Elements of Microbial Growth, Nutrition and Environment

Do different organisms require specific diets and environments?

• To Encourage the microbes we want
• Brewery, winery, food production
• Vaccine and drug production
• Microbial fuel cells
• Bioremediation, Sewage treatment plant, oil spill clean up
• Resident microbiota-probiotics to aid microbial antagonism and perform other functions
• To Discourage the microbes we don’t want
• Pathogens

• In microbiology, we define growth in relation to the number of cells, not the size of cells.
• Concentrate on population growth
• Bacterial cells divide via binary fission, not mitosis.

• The division of a bacterial cell
• Parental cell enlarges and duplicates its DNA
• Septum formation divides the cell into two separate chambers
• Complete division results in two identical cells

• The time required for a complete division cycle
(doubling)
• Length of the generation time is a measure of the growth rate
• Growth is exponential not arithmetic
• Dependent on chemical and physical conditions

• Average generation time is 30 – 60 minutes
• shortest generation times can be 10 – 12 minutes
• E. coli GT=20 min.
• Mycobacterium leprae has a generation time of 10 – 30 days
• 1  1 million cells (20 generations) in 7 hours
• most pathogens have relatively short generation times

Which is bacterial growth curve?

Four phases of growth in a bacterial culture

1. Lag Phase
• Cells are adjusting, enlarging, and synthesizing critical proteins and metabolites
• Not doubling at their maximum growth rate

2. Exponential
Growth Phase
• Maximum exponential growth rate of cell division
• Adequate nutrients
• Favorable environment
• Most sensitive to antibiotics. Why?

• A person actively shedding bacteria in the early and middle stages of infection is more likely to spread it than a person in the later stages.
Why?
MRSA

3. Stationary Phase
• Cell birth and cell death rates are equal
• Survival mode – depletion in nutrients, released waste can inhibit growth

4. Death Phase
• A majority of cells begin to die exponentially due to lack of nutrients or build up of waste
• Slower than the exponential growth phase

How do we measure microbial growth?
• Direct measurement
– Standard Plate counts
• most common, need to
DILUTE to get individual, countable colonies
– Microscopic Count
• count with microscope
– Filtration
• when # microbes small,
• water run thru filter and filter applied to TSA plate and incubated
– Coulter Counter
• Automated cell counter
• Indirect (Estimation)
– Turbidity
– more bacteria, more cloudiness
– can measure w/ spectrophotometer or eye
– Metabolic Activity
– assumes amount of metabolic product is proportional to #
– Dry Weight
– used for filamentous organisms, like molds
– Genetic Probing
– Real-time PCR

• Advantages
– Easy and fast
• Disadvantages
– Uses special microscope counting slide
– Does not differentiate between live and dead bacteria

Direct: Membrane Filtration

Uses an electronic sensor to detect and count the number of cells.

The greater the turbidity, the larger the population size.
Which culture (left or right) has more bacteria?

• The metabolic output or input of a culture may be used to estimate viable count.
• Examples:
• Measure how fast gases and/or acids are formed in a culture
• Or the rate a substrate such as glucose or oxygen is used up

• To calculate the dry weight of cells
– cells must be separated from the medium
– then dried
– the resulting mass is then weighed

• Use real-time PCR to “count” how many bacterial genes there are in a sample.

Which techniques distinguish between live and dead cells?
– Standard Plate counts
– Direct Microscopic
– Filtration
– Coulter counter
– Turbidity
– Metabolic activity
– Dry weight
– Genetic Probing

Which techniques distinguish between live and dead cells?
– Standard Plate counts
– Direct Microscopic
– Filtration
– Coulter counter
– Turbidity
– Metabolic activity
– Dry weight
– Genetic Probing

What are the requirements for microbial growth?

