Part 1

Basic
microbiology
1
Basic bacteriology
Peter Lambert
Aston University, Birmingham, UK
Bacterial structure
Bacteria are single-celled prokaryotic microorgan-
isms, and their DA is not contained !ithin a
separate nucleus as in eukaryotic cells" #hey are
appro$imately %"1&1%"% mm in si'e ()igure 1"1* and
e$ist in various shapes, including spheres (cocci*,
curves, spirals and rods (bacilli* ()igure 1"+*" #hese
characteristic shapes are used to classi,y and iden-
ti,y bacteria" #he appearance o, bacteria ,ollo!ing
the -ram stain is also used ,or identi,ication" Bac-
teria !hich stain purple.blue are termed -ram-
positive, !hereas those that stain pink.red are
termed -ram-negative" #his di,,erence in response
to the -ram stain results ,rom the composition o,
the cell envelope (!all* ()igure 1"/*, !hich are
described belo!"
0ell envelope
0ytoplasmic membrane
A cytoplasmic membrane surrounds the
cytoplasm o, all bacterial cells and are composed
o, protein and phospholipid1 they resemble the
membrane surrounding mammalian
(eukaryotic* cells but lack sterols" #he
phospholipids ,orm a bilayer into !hich proteins
are embedded, some spanning the membrane"
#he membrane carries out many ,unctions,
including the synthesis and e$port o, cell-!all
components, respiration, secretion o,
e$tracellular en'ymes and to$ins, and the uptake
o, nutrients by active transport mechanisms"
2esosomes are intracellular membrane struc-
tures, ,ormed by ,olding o, the cytoplasmic
membrane" #hey occur more ,re3uently in
-ram-positive than in -ram-negative bacteria"
2esosomes present at the point o, cell division
o, -ram-positive bacteria are involved in chromo-
somal separation1 at other sites they may be asso-
ciated !ith cellular respiration and metabolism"
0ell !all
Bacteria maintain their shape by a strong rigid
outer cover, the cell !all ()igure 1"/*"
-ram-positive bacteria have a relatively thick,
uni,orm cell !all, largely composed o, peptidogly-
can, a comple$ molecule consisting o, linear re-
peating sugar subunits cross-linked by peptide
side chains ()igure 1"4a*" 5ther cell-!all polymers,
including teichoic acids, teichuronic acids and
proteins, are also present"
-ram-negative bacteria have a thinner peptido-
glycan layer and an additional outer membrane
that di,,ers in structure ,rom the cytoplasmic
membrane ()igure 1"4b*" #he outer membrane
contains lipo- polysaccharides on its outer ,ace,
phospholipids on its inner ,ace, proteins and
lipoproteins !hich an- chor it to the
peptidoglycan" Porins are a group o, proteins that
,orm channels through !hich small hydrophilic
molecules, including nutrients, can cross the outer
membrane" Lipopolysaccharides are
2edical 2icrobiology and 6n,ection Lecture otes, )i,th 7dition" 7dited by #om 7lliott, Anna 0asey,
Peter Lambert and 8onathan 9andoe"
+%11 Black!ell Publishing Ltd" Published +%11 by Black!ell Publishing Ltd"
4 Basic bacteriology
Red blood cell
Bacillus anthracis
Clostridium perfringens
Escherichia coli Borrelia
recurrentis Treponema
pallidum Staphylococcus
aureus Streptococcus
pneumoniae
Chlamydia trachomatis
Mycoplasma pneumoniae
0 5 10
Size (:m)
)igure 1"1 9hape and si'e o, some clinically important bacteria"
a characteristic ,eature o, -ram-negative bacteria
and are also termed ;endoto$ins< or ;pyrogen<"
7ndoto$ins are released on cell lysis and have
important biological activities involved in
the
pathogenesis o, -ram-negative in,ections1 they
activate macrophages, clotting ,actors and com-
plement, leading to disseminated intravascular
coagulation and septic shock (0hapter //*"
Basic bacteriology =
Coccus
Bacillus (rod) Curve Spiral
)igure 1"+ 9ome bacterial shapes"
DNA
Ribosomes Cross-wall formi! C"#oplasmic membrae
C"#oplasm Capsule Cell wall
$la!ellum
