Standard Growth Conditions and Measurement of Growth

Binary fission in bacteria

Scanning electronmicrograph

Geometric progression in the number of

bacteria in a population resulting from binary fission

Generation time length of time it takesa single bacterium to double

E. coli 25 minutesMycobacterium spp. 1-3days.

Binary fission requires the addition of new material at the growing sites of bacteria

Gram positive cells such asthis coccus, new material is added at the division plane

Gram negative cells such asthis bacillus, new material isadded at the division plane and also throughout the length ofthe cell

Bacteria need to synthesize the macromolecules that allow for theirgrowth and reproduction.

Bacteria need to synthesize macromolecules that allow for their growth

and reproduction— what are bacterial cells made up of ?

Cells consist of WATER and MACROMOLECULES

Macromolecules are made up of smaller monomeric molecules

Small monomeric molecules are made up of atoms

Macromolecules of the bacterial cell

Proteins—The most abundant class of macromolecules and comprisemost of the structures of the cell as well as enzymes

amino acids—monomeric subunits of proteinsconsists of carbon, hydrogen, oxygen, nitrogen, sulfur andsometimes seleniumamino acids are covalently linked to form a peptide bond

NH3 C COOH

H

R

NH3 C C

H

R1

C COOH

H

R2O

N

H

Peptide bondWhere have you heard theword tetra-peptide?

Macromolecules of the bacterial cell (cont’d)

Polysaccharides—2nd most abundant of the bacteral macromoleculessugars (monosaccharides)—monomeric units consists of Carbon, Hydrogen and Oxygen atoms at a ratioof 1:2:1individual sugars are linked by a glycosidic bond

Polysaccharides form covalent linkages with other macromoleculeswith proteins—glycoproteinswith lipids—glycolipids, lipopolysaccharides

Macromolecules of the bacterial cell (cont’d)

Nucleic Acids DNA—deoxyribonucleic acidRNA—ribonucleic acid

Backbone of nucleic acids= polymer of phospho-ribose (RNA)or phospho-deoxyribose (DNA)The sugars are covalently attached to each other by phospho-diester bondsBases are attached to a carbon atom of the sugar moiety

cytosine, adenine, guanine (DNA/RNA); thymine (DNA)uracil (RNA)

Comprised of the atoms Phosphorous, Carbon, Hydrogen, Oxygenand Nitrogen

Macromolecules of the bacterial cell (cont’d)

Lipids—made up of a long carbon chain—fatty acids—(14-20carbons and one carboxylic acid group)

Saturated—Hydrogen atoms attached to most or all carbonmoieties.Unsaturated—fewer Hydrogen atoms associated with carbons

Complex lipids are attached to simple sugars like phoshoglycerol (ie phospholipids) or complex polysaccharides (LPS)

C, H, O, P

Bacterial Nutritional Requirements

NUTRITION::act of supplying microorganisms with the moleculesand atoms they require for the biosynthesis of small moleculesand macromoleculesMacronutrients: nutrients required in high amounts

Carbon, Nitrogen, Phosphorous, SulfurALSO

Potassium, Magnesium, Calcium, SodiumMicronutrients: nutrients required in small or even trace amounts

Chromium, Cobalt, Copper, Manganese, MolybdenumNickel, Selenium, Tungsten, Vanadium, Zinc, Iron

Growth Factors::organic compounds required in very small amounts

Vitamins, amino acids, purines and pyrimidines

Bacterial Nutritional RequirementsAutotroph: an organism capable of biosynthesizing all cellmaterial from CO2 as a sole carbon sourceHeterotroph: an organism that requires carbon from preexistingorganic material

Photo--: solar energy converted to chemical energyChemo--: energy derived from chemical compounds

Catabolism: Act of breaking down complex molecular materialfor energy or biosynthetic material Anabolism: The act of biosynthesizing complex material from simplerorganic compoundsCulture media for artificial cultivation of bacteria in the lab

1. Complex (undefined)—enzymatic digests of milk protein(casein), beef, yeast2. Chemically defined—precise amounts of purified organicand inorganic compounds are added to distilled water

Types of culture media

FastidiousBrock, 10th edition

Other considerations with respect to bacterial growth

1. pH—optimum pH of most organisms is 7.02. Water activity—most bacteria require a water activity between 0.9 and 1.03. Osmolarity—The osmolarity of the bacterial cell cytoplasm

must be greater than that of its environment for cell growth—turgor pressure

4. Oxygen—bacteria have a great variety of specifications with respect to the amount of oxygen they require5. Temperature—most organisms like 37oC

About oxygen

Aerobes—capable of growth at full oxygen tensionsMicroaerophiles—can only grow when oxygen tensions are lowerthan that found in air (soil, water bacteria)Anaerobes—

obligate anaerobes—oxygen kills the organismfacultative anaerobes—prefer oxygen but can grow in its absenceaerotolerant anaerobes—can grow in the presence of oxygenbut they don’t use it.

Quantifying bacteria

Direct microscopic cell count

Viable cell count (plate count or colony count)

Indirect measurement::microbial turbidity

Use of the hemocytometer or Petroff-Hausser counting chamber (direct microscopic count)

Sample added to the surface ofthe grid, the whole grid has 25large squares, the total volumethat can be added is 0.02mm3

12 bacteria in one square(assume 12 in each large square) 12 X 25 = 300 bacteria

Total volume held is 0.02 mm3

300/.02 = 15000 or 1.5 X 104

Bacteria per mm3

1cm3 = 1000mm3 (10 x 10 x 10)1.5 X 104 x 1000 = 1.5 X 107/cm3 or mL

Use of dilution and direct plating to measure viable bacteria (viable cell count)

Use of the spectrophotometer to quantify bacteria in a population

OD= Log Io/IIo incident lightI unscattered light

Use of the spectrophotometer in measuring the number of cells in a population

By using both spectro-photometry and dilutionsand plate counts one can make a good correlationbetween optical densityand cell population numbers

Stages of bacterial growth::typical growth curve

Biphasic Growth Curve

Premature stationary phasecells have exhausted available glucose

2nd stationary phasecells have exhaustedlactose

2nd log phase, cells prepareenzymes required for the catabolism of lactose