MICROBIAL NUTRITION AND GROWTH

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MICROBIAL NUTRITION

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Biochemical Components of Cells

Water: 80 % of TOTAL BIOMASS Dry weight
Protein 40-70 % Nucleic acid 13-34% Lipid 10-15 % Also monomers, intermediates and inorganic ions

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Macronutrients
These are used to synthesize biomolecules (C, O, H, N, S, P) and to regulate many important metabolic and structural functions (K+, Ca2+, Mg2+, Fe2+)
proteins, carbohydrates, lipids and nucleic acids are primarily made up of (COHNSP) ions have variable roles (eg. K+ is needed to activate enzymes during photosynthesis; Ca2+ is responsible for the heat resistance of bacterial endospores; Mg2+ is a cofactor for many enzymes; Fe2+/3+ is an important component of electron transport systems)
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Micronutrients
These are needed in trace quantities that even very slight contaminants in aqueous environments are enough to meet the demands of microbes.
Trace elements include Ni, Mo, Co, Cu, Mn, and Zn Growth factors such as vitamins and some enzymes are also essential but are needed only in trace quantities.

NUTRITIONAL TYPES

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NUTRIENT UPTAKE
Passive Transport does not require energy; substances exist in a gradient and move from areas of higher concentration towards areas of lower concentration diffusion osmosis (diffusion of water) facilitated diffusion (permeases) Active Transport requires energy and carrier proteins; gradient independent active transport (symport, antiport) group translocation (transported molecule chemically altered) bulk transport (endocytosis, exocytosis, pinocytosis)
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DIFFUSION ACROSS A MEMBRANE

PASSIVE

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PASSIVE

TONICITY

PASSIVE

FACILITATED DIFFUSION -utilizes channel and carrier proteins collectively called as permeases

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ACTIVE

ACTIVE TRANSPORT IN PROCARYOTES

ACTIVE

ACTIVE TRANSPORT USUALLY UTILIZES TRANSPORT PROTEINS

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ACTIVE

Group Translocation

5 proteins involved. transported substance (glucose) is chemically altered (glucose-6-P) during transportation
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ACTIVE

Group Translocation

SIDEROPHORES

ACTIVE

ENDOCYTOSIS

MICROBIAL GROWTH
Microbes especially bacteria divide asexually via binary fission. Generation Time - the time required for parent cell to form two new daughter cells. Microbial growth is always measured by number, not by biomass.
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MICROBIAL GROWTH CURVE

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Calculating Growth of Cells

Nf = (Ni)2

Nf = Final population Ni = Initial population 2n = # cells in generation

n

= generation number
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Doubling times for some common bacteria under optimal conditions of growth
Bacterium Escherichia coli Bacillus megaterium Streptococcus lactis Streptococcus lactis Staphylococcus aureus Lactobacillus acidophilus Medium Glucose-salts Sucrose-salts Milk Lactose broth Heart infusion broth Milk Generation Time (minutes) 17 25 26 48 27-30 66-87 344-461 (6hr) 792-932 (15hr)
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Rhizobium japonicum Mannitol-salts-yeast extract Mycobacterium tuberculosis Synthetic

Measuring Growth: Direct Count, Serial Dilutions, Plate Counts and Turbidity

A. Direct Count

B. Viable Count
Spread-plate method Pour-plate method Pour-plate method with serial dilution

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C. Indirect Count: Turbidity Measurement

Calculation of the Number of Bacteria per milliliter of Culture Using Serial Dilution

Pour plate: made by first adding 1.0ml of diluted culture to 9ml of molten agar

Spread plate: made by adding 0.1ml of diluted culture to surface of solid medium

Colony Counter

Plate as viewed under the colony counter grid.

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Countable number of colonies (30 to 300 per plate)

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The Petroff-Hausser Counting Chamber

Turbidity, or a cloudy appearance, is an indicator of bacterial growth in urine in the tube on the left

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A Spectrophotometer: This instrument can be used to measure bacterial growth by determining the degree of light transmission through the culture

STREAK PLATE TECHNIQUE

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A Streak Plate of Serratia marcescens. Note the greatly reduced numbers of growth /colonies in each successive region.

Types of Culture Media

Natural Media: In nature, many species of microorganisms grow together in oceans, lakes, and soil and on living or dead organic matter Synthetic medium (Defined): A medium prepared in the laboratory from material of precise or reasonably welldefined composition Complex medium (Undefined): contains reasonably familiar material but varies slightly in chemical composition from batch to batch (e.g. peptone, a product of enzyme digestion of proteins)
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Selective, Differential, and Enrichment Media

Selective Medium: encourages growth of some organisms but suppresses growth of others (e.g. antibiotics) Differential Medium: contains a constituent that causes an observable change that can differentiate one population from the other (e.g. MacConkey agar) Enrichment Medium: contains special nutrients that allow growth of a particular organism that might not otherwise be present in sufficient numbers to allow it to be isolated and identified
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Bristol's Medium for Algae (mg/liter) NaNO3 K2HPO4 KH2PO4 CaCl2 NaCl MgSO4.7H2O FeCl3 MnSO4.4H2O ZnSO4.7H2O H3BO3 CuSO4.5H2O 250 mg 75 mg 175 mg 25 mg 25 mg 75 mg 0.3 mg 0.3 mg 0.2 mg 0.2mg 0.06 mg

