Iden%fica%on

 of  medically  important  bacteria  by  

conven%onal  methods  
•   Based  on  morphology  (staining   Diagnos%c  tests  are  performed  on  
proper%es)   the  following  specimens  
•  Cultural  characteris%cs,  colony   •  Blood  
morphology    on  agar  media   •  CSF  
•  Tests  for  produc%on  of  enzymes   •  Exudates  (pus,  sputum,  bronchial  
•  Tests  for  metabolic  end  products   washings)  
•  Sensi%vity  to  chemicals   •  Tissue  
•  Serotyping  (agglu%na%on);   •  Urine  
detec%on  of  an%gens   •  stool  
•  An%bio%c  suscep%bility  profile  
Microscopy:   Culture    
–  Gram  stain   •  Cul%va%on  and  growth  of  
bacteria  required  for  defini3ve  
–  Acid  fast  stain   iden3fica3on  and  
–  Flurorescent  stain   characterisa3on  of  the  infec3ous  
–  Immunofluorescence   agent  and  for  performing  
an3bio3c  suscep3bility  tes3ng  
–  Dark  ground  microscopy  
–  Simple  stains   •  Achieved  by  isola%ng  the  bacteria  
–  Wet  mount   from  the  clinical  specimens  
collected  from  the  infec%ous  site  
on  ar3ficial  culture  media.    

•  Ar%ficial  media  used  for  growing  

bacteria  should  contain  the  basic  
nutri%onal  requirements  for  non  
fas3dious  bacteria  and  special  
nutri%ve  supplements  for  
fas3dious  bacteria  
 
 Culture  media  
Growth  media  are  used  in  either  of   •  Solid  agar  media:  
two  phases:     •  These  media  are  made  by  adding  a  
•  Liquid  (broth)     solidifying  agent  like  agar  to  the  
•  Solid  (agar)     nutrients  and  water.  
•  A  biphasic  medium  that  contains  
both  liquid  and  solid  (blood  
culture  medium)  

Types  of  media:  

•  Liquid  or  broth  media  
•  Nutrients  dissolved  in  water;  
bacterial  growth  indicated  by  
change  in  the  broth’s  appearance  
from  clear  to  cloudy  or  turbid.    
•  Due  to  light  deflected  by  bacteria  
present  in  the  culture  
Examples  of  broth  media  and  their  
use  
•  Tryp%c  soya  broth  (TSB):  for  
blood  culture  
•  Solid  media  are  used  to  isolate  bacteria  
in  a  “pure  culture”  which  is  absolutely  
essen3al  for  iden3fying  and  
characterising  the  pathogens.  

•  Clinical  specimens  containing  suspected  

bacteria  are  spread  (streaked)  on  the  
surface  of  appropriate  solid  agar  media,  
incubated  under  appropriate  
condi%ons,    

•  Each  bacterial  cell  from  the  specimen  

inoculated  on  the  surface  of  agar  
medium  will  proliferate  to  sufficiently  
large  numbers  to  be  observed  under  
the  naked  eye.    
•  The  resul%ng  bacterial  popula%on  is  
considered  to  be  derived  from  a  single  
bacterial  cell  and  is  known  as  a  colony;    
•  Bacteria  within  a  single  colony  are  the  
same  genus  and  species,  having  
iden%cal  gene%c  and  phenotypic  
characteris%cs.    

•  Bacterial  cultures  derived  from  a  single  

colony  are  considered  pure  
  
Media  classifica3on  and  func3ons:  
•  Media  categorised  according  to     •  Basal  suppor3ve  media:  contain  
their  func%on  and  use.   nutrients  that  support  growth  of  
Five  general  categories  of  media   most  non  fas%dious  organisms  
•  Basal  suppor%ve  media   without  giving  any  par%cular  
organism  a  growth  advantage  
•  Enriched  media  
•  Example;  Nutrient  agar  
•  Enrichment  media  
•  Selec%ve  media  
•  Differen%al  media  
 Alpha hemolysis,
Streptococcus pneumoniae,
S.mutans •  Enriched  media:  contain  enriched  
substance  like  blood,  serum,  egg  
to  support  the  growth  of  
fas%dious  bacteria  