Chemical Composition of an Escherichia coli Cell

• Macronutrients:
carbon, hydrogen, and oxygen
required in relatively large quantities and play principal roles in cell structure and metabolism
• Micronutrients:
present in much smaller amounts
manganese, zinc, nickel
• Inorganic nutrients:
Can have carbon OR hydrogen, but not both
• Organic nutrients:
Contain carbon and hydrogen

• All cells require the following for metabolism and growth:
– Carbon source
– Energy source
• Growth factors (some bacteria are fastidious/picky and require extra supplements)

• Heterotroph: Organic carbon is carbon source
• Autotroph: inorganic CO
2 as its carbon source
has the capacity to convert CO
2 into organic compounds
not nutritionally dependent on other living things
• Phototroph: microbes that photosynthesize
• Chemotroph: microbes that gain energy from injesting chemical compounds

• Photoautotrophs:
Photosynthetic
Produce organic molecules using CO
2
Ex: Cyanobacteria, algea
• Chemoautotrophs:
Ingest organic compounds for energy
Produce organic molecules using CO
2

• Chemoheterotrophs:
organic compounds for both carbon and energy source
derive both carbon and energy from processing these molecules through respiration or fermentation
The vast majority of microbes causing human disease are chemoheterotrophs
Ex: Most bacteria, all, protists, all fungi, and all animals

• Transport of necessary nutrients occurs across the cell membrane, even in organisms with cell walls
• Diffusion:
• Atoms or molecules move in a gradient from an area of higher concentration to lower concentration
• Diffusion of molecules across the cell membrane is largely determined by the concentration gradient and permeability of the substance

• Osmosis: the diffusion of water through a selectively permeable membrane
• Isotonic: Equal solutes in cell and in environment
parasites living in host tissues are most likely to be living in isotonic habitats
Hypotonic: More solutes in cell than in environment
A slightly hypotonic environment can be favorable to bacteria cells
• Hypertonic: Less solutes in cell than in environment
• hypertonic solutions such as concentrated salt and sugar solutions act as preservatives for food(salted ham is an example)

37

Environmental (Physical) Factors
Effecting Bacterial Growth
• Temperature
• Gas
• pH
• Osmotic pressure
• Other factors
• Microbial association
Survival in a changing environment is largely a matter of whether the enzyme systems of microorganisms can adapt to alterations in their habitat

• Effect of temperature on proteins:
– Too high, proteins unfold and denature
– Too low, do not work efficiently
• Effect of temperature on membranes of cells and organelles:
– Too low, membranes become rigid and fragile
– Too high, membranes become too fluid

Minimum
Maximum
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Optimum
Psychrophile
Psychrotroph
Mesophile
Thermophile
Extreme thermophile
-20 -10 0 10 20 30 40 50 60
Temperature °C
70 80 90 100 110 120 130
Which category do human pathogens usually fall into? Why?

• Two gases that most influence microbial growth
– Oxygen
• O
2
• O
2 has the greatest impact on microbial growth is an important respiratory gas and a powerful oxidizing agent
– Carbon dioxide

• As oxygen enters cellular reactions, it is transformed into several toxic products
– highly reactive and excellent oxidizing agents
• Resulting oxidation causes irreparable damage to cells by attacking enzymes and proteins

• As oxygen enters cellular reactions, it is transformed into several toxic products:
singlet oxygen (O)
superoxide ion (O
2
)
hydrogen peroxide (H
2
hydroxyl radicals (OH )
O
2
)
• Most cells have developed enzymes that scavenge and neutralize reactive oxygen byproducts
• Two-step process requires two enzymes:

If bacteria do not have superoxide
dismutase or
catalase they can not tolerate oxygen.
Catalase Test

Oxygen Requirements
• Aerobes
• Anaerobes
• Facultative anaerobes
• Aerotolerant anaerobes
• Microaerophiles

• Requires oxygen for metabolism
• Have enzymes that neutralize toxic oxygen metabolites
• Ex. Most fungi, protozoa, and bacteria, such as
Bacillus species and
Mycobacterium tuberculosis

