Chapter 7 Microbial Growth

(Plus A Small Portion of Chapter 3!)

Prokaryotic Cell Division

Binary Fission

Generation Time
Time required by a cell to ________? In each generation, the cell number __________?

If Conditions are Ideal - Bacteria Undergo Exponential Growth

Solve These!
8 cells land on a hamburger Generation time is 30 mins How many cells after 4 hours? __________________________________ If 5 cells were to fall onto a wound How long before there were over 1000 cells if the generation time is 15 minutes? __________________________________
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Solve These!
If 4 bacteria are dividing logarithmically, how many bacteria will there be in the sixth generation? ___________________________________ How long will it take for 1 cell to turn into a million or more if the pathogen has a 20 minute generation time? ___________________________________
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Growth Curve - Phases of Growth

1. 2. 3. 4. Lag phase flat period of adjustment, enlargement; little ___________ Exponential growth phase a period of maximum __________ as long as cells have adequate ________ & a favorable ___________ Stationary phase rate of cell growth equals rate of cell _________ depleted nutrients & ____, excretion of organic acids & waste __________ Death phase limiting factors intensify, cells die exponentially in their own wastes. Log decrease.

Cell Division

Growth curve
Which phase would be most affected by an antibiotic targeting cell wall growth? When would endospores be most common? When would heat treatment be most/least effective?

Measuring Microbial Growth

Bacteria can grow so quickly, that a culture often needs to be diluted before it can be counted!

The Serial Dilution

PLATE COUNTS - The Serial Dilution Method

Will all cells be able to grow on this medium? What if 2 cells land on the same spot?

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Colony Forming Unit (CFU) = bacterial cell! Number of colonies x Dilution Factor = Number of CFU/mL
DF = reciprocal of the dilution!

Suppose, average number of colonies = 81 dilution was 1:100,000 (10-5)

DF = 100,000 (105)

81 x 100,000 = 8.1 x 106 CFU/mL

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Most Probable Number Tubes

Statistical Estimate

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Division

Direct microscopic count

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Division

Turbidity

Living cells versus dead cells. Can this technique distinguish between them? What could interfere with the results? Overestimates?
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Division

Electronic counting

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Metabolic Activity

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Dry Weight Estimates

How accurate would this be with variable cells instead of pennies?

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Microbial Numbers Too Low

Concentrate sample by filtration

Filter allows water to pass through not ____________

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Microbial Numbers Too Low

Centrifuge sample. Cells become concentrated into a pellet

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Microbial Numbers Too High

Serially dilute the sample until countable.

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Environmental Influences

Environmental influences on microbial growth

T________________ O________ requirements ____ E____________ radiation B____________ pressure

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Environmental Influences

3 Cardinal Temperatures
Minimum temperature ______ temperature permitting a microbes growth & metabolism Maximum temperature _____ temperature permitting a microbes growth and metabolism Optimum temperature ______ & fastest rate of growth and metabolism
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Environmental Influences

Main temperature adaptation groups

1. Psychrophiles optimum temperature below ___oC, capable of growth at 0oC 2. Mesophiles optimum temperature ____-____oC, most human pathogens 3. Thermophiles optimum temperature greater than ___oC

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3 Main Temperature Adaptation Groups

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Environmental Influences

Oxygen Requirements

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Optimum pH Growth Curves

Acidophile Neutrophile Alkalophile

Use buffers to stabilize the medium pH

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Minimum, maximum and optimum pH.

