Microbiology 2

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4.3 Culturing and Counting Bacteria

Microbes in nature exist in complex, multispecies communities, but for detailed studies they must be grown separately in pure culture.

After 120 years of trying, we have succeeded in culturing less than 1% of the microorganisms around us.

The vast majority has yet to be tamed.

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Bacteria Are Grown in Culture Media – 1

Bacteria are grown in culture media, which are of two main types:

Liquid or broth

Useful for studying the growth characteristics of a pure culture

Solid (usually gelled with agar)

Useful for trying to separate mixed cultures from clinical specimens or natural environments

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Bacteria Are Grown in Culture Media – 2

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FIGURE 4.12 ■ Separation and growth of microbes on an agar surface. A. Colonies (diameter 1–5 mm) of Acidovorax citrulli separated on an agar plate. A. citrulli is a plant pathogen that causes watermelon fruit blotch. B. A mixture of yellow-pigmented bacterial colonies, wrinkled bacterial colonies, and fungus separated by dilution on an agar plate. As time passed, the fungal colony overgrew adjacent bacterial colonies.

Dilution Streaking and Spread Plates – 1

Pure colonies are isolated via two main techniques:

Dilution streaking

A loop is dragged across the surface of an agar plate.

Spread plate

Tenfold serial dilutions are performed on a liquid culture.

A small amount of each dilution is then plated.

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Dilution Streaking and Spread Plates – 2

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FIGURE 4.13 ■ Dilution streaking technique. A. A liquid culture is sampled with a sterile inoculating loop and streaked across the plate in three or four areas, with the loop flamed between areas to kill bacteria still clinging to it. Dragging the loop across the agar diminishes the number of organisms clinging to the loop until only single cells are deposited at a given location. B. Salmonella enterica culture obtained by dilution streaking.

Dilution Streaking and Spread Plates – 3

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FIGURE 4.15 ■ Tenfold dilutions, plating, and viable counts. A. A culture containing an unknown concentration of cells is serially diluted. One milliliter (ml) of culture is added to 9.0 ml of diluent broth and mixed, and then 1 ml of this 1/10 dilution is added to another 9.0 ml of diluent (10–2 dilution). These steps are repeated for further dilution, each of which lowers the cell number tenfold. After dilution, 0.1 ml of each dilution is spread onto an agar plate. B. Plates prepared as in (A) are incubated at 37°C to yield colonies. By multiplying the number of countable colonies (107 colonies on the 10–5 plate) by 10, you get the number of cells in 1.0 ml of the 10–5 dilution. Multiplying that number by the reciprocal of the dilution factor, you can calculate the number of cells (colony-forming units, or CFUs) per milliliter in the original broth tube (107 x 101 x 105 = 1.1 x 108 CFUs/ml). TNTC = too numerous to count.

Types of Media – 1

Complex media are nutrient rich but poorly defined.

Minimal defined media contains only those nutrients that are essential for growth of a given microbe.

Enriched media are complex media to which specific blood components are added.

Selective media favor the growth of one organism over another.

Differential media exploit differences between two species that grow equally well.

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Types of Media – 2

Medium	Ingredients	Amount per liter	Organisms cultured
Luria Bertani (complex)	Bacto tryptone, a pancreatic digest of casein (bovine milk protein)	10 grams	Many Gram-negative and Gram-positive organisms (such as Escherichia coli and Staphylococcus aureus, respectively)
Luria Bertani (complex)	Bacto yeast extract	5 grams	Empty cell
Luria Bertani (complex)	Upper N a upper C l Adjust to pH 7	10 grams	Empty cell
M 9 medium (defined)	Glucose	2.0 grams	Gram-negative organisms such as E. coli
M 9 medium (defined)	Upper N a 2 upper H upper P upper O 4	6.0 grams (42 millimolar)	Gram-negative organisms such as E. coli
M 9 medium (defined)	Upper K upper H 2 upper P upper O 4	3.0 grams (22 millimolar)	Gram-negative organisms such as E. coli
M 9 medium (defined)	Upper N upper H 4 upper C l	1.0 grams (19 millimolar)	Gram-negative organisms such as E. coli
M 9 medium (defined)	Upper N a upper C l	0.5 grams (9 millimolar)	Gram-negative organisms such as E. coli
M 9 medium (defined)	Upper M g upper S upper O 4	2.0 millimolar	Gram-negative organisms such as E. coli
M 9 medium (defined)	Upper C a upper C l 2 Adjust to pH 7	0.1 millimolar	Gram-negative organisms such as E. coli
Sulfur oxidizers (defined)	Upper N upper H 4 upper C l	0.52 grams	Acidithiobacillus thiooxidans
Sulfur oxidizers (defined)	Upper K upper H 2 upper P upper O 4	0.28 grams	Acidithiobacillus thiooxidans
Sulfur oxidizers (defined)	Upper M g upper S upper O 4 times 7 upper H 2 upper O	0.25 grams	Acidithiobacillus thiooxidans
Sulfur oxidizers (defined)	Upper C a upper C l 2	0.07 grams	Acidithiobacillus thiooxidans
Sulfur oxidizers (defined)	Elemental sulfur	1.56 grams	Acidithiobacillus thiooxidans
Sulfur oxidizers (defined)	Upper C upper O 2 Adjust to pH 3	5 percent	Acidithiobacillus thiooxidans

