Chapter 4 Microbial Growth and Control

Microbio

Microbial Growth

• Growth: increase in cell number and biomass (population growth)

1. Enlargement: increase cell body, replication of DNA, separation of intracellular molecules into the daughter cells

2. Septum: partition between divine cells, membrane and wall pinch off between the two daughter cells

3. Division: each daughter cells receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell

Reproductive strategies (Binary Fission & Budding)

• Binary fission: most common cell division process

  • everything in the cell is doubled

  • cell wall is produced throughout the whole cell

  • yields two equivalent cells

• Budding: many bacteria, but also very common in yeast (single-celled fungi)

  • only genetic information and small molecules are located in the bud

  • cell wall is produced on one pole only

  • the daughter cell is not equivalent to the mother cell, does not contain larger cytoplasmic structures such as complex membrane systems (phototrophic species)

Biofilms

• on surfaces, typically with a nutrient flow/current going by

• complex picture of different bacterial species and types of living

• exopolysaccharide matrix (slime) embeds the organisms

• the formation and maturation is a dependent on surface, substrate

• first colonizers are planktonic cells

• ex: plaque on teeth

Generation time

• every time one cell becomes two we consider as a new generation

• the time, a given species or strain needs to divide, is highly variable: among the fastest is E. coli with 20 minutes, others can take days, weeks, months, years, etc.

• Factors that determine the speed of reproduction:

  • type of metabolism

  • bioavailability and/or concentration of substrate

  • competition with other microbes

Bacteria Grow Exponentially (Graphing)

• there is a mathematic relationship between the initial number of cells present in a culture and the number present after a period of exponential growth

• when the line is straight on the semi-logarithmic graph it means that the cells are growing exponentially, cells are doubling at constant time intervals. single cell to colony over night

Growth Media and Lab Cultures

• invented by Robert Koch and Louis Pasteur

• Growth media

  • nutrient solutions used to grow microbes in the laboratory

  • sterilized in an auotcave

  • liquid (broth), or addition of solidifying agent (agar)

  • supply of macronutrients; C, N, P, S

  • supply of micronutrients: minerals and vitamins

  • Recipes can be very complicated to serve the needs of your study organism

2 general classes of medium

1. Defined media: exact chemical composition know, you “design” it, weigh out or measure the components to add

• for specific physiological tests of a bacterial strain

• need to know exact nutritional requirements

2. Complex media: composed of digest of microbial, animal, or plant products (yeast & meat extracts)

• grow the bacterial strain to do other tan metabolic studies

• grow cup biomass to extract to certain product from the cells (harvest)

• one one hand, high concentration of undefined nutrients prevents many free-living bacterial from growing; not natural

• other hand, pathogenic bacteria you have to add (enrich) medium with essential ingredients such as blood or serum.

selective and differential additions

• defined media: water + only measured/weighed out substances and chemicals that you want to have in the media

• complex media: undefined content, buy from shelf to suit most bacteria

• either one can be:

  • Selective: add substance(s) to inhibit the growth of particular, unwanted microbes (bile & salts)

  • Differential: add substance(s) to visualize the biochemical reactions of particular microbes (pH-senstivie dyes, blood)

Blood Agar

• selective: (rich) media

• Differentials: extent of hemolysis

• distinguishes between types of staphylococcus

Mannitol Salt Agar (MSA)

• selective: high salt content; 6.5%

• differential: if mannitol is fermented, acidic end products are made. These turn the pH-senstive dye in agar from red to yellow

  • staph aureus = yellow

  • staph epidermidis = red

Growing Bacteria in the Lab

1. sterile growth medium - solid or liquid

2. aseptic/sterile technique

3. inoculation

4. growth - colonies or turbidity

Sterile Technique

• procedures implemented and special equipment available to avoid the spread of microorganisms

• creates a sterile work area (bunsen burner, biosafety cabinet)

• sterilize surfaces and instruments, buy sterile consumables

• wear gloves, face mask, or other protective equipment

• avoid incoming contamination - from the environment

• avoid outgoing contamination - into the environment

Obtaining a “pure culture” - descendants of a single cell

streak for isolation:

• mixed sample/culture - multiple species present

• streak for isolated colonies, pick one

• re-streak

• pure culture - one species present

Cell Counts

• total cell counts - culture-dependent and independent, everything including dead cells (microscope, turbidity)

• Viable cell counts - culture-dependent, only what grows in your medium (dilution-to-extinction, plate counts)

Pros and cons of viable cell counts

Pros:

• used in food, dairy, medical, and water analyses

• only this bacteria that live can produce toxins or other waste products harming humans or causing fouling

• high sensitivity

• can target particular species in mixed samples

Cons:

• depends on ability to culture

• standardize plating inconsistencies; inoculum size, viability, culture medium, incubation conditions

• mixed cultures grow at different rates

• report in colony-forming units instead of number of viable cells, accounts for clumps

The great plate count anomaly

• direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates/in cultures

• why?