Oxygen Requirements: Facultative Anaerobe
• Does not require oxygen, but can grow in its presence
• During minus oxygen states, anaerobic respiration or fermentation occurs
• Possess superoxide dismutase and catalase
• Ex. Many Gram-negative pathogens
Prefer oxygenated environments because more energy is produced during aerobic respiration compared to anaerobic respiration or fermentation

Oxygen Requirements: Obligate Anaerobes
• Cannot use oxygen for metabolism
• Do not possess superoxide dismutase and catalase
• The presence of oxygen is toxic to the cell and will kill it
• Ex. Many oral bacteria, intestinal bacteria

Thioglycollate broth enables the identification of aerobes, facultative anaerobes, and obligate anaerobes.
Use of thioglycollate broth to demonstrate oxygen requirements.

Culturing Technique for Anaerobes
Anaerobes must grow in an oxygen minus environment, because toxic oxygen metabolites cannot be neutralized.

• Most cells grow best between pH 6-8
– strong acids and bases can be damaging to enzymes and other cellular substances
• Pathogens like our neutral pH
• Yeast & Molds like acidic conditions

• Acidophiles
– thrive in acidic environments.
– Ex. Helicobacter pylori
• Alkalinophiles
– thrive in alkaline conditions
– Ex. Proteus can create alkaline conditions to neutralize urine and colonize and infect the urinary system

Example of the use of a selective medium for pH
Bacterial colonies Fungal colonies pH 7.3
pH 5.6

• Microbes require water to dissolve enzymes and nutrients
• Water is important reactant in many metabolic reactions
• Most cells die in absence of water
– Some have cell walls that retain water
– Endospores and cysts cease most metabolic activity
• Two physical effects of water
– Osmotic pressure
– Hydrostatic pressure

Osmotic pressure:
• Halophiles (Salt lovers)
– Requires high salt concentrations
– Withstands hypertonic conditions
• Ex. Halobacterium
• Facultative halophiles
– Can survive high salt conditions but is not required
– Ex. Staphylococcus aureus

Other Physical Factors
Influencing Microbial Growth
• Radiation- UV, infrared
• Barophiles – withstand high pressures
• Spores and cysts- can survive dry habitats
Microbes require different nutrients and different environments specific to survive. They have specialized over the years!

Associations Between Organisms
– Organisms live in association with different species
– Often involve nutritional interactions
• Antagonistic relationships
• Synergistic relationships
• Symbiotic relationships
Associations Between Organisms
Symbiotic
Organisms live in close nutritional relationships; required by one or both members.
Non symbiotic
Organisms are free-living; relationships not required for survival.
Mutualism
Obligatory, dependent; both members benefit.
Commensalism
The commensal benefits; other member not harmed.
Parasitism
Parasite is dependent and benefits; host harmed.
Synergism
Members cooperate and share nutrients.
Antagonism
Some members are inhibited or destroyed by others.

• Antagonism: free-living species compete
Antibiosis: the production of inhibitory compounds such as antibiotics
The first microbe has a competitive advantage by increasing the space and nutrients available to it
Remember importance of microflora?!
A biocontrol agent on the right (a bacteria) is making a material that is keeping the pathogen on the left (a fungus) from growing.

• Synergism: free-living species benefits together but is not necessary for survival
• Together the participants cooperate to produce a result that none of them could do alone
• Gum disease, dental caries, and some bloodstream infections involve mixed infections of bacteria interacting synergistically

Associations and Biofilms
– Biofilms
• Complex relationships among numerous species of microorganisms
• Develop an extracellular matrix
– Adheres cells to one another
– Allows attachment to a substrate
– Sequesters nutrients
– May protect individuals in the biofilm
• Form on surfaces often as a result of quorum sensing
• Many microorganisms more harmful as part of a biofilm
Plaque
(biofilm) on a human tooth

Biofilms: Quorum Sensing
• Quorum sensing: used by bacteria to interact with members of the same species as well as members of other species that are close by
• Structure of the biofilm
large, complex communities form with different physical and biological characteristics
the bottom may have very different pH and oxygen conditions than the surface
partnership among multiple microbial inhabitants
cannot be eradicated by traditional methods

Now that you know more about the nutritional needs of bacteria let’s look at using this information to ID bacteria!