DEFINED MEDIA
-all components are known

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e st O f COMPLEX MEDIA - components C a Peptone/Tryptone/Yeast Extract s Bacteriological Media Ingredients ei ANIMAL BASED & ANIMAL FREE VEGETABLE n PEPTONES:- Wide gamut of peptones for cell nutrition, fermentation and media ingredients. N Peptones are low-cost growth promoting nutrients ut extensively as Dehydrated Culture media used ri ingredients,fermentation ingredients as animal cell culture nutrients. P e Animal Free Peptones p VegiPep S : Soy Peptone NON ANIMAL,NON GMO A VegiPep G : Peanut Peptone NON ANIMAL, NON GMO Animal Origin Peptones ci NutriPep Bacto Peptone d Nutri Pep Casein Peptone H Nutri Pep Meat Peptone y NutriPep Proteose Peptone dr NutriPep Pancreatic Digest Of Casein Nutri ol Pep Acid Hydrolyzate of Casein Nutri Pep Acid Hydrolyzate of Soy y NutriPep Tryptose Nutri Pep Tryptone. z NutriPep Meat Extract Powder at Pep Yeast Extract Powder Nutri e

are unknown

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SELECTIVE MEDIA favor growth of organism of interest

XLT-4 Agar
The detection of Salmonella species is often complicated by the presence of background flora and other Enterobacteriaceae on an agar plate. The presence of the selective agent, Tergitol 4, in XLT-4 Agar inhibits many organisms that can be problematic on other plating media. In addition, biochemical and pH changes within the medium allow Salmonella spp. (black colonies) to be differentiated from organisms, such as E. coli (yellow colonies) and Shigella spp. (red colonies).

DIFFERENTIAL MEDIA show comparative changes Blood Agar is a bacterial differential medium that can distinguish normal from pathogenic bacteria based on the interaction of sheep's blood and bacterial hemolytic enzymes.

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Identification of urinary tract pathogens with differential media (CHROMagar)

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FACTORS AFFECTING GROWTH Temperature pH Oxygen Pressure Salinity

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Effects of Temperature on Growth

40oF

77oF

95oF

Most of our plates are incubated at 37oC (98.6oF).

Conversion C to F = 1.8xC + 32 http://www.sciencemadesimple.com/conversions.html
Image: Pearson Education Inc. (2004) publishing as Benjamin Cummings

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Categories of Microbes Based on Temperature Range

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Psychrophiles (< 20 oC)

Polar regions are the usual habitat of psychrophiles.

A form of psychrophile named Strain34h, stained fluorescent blue for better viewing.

Mesophiles (15-45oC)
The habitats of these organisms include soil, the human body, animals, etc. The optimal temperature of many pathogenic mesophiles is 37 C (98 F), the normal human body temperature. Mesophilic organisms have important uses in food preparation especially in cheese and yogurt making and in beer and wine making.

E. coli

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Thermophiles (45-80oC)

Thermophiles produce some of the bright colors of Grand Prismatic Spring, Yellowstone National Park

Hyperthermophiles (65-110oC)

EXTREMELY HOT!!!

Mostly Archaeans believed to be the first microbes on earth!!!

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pH and Growth

These unknown bacteria were taken from the film on the surface of sulfuric acid.

Acidobacterium -abundant in soils (pH 3-5)

Meet the Microbe! Vibrio cholerae (Cholera bacteria) will grow outside the body at a pH of 9.0.

Oxygen Requirements
Obligate Aerobes need oxygen to stay alive Obligate Anaerobes die in presence of oxygen Facultative Anaerobes not strict anaerobes; optimum growth in anaerobic conditions but can still grow in the presence of oxygen Microaerophillic Bacteria require oxygen levels lower that that found under normal atmospheric conditions; aerobic but optimum growth is when oxygen is minimal Aerotolerant Anaerobes - can tolerate a small amount of oxygen by being able to detoxify the poisonous forms of oxygen present
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1: Obligate aerobic bacteria gather at top of test tube to absorb maximal amount of O2. 2: Obligate anaerobic bacteria gather at bottom to avoid oxygen. 3: Facultative bacteria gather mostly at the top, since aerobic respiration is beneficial; but as lack of oxygen does not hurt them, they can be found all along the test tube. 4: Microaerophiles gather at upper part of test tube. They require O2, but at low concentration. 5: Aerotolerant bacteria are not affected by oxygen, and they are evenly spread along the test tube.

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Osmotic Pressure
Some bacteria can be plasmolyzed by high concentrations of solutes, which results in the formation of plasmolysis bays throughout the length of the cell. These bays result from the contraction of the cytoplasmic membrane in response to hyperosmotic shock.

Why can you keep honey on the cupboard for months, even years, without it spoiling?
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Salinity

Seawater: 3% NaCl Nonhalophile: less than 1% Halotolerant: less than 5% Halophile: 1 to 15 % Extreme halophile: more than 15%

Pressure
Barophiles
- organisms that grow at elevated pressure (3-1000 x air pressure). (Found in ocean depths often in thermal vents)

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THANKS!!!

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