Microbial growth

Microbial Growth

Increase in number of cells rather than an increase in size

Understanding the

requirements for microbial

growth

Allow us determine how to

control the growth of

microbes

Specifically, of those

microbes that cause

disease and food

spoilage

The Requirements for Growth

Physical requirements include Temperature pH Osmotic pressure

Microorganisms have physical, chemical, and

energy requirements for growth

Effect of temperature

Minimum growth temperature - microbe is able to conduct metabolism

Maximum growth temperature – microbe continues to metabolize

Optimum growth temperature – highest growth rate

Growth rate plotted against temperature

Growth of Escherichia coli on nutrient agar at three different temperature

Categories of microbes based on temperature ranges for growth

Human pathogens are mesophiles (Optimum growth temperature is ~ 37C)

Effect of temperature

Treponema pallidum (the

causative agent of

syphilis) likes lower

temperatures

Lesions are first seen on

exterior parts of the body

including lips, tongue, and

genitalia

Variable temperature requirements are seen in certain diseases

Chancroids

Temperature and bacterial growth

Temperature and bacterial growth

Variable temperature requirements are seen in certain diseases

Mycobacterium leprae (the

causative agent of leprosy)

also likes lower temperatures

Leprosy is initially seen on

the extremities of the body,

like face, ears, hands, feet,

and fingers

Effect of pH

Neutrophiles Grow best in a narrow range

around neutral pH (pH 6.5-7.5) Acidophiles

Grow best in acidic habitats Alkalinophiles

Live in alkaline soils and water

Most pathogens are neutrophiles

Helicobacter pylori (causative agent of gastric ulcers) is not an acidophile but an acid-tolerant (secretes bicarbonate and urease)

Vibrio cholerae, the cause of cholera, can thrive at a pH as high as 9.0.

Effect of Osmotic Pressure

Isotonic Hypertonic (plasmolysis)

Osmotic pressure is the pressure exerted on bacterial cells by their environment

Hypotonic: the bacterial cell gains water and swells to the limit of its cell wall

Some opportunistic pathogens are facultative halophiles Staphylococcus aureus - colonizes the surface of the skin (salt)

Chemical Requirements Microorganisms use a variety of chemicals (nutrients) as a source

of energy to build organic molecules and cell structures

Several core chemicals are required for bacterial growth

Chemoheterotrophs, which include pathogenic bacteria, use organic molecules as a source of carbon and energy

Trace elements or micronutrients are minerals essential for the function of certain enzymes

Include copper zinc manganese molybdenum

Trace elements and growth factors

Oxygen Requirements

Capnophiles are microbes that require higher concentration of carbon dioxide (3-10%) in addition to low oxygen levels

Superoxide dismutase (SOD) converts superoxide radicals (O2-) to

molecular oxygen and hydrogen peroxide, which is also toxic

Catalase converts hydrogen peroxide (H2O2) to water and oxygen

Catalase test Phagocytic cells use toxic forms of oxygen to kill ingested pathogens

Hydrogen peroxide can be used as an antimicrobial agent

Many of the bacteria that form our normal flora and many pathogens are facultative anaerobes

Some pathogens can be obligate anaerobes

Gas gangrene is caused by Clostridium perfringens

Exposure of this organism to air is a lethal event for the bacterium

How do we culture microbes?

To cultivate (or culture) microorganisms

A sample (inoculum) is placed into/on broths (liquid media) and solid media

Microorganisms that grow from an inoculum are called a culture Cultures visible on solid

media as discrete units are called colonies

Petri plate

Deeps

Slants

What criteria must a culture medium meet?

All nutrients required by bacteria in the specimen including growth factors

Sufficient moisture, properly adjusted pH of the medium, oxygen requirements

Proper temperature of incubation for growth

Sterilization and aseptic techniques are designed to minimize contamination of the specimen

A chemically defined medium (synthetic medium)

A complex medium

Nutrient broth is the liquid version of the medium - without agar (another example is TSB/TSA: Trypticase soy broth/agar)

Anaerobic microbial cultures, media, and systems

Stab cultures

Reducing media are used to culture anaerobes

Contain chemicals such as thioglycollate that combines with oxygen and removes it from the medium

Anaerobic culture system (anaerobic jar or GasPakTM jar)

An Anaerobic Chamber

Handling and culturing clinical specimens

Clinical specimens are collected to identify a suspected

pathogen

Specimens often include microorganisms associated with

the normal microbiota

Suspected pathogen in the clinical specimen must be

isolated from the normal microbiota in culture

Several techniques can be used to isolate organisms in pure

cultures (axenic cultures)

Handling and culturing clinical specimens

Properly collected and labeled

placed in sterile containers

promptly transported to a clinical laboratory to

avoid death of the pathogen

minimize the growth of members of the normal microbiota

transport media are often used to move specimens from one

location to another

If clinical specimens are not handled or cultured properly

Pathogenic bacteria may be missed or may not survive

leading to wrong diagnoses!!!!

