MICROBIOLOGY SPOTTERS
PART-III
-Dr. Babita Fageria
UREASE TEST
PRINCIPLE: To determine the ability of an organism to produce an enzyme urease
which splits urea to ammonia. Ammonia makes the medium alkaline & thus phenol red
indicator changes to pink/red in colour.
Urease
Urea CO2+ H2O+2NH3—(NH4)2CO3
INTERPRETATION:
Urease positive: Purple pink colour
e.g. Klebsiella sp., Proteus sp., Yersinia enterocolitica, Helicobacter pylori
Urease negative: Pale yellow colour
e.g. E. coli., Providencia sp., Yersinia pestis
TRIPLE-SUGAR IRON (TSI)
PRINCIPLE: Triple-Sugar iron agar is used to determine whether gram –negative rod utilizes glucose
and lactose or sucrose fermentatively and forms hydrogen sulfide (H 2S ).TSI medium facilitates
preleminary identification of gram- negative bacilli.
INTERPRETATION :
Red color (alkaline )-no fermentation
Yellow color (acidic)-fermentation of carbohydrate .
Bubbles in the butt-gas are also produced during fermentation of carbohydrate .
Blackening of the medium –H2S PRODUCTION .
Examples: A/A without gas- Vibrio Cholerae
A/A with gas- E. coli., Klebsiella pneumoniae
K/A with abundant H2S- Proteus spp
K/A with scanty H2S at the junction of butt and slant- Salmonella Typhi
K/K- Pseudomonas aeruginosa, Acinetobacter spp
LOWENSTEIN- JENSEN MEDIUM
COMPOSITION
•1. Coagulated hen’s egg
2.Mineral salt solution
3. Asparagine
4. Malachite green
CHARACTERSTICS
Acting as a selective agent inhibiting other bacteria and to provide a
contrasting color against which colonies of mycobacterium are early
seen.
LOEFFLER’S SERUM SLOPE
COMPOSITION
Nutrient broth serum (Ox ,sheep or horse )glucose
CHARACTERSTICS
Culture of corynrebacterium diphtheriae
ANTIBIOTIC DISC DIFFUSION BY KIRBY –BAUER METHOD
COMPOSITION
1. Beef extract
2. Acid hydrolysate of casein
3. Starch
4. Agar
5. Distilled water
CHARACTERISTICS
Commonly used for the routine susceptibility testing of non-fastidious microorganisms
by the kirby-bauer disc diffusion technique.
Blood culture bottles
Glucose Broth
• Composition:
Glucose 0.5%
Yeast extract base as a source of
protein
Sodium Chloride
SPS (Sodium Polyanethol
Sulfonate) which acts as
anticoagulant.
• Use: It was being used in the past
as essential medium to grow most
of the pathogenic bacteria including
fastidious ones from blood except
Salmonella, now replaced with the
more advanced alternatives.
Bile Broth
• Composition:
Peptone 2%
Sodium taurocholate
0.5% - bile salt
Sodium Chloride
• Use: It was being used
in the past for culture of
Salmonella Typhi &
Salmonella Paratyphi
from blood.
Brain-Heart Infusion (BHI) Broth
• Composition:
Calf brain, 20%
Beef heart, 25%
Proteose peptone, 1%
Dextrose, 0.2%
Sodium chloride
Disodium phosphate
• Uses:
For blood culture (commonly used now a days for
conventional blood culture)
To provide enrichment for sterile fluids with low
microbial load, such as CSF, pleural fluid etc.
BHI with hemin & vit K- for anaerobes
To prepare inoculum for antibiotic susceptibility
and detection of MIC
For fastidious fungi such as Histoplasma
capsulatum
Castaneda’s Biphasic Medium
COMPOSITION
1. Bile broth
2. Nutrient agar slant
3. Brain heart infusion broth
CHARACTERSTICS
Used for blood culture
Automated Blood culture bottle
• Composition:
Soybean-casein digest broth, 0.05%
Sodium polyanethol sulfonate, 16.0%
Nonionic adsorbing resins
1.0% cationic-exchange resins
• Use: this is used in automated blood culture
systems such as
BACTEC
BacTALERT
VersaTREK
• These systems are most commonly used now
a days for blood culture, as they have a less
turnaround time due to continuous monitoring
of bacterial growth based on clorimetric,
fluorometric, or manometric signals.
Catalase Test
Principle: when the colony of a catalse enzyme producing bacteria is mixed with
a drop of catalase reagent (3% Hydrogen Peroxide), effervescence or bubbles
appear due to breakdown of H2O2 to produce oxygen. This enzyme is produced by
bacteria to counteract toxic effects of oxygen.
Use: rapid biochemical test commonly used to differentiate Staphylococcus from
Streptococcus
Positive test- Staphylococcus, most of the pathogenic aerobic and facultative
anaerobic bacteria
Negative test- anaerobes, Streptococcus, Shigella dysenteriae type 1
Slide Coagulase Test
• This test is done to detect presence of clumping factor (bound coagulase) in the wall of
Staphylococcus species.
•Principle: clumping factor reacts directly with fibrinogen converting this to fibrin,
causing the staphylococcal cells to precipitate due to cross linking of fibrin strands.
•Procedure: a milky white suspension of bacterial colony is formed in normal saline, in
which a loopful of plasma is added.
•Interpretation:
Positive test- formation of coarse clumps- Staphylococcus aureus, Staph lugdunensis
Negative test- no change in the milky white suspension- other species of Staphylococcus
Tube Coagulase Test
• This test is done to detect presence of free coagulase
secreted by Staphylococcus aureus.
• Principle: free coagulase reacts with a substance in
serum called as CRF (coagulase reacting factor) to
activate prothrombin, which gets converted into
thrombin activating the clotting pathway
• Procedure: a mixture of plasma with normal saline
is formed in a test tube in a ratio of 1:5. colony from
bacterial culture is added and incubated at 37°C for
4 hours.
• Interpretation:
• Positive test- formation of a clot that does not
disrupt when test tube is tilted. e.g.Staphylococcus
aureus
• Negative test- no clot formation, extend the
incubation to overnight as some strands may give a
late result- species of Staphylococcus other than
aureus.
Negative Staining
• Principle: unstained
bacterial/yeast cells having
capsules stand refractile
against dark background.
• Dyes used: India ink, nigrosin,
congo red, eosin etc.
• Use: demonstration of capsule
of organisms e.g.
Pneumococcus,
Meningococcus, Haemophilus
Cryptococcus capsule demonstration influenzae, Cryptococcus
on India ink staining neoformans etc.
