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
| CLASS | CHARACTERISTIC | EXAMPLES |
| Psychrophiles | Grow well at 0°C and have an optimum growth temperature of 15°C or lower; the maximum is around 20°C | Found in such genera as Pseudomonas, Vibrio, Alcaligenes, Bacillus, Arthrobacter |
| Psychrotrophs or facultative psychrophiles | Grow at 0 to 7°C even though they have optima between 20 and 30°C, and maxima at about 35°C. | Organisms that are Involved in spoilage of refrigerated foods |
| Mesophiles | Growth optima around 20 to 45°C; they often have a temperature minimum of 15 to 20°C. | Almost all human pathogens are mesophiles |
| Thermophiles | Grow at temperatures of 55°C or higher. Growth minimum is usually around 45°C and they often have optima between 55 and 65°C | Organisms that are found in composts, self-heating hay stacks, hot water lines, and hot springs |
| Hyperthermophiles | Procaryotes that have growth optima between 80°C and about 113°C | Pyrococcus abyssi and Pyrodictium occultum are examples of marine hyperthermophiles found in hot areas of the seafloor |
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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