•  Blood  agar:  used  for  the  

cul%va%on  of  fas%dious  
organisms,  determina%on  of  
Gamma hemolysis, hemoly%c  proper%es  especially  
Enterococcus spp., Streptococcus  spp;  Bacteria  may  
show  beta  hemolysis  (complete  
lysis  of  RBC),  alpha  hemolysis  
(par%al  lysis  of  RBC)  or  gamma  
hemolysis  (no  hemolysis)  
Beta hemolysis,
Streptococcus pyogenes
 Culture  media  

•  Chocolate  agar:  prepared  with  

lysed  or  heated  blood.  Lysed  RBC  
release  growth  factors  like  
haemin  and  NAD  which  is  
required  by  fas%dious  bacteria  
like  Haemophilus  influenzae  and  
pathogenic  Neisseria  spp.,  

Chocolate agar
•  Loeffler’s  coagulated  serum:  
growth  of  Corynebacterim  
diphtheria  and  produc%on  of  
metachroma:c  granules  
 Buffered charcoal
yeast extract agar
with Legionella Enrichment  media:  contain  specific  
colonies nutrients  required  for  the  
growth  of  a  par%cular  pathogen    
•  used  to  enhance  the  growth  of  
a  par%cular  pathogen  from  a  
mixture  of  organisms  by  taking  
advantage  of  its  nutri%on  
specificity  

•  Buffered  charcoal  yeast  extract  

agar:  used  for  isola%on  and  
enrichment  of  Legionella  
pneumophila  

•  Regan  Lowe:  enrichment  agar  

for  Bordetella  pertussis  
(selec%ve  agar)  

Regan Lowe with Bordetella pertussis

colonies
 Selec3ve  media:  one  or  more  
agents  that  are  inhibitory  to  all  
organisms  except  those  being  
isolated.  Inhibitory  agents  
used  are  an%bio%cs,  
S.AUREUS chemicals,  dyes,  bile  salts.  
•  Mannitol  salt  agar:  selec%on  
of  Staphylococcus  aureus  from  
clinical  specimens  

•  Cysteine  tellurite  blood  agar:  

for  selec%on  and  isola%on  of  
Corynebacterium  diphtheria  
from  throat  and  
nasopharyngeal  swabs  

Black colonies of C.diphtheria on

cysteine tellurite blood agar
 •  New  York  City  agar  or  Thayer  
Mar3n  Agar:  Selec%ve  agar  for  
Neisseria  gonorrhoea  from  genital  
swabs  rectal,  throat  swabs  and  also  
for  N.meningi:dis  from  throat  
swabs  

•  TCBS:  selec%ve  and  differen%al  for  

Vibrio  cholerae  from  stool  
specimens  

TCBS agar
•  MacConkey  sorbitol  agar:  
selec%on  and  differen%a%on  of  
E.coli  O157:H7  in  stool  
specimens  
•  EHEC  (E.coli  O157:H7)  does  not  
ferment  sorbitol  
 Pink lactose Pale non lactose
fermenting colonies fermenting colonies
Differen3al  media:  contain  some  
nutrient  or  chemical  which  allows  
colonies  of  bacterial  species  or  
type  to  exhibit  certain  metabolic  
or  culture  characteris%cs  which  
can  be  used  to  dis%nguish  them  
from  other  bacteria  in  the  same  
plate  

•  MacConkey’s  agar:  most  

A; Lactose and B; non lactose fermenting colonies
commonly  used  differen%al  
medium  for  differen%a%ng  
lactose  fermen%ng  Gram  nega%ve  
rods  from  non  lactose  fermen%ng  
Gram  nega%ve  rods;  from  stool  
specimens,  urine  and  other  
specimens.  It  is  also  a  selec%ve  
medium  which  does  not  allow  
most  Gram  posi%ve  bacteria  to  
grow.    
 •  EMB  (eosin  methylene  blue)  agar:  
differen%al  agar  for  differen%a%ng  
lactose  fermen%ng  and  non  lactose  
fermen%ng  Gram  nega%ve  rods  
from  urine  specimens  

•  Hecktoen  enteric  agar:  defferen%al  

and  selec%ve  medium  for  the  
isola%on  and  differen%a%on  of  
Salmonella  and  Shigella  from  other  
Gram  nega%ve  enteric  bacilli  in  
stool  

