test Starch Hydrolysis Gelatin

Liquefaction
X-ray film Catalase production
Purpose Detect bacteria that produce
-amylase
Detect bacteria
that produce
Gelatinase
Detect bacteria
that produce
Gelatinase
Detect bacteria that produce
Catalase
Principle In the presence of starch
iodine will form complex
with it purple or or black
color
Hydrolysis of starch - No
starch to react with iodine
clear zone after adding
iodine
Gelatin solid at a
temperature of
25
o
C or lower.

gelatinase
breaks up the
gelatin the
medium cannot
solidify at cold
temperatures
X-ray film is
coated with
gelatin emulsion.

gelatinase
breaks up the
gelatin on X-ray
clear zone on
x-ray film

Catalase hydrogen peroxide
into water and oxygen
bubbles
Media
contents
Nutrient Agar + starch Gelatin tube of 0.5 ml
saline.
Tube method : Nutrient Agar
Slide method : Add H2o2 Drop
Add the organism on it !
Media
type
Agar plate Gelatin Tube Liquid tube agar slant tube
Media
color
Black after adding iodine transparent --- Trasnparent
Reagents Iodine None X-ray film strip 3% Hydrogen peroxide
Results +ve clear zone after
adding iodine


-ve No clear zone , plate
remain black
After 30 days
+ve Reamain
liquid after
cooling
-be Become
sold after cooling
+ve clear
zone on x-ray

-ve no change
of X-ray film
+ve Gas bubbles


-ve No Bubbles
Examples +ve : Bacillus subtilis
-ve : E. Coli
+ve : Serratia, Pseudomonas, and
Vibrio

+ve : -ve :
Staphylococci Streptococci
Listeria
monocytogenes
beta
hemolytic
streptococci.
Neisseria
Bacillus Clostridium
Biochemical tests
test Coagulase Test MR-VP tests Oxidase Test Tryptophan
hydrolysis
Purpose Detect bacteria that
Have Coagulase
MR portion : Detect
bacteria that ferment
glucose to acidic products
VP portion : Detect
bacteria that ferment
glucose to acetoin
Detect bacteria that have
Cytochrome oxidase
Detect bacteria
that have
tryptophanase
Principle Plasma " contain
fibrinogen "

CoagulaseFibrinog
en
To fibrin clump
the plasma
MR portion :
Dextrose ferment to
acid products pH drop
below pH 5 methyl red
"indictor " turn red

VP portion : Dextrose
ferment to Acetoin Add
Barritt's reagents " alpha-
naphthol & potassium
hydroxide " red color

Cytochrome oxidase
O2 & reduced will be
oxidized to to form water
and oxidized cytochrome c.
cytochrome oxidase will
oxidase the test reagent
purple color
Tryptophan by
tryptophanase
indole and other
products

Indole + Kovac's
reagent red
color
test
contents
Test slide : plasma +
organism
Media : MR-VP Broth :
Liquid media contain
Dextrose

transfer the bacteria to the
filter paper Add oxidase
reagent Purple color
Media : Tryptone
broth "liquid
media contain
tryptophan "
Media type None Broth "liquid" None Broth "liquid"
Media
color
---- transparent ---- transparent
Reagents None MR portion : Methyl Red
VP portion : Barritt's
reagents
A: 5% Alpha-Naphthol
B: Potassium Hydroxide
oxidase test reagent :
(tetramethyl-p-
phenylenediamine
dihydrochloride or an
Oxidase Disk, p
aminodimethylaniline
Kovacs reagent
contains HCL ,
dimethylaminoben
zaldehyde, and
amyl alcohol
yellow in color.

Results +ve plasma
clumps
-ve No clumps
MR portion :
+ve Red color

-ve yellow color

VP portion :
+ve Red color
-ve copper in colo
+ve purple color
-ve No color change
+ve red/pink
layer "red ring "

-ve No Layer
Examples +ve :
Staphylococcus
aureus
-ve : the rest of
Staphylocci
MR +ve : E. Coli
MR ve : Enterobacter

VP +ve : Enterobacter
VP ve : E. Coli
+ve : many gram negative
and gram positive
-ve : Enterobacteriaceae
+ve : E. Coli
ve : Enterobacter



test Citrate Utilization
test
Urease Test Nitrate reduction Zinc test
Purpose Determine ability of
an organism to use
citrate as sole
carbon source.
Detect bacteria that
have urease
bacteria that have nitrate
reductase , so it can utilize
organic compounds as
sources of carbon and use
nitrate as a terminal
electron acceptor during
anaerobic respiration.