$imbriae
%clusio
!raules
)igure 1"/ A section o, a typical bacterial cell"
2ycobacteria have a distinctive cell !all struc-
ture and composition that di,,ers ,rom that o,
-ram-positive and -ram-negative bacteria" 6t
contains peptidoglycan but has large amounts
o, high molecular !eight lipids in the ,orm o,
long chain length ,atty acids (mycolic acids*
attached to polysaccharides and proteins" #his
high lipid content gives the mycobacteria their
acid ,ast properties (retaining a stain on
heating in acid*, !hich allo!s them to be dis-
tinguished ,rom other bacteria (e"g" positive
>iehl-eelsen stain*"
#he cell !all is important in protecting bacteria
against e$ternal osmotic pressure" Bacteria !ith
damaged cell !alls, e"g" a,ter e$posure to b-lactam
antibiotics such as penicillin, o,ten rupture" ?o!-
ever, in an osmotically balanced medium, bacteria
de,icient in cell !alls may survive in a spherical
,orm called protoplasts" Under certain conditions
some protoplasts can multiply and are re,erred to
as L-,orms" 9ome bacteria, e"g" mycoplasmas, have
no cell !all at any stage in their li,e cycle"
#he cell !all is involved in bacterial division"
A,ter the nuclear material has replicated and sep-
arated, a cell !all (septum* ,orms at the e3uator o,
the parent cell" #he septum gro!s in, produces a
cross-!all and eventually the daughter cells may
separate" 6n many species the cells can remain
attached, ,orming groups, e"g" staphylococci ,orm
clusters and streptococci ,orm long chains
()igure 1"=*"
0apsules
9ome bacteria have capsules e$ternal to their cell
!alls ()igure 1"/*" #hese structures are bound
@ Basic bacteriology
)igure 1"4 0ell !all and cytoplasmic membrane o, (a* -ram-positive bacteria, (b* -ram-negative bacteria and
(c* mycobacteria" #he -ram-positive bacterial cell !all has a thick peptidoglycan layer !ith associated molecules
(teichoic acids, teichuronic acids and proteins*" #he -ram-negative bacterial cell !all contains lipopolysacchar-
ides, phospholipids and proteins in an outer membrane linked to a thin inner peptidoglycan layer" #he
mycobacterial cell !all contains long chain length ,atty acids (mycolic acids*"
S#ap&"lococci Neisseriae 'eumococci S#rep#ococci
)igure 1"= 9ome groups o, bacteria"
to the bacterial cell and have a clearly de,ined
boundary" #hey are usually polysaccharides !ith
characteristic compositions that can be used to
distinguish bet!een microorganisms o, the same
species (e"g" in serotyping*" 0apsular antigens can
be used to di,,erentiate bet!een strains o, the
same bacterial species, e"g" in the typing o,
9treptococcus pneumoniae ,or epidemiological
purposes" #he capsules are important virulence
determinants in both -ram-positive and -ram-
negative bacteria, because they may protect the
bacteria ,rom host
de,ences and, in some bacteria, aid attachment to
host cells"
Bacterial slime and bioAlm
7$tracellular slime layers are produced by some
bacteria" #hey are more loosely bound to the cell
sur,ace than capsules and do not ,orm a clearly
de,ined sur,ace boundary" #he slime layer is
composed predominantly o, comple$ polysac-
charides (glycocaly$*, !hich acts as a virulence
Basic bacteriology B
)igure 1"@ 9canning electronmicrograph o,
9taphylococcus epidermidis embedded in slime
attached to a catheter"
,actor through the ,ormation o, bio,ilm, e"g" by
,acilitating the attachment o, 9taphylococcus epi-
dermidis onto arti,icial sur,aces, such as intra-
vascular cannulae ()igure 1"@*, replacement
Coints and heart valves" 5nce ,ormed, bio,ilms
present a maCor problem ,or treatment and may
re3uire removal o, the biomedical device"
)lagella
Bacterial ,lagella are spiral-shaped sur,ace ,ila-
ments consisting mainly o, the protein, ,lagellin"
#hey are attached to the cell envelope as single
