Elements of Microbial Growth, Nutrition and Environment

Do different organisms require specific diets and environments?

Why do we care about growth?

• 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

What is Growth?

• 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.

Binary fission• 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

Generation Time• 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

Generation Time

• 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• 11 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

(Log

)

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?

Exponential Growth Phase

• 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

Direct: Standard Plate Counts

Direct: Microscopic Count• Advantages

– Easy and fast

• Disadvantages– Uses special

microscope counting slide

– Does not differentiate between live and dead bacteria

Direct: Membrane Filtration

Direct: Coulter Counter

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?

Indirect: Turbidity Using Spectrometer

Indirect: Metabolism Activity

• 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

Indirect: Dry Weight

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

Indirect: Genetic Methods

• 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

Microbial Nutrition

Microbial Nutrition

• 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 CO2 as its carbon source-has the capacity to convert CO2 into organic

compounds-not nutritionally dependent on other living

things

•Phototroph: microbes that photosynthesize

•Chemotroph: microbes that gain energy from injesting chemical compounds

Microbial Nutrition

• Photoautotrophs:-Photosynthetic-Produce organic molecules using CO2

-Ex: Cyanobacteria, algea

• Chemoautotrophs:-Ingest organic or inorganic

compounds for energy -Carbon source is CO2

Microbial Nutrition: Autotrophs

• 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

Microbial Nutrition: Heterotrophs

•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

Diffusion: Review

•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)

Osmosis: Review

Osmosis: Review

37

Chemoautotrophschemoorganic autotrophs: use organic

compounds for energy and inorganic compounds as a carbon source

lithoautotrophs: rely totally on inorganic minerals and require neither sunlight nor organic

nutrients

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

Environmental Factors: Temperature

• 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

Temperature and Bacterial Growth

Five categories of microbes based on temperature ranges for growth

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

0

Rat

e of

Gro

wth

Temperature °C

PsychrophilePsychrotrophMesophileThermophileExtreme thermophile

Optimum

MinimumMaximum

-20 -10 10 20 30 40 50 60 70 80 90 100 110 120 130

Which category do human pathogens usually fall into? Why?

Environmental Factors: Gases

• Two gases that most influence microbial growth– Oxygen

• O2 has the greatest impact on microbial growth

• O2 is an important respiratory gas and a powerful oxidizing agent

– Carbon dioxide• Waste for bacteria• Carbon source for others (non-pathogens)

Oxygen Requirements

• 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

Oxygen Requirements•As oxygen enters cellular reactions, it is transformed into several toxic products:

-singlet oxygen (O)-superoxide ion (O2

-)-hydrogen peroxide (H2O2)-hydroxyl radicals (OH-)

•Most cells have enzymes that scavenge and neutralize reactive oxygen byproducts

•Two-step process requires two enzymes:

Catalase Test

Oxygen Requirements

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

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

Determining Oxygen Requirements• Thioglycollate broth to

demonstrate oxygen requirements.

• Oxygen levels throughout the media are reduced via reaction with sodium thioglycolate.

• Producing a range of oxygen levels in the media that decreases with increasing distance from the surface

Oxygen Requirements: Obligate Aerobe• 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 oxygen free 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

Why is this the “best” for pathogens?

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

Oxygen Requirements: Aerotolerant• Can live with, but do not use

oxygen• Able to break down

peroxides (not using catylase)

• Ex. Some lactobacilli and streptococci

Oxygen Requirements: Aerotolerant• Can live with, but do not use

oxygen• Able to break down

peroxides (not using catylase)

• Ex. Some lactobacilli and streptococci

Oxygen Requirements: Microaerophiles• Require small amounts of

oxygen

• Ex. H. pylori

Culturing Technique for Anaerobes

• 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

Environmental Factors: pH

• 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

Environmental Factors: pH

Example of the use of a selective medium for pH

Fungal coloniesBacterial 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 cease most metabolic activity

• Two physical effects of water–Osmotic pressure–Hydrostatic pressure

Environmental Factors: Water

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

Environmental Factors: Water

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 are very

specialized!

Associations Between Organisms – Organisms live in association with different species– Often involve nutritional interactions

• Antagonistic relationships• Synergistic relationships• Symbiotic relationships

Associations Between Organisms

Organisms live in closenutritional relationships;

required by one or both members.

MutualismObligatory,dependent;

both membersbenefit.

CommensalismThe commensalbenefits; othermember not

harmed.

ParasitismParasite isdependent

and benefits;host harmed.

SynergismMemberscooperateand sharenutrients.

AntagonismSome members

are inhibitedor destroyed

by others.

Organisms are free-living;relationships not required

for survival.

Symbiotic Non symbiotic

Associations Between Organisms •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 the competitor

-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.

Associations Between Organisms

• 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

• Complex relationships among numerous species of microorganisms

• Many microorganisms more harmful as part of a biofilm

• Quorum sensing: used by bacteria to interact with members of the same species as well as members of other species that are close by

Plaque (biofilm) on a human tooth

•Benefits of 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 species

– cannot be eradicated by traditional methods

Associations and Biofilms

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? Or the MM project;)

-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

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

Survey of Microbial Diseases: Phenotypic Methods

Blood agar as a differential medium

Beta-hemolysis

Alpha-hemolysis

No hemolysis(gamma-hemolysis)

Tests for fermentation and

gas production

No fermentation Acid fermentationwith gas

Durham tube(inverted tubeto trap gas)

Survey of Microbial Diseases: Phenotypic Methods

•Direct observation of fresh or stained specimen

•Stains most often used-Gram stain-acid-fast stain

Phenotypic Methods: Direct Examination of Specimen

• 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

Survey of Microbial Diseases: Phenotypic Methods

MacConkey Agar: Selective and Differential

Selects for Gram-negative and tells you if the bacterium ferments lactose

• Physiological reactions: indirect evidence of enzymes present in a species. If bacteria tests + for cytochrome c oxidase what does that tell you?

Phenotypic Methods: Biochemical Testing

– – + – – ++ – – – – + – – – – – – – –

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Unknownmicrobe +differentsubstrates

Results (+/–)

• 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

Phenotypic Methods: Biochemical Testing

Flowchart: We will use this to ID our MM!

Gram (–)

Cocci

Anaerobic,oxidase (–),catalase (–)

VeillonellaNeisseriaBranhamella

Moraxella

Aerobic,oxidase (+),catalase (+)

Catalase (–),pairs, chainarrangement

Gram (+)

Catalase (+),irregular clusters,

tetrads

Strictlyaerobic

Micrococcus StaphylococcusPlanococcus

Facultativeanaerobic

Streptococcus

-Testing for sensitivity to various phage groups

-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

-Ex. S. aureus Phage Group I vs. Group II

Phenotypic Methods: Phage Typing

•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

Determining Clinical Significance of Cultures