(MMI) Lecture 11 (Chapter 11)

What is Microbial Growth?

The increase in number cells, not cell size. Includes population and colonies.

What are the requirements for microbial growth? (The categories)

1. Physical Requirements

2. Chemical Requirements

What are the physical requirements for microbial growth?

 Temperature

 pH

 Osmotic pressure

What are the chemical requirements for microbial growth?

 Carbon

 Nitrogen, sulfur, and phosphorous

 Trace elements

 Oxygen

 Organic growth

In terms of temperature how is it displayed on a graph?

There is…

 Minimum growth temperature

 Optimum growth temperature

 Maximum growth temperature

Rank the organisms based on their growth dependent on heat.

1. Psychrophile

2. Mesophile

3. Thermophile

4. Hyperthermophile

Psychrophiles

Extremophilic microorganisms that thrive and reproduce in permanently cold environments, Grow best at 0-25^oC and cause food spoilage.

Mesophile

An organism that grows best in moderate temperatures, typically between 20°C and 45°C (68°F and 113°F). Most typical disease causing microbes in this category,

Thermophile

Thermophiles are heat-loving microorganisms that thrive and grow optimally at exceptionally high temperatures, typically between 41°C and 122°C (106°F to 252°F).

Hyperthermophiles

an organism that thrives in extremely hot environments, growing optimally at temperatures of 80 °C (176 °F) or higher

0 to -30 degrees

There is no significant growth below freezing

0-5 and 5-15 degrees

• Refrigerator temperatures; may allow slow growth of spoilage bacteria, very few pathogens

• Many bacteria survive and may grow

15-55 degrees

DANGER ZONE. Rapid growth of bacteria; some may produce toxins

55-65

Very sow bacteria growth

65-130 degrees and above

Temperatures in this range destroy most microbes, although lower temperatures take more time

nMost bacteria grow between pH _____ and _____.

6.5 and 7.5, variations out of the range slow growth.

Moulds and yeasts typically grow optimally between pH ____ and ____.

5 and 6

Acidophiles

microorganisms that thrive in acidic environments, showing optimal growth at a pH of 5.5 or below, with extreme acidophiles preferring a pH of 3 or lower.

What is plasmolysis?

the process where a plant cell or bacterial cell loses water in a hypertonic solution causing the cytoplasm and plasma membrane to shrink and pull away from the rigid cell wall

What causes plasmolysis?

Hypertonic environments, or an increase in salt or sugar, cause plasmolysis

Extreme or obligate halophiles

require high osmotic pressure

Facultative halophiles

tolerate high osmotic pressure

Chemical Requirements (Carbon)

• Structural organic molecules, energy source

• Chemoheterotrophs use organic carbon sources

• Autotrophs use CO₂

Chemical Requirements (Nitrogen)

• In amino acids and proteins

• Most bacteria decompose proteins

• Some bacteria use NH₄⁺ or NO₃^–

• A few bacteria use N₂ in nitrogen fixation

Chemical Requirements (Sulfur)

• In amino acids, thiamine, and biotin

• Most bacteria decompose proteins

• Some bacteria use SO₄^2– or H₂S

Chemical Requirements (Phosphorus)

• In DNA, RNA, ATP, and membranes

• PO₄^3– is a source of phosphorus

Chemical Requirements (Trace Elements)

• Inorganic elements required in small amounts

• Usually as enzyme cofactors

A

Obligate Anaerobes

B

Microaerophiles

C

Facultative Anaerobes

D

Aerotolerant Anaerobes

E

Obligate Anaerobes

pH Environmental Classification of a Microorganism

• Alkaliphile (Above pH 9)

• Neutralophile (between pH 5 and pH 8)

• Moderate acidophiles (between pH 3 and 5 pH)

Osmolarity Environmental Classification of a Microorganism

• Halophile (High salt, greater than 2 M NaCl)

• Halotolerant (up to 2 M NaCl)

• Non Halophiles (up to 0.2 M NaCl)

Pressure Environmental Classification of a Microorganism

• Barophile (at high pressure, greater than 380 atm)

• Barotolerant (between 10 and 500 atm)

What are organic growth factors?

They are obtained from the environment

• Examples include Vitamins, amino acids, purines, and pyrimidines

Steps of Bacterial Binary Fission

• DNA Replication

• Cell Elongation

• Contractile Ring Formation

• Septum Formation

• Division

The Phases of Growth

1. Lag phase

2. Logarithmic (exponential) growth phase (log phase)

3. Stationary phase

4. Death phase

What is the lag phase?

The first. Bacteria are preparing their cell machinery for growth.

What is the log phase?

The second. Growth approximates an exponential curve (straight, on a logarithmic scale).

Why do microbiologists use logarithmic scales instead of arithmetic scales to graph bacterial growth during the exponential (log) phase?