(monotrichous* or multiple (peritrichous* ,orms
()igure 1"B*"
)lagella ,acilitate movement (motility* in bacte-
ria by rapid rotation" #hey can be observed under
the light microscope !ith special stains" )lagella
are usually detected ,or diagnostic purposes by
observing motility in a bacterial suspension or by
spreading gro!th on solid media" #he antigenic
nature o, the ,lagella may be used to di,,erentiate
bet!een and identi,y strains o, 9almonella spp"
)imbriae
)imbriae (also termed pili* are thin, hair-like ap-
pendages on the sur,ace o, many -ram-negative,
and some -ram-positive, bacteria ()igure 1"/*"
#hey are appro$imately hal, the !idth o, ,lagella,
and are composed o, proteins called pilins" 6n
some bacteria they are distributed over the entire
cell sur,ace"
)imbriae are virulence ,actors enabling bacteria
to adhere to particular mammalian cell sur,aces,
an important initial step in colonisation o, mucosal
sur,aces, e"g" eisseria gonorrhoeae produce ,im-
briae that bind to speci,ic receptors o, cervical
epithelial cells, !hereas 9treptococcus pyogenes
have ,imbriae containing ;2< protein, !hich ,acil-
itates adhesion to human cells in the pharyn$"
9pecialised ,imbriae are involved in genetic ma-
terial trans,er bet!een bacteria, a process called
conCugation"
(oo#ric&ous 'eri#ric&ous
)igure 1"B Arrangements o, bacterial ,lagella"
D Basic bacteriology
Ce#ral )ermial Sub#ermial
)igure 1"D 9i'e, shape and position o, bacterial spores (,rom le,t to right*E non-proCecting, oval, central, e"g"
Bacillus anthracis1 proCecting, spherical, terminal, e"g" 0lostridium tetani1 non-proCecting, oval, subterminal, e"g"
0" per,ringens"
6ntracellular structures
uclear material
#he bacterial chromosome consists o, a single
circular molecule o, double-stranded DA, !hich
is maintained in a compact ,orm !ithin the cell by
supercoiling" Fhen released ,rom the cell and
uncoiled the DA !ould be about 1 mm long
(1% to 1%%-times the length o, the cell*" Additional
smaller e$tra-chromosomal DA molecules,
called plasmids, may also be present in bacteria"
#he chromosome usually codes ,or all the essential
,unctions re3uired by the cell1 some plasmids
con- trol important phenotypic properties o,
pathogen- ic bacteria, including antibiotic
resistance and to$in production" 7$tracellular
nuclear material ,or encoding virulence and
antibiotic resistance may also be trans,erred
bet!een bacteria and incorporated into the
recipient<s chromosome or plasmid" #rans,er o,
genes encoding ,or viru- lence or antibiotic
resistance may account ,or bacteria becoming
resistant to antibiotics and ,or lo!-virulent
bacteria becoming pathogenic"
Gibosomes
#he cytoplasm has many ribosomes, !hich con-
tain both ribonucleic acid (GA* and proteins"
Gibosomes are involved in protein synthesis"
6nclusion granules
Harious cellular inclusions, !hich serve as energy
and nutrient reserves, may be present in the
bacterial cytoplasm" #he si'e o, these inclusions
may increase in a ,avourable environment
and decrease !hen conditions are adverse, e"g"
0orynebacterium diphtheriae may contain high-
energy phosphate reserves in inclusions termed
;volutin granules<"
7ndospores
7ndospores (spores* are small, metabolically dor-
mant cells !ith a thick, multi-layered coat, ,ormed
intracellularly by members o, the genera Bacillus
and 0lostridium ()igure 1"D*" #hey are highly re-
sistant to adverse environmental conditions and
may survive desiccation, disin,ectants or boiling
!ater ,or several hours"
9pores are ,ormed in response to limitations o,
nutrients by a comple$ process (sporulation* in-
volving at least seven stages" Fhen ,ully ,ormed,
they appear as oval or round cells !ithin the veg-
etative cell" #he location is variable, but is constant