Organism Thiobacillus thiooxidans Sulfolobus acidocaldarius Bacillus acidocaldarius Zymomonas lindneri Lactobacillus acidophilus Staphylococcus aureus Escherichia coli Clostridium sporogenes Erwinia caratovora Pseudomonas aeruginosa Thiobacillus novellus Nitrobacter spp Minimum pH Optimum pH Maximum pH 0.5 1.0 2.0 3.5 4.0-4.6 4.2 4.4 5.0-5.8 5.6 5.6 5.7 6.6 2.0-2.8 2.0-3.0 4.0 5.5-6.0 5.8-6.6 7.0-7.5 6.0-7.0 6.0-7.6 7.1 6.6-7.0 7.0 7.8 7.6-8.6 4.0-6.0 5.0 6.0 7.5 6.8 9.3 9.0 8.5-9.0 9.3 8.0 9.0 8.3 10.0
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Streptococcus pneumoniae 6.5

Osmotic Conditions - Halophiles

Hypersaline Lakes ___-___% sodium chloride
Eg ______________City _________ food

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Microbial nutrition
Macronutrients required in _______ quantities; play principal roles in cell structure & metabolism Micronutrients or trace elements required in _______ amounts; involved in enzyme function & maintenance of protein structure
Man_________, z_____, n_______ p________, c___________________

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Nutrients
Inorganic nutrients atom or molecule that contains a combination of atoms other than c___________ and h__________

Organic nutrients - contain carbon and hydrogen atoms and are usually the products of l________ things
M_________ (CH4), c______________, lipids, p______________, and nucleic acids

metals and their salts (mag________ sulfate, ferric nitrate, sodium phosphate), gases (o______, carbon d_________) and w________

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Chemical Composition of Cytoplasm

______% water proteins 96% of cell is composed of 6 elements
Carbon Hydrogen Oxygen Nitrogen Phosphorous Sulfur

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Sources of Essential Nutrients

Obtaining Carbon
Heterotroph an organism that must obtain carbon in an ________ form made by other living organisms eg proteins, _____________, ________ and _________ acids Autotroph - an organism that uses CO2, an _____________ gas as its carbon source
not dependent on other ________ things
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Carbon & Energy Source

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Carbon Energy source source Photoautotrophs Chemoautotrophs _____ _____ _____ _____ _______ _______ _______ _______ _______ _______ _______ _______

Examples?

Photoheterotrophs _____ _____ Chemoheterotroph _____ s _____

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Carbon source
Photoautotrophs Chemoautotrophs

Energy source sunlight Simple inorganic chemicals sunlight

Metabolizing organic cpds

Examples?

CO2 CO2

Photoheterotrophs organic Chemoheterotroph s

organic

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Environmental Influences

Microbial Associations
Symbiotic organisms live in close nutritional relationships; required by one or both members
Mutualism co-dependent; both members b_________ Commensalism commensal member b________, other member not harmed Parasitism parasite is dependent and b________; host is __________
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Microbial Lifestyles
Saprobes decompose _______ organisms, recycle elements, release __________ to digest materials Parasites utilize tissues and fluids of a _________ host and cause harm Ectoparasites live ___ the body Endoparasites live ___ organs/tissues Intracellular parasites live within ______
Obligate parasite cannot grow outside host Obligate ______________ parasite need to live inside host cell

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Environmental Influences

Microbial Associations
Non-symbiotic organisms are freeliving; relationships not required for survival
Synergism members cooperate and share nutrients Antagonism some members are inhibited or destroyed by others

(Free-Living: Primary Producers, Herbivores, Carnivores, Omnivores)

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Sources of Essential Nutrients

Nitrogen
_____% of earths atmosphere is N2 Nitrogen is part of the structure of ________, ______ acids, DNA, RNA & ATP primary source of N for ________trophs. Some bacteria & algae use inorganic N nutrients (______, ______, ______) Some bacteria can fix _______ N must be converted to _______, the only form that can be combined with carbon to synthesis amino acids, etc.
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Sources of Essential Nutrients

Hydrogen
major element in all _________ compounds & several inorganic ones (water, salts & gases) gases are produced & used by microbes roles of hydrogen
maintaining pH forming H bonds between molecules serving as the source of free energy in oxidationreduction reactions of respiration

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Sources of Essential Nutrients