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Types of Media – 3

Several media used in clinical microbiology are both selective and differential.

e.g., MacConkey medium

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FIGURE 4.16 ■ MacConkey medium, a culture medium both selective and differential. Only Gram-negative bacteria grow on lactose MacConkey (selective). Only a species capable of fermenting lactose produces pink colonies (differential), because only fermenters can take up the neutral red and peptones that are also in the medium. Gram-negative nonfermenters appear as uncolored colonies.

Growth Factors, Unculturable Microbes, and Obligate Intracellular Bacteria – 1

Microbes can evolve to require specific growth factors depending on the nutrient richness of their natural ecological niche.

Growth factors are specific nutrients not required by other species.

A microbe needs them in order to be able to grow in laboratory media.

e.g.: Streptococcus pyogenes requires glutamic acid and alanine because it can no longer synthesize them.

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Growth Factors, Unculturable Microbes, and Obligate Intracellular Bacteria – 2

Organism	Diseases	Natural habitats	Growth factors
Shigella	Bloody diarrhea	Humans	Nicotinamide, which is derived from upper N upper A upper D, nicotinamide adenine dinucleotide
Haemophilus	Meningitis, chancroid	Humans and other animal species, upper respiratory tract	Hemin, upper N upper A upper D
Staphylococcus	Boils, osteomyelitis	Widespread	Complex requirement
Abiotrophia	Osteomyelitis	Humans and other animal species	Vitamin K, cysteine
Legionella	Legionnaires’ disease	Soil, refrigeration cooling towers	Cysteine
Bordetella	Whooping cough	Humans and other animal species	Glutamate, proline, cysteine
Francisella	Tularemia	Wild deer, rabbits	Complex, cysteine
Mycobacterium	Tuberculosis, leprosy	Humans	Nicotinic acid, which is derived from upper N upper A upper D, nicotinamide adenine dinucleotide, and alanine (M. leprae is unculturable)
Streptococcus pyogenes	Pharyngitis, rheumatic fever	Humans	Glutamate, alanine

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Growth Factors, Unculturable Microbes, and Obligate Intracellular Bacteria – 3

Some species have adapted so well to their natural habitats that we still do not know how to grow them in the lab.

Some of these “unculturable” organisms depend on factors provided by other species that cohabit their niche.

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FIGURE 4.17 ■ “Unculturable” marine organism MSC33. To grow in natural environments, many bacterial species rely on factors produced by other species within their niche. The microbe shown, MSC33, will not grow in laboratory media unless a peptide growth factor from another species is included.

Growth Factors, Unculturable Microbes, and Obligate Intracellular Bacteria – 4

Obligate intracellular bacteria are also unculturable.

e.g.: Rickettsia prowazekii, the cause of epidemic typhus fever, has adapted to grow within the cytoplasm of eukaryotic cells and nowhere else.

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FIGURE 4.18 ■ Rickettsia prowazekii growing within eukaryotic cells. A. R. prowazekii growing within the cytoplasm of a chicken embryo fibroblast (SEM). B. Fluorescent stain of R. prowazekii (approx. 0.5 μm long), growing within a cultured human cell (outline marked by dotted line). The rickettsias are green (FITClabeled antibody, arrow), the host cell nucleus is blue (Hoechst stain), and the mitochondria are red (Texas Red MitoTracker). The bacterium grows only in the cytoplasm, not in the nucleus.

Techniques for Counting Bacteria

There are many reasons why it is important to know the number of organisms in a sample.

Counting or quantifying organisms invisible to the naked eye is surprisingly difficult.

Each of the available techniques measures a different physical or biochemical aspect of growth.

Thus, cell density values derived from these techniques may not necessarily agree with one another.

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Direct Counting of Living and Dead Cells – 1

Microorganisms can be counted directly by placing dilutions on a special microscope slide called a Petroff-Hausser counting chamber.