in any one bacterial species ()igure 1"I*" 9pores can
remain dormant ,or long periods o, time" ?o!ever,
they are able to revert to actively-gro!ing cells (i"e"
germinate* relatively rapidly in response to certain
conditions such as the presence o, speci,ic sugars,
amino acids or bile salts"
9pores also have an important role in the epi-
demiology o, certain human diseases, such as
anthra$, tetanus, gas gangrene and in,ection
caused by 0lostridium di,,icile"
#he eradication o, spores is o, particular im-
portance in some processes, e"g" the production
o, sterile products including pharmaceuticals
and surgical instruments, in routine hospital
!ard and care centre cleaning, and in ,ood
preservation"
Bacterial gro!th
2ost bacteria !ill gro! on arti,icial culture media
prepared ,rom e$tracts ,rom animal or plant
tissues, !hich supply pre-,ormed nutrients and
vitamins" ?o!ever, some bacteria, e"g" 2ycobac-
terium leprae (leprosy* and #reponema pallidum
Basic bacteriology I
)igure 1"I -ram-stain o, 0lostridium sporogenes
(sho!ing oval subterminal spores* and a 0lostridium
tetani !ith a terminal spore (arro!ed*"
(syphilis*, cannot yet be gro!n in vitro1 other
bacteria, e"g" 0hlamydia spp" and Gickettsia spp",
only replicate intracellularly !ithin host cells and
are there,ore gro!n in tissue culture"
Under suitable conditions (nutrients, temper-
ature and atmosphere* a bacterial cell !ill in-
crease in si'e and then divide by binary ,ission
into t!o identical cells" #hese t!o cells are able
to gro! and divide at the same rate as the parent
cell, provided that conditions including nutrient
supply remain stable" #his results in an e$po-
nential or logarithmic gro!th rate" #he time
re3uired ,or the number o, bacteria in a culture
to double is called the generation time, e"g"
7scherichia coli has a generation time o, about
+% minutes under optimal conditions" By con-
trast, 2ycobacterium tuberculosis has a genera-
tion time o, +4 hours"
Ge3uirements ,or bacterial
gro!th
2ost bacteria o, medical importance re3uire car-
bon, nitrogen, !ater, inorganic salts and a source
o, energy ,or gro!th" #hey have various gaseous,
temperature and p? re3uirements, and can utilise
a range o, carbon, nitrogen and energy sources"
9ome bacteria also re3uire special gro!th ,actors,
including amino acids and vitamins"
-ro!th re3uirements are important in selecting
the various culture media re3uired in diagnostic
microbiology and in understanding the tests ,or
identi,ying bacteria"
0arbon and nitrogen sources
Bacteria are classi,ied into t!o main groups ac-
cording to the type o, compounds that they can
utilise as a carbon sourceE
1 Autotrophs utilise inorganic carbon ,rom carbon
dio$ide and nitrogen ,rom ammonia, nitrites
and nitrates1 they are o, minor medical
importance"
+ ?eterotrophs re3uire organic compounds as
their maCor source o, carbon and energy1 they
include most bacteria o, medical importance"
Atmospheric conditions
0arbon dio$ide
Bacteria re3uire 05
+
,or gro!th1 ade3uate
amounts are present in the air or are produced
during metabolism by the microorganisms them-
selves" A ,e! bacteria, ho!ever, re3uire addi-
tional 05
+
,or gro!th, e"g" eisseria meningitidis,
0ampylobacter CeCuni"
5$ygen
Bacteria may be classi,ied into ,our groups accord-
ing to their 5
+
re3uirementsE
1 5bligate (strict* aerobesE gro! only in the pres-
ence o, o$ygen, e"g" Pseudomonas aeruginosa"
+ 2icroaerophilic bacteriaE gro! best in lo!
o$ygen concentrations, e"g" 0ampylobacter
CeCuni"
/ 5bligate (strict* anaerobesE gro! only in the ab-
sence o, ,ree o$ygen, e"g" 0lostridium tetani"
4 )acultative anaerobesE gro! in the presence or
absence o, o$ygen, e"g" 7scherichia coli"
#emperature
2ost pathogenic bacteria gro! best at /B