Health care professionals collect specimens according to the CDC - Standard precautions

Sterile swabs Needle aspiration

Intubation

Catheter Clean catch method Sputum

(coughing/catheter) Biopsy

Streak-plate technique of isolation

The method of serial dilutions

Techniques to isolate microorganisms in pure cultures or axenic cultures

Pour-plate/spread-plate techniques of isolation

Plating serial dilutions of the specimen Pour plate method Spread plate

method

Characteristics of bacterial colonies can help in the process of identification

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Mixed culture

Pure culture

Clinical implications of bacterial growth and culture media

Bacteria can be “fastidious” in a laboratory setting, Nesseria gonorrhoeae or Haemophilus influenzae

Some cannot be grown on culture media: Mycobacterium leprae (armadillos) or Treponema pallidum (rabbits)

Some others are obligate intracellular parasites (chlamydias and richettsias) and require cultures of living cells

Chocolate agar used to culture H. influenzae and N. gonorrhoeae

Enriched medium

Enriched, selective, and differential media help establish the presence of pathogens

A selective medium contains ingredients that inhibit the growth of some organisms while encouraging the growth of others

Sabouraud dextrose agar selects for the growth of fungi while inhibits the growth of bacteria

Nutrient agar - pH 7.3 Sabouraud agar - pH 5.6

Enriched, selective, and differential media help establish the presence of pathogens

Blood agar plate (BAP) is an enriched and differential medium, which is usually used to detect hemolytic activity

No hemolysis (gamma-hemolysis)

Alpha-hemolysis

Beta-hemolysis

Streptococcus pyogenes Streptococcus pneumoniae Enterococcus faecalis

Beta-hemolysis

Enriched, selective, and differential media help establish the presence of pathogens

Many selective media are also differential media

Enriched, selective, and differential media help establish the presence of pathogens

Many selective media are also differential media

MSA (Mannitol salt agar)

High salt concentration (7.5%) to select for Staphylococcus species while inhibiting the growth of other species

Mannitol sugar in MSA helps differentiate Staphylococcus species

Bacterial growth by binary fission – asexual reproduction

Generation time is the time required for a bacterial cell to grow and divide

Under optimal conditions, E. coli or S. aureus have a generation time of ~ 20 min

Typical microbial growth curve

Stationary phase

Death (decline) phase

Log (exponential) phase

Lag phase

Time

Nu

mb

er

of

live

ce

lls (

log)

When bacteria are grown in a broth, the bacterial growth curve has four distinct phases

How do we measure microbial growth?

Direct Methods

• Plate counts*

• Filtration*

• MPN

• Direct microscopic count

Indirect Methods

• Turbidity*

• Metabolic activity

• Dry weight

Working with clinical specimens can involve quantitative analysis such as assessing a significant bacteriuria - urine samples

Assessing effectiveness of disinfectants, antibiotics …… requires quantitative analysis!!!!

Direct Method: Viable Plate Counts

After incubation, count colonies on plates 25 to 250 colonies - CFUs: colony-forming units

Serial dilutions of the specimen

Direct Method Counting Bacteria by Membrane Filtration

Direct Method: Counting Bacteria by Membrane Filtration

Bacteria are filtered out and retained on the surface of the filter

The filter is transferred to a culture medium

Colonies arise from the bacterial cells on the surface of the filter

Indirect Methods Turbidity

This method uses an instrument called spectrophotometer

The amount of light hitting the detector is inversely proportional to the number of bacteria

The less light transmitted, the more bacteria in the sample

Preserving Bacterial Cultures

Bacterial cultures are stored by slowing the cell’s metabolism Prevent exhaustion of all nutrients and excessive accumulation

of waste products

Storage for short period of time Refrigeration (weeks to months)

Long-term storage

Deep-freezing (years) Lyophilization (freeze-drying) (decades)

Involves removing water from a frozen culture using an intense vacuum. Lyophilized cultures are restored by adding them to liquid media