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FIGURE 4.19 ■ The Petroff-Hausser chamber for direct microscopic counts. A precision grid is etched on the surface of the slide. The organisms in several squares are counted, and their numbers are averaged. Knowing the dimensions of the grid and the height of the coverslip over the slide makes it possible to calculate the number of organisms in a milliliter.

Direct Counting of Living and Dead Cells – 2

Living cells may be distinguished from dead cells by fluorescence microscopy using fluorescent chemical dyes.

Dead bacterial cells fluoresce orange or yellow because propidium (red) can enter the cells and intercalate the base pairs of DNA.

Live cells fluoresce green because Syto-9 (green) enters the cell.

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FIGURE 4.20 ■ Live/dead stain. Live and dead bacteria visualized on freshly isolated human cheek epithelial cells using the LIVE/DEAD BacLight Bacterial Viability Kit. Dead bacterial cells fluoresce orange or yellow because propidium (red) can enter the cells and intercalate the base pairs of DNA. Live cells fluoresce green because Syto-9 (green) enters the cell. The faint green smears are the outlines of cheek cells.

Direct Counting of Living and Dead Cells – 3

Direct counting without microscopy can be done using an electronic technique that not only counts but also separates populations of bacterial cells according to their distinguishing properties.

The instrument is called a fluorescence-activated cell sorter (FACS) or flow cytometer.

Fluorescent cells are passed through a small orifice and then past a laser.

Detectors measure light scatter in the forward direction (measure of particle size) and to the side (particle shape or granularity).

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Direct Counting of Living and Dead Cells – 4

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FIGURE 4.21 ■ Fluorescence-activated cell sorter (FACS). A. Schematic of a FACS apparatus (bidirectional sorting). B. Separation of GFP-producing E. coli from non-GFP-producing E. coli. The low-level fluorescence in the cells on the left is baseline fluorescence (autofluorescence). The scatterplot displays the same FACS data, showing the size distribution of cells (x-axis) with respect to the level of fluorescence (y-axis). The larger cells may be cells that are about to divide.

Other Techniques

A viable bacterium is defined as being capable of replicating and forming a colony on a solid medium.

Viable cells can be counted via the pour plate method.

Microorganisms can be counted indirectly via biochemical assays of cell mass, protein content, or metabolic rate.

Also by measuring optical density

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4.4 The Growth Cycle

Most bacteria divide by binary fission, where one parent cell splits into two equal daughter cells.

However, some divide asymmetrically.

Hyphomicrobium divides by budding.

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FIGURE 4.22 ■ Symmetrical and asymmetrical cell division. A. Symmetrical cell division, or binary fission, in Lactobacillus sp. (SEM). B. Asymmetrical cell division via budding in the marine bacterium Hyphomicrobium (approx. 4 μm long).

Exponential Growth

The growth rate, or rate of increase in cell numbers or biomass, is proportional to the population size at a given time.

Such a growth rate is called “exponential” because it generates an exponential curve, a curve whose slope increases continually.

If a cell divides by binary fission, the number of cells is proportional to 2n.

Where n = number of generations

Note: Some cyanobacteria divide by multiple fission.

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Generation Time

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Stages of Growth in a Batch Culture

Exponential growth never lasts indefinitely.

The simplest way to model the effects of a changing environment is to culture bacteria in a batch culture.

A liquid medium within a closed system

The changing conditions in this system greatly affect bacterial physiology and growth.

This illustrates the remarkable ability of bacteria to adapt to their environment.

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Bacterial Growth Curves

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FIGURE 4.23 ■ Bacterial growth curves. A. Theoretical growth curve of a bacterial suspension measured by optical density (OD) at a wavelength of 600 nm. B. Phases of bacterial growth in a typical batch culture.

Continuous Culture – 1

In a continuous culture, all cells in a population achieve a steady state, which allows detailed study of bacterial physiology.

The chemostat ensures logarithmic growth by constantly adding and removing equal amounts of culture media.

Note that the human gastrointestinal tract is engineered much like a chemostat.

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Continuous Culture – 2

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FIGURE 4.25 ■ Chemostats and continuous culture. A. The basic chemostat ensures logarithmic growth by constantly adding and removing equal amounts of culture media. B. The human gastrointestinal tract is engineered much like a chemostat, in that new nutrients are always arriving from the throat while equal amounts of bacterial culture exit in fecal waste. C. A modern chemostat.

Continuous Culture – 3

The complex relationships among dilution rate, cell mass, and generation time in a chemostat are illustrated here.

