MCB CH6

Microbial growth

Increase in population of microbes

Reproduction of individual microbes

Results:

Discrete colony - aggregation of cells arising from single parent cell

Biofilm - collection of microbes living surface in complex community

TEACHER: GROWTH = SIZE REPRODUCTION = NUMBER

Growth Requirements

Organisms use variety of nutrients - energy needs- to build organic molecules & cell struct

Most common nutrients: necessary elem: carbon, oxygen, nitrogen, hydrogen, sulfur, phosphorus

Microbes obtain nutrients from variety of sources

Nutrients: Chem & Energy Requirements

Sources of carbon, energy, and electrons

2 groups of organisms based on source of carbon:

Autotrophs — using INORGANIC carbon (CO2)

Heterotrophs — using ORGANIC carbon (Glucose, Sucrose, Amino Acid, Protein, Fat)

2 groups of organisms based on source of energy:

Chemotrophs — Oxidation of organic / inorganic molecules

Phototrophs — Light

E- Source (LAB):

Litho — INORGANIC E- (Chromium, Iron, Zinc)

Organo — NADH, FADH2, NADPH

BE ABLE TO IDENTIFY THESE TERMS

Nutrients Chem, Energy

2 groups of organisms based on source of electrons:

Organotrophs

Lithotrophs

“Belong together, order doesn’t matter as long as correct”

Oxygen Requirements

Oxygen essential for obligate aerobes

Oxygen deadly for obligate anaerobes

How true?

Toxic forms of oxygen high reactive + excellent oxidizing agents

Resulting oxidation causes irreparable damage to cells

4 Toxic Forms of Oxygen

Singlet Oxygen ½ O

Superoxide Radicals o3

Peroxide anion H2O2

Hydroxyl radical [-OH]

SOD’s NEEDED TO GROW IN PRESENCE OF OXYGEN

CATALASE — enzyme breaks down peroxide into water & free oxygen

H2O2 → H2O + O2

Organisms & Oxygen Concentrations

Aerobes

Anaerobes

Facultative anaerobes → grow w/ or w/o oxygen

Aerotolerant anaerobes = w/ or w/o oxygen

Microerophiles → require oxygen 2-10%

Liquid Identify Oxygen Req

Resazurin → Oxygen Indicator

Nitrogen Requirements

Anabolism often ceases due to insufficient nitrogen

Nitrogen acquired from organic + inorganic nutrients

All cells recycle nitrogen for amino acids + nucleotides

Nitrogen fixation by certain bacteria essential to life on Earth.

Other Chem Requirements

Phosphorus — DNA / RNA / cell membrane

Sulfur — met, cys (A.A.)

Trace elements - only req in small amts

Growth factors — necessary organic chemicals that cannot be synthesized by certain organisms

Growth Factors & Funct

Physical Requirements (Temp)

Temperature affects 3d structure of proteins

Lipid containing membranes of cells and organelles are temperature sensitive

— If too low, membranes rigid & fragile

— If too high, membranes too fluid

Temperature & Growth

37 is body temp

Organisms that grow best at body temperature = Mesophiles

5 Cat of Microbes - Temp

Physical Requirements (pH)

Organisms sensitive to changes in acidity

Hydrogen ions interfere w/ H bonding

Neutrophiles grow best in narrow range around neutral pH 6-8

Acidophiles grow best in acidic habitats <6

Alkalinophiles live in alkaline soils and water 8+

Phys effect of water

Microbes require water to dissolve enzymes and nutrients

Water important reactant in many metabolic reactions

Most cells die in absence of water

Some have cell walls retain water

Endospores and cysts cease most metabolic activity

Two physical effects of water:

Osmotic pressure

Hydrostatic pressure — Barophiles

Osmotic Pressure Phys effect of water

Osmotic pressure

Pressure exert on semipermeable memb by solution containing solute cannot freely cross memb

— Hypotonic solutions have lower solute concent

Cell placed in hypotonic solution swells

— Hypertonic solutions have greater solute concentrations

Cell placed in hypertonic solution shrivels

— Restricts organisms to certain environments

Obligate and facultative halophiles—(NEED SODIUM TO GROW)

Hydrostatic pressure Phys effects of water

Hydrostatic pressure

Water exert pressure proportion to depth

Barophiles live under extreme pressure

Membranes + enzymes depend on pressure to maintain 3d shape

Associations & Biofilm

Organisms live assoc w/ diff species

Antagonistic relationships — compete against each other

Synergistic relationships — mutualistic (benefit)

Symbiotic relationships — grow together

Biofilms

Complex relationships among numerous microorganisms

Form on surfaces, medical devices, mucous membranes of digestive system

— Form as result of quorum sensing (small molecule communication) IMPT

Many microorganisms more harmful as part of biofilm

Scientists seek ways to prevent biofilm form

Culturing

Culture — cultivating microorganisms / that are cultivated

inoculum — microoorganisms intro to medium containing nutritents

Inocula obt from various soruces

Environmental specimens

Clinical specimens

Stored specimens

Obt pure cultures

Cultures comp of cells arise from single progenitor

Progenitor termed colony forming unit CFU

Aseptic technique prevents contam of sterile subs / obj

2 common iso techniques:

Streak plate

Pour plate

Culture Media

Majority of prokaryotes have not been grown in culture medium

Variety of liquid & solid media used to culture microbes

Nutrient broth — common liquid medium

Agar — common addition to make media solid

• used to make petri plates & slant tubes

6 types of culture media

Defined — every single component in media & concentration

Complex — remains unknown, concent / agent

Selective — inhibits growth of one while promoting other

Differential — distinguishing different microbes & phenotype

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Anaerobic — allows growth for obligate anaerobes (reducing agent to prevent oxygen getting to sample)

Transport — swab & transport, keep stable for lab

Culture Media

Types of media:

EMB, MAC, SS, MSA

Know all 4 + Differential

Blood Agar (differential & enriched) VS Chocolate Agar (only enriched)

REVIEW changes in color (its meaning)

MAC agar selective & diff

Preserving Culture

Refrigeration

Stores for short periods of time —30 days

Deep-freezing

Stores for years -80c* DMSO

Lyophilization

Stores for decades

Growth of microbial populations

Most microorganisms reproduce by binary fission

One divides in half to produce 2 daughter cells

Involves 4 steps

Math Cells & Time

Cells present after time

At end use calculator to input formula

End result, 51,200

Formula Nt (cells present after time)= N(number of cells begin) x 2^n

***ALWAYS ROUND AT 2nd decimal place for TEST

Typical Population Growth Curve

BE FAMILIAR WITH GROWTH CURVE — EXAM

Continuous culture in chemostat

Chemostat — maintains microbial population in particular phase of growth

Open system

Requires addition of fresh medium + removal of old medium

Used in several industrial settings

Estimating # of microorganisms microbial growth

-Estimating number of microorganisms useful

Determine severity of certain infections

Determine effectiveness of food preservation techniques

Measure degree of contamination of water supp

Eval disinfectants & abx

-Direct methods not req incubation

• Microscopic counts

Direct methods not req incubation microbial growth

Electronic counters

Coulter counter

Counts cells as they interrupt electrical current

Flow cytometry

Detects changes in light transmission as cells pass detector

Direct methods requiring incubation microbial growth

Serial dilution and viable plate counts ← titer

Membrane filtration

Most probably number

Serial dilution & viable plate counts

Indirect methods microbial growth

Turbidity

Often more turbid a culture, greater the bacterial population