Microbial Growth

• Microbial Growth Defined:
• Microbial growth is the increase in number of cells, not cell size.( Mother cells, Binary fission, Daughter cells).

• How growth is seen in Laboratory
• Colony formation: By development of colonies.
• Turbidity: By the transformation of a clear broth medium to a turbid suspension.

Growth in Colonies

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• A pure culture contains only one species or strain.
• A colony is a population of cells arising from a single cell or spore or from a group of attached cells.
• A colony is often called a colony-forming unit (CFU).

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• Methods of measuring microbial growth

A)Total bacterial count: includes living as well as dead bacteria. It can be obtained by following methods.

• Microscope count (using microscope slide with special grid)

1)Direct Measurements of Microbial Growth

Direct Measurements of Microbial Growth

• Direct microscopic count

2)Counting in an electronic device as coulter counter.

3)Direct counting using stained smears prepared by spreading known volume of culture over measured area of a slide.

4)Turbidimtric measurement using spectrophotometer.

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How do we measure growth of bacteria in a growth curve?

Coulter counter. Electronically counts number of bacteria as bacteria pass through a tiny tube. Expensive.

• Turbidity

5)Measuring wet and dry weight of cells after centrifugation or filtration.

• B)The viable count: measures number of living organism. It can be obtained as under.
• Dilution method: several tubes are inoculated with varying dilution and appropriate dilutions are inoculated on solid media. The number of colonies that develop after incubation gives an estimate of viable count.

Measuring Bacterial Growth Serial Dilutions

• Direct Measurements of Microbial Growth

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Plate Count

After incubation, count colonies on plates that have �25-250 colonies (CFUs)

Factors Affecting Bacterial Growth ��The Requirements for Growth: �Physical Requirements

1)Temperature

• Minimum growth temperature
• Optimum growth temperature
• Maximum growth temperature

A)Psychrotrophs/Psychrophiles

• Grow between 0°C and 20-30°C
• Cause food spoilage

B)Mesophiles & Thermophiles

• Mesophiles
• grow best between 25 degrees C and 40 degrees C
• Thermophiles
• Heat loving- grow best at 50 - 60 degrees C
• Obligate thermophiles
• Facultative thermophiles
• Extreme Thermophiles (Hyperthermophiles)

Optimum growth at 80oC or higher. Archaebacteria. Most live in volcanic and ocean vents.

Psychrotrophs/Psychrophiles

Figure 6.2

2) hydrogen ion concentration (pH): Organisms can be classified as:

• A. Acidophiles: “Acid loving”.
• Grow at very low pH (optimum 3)
• Lactobacillus produces lactic acid, tolerates mild acidity.
• B. Neutrophiles:
• Grow at pH 6 to 8.
• Includes most human pathogens.
• C. Alkaliphiles: “Alkali loving”.
• Grow at alkaline or high pH (7 to 12 or higher)
• Vibrio cholerae and Alkaligenes faecalis optimal pH 9.
• Soil bacterium Agrobacterium grows at pH 12.

3) Osmotic Pressure

• Halophiles: Require moderate to large salt concentrations. Ocean water contains 3.5% salt.
• Most bacteria in oceans.
• Extreme or Obligate Halophiles: Require very high salt concentrations (20 to 30%).
• Bacteria in Dead Sea.
• Facultative Halophiles: Do not require high salt concentrations for growth, but tolerate 2% salt or more.

4)Gaseous Requirement�Role of Oxygen. Bacteria may be classified into four groups on oxygen requirement :�(i)    Aerobes. They cannot grow without oxygen, e.g. Mycobacterium tuberculosis.�(ii)   Facultative anaerobes. These grow under both aerobic and anaerobic conditions. Most bacteria are facultative anaerobes, e.g. Enterobacteriaceae.�(iii)Anaerobes .They only grow in absence of free oxygen, e.g. Clostridium, Bacteroides.�(iv)   Microaerophils grow best in oxygen less than that present in the air, with carbon dioxide (5-10% CO2) e.g. Campylobacter.

Oxygen (O2)

Using oxygen (1/2 O₂) in metabolism creates toxic waste.

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Microbes that are able to use aerobic respiration produce enzymes to detoxify oxygen:

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Catalase: H₂O₂ ---🡪 H₂0 and 0₂

Superoxide dismutase (SOD): oxygen radical ---🡪 H₂0 and O₂

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Microbes that don’t make these enzymes cannot exist in the presence of oxygen.

Microbes & Oxygen

Image: Catalase enzyme structure, Vossman; Superoxide dismutase enzyme structure, Fvasconcellos

From the Virtual Microbiology Classroom on ScienceProfOnline.com

Anaerobic Culture Methods

1)Anaerobic jar

Figure 6.5

Anaerobic Culture Methods

2)Anaerobic chamber

Figure 6.6

3)Candle jar

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4)CO₂-packet

Figure 6.7

• Many aerobes and aerotolerant anaerobes are protect from the reactive O compound by the presence of superoxide dismutase and catalase. While strict anaerobes lack both superoxide-dismutase and catalase. As a result obligate anaerobes are not inhibited but rapidly killed by air.

• In laboratory cultures of aerobic organisms are maintained in Petri dishes or test tubes under aerobic conditions by using ordinary incubators, while microaerophiles cultures are incubated in candle jar or CO2 incubator. For anaerobic cultures two main methods may be used such as thioglycollate broth method, this is one of the most common methods of producing an anaerobic environment for microbes by introducing a reducing agent (sodium thiogLycollate) into a liquid medium. The reducing agents remove oxygen from the liquid medium through a chemical reaction. The other method by using the Brewer anaerobic jar method and gas-pak system, the anaerobic condition of the Brewer jar is created by physically evacuating the air out with the help of a vacuum pump and avacuated space is filled with an inert gas like hydrogen, nitrogen or carbon dioxide whereas in gas pak system, oxygen is chemically removed from the chamber.

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