• How to identify bacteria in patient specimens or in samples from nature?
phenotypic: considers macroscopic and microscopic morphology, physiology, and biochemistry
immunologic: serological analysis
genotypic: genetic techniques increasingly being used as a sole resource for identifying bacteria
• Data from these methods can provide a unique profile for any bacterium

Survey of Microbial Diseases:
Phenotypic Methods
Physiological/Biochemical Characteristics
• Traditional mainstay of bacterial identification
• Enzyme production and other biochemical properties are reliable ways to ID microbes
• Dozens of diagnostic tests exist for determining the presence of specific enzymes and to assess nutritional and metabolic activities
fermentation of sugars
capacity to digest complex polymers
production of gas
sensitivity to antibiotics
nutrient sources

Beta-hemolysis
Blood agar as a differential medium
Alpha-hemolysis
No hemolysis
(gamma-hemolysis)

Survey of Microbial Diseases:
Phenotypic Methods
Tests for fermentation and gas production
Durham tube
(inverted tube to trap gas)
No fermentation
Acid fermentation with gas

Phenotypic Methods: Direct Examination of Specimen
• Direct observation of fresh or stained specimen
• Stains most often used
Gram stain
acid-fast stain

Survey of Microbial Diseases:
Phenotypic Methods
• Isolation Media and Morphological Testing
Selective media: encourage the growth of only the suspected pathogen
Differential media: used to identify definitive characteristics and fermentation patterns

MacConkey Agar: Selective and Differential
Selects for Gram-negative and tells you if the bacterium ferments lactose

Phenotypic Methods: Biochemical Testing
• Physiological reactions: indirect evidence of enzymes present in a species. If bacteria tests + for superoxide dismutase
(an oxidase) what does that tell you?

Phenotypic Methods: Biochemical Testing
Unknown microbe + different substrates
Results (+/
–)
DNPG ADH LDC ODC | CIT | H2S URE TDA IND | VP | | GEL | GLU MAN INO SOR RHA SAC MEL AMY ARA
– –
+ +
– – – –
+
– –
+
– – – – – – – –
• Enzyme-mediated metabolic reactions often visualized by a color change
microbe is cultured in a medium with a special substrate, then tested for a particular end product
microbial expression of the enzyme is made visible by a colored dye

Flowchart: We will use this to ID our MM!
Cocci
Gram (+)
Strictly aerobic
Micrococcus
Catalase (+), irregular clusters, tetrads
Facultative anaerobic
Staphylococcus
Planococcus
Catalase ( –), pairs, chain arrangement
Streptococcus
Aerobic, oxidase (+), catalase (+)
Neisseria
Branhamella
Moraxella
Gram (
–)
Anaerobic, oxidase (
–), catalase ( –)
Veillonella

Phenotypic Methods: Phage Typing
used when morphological and biochemical tests are insufficient. Ex. S. aureus Phage Group I vs. Group II
bacteriophage infect bacteria in a species-specific and strain-specific way, which is useful in identifying some bacteria
a lawn of bacterial cells is inoculated onto agar, mapped off into blocks, and phage are exposed to each block
cleared areas corresponding to lysed cells indicate sensitivity to that phage

Determining Clinical Significance of Cultures
• Important to rapidly determine if an isolate from a specimen is clinically important or if it is merely a contaminant or normal biota
a few colonies of E. coli in a urine sample can indicate normal biota, but several hundred can mean an active infection
a single colony of a true pathogen such as
Mycobacterium tuberculosis in a sputum culture, or an opportunist in a sterile site, is highly suggestive of disease
repeated isolation of a relatively pure culture of any microorganism can mean it is an agent of disease