0" ?o!-
ever, the optimum temperature ,or gro!th is oc-
casionally higher, e"g" ,or 0" CeCuni, it is 4+

0" #he
ability o, some bacteria to gro! at lo! tempera-
tures (%&4

0* is important in ,ood microbiology1
Listeria monocytogenes, a cause o, ,ood poisoning,
!ill gro! slo!ly at 4

0 and has resulted in out-
breaks o, ,ood poisoning associated !ith cook-
chill products"
p?
2ost pathogenic bacteria gro! best at a slightly
alkaline p? (p? B"+&B"@*" #here are a ,e! e$cep-
tionsE Lactobacillus acidophilus, present in the
C
D
B
A
*
i
a
b
l
e

c
o
u

#

(
p
e
r

m
+
)
1% Basic bacteriology
vagina o, post-pubescent ,emales, pre,ers an acid
medium (p? 4"%*" 6t produces lactic acid, !hich
keeps the vaginal secretions acid, thus preventing
many pathogenic bacteria ,rom establishing
in,ec- tion" Hibrio cholerae, the cause o, cholera,
pre,ers an alkaline environment (p? D"=*"
-ro!th in li3uid media
Fhen bacteria are added (inoculated* into a li3uid
gro!th medium, subse3uent multiplication can be
,ollo!ed by determining the total number o, live
microorganisms (viable counts* at various time
intervals" #he gro!th curve produced normally has
,our distinct phases ()igure 1"1%*E
1 Lag phase (A*E the interval bet!een inoculation
o, a ,resh gro!th medium !ith bacteria and the
commencement o, gro!th1
+ Log phase (B*E the phase o, e$ponential gro!th1
the gro!th medium becomes visibly turbid at
appro$imately 1 1%
@
cells.ml1
/ 9tationary phase (0 *E the gro!th rate slo!s as
nutrients become e$hausted, !aste products
accumulate, and the rate o, cell division e3uals
the rate o, death1 the total viable count remains
relatively constant1
4 Decline phase (D*E the rate o, bacterial division is
slo!er than the rate o, death, resulting in a
decline in the total viable count"
ote that the production o, !aste products by
bacteria, particularly 05
+
, and the uptake o, 5
+
have been utilised in the development o, semi-
automated instruments to detect bacterial gro!th
in blood samples obtained ,rom patients !ith
suspected bloodstream in,ection"
-ro!th on solid media
Li3uid gro!th media containing the nutrients
needed ,or bacterial gro!th can be solidi,ied !ith
agar, a polysaccharide e$tracted ,rom sea!eed"
?eating during sterilisation o, the medium melts
the agar, !hich then remains li3uid until the tem-
perature ,alls to appro$imately 4%

0, !hen it pro-
duces a transparent solid gel" 9olid media are
normally set in Petri dishes (;agar plates<*" Fhen
spread across the sur,ace o, an agar plate, most
bacteria gro! as visible colonies" 7ach colony
comprises millions o, bacterial cells that emanated
,rom either a single cell or a cluster o, cells" #he
appearance o, the bacterial colony (colonial mor-
phology* assists in identi,ication"
-ro!th on laboratory media
#o gro! bacteria in vitro, the microbiologist has to
take into account the physiological re3uirements"
Harious types o, li3uid and solid media have been
10
10
10
,
10
-
10
.
10
/
0 1/
)ime (&)
)igure 1"1% Bacterial
gro!th curve sho!ing
the ,our phasesE
/.
(A* lag1 (B* log or
e$ponential1
Bac#eria iocula#ed
(0* stationary1 and
(D* decline (death*"
Basic bacteriology 11
developed ,or the diagnostic microbiology
laboratory"
9imple media
2any bacteria !ill gro! in or on simple media, e"g"
nutrient broth.nutrient agar that contains
;peptone< (polypeptides and amino acids ,rom the
en'ymatic digestion o, meat* and ;meat e$tract<
(!ater-soluble components o, meat containing
mineral salts and vitamins*"
7nriched media
#hese contain additional nutrients ,or the isolation
o, more ,astidious bacteria that re3uire special
conditions ,or gro!th, e"g" agar containing !hole
blood (blood agar* or agar containing lysed blood
(chocolate agar*"
9elective media
#hese are designed to ,acilitate gro!th o, some
bacteria, !hile suppressing the gro!th o, others,
and includeE
"
mannitol salt agar !hich contains increased
a0l (salt* concentration ,or the recovery o,
staphylococci1
"
2ac0onkey agar, !hich contains bile salts and
allo!s the gro!th o, bile-tolerant bacteria only1
and
"
antibiotics, !hich are ,re3uently added to media
to allo! only certain bacteria to gro! !hile sup-
pressing or killing others"
6ndicator media
#hese are designed to aid the detection and rec-
ognition o, particular pathogens" #hey are o,ten
based on sugar ,ermentation reactions that result
in production o, acid and the subse3uent colour
change o, a p? indicator, e"g" 2ac0onkey agar
contains lactose and a p? indicator (neutral red*1
lactose-,ermenting bacteria (e"g" 7scherichia coli*
produce acid and ,orm pink colonies, !hereas
non-lactose ,ermenting bacteria (e"g" 9almonella
spp"* do not produce acid and ,orm pale yello!
colonies" #his property ,acilitates the recognition
o, possible 9almonella colonies among normal
bo!el ,lora" ote that indicator media may also
contain selective agents including antibiotics or
substances such as bile salts and crystal violet to
suppress gro!th o, most -ram-positive microor-
ganisms" 2ac0onkey agar is there,ore both a se-
lective medium and an indicator medium"