ENCYCLOPEDIA OF ENVIRONMENTAL SCIENCE AND ENGINEERING - MICROBIOLOGY ppt

Microorganisms include viruses, bacteria including rickettsia, mycoplasma, fungi yeast and molds, most algae, protozoa and, if one inter-prets “micro” broadly, certain tiny multicellular

INTRODUCTION

Microbiology is the study of organisms which are small

enough to require the aid of a microscope to be seen In a

few cases, organisms are included in this group which can

be seen by the unaided eye because these organisms are

clearly related to the smaller ones Microorganisms include

viruses, bacteria including rickettsia, mycoplasma, fungi

(yeast and molds), most algae, protozoa and, if one

inter-prets “micro” broadly, certain tiny multicellular plants and

animals The study of cells and tissues from higher plants

and animals ( tissue culture ) uses techniques common to the

microbiologist and is frequently considered part of modern

microbiology

Cells in general vary greatly in size but have many

simi-larities in internal organization Among the most primitive type

of cells, it is impossible to clearly distinguish whether they are

distinctly “plants” or “animals” since they may have some of

the properties of each type Viruses, on the other hand, are not

cells at all Instead of arguing endlessly about whether a

micro-organisms is more plant-like or more animal-like and worrying

how to assign viruses, many scientists have divided organisms

in general into those which have (1) only animal characteristics,

(2) only plant characteristics and (3) the Protista (Table 1),

which have some characteristics of both plants and animals

Some protists, viruses, may have characteristics not shared by

either plants or animals, that is, crystallizability and ability

to reproduce only by infecting some cell and using the cell’s manufacturing machinery

PHYSICAL CHARACTERISTICS OF MICROORGANISMS

Protists vary greatly in size, shape and internal architecture

Protists are subdivided into prokaryotes, and eukaryotes

Prokaryotes do not have their genetic material

(chromo-somes) separated from the rest of the cell by a membrane

whereas eukaryotes have a true nucleus ( eu —true, karyo —

nucleus) separated from the rest of the cell by a nuclear membrane Viruses (virions) are usually included among the prokaryotes There are 9 types of prokaryotes

Prokaryotes

1) Viruses are the smallest protists They range in

size from about 30–300 nm The smallest viruses can only be visualized with an electron micro-scope while the largest can be seen with a light microscope Viruses are composed of two general

molecular types (1) only one nucleic acid, either

ribonucleic acid (RNA) or deoxyribonucleic acid

(DNA), and (2) a group of proteins also called

pro-tein subunits or capsomeres, which surround the

TABLE 1 Characteristics of the Protista

Virion (virus) 30–300 nm icosahedron, hollow cylinder

icosahedral head ⫹ tail RNA, DNA requires participation of host machinery

True bacteria 250–3000 nm spherical, rod, spiral rods,

prokaryotes

Higher bacteria 500–5000 nm spherical, rod, spiral rods,

filamentous, prokaryotes

DNA fission, budding Prokaryotic algae 500–5000 nm spherical, rods in chains, spiral

rods in chains

DNA fission, internal septation,

gonidia Eukaryotic algae 500 nm to macroscopic unicellular or multicellular,

filamentous, leafy

DNA in nucleus, chloroplasts, mitochondria

asexual or sexual simple fission

to complex life cycles Protozoa 500–500,000 nm unicellular or colonial various

forms

DNA in nucleus, mitochondria

asexual or sexual simple fission

to complex life cycles

nucleic acid and form a protective coat or capsid

The smallest viruses appear spherical but magnifi-cation in the order of 150,000–700,000 ⫻ reveals

that they are icosahedrons (20 triangular faces and

12 corners) for example, wart virus Other viruses, for example, tobacco mosaic virus (TMV), the first virus crystallized in 1935 by Wendell Stanley, is grossly rodlike Tobacco mosaic virus is composed

of a central, spirally-attached RNA to which capso-meres are attached to form the outside of a cylinder

The center of the RNA spiral of TMV is hollow

Structurally, the most complicated viruses are some which attack bacteria and blue-green algae

These complicated viruses are composed of an icosahedral

head, containing DNA, a protenaceous tail and sometimes

accessory tail structures which are important for the

attach-ment of the virus to its host cell

2) Mycoplasma are prokaryotes which overlap viruses

in size They range from 100–300 nm in size They

are highly pleomorphic: they do not have one

typi-cal shape but rather can appear coccoid, filamentous,

or highly branched Unlike most other prokaryotes, they do not have cell walls external to their cell membranes Their cell membranes usually contain sterols, which are thought to lend strength to these cell-limiting membranes (see also Table 2)