• Logarithmic Scale: Produces a clear, straight line that makes it easy to track the growth rate. (calculates exact generation (doubling) time

• Arithmetic Scale: Produces a steep curve where early growth looks flat and late growth spikes off the chart. (makes early generations looks flat and late ones spike)

What is the Stationary phase?

The third. Cells stop growing and shut down their growth machinery while turning on stress responses to help retain viability.

What is the Death Phase?

Cells begin to die at an exponential rate.

What are Biofilms?

Microbial communities where cells are enclosed within an EPS (extracellular polymeric substance) what are widely found in nature.

Formation of Biofilms

Both bacteria and fungi form them, and bacteria are attracted to the community via chemical signals called quorum sensing

Why are Biofilms Clinically significant

• They frequently grow on catheters and implanted medical devices.

• Those formed by Candida albicans are resistant to common treatments, such as azole antifungal drugs.

What are two functional advantages for microbes living in a biofilm?

• They are able to share nutrients.

• They are sheltered from harmful environmental factors and host defenses.

Culture Medium

mixture of nutrients prepared to enable microbial growth

Inoculum

a small sample of microbes placed into the medium to initiate a culture

What are the key properties of agar?

• a complex polysaccharide used as a solidifying agent for culture media (plates, slants, and deeps).

• It is generally not metabolized by microbes, leaving the structure intact.

• It liquefies at 100°C and solidifies at ~40°C.

Reducing Media

• Contain chemicals that combine with O₂ and remove it from air

• Heated to drive off O₂

Functions of the Gasket, GasPak envelope, and Catalyst

• Gasket: It seals the lid of the jar, keeping atmospheric oxygen out.

• GasPak envelope: It generates hydrogen and carbon dioxide

• Catalyst: Hydrogen combines with oxygen to form water

BSL-3

Biosafety cabinets to prevent airborne transmission

• Inhaled infectious particles

• Primary pathogens (infect healthy hosts)

• E.g. Coccidiodes fungus, Mycobacterium tuberculosis

BSL-4

• sealed, negative pressure

• Exhaust air is filtered twice

Cultures in Clinical Practice

• Allow for accurate diagnosis and assist in developing treatment plans

• Require proper specimen collection, transportation, culturing, and analysis

Differential Media

• Used to distinguish colonies of different microbes (blood agar)

Streptococcus pyogenes

a common cause of “strep throat” produces a halo on blood agar plates as they secret an enzyme which lyses red blood cells (they are b-hemolytic)

Mannitol Salt Agar (Differential Media)

• Bacteria which can metabolize (ferment) the mannitol produce acid

• This acid can be detected by adding an indicator to the medium

(S. aureus)

Selective Media

• Suppress growth of unwanted microbes and encourage growth of desired microbes

• Bismuth Sulfite Agar

Bismuth Sulfite Agar

• Used to isolate Salmonella typhi (typhoid)

• Gram positive bacteria are inhibited

• Most gram positive enterics also inhibted

• S. typhi produces black colonies

Some media are both selective and differential

• MacConkey medium

• Only gram-negative bacteria grow (= selective)

• Only bacteria that can use lactose (Lac⁺) produce red colonies (= differential)

When is the serial dilution and plate count method typically used?

It is used as a direct measurement of the number of microbes in a sample when there are very high numbers of microbes present

In a standard serial dilution, what volumes are transferred to achieve a 1:10 (10^-1) dilution step?

1.0 ml of the sample/inoculum is transferred into 9.0 ml of broth.

When reading early dilution plates, what does the abbreviation TNTC stand for?

Too numerous to count

What is the standard calculation formula to determine the concentration of bacteria in the original sample?

Number of colonies on the plate X the reciprocal of the dilution of the sample = number of bacteria/ml

When are Serial Dilutions and plate counts + Filtrations + haemocytometer used?

Serial dilutions and plate counts

• Used when have high numbers in test sample

Filtration

• Used when likely have low numbers in large test sample

Haemocytometer

• Actual count of cells under a microscope

How does the filtration method quantify bacterial growth in a liquid sample?

• Sample to be tested is passed through a filter with pores too small to enable microbes to pass

• Filter then placed on a solid media plate (often selective in environmental testing) and incubated to allow colony formation

When using the filtration method to test a water sample, what do colonies that develop a green metallic sheen indicate?

Contamination by fecal material

Direct cell count (haemocytometer)

• Add (diluted) microbial suspension

• Count number of cells in 4 x4 grid

• Multiply by volume factor (usually 10⁴) and any dilution factor to calculate actual cell numbers per ml

Indirect Measurements of Numbers of Microbes in a Sample

• Turbidity

• Metabolic activity

• Dry weight

Estimation of bacterial numbers in a culture by spectrophotometry/turbidity.

• When there are no bacteria in the medium, all the light passes through it (= zero absorbance)

• Microbes in the culture block the light from reaching the detector

• More blockage (absorbance) = more microbes