Transcript Enterobacteriaceae

LAB EXERCISE 5
Identification of
Unknown Bacteria
（Enteric Gram Negative Rods）
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Enterobacteriaceae
Lactose
fermenters (LF)
E. coli
Klebsiella
Non-lactose
fermenters (LNF)
Salmonella
Shigella
Enterobacter
Proteus
……
……
• There are several selective and differential
media used to isolate distinguishes between
LF & LNF.
• The most important media are:
– MacConKey agar
– Eosin Methylene Blue (EMB) agar
– Salmonella Shigella (SS) agar
– Triple Sugar Iron (TSI) agar
• Kligler Iron Agar (KIA)
Differentiate between LF & LNF by
Growth on SS agar
• SS agar is selective & differential medium
for Enterobacteriaceae.
Differentiate between LF & LNF by
Growth on KIA slant
Unpathogenic
Pathogenic
SOME IDENTIFICATION TEST
• Biochemical test
– Carbohydrate Fermentation
– Indole test (Enzyme test)
• Other
– Motility
• The indicator is RED at alkaline pH, and
YELLOW at acidic pH.
• Interpretation:
– Red slant /yellow butt → only glucose is
fermented.
– Yellow slant /yellow butt →glucose and lactose
both are fermented.
– Bubbles or crack present → gas production.
– Black precipitate present → H2S production.
Control
LNF
LNF
H2S produced
LF
Gases produced
• Aim: To determine the ability of microbe to
ferment a specific carbohydrate with or
without the production of gas.
• Material:
Carbohydrate
Carbohydrate
Glucose (Red cap) /golden
Mannitol (White cap)
Lactose (Yellow cap)
Sucrose (Black cap) /green
Maltose (Blue cap)
* Indicator: Phenol Red (acid → yellow)
• Interpretation:
– Acid production: Changes the medium into yellow
color- organism ferments the given carbohydrate and
produce organic acids there by reducing the pH of the
medium into acidic.
– Acid and Gas production: Changes the medium into
yellow color. Gas production can be detected by the
presence of small bubbles in the inverted Durham
tubes.
– Absence of fermentation: No change observed in the
colour of medium. The organism cannot utilize the
carbohydrate but the organism continues to grow in
the medium using other energy sources in the
medium.
• Aim: To determine the ability of microbe to
degrade the amino acid tryptophan.
• Interpretation:
– Development of cherry red colour at the
interface of the reagent and the broth, within
seconds after adding the Kovacs’ reagent
indicates the presence of indole and the test is
positive.
– If no colour change is observed, then the test is
negative and so organisms are not capable of
producing tryptophanase.
• Aim: To determine the ability of microbe to
migrate (motile).
• Interpretation:
– Organism growing only in line of
inoculation → non-motile.
– Organism appears as haze beyond line of
inoculation → motile
• Object of study :
– Use known antiserum (antibodies) to identify
unknown antigens.
• Materials and equipment：
• 1:20 Typhoid serum
• 1:20 Shiga serum
• Practise：
– Using a wax marker, draw 3 circles on 2 clean glass
slides. Label the circles A, B, and C.
– Add 1 drop of NS to the “A” circle, 1 drop of known
Typhoid serum to the “B” circle, and 1 drop of
known Shiga serum to the “C” circle.
– Emulsify one or two colonies on each drop to make
a smooth suspension.
• Interpretation:
– Agglutination of the bacteria indicates a
positive reaction.
– No agglutination is negative.
• Object of study :
– This is a tube agglutination test employed in the
serological diagnosis of enteric fever.
• Materials and equipment：
• S.TYPHI "O" antigen
• S.TYPHI "H" antigen
• S.PARATYPHI "AH" antigen
• S.PARATYPHI "BH" antigen
• Patient serum (1:10 dilution)
• 200μL pipettor, Pipettor tips,
40-well Immunoplate
• Practise：
– An immunoplate having 40 wells in 4 rows is
taken. The rows are labeled as O, H, AH, BH.
– Using a micropipettor add 50μL normal saline to
all the wells in the 40-well immunoplate.
– Add 50μL diluted patient serum (1:10) into well
O1 containing 50μL saline, mixed by sucking and
pumping the fluid with tip for 3 times.
• Practise：
– Transfer 50μL from well O1 to O2 , mixed by
gently pipetting. Use the same tip to transfer
50μL from well O2 to O3. Avoid bubbles. Using
the same tip, repeat these twofold dilution down
the entire row, discarding 50μL from A9 so that it
ends up with 50μL of diluted serum. The dilution
from well A1 to A9 is 1:20, 1:40……1:1280.
• Practise：
– Add 50μL antigen S.TYPHI "O" to each well (from
“9” to “1”), mix well. The final dilution from
well O1 to O9 is 1:40, 1:80……1:2560; Well “10”
is the control tube.
– Repeat this doubling dilution with different
antigen (H /AH /BH) in defined rows.
– Mix each well and incubate for 1h at 45℃. And
place the plate at RT standing for 15 minutes to
wake up.
• Practise：
– Observe the results and determine the titer of the
serum ( + + ) .The highest dilution of serum causing
obvious agglutination of bacteria ( + + ) is defined
as the titer of the serum.
• Results：
– First observe the control well 10. They should
show a uniform turbidity in contrast to the
aggregates seen in the wells containing the serum
dilutions . it is represented as “ - ”.
– The agglutination is represented as“+”:
• “+ + + +”represents all of the bacteria agglutinated.
Big agglutin appears at the bottom of the tube, while
the supernatant is clear.
• “+ + +”represents part of the bacteria agglutinated.
Middle agglutin appears at the bottom of the tube,
while the supernatant is a little turbid.
• “+ +” represents small part of the bacteria
agglutinated. Half of the supernatant is turbid.
• Results：
– The agglutination is represented as“+”:
• “+”represents very small amount of the bacteria
agglutinated, while the supernatant is turbid.
• “ - ”represents no agglutination. The phenomenon is
same as that of the control tube.
• Attentions
– Gently manipulate the pipettes. Do not break the test
tubes with the pipettes. Be careful when you add and
transfer the samples one by one, to avoid confusion.
– Do not shake the test tubes when you observe the
results , to avoid the spreading of the agglutinates
during observation.
O<1:80,TH<1:160, PH<1:80
Normal value
O ≥1:80 & TH ≥1:160 or
Typhoid fever
O ≥1:80 & PH ≥1:80
Paratyphoid fever
O ≥1:80 & TH <1:160 or
Early infection or other
O ≥1:80 & PH <1:80
salmonella infections
O <1:80 & TH ≥1:160 or
Vaccination or nonspecific
O <1:80 & PH ≥ 1:80
memory reaction