3) The true bacteria or Eubacteriales are prokaryotes

which are built on three general geometric forms:

spheres or cocci, rods, and spirals (including

spi-ral helices) All true bacteria have rigid cell walls

They are either permanently immotile or move

by means of one to many flagella They may be aerobes or anaerobes Some of the anaerobes are photosynthetic Their sizes and shapes are usually

constant except among the rods, in which rapidly-multiplying cells may be somewhat smaller than usual When the cells divide, they often remain attached to each other and form characteristic, multicellular clusters The shape of the cluster is determined by the number of division planes

When cocci divide in only one plane, they form chains which may be as much as 20 cells long Diplococcus pneumoniae forms chains only two cells long while Streptococcus is

an example of the long-chain forming type On the other hand, cocci which divide along two planes, at right angles

to each other, form sheets of cells, and cocci which divide in three planes form cube-shaped packets If there is no regu-lar pattern of the orientation of successive division planes,

a randomly-shaped cluster is formed Staphylococcus is an

example of a coccus which forms random clusters A typical coccus is in the size range of 0.15–1.5 ␮ m in diameter

Rods always divide in only one plane They may appear

as single cells or groups of only two when they separate

rap-idly The common intestinal bacterium Escherichia coli (size

0.5 ⫻ 2.0 ␮ m) is an example of this type Frequently rods

form long chains or streptobacilli Bacillus megaterium (size

1.35 ⫻ 3.0 ␮ m), the organism responsible for the “bloody bread” of ancient times, is an example of a chain forming rod Some basically rodshaped bacteria are either curved or helical rods Their sizes range from almost as small as the smallest straight rod shaped form to close to twice the length

of the largest straight rod

True bacteria always divide by binary fi ssion after their single circular chromosome replicates in a semiconservative fashion

Some true bacteria have complicated life cycles which

includes spore-formation Spore-formers are all rods but

belong to diverse genera They are ecologically related

in that they are found primarily in soil Since that natural

TABLE 2 Some characteristics of prokaryotic and eukaryotic cells

Weight Chromosome 0.001–1.0 pg

one, single circular DNA double helix not complexed with histones

10–10,000 pg several linear DNA double helices (several chromosomes usually complex with histones) Nucleus No true nucleus Chromosomes not separated from

cytoplasm by a membrane

True nucleus Chromosomes enclosed in a nuclear membrane

Reproduction Usually asexual, conjugation takes place rarely, no

mitosis or meiosis

Asexually by mitosis or sexually after meiosis Membranes Only cell limiting membrane present Usually lacks

sterols (except for mycoplasma)

Cell limiting membrane plus membrane limited organelles present Composition includes sterols

only), Golgi apparatus, lysosomes, etc.

Apparatus for protein synthesis Ribosomes, 70 S type usually not associated with

membranes

Ribosomes, 80 S type in cytoplasm associated with endoplasmic reticulum 70 S type in mitochondria and chloroplasts not associated with membranes

environment is rather variable in that it can range from very

hot to very cold and from very wet to very dry, the heat- and

cold-resistant dormant spores offer the bacteria a means of

surviving adverse environmental conditions for months or

even years Many important pathogens and commercially

important organisms are spore formers, e.g Bacillus

anthra-cis which causes anthrax, Clostridium tetani which causes

tetanus and Clostridium acetobutylicum which can ferment

corn or potato mash into acetone, ethanol and butanol

Corynebacteria are also rod-shaped bacteria but they

are pleomorphic and often look club-shaped One of the

best known members of the genus is C diphtheriae, which

causes diphtheria Other members of the genus are

commer-cially important as producers of the vitamin folic acid

Arthrobacter species are found widely in soil and water

Depending upon the nutrients supplied, they can appear as

cocci or pleomorphic rods

4) Spirochetes are NOT true bacteria though they

resemble Eubacteriales in that they are spirally curved, unicellular and multiply by binary fission

They differ from eubacteria by the absence of

a rigid cell wall which allows them to be quite

flexible They are all motile by means of axial

filaments attached at the cell poles and spirally

wrapped around the cell The smallest spiro-chete is 0.1 ⫻ 5 nm while the largest is 3.0 ⫻

