aerobe, whereas one that can grow in its absence is an anaerobe

• Organisms that are completely dependent on atmospheric O2 for
growth are obligate aerobes

• Facultative anaerobes do not require O2 for growth but do
grow better in its presence. In the presence of oxygen they will use aerobic
respiration.

• Aerotolerant anaerobes such as Enterococcus faecalis
simply ignore O2 and grow equally well whether it is present or not

• Strict or obligate anaerobes (strict or obligate anaerobes
(e.g., Bacteroides, Fusobacterium, Clostridium pasteurianum, Methanococcus) do
not tolerate O2 at all and die in its presence

• Aerobes such as Campylobacter, called microaerophiles,
that are damaged by the normal atmospheric level of O2 (20%) and require O2
levels below the range of 2 to 10% for growth

• Obligate aerobes and facultative anaerobes usually contain
the enzymes superoxide dismutase (SOD) and catalase, which catalyze the
destruction of superoxide radical and hydrogen peroxide, respectively.

2O ^–+ 2H⁺—-> H2O2 + O2………………… SOD catalysed

2H2O2 —> 2H2O + O2………………………..Catalase catalysed

• Peroxidase also can be used to destroy hydrogen peroxide.

Representative
Anaerobic Pathogens:

1. Clostridium tetani
– agent of tetanus, puncture wounds, produces a toxin which enters the
spinal column and blocks the inhibitory spinal motor neurons. This produces
generalized muscle spasms or spastic paralysis. The muscle of the jaw                                                     Representative Anaerobic Pathogens:

2. Clostridium
botulinum – this soil organism is the causative agent of botulism which
typically occurs after eating home canned alkaline vegetables which were not
heated enough during canning. The neurotoxin blocks transmission across
neuromuscular junctions and this results in flaccid paralysis.

3. Clostridium
perfringes and Clostridium sporogenes – these organisms are associated with
invasive infections known as GAS GANGRENE.

4. Clostridium
difficile – the causative agent of pseudomembranous colitis, a side effect
of antibiotic treatment which eliminates the normal flora.

E. Pressure

• Barotolerant – Increased pressure does adversely affect
these microorganisms

• Barophilic—Bacteria they grow more rapidly at high
pressures

Cultivation
of anaerobes

• Anaerobic media containing reducing agents

• Wright’s tube

• Anaerobic jar

• Pre-reduced media

• Anaerobic glove box

• Candle jar method

A. Anaerobic media
containing reducing agents

• Most common adaptation of media is the addition of a
reducing agent, e.g. thioglycollate, cysteine

• Acts to reduce the oxygen to water, brings down the redox
potential -300mV or less.

• Can add a redox indicator such as rezazurin, pink in the
presence of oyxgen – colourless in its absence

• Deep culture tubes can be used to test whether an unknown organism
is anaerobic/facultative or aerobic

• Thioglycollate added to culture medium

• Oxygen only found near top where it can diffuse from air

B. Wright’s tube Pyrogallic
acid-sodium hydroxide method

C. Anaerobic jars
(GasPak System)

D. Prereduced media

• For culture of strict anaerobes all traces of oxygen must
be removed from medium and for many organisms sample must be kept entirely
anaerobic during manipulations

• Methanogenic archaea from rumen and sewage treatment
plants killed by even a brief exposure to O2

• Medium usually boiled during preparation and reducing agent
added, stored under O2-free atmosphere

• Manipulations usually carried out under a jet of O2-free
N2 or N2/CO2 to exclude O2

• Roll-tube (Hungate) method often used instead of
conventional plates for isolation and culture of strict anaerobes

1. Exclude oxygen by flushing the tube with the desired gas

2. Place 4.5ml of pre- reduced anaerobic agar medium into
tube

3. Seal the tube with the butyl rubber stopper and screw cap

4. Autoclave the
tube

5. Inoculate with a syringe

6. Prepare on roll tube spinner

7. Incubate in water bath

E. Anaerobic glove
box

• Use of anaerobic cabinet/glove box allows conventional
bacteriological techniques e.g. replica plating, antibiotic sensitivity testing
etc. to be carried out anaerobically

F. Candle jar method

Summary

• Different environmental factors affecting bacterial growth
are – temperature, osmotic pressure, oxygen and pH

• Based on temperature requirement organisms are classified
as Psychrophiles, Psychrotrophs or facultative psychrophiles, Mesophiles,
Thermophiles and Hyperthermophiles

• Based on oxygen requirement they are classified as aerobic
anaerobic, facultative anaerobes, obligate anaerobe and microaerophillic

• Based on pH – aidophiles, neutrophiles ans alkaliphiles

• Methods for the cultivation of anaerobes – anaerobic
media, prereduced media, wright’s tube, anaerobic jar, anaerobic chamber

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