Distinguish between
bacteria didn't utilize
nitrate or that
reduced nitrite
further to ammonia or
molecular nitrogen
- Done after nitrate
test if it was
negative -
Principle Metabolize citrate
& utilize ammonium
relaes ammonia
increasing the pH
Bromthymol blue
"indicator " turns
from green to blue
Urease convert
urea ammonia,
CO2, and water
ammonia increase pH
phenol red" pH
indicator " change
from red to bright pink
"
nitrate reductase reduce
nitrate to nitrite add
Sulfanic acid and
naphthylamine red color

No color change : either
bacteria didn't utilize nitrate
or it reduced nitrite further
to ammonia or molecular
nitrogen
-- use zinc to distinguish it -
If nitrate wasn't
reduced :
Zinc reduce nitrate
to nitrite the
Sulfanic acid and
naphthylamine that
were added in the
nitrate test change
to red color
If nitrate was further
reduced : no color
change
test
contents
Simmons Citrate
Agar : contains
sodium citrate
(carbon source),
ammonium ion ,
PH indicator
bromthymol Blue
Media :
urea slant agar :
contain urea + agar
pH indicator phenol
red
Nitrate broth containing
0.5% potassium nitrate

Nitrat test
Media type Agar slant tube Broth "liquid" Broth "liquid" Broth "liquid"
Media
color
Green

Yellow

Transparent Transparent
Reagents None None Reagent A (Sulfanic acid) and
reagent B (naphthylamine)
Zinc powder
Results +ve blue

-ve green
+ve bright pink
color

+ve red color

-ve No color change
+ve red color
-ve No color
change

-ve media remain
yellow
Examples +ve : Enterobacter
ve : E. Coli
+ve Proteus

+ve : Escherichia coli
Klebsiella pneumonia
Morganella morganii and
Proteus mirabilis

+ve complete : (full
reduction ): Pseudomonas
aeruginosa must use zinc
to prove that NO color
change !!!!!

-ve : Acinetobacter
Staphylococcus epidermidis
+ve : Acinetobacter
Staphylococcus
epidermidis

ve : Pseudomonas
aeruginosa



test Motility test SIM agar
Purpose Detect motile bacteria 1-Determine ability of an
organism to produce H2S
2- Determine the abilitry to
produce Indole
3- Determine motility



Principle Stab quickly a tube of
semi solid media if
motile, there will be
growth going out
away from the stab
line, and test is
positive

If bacteria is not
motile, there will only
be growth along the
stab line.
Media contain Cysteine and
sodium thiosulfate
bacteria metabolized these
amino acids Produce H2S
ferrous sulfate "
indicator" react with H2S
black color

If motile the entire tube
become black
If non motile black
pericipitate only around the
stab line

To test For indole add
Kovac's reagent red color

test
contents
Semi solid media
contains tryptose,
sodium chloride, agar,
and a color indicator
"tetrazolium chloride"
SIM media : sulfur
containing amino acid
"cysteine, sodium
thiosulfate" ,
ferrous sulfate"indicator"

Media
type
Semi solid Agar semisolid
Media Transparent Transparent
Differential and selective Media
color
Reagents None Kovac's reagent
Results +ve = Motile : growth
going out away from
the stab line

-ve = Non Motile =
growth along the stab
line


H2S and motile the whole
tube become black
H2S and non motile black
around stab line
Non H2S No color change

After addition of Kovac's
Indole +ve : +ve red/pink
layer "red ring "
-ve No Layer

Examples +ve : Salmonella
Proteus


-ve : Klebsilla
H2S and motile:
Salmonella
Proteus
H2S and non motile :
Citrobacter

Non H2S and non motile :
Klebsiella


















test Mannitol Salt Agar
(MSA)
Eosin Methylene Blue
Agar
MacConkey Agar Blood agar
Purpose 1-Allow growth of
staphylococci
2- Differentiate
between
Staphylococcus
aureus & other
staphylococci
1-allow growth of Gram-
negative bacteria.
2- Differentiate between of
Gram-negative
"Enterobactericae"


1-allow growth of
Gram-negative bacteria.
2- Differentiate
between Gram-
negative "


Detect bacteria that
produce enzymes
hemolysins (exotoxin)
that act on the red
cells
Principle high salt
concentration inhibits
the growth of most
bacteria other than
staphylococci

Staphylococcus
aureus ferment
ferments the mannitol
acid lower pH
changes the
indicator "phenol red "
from red to yellow
contains methylene blue and
eosin inhibit the growth
of Gram-positive

contains lactose & sucrose

Lactose and sucrose
fermenters dark metallic
green colonies " E. coli "
Only Lactose fermenters
pink colonies "Enterobacter"
Non-fermenters produce
no color and take the color
contains bile salts and
crystal violet inhibit
the growth of Gram-
positive


Lactose fermenters
Pink colonies
" E. coli , Enterobacter "
Non-fermenters
produce no color and
take the color of the
media "only growth" -
Beta hemolysis:
Enzymes lyse the
blood cells completely,
producing a clear area
around the colony.
Alpha hemolysis:
Incomplete hemolysis
produces a greenish
discoloration around
the colony.
Gamma hemolysis: No
effect on the red cells.



of the media " Salmonella " Salmonella
Media
contents
Mannitol
7.5% sodium chloride
phenol red "indicator
"
methylene blue
eosin

bile salts and crystal
violet
Nutrient agar + 5%
sheep blood


Media
type
Agar plate Agar plate Agar plate Agar plate
Media
color




Results S. aureus
- growth + color
change from red to
yellow

Other staphylococci
-only growth , color
remain red

Other bacteria
No growth or color
change
dark metallic green colonies
Lactose and sucrose " E.
coli
&
K. pneumonia"Mucoid "

pink colonies lactose only
" Enterobacter"

No color change , only
growth Non-fermenters
" Salmonella"


pink colonies
lactose " E. coli ,
Enterobacter , K.
pneumonia"Mucoid "

No color change , only
growth
Non-fermenters
" Salmonella"

Clear zone beta
hemolysis


Greenish discoloration
alpha hemolysis

No effect gamma
hemolysis