120 ␮ m One of the most important spirochetes is

Treponema pallidum, which causes syphilis

5) Actinomycetes are NOT true bacteria Rather,

they are naturally-branching, filamentous,

spore-forming organisms which have a mycelial

struc-ture similar to that of filamentous fungi Many

actinomycetes, especially those from the genus

Streptomyces, are commercially important sources

of antibiotics

6) Mycobacteria are rods which can form a

rudimen-tary mycelium which resembles actinomycetes, but they differ in that their cell walls are particu-larly rich in waxes, which allows them to retain

stain imparted by such dyes as basic fuchsin even after treatment with dilute acid This property,

called acid fastness, is characteristic of

myco-bacteria Many species occur in soil but the best

known are the human pathogens M tuberculosis and M leprae , which cause tuberculosis and

lep-rosy respectively

7) Budding bacteria are NOT true bacteria They

possess a complicated life cycle which includes

multiplication by budding rather than binary

fis-sion Their type of budding can be readily dis-tinguished from that of true fungi such as yeast

The budding bacterium Hyphomicrobium exists

for part of its life cycle as a flagellated, slightly curved rod For multiplication, the flagellum is lost, the chromosome replicates, and one chro-mosome migrates to one end of the cell where

a hypha-like lengthening takes place When the hyphal extension ceases, it becomes a rounded bud which contains the chromosome The bud grows in length and diameter until it reaches the size of the mother cell, grows a new flagellum, and separates from the hyphal extension

8) Gliding bacteria are diverse group of prokaryotes

which are motile without having flagella They have very close affinities to blue-green algae although gliding bacteria are not themselves pho-tosynthetic They may be unicellular rods, helical

or spiral-helical, or filamentous

9) Blue-green algae or Cyanophyta are the only

prokaryotic algae They are a diverse group that include both unicellular and filamentous forms

They have cell walls that resemble Gram-negative bacteria but their photosynthesis more closely resembles that of eukaryotes in that it is aerobic rather than anaerobic (as in photosynthetic bacte-ria) They are among the most complex prokary-otes Even though they lack defined organelles,

e.g they lack chloroplasts, many species have

complex membranous or vesicular substructures which are continuous with the cell membrane

Some fi lamentous forms contain specialized structures such

as gas vacuoles, heterocysts, or resting spores ( akinetes )

Gas vacuoles are frequently found in planktonic species, i.e

those which live in open water These vacuoles are thought

to provide the algae with a means of fl oating and sinking

to the depth most appropriate to support photosynthesis

Heterocysts arise from vegetative cells and are thought to

function in N 2 fi xation Some blue-green algae show gliding motility None are fl agellated They are very widely distrib-uted either in terrestrial or aquatic habitats from the arctic

to the tropics Some forms are found in hot springs Other

Cyanophyta are symbionts in a variety of plants and animals

For example a species of Anabaena fi xes N 2 for its host the

water fern, Azolla Many blue-green algae form especially luxuriant mats of growth called blooms which clog

water-ways and limit their use for navigation, etc

FIGURE 1 Animal viruses are often grown in

embry-onated eggs The position of the hypodermic needles

indicates three common inoculation places.

The prokaryotic blue-green algae, Cyanophyta, are

usually divided into 5 groups:

Chooccocales are unicellular They sometimes occur in

irregular packets or colonies Cells multiply by binary fi ssion

Chamaesiphonales are unicellular, fi lamentous, or

colo-nial epiphytes or lithophytes Cells show distinct polarity

from apex to base The base usually has a holdfast which

permits attachment to the substrate Cells multiply by

inter-nal septation or by formation of spherical cells ( gonidia ) at

the ends of fi laments

Pleurocapsales are fi lamentous with differentiation into

aerial and nonaerial elements Cells multiply by crosswall

formation or by internal septation

Nostocales are fi lamentous without differentiation into

aerial and nonaerial elements They are unbranched or falsely

branched and frequently have pale, empty-looking cells called

heterocysts and resting spores ( akinetes ) Reproduction is by

liberation of a short fi lament only a few cells long, called a

hormogonium, which then elongates

a) Nostacaceae are unbranched and produce

hetero-cysts They frequently produce akinetes

b) Rivulariaceae are unbranched or falsely branched

Filaments taper from base to tip Heterocysts are usually present at the base There is some akinete formation

c) Scytonemataceae are false branched Heterocysts

are frequently found at branch points

d) Stigonematalis are filamentous with aerial and

nonaerial differentiation Hormogonia and hetero-cysts are present They often show true branching

and have pit connections between cells Akinetes are rare

Eukaryotes Eukaryotic microorganisms include all the algae (except the Cyanophyta ), all the protozoa, and most fungi All are

microscopic in size

The eukaryotic algae are separated into nine

divi-sions based upon their pigment and carbohydrate reserves

(Table 3) They are all photosynthetic and, like higher plants,

evolve oxygen during photosynthesis Many algae are

obli-gate phototrophs That is, they are completely dependent

upon photosynthesis: they can not use exogenously supplied organic compounds for growth in either the dark or light