Black colonies of Salmonella and pale

colonies of Shigella and E.coli
 Differen%a%ng  Staphylococci  from  Streptococci  
•  Gram  stain  and  morphology   •  Enzyme  tests  
–  Both  Gram  posi%ve   •  Catalase  test:  
–  Staphylococci:  clustered  cocci   –  Enzyme  catalase  breaks  down  
hydrogen  peroxide  into  H2O+O2  
(grape  like)  
–  Streptococci:  chained  cocci     –  Staphylococci:  catalase  +  
–  Streptococci:  catalase  -­‐  
•  S.  pneumoniae:  
diplococcus  
•  Growth  
–  Staph.:  large  colonies  (non-­‐
fas%dious),  some  hemoly%c  
–  Strep.:  small  colonies  
(fas%dious),  many  hemoly%c  
 Gram positive cocci in clusters Gram positive cocci in chains

Catalase positive Catalase negative

BETA HEMOLYSIS ON BLOOD AGAR

Gram positive cocci in clusters

Staphylococcus colonies grown

on Blood agar showing beta
hemolysis

Golden yellow White

Coagulase
test

Cagulase + S.aurues
 Gram positive cocci

clusters chains

Staphylococci Streptococci

Beta hemolysis on blood agar

Staphylococci
Streptococci

Catalase test

positive negative

Staphylococci Streptococci

coagulase
positive negative
Staphylococcus aureus Staphylococcus epidermidis/
saprophyticus
 Streptococci  
streptococci Growth on blood agar identification

Streptococcus pyogenes Beta hemolysis Sensitive to Bacitracin

Group A

Streptococcus agalactiae Beta hemolysis Resistant to Bacitracin

Group B

Streptococcus pneumoniae Alpha hemolysis Sensitive to optochin

Viridans streptococci Alpha hemolysis Resistant to optochin
Enterococci Alpha hemolysis or no Resistance to 40% bile
hemolysis
Gram positive cocci in pairs Gram positive cocci in chains
Alpha hemolysis,
Streptococcus pneumoniae,
S.mutans

Gamma hemolysis,
Enterococcus spp., Beta hemolysis,
Streptococcus pyogenes
Streptococcus agalactiae
 Gram positive cocci in chains

Beta hemolysis
Group A? group B?

GROUP A BACITRACIN GROUP B BACITRACIN

SENSITIVE RESISTANT

CATALASE
negative

PYR POSITIVE
PYROGLUTAMYL
Alpha hemolysis, Streptococcus pneumoniae,?
S.mutans ? Viridans streptococci
 Optochin resistant
S.mitis

Optochin sensitivity
S.pneumoniae

Quellung (capsular typing)

SEROTYPING
 Streptococci

Alpha hemolysis
Beta hemolysis on blood agar
on blood agar

Streptococcus Viridans
pneumoniae streptococci

Optochin
Group A or Group B

sensitive resistant Bacitracin

Bile solubility
Sensitive resistant
soluble insoluble
Group A Group B

Streptococcus Viridans
pneumoniae streptococci
 Enterococcus
• No hemolysis on
blood agar
• 40% bile tolerance
• Grow in 6.5%
sodium chloride

Gamma hemolysis,
Enterococcus spp.
GRAM NEGATIVE COCCI
 Growth on
Thayer Martin
medium

Oxidase test positive

Fermentation of sugars

Neisseria gonorrhoea

Neisseria meningitidis
 Gram negative cocci in pairs

Growth on chocolate agar

Oxidase test

positive

Neisseria

Ferments glucose and

Ferments glucose
maltose

Neisseria gonorrhea Neisseria meningtidis

 Differen%a%ng  rods  
Metabolism   •  Pathogenic  enteric  are  Lactose  
•  U%liza%on  of  specific  substrates   non  fermen%ng-­‐  (important  
–  Lactose     dis%nc%on  from  non-­‐pathogenic  
–  Citrate     enteric)  
•  Produc%on  of  certain  end   •  Citrate  slant  agar:  dis%nguish  two  
products   commonly  seem  Gram  nega%ve  
–  Fermenta%on  end  products     rods  (E.coli  vs.  Klebsiella)  
–  Acid  (acetate,  propionic  acid,  butyric   •  H2S:  important  in  dis%nguishing  
acid  etc.)  
Salmonella(+)  from  Shigella(-­‐)  
–  Acetoin    
–  Alcohol  
•  Urease:  differen%ate  Proteus  
–  Amine  
from  othe  non  lactose  fermenters  
•  Produc%on  of  enzymes  
–  Urease  
•  Specialized  tests  
–  Immunological  
•  O-­‐,  H-­‐  &  K-­‐Ag  (serotype)  
•  Agglu%na%on  
–  An%biogram  pabern  
–  Phage  typing  
 Gram negative rods with simple growth requirements