  • microscopic methods count dead cells, whereas viable methods do not

  • scientist estimate and to-date we are able to cultivate about 1% of the bacterial diversity out there in nature

Controlling cell growth (factors other than growth medium components)

• temperature (revisit graph)

• pH

• oxygen

• salt

Life at low temperatures

• global oceans - covers 70% of earth’s surface, temp is about 1-5C

• all of arctic and Antarctica for most of the year

• even in solid frozen natural material are pockets of liquid water with concentrated solutes

• microorganisms from permanently cold environments are typically obligate psychophriles, those whose optimal growth is yet around 20C are pyschotolerant

• adaptation of membranes (more unsaturated fatty acids) and enzymes (more alpha-helices) to stay fluid at cold temperatures

• enzymes for colds wash laundry detergents

life at high temperatures

• sun0heated soil and sand, hot springs, hydrothermal systems

• specific organisms along the natural thermal gradient

• microorganisms from environments around 60C are typically obligate thermophiles, those from places around 80C or more are hypermophiles

• many of those are Archie, “hottest” bacterium found at 80C

• membranes have more saturated fatty acids to stay “firm”

• enzymes have increased ionic bonds between acidic and basic bonds, highly hydrophobic cores to prevent denaturing

• enzymes for PCR

pH effects on bacteria

• high pH = alkalic = high OH- concentration

• alkaline soils, lakes (soda lakes)

• microorganisms growing optimally at pH > 8 are alkaliphiles

• bacteria and archaea

• enzymes for laundry detergents

• Low pH = acidic = high H+ concentrations

• acidic soils, acid mine drainage

• microorganisms growing optimally at pH <5.5 are acidophiles

• bacteria and fungi

• use organisms for pickling food

Oxygen or no Oxygen

• obligate aerobes: require oxygen and grow at full oxygen tension - about 21%

• obligate anaerobes: oxygen is harmful or lethal

• facultative aerobes: can respire oxygen when present, uses alternative ways when absent

• microaerophiles: can use oxygen only when it is present at levels reduced from that in air due to limited respiration or oxygen sensitivity

• aerotolerant: do not use oxygen for growth at all, but are also not harmed by its presence

**Chart**

why can oxygen be toxic?

O2 is not

• exposure to oxygen yields toxic byproducts:

  • superoxide anion

  • hydrogen peroxide

  • hydroxyl radical

• many cells have enzymes to detoxify

  • Catalase and peroxidase convert H2O2 to O2 and H2O

  • superoxide dismutase converts 2O2- to H2O2 and O2

  • superoxide reductase in some strict anaerobes converts O2- to H2O2 without producing O2

Osmolarity

• Osmosis: water diffuses from high to low (solute) concentrations

• typically the cytoplasm has a higher solute concentration than the surrounding environment; thus, the tendency is for water to move into the cell (positive water balance)

• when a cell is in an environment with a higher external solute concentration (high salt), water will flow out

• in order to not loose water, cells produce compatible solutes: highly water-soluble organic molecules, sugars, alcohols, amino acid, that do not interfere with metabolism

Control bacteria

• sterilization, disinfecting.. (all methods are not specific to any bacterial species, gram-type, etc.)

1. heat

• autoclaving, pressurized saturated steam (121C) for 30 minutes destroys all biomolecules, FULL sterilization of all types of materials

• pasteurization, heating to 75C for 30 seconds destroys most microorganisms, but not endospores, sterilization of liquids

2. physical

• radiation, UV, X-rays, destroys mainly DNA

  • useful for decontaminating surfaces

  • some bacteria, viruses and endospores survive

• filtration, for liquids or air

3. chemical

• alcohols - ethyl and isopropyl

  • used as disinfectant and antiseptic, denatures proteins and solubilize lipids

• sodium hypochlorite - bleach

  • disinfectant, used to sanitize surfaces

  • denatures proteins and oxidizes macromolecules

• cresols - lysol, and phenols - vesphene

  • disinfectant, used to sanitize surfaces, dentures proteins

• soaps and detergents

  • weak antiseptics, lower surface tension, disrupt cell membranes/permeability

• heat & physical only for inanimate objects

• chemical disinfectants (inanimate objects), antiseptics (living tissue)

**revisit charts**

antimicrobial agent susceptibility assay using diffusion method

• inoculate plate with a liquid culture of a test organism

• disks containing antimicrobial agents are placed on surface

• incubate for 24 to 48 hours

• test organism shows susceptibility to some agents, indicated by inhibition of bacterial growth around disks (zones of inhibition)