Some algae are facultative phototrophs; they are able to

uti-lize organic compounds for growth in the dark but fi x carbon dioxide photosynthetically in the light

Occasionally algae, especially unicellular forms, per-manently lose their chloroplasts by exposure to any one

of several adverse conditions, e.g heat or chemicals If the organism had been a facultative phototroph, before the loss of the chloroplasts, it has the enzymatic machinery necessary to survive except that now, in its chloroplastless state, it is indis-tinguishable from certain other unicellular organisms more commonly called protozoa The ease with which an organ-ism at this primitive level of evolution may be interchanged between groups containing a preponderance of plant-like or

animal-like attributes underlines the need for the term protist

rather than plant or animal to describe them Indeed both botanists and zoologists claim the protists Some algae e.g

Euglena spp., normally only form chloroplasts when they

grow in the light while others e.g Chlorella spp form

chlo-roplasts regardless of the presence of absence of light There

is great diversity in size, shape, presence or absence of life cycles, type of multiplication, motility, cell wall chemistry, and chloroplast structure Although these parameters are of great assistance in defi ning affi nities among algae, there are still groups whose proper place is debated

Many algae are important as sources of food, chemi-cal intermediates of industrial and medichemi-cal importance, and research tools Others are nuisances which clog waterways

or poison other aquatic life with their potent toxins

Eukaryotic Algal Groups

The eight groups are:

1) Chlorophyta (green algae) are either marine or

fresh-water forms This large and diverse group includes forms which are either unicellular, colonial,

filamen-tous, tetrasporal (cells separated but held together

in groups of four in a mucilaginous material),

coe-nobial (cells more or less attached to each other in

an aggregate), or siphonaceous (simple, nonseptate

filaments) They frequently have life cycles which

RELATIVE SIZE OF BACTERIA

Clostridium 1x3.10m

Salmonella 0.6x2.3m

Hemophilus 0.3x0.6–1.5m

Pseudomonas 0.5x1.3m

Fusibacterium 0.75–1.5x8.80m Neisseria 0.6x0.8m

Streptococcus 0.5–0.75m

Staphylococcus 0.8–1m Erythocyte 7m diameter

FIGURE 2 Relative sizes of bacteria.

include motile, flagellated stages Both asexual and sexual reproduction occurs

2) Euglenophyta differ from the other algae by

pos-sessing a rather flexible cell wall which allows con-siderable plasticity of form They are either fresh water or marine forms They all have two flagella but in some genera the second flagellum is often

rudimentary Many forms are phagotrophic (can

ingest particles) Chloroplastless forms are fairly common Multiplication is only by asexual means

3) Xanthophyta are mostly freshwater forms They

may be unicellular, colonial, filamentous or siphonaceous Some forms have life cycles which include both asexual and sexual reproduction

Motile anteriorly flagellated cells are found

4) Chrysophyta are mainly freshwater forms but

important marine forms are known Most genera are unicellular but there are some colonial forms

Cell walls are often composed of siliceous or cal-careous plates Some form siliceous cysts They

are mainly found in fresh water but some impor-tant marine forms exist Reproduction is asexual

5) Phaeophyta (diatoms) are unicellular or colonial

forms with distinctly patterned siliceous cell walls

Both asexual and sexual multiplication is found

Freshwater, marine, soil and aerial forms exist

6) Pyrrophyta are unicellular flagellates with

cel-lulose cell walls which are sometimes formed in plates Reproduction is asexual Sexual reproduc-tion is rare

7) Cryptophyta are unicellular, usually flagellated

forms which produce asexually

8) Rhodophyta (red algae) are unicellular, filamentous

or leafy forms with complex sexual cycles Most are marine but there are a few freshwater forms

Fungi The “true” fungi or Eumycota are eukaryotes which are

related to both protozoa and algae They are divided between

Reserve material (cont.)

alcohols Mannitol Division Laminarin Paramylon Chrysolamainarin Floridoside Sucrose Lipid