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FIGURE 4.26 ■ Relationships among chemostat dilution rate, cell mass, and generation time. As the dilution rate (x-axis) increases, the generation time decreases and the mass of the culture increases. When the rate of dilution exceeds the division rate, cells are washed from the vessel faster than they can be replaced by division, and the cell mass decreases. The y-axis varies depending on the curve, as labeled.

4.5 Biofilms – 1

In nature, many bacteria form specialized, surface-attached communities called biofilms.

These can be constructed by one or multiple species and can form on a range of organic or inorganic surfaces.

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FIGURE 4.27 ■ Biofilms. A. A greenish-brown slime biofilm found on cobbles of the streambed in High Ore Creek, Montana. B. The biofilm that forms on teeth is called plaque.

4.5 Biofilms – 2

Bacterial biofilms form when nutrients are plentiful.

Once nutrients become scarce, individuals detach from the community to forage for new sources of nutrients.

Biofilms in nature can take many different forms and serve different functions for different species.

The formation of biofilms can be cued by different environmental signals in different species.

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4.5 Biofilms – 3

Chemical signals enable bacteria to communicate (quorum sensing) and in some cases to form biofilms.

Biofilm development involves:

The adherence of cells to a substrate

The formation of microcolonies

Ultimately, the formation of complex channeled communities that generate new planktonic cells

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Biofilm Development – 1

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FIGURE 4.28 ■ Biofilm development. The stages of biofilm development in Pseudomonas, which generally apply to the formation of many kinds of biofilms. Inset: A mucoid environmental strain of P. aeruginosa produces uneven, lumpy biofilms in an experimental flow cell (see Fig. 2.18). Cells in the biofilm were stained green with the fluorescent DNAbinding dye Syto-9 (3D confocal laser scanning microscopy). Source: H. C. Flemming and J. Wingender. 2010. Nat. Rev. Microbiol. 8:623–633.

Biofilm Development – 2

For many bacteria, sessile (nonmoving) cells in a biofilm chemically “talk” to each other in order to build microcolonies and keep water channels open.

Bacillus subtilis also spins out a fibril-like amyloid protein called TasA, which tethers cells and strengthens biofilms.

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FIGURE 4.29 ■ Floating biofilm (pellicle) formation of Bacillus subtilis. Cells were grown in a broth for 48 hours without agitation at 30°C. The pellicles formed by wild-type and tasA mutant B. subtilis are strikingly different. Wild-type pellicles are extremely wrinkly (A), whereas tasA mutant pellicles are flat and fragile (B). Insets: Electron micrographs of wild-type (A) and tasA mutant (B) cells.

Background

Is a non-pathogenic bacterium, which is the model organism for studying the formation and growth of bacterial biofilms.

B. subtilus is non-pathogenic, which means does not cause disease.

Several bacterial species, prefer to live under B. subtilus biofilms, as they are robust, and provide a barrier of protection from environmental stressors (chemicals, or protection from other microorganisms).

Pathogenic (disease causing) bacteria in the wild primarily prefer B. subtilus biofilms.

An example of a pathogen frequently found in B. subtilus biofilms is Bacillus cereus.

In the same genus as Bacillus

A well known food poisoning bacterium

Forms its own biofilm, but when grown together with B. subtilus, will send signals to have B. subtilus create the biofilm.

Bacillus subtilus

Bacillus cereus

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B. subtilus biofilm formation is possible by adding 1% glycerol/0.1mM MnSO4 to LB media

LB only

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Biofilm composition in B. subtilus and B. cereus

B. subtilus and B. cereus share homology between genes of the epsA-epsO and tapA operons that are involved in biofim formation.

The epsA-epsO is an operon composed of 15 genes, which encode proteins responsible for the formation of a exopolysaccharide (sugars) in the extracellular matrix that hold bacteria (not only B. subtilus and B. cereus) in these biofilms together.

The tap A operon contains a gene known as tasA, which encodes a protein that produces amyloid fibers.

The epsA-epsO and tapA are inducible operons, meaning the genes are not expressed until needed.

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Triggers of biofilm formation in B. subtilus

Biofilm formation requires specific nutrients in order to create the exopolysaccharide (EPS), and amyloid fibers.

The two nutrients needed by B. subtilus and B. cereus to create biofilms are glycerol (C3H8O3), and manganese (Mn++).

The picture on the right is a depiction of a signal transduction pathway in B. subtilus and B. cereus, which ultimately leads to the induction of the epsA-epsO and tapA operons.

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1% Glycerol/0.1mM MnSO4 is necessary and sufficient for complete biofilm formation

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