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Microscopic Identification (Intracellular enzyme biochemical test (Triple…
Microscopic Identification
Acid-Fast stain
Ziehl-Neelsen stain
(heat is used)
Stain with
Carbolfuchsin
, steam over boiling water
Decolourised with
acid-alcohol
for 20 seconds, then rinse
Counterstain with
methylene blue
for 30 seconds, then rinse
Dry and examine
Kinyoun stain
(cold- heat is not used)
Mycobacterium
Nocardia
sp.
Capsule stain
Anthony's capsule stain
Crystal violet
air dry
3.
copper sulphate
Maneval's capsule stain
Congo red
air dry
Maneval's solution
(acetic acid +fuchsin)
Capsules are seen as
clear halo
Without heat
Eg:
Bacillus anthraxis, Streptococcus pneumoniae, Klebsiella
spp.
Spore stain
Heat
is employed -so that dyes can penetrate into spores
Alcohol decolourises vegetative cells
Spores are stained with
malachite green
, vegetative cells are counter-stained with
safranin
Extracellular enzyme
biochemical test
Coagulase test
:star:
Bound coagulase
Free and bound coagulase
Starch hydrolysis
Lipid hydrolysis
Casein hydrolysis
Gelatin hydrolysis
Intracellular enzyme
biochemical test
Carbohydrate fermentation
test
Phenol red
as pH indicator
Fermentation occurs when there is
acid (yellow colour) and gas
Triple Sugar-Iron (TSI) Agar Test
To differentiate
enterobacteriaceae
:<3:
Contains triple sugars (dextrose,sucrose and lactose),phenol red (pH indicator) and ferrous ammonium sulphate (detect H2S) -
HS2 appear as darkening
Acid slant,acid butt, no H2S
Escherichia, Klebsiella,Enterobacter
Acid slant, acid butt,
H2S present
Proteus
spp
, Arizona, Citrobacter
Alkaline slant, acid butt, no H2S
Shigella spp, Proteus spp.
Alkaline slant,acid butt,
H2S present
Salmonella, Arizona,Citrobacter
Alkaline slant, alkaline butt
Pseudomonas, Acinetobacter, Alcaligenes
IMViC Test
Indole test
Kovac's reagent
To differentiate
Enterobacteriaceae
:<3:
Methyl Red test
Ability to
ferment glucose
and maintain
stable acid end products
methyl red
as pH indicator (
red:acidic
, yellow:basic)
Positive: without acetoin,
Negative: acetoin is present
Voges-Proskauer test
Test the
presence of acetoin
+ve for methyl red test will be
-ve for Voges-Porskauer test
Citrate Utilisation test
ability to
ferment citrate
Simmons citrate agar -
cirate
as source of carbon,
bromothymol blue
as pH indicator
Litmus milk reaction
Ability to
metabolise milk
Lactose fermentation -pink
Curd formation
Nitrate Reduction
Ability to reduce nitrate to nitrite
Negative result - after adding
sulfanilic acid and alpha-naphtylamine
Urease Test
Differentiate
Proteus
from the
non-lactose fermenting Enterobacteriaceae
Ability to hydrolyse urea into ammonia and carbon dioxide
Phenol red as pH indicator
Catalase Test
Produced in
aerobic respiration
Break down hydrogen peroxide into water and oxygen
Oxidase test
Cytochrome C oxidase
TMPD / Wurster's Blue
becoming oxidsed into
dark blue colour
in the presence of cytochrome C oxidase and free oxygen
Oxidase
+ve
:
Pseudomonas,Neisseria,Moraxella
Oxidase
-ve
:
Enterobacteriaceae
tetra-methyl-p-phenylenediamine dihydrochloride (TMPD) is the
artificial electron donor
Multitest Microsystem
Rapid and automated
API Rapid 20E
Enterobacteriaceae
Enterotube II
Enterobacteriaceae
Crystal Gram+ve
Gram +ve cocci and bacilli
Rapid NH
Neisseria, Haemophillus
,and some Gram -ve
Immunodiagnostic procedures/Antigen-antibody reaction
Agglutination techniques
Precipitation techniques
EG: ELISA
Complement and complement fixation techniques
Fluorescent antibody techniques
For bacteria that
do not grow on artificial media
Molecular diagnostic procedures
DNA Polymerase Chain Reaction (PCR)
For slow-growing or non-cultured bacteria
Quickly diagnose bacterial infection disease