Chlorophyta (green algae) ⫹

Euglenophyta

Xanthophyta ⫹ ⫹

Chrysophyta

Phaeophyta (brown algae) ⫹ o ⫹ ⫹

Bacillariophyta (diatoms) ⫹ ⫹

Cryptophyta

Rhodophyta (red algae) ⫹

TABLE 3 Divisions and characteristics of the eukaryotic algae

Chlorophyll Biliproteins Starches (a-1,4-glucans)

starch

Floridian starch

Chlorophyta (green algae) ⫹ ⫹ ⫺ ⫺ ⫺ ⫹

Euglenophyta ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺

Xanthophyta ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺

Chrysophyta ⫹ ⫺ ⫺ ⫺

Phaeophyta (brown algae) ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺

Bacillariophyta (diatoms) ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺

Pyrrophyta ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹

Cryptophyta ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹

Rhodaphyta (red algae) ⫹ ⫺ ⫺ ? ⫺ ⫹ ⫹ ⫹

microscopic and macroscopic and macroscopic groups In

general, they have rigid cell walls, lack chlorophyll, and are

usually immotile Most fungi reproduce asexually or

sexu-ally by means of spores though important budding groups

such as yeasts are well known Since fungi are classifi ed by

the pattern of their sexual structures, fungi whose sexual

stages are unknown are placed into a group called Fungi

Imperfecti and assigned genera on the basis of their asexual

structures They are further subdivided into the so-called

lower and higher fungi The lower fungi, Phycomycetes, are

also called water molds but not all are aquatic (e.g black bread molds) Some species multiply by means of fl agellated gametes or fl agellated spores i.e more like certain green algae than other fungi; Most, but not all, Phycomycetes have

COCCI BACILLI

VIBRIOS SPIRILLA

SPIROCHAETES ACTINOMYCETALES

(A) MORPHOLOGICAL CHARACTERIZATION OF BACTERIA

(B)

(C)

FIGURE 3 A General morphological characteristics of bacteria; B Variety of morphological types among the cocci; C Variety of morphological types among the bacilli (rods).

EDGE

Flat Raised Low Convex

High Convex

Entire

Umbonate Convex with

papillate surface

Erose Crenated

Undulate Lobate Rhizoid

FIGURE 5 Diagrammatic representa-tion of types of bacterial colonies These shapes are specific for individual types and are therefore quite useful as a step in the process of identification of unknown organisms.

SPORE FORMS

FIGURE 4 Diagrammatic represen-tation of spores (clear areas) inside rod-shaped bacteria Note (bottom row) that free spores may be ball or egg-shaped.

hyphae, microscopic cytoplasm-fi lled tube-like branches

(lacking crosswalls), which together make a felty mat called

a mycelium Individual hyphae are microscopic but the

mycelium, equivalent to a bacterial colony, is macroscopic

Growth takes place by extension of the hyphae Specialized

spore-containing bodies called sporangia can form at the

ends of some hyphae Sexual reproduction requires fusion

of hyphae from two different mycelia to form a specialized

zygospore

It is more common now to discard the term Phycomyetes

and instead subdivide the group into 4 classes in which affi

n-ities are much clearer However, at present, the literature is

divided in its use of the older and newer terminology As

with bacteria, chemical analyses of structures and metabolic

pathways followed are important in defi ning the classes

These four classes are:

1) Chytridiomycetes lack true mycelia They are aquatic,

have posteriorly uniflagellated zoospores and cell walls composed of chitin

2) Hyphochytridiomycetes have true mycelia They are

aquatic, have anteriorly uniflagellated zoospores and cell walls composed of chitin

3) Oomycetes have true well developed mycelia and

cell walls composed of cellulose

a) Saprolegniales are generally aquatic and have

asexual spores on specialized mycelear structures

Only male gametes are motile

b) Peronosporales are generally terrestrial

Sporan-gia either produce asexual zoospores or may

germinate directly to form hyphae Both gametes are nonmotile

4) Zygomycetes are terrestrial and have large and

well developed mycelia and nonmotile spores

Asexual spores are produced in sporangia Cell walls are made of chitosan or chitin