Gram negative bacilli with capsule

E.coli/ Klebsiella/ Salmonella/ Shigella/

Proteus

Klebsiella pneumoniae
Gram negative bacteria; A lactose fermenting; (E.coli or Klebsiella)
B. non lactose fermenting colonies (Salmonella or Proteus or
Pseudomonas or Shigella on Mac Conkey’s medium
 Lactose fermenting Gram negative bacilli
E.coli ??
Klebsiella ??

Indole test

Positive; E.coli Negative; Klebsiella

Indole Test  
CITRATE UTILISED CITRATE NOT
KLEBSIELLA UTILISED
E.COLI
 Identifying Non lactose fermenting colonies B

Oxidase test

Oxidase positive
Oxidase negative
Pseudomonas spp;
Salmonella spp;
Vibrio cholerae
Shigella spp;
Proteus spp;
 Oxidase negative: PROTEUS AND
SALMONELLA

Urease +ve

swarming

Proteus spp; swarming and urease positive

Salmonella producing H2S
 Urease Test  
H2 O
Urea CO2 + NH3 NH4+ + OH-
Triple Sugar Iron Agar Slants  

TSI
• Fermentation of glucose,
lactose, and/or sucrose
• Reduction of sulfur to
hydrogen sulfide
• Gas formation

Used for
Enterobacteriaceae
 API system for identification

API test strip

 OXIDASE  POSITIVE  
VIBRIO  CHOLEARAE  

COMMA SHAPED GRAM NEG

BACILLI
 CAMPYLOBACTER

CAMPYLOBACTER COLONIES
GROWN AT 420c MICROAEROPHILIC

CURVED RODS (SEA GULL

WING APPEARANCE )
 Gram  nega%ve  rods  
Straight  rods   Curved  rods  
Growth  on  Mac  Conkeys  agar   oxidase    
posi%ve  
Lactose  fermen%ng   Non  Lactose  fermen%ng  
OXIDASE   TCBS  agar   Campy  Blood  
E.  coli  
posi%ve   nega%ve   agar  
Klebsiella   Yellow     42oC+    
Pseudomonas   Salmonella  
Vibrio  
Citrate   Shigella   Campylobacter  
Indole   Proteus  
Urease  test  
Citrate  not   Citrate    
u%lized   u%lized   posi%ve   Nega%ve  
Indole  +   Indole  -­‐   Salmonella  or  Shigella  
Proteus  
E.  coli   Klebsiella  
 Salmonella or Shigella

Non lactose fermenting

Motility

Motile Non Motile

H 2S
No H2S
producer

Salmonella Shigella
 Gram positive rods
Aerobic Gram positive rods
Bacillus anthracis Listeria monocytogenes

Corynebacterium diphtheriae Nocardia spp.,

 Gram positive anaerobic rods

Clostridium spp., with spores

Actinomycetes spp., Clostridium spp., with spores

 Gram negative rods

Anaerobic rods;
Bacteroides fragilis
Gram negative cocco
bacilli with fastidious
growth requirements

Haemophilus influenzae
 Culture performed using chocolate
agar

Satellitsm on blood agar (growth factors X and V)

BORDETELLA PERTUSSIS

COLONIES ON REAGAN LOWE

LEGIONELLA PNEUMOPHILA

BUFFERED CHARCOAL YEAST EXTRACT

AGAR
 BRUCELLA SPP;

Gram negative cocco bacilli

SPIROCHAETES
Silver stained Treponema pallidum,
FUSOBACTERIUM
MYCOBACTERIA ;ACID FAST BACTERIA (ZIEHL NEELSEN STAIN)

COLONIES ON
LOWENSTEIN
JENSEN MEDIUM