Phosphorous
main inorganic source is ________ (PO4-) derived from phosphoric acid (H3PO4) found in rocks & oceanic mineral deposits key component of _________ acids, essential to genetics serves in _________ transfers (ATP)

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Sources of Essential Nutrients

Sulfur
widely distributed in environment, rocks, sediments contain ______, sulfides, hydrogen sulfide gas and sulfur essential component of some vitamins and the amino acids: m__________ & c__________ contributes to stability of proteins by forming disulfide bonds
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Essential Mineral Ions

Trace Elements:
Potassium Sodium Calcium Magnesium Iron

Why is each of these needed?

Usually supplied by t___ w________ in lab media!

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Essential Growth factors

Organic Compounds that cannot be s_________ by an organism
essential amino acids, vitamins

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Sources of Essential Nutrients

Oxygen
____% of the atmosphere plays an important role in structural & enzymatic functions of cell component of organic molecules, inorganic salts (sulfates, phosphates, nitrates) & water essential to metabolism of many organisms
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Oxygen Poison or Evil Necessity?

Obligate/strict aerobe? Obligate anaerobe? Facultative anaerobe? Microaerophile? Aerotolerant anaerobe?

Metabolism Generates
Free Radicals O2Superoxide ions

O2 is very reactive, free radicals destroy m____________ 1) 2O2+ 2H+

Protective Enzymes

OHHydroxyl radicals

Superoxide --------------------> Dismutase (SOD)

H2O2

+ O2

2) 2H2O2

----------------------->

Catalase

2H2O

O2 47

Special Incubators: Anaerobic chambers

Anaerobes e.g. Clostridium sp.

CO2 incubators

Capnophiles e.g. Neisseria

Strep pneum.

Some pathogens require high CO2 concentrations

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Tools of the Laboratory Culturing Microbes

Ch 3 Contd.

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The 5 Is of Culturing Microbes

1. Inoculation introduction of a sample into a
container of media

2. Incubation under conditions that allow growth 3. Isolation separating one species from another 4. Inspection 5. Identification

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1. Inoculate and 2. Incubate

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3. Isolate,

4. Inspect,

and 5. Identify

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Isolation
Individual bacterial cell is separated from other cells & has space on a nutrient surface, it grows into a mound of cells - a pure colony A pure colony consists of one species
Streak Plating

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Media providing nutrients in the laboratory

Most commonly used: Nutrient broth liquid medium containing beef extract & peptone Nutrient agar nutrient broth solidified with agar Agar = complex polysaccharide isolated from red algae Solid at room temp, liquefies at boiling (100oC) Solid matrix for moisture & nutrients Not digested by most microbes

Types of Media

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Types of Media Chemical Composition

listed with their exact c_______ f________ e.g. sodium citrate broth.

Synthetic (Chemically Defined), all compounds known and Nonsynthetic (Complex)

has no f_________ e.g. beef.

contains at least one ingredient that

1.

General Purpose Media

Specific Function

- grows a b________ range of microbes, usually N______________ ingredients

2.

Enriched Media - contains complex o_________ substances such as

____________, _______, serum, hemoglobin or special growth factors required by fastidious microbes

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Media with a Specific Function

3.

- continued

Selective Media - contains one or more agents that i___________

growth of some microbes and ____________ growth of the desired microbes

4.

Differential Media allows growth of several types of microbes

but displays visible d____________ among desired & undesired microbes

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Selective Media

Differential Media

Miscellaneous Media
Reducing Medium contains a substance that absorbs o_____________; used for growing a__________ bacteria Carbohydrate Fermentation - contains sugars that can be fermented, converted to acids, which change the color of a ____________ to show the reaction; Fermentation Tubes - bacterial/fungal ID
Thioglycolate Broth

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Obligate Intracellular Media

What lab media would you use to grow an obligate intracellular pathogen? ___________________________

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Bubble, bubble, toil and trouble, Until those microbes grow!

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