There are two classes included in the higher fungi

1) The Ascomycetes are the best known and largest

class of fungi Ascomycetes have hyphae divided by porous crosswalls Each of these hyphal compart-ments usually contains a separate nucleus Asexual

spores called conidia, form singly or in chains at

the tip of a specialized hypha The sexual structure

called ascus, is formed at the enlarged end of a

spe-cialized fruiting structure and usually contains eight

ascospores Some important microscopic members

of this group include yeasts, mildews, the common red bread mold and many species which produce antibiotics On the other hand macroscopic forms

include Morchella esculenta or morels which are

highly regarded as a delicacy by gourmets

2) The Basidiomycetes are entirely macroscopic and are

commonly known as mushrooms and toadstools

Slime Molds The slime molds, Myxomycetes, are at times classifi ed with

either true fungi or protozoa or, as here, treated separately

They produce vegetative structures which look like ameboid

protozoa and fruiting bodies which produce spores with cell

walls like fungi There are two major subdivisions (a) Cellular

and (b) Acellular They both primarily live on decaying plant

material and can ingest other microorganisms, such as

bacte-ria, phagocytically Both have life cycles, but that of the

acel-lular slime molds is more complicated

Cellular slime molds have vegetative forms composed of

single ameboid cells Cyclically, ameboid cells aggregate to

form a slug-shaped pseudoplasmodium that begins to form

fruiting bodies when the slug becomes immotile Spores are

fi nally produced by the fruiting bodies

Acellular slime molds have vegetative forms called

plas-modia which are composed of naked masses of protoplasm

of indefi nite size and shape and which travel by ameboid movement (protoplasmic streaming) Two kinds of nesting

structures are produced: fruiting bodies (part of the sexual cycle) and sclerolia

Protozoa

The last major group of microorganisms are the protozoa

As already stated, it is very hard to distinguish plants from animals at this primitive stage in evolution where organisms have some attributes of each Most workers therefore are less interested in whether protozoa should be claimed by bota-nists or zoologists as they are in studying the group as the root of a phylogenetic tree which gave rise to clearly sepa-rable plants and animals Protozoa range in size from that

of large bacteria to just visible without a microscope They have a variety of shapes, multiplication methods and associ-ations which range from single cells to specialized colonies

They are variously found in fresh water, marine, terrestrial, and occasionally, aerial habitats Both freeliving and para-sitic forms are included Most are motile but there are also important nonmotile forms The protozoa are divided into four subphyla (I–IV)

I Sarcomastigophora include forms which have either fl

a-gella, pseudopodia or both Usually a single-type of nucleus (though opalinids contain multiples of this one type) is pres-ent except in developmpres-ent stages of a few forms Asexual reproduction by binary fi ssion is common One whole class contains chloroplasts and are claimed by both protozoolo-gists and algoloprotozoolo-gists (they are considered here in detail with the eukaryotic algae) Many important parasites of diverse animal and some plant groups are found here Sexual repro-duction is present in a few forms

The Sarcomastigophora are divided into three super-classes

A Mastigophora ( fl agellates ) Are further sub-divided into

Phytomastigophorea or plant-like fl agellates (see eukaryotic

FIGURE 6 Bacterial motility Motility is tested by stabbing an inoculated needle into a tube of very

vis-cous growth medium The motile organisms (S typhi and P vulgaris) grow away from the stab mark.

4 3

2

FIGURE 7 Isolation of single bacterial colonies

on agar plates by dilution streaking A diagrammatic representation of method of streaking inoculated needle across nutrient-containing plate Stippled area is the primary inoculation The inoculation needle is then flamed to sterilize and is then drawn across the stippled areas as indicated for area 1

The needle is then resterilized and drawn across area 2, etc.

FIGURE 8 Isolation of single colonies by pour plate technique.

algae) and Zoomastigophorea or animal-like fl agellates which

are divided into nine orders

1) Choanoflagellida have a single anterior flagellum

surrounded posteriorly by a collar Some forms are attached to substrates They are solitary or colonial and are all free-living

2) Bicosoecida have 2 flagella (one free, the other

attached to the posterior of the organism) They are free-living

3) Rhizomastigida have pseudopodia and 1–4 or

more flagella Most species are free-living

4) Kinetoplastida have 1–4 flagella and all have a

kinetoplast (specialized mitochondrion) Many important pathogens (e.g trypanosomes) and some free-living genera are included

5) Retortamonadida have 2–4 flagella The cytostome

is fibril-bordered All are parasitic

6) Diplomonadida have 2 karyomastigonts, each with

4 flagella and sets of accessory organelles Most species are parasitic

7) Oxymonadida have one or more karyomastigonts,

each with 4 flagella All species are parasitic

8) Trichomonadida have mastigont systems with

4–6 flagella Some have undulated membranes

Many important pathogens (e.g Trichomonas )

are included

9) Hypermastigida have mastigont systems with

numerous flagella and multiple parabasal apparatus

All are parasitic Some forms reproduce sexually

B Opalinata Are an intermediary group related to both

ciliates and fl agellates and are entirely parasitic Opalinics

have many cilia-like organelles arranged in oblique rows over

their entire body surface They lack cytosomes (oral

open-ings) They have multiple nuclei (ranging from 2 to many)

which divide acentrically The whole organism divides by

binary fi ssion Life cycles are complex

C Sarcodina Or ameboid organisms have Pseudopodia

which are typically present but fl agella may be present

during certain restricted developmental stages Some forms

have external or internal tests or skeletons which vary widely

in type and chemical composition All reproduce asexually

by fi ssion but some also reproduce asexually Most species

are free-living (in both aquatic and terrestrial habitats) but

some are important pathogens; for example, Entameba

his-tolytica , which causes amebic dysentary The sarcodinids are

further divided into three classes

1) Rhizopodae, a free-living, mostly particle-eating

(phagotrophic) group which includes both naked and shelled species The specialized pseudopodia are called lobopodia, filopodia, or reticulopodia

2) Piroplasmea These parasitic small, piriform,

round, rod-shaped or ameboid organisms do not form spores, flagella or cilia Locomotion is by body-flexing or gliding They reproduce by binary fission or schizogony

3) Actinopodea are free-living, spherical, typically

floating forms with typically delicate and radiose pseudopodia Forms may be naked or have mem-braneous, clutenoid, or silicated tests Both asex-ual and sexasex-ual reproduction occurs Gametes are usually flagellated

II Sporozoa typically form spores without polar fi laments

and lack fl agella or cilia Both asexual and sexual reproduc-tion takes place All species are parasitic Some have rather complicated life cycles

The Sporozoa are divided into three classes:

A Telesporea Can reproduce sexually or asexually, have

spores, move by body fl exion or gliding and generally do not have pseudopodia

B Toxoplasmea Reproduce asexually, lack spores,

pseudo-podia or fl agella, and move by body fl exion or gliding

C Haplosporea Reproduce asexually and lack fl agella They

have spores and may have pseudopodia

III Cnidospora have spores with one or more polar fi laments

and one or more sporoplasms All species are parasitic There are two classes

IV Ciliophora have simple cilia or compound ciliary

organ-elles in at least one stage of their life cycle They usually have two types of nucleus Reproduction is asexually by

fi ssion or sexually by various means Most species are free-living but parasitic forms are known

ENERGY AND CARBON METABOLISM All cells require a source of chemical energy and of carbon for building protoplasm Regardless of whether the cell type

is prokaryote or eukaryote or whether it is more plant-like

or more animal-like, this basic requirement is the same The

most basic division relates to the source of carbon used to

build protoplasm Organisms which can manufacture all their carbon-containing compounds from originally ingested inor-ganic carbon (CO 2 ) are called autotrophs while those which

require ingestion of one or several organic compounds for use

in the manufacture of cellular carbon compounds are called

heterotrophs Some organisms are nutritionally versatile and

may operate either as autotrophs or heterotrophs and are

therefore referred to as autotrophs or

facultative-heterotrophs (depending upon which mode of nutrition

usu-ally predominates)

Autotrophs are further divided according to the manner

in which they obtain energy Chemoautotrophs (also called chemotrophs or chemolithotrophs oxidize various inorganic compounds to obtain energy while photoautotrophs (also called phototrophs or photolithotrophs ) convert light to chemical energy via the absorption of light energy by special

pig-ments (chlorophylls and carotenoids) In both cases, chemical energy is stored in the form of chemical bond energy in the compound adenosine triphosphate (ATP)

When bonds of ATP indicated by ~ are broken, a

con-siderable amount of energy is released This ~ bond

cleav-age energy operates the biological engines: it is the universal

chemical power which operates in all cells, autotroph or

het-erotroph

Chemolithotrophic nutrition is only used by certain true

bacteria These bacteria are of ecological importance in that

they are used to convert one form of nitrogen to another (i.e

in the nitrogen cycle) or industrially to oxidize low grade

metallic or non-metallic ores There are six bacterial groups

which are chemolithotrophic

1) The ammonia oxidizers such as Nitrosomonas,

Nitrosococcus, Nitrosocystis, Nitrosogloea and Nitrosospira

One scheme for ammonia oxidation had

hydrox-ylamine as an obligate intermediate and has been

proposed for Nitrosomonas

2) The nitrite oxidizers such as Nitrobacter and

Nitrocystis One proposed scheme for nitrite

oxi-dation for Nitrobacter is:

NO cytochrome reductase cytochrome C

cytochrome oxi

2

d dase O ATP ADP

2

3) Hydrogen oxidizers Hydrogenomonas One

pro-posed hydrogen oxidation scheme is:

H 2 →2H⫹ ⫹ 2e→unknown→fl avor protein compound

→ubiquinone→O 2 cytochrome

b compex Nicotinamide adenine→menadione

→cytochrome C→cytochrome a

→O 2 dinucleotide (NAD) 4) Ferrous compound oxidizing bacteria such as

Ferrobacillus and Thiobacillus ferroxidans

One proposed ferrous oxidizing scheme for

F ferrooxidans is:

4FeCO 3 ⫹ O 2 ⫹ 6H 2 O→4Fe(OH) 3 ⫹ 4CO 2

5) Methane oxidizers such as Methanomonas

methano-oxidans and Pseudomonas methanica are common

in the upper layers of marine sediments and soil

Methane is oxidized in the following manner:

CH 4 →CH 3 OH→HCHO→HCOOH→CO 2

6) The sulfur-compound oxidizing bacteria

Thioba-cillus

Four pathways for oxidation of thiosulfate (S 2 O 3 ⫹2 ) by

different Thiobacillus species are known These are:

a) 6Na 2 S 2 O 3 ⫹ SO 2 →4Na 2 SO 4 ⫹ 2Na 2 S 4 O 6 2Na 2 S 4 O 6 ⫹ 6H 2 O ⫹ 7O 2 →2Na 2 SO 4 ⫹ 6H 2 SO 4 b) Na 2 S 2 O 3 ⫹ 2O 2 ⫹ H 2 O→Na 2 SO 4 ⫹ H 2 SO 4 c) 5Na 2 S 2 O 3 ⫹ H 2 O ⫹ 4O 2

→5Na 2 SO 2 ⫹ H 2 SO 4 ⫹ 4S 2S ⫹ 3O 2 ⫹ 2H 2 O→2H 2 SO 4 d) 2Na 2 S 2 O 3 ⫹ H 2 O ⫹ 1/2O 2 →Na 2 S 4 O 6 ⫹ 2NaOH

Photolithotrophic nutrition is used by photosynthetic

bacteria, blue green algae and eukaryotic algae The general reaction in which both utilization of CO 2 (carbon dioxide

fi xation) and energy generation is summarized is:

CO H A CH O A H O2⫹ 2 ⎯ →nv 2⎯ ( )⫹2 ⫹ 2 Where A is either oxygen for all eukaryotic algae and the prokaryotic blue-green algae (H 2 A = H 2 O), or sulfur for green

sulfur bacteria, Chlorobacteriaceae, and purple sulfur bacte-ria, Thiorhodaceae (H 2 A = H 2 S) or any one of several organic

compounds for nonsulfur purple bacteria, Athiorhodaceao

(H 2 A = H 2 -organic compound which is oxidizable)

Both green and purple sulfur bacteria are obligate anaerobes whereas the non-sulfur purple bacteria are facul-tative anaerobes (they are anaerobic when growing hetero-trophically) In all cases, photosynthetic organisms operate

by the initial transduction of light to chemical energy In this transduction, chlorophyll ⫹ light quanta Ch1⫹ (excited chlorophyll) ⫹ e⫺ (electron driven off of Ch1) Many such events take place simultaneously and electrons released during these reactions migrate through the photosynthetic unit to the reaction center and transfer energy to a special

reaction-center chlorophyll At the reaction center, a charge

separation of the oxidant and reductant occurs Electron

fl ow after this event differs in photosynthetic bacteria as compared with algae and higher plants (Figures 9 and

10).In addition, differences in photosynthetic ability exist among organisms based upon the absorption maxima of their light-transducing pigments (primarily chlorophylls)

The combination of light intensity, wavelength of available light, wavelength of operation of principal energy trans-ducing pigment, degree of aerobiasis, and availability of oxidizable compound (H 2 O, H 2 S, or H 2 -organic compound) all infl uence the effi ciency of photosynthesis These factors should be borne in mind when one looks for the ecological niche occupied by these various organisms

Ecology of Microorganisms

One should understand the physiological requirements of

microorganisms before investigating the effects of

environ-mental changes on the distribution and activity of diverse